DEVELOPMENT AND APPLICATION OF BOVINE IN VITRO FERTILIZATION · DEVELOPMENT AND APPLICATION OF BOVINE IN VITRO ... DEVELOPMENT AND APPLICATION OF BOVINE IN VITRO FERTILIZATION by

DEVELOPMENT AND APPLICATION OF BOVINE IN VITRO FERTILIZATION

by

SAKSIRI SIRISATHIEN

(Under the direction of BENJAMIN G. BRACKETT)

ABSTRACT

Improved understanding of in vitro fertilization, in vitro oocyte maturation, and embryo culture

will allow laboratory production of bovine blastocysts to become a well-established procedure. However,

data related to production of bovine blastocysts in chemically defined media, i.e., no component of

animal-origin included, are limited. Bovine blastocyst production in chemically defined media is an

important technology that remains in need of improvement. The objective of this study was to examine

the influence of growth factors or cytokines supplemented to embryo culture media on bovine embryonic

development. A better means for handling of ovaries to maximize the efficiency of this procedure was

also evaluated. Experimentation was also conducted to assess the feasibility of using bovine spermatozoa

as a gene transfer vector to produce transgenic bovine embryos through in vitro fertilization or through

intracytoplasmic sperm injection.

Results presented here demonstrated that the way in which bovine ovaries were handled

had a great impact on developmental competence of the harvested oocytes. Two hours of

postmortem delay prior to oocyte aspiration benefited in vitro bovine embryo production.

An effectect of adding leukemia inhibitory factor to embryo culture media was shown to increase

numbers of inner cell mass nuclei without improving blastocyst yields or survival after

cryopreservation. Also, this treatment stimulated in vitro hatching of blastocysts from their

surrounding zonae pellucidae. Both epidermal growth factor and insulin-like growth factor-I

improved rates of blastocyst development (from inseminated oocytes) when added to defined

embryo culture media at a concentration of 5 and 50 ng/mL, respectively. No significant

interaction resulting from combined treatment with those growth factors was detected. Insulin-

like growth factor-I also increased numbers of inner cell mass nuclei and reduced numbers of

DNA fragmented nuclei while epidermal growth factor had no effect either on numbers of inner

cell mass nuclei or numbers of DNA fragmented nuclei when compared to untreated controls.

Only bovine epididymal spermatozoa, not ejaculated spermatozoa, possess an ability to

take up exogenous DNA. However, in contrast to recent findings with murine spermatozoa, there

remains a barrier in efforts to adopt this approach for bovine spermatozoa as gene transfer vectors

either in conjunction with in vitro fertilization or intracytoplasmic sperm injection. This body of

research has contributed to a better understanding of requirements for laboratory production of

bovine blastocysts and provides encouragement for realistic optimization of this reproductive

biotechnology.

INDEX WORDS: Bovine blastocyst, In vitro fertilization, Epidermal growth factor,

Insulin-like growth factor-I, Leukemia inhibitory factor,

Intracytoplasmic sperm injection, DNA uptake, Transgenic,

Chemically defined media, Nuclear DNA fragmentation.


by

SAKSIRI SIRISATHIEN

D.V.M., Kasetsart University, Thailand, 1993

A Dissertation Submitted to The Graduate Faculty of The University of Georgia in Partial

Fulfillment of the Requirements for the Degree

DOCTOR OF PHILOSOPHY

ATHENS, GEORGIA

2002

© 2002

Saksiri Sirisathien

All Rights Reserved


by

SAKSIRI SIRISATHIEN

Major Professor: Benjamin G. Brackett Committee: Hugh D. Doowah

Oliver W. Li Royal A. McGraw Steven L. Stice

Electronic Version Approved:

Maureen Grasso

Dean of The Graduate School

The University of Georgia

December 2002

iv

TABLE OF CONTENTS

CHAPTER Page

1 INTRODUCTION……………………………………………………………1

2 LITERATURE REVIEW

A. EXPERIMENTAL FINDINGS LEADING TO

IMPROVEMENT OF IN VITRO PRODUCTION OF BOVINE

BLASTOCYSTS IN CHEMICALLY DEFINED CONDITIONS……...7

B. GENE TRANSFER METHODOLOGIES FOR

MAMMALS……………………………………………………………39

3 EFFECT OF LEUKEMIA INHIBITORY FACTOR ON

BOVINE EMBRYOS PRODUCED IN VITRO UNDER

CHEMICALLY DEFINED CONDITIONS……………………………..…95

4 BENEFICIAL POSTMORTEM INFLUENCE ON PRODUCTION

OF BOVINE BLASTOCYSTS IN VITRO…………………………..…...122

5 INFLUENCES OF EPIDERMAL GROWTH FACTOR AND

INSULIN-LIKE GROWTH FACTOR-I ON BOVINE

BLASTOCYST DEVELOPMENT IN VITRO……………………..…….131

6 TUNEL ANALYSES OF BOVINE BLASTOCYSTS AFTER

CULTURE WITH EPIDERMAL GROWTH FACTOR AND

INSULIN-LIKE GROWTH FACTOR………………………………..…..154

7 BULL SPERM UPTAKE OF EXOGENOUS DNA AND EFFORTS

TO OBTAIN TRANSGENIC EMBRYOS………………………..…...…175

8 CONCLUSIONS………………………………………………..……..….197

APPENDICES……………………………………………………………………………...200

v

1

CHAPTER 1

INTRODUCTION

In vitro production of bovine blastocysts is a multi-step procedure consisting of the

maturation of immature oocytes, fertilization of mature oocytes, and culture of embryos until the

blastocyst stage. The production of bovine blastocysts in vitro is a promising approach to

maximize the use of bovine gametes. For in vivo bovine blastocyst production, it is conceivable

that the average numbers of 4.5 to 5 blastocysts are expected from each superovulated donor

(Hasler, 1992). Bousquet et al (1999) showed that, during a 60-day period, in vitro production of

bovine blastocysts in conjunction with transvaginal oocyte recovery resulted in 18.8 transferable

blastocysts whereas the in vivo production approach resulted in 4.3 transferable blastocysts.

Using media containing serum and somatic cells, bovine blastocyst development in vitro at the

rates above 70 % of oocytes has been achieved after proper ovarian stimulation procedures

(Blondin et al., 2002, Rizos et al., 2002).

Enormous progress in the production of bovine blastocysts in vitro has been made since

the first calf was born from an in vitro fertilized embryo in 1981 (Brackett et al., 1982).

Improved fertilization rates, as represented by the cleavage of over 80 % of oocytes followed the

use of heparin treatment for sperm capacitation (Parrish et al., 1985). Improvement in maturation

of immature oocytes in vitro has been made mainly by the introduction of gonadotropins,

estradiol, and several growth factors into the media. Formulation of synthetic oviductal fluid

(Tervit et al., 1972) for bovine embryo culture medium allows blastocyst development without

using somatic cells support. Currently, it has become feasible to obtain in vitro produced bovine

2

blastocysts in chemically defined media beginning with the maturation of immature oocyte

collected from the slaughterhouse (Keskintepe and Brackett, 1996, Holm et al., 1999,

Dinkins and Brackett, 2000, Hernandez et al., 2002). In general, chemically defined media are

formulated with the use of synthetic macromolecules such as polyvinyl alcohol (PVA) or

polyvinyl pyrolidone (PVP) to replace serum, serum albumin, or other biological components.

PVA, initially found to be an acceptable BSA replacement in hamster fertilization medium

(Bavister, 1981), has been proven to be a suitable BSA substitute in a variety of chemically

defined bovine embryo culture media (Carney and Foote, 1991, Pinyopummintr and Bavister,

1991, Takahashi and First, 1992, Kim et al., 1993, Rosenkrans and First, 1994, Lee and Fukui,

1996, Keskintepe and Brackett, 1996, Olsen and Seidel, 2000). Although the production of

bovine blastocysts in chemically defined medium affords development of lower blastocyst yields

(Pinyopummintr and Bavister, 1991, Takahashi and First, 1992, Holm et al., 1999, Krisher et al.,

1999), in turn, it is a more suitable approach to elucidate essential factors for blastocyst

development (Bavister, 1992). Chemically defined medium is also a more desirable approach

regarding repeatability among laboratories and hygienic concerns pertaining to transmission of

infectious pathogens (Stringfellow and Givens, 2000). Therefore, more studies are needed to

improve blastocyst yields as well as to bring the viability of in vitro produced blastocysts up to

that of blastocysts obtained in vivo if embryo production in chemically defined medium is to be

widely used for both research and commercial applications.

The ultimate goal in formulating embryo culture medium is to simulate the

microenvironment resemble to that in female reproductive tract that these embryos experience.

Among that, a variety of growth factors and cytokines are now widely recognized to be present in

the female reproductive tract. For past few years, the roles of growth factors and cytokines in

controlling preimplantational embryonic development have been a subject of great research area

of interest not only for fundamental scientific merits but also in the relation to be used to improve

the quality of embryos produced by in vitro fertilization. The latter is the primary goal in this

3

work. It is in agreement that the ultimate measurement of blastocyst quality is the ability to

initiate pregnancy with resulting healthy offspring. However, in most circumstance, it is

impractical to transfer every blastocyst produced for quality assessment. Additionally, the

selection of the best blastocysts from the blastocyst pools for embryo transfer may not truly

represent the overall quality of the entire population (Bavister, 1995). Therefore, in vitro

assessment by indicators of embryo quality such as metabolic activity, ultrastructure, cell

numbers, or freezability provides an affordable alternative methodology for in vitro viability

assessment.

To date, the benefits from in vitro produced blastocyst methodology have gone beyond

the initial goals to provide low-cost embryos for the cattle industry or to obtain embryos from

problem cows. Production of transgenic embryo technology is another area that the author hopes

to expand by the application of knowledge of in vitro production of bovine blastocysts. Although

transgenic bovine embryo production via nuclear transfer approach has been shown to be an

outstanding improvement, several alternative transgenic methodologies have not been

overlooked. Among those, sperm mediated gene transfer is arguably the most attractive due to its

simplicity and less zygote manipulation required.

The present investigation addressed three major subjects. In brief, the purpose was to: 1)

Evaluate the putative benefits of supplementing specific growth factor or cytokine such as

epidermal growth factor, insulin-like growth factor-I, and leukemia inhibitory factor to

chemically defined embryo culture media; 2) Assess the quality of the resulting blastocysts by

employing several in vitro techniques including survival after cryopreservation, counting of inner

cell mass cells and trophoblast cells, and incidences of DNA fragmented nuclei; 3) Investigate the

feasibility of using spermatozoa as an alternative gene transfer method to produce transgenic

bovine embryos. A review of pertinent literature and original findings from this research follow.

4

REFERENCES

Bavister BD. Substitution of a synthetic polymer for protein in a mammalian gamete culture

system. J. Exp. Zool. 1981;217:45-51.

Bavister BD. Co-culture for embryo development: is it really necessary? Hum Reprod.

1992;7(10):1339-41.

Bavister BD. Culture of preimplantation embryos: facts and artifacts. Hum. Reprod. Update

1995;(1):91-148.

Blondin P, Bousquet D, Twagiramungu H, Barnes F, Sirard MA. Manipulation of follicular

development to produce developmentally competent bovine oocytes. Biol Reprod.

2002;66(1):38-43.

Bousquet D, Twagiramungu H, Morin N, Brisson C, Carboneau G, Durocher J. In vitro embryo

production in the cow: an effective alternative to the conventional embryo production

approach. Theriogenology. 1999;51(1):59-7.

Brackett BG, Bousquet D, Boice ML, Donawick WJ, Evans JF, Dressel MA. Normal

development following in vitro fertilization in the cow. Biol Reprod. 1982;27(1):147-58.

Carney EW, Foote RH. Improved development of rabbit one-cell embryos to the hatching

blastocyst stage by culture in a defined, protein-free culture medium. J. Reprod. Fertil.

1991;91:113-23.

Dinkins MB, Brackett BG. Chlortetracycline staining patterns of frozen-thawed bull spermatozoa

treated with beta-cyclodextrins, dibutyryl cAMP and progesterone. Zygote.

2000;8(3):245-56.

Hasler JF. Current status and potential of embryo transfer and reproductive technology in dairy

cattle. J Dairy Sci. 1992;75(10):2857-79.

Hernandez-Fonseca HJ, Sirisathien S, Bosch P, Cho HS, Lott JD, Hawkins LL, Hollett RB, Coley

SL, Brackett BG. Offspring resulting from direct transfer of cryopreserved bovine embryos

produced in vitro in chemically defined media. Anim Reprod Sci. 2002;69(3-4):151-8.

5

Holm P, Booth PJ, Schmidt MH, Greve T, Callesen H. High bovine blastocyst development in a

static in vitro production system using SOFaa medium supplemented with sodium citrate

and myo-inositol with or without serum-proteins. Theriogenology. 1999, 52(4):683-700.

Keskintepe L, Brackett BG. In vitro developmental competence of in vitro-matured bovine

oocytes fertilized and cultured in completely defined media. Biol Reprod. 1996;55(2):333-

339.

Kim JH, Niwa K, Lim JM, Okuda K. Effects of phosphate, energy substrates, and amino acids on

development of in vitro-matured, in vitro-fertilized bovine oocytes in a chemically defined,

protein-free culture medium. Biol. Reprod. 1993;48:1320-1325.

Krisher RL, Lane M, Bavister BD. Developmental competence and metabolism of bovine

embryos cultured in semi-defined and defined culture media. Biol Reprod.

1999;60(6):1345-52.

Lee ES, Fukui Y. Synergistic effect of alanine and glycine on bovine embryos cultured in a

chemically defined medium and amino acid uptake by vitro-produced bovine morulae and

blastocysts. Biol. Reprod. 1996;55:1383-1389.

Olson SE, Seidel GE Jr. Reduced oxygen tension and EDTA improve bovine zygote development

in a chemically defined medium. J Anim Sci. 2000;78(1):152-7.

Parrish JJ, Susko-Parrish JL, First NL. In vitro fertilization of bovine oocytes using heparin-

treated and swim-up separated frozen thawed bovine semen is repeatable and results in

high frequencies of fertilization. Theriogenology 1985;35:234 (Abstr).

Pinyopummintr T, Bavister BD. In vitro-matured/in vitro-fertilized bovine oocytes can develop

into morulae/blastocysts in chemically defined, protein-free culture media. Biol. Reprod.

1991;45:736-742.

Rosenkrans CF Jr, First NL. Effect of free amino acids and vitamins on cleavage and

developmental rate of bovine zygotes in vitro. J Anim Sci. 1994;72(2):434-7.

6

Stringfellow DA, Givens MD. Epidemiologic concerns relative to in vivo and in vitro production

of livestock embryos. Anim Reprod Sci. 2000;60-61:629-42.

Tervit HR, Whittingham DG, Rowson LE. Successful culture in vitro of sheep and cattle

ova. J Reprod Fertil. 1972, 30(3):493-7.

Takahashi Y, First NL. In vitro development of one-cell embryo: influence of glucose,

lactate, pyruvate, amino acids and vitamins. Theriogenology 1992;37:963-978.

7

CHAPTER 2

LITERATURE REVIEW

A. EXPERIMENTAL FINDINDS LEADING TO IMPROVEMENT OF IN VITRO

PRODUCTION OF BOVINE BLASTOCYSTS IN CHEMICALLY DEFINED CONDITIONS

IN VITRO OOCYTE MATURATION

Early Reports on In Vitro Bovine Oocyte Maturation

In vitro maturation is a process in which meiotically-arrested (prophaes, or germinal

vesicle stage) oocytes from small to medium antral follicles are cultured in the laboratory to

become ready for fertilization by reaching metaphase II with extrusion of the first polar body.

Pincus and Enzman (1935) first observed that rabbit oocytes resume meiosis spontaneously after

being removed from the follicular environment. Edwards (1965) reported similar findings with

mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Successful and repeatable

IVF results of bovine oocytes began with in vivo matured oocytes (Brackett et al., 1978, 1980)

with appropriate sperm capacitation treatments, leading to the first bovine IVF offspring

(Brackett et al., 1982). This was followed by reports of several more pregnancies (Brackett et al.,

1984, Sirard and Lambert, 1985, Sirard et al., 1985, Lambert et al., 1986, Leibfried-Rutledge et

al., 1987). The cost of retrieving in vivo matured oocytes is high, compared to collection of

immature oocytes from small follicles, making in vivo matured oocytes less attractive as starting

material than immature oocytes for IVF research and for large scale laboratory embryo

production. Early IVF results of in vitro matured cow oocytes were extremely low and

terminated at the pronuclear stage. Iritani and Niwa (1977) observed only 6 to 7% pronuclear

8

formation rates (i.e., percentages of inseminated oocytes that became zygotes) after IVF of cow

oocytes matured in vitro. Fulka et al (1982) reported 45 % pronuclear formation rates after IVF

of zona-free in vitro matured cow oocytes. Fukui et al (1983) capacitated frozen-thawed sperm

with high ionic strength (HIS) medium (Brackett and Oliphant, 1975) and obtained 27 %

pronuclear formation after insemination of in vitro matured oocytes. Ball et al (1983) observed

the crucial role of cumulus cells during oocyte in vitro maturation for high pronuclear formation

rates after IVF. Iritani et al (1984) capacitated bull sperm with isotonic medium (m-KRB) and

obtained up to 58 % fertilization rates. Hensleigh and Hunter (1985) matured cow oocytes for 48

h in vitro, inseminated with extended cooled sperm and obtained 15 % cleaved oocytes after IVF.

Eighty percent male pronuclear formation rates were obtained after IVF with heparin-treated

sperm (Leibfried-Rutledge et al., 1986, Parrish et al., 1986). Development to the blastocyst stage

of in vitro matured and fertilized bovine oocytes was initially obtained after transfer of zygotes or

early cleaved embryos into the oviducts which resulted in pregnancies after embryo transfer

(Critser et al., 1986, Xu et al., 1987, Lu et al., 1987, Sirard et al., 1988, Fayrer-Hosken et al.,

1989). This marked initiation of mass production of bovine embryos from immature oocytes.

An Overview of In Vitro Bovine Oocyte Maturation and Factors Improving In Vitro Oocyte

Maturation

Although more than 80 % of immature oocytes usually reach the metaphase II stage after

being incubated for a period of 20 h, in vitro fertilization following 24 h of in vitro maturation

period has been shown to be optimal (Monaghan et al., 1993, Long et al., 1994, Ward et al.,

2002). Dominko and First (1997) demonstrated that blastocyst development was higher when

insemination was delayed for 8 h after first polar body extrusion. Tissue culture medium 199

(TCM-199) with Earle’s salts is the most widely used basic medium for in vitro oocyte

maturation. Rose and Bavister (1992) compared seven commercially available complex media

and concluded that both TCM-199 and MEM were equally suitable for oocyte maturation while

9

use of Waymouth’s MB 752/1 and Ham’s F-12 to support maturation resulted in significantly

reduced cleavage rates and post-cleavage development of the mature oocytes after IVF to the

blastocyst stage. Gliedt et al (1996) compared TCM-199 vs. RPMI-1640 for maturation and

observed that blastocyst development from oocytes matured in TCM-199 was slightly higher than

for those from RPMI-1640. Oocyte maturation conditions strongly affect not only fertilizing

ability but also determine early embryonic developmental competence, this can be understood by

considering the fact that mRNA in early cleavage stage embryo predominantly arises from pools

within the oocytes. Several simple media have been used successfully for oocyte maturation such

as hamster embryo culture medium-6 (Rose-Hellekant et al., 1998), modified basic medium

(Krisher and Bavister, 1999), and synthetic oviductal fluid (Hashimoto et al., 2000, Gandhi et al.,

2000). The use of laboratory prepared media allows better understanding of factors that are

important for in vitro oocyte maturation.

Generally, TCM-199 is supplemented with a variety of substances that have been shown

to contribute to some improvements. Ten to twenty per cent of various types of bovine sera are

usually included in the maturation medium since early works uncovered advantages of including

estrus cow serum, pro-estrous serum, steer serum, or fetal bovine serum (Leibfried-Rutledge et

al., 1986, Parrish et al., 1986, Sirard et al., 1988, Younis et al., 1989, Sanbuissho and Threlfall,

1989). However, the presence of bovine sera during in vitro maturation is not crucial. Bovine

oocytes have been matured in serum-free medium (Zuelke and Brackett, 1990, Saeki et al., 1991,

Harper and Brackett, 1993, Lonergan et al., 1994) then fertilized, and cultured before resulting in

healthy offspring after embryo transfer of either fresh (Keskintepe et al., 1995) or cryopreserved

(Hernandez-Fonseca et al., 2002) embryos.

Gonadotropins and Prolactin

Gonadotropins are commonly included in maturation media to make mature oocytes

more effective in sustaining high fertilization and developmental rates. Ball et al (1983) observed

10

that inclusion of FSH during in vitro maturation improved pronuclear rates after IVF. Inclusion

of FSH also improved the degree of cumulus expansion over inclusion of hCG. In vitro

maturation with a combination of FSH, LH, and estradiol increased morula plus blastocyst yields

28%, compared to 18% of oocytes in vitro matured without hormones (Sirard et al., 1988).

Similarly, Younis et al (1989) founded that inclusion of FSH or LH improved cleavage and

development to 4- to 8-cell stages from 6% (without hormone) to 15% (plus FSH) or 20% (plus

LH) whereas inclusion of estradiol provided no improvement over maturation without hormones.

Despite the fact that resumption of meiosis in oocytes is known to be triggered by the LH

surge in vivo, benefits of including LH during vitro maturation has been controversial. Several

reports from our lab showed that LH supplementation enhanced development. Brackett et al

(1989) observed a positive dose dependent effect of LH and demonstrated that a high LH

concentration (100 µg/mL) was beneficial for oocyte maturation as reflected in subsequent

development to 6-to 8-cell stages. Zuelke and Brackett (1990) obtained 27% blastocysts when at

least 50 µg/mL of LH was included in serum-free oocyte maturation medium. Harper and

Brackett (1993) obtained both higher cleavage and blastocyst formation rates when LH (50

µg/mL) was included compared to serum-free maturation medium alone. Whether the positive

effects of LH were caused by an impurity of the LH preparation may have been answered by the

possibility that when thyroid stimulating hormone (TSH) was added at 0.5 µg/mL for maturation

improved IVF results also followed whereas prolactin (up to 1000 µg/mL) had no effect (Younis

and Brackett, 1992). Alternatively, some other contaminant in both of the biological preparations

i.e., LH and for TSH derived from the same region of the anterior pituitary glands, may have been

responsible for the positive effect. Although not a direct comparison, Harper and Brackett (1993)

also observed that FSH at 1 µg/mL resulted in a comparable blastocyst yield to that of LH at 100

µg/mL. Gliedt et al (1996) included equine LH (up to 30 µg/mL) in maturation medium

containing 20 % estrous cow serum and reported no positive effect of LH on either cleavage or

11

blastocyst formation rates. However, this was not surprising since serum most likely had a high

LH content as shown for proestrous serum by Younis et al (1989). Bevers et al (1997) reported

that hCG (0.05 IU/mL) during in vitro maturation had no positive effect whereas FSH (0.05

IU/mL) significantly improved blastocyst formation rates. Similarly, Martins et al (1998),

following the lead of Harper and Brackett (1993), supplemented chemically defined maturation

medium with recombinant gonadotropins to and concluded that FSH (10 ng/mL) significantly

improved both cleavage and blastocyst development over maturation medium without hormone

or with LH (1 ng/mL) alone whereas LH alone slightly improved blastocyst yields over those

following maturation without any added hormone. Further there was no additive effect of FSH

and LH supplementation for maturation. Unfortunately, only one concentration of LH was

investigated at that moment. In apparent contrast, Anderiesz et al (2000) added recombinant

gonadotropins in maturation medium containing 10 % fetal bovine serum and observed an

additive effect of LH (10 IU/mL) plus FSH (1 IU/mL) over either FSH or LH alone. This result

probably reflected a lower gonadotropin contribution to the basic medium by the included serum

preparation selected by those investigators to “cloud the issue”

A beneficial effect of including FSH during in vitro oocyte maturation is firmly

established. Fukushima and Fukui (1985) observed that inclusion of FSH but not LH nor estradiol

in maturation medium improved fertilization rates. Eyestone and Boer (1993) used serum-free

maturation medium and demonstrated a positive effect of FSH on blastocyst development from 2-

cell stage embryos. Martins et al (1998) employed recombinant FSH (10 ng/mL) in chemically

defined oocyte maturation and obtained improvements in both cleavage and blastocyst formation

rates. Ali and Sirard (2002) also reported similar findings of positive effects on blastocyst

development but without affecting cleavage rates recombinant after using FSH (5 to 500 ng/mL)

for maturation. Van Tol et al (1996) isolated oocytes that remaining connected to membrana

granulosa to study oocyte maturation and demonstrated that membrana granulosa inhibited oocyte

resumption of meiosis, i.e., germinal vesicle breakdown. The inhibitory effect produced from

12

membrana granulosa could be overcome by adding recombinant FSH to the maturation medium

but could not be overcome by adding hCG (which was predominantly LH with some FSH-like

activity). The authors concluded that in vitro resumption of meiosis of oocytes, originating from

antral follicles between 2 to 8 mm, was triggered by FSH and not by LH. FSH and LH receptor

mRNA expression in small antral follicles, i.e., smaller than 9 mm, has been characterized. LH

receptor mRNA was detectable exclusively in the theca cells whereas FSH receptor mRNA was

present in both granulosa and cumulus cells; LH receptor in granulosa cells was only detectable

in follicles larger than 9 mm (Xu et al., 1995, van Tol et al., 1996). However, Baltar et al (2000)

employed 125I-LH to demonstrate that cumulus oocyte complexes were able to specifically bind

LH, indicating the presence of LH receptors. Interestingly, the authors also showed that small

antral follicles (2-3 mm) had higher LH binding capacity than larger follicles (> 5 mm).

Nonetheless, the positive effect of gonadotropins is not a definitive finding. Several

investigators have failed to observe any positive effect of supplementing gonadotropins in oocyte

maturation medium either in serum-containing media (Fukui and Ono, 1989, Goto and Iritani,

1992, Keefer et al., 1993) or serum-free medium (Lonergan et al., 1994, Choi et al., 2001). There

are few documentations regarding any effect of prolactin. Early reports showed no benefit of

supplementing prolactin in either serum-free (Saeki et al., 1991) or serum-containing maturation

medium (Younis and Brackett, 1992). A recent report showed that prolactin had some regulatory

role on intracellular stored calcium during bovine oocyte maturation (Kuzmina et al., 1999),

whether that role can affect IVF result remains to be demonstrated.

Estradiol and Progesterone

Estradiol is widely added to maturation media at a level of 1 µg/mL based on found in

preovulatory follicles (Dieleman et al., 1983, Fortune and Hansel, 1985). Several investigators

showed no benefits of including estradiol in maturation medium containing fetal bovine serum

(Fukushima and Fukui, 1985, Younis et al., 1989, Saeki et al., 1991) or estrous cow serum

13

(Brackett et al., 1989, Gliedt et al., 1996). Data on benefit of including estradiol during oocyte

maturation remain controversial. Beker et al (2002) confirmed no benefit of adding estradiol (1

µg/mL) to serum-free maturation medium . In contrast, Ali and Sirard (2002) observed a positive

effect of estradiol (1 µg/mL) in serum-free maturation medium on cleavage rates while having no

effect on post-cleavage development to the blastocyst stage. Guler et al (2000) reported a higher

blastocyst development when sheep oocytes were matured with estradiol (0.1 µg/mL) in serum-

free medium and showed no benefit of estradiol when maturation medium was supplemented

with 10% follicular fluid that had been treated to contain only 0.06 ng/mL of estradiol. Mingoti

et al (2002) detected a significant increase in estradiol concentration (120 ng/mL) in maturation

medium after a 16 h culture period for bovine cumulus oocyte complexes matured with FSH and

hCG (10 oocytes in 3 mL medium). This indicates that exogenous estradiol might not be required

owing to the ability of cumulus oocyte complexes to secrete estradiol while being cultured with

gonadotropins.

Data on the effect of progesterone during oocyte maturation on fertilization and

embryonic development are limited. Progesterone has been included in maturation medium due

to the fact that follicular fluid of follicles approaching ovulation contain increasing levels of

progesterone when luteinzation of granulosa cells occurs (Dieleman et al., 1983). Fukushima and

Fukui (1985) observed a slight reduction in fertilization rates when progesterone was included in

maturation medium. Silva and Knight (2001) observed a significant reduction in blastocyst

development when oocytes were matured in maturation medium supplemented with 300 nM

progesterone compared to control medium containing 10% estrous cow serum and this negative

effect was partially reversed by adding RU486 (an anti-progestin) to the maturation medium

while RU486 by itself had no effect either fertilization of blastocyst formation rates.

14

Activin A

Activin A, measured from dominant follicles, is present in follicular fluid at

approximately 3 µg/mL (Knight et al., 1996). Activin A is one of the ligands reported to have a

positive effect. Izadyar et al (1996) observed positive effect of activin A (10 ng/mL) in

conjunction with gonadotropins on cleavage rates without a further effect on blastocyst

development while activin alone produced no effect. Stock et al (1997), however, observed

higher blastocyst formation rates from 2-cell stage embryos when activin A alone (1 and 10

ng/mL) was included compared to serum-free maturation medium alone. Similarly, Silva et al

(1998) employed 500 ng/mL activin A in serum-included maturation medium and obtained higher

blastocyst development. Activin A also produced a similar effect on cumulus-free oocytes

indicating the presence of activin receptors on oolemma which was later confirmed by Izadyar et

al (1998).

Growth hormone (GH)

Several reports, mostly from one laboratory, have shown that supplementation of growth

hormone in serum-free maturation medium improved IVF outcomes. Izadyar et al (1996) found

that GH at 0.1 and 1 µg/mL accelerated the nuclear maturation process by increasing the

proportions of oocytes reaching metaphase II stage by 16 h while total oocytes reaching the

metaphase II stage at 24 h remained similar to controls. Growth hormone also improved both

cleavage rates (from 50-60% to 73-78%) and blastocyst formation rates (from 18-20% to 25-

30%). These findings support the report of Dominko and First (1997) who found that the sooner

oocytes reach metaphase II, the higher the blastocyst formation rates that would be obtained.

Oocytes matured in the presence of GH (0.1 µg/mL) had more cortical granules evenly disperse

in the cortical cytoplasm aligning the oolemma than oocytes matured in maturation medium alone

(Izadyar et al., 1998). Recently, Moreira et al (2002) added GH (10 µg/mL) to control maturation

15

medium consisting of steer serum and FSH and obtained higher cleavage rates but without

improvement on blastocyst development from 2-cell stage embryos.

Growth Factors

Additions of epidermal growth factor (EGF) at concentrations of 10-50 ng/mL are

frequently included in maturation medium. EGF has been shown to stimulate nuclear maturation

of bovine oocytes in vitro (Lorenzo et al., 1994). Coskun et al (1991) improved fertilizing and

developmental ability of in vitro matured bovine oocytes by adding EGF to the maturation

medium. Harper and Brackett (1993a) observed positive effects of EGF (1-100 ng/mL) on

morula and blastocyst development when added in combination with FSH (10 µg/mL) while EGF

by itself or in combination with LH produced no effect. The positive effect of EGF was

confirmed in a subsequent report of Harper and Brackett (1993b). Kobayashi et al (1994)

obtained higher cleavage rates and blastocyst development when either EGF or Transforming

growth factor-α (TGF-α), a growth factor that is closely related to EGF, alone were included in

maturation medium but no additive effect of EGF was detected when it was combined with FSH

plus LH. Lonergan et al (1996), however, observed that EGF alone (1-100 ng/mL) improved

only cleavage rates without any improvement on blastocyst development. Park et al (1998)

obtained significantly higher cleavage and blastocyst formation rates when EGF (10-50 ng/mL)

was included compared to serum-free maturation medium alone. Similarly, Watson et al (2000)

reported that EGF alone (100 ng/mL) supplemented in serum-free maturation medium improved

both cleavage rates and blastocyst formation rates. Palasz et al (2000) obtained a higher cleavage

rate but found no benefit on blastocyst development of EGF (20 ng/mL) compared to that of

serum-free maturation medium alone. Recently, Sakaguchi et al (2002) observed that EGF

significantly accelerated the proportions of oocytes extruding their first polar bodies after 16 h of

maturation when compared to controls.

16

Levels of Insulin-like growth factor-I (IGF-I) in bovine follicular fluid from either small

or perovulatory follicles were reported to be at approximately 100 ng/mL (Funston et al., 1995).

Herrler et al (1992) observed no benefit of adding IGF-I (50 ng/mL) into serum-included

maturation medium except improved degree of cumulus expansion. Martins and Brackett (1998)

obtained significantly higher blastocyst yielded, without observing any effect on cleavage rates,

by supplemented IGF-I (1-100 ng/mL) in conjunction with gonadotropins into chemically defined

maturation medium. Reiger et al (1998) observed an additive effect of combining IGF-I (100

ng/mL) and EGF (50 ng/mL) on cleavage and blastocyst formation rates while EGF and IGF-I

alone produced no improvement compared to chemically defined maturation medium alone.

Makarevich and Markkula (2002), however, found no benefit of supplementing IGF-I (100

ng/mL) into control medium consisting of fetal bovine serum and gonadotropins.

Positive effect of platelet-derived growth factor (PDGF) at a level of 10 ng/mL in

conjunction with FSH on blastocyst formation rates has been demonstrated in serum-free

maturation medium (Harper and Brackett, 1993). Vascular endothelial growth factor (VEGF) at

a level of 100 ng/mL has been shown to be benefit to blastocyst production in vitro compared to

control maturation medium alone (Einspanier et al., 2002).

Importance of Increased Glutathione During Oocyte Maturation

An inability of in vitro matured oocytes to support male pronuclear formation was

apparent in early experiments. The majority (85%) of in vitro matured golden hamster oocytes

were unable to cause decondensation of sperm nuclei after 6 h of sperm/egg incubation while

98% of ovulated oocytes were fertilized normally (Leibfried and Bavister, 1983). The same

problem also occurred with bovine in vitro matured oocytes. Leibfried-Rutledge et al (1987)

reported that frequencies of sperm penetration were not different for in vitro matured oocytes vs.

in vivo matured oocytes. However, formation of male pronuclei was reduced for oocytes matured

in vitro compared to in those matured vivo. Only 3% of in vitro matured oocytes developed to

17

the 2-cell stage whereas 40% of oocytes matured in vivo showed normal development to the 4-

cell stage after in vitro fertilization.

The role of a disulfide bond reducing agent for sperm nuclear decondensation has long

been recognized (Perreault et al., 1984, Perreault et al., 1987). Oocyte glutathione has been

shown to play an important role for the sperm decondensation process in forming male pronuclei.

Sperm nuclear decondensation was prevented or delayed by blocking glutathione synthesis with

buthionine sulfoximine during the early stages of oocyte maturation in the hamster (Perreault et

al., 1988) and cow (Sutovsky and Schatten, 1997). In vitro matured hamster oocytes were found

to contain significantly lower amounts of intracellular glutathione than in vivo matured oocytes

(Kito and Bavister, 1997). The need to increase oocyte glutathione was reported in porcine

oocyte. The rates of male pronuclear formation were higher in porcine oocytes matured in

Waymouth MB 752/l, a cysteine rich medium, than in TCM 199 (Yoshida et al., 1992).

However, this finding was in contrast to a report by Rose and Bavister (1991) who found bovine

oocytes that were matured in Waymouth's medium MB 752/l had a significantly reduced

incidence of cleavage and blastocyst development compared to TCM 199.

As somatic cells, mammalian oocytes are unable to take up free glutathione. Glutathione

is synthesized during maturation from three amino acid precursors, i.e. glutamine, cysteine, and

glycine. Therefore, concentrations of glutathione within oocytes depend on the availability of the

precursors. Male pronuclear formation rates were improved when compounds like cysteamine or

β-mercaptoethanol were added to maturation media. Supplementation of oocyte maturation

medium with 0.1 mM cysteamine significantly increased oocyte glutathione content and

subsequently improved blastocyst yields (De Matos et al., 1995). Similarly, improvement could

be achieved by adding cystine or cysteine as well as glutamine to the IVM medium (De Matos et

al., 1996, De Matos and Furnus, 2000). In vitro fertilization of bovine oocytes matured in the

presence of 2 mM glutamine resulted in the highest cleavage and blastocyst development rates

compared to those matured in medium supplemented with 0, 1, or 3 mM glutamine (Furnus et al.,

18

1998). Glutathione has been shown to be synthesized predominantly within cumulus cells and

transported into oocytes through gap junctions. Experimentally, the amount of glutathione in

cultured oocytes tended to decrease as the concentration of heptanol, a gap junction inhibitor, in

the maturation medium was increased. Although there were no differences in the rates of sperm

penetration after porcine oocytes were matured in maturation medium with different

concentrations of heptanol, proportion of porcine oocytes forming male pronuclei decreased

significantly (by comparison to controls without heptanol) at all heptanol concentrations tested

(Mori et al., 2000).

IN VITRO FERTILIZATION

An understanding of sperm capacitation was initiated by Chang (1951) and Austin (1951)

who simultaneously observed that spermatozoa must be exposed to the female reproductive

environment for a period of time before gaining the ability to penetrate oocytes. The term sperm

capacitation was coined to describe the process by which sperm cells achieve the capacity to

fertilize (Austin, 1952). This knowledge made efforts to fertilize mammalian oocytes feasible

and led to in vitro fertilization (IVF) of rabbit oocytes (Chang, 1959). Efforts to fertilized bovine

oocytes in vitro were first successfully in late 1970’s (Shea et al., 1976, Iritani et al., 1977,

Brackett et al., 1977). Several approaches have been employed to capacitate bull spermatozoa in

vitro. Iritani et al (1977) incubated washed spermatozoa in Kreb’s-Ringer bicarbonate modified

to contain lactate and BSA (4 mg/mL) for 12-14 h at 37°C prior to expose to ova in vitro. A brief

high ionic strength treatment (Brackett and Oliphant, 1975) of bull sperm proved successful with

embryos developing to 2-cell stage (Brackett et al., 1977, 1978), 4-cell stage (Brackett et al.,

1980), and eventually, birth of live offspring (Brackett et al., 1982). The use of calcium

ionophore to capacitate bull spermatozoa (Jiang et al., 1991, 1992) has been useful in several

labs.

19

Flushings from bovine oviducts revealed a high concentration of glycosaminoglcans (Lee

and Ax, 1984). Heparin has been shown to be the most potent glycosaminoglcan in its ability to

induce the acrosome reaction of epididymal bull spermatozoa (Handrow et al., 1982). Based on

that, Parrish et al (1986) employed tyrode albumin lactate pyruvate (TALP) medium and

observed that pretreating cryopreserved bull spermatozoa with heparin (10 µg/mL) for 15 min

prior to IVF increased fertilization rates from 40 % to 79 %; more than 70 % fertilization rates

were achieved with four different bull semen. To date, heparin is most widely used, and probably

is the most effective way currently known for capacitating bull spermatozoa. Other capacitation

methods have been reported such as the use of progesterone (Dinkins and Brackett, 2000),

cyclodextrins (Choi and Toyoda, 1998, Dinkins and Brackett, 2000), or xanthine-xanthine

oxidase (O’flaherty et al., 1999) but the results have not surpassed those provided by the use of

heparin.

Swim-up (Parrish et al., 1985, Keefer et al., 1985) and Percoll density gradient are the

two most widely used sperm treatment, usually in conjunction with heparin-induced capacitation.

A modified swim-up with the use of hyarulonic acid was shown to provide better results with

cryopreserved bull spermatozoa (Shamsuddin et al., 1993). Either two discontinuous layers of 30

% and 45 % Percoll (Utsumi et al., 1991) or 45 % and 90 % Percoll (Saeki et al., 1991) have been

used successfully. However, the 45 % and 90 % Percoll density gradient has gained the most

popularity. Parrish et al (1995) obtained higher percentages of ova penetrated and cleavage rates

but similar percentages of blastocysts using swim-up vs. Percoll for fractionating motile

spermatozoa. However, the use of Percoll resulted in almost six-fold greater motile sperm

recovery compared to the swim-up method.

Several chemicals have been included to improve fertilization rates. Phosphodiesterase

inhibitors, caffeine, are commonly used to increase sperm cAMP levels. Caffeine has been shown

to improve sperm motility in poor ejaculates (Critser et al., 1984). Niwa et al (1991) reported a

synergic effect of a high level of caffeine (5 mM) with heparin on sperm penetration rates. Lower

20

concentrations of caffeine (2.5-7.5 µM) have been shown to be ineffective (Coscioni et al., 2001).

Numabe et al (2001) obtained similar results when using pentoxifylline (5 mM) in conjunction

with heparin as following caffeine plus heparin. Slaweta and Laskowska (1987) showed an

improvement by using glutathione (5 mM) during IVF. Kim et al (1999), however, obtained

positive effects as well as negative effects with glutathione (1 mM) supplementation of the IVF

medium, depending on the bull semen. A PHE mixture, i.e., penicillamine (2 mM), hypotaurine

(10 mM), and epinephrine (1 mM) in IVF medium first shown effective in hamster experiments

(Leifried and Bavister, 1983) has been commonly used for bovine IVF. Reports involving PHE

either show positive effect (Susko-Parrish et al., 1990, Miller et al., 1992) or no effect (Long et

al., 1993, Palma et al., 1993) on bovine IVF. Penicillamine alone has been shown to improve

IVF results (Keskintepe and Brackett, 1995). The sperm-oocyte ratios for IVF are far greater

than those that occur in vivo which are close to 1:1 (Hunter, 1993). A ratio of 5,000 to 10,000

spermatozoa per oocyte are common for bovine IVF systems. A sperm: oocyte ratio below 500:1

significantly reduced cleavage rates (Long et al., 1994, Ward et al., 2002).

IN VITRO EMBRYO CULTURE

Reduced Oxygen During Embryo Culture

The oxygen tension in the mammalian female reproductive tract is lower than in the

atmospheric oxygen (Mastroianni and Jones, 1965, Mitchell and Yochim, 1968, Maas et al.,

1976, Garris and Mitchell, 1979, Fischer and Bavister, 1993). Reduced tension condition was

beneficial for murine embryonic development in vitro (Whitten 1957, Auerbach and Brinster,

1968, Brinster and Troike, 1979). Tervit et al (1972) first reported a low O2 atmosphere to be

beneficial for ovine and bovine embryo culture with birth of lambs following culture of in vivo

fertilized embryos (Tervit and Rowson, 1974). The benefit of low O2 was confirmed by their later

work with bovine embryos (Wright et al., 1976). When bovine embryos at 2-to 4-cell and 8-cell

stages were cultured in synthetic oviduct fluid previously equilibrated with one of several O2

21

concentrations ranging from 0 to 20 % the proportions of embryos reaching at least the morula

stage were higher at 4% and 8 % O2, compared to those at 20 % or without O2 (Thompson et al.,

1990). Their results confirmed that, under lowered oxygen levels, development of bovine

embryos can occur through the 8- to 16-cell block in a simple medium without somatic cell

support.

Clearly, a reduced O2 atmosphere from approx 20% in air to 5% positively influences

numbers of zygotes developing to the blastocyst stage in ruminants (Batt et al., 1991, Voelkel and

Hu, 1992, Liu et al., 1995, Lonergan et al., 1999) as well as for other mammalian species

including the rabbit (Li and Foote, 1993), goat (Berthelot and Terqui, 1996), and human

(Dumoulin et al., 1999). Culture under a low O2 atmosphere also eliminates the need for co-

culture (Xu et al., 1992, Watson et al., 1994, Carolan et al., 1995, Rizos et al., 2001).

In contrast, reduced oxygen tension may not benefit the discrete events of bovine oocyte

maturation and fertilization. Thus, lower percentages of bovine oocytes reached the metaphase II

stage after in vitro maturation under 5% oxygen tension compared to that under 20% oxygen

tension (Pinyopummintr and Bavister, 1995, Hashimoto et al., 2000). Similarly, in vitro

fertilization rates of bovine in vitro matured oocytes have been shown to be higher at 20% oxygen

tension than that of 5% oxygen tension under certain culture conditions (Pinyopummintr and

Bavister, 1995, Watson et al., 2000).

Inclusion of Amino Acids in Culture Media

In 1972, Tervit et al (1972) reported the first somatic cell-free medium formulated for

ruminant embryo culture based on the composition of sheep oviductal fluid and named the

medium “synthetic oviductal fluid” (SOF). The use of SOF (supplemented with BSA) failed to

reproduce results reported by Tervit et al (1972). Efforts to improve results of embryo culture led

to a variety of co-culture systems including cumulus cells (Goto et al., 1988), trophoblastic

vesicles (Camous et al., 1984, Heyman et al., 1987), and oviductal cell co-culture or conditioned

22

medium (Gandolfi and Moor, 1987, Rexroad and Powell, 1988, Eyestone and First, 1989).

However, those approaches might be considered as a draw back in efforts toward developing

defined media. Several factors such as the estrous cycle stage from which oviducal tissue was

obtained, and the conditioning period of medium must be better defined for repeatable results

(Eyestone et al., 1991). More important, exactly how co-culture or conditioned media benefit

embryonic development remains unknown. Some investigators have suggested that some peptide

growth factors or cytokines were secreted into the medium (Gandolfi et al., 1994, Gandolfi, 1995)

while other investigators argued that co-culture and conditioned medium simply reduced some

inhibitory components such as high oxygen (Bavister, 1992, Watson et al., 1994) or high glucose

(Reiger et al., 1995, Edward et al., 1997). The complex nature of these approaches makes it

impossible to identify specific factors critical for embryonic development.

The successful use of SOF re-emerged when BSA was replaced with human serum

(McLaughlin et al., 1990), still a few steps away from being a defined medium. It was found that

the need for serum could be replaced with amino acids. Takahashi and First (1990) reported

benefits of adding a pool of amino acids (both essential and non-essential) to embryo culture

media (SOF) supplemented with BSA in improving blastocyst development while additions of

vitamins had no such effect. Kim et al (1993) also reported the benefit of adding amino acids in

chemically defined medium (TALP-PVA). Rosenkrans and First (1994) confirmed these findings

with amino acids incorporated into their CR1 culture medium. Interestingly, amino acids have

long been known to be abundant in female reproductive fluids (Fahning et al., 1967, Leese et al.,

1979, Miller and Schultz, 1987, Casslen, 1987). Why the need for amino acids in culture media

was overlooked for so many years is not clear. This may be due to some early findings (Brinster,

1965, Whitten and Biggers, 1968, Cholewa and Whitten, 1970) in which murine 2-cell embryos

developed to blastocysts in culture media without amino acid other than those provided by

inclusion of BSA. The effects of each individual amino acid on bovine embryonic development

has not been extensively investigated. In hamster embryonic development, Bavister and Arlotto

23

(1990) found 6 amino acids (phe, val, isoleu, tyr, trp, and arg) to inhibit whereas 3 amino acids

(gly, cys, and lys) were found to stimulate embryonic development. Addition of non-essential

amino acids has been found to be superior to addition of essential amino acids for bovine embryo

culture (Thompson et al., 1992, Gardner et al., 1994, Lui and Foote, 1995, Keskintepe et al.,

1995, Pinyopummintr and Bavister, 1996, Lee and Fukui, 1996), especially for the early cleavage

stages (Steeves and Gardner 1999). In studies with bovine embryos, uptake of individual amino

acids from the culture medium has been reported (Lee and Fukui, 1996, Partridge and Leese,

1996, Jung et al., 1998). Threonine was the only amino acid found to be depleted from medium

at all stages of development and glutamine was depleted from the presumptive zygote stage until

the 4-cell stage (Partridge and Leese, 1996) whereas alanine was highly increased in the culture

media (Lee and Fukui, 1996, Partridge and Leese, 1996, Jung et al., 1998) indicating that alanine

was secreted into culture medium. Interestingly, alanine is also the most abundant amino acid

found in bovine oviductal and uterine fluid (Elhassan et al., 2001). Metabolism (Reiger et al.,

1992) and uptake (Partridge and Leese, 1996) of glutamine was high in 2- and 4-cell embryos.

Those findings agreed with the developmental results of Steeves and Gardner (1999) on the

beneficial effect of glutamine for early cleavage stages.

Energy Substrates for Culture Media

Glucose, pyruvate , and lactate are the most common energy substrates present in culture

media. Bovine embryos, unlike somatic cells, do not use glucose as a major source of energy

substrates. Bovine zygotes were able to develop to the blastocyst stage in a glucose free medium

(Holm et al., 1999, Van Langendonckt et al., 1997, Gomez and Diez, 2000, Holm et al., 2002)

and also resulted in healthy calves born at a 50% pregnancy rate (Holm et al., 1999). In fact, the

plasma-like concentrations of 5-6 mM were found to inhibit early embryonic development,

reducing zygotes reaching the morula stage compared to culture without glucose or lower

concentrations e.g. 1.5 or 0.5 mM (Takahashi and First, 1990, Kim et al., 1993, Lim et al., 1993,

24

Matsuyama et al., 1993). Glucose can be metabolized by the pentose phosphate pathway to

generate ribose and nicotinamide adenine dinucleotide phosphate required for the biosynthesis of

several complex molecules. Therefore, inclusion of glucose in culture media at low

concentrations i.e. 1.5 mM (ovine oviductal fluid, Tervit et al., 1972) or 0.5 mM (human

oviductal fluid, Gardner et al., 1993) is generally recommended. Although not required beyond

initial cleavage, inclusion of pyruvate to culture media improve bovine embryonic development

to the blastocyst stage (Takahashi and First, 1992, Rosenkrans et al., 1993). Unlike glucose and

pyruvate, lactate was found to be essential (Takahashi and First, 1992, Rosenkrans et al., 1993)

and lactate alone can support bovine zygotes to develop to the morula stage (Pinyopumintr and

Bavister, 1996). The need for lactate originated from the work of Whitten (1957) in which

murine 2-cell embryos were able to develop to the blastocyst stage when calcium chloride was

replaced with calcium lactate

Elimination of Albumin in Culture Media

Embryo production in chemically defined media has become feasible in several

laboratories (Keskintepe and Brackett, 1996, Krisher et al., 1999, Holm et al., 1999, Kuran et al.,

2001). Repeatability and avoidance of the risk of introducing contaminated pathogens into the

system are two driving forces to eliminate all undefined or incompletely defined biological

components, e.g. BSA, from being included as constituents media. Chemically defined media

are usually formulated by replacing BSA with polyvinyl alcohol (PVA). It is now clear that

bovine blastocyst can be produced in vitro without using BSA at all (Keskintepe and Brackett,

1996, Holm et al, 1999). Removal of BSA from oocyte maturation media or fertilization media

had no major influence on blastocyst yields (Zuelke and Brackett, 1990, Harper and Brackett,

1993, Lonergan et al., 1994, Keskintepe and Brackett, 1996, Holm et al, 1999, Ali and Sirard,

2002). However, blastocyst yields were lower in several labs when zygotes/embryos were

cultured in protein-free media (Lui and Foote, 1995, Eckert et al., 1998, Krisher et al., 1999,

25

Lonergan et al., 1999, Holm et al., 1999, Holm et al., 2002, Kuran et al., 2001, Wrenzycki et al.,

2001). The impact of protein-free embryo culture media on the resulting blastocysts has been a

subject of increasing interest. Culture in protein-free media has a very slight impact on several

parameters such as speed of development, blastocyst diameters, hatching rates, and total cell

numbers (Carolan et al., 1995, Holm et al., 1999, Holm et al., 2002, Khuran et al., 2002).

To the contrary, metabolic parameters have been significantly disturbed by protein-free

media. Total protein content of blastocysts produced in SOF with PVA replacing BSA was

significantly lower than that after use of BSA-containing SOF which had a similar total protein

content to those of in vivo blastocysts (Thompson et al., 1998). Interestingly, the reduction in

total protein content was not associated with rates of protein synthesis. In vivo blastocysts,

blastocysts from SOF with BSA or blastocysts from SOF with PVA and no BSA had comparable

rates of protein synthesis (Thompson et al., 1998, Kuran et al., 2001, Kuran et al., 2002).

Consumption indices of amino acids except that of aspartate were similar between blastocysts

produced in SOF with BSA or SOF with PVA and no BSA (Kuran et al.,2002). Therefore, it can

be speculated that blastocysts produced in protein-free culture media probably were unable to

maintain their intracellular amino acid pools as well as blastocysts produced in protein-

supplemented culture media.

Eckert et al (1998) studied metabolic activity in blastocysts cultured in SOF medium

prepared with either BSA or PVA. Only pyruvate uptake was significantly increased in

blastocysts cultured in SOF with PVA compared to SOF with BSA while uptake of glucose and

lactate were comparable. However, lactate production of blastocysts from SOF with PVA was

correlated with glucose uptake whereas lactate production of blastocysts from SOF with BSA was

correlated with pyruvate uptake. Oxygen uptake was also significantly reduced in blastocysts

cultured in SOF with PVA compared to SOF with BSA. Lee et al (1998) reported a reduction in

CO2 production from pyruvate metabolized in blastocysts cultured in protein-free medium.

Krisher et al (1999) also reported a significant reduction in pyruvate oxidation when blastocysts

26

were cultured in protein-free medium. Therefore, there is consensus indicating that blastocysts

cultured in protein-free media increase their pyruvate uptake for other purposes, not for ATP

production via oxidation. For example, Eckert et al (1998) suggested that blastocysts from SOF

with PVA might have increased their pyruvate uptake for the maintenance of intracellular amino

acid pools. Certainly, albumin has some nutritional role in bovine preimplantational development

as has been shown to be the case in murine preimplantational development (Brinster, 1965,

Dunglison and Kaye, 1993, Dunglison and Kaye, 1995); murine blastocysts took up BSA by

endocytosis and the uptake was stimulated by adding insulin to the medium.

In contrast to metabolic disturbance, Wrenzycki et al (2001) used semi-quantitative RT-

PCR and reported no differences in mRNA levels of glucose transporter-I, plakophilin, heat

shock protein 70.1, and interferon-tau between blastocysts produced with SOF with PVA and

SOF with BSA but all of the transcripts were significantly different from those of in vivo

blastocysts. To date, few healthy calves have been born from blastocysts produced in protein-free

media (Keskintepe et al., 1995, Holm et al., 1999, Hernandez-Fonseca et al., 2002); this does not

tell us that protein-free media are highly efficient or optimal. Increased rabbit blastocyst yields

by presence of BSA was shown to be due to contaminants of the BSA. Thus, citrate was

identified as the embryotrophic substance found in BSA (Gray et al., 1992). Supplementation of

embryo culture media with citrate has been shown to improve blastocyst yields (Keskintepe et al.,

1995, Keskintepe and Brackett, 1996, Holm et al., 1999). The possibility that metabolic

disturbances were caused by lacking of BSA contaminants in protein-free media cannot be ruled

out. Certainly, the need for BSA for blastocyst production does not end at blastocyst yields.

How those metabolic disturbances might affect viability after embryo transfer or survival after

cryopreservation remain largely unknown. If BSA were proven to be crucial for vitro production

of bovine blastocysts, then PVA should be used only for specific purposes (Krisher et al., 1999)

and recombinant BSA, if available, should be incorporated in the culture media instead.

27

Impact of Oocyte Maturation and Embryo Culture In Vivo

Generally, only half or less of oocytes inseminated develop to blastocyst stage despite

tremendous efforts for improvement in IVF laboratories worldwide. Among three steps for

blastocyst production in vitro, i.e., in vitro maturation, in vitro fertilization, and in vitro culture,

several authors have agreed that the intrinsic oocyte developmental competence determines

blastocyst yields. Embryo culture media are not the only reason for low blastocyst yieldeds

although these are usually interpreted as suboptimal culture conditions. By the late 1980s, it was

quite clear that lower proportions of in vitro matured oocytes developed to the blastocyst stage

compared to in vivo matured or ovulated oocytes despite having a relatively high fertilizing

capability (Leibfried-Rutledge et al., 1987, Greve et al., 1987, Marquant-Le Guienne et al., 1989).

Several recent studies have confirmed this findings. Blondin et al (2002) were able to obtain 80%

blastocysts from inseminated oocytes when oocyte donor cows were treated with 6 injections of

FSH with a 48-h coasting period followed by LH 6 h before oocytes were recovered via an

ultrasound-guided transvaginal aspiration. Similarly, Rizos et al (2002) obtained 74% blastocysts

after in vitro culture of in vivo presumptive zygotes (collected from oviducts) using a serum-

containing SOF medium whereas only 40% of IVF zygotes reached the blastocyst stage in the

same medium. Van de Leemput et al (1999) reported a similar trend but with lower figures.

Only 49% of in vivo matured oocytes developed to the blastocyst stage compared to 26% of in

vitro matured oocytes. One can speculate that oocytes might continue to acquire their competence

until ovulation. To support this, Rizos et al (2002) demonstrated that only 58% of oocytes

developed to blastocysts if oocytes were matured in vivo but not ovulated, i.e. recovered 18 h

after the expected LH surge, while 74% of in vivo presumptive zygotes reached the blastocyst

stage. Hendriksen et al (2000), suggested that the enhancing effect of in vivo maturation is not

yet established within the first 6 h of the process.

Efforts to improve culture systems have not led to making more than 50% of in vitro

matured/fertilized oocytes to reach the blastocyst stage. Various in vitro culture systems seem to

28

reach a limit in their ability to support development as indicated by results from in vivo culture

experiments. Similar proportions of presumptive IVF zygotes developed to the blastocyst stage

whether they were cultured in vitro or in vivo, i.e. ligated sheep oviducts (Galli and Lazzari,

1996, Gutiérrez-Adán et al., 1996, Enright et al. 2000, Rizos et al., 2002). In fact, in vivo culture

in ligated sheep oviducts is a rather inefficient approach. No more than 60% of initial

embryos/zygotes have been recovered. Pregnancy rates after fresh embryo transfer were similar

between blastocysts produced from in vitro or in vivo culture (Enright et al. 2000). In contrast,

blastocysts recovered from in vivo culture had significantly higher survival after cryopreservation

either with slow freezing or vitrification than those from in vitro culture (Enright et al. 2000,

Pugh et al., 2001, Rizos et al., 2002). However, this improvement in survival after

cryopreservation have been achieved in vitro by employing co-culture system (Massip et al.,

1993, Massip et al., 1996, Rizos et al., 2001).

INFLUENCES OF EPIDERMAL GROWTH FACTOR, INSULIN-LIKE GROWTH

FACTOR-I, AND LEUKEMIA INHIBITORY FACTOR ON BOVINE

PREIMPLANTATIONAL DEVELOPMENT

Mammalian preimplantation embryos is greatly enhanced when they are cultured in

groups in a small volume of medium compared to when they are cultured singly in an equal

volume of medium or in groups but with a larger volume of medium (Wiley et al., 1986, Schini

and Bavister, 1988, Paria and Dey, 1990, Lane and Gardner, 1992, Gardner et al., 1994, Keefer et

al., 1994, Carolan et al., 1995, Moessner and Dodson, 1995, Brison and Schultz, 1997, O’Neil,

1997, Paula-Lopes et al., 1998). This suggested that in vitro development of preimplantation

embryos is under the influences of growth-promoting substances including growth factors and

cytokines secreted from embryos themselves. In fact, the presence of both ligands and receptors

for several growth factors and cytokines have been well characterized in mammalian embryos

and female reproductive tracts, and several exogenous growth factors and cytokines have been

29

shown, mostly in murine species, to improve in vitro embryonic development (for reviews, see

Kaye and Harvey, 1995, Harvey et al., 1995, Kane et al., 1997, Martal et al., 1997, Sargent et al.,

1998, Teruel et al., 2000). Although there are numerous studies relevant to the roles of growth

factors and cytokines in preimplantational embryonic development, no known growth factor or

cytokine has been identified to be absolutely crucial for preimplantational growth by abolishing

development to the blastocyst stage in murine gene knock out studies, or any other way.

However, failure to inhibit preimplantational development in absence of a particular growth

factor or cytokine does not mean that it has no role in preimplantation growth; there may be

redundancy of functions allowing one factor to be substituted for another.

Epidermal Growth Factor (EGF)

EGF is an acidic polypeptide molecule containing 53 amino acid residues with an

approximate molecular weight of 6 kDa. At least six other ligand members in the EGF

superfamily are known to directly activate EGF receptors; these include transforming growth

factor-α, heparin binding EGF-like growth factor, amphiregulin, betacellulin, and epiregulin.

These growth factors contain a common motif of around 40-45 amino acids with highly

conserved six cysteine residues known as the EGF-like domain (Carpenter and Cohen, 1990).

These growth factors are synthesized as transmembrane precursor molecules which are then

proteolytically cleaved by metalloproteases to be released from the cell surface as mature soluble

growth factors (Massague and Pandiella, 1993). The EGF receptor is a single transmembrane

polypeptide with an approximate molecular weight of 170 kDa. The cytoplasmic domain of EGF

receptor has tyrosine kinase activity and is a homologue to the avian oncogene erbB which

encodes a truncated EGF receptor that lacks the extracellular EGF binding domain (Ullrich et al.,

1984). Four members of the EGF receptor family have been identified and designated by either

HER (human EGF receptor) or erbB nomenclature. The classical EGF receptor is known as

HER1 or erbB1. The unstimulated EGF receptors are present as monomeric receptors. Upon

30

EGF ligand-receptor binding, the monomeric receptors could undergo either homodimerization or

heterodimerization with other members of the EGF receptor family, a process known as

transmodulation, resulting in autophosphorylation of specific tyrosine residues within their

cytoplasmic domains. Those phosphorylated tyrosine residues would serve as docking sites of

recognition for the SH2 (Src homology 2) and phosphotyrosine binding domains of numerous

down stream signaling molecules that have been shown to be activated by EGF receptors. The

lysine residue 721 of EGF receptor has been found to be functionally essential as this residue

participates in ATP binding that is required as a source of phosphate groups for the kinase

reaction. Mutation at this lysine residue has been shown to abolish the cellular responses to EGF

(Chen et al., 1987, Honegger et al., 1987, Gill et al., 1988). The ability of the EGF receptor

family to undergo heterodimerization allows them to diversify signaling potential. However, not

any combination of the heterodimers is equally potent in generating signals. For instance, a

heterodimer containing erbB2 is usually associated with a prolonged signal activation (Beerli et

al., 1995, Graus-Porta et al., 1996, Spencer et al., 2000) or erbB3 is the most potent in activating

phosphatidyl inositol 3-kinase (Soltoff et al., 1993, Prigent and Gullick, 1994).

Signal transduction of the EGF receptor (erbB1) has been well characterized, although not

completely elucidated (see Burgering and Bos, 1995, Carpenter G, 2000, Laserer et al., 2000,

Olayioye et al., 2000, Schlessinger, 2000 for review). In brief, two major signaling pathways are

involved, MAP kinase cascade and phosphoinositol-related cascade. The key molecules are the

follows. Phosplolipase C-γ (PLC-γ) has been the best characterized down stream molecule of the

EGF receptor (Kim et al., 1990, Todderud et al., 1990) in which more than half of total

intracellular PLC-γ has been shown to be activated (phosphorylated) within a few minutes after

addition of EGF (Wahl et al., 1989). Activation of PLC-γ results in hydrolysis of

phosphatidylinositol bisphosphate (PIP2) into inositol triphosphate (IP3) and diacylglycerol

(DAG) leading to the activation of Ca2+ related activated-protein cascade and protein kinase C

31

(PKC), respectively, which in turn, lead to a numerous cellular responses (see Czech, 2000,

Hunter T, 2000, Newton, 2001, Ventura and Maioli, 2001, Swannie and Kaye, 2002 for review).

The MAP kinase cascade has been shown to be activated through the EGF receptor. Briefly,

activated EGF receptors activate Grb2 leading to an activation of SOS, a guanine nucleotide

exchange factor. The SOS then stimulates Ras, a small G-protein, leading to an activation of

MAP kinase. This lead to stimulation of cell proliferation and differentiation (see Su et al., 1996,

Davis, 2000, Enslen and Davis, 2001, Chang and Karin, 2001, for reviews).

Inhibition of EGF receptor production by injection of EGF receptor antisense RNA into

murine 2-cell stage embryos resulted in delayed onset of blastocyst formation without reducing

blastocyst cell numbers (Brice et al., 1993). This indicated that EGF signaling has a role in

embryonic cell differentiation rather than cell proliferation. On the contrary, the same study has

shown that embryo culture with polyclonal antibody against EGF receptor resulted in murine

blastocysts with higher cell numbers; presumably antibody binding mimicked natural ligand-

receptor binding and induced the signal transduction cascade for the EGF receptor. Target gene

disruption of EGF receptor of CF-1 background mice resulted in death of embryos at the time of

implantation due to degeneration of the inner cell mass while a similar experiment with CD-1

background mice resulted in offspring born but with several severe abnormalities (Threadgill et

al., 1995). In bovine blastocysts, numbers of inner cell mass cells and trophoblast cells have been

shown to be unaffected by addition of exogenous EGF (Yang et al., 1993, Lonergan et al., 1996).

Lee and Fukui (1995), however, obtained bovine blastocysts with higher total cell numbers after

EGF supplementation.

EGF ligand were not detected in any stage of in vitro derived bovine embryos from one-

cell zygotes to blastocysts (Watson et al., 1992). In another study, transcripts of EGF ligand were

shown to be presence in every stage of in vitro derived bovine embryos while transcripts of

erbB3, one of the EGF receptor family, were detected only at the 2-cell stage and at the blastocyst

stage (Yashiyoda et al., 1997). Nonetheless, transcripts of both EGF and EGF receptor were

32

detected in the elongating bovine blastocyst stage recovered between days13 to 16 after

insemination (Kliem et al., 1998). Transcripts of EGF have not been detected in bovine oviductal

tissue (Watson et al., 1992) but have been detected in bovine endrometrial tissue (Kliem et al.,

1998).

Exogenous EGF has been shown to stimulate bovine embryonic development in vitro to

the blastocyst stage (Yang et al., 1993, Lonergan et al., 1996). Supplementation of serum-free

culture medium with EGF improved in vitro bovine blastocyst development to equal that

supplemented with fetal calf serum (Palasz et al., 2000). However, addition of EGF to a certain

chemically defined bovine embryo culture medium produced no positive effects on blastocyst

development (Flood et al., 1993; Shamsuddin, 1994, Lee and Fukui, 1995).

Insulin-like Growth Factor-I (IGF-I)

Insulin-like Growth Factor-I, or IGF-I, is a member of the IGF system which is

comprised of IGF-I itself, IGF-II, two types of receptors designated as type I and type II IGF

receptors, a group of six distinct IGF-binding proteins (IGFBP) identified (Baxter et al., 1998),

and IGF-binding protein proteases. IGF-I is a single chain basic protein containing 70 amino

acids with three disulfide bond sites to maintain tertiary structure. IGF-II is a slightly acidic

single chain peptide of 67 amino acid residues. Both are approximately 7 kDa and exibit 70 %

homology to one another. IGFBPs have greater affinity to both of the IGF ligands than those of

IGF receptors (Rechler, 1993, Rechler and Clemmons, 1998). In biological fluids, both IGFs are

normally bound to IGFBP. This serves as a means to regulate IGF bioavailability, i.e., half-life,

distribution, and target sites in which IGF ligands would be released from IGFBP via IGF-

binding proteins proteolytic activity (Clemmons et al., 1998). Both IGF ligands can interact with

both types of their cell surface receptors. Type II IGF receptors not only interact primarily to

IGF-II but also serve as the receptor for manose-6-phosphate containing ligands but at a distinct

binding site (Morgan et al., 1987, Kornfeld, 1992). Signal transduction and function of type II

33

IGF receptor has not been elucidated (LeRoith et al., 2001); only that binding of IGF-II to type II

receptor results in internalization leading to degradation of IGF-II thereby reducing the

circulatory level of IGF-II has been reported (Baker et al., 1993). The effects of IGF-I are

mediated mainly through type I IGF receptor. Type I IGF receptor comprises two extracellular

α- subunits and two transmembrane β-subunits. The α-subunits serve as binding sites while the

β-subunits possess tyrosine kinase activity. Activation of type I IGF receptors leads to auto

tyrosine phosphorylation of the intracellular domain of the β-subunits.

Several molecules, especially insulin receptor substrate (IRS) type 1 to 4 and src

homology 2 (SH2)-containing proteins, can be activated by means of recruiting to IGF receptors.

Once activated, IRS and SH2-containing proteins can interact with a variety of down stream

molecules to exert biological effects of IGF-I. Those molecules include, phosphatidyl (PI) 3-

kinase, and proteins in the mitogen-activated (MAP) pathway (see Stewart and Rotwein, 1996, Le

Roith et al., 2001, for reviews).

The significances of IGF-I and other components in the IGF system relevant to bovine

preimplantational development is well characterized. IGF-I transcripts are detectable in both

bovine oviductal epithelium all three oviductal regions (Schmidt et al., 1994; Xia et al., 1996,

Pushpakumara et al., 2002), and endometrium (Geisert et al., 1991, Kirby et al., 1996). The

mRNA expression of IGF-I in the bovine uterus has been shown to be highest at estrus and lowest

during the early and late luteal phases (Robinson et al., 2000). Bovine oviductal cell primary

cultures have been shown to be able to secrete both IGF-I and IGF-II into culture medium, with

ten times more IGF-II than IGF-I (Winger et al., 1997). IGF-I is estimated to be present in

bovine uterine fluid at concentrations of 4 to 5 pg/µg protein (Funston et al., 1995). Expression

of IGFBPs in bovine reproductive tissue has been well characterized (Geisert et al., 1991, Keller

et al., 1998). In vitro derived bovine embryos from 2-cell to the blastocyst stage have been

shown to express IGF-I and type I IGF receptor mRNA (Watson et al., 1992, Schultz et al., 1992,

34

Yashiyoda et al., 1997, Yaseen et al., 2001). Messenger RNA of IGFBP-2, -3, and -4 have been

detected throughout bovine embryonic development to the blastocyst stage while transcripts of

IGFBP-5 have been detected only in blastocysts and IGFBP-1 and -6 transcripts have not been

detected (Winger et al., 1997). IRS type 1 mRNA has been shown to be highly expressed in

murine blastocysts (Puscheck et al., 1998).

Effects of exogenous IGF-I on in vitro development of bovine preimplantation embryos

have been investigated by several authors. Data are available ranging from no effect of IGF-I

(Flood et al., 1993, Shamsuddin, 1994, Lee and Fukui, 1995), or positive effects only when IGF-I

was used in conjunction with granulosa cell co-culture (Herrler et al., 1992) or only when bovine

serum was included in the culture medium (Palma et al, 1997) to positive effects in serum-free

culture medium (Matsui et al., 1997, Prelle et al., 2001, Moreira et al., 2002, Makarevich and

Markkula, 2002). IGF-I has been shown to stimulate the proliferation of murine and human

blastocysts, mainly in the inner cell mass (Harvey and Kaye, 1992; Kaye et al., 1992, Rappolee et

al., 1992, Lighten et al., 1998). In bovine blastocysts, however, IGF-I has been shown to

increase numbers of trophoblast cells without affecting cell numbers in the inner cell mass (Prelle

et al., 2000; Makarevich and Markkula, 2002). Protein synthesis in mouse embryos has been

shown to be stimulated by exogenous IGF-I (Harvey and Kaye, 1991). IGF-I also has been

shown to have an anti-apoptotic action during in vitro development of rabbit (Herrler et al., 1998,

Makarevich et al., 2000), murine (Chi et al., 2000), human (Spanos et al., 2000), and bovine

embryos (Makarevich and Markkula, 2002, Byrne et al., 2002). The use of IGF-I and IGF-I

receptor mRNA expression as a potential markers of embryo viability have been reported in

mouse (Kowalik et al., 1999) and human embryos (Liu et al., 1997).

Leukemia Inhibitory Factor (LIF)

Leukemia Inhibitory Factor, or LIF, is a multifunctional cytokine belonging to the

interleukin-6 (IL-6) cytokine family. Cytokines in the IL-6 family share gp 130 receptor subunit

35

in their signal transduction mechanisms. Bovine and human LIF contain 187 amino acids and

show 80 % sequence homology. Murine LIF has been shown to contain a variety of degrees of

glycosylation resulting in a range of molecular weights from 37 to 60 kDa (Hilton et al., 1988).

Crystal structure analyses of human LIF reveal that LIF is a four-α-helix bundles cytokine, a

common feature of cytokines in the IL-6 family (Robinson et al., 1994). Actions of LIF are

mediated through a cell-surface LIF receptor that subsequently heterodimerizes with gp 130.

LIF initially binds to LIF receptor at a low affinity level, but its subsequent association with gp

130 results in formation of a high affinity complex (Gearing et al., 1992).

Signal transduction of LIF has been shown to be involved with both JAK-STAT and

MAP kinase pathway. Janus kinase (Jak) could be activated after heterodimerization of LIF

receptor and gp 130 complex resulting in phosphorylations at tyrosine residues of both LIF

receptor and gp 130. Phosphorylations of LIF receptor and gp 130 lead to an activation of signal

transduction and activator of transcription (STAT) proteins. Once activated, STAT would

undergo dimerization leading to their translocation into nucleus and resulting in transcription

activation of numerous genes (see Karin and Hunter, 1995 for details). Both Jak 1 and Jak 2 have

been shown to be activated by gp 130-LIF receptor complexes. Tyrosine phosphorylation of Jak

1 and Jak 2 have been observed after LIF stimulation in rodent cardiac myocytes (Kunisada et al.,

1996, Kodama et al., 1997), murine osteoblast cells (Lowe et al., 1995), and also in murine

embryonic stem cells (Ernst et al., 1996). However, only Jak 1 appears to be the main down

stream pathway of LIF actions. Lowe et al (1995) showed that LIF treatment of murine

osteoblast cells predominantly phosphorylates Jak 1 compared to that of Jak 2. Accordingly, it

has been shown that the absence of Jak1 markedly reduced the effects of IL6 compared to that of

Jak 2 in human fibrosarcoma cells (Guschin et al., 1995). Target disruption of Jak 1 gene in

mice has shown that Jak1 plays an essential and nonredundant role in promoting biological

responses of cytokines that depend on the gp130 subunit for signaling, including LIF (Rodig et

al., 1998). In fibroblast cells derived from Jak2-deficient mice, the responses to IL-6 have been

36

shown to be undisturbed (Parganus et al., 1998). Similarly, murine embryonic stem cells derived

from Jak 2–deficient embryos have been shown to be normally responsive to LIF signaling

(Neubauer et al., 1998). Among all of STAT isoforms, LIF predominantly activates STAT3 in

several cell types such as in myoblast cells (Megeney et al., 1996), adipocytes (Balhoff et al.,

1998), corticotroph cells (Bousquet and Melmed, 1999), and Sertoli cells (Jenab and Morris,

1998). Overexpression of the STAT 3 dominant negative mutants has been shown to inhibit the

effect of LIF on maintaining pluripotentcy of embryonic stem cells (Ernst et al., 1997, Niwa et

al., 1998). Similarly, overexpression of STAT 3 dominant negative mutants also inhibit the effect

of LIF on induction of differentiation in M1 leukemic cells (Minami et al., 1996, Nakajima et al.,

1996).

In addition to activating the JAK-STAT cascade, LIF has been shown to stimulate several

molecules in the MAP kinase pathway including Ras, Raf, MAPKK, and Erk (Schwarzschild et

al., 1994, Schiemann and Nathanson, 1994, Ernst et al., 1996, Kunisada et al., 1997, Schiemann

and Nathanson, 1998, Raz et al., 1999). Phosphatidylinositol (PI) 3-kinase also appears to be

involved in this pathway of LIF signaling since wortmanin, a PI 3-kinase, inhibits LIF-induced

activation of MAPK activity (Oh et al., 1998).

Striking evidence for the roles of LIF in reproduction have been provided by Stewart et al

(1992) who showed that female knockout LIF¯/LIF¯ mice were infertile due to defective

implantation while transfer of LIF¯/LIF¯ blastocysts to wild type female mice resulted in

successful pregnancy. Treatment of LIF¯ /LIF¯ female mice with recombinant human LIF

resulted in successful implantation and pregnancies. LIF has been shown to stimulate the

differentiation of human trophoblast cells as determined by the decreasing of hCG and increasing

of fibronectin expression (Nachtigall et al., 1996). High concentrations of LIF and LIF receptor

proteins have been found in the uteri of day 4 pregnant mice, which is just prior to blastocyst

implantation (Bhatt et al., 1991) and a similar finding has been observed in rabbit uteri at 5 to 6

days post ovulation (Yang et al., 1994). In human, endometrial LIF expression is peak during

37

mid and late secretory phases while LIF expression during the perovulatory phase is almost

undetectable (Charnock-Jones et al., 1994, Chen et al., 1996, Vogiagis et al., 1996, Cullinan et al.,

1996). In cow, uterine LIF expression has been found to be relatively constant throughout the

estrous cycle and early pregnancy (Vogiagis et al., 1997a). However, immunizations against

LIF in recipient cows and ewes around the time of implantation have resulted in reduced

pregnancy rates following embryo transfer (Vogiagis et al., 1997b). In human, LIF expression in

the endometrium derived from unexplained infertile women has been shown to be only 30-40%

of that in fertile women (Hambartsoumian, 1998). Concentrations of LIF in follicular fluid of

preovulatory follicles were significantly increased after hCG treatment and LIF was undetectable

in follicular fluid of small follicles (Arici et al., 1997, Coskun et al., 1998). HCG has been

demonstrated to stimulate LIF production from granulosa cells of preovulatory follicles. This

indicates that LIF may also have some role during the final oocyte maturation event and/or in the

ovulatory process.

LIF and expression of its receptors have been detected in human (Sharkey et al., 1995,

Chen et al., 1999), murine (Murray et al., 1990, Bhatt et al., 1991), and bovine blastocysts (Eckert

and Niemann, 1998). In rabbit blastocysts, an in situ hybridization study revealed that LIF

receptors predominantly localize in the inner cell mass while LIF ligands predominantly localize

in the trophoblast cells (Yang et al., 1995). Supplementing culture media with LIF has been

shown to stimulate murine embryonic development to the hatched blastocyst stage (Mitchel et al.,

1994, Lavarnos et al., 1995). Addition of LIF has been shown to improve human embryonic

development to the blastocyst stage (Dunglison et al., 1996). Similarly, the presence of LIF in

endometrium co-culture of human IVF embryos has been shown to be associated with

improvement in embryonic development and pregnancy rates (Spandorfer et al., 2001).

Incubation of sheep embryos with LIF for the preceding 2 days prior to embryo transfer has been

shown to increase pregnancy rates from 16% to 50% (Fry et al., 1992). Addition of LIF to

culture media has been shown to improve in vitro bovine embryonic development to the

38

blastocyst stage by several authors (Fukui and Matsuyama, 1994, Han et al., 1995, Funstun et al.,

1997). Nonetheless, the LIF system is not crucial for preimplantational embryonic development,

at least in murine species. Not only murine embryos lacking LIF but also those either lacking gp

130 or LIF receptor have been shown to develop beyond the implantational period (Ware et al.,

1995, Yoshida et al., 1996).

Embryonic stem (ES) cells are pluripotent cells established from the early embryo which

can be differentiated to all adult tissues when they are reintroduced into developing embryos,

usually at the blastocyst stage. It has long been known that murine embryonic stem cells can be

maintained in an undifferentiated state in the presence of LIF (Smith et al., 1988, Williams et al.,

1988). LIF actions in ES cells is mediated predominantly through STAT3. By employing the

dominant negative mutant of STAT3 approach, Boeuf et al (1997) and Niwa et al (1998) have

demonstrated that LIF was unable to maintain ES cells overexpressing dominant negative mutant

of STAT3 in an undifferentiated state. Ernst et al (1999) have shown that STAT3 antisense

oligonucleotides impaired the effect of LIF on inhibition of ES cell differentiation. In fact, it has

been demonstrated that activation of STAT 3 alone without the presence of LIF is sufficient to

maintain murine ES cells (Matsuda et al., 1999). Similarly, it has been shown that murine ES

cells harboring the abrogated STAT3 gene were unable to be maintained in an undifferentiated

state (Raz et al., 1999). Those findings indicate that the MAP kinase pathway is not essential for

LIF signaling in ES cells. However, LIF does not appear to be as effective for ES cells from

other species compared to murine ES cells. Thomson et al (1995) observed that rhesus monkey

ES cells could not survive or remain in the undifferentiated state in the presence of LIF alone

without the presence of fibroblast feeder cells. Similarly, LIF without the presence of fibroblast

feeder cells could not sustain human ES cells (Thomson et al., 1998, Reubinoff et al., 2000).

39

B. GENE TRANSFER METHODOLOGIES FOR MAMMALS

The first transgenic experiment in a mammalian system was described by Brackett et al

(1971) , a full decade before the word “transgenic” was coined. Rabbit spermatozoa depleted

from seminal fluid were able to incorporate exogenous DNA (SV40 DNA) in the presence of

dimethyl sulfoxide (DMSO) and to transfer the foreign DNA into ova at fertilization. Expression

of the viral activity was then recovered from resulting embryos. Jaenisch and Mintz (1974)

injected SV40 DNA into murine blastocysts to show the incorporation of exogenous DNA in the

offspring. Later, Moloney murine leukemia virus was used and resulted in offspring developing

leukemia after birth (Jaenisch et al., 1975) as well as germline transmission (Jaenisch, 1976).

Those findings indicated that genetic information of mammalian embryos could be manipulated

with foreign DNA and those embryos could still develop to term resulting in live transgenic

animals. A transgene is a recombinant DNA molecule that consists of at least two elements,

regulatory element and structural element. The regulatory element may consist only of the

promoter sequences or include, in several circumstances, the enhancer sequences for better

expression. The structural element is comprised of DNA sequences encoding the gene product.

This could include either genomic sequences containing exons and introns, or cDNA sequences

which contain only exons. However, minigenes (DNA sequences containing some but not all of

the introns of a particular gene) are usually more preferable.

Brinster et al (1980) reported appropriate translation of rabbit globin mRNA after

transferring the exogenous RNA into fertilizing mouse ova. The word “transgenic” was first used

by Gordon and Ruddle (1981) who reported genetically modified mouse embryos created by the

pronuclear injection technique (Lin, 1966). It was demonstrated that a transgene not only

integrated into embryonic genomes but also was transmitted to the offspring (Gordon and Ruddle,

1981). However, it was the works of Palmiter and Brinster who made the scientific community

realize the powerful impact of transgenic animals Palmiter et al., 1982, 1983). First, they

demonstrated that the transgene expression could drastically change the phenotype in mice

40

overexpressing human growth hormone, the transgenic mice grew to almost twice size of normal

mice. Second, the transgene expression was controllable by using the metallothionein (zinc

inducible gene) and growth hormone fusion sequences. Therefore, those transgenic mice grew

much faster after being provided zinc-containing water to induce overexpression of growth

hormone. Methodologies to produce transgenic animals include pronuclear injection, nuclear

transfer, and a variety of alternative gene transfer approaches including sperm-mediated gene

transfer which will be the main focus of this review.

PRONUCLEAR INJECTION

Pronuclear injection has been working reasonably well with murine zygotes. Transgenic

mice have been used to answer tremendous numbers of basic research questions as well as to

provide very useful models for study of human diseases. Wall (1996) stated that more than 6,000

literature reports are related to transgenic mice whereas less than 300 are related to livestock

species. From the 300 livestock studies, only 37 gene construcs were involved. More than

18,000 Medline database searches have been related to transgenic animals; most dealt with

transgenic mice (Wall RJ, 2001). Success in producing transgenic livestock species after

pronuclear microinjection has been extremely low due to two related problems. First, pronuclei

of livestock zygotes are not easy to visualize compared to those of murine zygotes. For instance,

in less than 60% of bovine zygotes pronuclei could be visualized after centrifugation to displace

opaque cytoplasmic material (Bondioli et al., 1992, Eyestone, 1999). Transgene integration is

believed to occur during the first round of DNA replication, S-phase (Bishop & Smith, 1989,

Coffin, 1990). The optimal time for bovine pronuclear injection has been reported to be between

18 to 26 h post in vitro insemination (Krisher et al., 1994, Gagne et al., 1995, Chan et al., 1999)

which is near the end of S-phase. This would result in low incidences of transgene integration.

In addition, survival of injected zygotes is somewhat low in large domestic animals; only 1 to 4 %

41

transgenic offspring are expected from offspring born after pronuclear injection (Niemann &

Kues, 2000).

Eventhough IVF has become a widely practiced technique and expenses to produce

bovine zygotes has been drastically reduced, pronuclear injection of livestock species remains an

inefficient approach for the production of transgenic animals. Eyestone (1999) reported

fascinating but realistic figures regarding the production of transgenic calves. He found only

35,000 out of 70,000 zygotes to be injectable. Of those, 2,200 developed to morula or blastocyst

stages. Only 1,700 were transferable and these resulted in 370 pregnancies followed by births of

226 calves. Only 18 of the calves (almost 8%) were transgenic. Interestingly, if transgenic

embryos could have been identified prior to embryo transfer only 30 embryos would have been

needed to produce 18 transgenic calves. With the advent of judicious use of green fluorescent

protein, this demand should be possible to fulfill (Chan et al., 2002).

NUCLEAR TRANSFER

Campbell et al (1996) reported birth of lambs after using differentiated cells derived from

the inner cell mass as donor cells for nuclear transfer. Soon afterward, Wilmut et al (1997)

employed adult sheep somatic cells as donor cells for nuclear transfer resulting in the birth of

Dolly. Now, that several groups have reported birth of live offspring after nuclear transfer from

adult somatic cells of several species including bovine (Kato et al., 1998), murine (Wakayama et

al., 1998), caprine (Baguisi et al., 1999), and porcine (Polejaeva et al., 2000). Major obstacles to

utilizing this technology to produce transgenic animals are the high incidences of fetal, perinatal,

and postnatal loses. However, nuclear transfer now tends to be a more efficient methodology for

producing transgenic animals than pronuclear injection. Donor cells can be transfected and

selected in vitro prior to nuclear transfer. Therefore, all offspring born should be transgenic, with

uniform gene integration and without mosaicism. Schnieke et al (1997) reported transgenic sheep

producing human factor IX that were obtained by the nuclear transfer approach. Soon thereafter,

42

birth of transgenic calves after nuclear transfer was reported (Cibelli et al, 1998). A major

breakthrough in the production of genetically modified animals that is impossible to achieve via

pronuclear injection is the currently available means for livestock production that incorporates

gene-targeting. Transgenic lambs harboring human α1-anti-trypsin at their α1-procollagen locus

was reported by McCreath et al (2000). Lambs with prion protein (PrP) gene deleted have also

been reported (Denning et al., 2001).

ALTERNATIVE GENE TRANSFER METHODOLOGIES

Viral-mediated gene transfer

Most recent reports with viral methodology involve use of retroviruses. The endogenous

retroviruses (ERVs) are sequences with similarity to infectious retrovirus found in mammalian

genome (Temin, 1989). ERVs represent the remnants of ancestral retroviral infections that

became fixed in the genome. ERVs are being estimated to occupy up to 1% of the human

genome (Taruscio and Mantovani, 1998, Paces et al., 2002). Based on that, retroviral vector

could be considered as a natural means for gene delivery into mammalian genomes. A transgene

carried within the retroviral genome will be reverse transcribed into DNA, then host genomic

integration is mediated through retroviral integrase and specific sequences located at both ends of

the retroviral genome (Brown, 1990). Indeed, viral-mediated gene transfer was one of the very

early transgenic experiments reported (Jaenisch et al., 1975, Jaenisch, 1976). With the

introduction of replication defective retroviral vectors (Shimotohno & Temin, 1981), the

retroviral vector became one of the potentially most powerful gene transfer methodologies. Due

to species specificity, the retoviral vector approach was not tried on bovine species until 1993

(Kim et al.,1993; Haskell & Bowen,1995) but results were not very successful due to low

developmental rates, possibly caused by multiple insertions from continuous infections. Several

modern retroviral vectors have been developed, including a Moloney murine leukemia virus

pseudotyped with the envelope glycoprotein of vesicular stomatitis virus (VSV-G). This vector

43

has been used to produce transgenic calves via mature oocyte infection (Chan et al., 1998).

Efficiencies of viral vector infection to produce transgenic offspring compare favorably with

those after nuclear transfer. Four calves born from retroviral infected oocytes were transgenic

(Chan et al., 1998). Other transgenic offspring that have been produced by this approach are

those of rhesus monkeys (Chan et al., 2001), pigs (Cabot et al., 2001), and mice (Nagano et al.,

2001). The use of Lentiviral vector, another subclass of retrovirus, has been reported (Nagano et

al., 2002).

Few disadvantages of retroviral vectors have been recognized. First, the viral genome

could not harbor large transgenes; the total viral genome must be less than 10 kB in order to be

packaged to form an infectious particle as well as to be efficiently reverse transcribed. Second,

several steps are required from transgene insertion through recovery of viral particles. Third,

currently low concentrations of viral vector preparations require large volumes for injection. In

general, viral vector-mediated gene transfer seems to be a very promising approach for gene

integration (Wells et al., 1999).

The uses of adenovirus, another viral vector, has been reported (Tsuzuki et al., 1995,

Tsuzuki et al., 1996, Blanchard and Boekelheide K, 1997, Kubisch et al., 1997). Kubisch et al

(1997) injected the replication-defective human adenovirus harboring the lacZ reporter gene

(AdCMVLacZ/sub360) into the perivitelline space of mouse, rat, and cow zygotes and observed

lacZ expression in resulting blastocysts; 68% for cow, 18% for mouse, and 9% for rat blasocysts.

However, compared to retroviral vectors adenoviral vectors do not ensure transgene integration,

but rather just, deliver foreign DNA into the nuclei.

Sperm-mediated gene transfer

Due to its relative simplicity compared to pronuclear injection, the use of spermatozoa as

vectors to deliver transgenes into oocytes during fertilization has attracted several investigators.

Brackett et al (1971) first described this approach in rabbit experiments. Later, sperm-mediated

44

gene transfer gained prominence with the success in the mouse reported by (Lavitrano et al

(1989). The latter authors reported the following findings: mouse epididymal spermatozoa were

able to take up pSV2CAT plasmid DNA spontaneously; exogenous DNA was delivered into

oocytes during fertilization resulting in transgenic offspring expressing the chloramphenicol

acetyl transferase (CAT) reporter gene; and, the transgenes were transmitted to F1 progeny. To

date, DNA association with spermatozoa has been reported in a variety of organisms from insects

to mammals (Sadafora C, 1998, Gandolfi F, 1998). Work with mouse spermatozoa has been

shown to be more repeatable (Maione et al., 1998), or mere nearly so, compared to work in

domestic species (Shellander et al., 1995, Sperandio et al., 1996). Besides species differences, an

explanation could be that most investigators have applied this technique to ejaculated

spermatozoa which are known to be mostly protected from DNA association by proteins present

in seminal fluid (Lavitrano et al., 1992, Zani et al., 1995).

Some modifications have been applied to ejaculated spermatozoa to improve binding and

internalization of DNA. These include the use of DNA liposome complexes. Bachiller et al

(1991) used a commercial liposome preparation (Lipofecin) with epididymal mouse spermatozoa

prior to IVF but no transgenic mice were found among 458 pups born. However, Rottman et al

(1991) reported three transgenic calves from three calves born after mixing bull spermatozoa with

DNA liposome complex prior to artificial insemination. Shemesh et al (2000) also reported four

transgenic of four calves born after using another liposome preparation (Lipofectamine)

combined with restriction enzyme. Electroporation has been applied to bull spermatozoa as well,

but transgenic offspring after this have never been achieved (Gagne et al., 1991, Reith et al.,

2000). The use of adenoviral vectors to infect spermatozoa has been reported. Blanchard and

Boekelheide, (1997) injected an adenovirus vector carrying a lacZ transgene into rat testes and

observed lacZ expression in Leydig and Sertoli cells but not in germ cells. Farre et al (1999)

adopted that approach by exposing boar spermatozoa with adenoviral vector carrying a lacZ

transgene prior to artificial insemination and obtained 4 transgenic of 56 total piglets born.

45

However, germline transmission was not achieved as offspring obtained after the mating of two

positive animals did not carry the transgene.

Other investigators have chosen to use an in vivo sperm transfection approach. This was

carried out by injecting either DNA solution alone into the vas deferens (Sato et al., 1994, Huguet

and Esponda, 2000) or into seminiferous tubules (Yamazaki et al., 1998, Huang et al., 2000,

Yamazaki et al., 2000). Transgenic mice were produced from the work of Huang et al (2000).

Some investigators combined DNA with liposome then injected it into seminiferous tubules

(Yonezawa et al., 2001, Celebi et al., 2002). In this way transgenic mice resulted in the work of

Celebi et al (2002). No reports are available from similar work in other species.

Another promising approach was to isolate male germ cells, transfect them in vitro, then

reimplant them into testes depleted of germ cells (Kim et al., 1997). Transgenic mice have been

produced by this approach with a retroviral vector (Nagano et al., 2001). Perry et al (1999)

combined sperm-mediated gene transfer with ICSI to produce transgenic mice. The results were

impressive, 10 (19%) out of 57 live pups were transgenic. This approach was then named

metaphase II-mediated transgenesis by Perry et al (2001). There has been some level of success

with other species as well: rhesus monkey (Chan et al., 2000) and pig (Lai et al., 2001); but apart

from mice, live transgenic offspring have not yet been produced.

Another approach under development is to raise monoclonal antibodies against

spermatozoa, then link transgenes to the antibodies, and to mix transgene-antisperm antibody

complexes with spermatozoa prior to insemination (Chang et al., 2002). By this means not only

were transgenic offspring produced, but expression of the transgene was also demonstrated in

61% (35/57) of transgenic pigs. Interestingly, this approach provide strong evidence that there is

no need for DNA internalization, only tight external association of transgene and spermatozoa is

required. However, there remains a possibility that spermatozoa might take up DNA-antisperm

complexes much better than naked DNA alone.

46

MECHANISMS OF SPERM DNA ASSOCIATION

Binding DNA to spermatozoa

The binding of exogenous DNA to murine epididymal spermatozoa seems to be mediated

by a 30-35 kDa sperm membrane protein identified by using Southwestern blot analysis

(Lavitrano et al., 1992). This 30-35 kDa sperm membrane protein was also found in human,

bovine, and porcine spermatozoa (Camaioni et al., 1992). Lavitrano et al (1992) also found that

spermatozoa bind exogenous DNA in a non-specific manner. High negatively charged molecules

such as heparin or dextran sulfate can compete and displace exogenous DNA from binding to

spermatozoa indicating that the interaction is ionic, reversible, and not restrict to DNA.

Interestingly, high positively charged molecules such as polylysine facilitated DNA binding

instead of sequestering DNA away from spermatozoa. It turned out that ejaculated spermatozoa

could not bind exogenous DNA as well as epididymal spermatozoa could. A glycoprotein

presence in the seminal fluid was found to act as a barrier preventing ejaculated spermatozoa

from binding to exogenous DNA. In the presence of these inhibitory glycoproteins, the putative

30-35 kDa DNA binding proteins lost their ability to bind to exogenous DNA (Zani et al., 1995).

It can be speculated that factor(s) in seminal plasma might protect ejaculated spermatozoa from

being transfected by foreign DNA that may be present in the genital tracts , e.g. from bacterial or

viral sources.

Epididymal spermatozoa from major histocompatability (MHC) class II knock-out mice

have reduced (50% reduction compared to wild-type) ability to bind to exogenous DNA

(Lavitrano et al., 1997). Whether MHC class II molecules are present on mouse spermatozoa

remains inconclusive. MHC II molecules have been reported to be present on mouse

spermatozoa (Mori et al., 1990, Wu et al., 1990) but Lavitrano et al (1997) were unable to

confirm those findings. Therefore, it can only be concluded that MHC class II gene product is

required for the production of normal mouse spermatozoa capable of binding to exogenous DNA.

47

It would be very interesting to investigate whether, or not, spermatozoa from MHC class II

knock-out mice possess the putative 30-35 kDa sperm DNA binding protein.

Internalization and chromosomal integration of bound DNA

Nuclear internalization of bound plasmid has been reported in murine, bovine, porcine,

and human spermatozoa (Atkinson et al., 1991, Camaioni et al., 1992). Fifteen to twenty percent

of bound DNA was internalized into murine sperm nuclei within 60 min of incubation

(Francollini et al., 1993). There are some concerns suggesting that nuclear internalization could

be an artifact caused by the preparation process (Kim et al., 1997). However, a preliminary

study of McCarthy and Ward (2000) with murine spermatozoa seems to be convincing that

exogenous DNA could be taken up and become tightly associated with the nuclear matrix. CD4

molecules have been shown to play a role in DNA internalization (Lavitrano et al., 1997).

Spermatozoa from CD4 knock-out mice were unable to take up bound DNA. CD4 molecules are

detectable on spermatozoa and anti-CD4 antibodies can prevent the internalization of bound

DNA.

After exogenous DNA was taken up into the nuclear matrix could sperm genomic

integration occur? It is unlikely for that to happen but yet possible. Sperm chromatin, unlike that

of somatic cells, is tightly packed into a small volume, six times more condensed than in mitotic

chromatin (Ward and Coffey, 1991, Ward, 1993, Barone et al., 1994, McCarthy and Ward,

1999). For example, the basic DNA packing unit of spermatozoa is in a doughnut shape (toroid)

containing around 60 kb of DNA; each unit is linked by uncoiled DNA stretched out (Hud et al.,

1993). Therefore, it is common to suspect that exogenous DNA will not integrate into sperm

DNA. However, an integration of foreign DNA into the sperm genome has been documented

(Zoraqi et al., 1997). Genomic libraries were constructed from mouse spermatozoa after being

incubated with plasmid DNA. From two selected clones, plasmid DNA sequences were found to

be integrated with the sperm genome. Interestingly, topoisomerase II consensus sequences were

48

found in one end of both clones. This would indicate integration events of exogenous DNA was

mediated by the enzyme topoisomerase II. Whether the integration site is located within the

toroid units or within the linkers was not known. Nonetheless, spermatozoa are not completely

depleted of histones. A certain amount of histones was estimated to remain with sperm DNA,

approx 15% in human spermatozoa (Gatewood et al., 1987). Therefore, those histone associated

sequences would also be good target sites for integration mediated by topoisomerase.

49

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CHAPTER 3

EFFECT OF LEUKEMIA INHIBITORY FACTOR ON BOVINE EMBRYOS PRODUCED IN

VITRO UNDER CHEMICALLY DEFINED CONDITIONS1

1S. Sirisathien, H. J. Hernandez-Fonseca, P. Bosch, B. R. Hollett, J. D. Lott, and B. G. Brackett .

Accepted by Theriogenology . Reprinted here with permission of publisher, 7/3/2002.

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ABSTRACT

The objective of these experiments was to assess putative embryotrophic effects of leukemia

inhibitory factor (LIF) on bovine preimplantation development in chemically defined media.

Recombinant human LIF was added to embryo culture media at a concentration of 100 ng/mL.

When added for culture of morulae LIF had no positive effect on the proportion of embryos

reaching the blastocyst stage. However, LIF significantly reduced development to the blastocyst

stage when added for culture of 4-cell stage embryos (P < 0.05). In contrast, a positive effect

was found for progression of blastocyst development. In vitro blastocyst hatching rates were

significantly improved in the presence of LIF (P < 0.02). Numbers of total cells and of inner cell

mass cells were increased in LIF-treated blastocysts. In vitro survival of frozen-thawed

blastocysts was not improved by adding LIF to morula stage embryos before cryopreservation.

The pregnancy rate after direct transfer of cryopreserved LIF-treated embryos was not different

from that for untreated control embryos. Data indicate that addition of LIF has no major

beneficial effect on bovine embryos produced in these chemically defined conditions.

Keywords: LIF, IVF, Blastocyst, Direct Transfer

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INTRODUCTION

During the past decade most IVF laboratories have employed serum for enrichment of culture

media at specific steps of production of bovine embryos in vitro. Serum-supplemented media are

consistently able to support development of a reasonable number of oocytes into blastocysts.

However, undefined conditions, such as inclusion of various sera, make it difficult to obtain

consistent results or to compare results among laboratories. Furthermore, in vitro conditions that

are not completely defined are not suitable for determining a definitive role for a defined

molecular species in gamete function or preimplantation development. Very few attempts have

been made to employ chemically defined conditions beginning with in vitro oocyte maturation for

the production of bovine embryos (1-4). In general, completely defined conditions result in lower

yields of blastocysts (5). Nevertheless, defined conditions enable more accurate assessment of

quality of the biological starting materials, i.e., oocytes and spermatozoa that are known to vary

among gamete donors. There are numerous reports involving addition of growth factors or

cytokines to improve the efficiency of bovine embryo production in vitro. However, studies

conducted with serum-containing media, or with serum albumin or other incompletely defined

components during segments of the embryo-production process make interpretation of the results

difficult in the absence of data from more refined approaches as afforded by the use of completely

defined media.

Leukemia inhibitory factor (LIF), a glycoprotein cytokine with a molecular weight of 38 to 67

kDa, has been shown to have an important role in murine implantation (6, 7). Leukemia

inhibitory factor mRNA and protein have also been detected in the endometrium of pregnant

ewes, with the highest levels of expression on peri-implantation days 16-20 (8). Addition of LIF

to undefined bovine embryo culture conditions has led to inconsistent results. Supplementation

of bovine embryo culture medium with LIF (5,000 U/mL) beginning at morula or early blastocyst

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stages resulted in improvement in further development to expanded blastocyst stages without any

affect on hatching rates (9). In contrast, addition of 4,000 U/mL of LIF to another bovine culture

system for several intervals following in vitro insemination on Day 0, for example Day 1 to 3,

Day 4 to 7 or Day 1 to 7, did not improve development of IVF embryos to the blastocyst stage but

resulted in higher numbers of cells per blastocyst (10). In another report, supplementation of

bovine embryo culture medium with LIF (5,000 U/mL) at morula or early blastocyst stages prior

to cryopreservation improved the survival rate after thawing as reflected by subsequent culture in

vitro, without improving blastocyst development (11). Use of a high LIF secreting coculture

system for bovine embryo production resulted in improvement of blastocyst formation and also in

a better pregnancy rate after transfer of cryopreserved embryos into recipient cows (12). Our

purpose was to investigate whether exposure of in vitro produced bovine embryos to LIF under

chemically defined conditions was able to improve blastocyst development or continuing

embryonic viability after cryopreservation or to markedly enhance pregnancy rates following

direct embryo transfer (ET) after cryopreservation.

MATERIALS AND METHODS

All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA; catalog numbers

are indicated in parentheses). Recombinant human LIF (L-5283, Sigma) was characterized for

biological activity to contain specific activity of no less than 100 units/ng, where 50 units is

defined as the amount of LIF required to induce differentiation in 50 % of M1 myeloid leukemic

cells.

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In vitro maturation

Ovarian follicles were aspirated within 15 min after cows were slaughtered. Cumulus oocyte

complexes (COCs) were harvested from follicles of surface diameters less than 6 mm using a 10

mL syringe and 18 g needle. Follicular fluids were pooled in a 50 mL test tube and maintained at

33°C for 3 h during transit to the lab. In an initial experiment a comparison of cleavage and

blastocyst results was carried out for COCs with at least 2 compact layers of cumulus cells vs.

COCs selected for homogeneous cytoplasm and a healthy cumulus complement (13).

Subsequently, COCs with at least 2 compact layers of cumulus were utilized. The COCs were

washed twice with maturation medium and incubated in groups of 20 to 22 in 100 µL drops of

maturation medium covered with light mineral oil (M-3516, Sigma). The maturation medium was

TCM-199 (M-3769, Sigma) supplemented with 50 µg/mL sodium pyruvate, 2.2 mg/mL NaHCO3,

1 mg/mL polyvinyl alcohol (PVA), 0.25 mM glutamine, 0.1 mM cystine, 0.1 mM cysteamine, 10

mM HEPES, 50 µg/mL gentamicin sulfate plus 0.1 IU/mL of recombinant human FSH (1.7 IU/

µg, Ares Advanced Technology Inc., Randolph, MA, USA) and 5 ng/mL of recombinant human

IGF-I (Promega, Madison, WI, USA). The COCs were incubated under moist 5 % CO2, 5 % O2

and 90 % N2 in a modular incubator chamber (Billups-Rothenberg Inc, Del Mar, CA, USA) for 24

h. The same atmosphere, incubator, and temperature of 39 °C were employed for all in vitro

cultures.

In vitro fertilization

Swim-up selected spermatozoa were prepared in modified defined medium (mDM; 14)

without hypotaurine or epinephrine. Frozen semen (of similar quality as determined by

preliminary IVF results in conditions described below) from two breeds, Holstein and

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Brahma was used. For each replication, two straws of frozen Holstein semen, specially

processed for our laboratory with 108 sperm/straw, or four straws of frozen Brahma

semen, commercially processed with 20×106 spermatozoa/straw, were thawed at 37 °C

for 30 sec; then 110 to 150 µL of semen was layered under 1.5 mL of mDM in each of

several 12×75 mm tubes. The tubes were held at a 45 ° angle for 45 min at 39 °C under

moist 5 % CO2 in air. The uppermost 850 µL aliquots from each tube were then pooled in

a 15 mL tube and centrifuged at 320 × g for 10 min. The sperm pellet was resuspended to

380 µL and 20 µL mDM containing heparin (100 µg/mL final concentration for Brahma

semen or 200 µg/mL final concentration for Holstein semen) was added for 15 min sperm

incubation before insemination. During this interval aliquots of spermatozoa were

counted in a hemacytometer, and checked for motility. Then, 12 to 14 µL of sperm

suspension was added to each 82 to 86 µL drop of mDM to provide a concentration of 2 ×

106 motile spermatozoa per mL in 100µL. Matured COCs were added to each drop with

minimal amounts of medium; co-incubation with spermatozoa for 18 h allowed initiation

of IVF.

In vitro culture

At 18 h post-insemination, loosely associated cumulus cells and spermatozoa were removed from

zonae by gentle pipetting of the oocytes. Presumptive zygotes were cultured in groups of 20 in 50

µL of synthetic oviductal fluid (SOF; 15) modified to contain 0.1 mM nonessential amino acids

(M-7145, Sigma), 0.5 mM glutamine, 0.4 mM threonine, 3 mg/mL PVA instead of BSA, and no

glucose. At 72 h post-insemination, embryos with at least 4 cells were selected for further culture

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and any cumulus cells remaining were completely removed. Data were recorded as percentages of

4-cell embryos from inseminated oocytes. From 72 to 144 h post- insemination, embryos were

cultured in the citrate-supplemented SOF-based medium (c-SOF+NEA; 2) plus 0.4 mM

threonine. At 144 h post-insemination, embryos were cultured in the maturation medium without

FSH or IGF-I. Proportions of embryos reaching blastocyst stages (early, full, and expanding)

were recorded at 192 h post-insemination.

Differential staining of blastocysts

Blastocysts were differentially stained as described by Van Soom et al (16). Briefly, zona intact

bovine embryos were incubated for 5 min in 10 mM picrylsulphonic acid (P-2297, Sigma) in cold

Ca2+-free PBS (4 °C). They were then washed and incubated in anti dinitrophenyl antibody (D-

9656, Sigma): Ca2+-free PBS (3:7) for 30 min at 39 °C. Embryos were then transferred into

guinea pig complement (55852, ICN): Ca2+-free PBS (1:4), containing 50 µg/mL propidium

iodide, and 10 µg/mL bisbenzemide, for 30 min at 39 °C. Finally, embryos were fixed in 2 %

paraformaldehyde in PBS for 1 min at 25 °C before mounting on slides with a 10 µL drop of 0.2

M 1,4 Diazabicyclo-octane diluted in 50 % glycerol (v:v) in PBS as an antifading solution.

Numbers of inner cell mass (ICM) blue-stained nuclei were immediately counted under an

epifluorescence microscope using a 340 to 380 nm excitation filter and a 430 nm barrier filter.

Total nuclei were subsequently counted under an epifluorescence microscope equipped with a

digital camera. Numbers of trophoblast cell nuclei were determined by subtracting ICM nuclei

from total nuclei of each blastocyst.

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Embryo cryopreservation

Day 7 (168 h post-insemination) and Day 8 (192 h post-insemination) blastocysts were classified

according to stage of development and quality as per the IETS manual (17). The freezing

procedure was adapted from the report of Dochi et al. (18). The freezing solution was PBS

containing 4 mg/mL BSA (fatty acid-free) and 1.8 M ethylene glycol. Excellent quality embryos

were placed into freezing solution and loaded via a 1 mL syringe into 0.25 mL straws (yellow-

DT, Agtech Inc., Manhattan, KS, USA) at room temperature (22 to 23 °C) as in Fig. 1. Seeding

was induced by applying forcep-held paper soaked with liquid nitrogen to straws immediately

before placing them (with plastic plug end upward) into the freezer (FREEZE CONTROL CL-

863, CryoLogic, Victoria, Australia) in which the temperature was maintained at –7.0 °C for 10

min after placing the last straw. Cooling was continued at a rate of 0.3 ° C/min to –30 °C before

plunging the straws into liquid nitrogen (-196 ° C). Embryo-containing straws were thawed , in

turn, with 2-step warming (19), i.e., 10 sec in air followed by immersion in water at 27 °C for 10

sec. The straw was then dried with a paper towel, the plastic plug removed, and the straw was

loaded into an ET gun for direct transfer. Embryos were also thawed in this way in the laboratory

for assessment of continuing development in culture.

Preparation of recipient cows and embryo transfer

Mixed breed lactating beef cows maintained on pasture at the farm of the College of Veterinary

Medicine were used as ET recipients. Their estrous cycles were synchronized by two treatments

with PGF2α (25 mg Lutalyse im, Pharmacia & Upjohn, Kalamazoo, MI, USA), 11 days apart.

Beginning 36 h after the last treatment the cows were observed for estrus twice daily for a

minimum of 30 min each time. All cows showing estrus (standing heat) within 96 h after the last

PGF2α treatment and with a good quality CL, determined by palpation per rectum at the time of

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embryo transfer, were used. Freshly thawed, single embryos were deposited into the uterine horn

ipsilateral to the CL. Early blastocyst (stage 5) and blastocyst (stage 6) embryos were thawed and

immediately transferred to Day 6 and Day 7 recipient cows (Day 0 = estrus) and expanded

blastocyst (stage 7) embryos were transferred into Day 8 or Day 9 recipients according to

availability. Pregnancy was diagnosed at Day 35 and Day 60 by ultrasonography and palpation per

rectum. Pregnancy rate was defined as the percentage of cows diagnosed pregnant based on all

cows into which an embryo was transferred. Subsequent pregnancy diagnoses were performed

around Day 100 of pregnancy to verify the presence of a fetus.

Experimental studies

Experiment 1. Effect of LIF on blastocyst formation and hatching.

Since recombinant bovine LIF is not commercially available, recombinant human LIF was

selected over murine LIF for this work due to its higher sequence homology to bovine LIF. A

concentration of 100 ng/mL was arbitrarily selected for addition to culture media following our

study of previous reports (9-11).

Experiment 1a was conducted to examine the effect of LIF when added at 144 h post-

insemination. On Day 6 post-insemination embryos transfered from c-SOF+ NEA to the

maturation medium without FSH or IGF-I were randomly assigned to culture drops containing 0

(control group) or 100 ng/mL LIF (treatment group). Cultures were carried out for each group

consisting of 20 embryos in several 50 µL drops of medium under oil within a petri dish (60 mm

diameter). Proportions of blastocysts were determined on Day 8 post-insemination. This

experiment was replicated 15 times. Each replication included all oocytes and embryos resulting

from a single (separate day) slaughterhouse collection. Some Day 8 expanding blastocysts were

randomly chosen for differential staining. To examine the effect of LIF on hatching, LIF-treated

blastocysts at 198 h post-insemination were further randomly divided into two groups. These

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groups received 0 or 100 ng/mL LIF until 240 h post-insemination (Day 10). At this time,

blastocysts were transferred into fresh medium (up to 20 per drop) and the volume of medium

was increased to 100 µL. Proportions of completely hatched blastocysts were recorded at 240 h

post insemination. Hatched blastocysts were expressed as percentages of total blastocysts

cultured. This experiment was replicated 5 times.

Experiment 1b was conducted to determine the effect of LIF when added at 72 h post-

insemination. At 72 h post-insemination when embryos with at least 4 cells transferred to c-

SOF+NEA were randomly assigned to culture drops containing either 0 or 100 ng/mL LIF.

Cultures were carried out in control and treatment groups as described in Experiment 1a.

Development to the blastocyst stage was determined at Day 8 post-insemination. The experiment

was replicated 6 times.

Experiment 2. Effect of LIF (100 ng/mL) on embryo survival after cryopreservation.

Day 7 and Day 8 bovine blastocysts were produced as in Experiment 1a for control and LIF-

treated embryos. Freezing and thawing were carried out as described above. Straws were

emptied into petri dishes for washing by agitation in culture medium. Zona-intact blastocysts

were cultured in 100 µL drops (10 to 15 blastocysts per drop) of the same medium, i.e.,

maturation medium without FSH or IGF-I . Proportions of embryos that completely hatched

(totally outside of zonae pellucidae) were recorded at 48 h post-thaw. Three replicates of this

experiment were carried out. Some of the cryopreserved embryos were directly transferred into

recipient cows and pregnancies were diagnosed as described previously.

105

Statistical analyses

Differences between proportions of the resulting blastocysts according to treatments and

differences between blastocyst cell numbers were analyzed by t-test. Proportions of hatched

blastocysts in Experiment 1a were analyzed by ANOVA and differences were determined by the

Bonferoni t-test. Chi-square analysis was employed in Experiment 2 for embryo survival after

cryopreservation. Significance was assigned at P< 0.05.

RESULTS

Insemination of COCs with at least two layers of cumulus cells surrounding zonae but without

more intensive selection resulted in lower (P < 0.01) percentages of cleavage (57.8 %, of which

50.9 % were at least 4-cell at 72 h post-insemination) compared to cleavage after insemination of

more carefully selected COCs (72.4 %, of which 68.2 % were at least 4-cell at 72 h). However,

the percentages of Day 8 blastocysts developing from 4-cell embryos were not improved by

intensive selection of COCs (44.9 % with intensive selection vs. 46.1 % without intensive

selection). In further work COCs were not stringently selected in order to maximize the

production of blastocysts from ovaries obtained; thus an average of 6.5 COCs per ovary were

used. The overall percentage of inseminated COCs that developed into 4-cell embryos by 72 h

post insemination in all subsequent experiments was 55.4 ± 2.5 %.

Effect of LIF on blastocyst formation and hatching

Addition of LIF to the culture medium from post-insemination Day 6 to Day 8 had no effect on

the percentage of 4-cell embryos developing to blastocysts (28.2 % vs. 28.7 %, Table 3.1).

106

However, when LIF was present from post-insemination Day 3 to Day 8 the percentage of 4-cell

embryos that developed to blastocysts was significantly reduced (28.1 % in contrast to 42.6 %)

(P < 0.05) for the control with no added LIF (Table 3.2). The presence of LIF in the culture

medium from post-insemination Day 6 to Day 10 (Fig 3.2) significantly increased the

percentage of blastocysts that hatched when compared to controls (76.1 ± 1.6 % vs. 56.9 ± 5.4 %,

P < 0.05). Addition of LIF from post insemination Day 6 to Day 8 slightly increased hatching

(65.4 ± 4.6 %) but this value did not reach significance when compared to controls. Numbers of

inner cell mass nuclei of Day 8 expanding blastocysts were also significantly increased (P < 0.05)

after exposure to LIF: 65 ± 6 vs. 82 ± 9 for control and LIF-treated, respectively, resulting in

significantly higher total cell numbers (148 ± 5 vs. 170 ± 7) (Fig. 3.3). Numbers of trophoblast

nuclei were similar between control and LIF-treated blastocysts: 90 ± 5 vs. 86 ± 4, respectively.

Effect of LIF on survival of cryopreserved embryos

The overall hatching rate of frozen-thawed embryos was significantly lower than of fresh control

embryos (37.0 % vs. 61.3 %, P < 0.01). When data for all Day 7 and Day 8 blastocysts were

combined without consideration of their developmental stage no significant effect of LIF on

survival after cryopreservation was detected. As shown in Table 3.3, embryos reaching the

expanded blastocyst stage on Day 7 after being treated with LIF yielded a significantly (P < 0.01)

higher percentage of hatched blastocysts (51.8 %) compared to control blastocysts at the same

developmental stage (7.4 %). Day 7 embryos frozen as early blastocysts also resulted in

significantly (P < 0.01) higher percentages of hatched blastocysts (57.7 %) than did embryos

frozen at the expanded blastocyst stage (7.4 %). Leukemia inhibitory factor had no detectable

effects on in vitro survival of frozen-thawed embryos in any other Day 7 (Table 3.3) or Day 8

(Table 3.4) blastocyst stages studied.

107

Overall, 3 of 23 recipient cows became pregnant after direct ET with confirmation by palpation

per rectum at 100 days of gestation. Two of these pregnancies resulted from seven control

embryos and one pregnancy resulted after direct transfer of 16 cryopreserved embryos following

prior culture with LIF.

DISCUSSION

In contrast to positive findings in other species (20-23), addition of LIF to culture media for

bovine embryos was not always beneficial. Better results have followed addition of LIF to the

media for later stages of development (morula or early blastocyst) than for earlier cleavage stages

(9). However, our present findings agree with other reports demonstrating no beneficial effects

on blastocyst development when LIF was added at the morula stage (10, 11). Numbers of total

nuclei and inner cell mass nuclei were higher in LIF-treated blastocysts as previously reported

(10). Leukemia inhibitory factor receptor mRNA was detected by RT-PCR from bovine embryos

produced in vitro (24) and in our bovine embryos as well (unpublished data). An unexpected

reduction in blastocyst development was observed when LIF was added at the 4- to 8-cell stages,

in Experiment 1b. This negative effect, for which no precedent was found in the literature, might

be a result of the high concentration of LIF employed in our experiments.

The positive effect of LIF on the ability of bovine blastocysts to completely hatch by 10 days

post-insemination, as reported here, was not observed in earlier work (9). The mechanism by

which LIF can affect the hatching process remains unclear. However, it is known that hatching is

a reflection of proteolytic activity of each individual embryo (25). Also, Menino and Williams

(26) demonstrated that plasmin, created from exogenous plasminogen, could be activated by

embryonic plasminogen activator to promote the hatching process of bovine embryos.

108

Furthermore, LIF and epidermal growth factor were found to be capable of increasing

plasminogen activator activity of murine blastocyst outgrowths (27). Thus, LIF may improve the

hatching process of bovine embryos by enhancing endogenous proteolytic activity at the

appropriate time.

The overall hatching rates of frozen-thawed blastocysts were significantly lower than those of

fresh blastocysts (34.5 % vs. 61.3 % for control and 39.5 % vs. 66.1 % for LIF treated, P < 0.01).

Day 7 early blastocysts displayed the highest in vitro survival after thawing. Our Day 7 expanded

blastocysts were unable to survive cryopreservation as well as the early blastocysts; this is in

marked contrast to findings reported by others (28-31). This discrepancy might reflect

differences in methodologies employed, especially the cryopreservation procedures.

Interestingly, in contrast to the survival rate of Day 7 expanded blastocysts, the survival rate of

Day 8 expanded blastocysts was not different from that of Day 8 early blastocysts (28.6 % vs.

31.2 %). Surprisingly, only Day 7 expanded blastocysts were significantly enhanced (i.e., in

ability to withstand cryopreservation) by the LIF treatment; the same treatment was not able to

increase in vitro survival of Day 7 and Day 8 embryos in any other stage of development. Our

results differ from those of earlier reports on the beneficial effect of LIF after co-culture with

cells secreting a high concentration of LIF (12), and on direct addition LIF prior to

cryopreservation (11). Clearly defined conditions employed in our work eliminated confusion

inherent with potential interactions with products of extraneous cells in co-cultures. Blastocysts

were not classified into precise stages before cryopreservation in the previous work of Han et al

(11) and their method of embryo production was different.

Unlike the previous study in sheep (21) where a more than 30 % increase in pregnancy rate was

seen after embryos were treated with 1000 U/mL LIF from Day 6 to 8 followed by transfer

(without cryopreservation), no beneficial effects on pregnancy rate were observed after addition

109

of LIF to culture media for bovine embryos before transfer to recipient cows when compared to

controls (6.25 % vs. 28.5 %, respectively). Our embryo transfer results were comparable to the

25 % overall pregnancy rate obtained after direct transfer of cryopreserved in vitro produced

bovine embryos reported by Sommerfeld and Niemann (32). However, pregnancy rates after

direct transfer of cryopreserved in vitro produced bovine embryos are lower than after direct

transfer of cryopreserved in vivo produced bovine embryos (18). Cryopreservation of

mammalian embryos has been shown to cause ultrastructural damage, such as ruptured plasma

membranes, mitochondrial damage and intercellular contact disruption, as well as metabolic

disturbances (33-36). In the freezing procedure employed in this study ice crystal formation

occuring during cooling or thawing could damage the embryos. In general, it seems unlikely that

any particular growth factor(s) or cytokine(s) can have a direct protective effect against such

physical damage.

The pregnancy rate obtained from in vitro produced embryos under the present conditions were

lower than anticipated from in vitro survival results. In vivo survival was not correlated with in

vitro survival as determined by hatching rate. In vitro survival rates following cryopreservation

are commonly high in various laboratories. Sommerfeld and Niemann (32) have shown that

bovine embryos produced in vitro with high in vitro survival rates and low percentages of dead

cells after cryopreservation still resulted in low pregnancy rates after transfer. Available data

suggests that in vitro survival, measured by hatching rates at 48 h post-thaw, is a poor indicator

for predicting pregnancy results. Present results demonstrated that chemically defined conditions

can support production of viable embryos as proven by continued normal in vivo development

after embryo transfer. The overall pregnancy rate (3 of 23 cows) indicates the need for additional

improvements in this approach before it can be used to enhance bovine reproductive efficiency.

The completely defined media employed here may be a good model for more precisely detecting

positive or negative effects that different treatments might have on embryonic development.

110

Acknowledgements

The authors gratefully acknowledge the support of The University of Georgia College of

Veterinary Medicine Award for Service, Research, or Teaching; Royal Thai Embassy

(Fellowship for S. Sirisathien); Ares Advanced Technology, Inc., Randolph, MA; Brown

Packing Co., Gaffney, SC; Genex CRI, Ithaca, NY; Santa Elena Ranch, Madisonville, TX; and

the assistance of Dr. H. Cho and Dr. L.L. Hawkins with cows.

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115

Table 3.1. Addition of leukemia inhibitory factor (100 ng/mL) to culture medium at

144 h post-insemination had no effect on development of in vitro produced bovine embryos to the

blastocyst stage. (Data from 15 replicates.)

4-cell Total % Blastocysts (from 4-cell)

embryos blastocysts† mean ± SEM

Control

1206

369

28.7 ± 3.1

+ LIF 1135 332 28.2 ± 2.5

† Day 8 post-insemination

116

Table 3.2. Effect of addition of leukemia inhibitory factor (100 ng/mL) to culture medium at 72

h post-insemination on development of bovine 4-cell stage embryos to blastocysts.

(Data from 6 replicates.)

4-cell Total % Blastocysts (from 4-cell)

embryos blastocysts† mean ± SEM

Control

142

55

42.6 ± 5.5a

+ LIF 276 80 28.1 ± 5.5b

† Day 8 post-insemination

ab Different superscripts denote significant differences (P < 0.05)

117

Table 3.3. Effect of leukemia inhibitory factor (100 ng/ml)† on post-thaw in vitro survival of

bovine embryos after cryopreservation on Day 7 post-insemination. (Data from 3 replicates.)

Blastocyst stage

Treatment

n

Hatched‡

Hatching

% Hatched

Early Control 33 19 5 57.6a

LIF 29 18 5 62.0a

Unexpanded Control 50 18 3 36.0b

LIF 43 14 6 32.6b

Expanded Control 27 2 9 7.4c

LIF 27 14 4 51.8d

† Exposure to LIF was during the 24 h culture interval prior to cryopreservation.

‡Blastocysts that completely escaped from their zonae pellucidae by 48 h were considered to have

survived.

abc Different superscripts denote significant differences (P < 0.05, Chi-square)

cd Significantly different (P < 0.01, Chi-square)

118

Table 3.4. Addition of leukemia inhibitory factor (100 ng/ml)† had no effect on post-thaw in vitro

survival of bovine embryos after cryopreservation on Day 8 post-insemination. (Data from 3

replicates.)

Blastocyst stage

Treatment

n

Hatched

Hatching

% Hatched

Early Control 49 14 11 28.6

LIF 51 21 6 41.1

Unexpanded Control 80 32 18 40.0

LIF 86 34 11 39.5

Expanded Control 80 25 15 31.2

LIF 87 29 11 33.3

† Exposure to LIF was during the 48 h culture interval prior to cryopreservation.

119

Seeding

cotton plug fluid air bubble embryo plastic plug

Figure 3.1. Embryo placement in a straw for cryopreservation and subsequently, for direct embryo

transfer.

120

0

20

40

60

80D

ay 8

bla

stoc

ysts

that

hat

ched

by

Day

10

(mea

n %

of 5

repl

icat

es)

56.9 %65.4 %

76.1 %

n 14

9/24

3

n 80

/121 n

103/

139

Day 6-8 Day 6-10Control

Postinsemination days of exposure to LIF

a

a,b

b

ab Different superscripts denote significant differences (P < 0.05).

Figure 3.2. Effect of leukemia inhibitory factor (LIF) on the mean percentage of Day 8 blastocysts

that completely hatched from their zonae pellucidae by Day 10 post-insemination. (Data from 5

replicates.)

121

0

50

100

150

200

Inner cell mass nuclei Total nuclei

Num

bers

of c

ells

in D

ay 8

exp

andi

ng b

last

ocys

ts

148

170

65

82a

b

c

d

ab, cd Different superscripts denote significant differences (P < 0.05).

Figure 3.3. Effect of leukemia inhibitory factor (LIF) on inner cell mass (ICM) and total nuclei

when added at 144 h post-insemination on cell numbers of Day 8 expanding blastocysts (Control

n = 27, LIF n = 32).

CHAPTER 4

BENEFICIAL POSTMORTEM INFLUENCE ON PRODUCTION OF BOVINE

BLASTOCYSTS IN VITRO1

1S. Sirisathien and B.G. Brackett, Submitted to Veterinary Record, 8/10/2002

123

In vitro production of bovine blastocysts in chemically defined media, i.e. with

synthetic macromolecules such as polyvinyl alcohol (PVA) instead of serum, serum albumin,

or other biological components represents the most desirable approach for investigating

influences of additives or modifications in protocol that might affect the outcome of embryo

production. Additionally, better definition of biological parameters related to female gametes,

e.g. oocyte diameter (Fair and others 1992, Otoi and others 1997), cumulus oocyte complex

(COC) morphology (Younis and others 1989, Madison and others 1992, Hazeleger and others

1994, Hawk and others 1994), follicular diameter (Pavlok and others 1992, Lonergan and

others 1994), and ovarian morphology (Gandolfi and others 1997), are of interest since many

are already known to affect embryo production outcome. Procedural influences including

temporal considerations in use of postmortem ovaries prior to oocyte aspiration represent

additional factors that can influence embryo production results. Protocols for bovine embryo

production in most in vitro fertilization (IVF) laboratories include transporting ovaries from

slaughterhouses prior to oocyte aspiration and incubation to achieve in vitro oocyte maturation

(IVM). Bovine ovaries can be maintained for up to 8 hours at 25°C prior to oocyte aspiration

without compromising IVF results (Yang and others 1990). Early work in our laboratory

revealed comparable morula development of oocytes fertilized in vitro after being aspirated

from ovaries within 15 minutes after slaughter with those similarly treated but taken from

ovaries after a delay of 1 to 3 hours at ambient temperature (Mackie,1994). Blondin and others

(1997) reported that bovine ovaries should be kept at 30°C for at least 3 hours before oocyte

aspiration in order to reach the highest proportions of morulae at 7 days post insemination.

However, in that study it was unclear whether the resulting morulae were capable of

developing further to blastocyst stages. The present study was carried out in chemically

defined media to determine whether holding bovine ovaries for a postmortem interval prior to

124

oocyte aspiration is beneficial for bovine blastocyst production when compared to the same

protocol but with oocytes recovered from follicles immediately after slaughter.

All chemicals used for embryo production were purchased from Sigma Chemical Co.

(St. Louis, MO, USA; appropriate catalog numbers are indicated in parentheses) except as

otherwise indicated. Within the same experimental replicates ovarian follicles were either

aspirated within 15 minutes after cows were slaughtered (0 hour) or ovaries were incubated for

2 hours (at ambient slaughterhouse temperature of approximately 25°C) prior to oocyte

aspiration. Cumulus oocyte complexes (COCs) were aspirated from follicles of surface

diameters less than 6 mm using a 10 ml syringe and an #18 gauge needle. Follicular fluids

were pooled in a 50 ml centrifuge tube and maintained at 33°C for 3 hours during transit to the

laboratory. In all cases, COCs with at least 2 compact layers of cumulus cells were utilized

without any further selection based on morphological considerations. They were incubated for

24 hours in groups of 20 to 22 in 100 µl drops of maturation medium covered with light

mineral oil (S894-00, J.T. Baker, Phillipsburg, NJ, USA). The maturation medium (Dinkins

and others 2001) was tissue culture medium 199 (M-3769, Sigma) supplemented with 50

µg/ml sodium pyruvate, 2.2 mg/ml NaHCO3, 1 mg/ml PVA, 0.25 mM glutamine, 0.1 mM

cystine, 0.1 mM cysteamine, 10 mM hydroxyethylpiperazine ethanesulfonic acid (HEPES)

plus 0.1 iu/ml of recombinant human follicle stimulating hormone (FSH, 1.7 iu/ µg, Ares

Advanced Technology Inc., Randolph, MA, USA) and 5 ng/ml of recombinant human insulin-

like growth factor I (IGF-I, Promega, Madison, WI, USA). Incubation for oocyte maturation,

fertilization (IVF) and embryo culture were under moist 5 per cent CO2, 5 per cent O2 and 90

per cent N2 in a modular incubator chamber (Billups-Rothenberg Inc, Del Mar, CA, USA).

Swim-up selected spermatozoa were prepared in modified defined medium (mDM)

without hypotaurine or epinephrine (Dinkins and Brackett 2000). Two straws of frozen semen

(108 sperm/straw) were thawed at 37°C for 30 seconds; then 115 µl of semen was layered under

125

1.5 ml of mDM in each of 7 tubes (12×75 mm). The tubes were held at a 45° angle for 45

minutes at 39°C under moist 5 per cent CO2 in air. Then, the uppermost 950 µl aliquots from

each tube were pooled in a 15 ml tube and centrifuged at 320 × g for 10 minutes. The sperm

pellet was resuspended to 380 µl to which 20 µl mDM containing 80 µg of heparin was added

(200 µg/ml final concentration) for a 15 minute incubation before insemination. Spermatozoa

were counted in a hemacytometer and checked for motility. Then, 12 to 14 µl of sperm

suspension was added to each 82 to 86 µl drop of mDM to provide a concentration of 2 × 106

motile spermatozoa per ml in 100 µl. Matured COCs were added to each drop with minimal

amounts, i.e. less than 5 µl, of medium.

After 18 hours of gamete co-incubation, loosely associated cumulus cells were

removed from COCs by gentle pipetting. Presumptive zygotes were cultured in groups of 20 in

50 µl of synthetic oviductal fluid (SOF; Tervit and others 1972) modified to contain 0.1 mM

non-essential amino acids (M-7145, Sigma), 0.5 mM glutamine, 0.4 mM threonine, 10 mM

HEPES, 3 mg/ml PVA instead of bovine serum albumin, and no glucose. At 72 hours post

insemination, embryos with at least 4 cells were selected for further culture and any cumulus

cells remaining were completely removed. Data were recorded as proportions of 4-cell

embryos that developed from inseminated oocytes. From 72 to 144 hours post insemination,

embryos were cultured in citrate-supplemented SOF-based medium (c-SOF+NEA; Keskintepe

and Brackett 1996) plus 0.4 mM threonine. After 144 hours post insemination, embryos were

cultured in the maturation medium without FSH or IGF-I. Proportions of embryos reaching

blastocyst stages were recorded at 192 hours post insemination. Statistical analyses were

performed with the SigmaStat software package (Jandel Scientific, San Rafael, CA).

Proportions of oocytes reaching the 4-cell stage and the blastocyst stage according to

treatments were transformed to their arc sine square roots. The transformed data were

126

subjected to one-way analysis of variance. Significant differences between groups was

determined by Bonferroni t-test and differences of P< 0.05 were taken as significant.

Fertilization competence was determined by proportions of oocytes reaching (at least)

the 4-cell stage by 72 hours post insemination, expressed as percentages. As shown in Table

4.1, the 2 hour postmortem delay prior to oocyte aspiration had no effect on fertilization

competence of the oocytes compared to that of oocytes aspirated immediately after slaughter

(55.2 vs. 54.9 per cent, respectively). However, 4-cell stage embryos derived from oocytes

recovered 2 hours post mortem developed to the blastocyst stage in significantly higher

percentages than did those derived from oocytes retrieved immediately (44.3 vs. 27.8 per cent

respectively, P< 0.05). Proportions of blastocysts from 4-cell embryos were not further

increased when ovaries were collected and transported back to the laboratory as a routine

procedure for a postmortem period of 4 to 5 hours prior to oocyte aspiration (45.3 ± 1.8 per

cent from 4-cell embryos).

Our results agree with those of Blondin and others (1997) showing developmental

competence of bovine oocytes can apparently be improved prior to the in vitro oocyte

maturation incubation. By contrast, our results indicated that 2 hours was a sufficient interval

for bovine oocytes to acquire this enhanced developmental competence provided by the

postmortem ovarian milieu. Oocyte developmental competence seemed to reach a plateau

earlier in our conditions, i.e. at 2 hours vs. 4 hours post mortem (Blondin and others, 1997). In

general, developmental competence of oocytes depends on nuclear maturation, i.e. ability to

reach the Metaphase II stage, and cytoplasmic maturation, i.e. ability to develop in vitro to the

blastocyst stage. How the postmortem incubation effected enhanced developmental

competence, presumably via cytoplasmic maturation of the oocytes, is unknown. The

postmortem treatment might mimic the intrafollicular environment to resemble the final in

vivo maturation physiologically effected by the luteinizing hormone surge prior to ovulation.

127

This positive postmortem effect merits further investigation to identify specific molecule(s)

present in the follicular milieu that exhibit the presumptive biological activity.

Present findings should be interpreted cautiously. More blastocysts are not

necessarily better quality blastocysts. Further experiments are required to evaluate whether

this post mortem treatment might improve or compromise the ability to establish pregnancies

after transfer into recipient cows.

ACKNOWLEDGEMENTS

Authors gratefully acknowledge support of The University of Georgia College of



Packing Co., Gaffney, SC; Genex CRI, Ithaca, NY.

References

BLONDIN, P., COENEN, K., GUILBAULT, L.A., SIRARD, M-A. (1997) In vitro production of

bovine embryos: developmental competence is acquired before maturation. Theriogenology

47,1061-1075.

DINKINS, M.B. & BRACKETT, B.G. (2000) Chlortetracycline staining patterns of frozen-thawed

bull spermatozoa treated with β-cyclodextrins, dibutyryl cAMP and progesterone. Zygote 8, 245-

256.

DINKINS, M.B., STALLKNECHT, D.E., HOWERTH, E.W. AND BRACKETT, B.G. (2001)

Photosensitive chemical and laser light treatments decrease epizootic hemorrhagic disease virus

associated with in vitro produced bovine embryos. Theriogenology 55:1639-1655.

FAIR, T., HYTTEL, P., GREVE, T. (1992) Bovine oocyte diameter in relation to maturational

competence and transcriptional activity. Molecular Reproduction and Development 42, 437-442.

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GANDOLFI, F., LUCIANO, A.M., MODINA, S., PONZINI, A., POCAR, P., ARMSTRONG, D.T.,

LAURIA, A. (1997) The in vitro developmental competence of bovine oocytes can be related to

morphology of the ovary. Theriogenology 48, 1153-1160.

HAWK, H.W. & WALL, R.J. (1994) Improved yields of bovine blastocysts from in vitro-produced

oocytes. I. Selection of oocytes and zygotes. Theriogenology 41, 1571-1583.

HAZELEGER, N.L., HILL, D.J., STUBBINGS, R.B., WALTON, J.S. (1995) Relationship of

morphology and follicular fluid environment of bovine oocytes to their developmental potential in

vitro. Theriogenology 43, 509-522.

KESKINTEPE, L. & BRACKETT, B.G. (1996) In vitro developmental competence of in vitro

matured bovine oocytes fertilized and cultured in completely defined media. Biology of

Reproduction 55, 333-339.

LONERGAN, P., MONAGHAN, P., RIZOS, D., BOLAND, M. P., GORDON, I. (1994) Effect of

follicle size on bovine oocyte quality and developmental competence following maturation,

fertilization and culture in vitro. Molecular Reproduction and Development 37, 48-53.

MACKIE, K.C. (1994) Temporal and hormonal influences on in vitro maturation of bovine oocytes.

MS Thesis, The University of Georgia, Athens, Georgia, 74p.

MADISON, V., AVERY, B., GREVE, T. (1992) Selection of immature bovine oocytes for

developmental potential in vitro. Animal Reproduction Science 27,1-11.

OTOI, T., YAMAMOTO, K., KOYAMA, N., TACHIKAWA, S., SUZUKI, T. (1997) Bovine

oocyte diameter in relation to developmental competence. Theriogenology 48, 769-774.

PAVLOK, A.A., LUCAS-HAHN, NIEMANN, H. (1992) fertilization and developmental

competence of bovine oocytes derived from different categories of antral follicles. Molecular

Reproduction and Development 31, 63-67.

TERVIT, H.R., WHITTINGHAM, D.G., ROWSON, L.E.A. (1972) Successful culture in vitro of

sheep and cattle ova. Journal of Reproduction and Fertility 30, 493-497.

129

YANG, N.S., LU, K.H., GORDON, I. (1990) In vitro fertilization (IVF) and culture (IVC) of bovine

oocytes from stored ovaries. Theriogenology 33, 352 (abstr).

YOUNIS, A.I., BRACKETT, B.G., FAYER-HOSKEN, R. A. (1989) Influence of serum and

hormones on bovine oocyte maturation and fertilization in vitro. Gamete Research 23, 188-201.

130

Table 4.1: Influence of delayed oocyte recovery on in vitro development of 4-cell stage

bovine embryos to the blastocyst stage (Data from 5 replicates)

Postmortem

Number

of

Per cent of

Per cent of 4-cell

embryos developed to

Per cent of oocytes

developed to delay (hours) oocytes 4-cell embryos* blastocysts blastocysts†

0

850

54.9 ± 3.4

27.8 ± 2.0a

15.1 ± 0.5c

2

918

55.2 ± 3.5

44.3 ± 4.3b

24.8 ± 3.5d

* 72 hours post insemination

† Day 8 post insemination

a,b,c,d Different superscripts within columns denote significant differences (P < 0.05)

CHAPTER 5

INFLUENCES OF EPIDERMAL GROWTH FACTOR AND INSULIN-LIKE GROWTH

FACTOR-I ON BOVINE BLASTOCYST DEVELOPMENT IN VITRO1

1S. Sirisathien, H.J. Hernandez-Fonseca, and B.G. Brackett. Submitted to Animal Reproduction

Science, 9/10/2002.

132

Abstract

Experiments were carried out to investigate putative beneficial effects of adding epidermal

growth factor (EGF) or insulin-like growth factor-I (IGF-I) for bovine embryo culture in

chemically defined media. Presumptive zygotes (18 h post insemination) were randomly

assigned to culture treatments. In experiment 1, treatments involved additions of recombinant

human EGF to provide concentrations of 0 ng (control), 1, 5, and 25 ng/ml. No differences were

seen in numbers of 4-cell embryos between groups. Concentrations of 1 and 5 ng/ml EGF during

culture improved percentages of 4-cell embryos reaching blastocysts compared to the control (P<

0.05). Ratios of cell numbers in the inner cell mass (ICM) to total cells for day 8 blastocysts were

similar for the control and 5 ng/ml EGF-treated groups. In experiment 2, culture with

recombinant human IGF-I in concentrations of 0 ng (control), 2, 10, and 50 ng/ml resulted in no

differences in numbers of 4-cell embryos between groups. When compared to controls, IGF-I

treatments at 10 and 50 ng/ml improved proportions of 4-cell embryos that reached blastocysts

(P< 0.05). Numbers of ICM cells of day 8 blastocysts were significantly higher after being

cultured with 50 ng/ml of IGF-I compared to those of the controls (P< 0.05). No additive effects

of combining EGF (5 ng/ml) and IGF-I (50 ng/ml) were seen when results were compared to

those following supplementation of the media with either EGF or IGF-I alone. These data

demonstrated that in vitro both EGF and IGF-I can enhance bovine preimplantational

development in chemically defined media.

Keywords: EGF; IGF-I; Bovine blastocyst; Defined media

133

1. Introduction

Bovine in vitro fertilization (IVF) technology has been greatly advanced during the last

two decades since the first calf was born from an IVF embryo (Brackett et al., 1982). Currently,

in vitro maturation, fertilization, and embryo culture (IVMFC) is a promising approach for

maximizing use of bovine gametes. To improve quality control and capability for comparing data

among laboratories, it is highly desirable to optimize completely defined media for embryo

production. From early studies onward it has become well-known that macromolecules are an

essential component of embryo culture media. Eight-cell mouse embryos successfully developed

to the blastocyst stage only if egg white was incorporated in the culture medium (Hammond,

1949). Whitten (1956) was eventually able to replace egg white with a more defined

macromolecule, bovine serum albumin (BSA). However, BSA has been shown to be ill-defined

due to its affinity for biological contaminants and the variability in quality of commercial

preparations (Kane, 1983; Gray et al., 1992; McKiernan et al., 1995). Furthermore, the risk of

spreading infectious diseases via laboratory-produced embryos resulting from contaminated

protein supplementation in culture media is well recognized (Stringfellow and Givens, 2000).

Therefore, embryo production in media with synthetic or recombinant macromolecules/proteins

promises the best approach for repeatability and for eliminating the risk of introducing infectious

pathogens to bovine IVF embryos.

Polyvinyl alcohol (PVA), a synthetic polymer, was found to be an acceptable BSA

replacement in hamster fertilization medium (Bavister, 1981). Subsequently, PVA has been

proven to be a suitable BSA substitute in a variety of chemically defined bovine embryo culture

media (Carney and Foote, 1991; Pinyopummintr and Bavister, 1991; Kim et al., 1993; Lee and

Fukui, 1996). In our laboratory, embryos have been produced in chemically defined media

beginning with oocyte maturation medium (Zulke and Brackett, 1990; Harper and Brackett, 1993)

and subsequently including fertilization and embryo culture media (Keskintepe et al., 1995;

134

Keskintepe and Brackett, 1996). Gonadotropins purified from pituitary extracts were replaced

with the recombinant forms for use during oocyte maturation (Martins and Brackett, 1998).

In general, chemically defined embryo culture media afford development of lower

blastocyst yields (Pinyopummintr and Bavister, 1991; Holm et al., 1999; Krisher et al., 1999). To

improve bovine blastocyst development, several common growth factors have been added to

embryo culture media as they are known to be involved in mammalian preimplantation

development (Adamson, 1993; Harvey et al., 1995). However, early studies of growth factors in

protein-free embryo culture media have led to equivocal results, i.e. lack of positive effects partly

due to suboptimal conditions reflected in poor development in a control medium (Flood et al.,

1993; Shamsuddin, 1994; Lee and Fukui, 1995). The purpose of this study was to further

investigate putative effects of two common growth factors, epidermal growth factor (EGF) and

insulin-like growth factor I (IGF-I), on improvement of bovine embryo production in well

established chemically defined media in which proportions of blastocysts accounting for 30% of

oocytes (matured and inseminated) have been achieved (Dinkins et al., 2001).

2. Materials and methods

2.1. In vitro oocyte maturation

Bovine ovaries collected at slaughter were transported to the lab without added medium

in a thermos (28 to 32°C) reaching the laboratory within three hours. Cumulus oocyte complexes

(COCs) were harvested from follicles of surface diameters less than 6 mm using 10 ml syringes

and 18G needles. Oocytes surrounded by at least two compact layers of cumulus cells were

utilized without any further selection of COCs. They were washed twice with maturation medium

and cultured in groups of 20-22 in 100 µl drops of maturation medium covered with light mineral

oil (S894-00, J.T. Baker, Phillipsburg, NJ, USA). The maturation medium was TCM-199 (M-

3769, Sigma Chemical Co., St. Louis, MO, USA) supplemented with 50 µg/ml sodium pyruvate,

135

2.2 mg/ml NaHCO3, 1 mg/ml PVA, 0.25 mM glutamine, 0.1 mM cystine, 0.1 mM cysteamine, 10

mM hydroxyethylpiperazine ethanesulfonic acid (HEPES), 50 µg/ml gentamicin sulfate plus 0.1

IU/ml of recombinant human FSH (1.7 IU/ µg, Ares Advanced Technology Inc., Randolph, MA,

USA) and 5 ng/ml of recombinant human IGF-I (Promega, Madison, WI, USA). Incubation of

COCs was under moist 5% CO2, 5% O2 and 90% N2 in a modular incubator chamber (Billups-

Rothenberg Inc, Del Mar, CA, USA) at 38.5°C for 24 h; this atmosphere and these physical

conditions were employed for subsequent in vitro embryo cultures.

2.2. Sperm preparation and in vitro fertilization

Frozen semen from a different bull was used for each of three experiments. In

experiments 1 and 2, swim-up selected spermatozoa were prepared in modified defined medium

(mDM) without hypotaurine or epinephrine (Dinkins and Brackett, 2000). Three straws of frozen

Holstein semen (108 sperm per 0.5 ml straw) were thawed at 37°C for 30 sec; then 135 µl of

semen was layered under 1.5 ml of mDM in each of ten test tubes (12×75 mm). The tubes were

held at a 45° angle for 60 min at 38.5°C under moist 5% CO2 in air. Then, 950 µL aliquots from

the top of each tube were pooled into a 15 ml tube and centrifuged at 320 × g for 10 min. After

discarding the supernatant the sperm pellet was resuspended to 380 µl; then, 20 µl mDM

containing 80 µg of heparin (H-3149, Sigma) was added (200 µg/ml final concentration) for a 15-

min sperm incubation before insemination. Spermatozoa were checked for motility and counted

in a hemacytometer. The volume needed (12 to 14 µl) to achieve a concentration of 2 × 106

motile spermatozoa per ml was added to each of several 82 to 86 µl drops (to complete 100 µl) of

mDM prepared as above but also without caffeine (IVF medium). Matured COCs were added to

each of these drops, and the gametes were co-incubated for 18 h to allow initiation of IVF.

In experiment 3, swim-up selection of motile spermatozoa was carried out as described

above except the medium was low bicarbonate-TALP (Parrish et al., 1988) modified to contain

136

5.15 mM caffeine, 3.35 mM D-penicillamine, and 1mg/ml PVA to replace BSA. After

centrifugation, the sperm concentration was adjusted to 20×106 motile sperm per ml. Then, 10 µl

of this sperm suspension was added to each 90 µl drop of Fert-TALP (Parrish et al., 1988)

containing 11 µg/ml heparin and further modified to contain 5 mM glucose, 5 mM HEPES, 3.35

mM D-penicillamine, and 1mg/ml PVA replacing BSA. Matured COCs were added to each drop

10 min later. This marked the beginning (0 h insemination) of an 18 h insemination (IVF)

interval.

2.3. In vitro embryo culture

After gamete co-incubation, loosely associated cumulus cells were removed from zonae

by gentle pipetting. Presumptive zygotes and embryos were cultured in groups of 20 in 50 µl

drops of culture media. Embryo cultures were in three sequential media as follows. From 18 to

72 hours post insemination, presumptive zygotes were cultured in synthetic oviductal fluid (SOF,

Tervit et al., 1972) modified by addition of 0.1 mM non-essential amino acids (M-7145, Sigma),

0.5 mM glutamine, 0.4 mM threonine, substitution of PVA (3 mg/ml) for BSA, and deletion of

glucose. At 72 hours post insemination, embryos of at least 4-cells were freed from any

remaining cumulus cells and were cultured in a citrate-containing modification of SOF, i.e. c-

SOF+NEA (Keskintepe and Brackett, 1996) plus 0.4 mM threonine. After 144 hours post

insemination, the basic medium for culture was the same as described above for oocyte

maturation medium but without FSH and IGF-I.

2.4. Differential blastocyst staining

Day 8 expanding blastocysts, characterized by zonae pellucidae showing signs of

thinning and with slightly increased embryo diameters, were stained as described by Van Soom et

al (1995). Briefly, zona intact blastocysts were incubated for 5 min in 10 mM picrylsulphonic

137

acid (P-2297, Sigma) in cold Ca2+-free PBS (4 °C). Then they were washed and incubated for 30

min at 39°C in anti-dinitrophenyl antibody (D-9656, Sigma) diluted to 30% (v/v) with Ca2+-free

PBS. Embryos were then transferred into guinea pig complement (55852, ICN biochemicals,

Irvine, CA, USA) diluted to 20% (v/v) in Ca2+-free PBS containing 50 µg/ml propidium iodide,

10 µg/ml bisbenzemide, and 50 µg/ml RNAse A for 30 min at 39°C. Finally, embryos were fixed

in 2% paraformaldehyde in PBS for 1 min at 25°C before mounting on slides with a 10 µL drop

of 0.2 M 1,4 diazabicyclo-octane in 50% glycerol (v/v) in Ca2+-free PBS as an anti-fading

solution. Numbers of inner cell mass (ICM) nuclei (blue-stained) were counted within 45 min

under an epifluorescence microscope (Leitz Laborlux 12; filter A code No. 513596, 340 to 380

nm excitation and 430 nm suppression). Total nuclei were subsequently counted under an

epifluorescence microscope equipped with a digital camera. Numbers of trophoblast cell nuclei

were the remainder following subtraction of ICM nuclei from total nuclei of each blastocyst.

2.5. Experimental studies

2.5.1 Experiment 1

At 18 hours post insemination, presumptive zygotes were randomly allocated to one of

the following treatment groups: control, 1, 5, or 25 ng/ml of EGF. Data were recorded as

proportions of 4-cell embryos resulting from inseminated oocytes at 72 hours post insemination

and proportions of embryos reaching blastocyst stages by 192 hours post insemination. In a

subsequent experiment, a concentration of 5 ng/ml EGF was selected to produce blastocysts for

differential staining to assess a putative effect on numbers of blastocyst nuclei.

2.5.2 Experiment 2

As in experiment 1, presumptive zygotes were randomly allocated into one of the

following culture groups: control, 2, 10, or 50 ng/ml of IGF-I. A concentration of 50 ng/ml IGF-I

was subsequently selected to study its putative effect on numbers of blastocyst nuclei.

138

2.5.3 Experiment3

Following the results from experiments 1 and 2, presumptive zygotes were randomly

allocated into one of the following embryo culture groups: control, 5 ng/ml EGF alone, 50 ng/ml

IGF-I alone, or 5 ng/ml EGF plus 50 ng/ml IGF-I.

2.6. Statistical Analyses

Proportions of oocytes reaching the 4-cell stage and, of oocytes and 4-cell embryos

reaching the blastocyst stage according to treatments were analyzed by Chi-square. Numbers of

blastocyst nuclei were analyzed by t-test. When P< 0.05, the differences were taken as

significant. Each experiment was carried out in 4 or 5 replicates.

3. Results

Data from experiment 1 are shown in Table 5.1. EGF treatments had no effect on

development of presumptive zygotes to the 4-cell stage compared to the control. A concentration

of 5 ng/ml of EGF supplementation during embryo culture significantly promoted blastocyst

formation from the 4-cell stage compared to the control (P< 0.05). Proportions of blastocysts

developing from oocytes in all EGF-treated groups were not different from the control. Numbers

of ICM nuclei and trophoblast cell nuclei of day 8 expanding blastocysts were similar for the

control and 5 ng/ml EGF-treated blastocysts (55 ± 3 and 127 ± 6 vs. 56 ± 3 and 125 ± 5

respectively, Fig.5.1).

In experiment 2, adding 2, 10, and 50 ng/ml of IGF-I to embryo culture media produced

no differences in proportions of zygotes developing to the 4-cell stage compared to the control

(Table 5.2). Percentages of 4-cell stage embryos developing to the blastocyst stage were

significantly improved by IGF-I supplementation in a dose dependent manner. IGF-I

supplementation at 10 and 50 ng/ml improved blastocyst development from 4-cell embryos

compared to the control (P<0.05). A concentration of 50 ng/ml of IGF-I also improved

139

proportions of blastocyst yields from oocytes (P<0.05). Numbers of total cells of day 8

expanding blastocysts were significantly higher (P<0.05) for embryos that were cultured with 50

ng/ml of IGF-I compared to those of the control (195 ± 6 vs. 160 ± 8). The higher blastocyst cell

numbers were predominantly due to significantly higher numbers of ICM nuclei not trophoblast

cell nuclei; counted ICM and trophoblast nuclei were 78 ± 3 and 115 ± 6 vs. 56 ± 4 and 105 ± 4,

for 50 ng/ml IGF-I treated and control blastocysts, respectively, (Fig.5.2).

Results of experiment 3 (Table 5.3) demonstrated that supplementation of culture media

with either 5 ng/ml EGF or 50 ng/ml IGF-I improved blastocyst development from 4-cell stage

embryos as well as from oocytes compared to the control (P<0.05). The combination consisting

of EGF and IGF-I resulted in higher proportions of blastocysts yields but this increase was not

significantly higher than the blastocyst yields after using either EGF or IGF-I alone for

supplementation of the media.

4. Discussion

In two previous studies addition of several growth factors, including EGF and IGF-I,

produced no positive effects on blastocyst development when TCM-199 was employed as the

chemically defined culture medium (Flood et al., 1993; Shamsuddin, 1994). A positive effect of

IGF-I on blastocyst development was found only when IGF-I was used in conjunction with

granulosa cell co-culture (Herrler et al., 1992) or only when bovine serum was included in the

culture medium (Palma et al, 1997). However, positive effects of IGF-I added to a chemically

defined medium were found when the simpler culture medium SOF was used instead of the

complex TCM-199. Matsui et al (1997) reported that morula development (their endpoint) from

oocytes was increased from 33% to 45% after adding IGF-I (2 to 200 ng/ml) to their mSOF.

Addition of IGF-I (100 ng/ml) to SOF was found to significantly improve blastocyst development

from 11 to 17 % of oocytes (Prelle et al., 2001). In our experiments supplementation of

140

chemically defined embryo culture media with IGF-I or EGF was beneficial to the in vitro

production of bovine blastocysts. IGF-I and EGF, however, had no effect on development of

presumptive zygotes reaching the 4-cell stage. Present results are in agreement with previous

reports of improvement in blastocyst yields after culture with added EGF (Lonergan et al., 1996)

or IGF-I without observing any positive effect on the cleavage rates (Matsui et al., 1997; Prelle et

al., 2001; Moreira et al., 2002; Makarevich and Markkula, 2002). Bovine embryos are known to

be capable of cleaving for 2 to 3 cell cycles followed by arrest at the 8-16 cell stage under inferior

culture conditions or when in the presence of a protein synthesis inhibitor (Barnes and First,

1991). Therefore, the cleavage rates are apparently a sole reflection of fertilization rates of

oocytes unlikely to be affected after sperm penetration by any exogenous growth factors.

Numbers of ICM nuclei and trophoblast cell nuclei were unaffected by adding exogenous

EGF which is in agreement with previous studies (Yang et al., 1993; Lonergan et al., 1996). Lee

and Fukui (1995), however, found higher blastocyst cell numbers after EGF supplementation.

Unlike EGF, IGF-I has recently been shown to increase total nuclei of bovine blastocysts by

increasing numbers of trophoblast cells (Prelle et al., 2000; Makarevich and Markkula, 2002). As

expected, IGF-I-treated day 8 expanding blastocysts were found to have higher total cell numbers

than control blastocysts. Surprisingly, IGF-I had little effect on proliferation of trophoblast cells

but selectively stimulated the proliferation of the ICM. This is also reported for human and

murine blastocysts, i.e. IGF-I stimulates the proliferation of ICM rather than trophoblast cells

(Harvey and Kaye, 1992; Lighten et al., 1998). In general, our results indicate that IGF-I not only

improved blastocyst yields from IVF embryos but also had a mitogenic effect on the resulting

blastocysts whereas EGF did not have such an effect despite its similar enhancement of blastocyst

development. Albeit a potent mitogen for several cell types, EGF seems to play a more important

role in embryonic cell differentiation than in cell proliferation. The onset of blastocoel formation

was delayed when mouse two-cell embryos were injected with antisense EGF receptor RNA

without significant reduction in blastocyst cell numbers (Brice et al., 1993). IGF-I has been

141

shown to have an anti-apoptotic action during preimplantation development in vitro (Makarevich

and Markkula, 2002; Spanos et al., 2000; Byrne et al., 2002; Herrler et al., 1998) especially

apoptosis tending to occur predominantly in the ICM (Byrne et al., 1999; Hardy, 1999). It can be

speculated that IGF-I could increase ICM cell numbers by reducing the incidence of apoptosis

within the ICM. Data suggest that IGF and EGF improve blastocyst yields via different

approaches. Blastocyst development from fertilized oocytes could be improved through either

proliferation or differentiation promoting factors.

Transcripts for IGF-I receptor are present in bovine embryos from zygote to blastocyst

stages (Watson et al., 1992; Yashiyoda et al., 1997; Yaseen et al., 2001). Transcripts of erbB3,

one of the EGF receptor family, were not detectable in bovine embryos between the 2-cell stage

and the blastocyst stage when transcripts were found (Yashiyoda et al., 1997). Unfortunately, it

was not determined whether transcripts of erbB3 were present at the morula stage. IGF-I

oviductal fluid concentrations are close to 15 ng/ml for human (Homburg et al., 1996; Lighten et

al., 1998), 20 to 40 ng/ml in porcine (Wiseman et al., 1992), and in the range of 100 ng/ml for

rhesus monkeys (Chi et al., 2002). Although IGF-I is present in uterine fluid at concentrations of

4 to 5 pg/µg protein (Funston et al., 1995), physiological concentrations of IGF-I in bovine

oviductal fluids are not known. Nonetheless, IGF-I transcripts are detectable in bovine oviductal

epithelium from intact oviducts (Schmidt et al., 1994; Xia et al., 1996) and bovine oviductal cell

primary cultures are known to be able to secrete IGF-I into culture medium (Winger et al., 1997).

In contrast to IGF-I, there is relatively little information regarding a significant role of EGF

during bovine preimplantational development in vivo. EGF transcripts were not detectable in

bovine oviductal epithelial cell primary cultures (Watson et al., 1992). However, EGF was found

in endometrial epithelium from the bovine uterus between day 13 to 14 of gestation (Kliem et al.,

1998).

In conclusion, present results demonstrated in chemically defined media that bovine

blastocyst production can be improved by exogenous IGF-I and EGF. The completely defined

142

media employed promise a good model to more precisely detect beneficial effects that different

treatments might have on embryonic development. Future studies to determine whether exposure

to IGF-I and EGF might alter bovine embryonic viability after embryo transfer or

cryopreservation and subsequent embryo transfer will be of great interest.

Acknowledgements




Packing Co., Gaffney, SC; and Genex/CRI, Ithaca, NY.

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Table 5.1. Results of bovine embryo development after addition of EGF to the culture media

from 18 to 192 h after in vitro insemination.

Added EGF

(ng/ml)

No.

Oocytes

No. 4-cell

embryos (%)

No. blastocysts from

4-cell embryos (%)

% Blastocysts from

oocytes

0 280 148 (52.8) 54 (36.5)a 19.3

1 197 110 (54.5) 47 (44.6)a,b 23.2

5 214 96 (44.9) 48 (50.0)b 22.5

25 180 100 (55.2) 30 (30.4)a 16.4

Different superscripts denote significant differences (P<0.05).

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Table 5.2. Results of bovine embryo development after addition of IGF-I to the culture media

from 18 to 192 h after in vitro insemination.

Added IGF-I

(ng/ml)

No.

Oocytes

No. 4-cell

embryos (%)

No. blastocysts from 4-

cell embryos (%)

% Blastocysts

from oocytes

0 286 178 (62.2) 70 (39.3)a 24.5d

2 221 140 (63.3) 64 (45.7)a,b 29.0d

10 238 136 (57.1) 74 (54.4)b 31.1d

50 221 134 (60.6) 86 (64.2)c 38.9e

Different superscripts within columns denote significant differences (P<0.05).

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Table 5.3. Effects of embryo culture from 18 to 192 h after oocyte insemination with IGF-I,

EGF, or EGF plus IGF-I (E+I) on bovine blastocyst production in vitro.

Treatment No.

oocytes

No. 4-cell

embryos (%)

No. blastocysts from 4-

cell embryos (%)

% Blastocysts from

oocytes

Cont 486 340 (70.0) 146 (42.9)a 30.0c

IGF 448 327 (73.0) 166 (50.8)b 37.1d

EGF 439 311 (70.8) 159 (51.1)b 36.2d

E+I 400 286 (71.5) 162 (56.6)b 40.5d

Different superscripts within columns denote significant differences (P<0.05).

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0

50

100

150

Control, n = 27+ EGF-I, n = 24

ICM Trophoblast

125127

5655

Figure 5.1. Numbers of ICM and trophoblast cells of day 8 expanding blastocysts after culture

with or without EGF (5 ng/ml).

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0

50

100

150

ICM Trophoblast

Control, n = 28+ IGF-I, n = 32

7856

115105

a

b

Figure 5.2. Numbers of ICM and trophoblast cells of day 8 expanding blastocysts after culture

with or without IGF-I (50 ng/ml).

ab Significant different (P<0.05, t-test)

CHAPTER 6

TUNEL ANALYSES OF BOVINE BLASTOCYSTS AFTER CULTURE WITH EPIDERMAL

GROWTH FACTOR AND INSULIN-LIKE GROWTH FACTOR-I1

1S. Sirisathien and B.G. Brackett. Submitted to Molecular Reproduction and Development,

9/28/2002

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ABSTRACT

Experiments were carried out to investigate beneficial effects of IGF-I or EGF on bovine

embryo development in chemically defined embryo culture media and resultant incidences of

nuclear DNA fragmentation as an indication of embryo quality. Presumptive IVF zygotes were

randomly cultured in either control (with no added growth factor) or treatment groups, i.e. with

50 ng/ml IGF-I (experiment1) or 5 ng/ml EGF (experiment 2). IGF-I supplemented to culture

media significantly improved proportions of blastocysts from oocytes inseminated compared to

untreated controls (38.0% vs. 28.5%). Only embryos reaching the blastocyst stage on day 8

showed significant effects of IGF-I treatment by resulting in higher blastocysts cell numbers (162

vs. 141) and lower percentages of TUNEL positive nuclei (2.1% vs. 3.3%) when compared to

controls. Blastocyst development from oocytes was also improved by EGF supplementation

compared to untreated controls (38.5% vs. 30.7%). Cell numbers of either day 7 or day 8

blastocysts were not affected by EGF treatment, nor were percentages of TUNEL positive nuclei

when compared with controls. Similar proportions of parthenogenetically activated oocytes

developed to blastocysts as for inseminated oocytes (28.8%). Parthenogenetic blastocysts

contained fewer cells (93) and an increased percentage of TUNEL positive nuclei (5.7%) than

were found for IVF embryos.

Key words: nuclear DNA, growth factors, embryo quality, IVF, parthenogenetic

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INTRODUCTION

An appreciable number of bovine blastocysts can be obtained from chemically defined

embryo culture media (Pinyopummintr and Bavister, 1991, Keskintepe and Brackett, 1996, Lee

and Fukui, 1996, Holm et al., 1999, Krisher et al., 1999). Addition of some growth factors to

chemically defined embryo culture media has been shown to improve bovine blastocyst

production (Lonergan et al., 1996, Prelle et al., 2001, Sirisathien et al., 2001). However, quality

of such embryos remains to be evaluated. Increased numbers of blastocyst yields may not be

correlated with higher developmental potential. Excessive concentrations of IGF-I could

triggered apoptosis in mouse blastocysts by down-regulating IGF-I receptors (Chi et al., 2000),

and resulted in higher resorption rates after being transferred into pseudo-pregnant females (Pinto

et al., 2002). Similarly, IGF-I receptor transcripts in bovine blastocysts were found to be

significantly reduced after embryo culture with 100 ng/ml IGF-I despite resulted in higher

blastocyst yield (Prelle et al., 2001). In vitro quality assessment cannot be based solely on

morphological examination. For example, parthenogenetic blastocysts look very similar to IVF

blastocysts but are unable to establish pregnancies (Fukui et al., 1992, Susko-Parrish et al., 1994).

Total numbers of blastomeres as well as ratios of inner cell mass (ICM) to trophoblast cells as

determined by differential staining method have limited value. Differential staining techniques

simply rely on the physical barrier properties of trophoblast cells protecting the ICM and enable

no further insight regarding the health of such blastocysts. Currently, in vitro produced bovine

blastocysts possess similar cell numbers and ratios of ICM to trophoblast cells to their in vivo

counterparts but result in lower pregnancy rates following embryo transfer, especially after

cryopreservation (Massip et al., 1995, Agca et al., 1998, van Wagtendonk-de Leeuw et al., 2000).

The incidences of DNA fragmented nuclei detected by applying terminal

deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) procedure may provide

more information regarding quality of in vitro produced bovine blastocysts. TUNEL has been

employed by several laboratories to assess apoptosis in mammalian blastocysts (Jurisicova et al.,

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1996, Brison et al., 1997, Byrne et al., 1999, Spanos et al., 2000, Paula-Lopes and Hansen, 2002).

Incidence of DNA fragmented nuclei might be a good indicator for the health of blastocysts. In

vitro produced blastocysts were found to have higher DNA fragmented nuclei than in vivo

produced blastocysts (Long et al., 1998, Jurisicova et al., 1998). The aims of this study were to

further assess the positive effects of EGF and IGF-I on increased blastocyst production by

studying the incidence of DNA fragmented nuclei determined by TUNEL staining and also, to

assess the incidence of DNA fragmented nuclei in blastocysts derived from parthenogenetically

activated oocytes.


In vitro production of bovine blastocysts

Bovine ovaries collected at slaughter were transported to the lab without added medium

in a thermos (28 to 32°C) reaching the laboratory within three hours. Cumulus oocytes complexes

(COCs) were harvested from follicles of surface diameters less than 6 mm using 10 ml syringes

and 18G needles. COCs with at least two compact layers of cumulus cells were utilized without

any further consideration of their cytoplasmic appearance. They were washed twice with

maturation medium and cultured in groups of 20-22 in 100 µl drops of maturation medium

covered with light mineral oil (S894-00, J.T. Baker, Phillipsburg, NJ, USA). The maturation

medium was TCM-199 (M-0650, Sigma Chemical Co., St. Louis, MO, USA) supplemented with

50 µg/ml sodium pyruvate, 25 mM NaHCO3, 1 mg/ml PVA, 0.25 mM glutamine, 0.1 mM cystine,

0.1 mM cysteamine, 10 mM hydroxyethylpiperazine ethanesulfonic acid (HEPES), 50 µg/ml

gentamicin sulfate plus 0.1 IU/ml of recombinant human FSH (1.7 IU/ µg, a gift from Ares

Advanced Technology Inc., Randolph, MA, USA), 5 ng/ml of recombinant human IGF-I

(Promega, Madison, WI, USA), and 5% fetal calf serum (Atlanta Biologicals, Atlanta, GA, USA).

COCs were incubated under moist 5% CO2, 5% O2 and 90% N2 in a modular incubator chamber

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(Billups-Rothenberg Inc, Del Mar, CA, USA) for 24 hours and this atmosphere was employed for

in vitro cultures of oocytes/embryos throughout this work.

Swim-up selected spermatozoa were prepared in low bicarbonate-TALP (Parrish et al.,

1988) modified to contain 5.15 mM caffeine, 3.35 mM D-penicillamine, and 1mg/ml PVA to

replace BSA. Three straws of frozen Holstein semen (108 sperm per 0.5 ml straw) were thawed at

37°C for 30 sec; then 135 µl of semen was layered under 1.5 ml of TALP in ten 12×75 mm tubes.

The tubes were held at a 45° angle for 60 min at 39°C under moist 5% CO2 in air. Then, 950 µL

aliquots from each tube were pooled into a 15 ml tube and then centrifuged at 500 × g for 10 min.

The sperm pellet was resuspended and the concentration was adjusted to be 20×106 motile sperm

per ml. Then, 10 µl of this sperm suspension was added to each of several 90 µl drops of Fert-

TALP (Parrish et al., 1988) containing 11 µg/ml heparin and further modified to contain 5 mM

glucose, 5 mM HEPES, 3.35 mM D-penicillamine, and 1mg/ml PVA. Matured COCs were

added to each drop 10 min after adding spermatozoa.

After 18 hours of gamete co-incubation, loosely associated cumulus cells were removed

from zonae by gentle pipetting. Presumptive zygotes and embryos were cultured in groups of 20

in 50 µl drops of culture media. Three-successive embryo culture media were employed. From

18 to 72 hours post insemination, presumptive zygotes were cultured in of synthetic oviductal

fluid (SOF) supplemented with 0.1 mM non-essential amino acid (M-7145, Sigma), 0.5 mM

glutamine, 0.4 mM threonine, 3 mg/ml PVA replacing serum albumin, without glucose. At 72

hours post insemination, embryos reaching at the least 4-cell stage were selected for further

culture after removal of any cumulus cells remaining. From 72 to 144 hours post insemination,

embryos were cultured in modified SOF containing citrate (c-SOF+NEA; Keskintepe and

Brackett, 1996) with threonine. After 144 hours post insemination, embryos were cultured in the

maturation medium but without FSH or IGF-I; (IGF-I was added only as described below).

159

To produce parthenogenetic bovine blastocysts, mature oocytes were striped of cumulus

cells by pipetting. Denuded oocytes were activated by being incubated with 5 µM ionomycin for

5 min followed by 5 min washing to stop the activation and then incubated with 2.5 mM dimethyl

aminopurine (DMAP) for 2.5 h (Susko-Parrish et al., 1994). All steps of the activation process

were carried out in the maturation medium prepared with 5 mM NaHCO3, without FSH and IGF-

I. Subsequently, cultures were carried out as IVF zygotes/embryos.

Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL)

The procedure for TUNEL analysis was adopted from published literature (Otoi et al.,

1999; Watson et al., 2000). Zona-pellucida intact blastocysts were fixed in 0.5%

paraformaldehyde in phosphate buffered saline (PBS) for 15 min at 25°C, then washed with

PBS. Blastocysts were either processed immediately or kept at 4°C overnight in PBS.

Blastocysts were permeabilized with PBS containing 0.1% Triton X-100 and 0.1% sodium

citrate for 30 min at 25°C and then washed. Blastocysts were placed into 30 µL drops of

TUNEL reagent (Roche Molecular Biochemicals, Indianapolis, IN, USA) covered with

paraffin oil and incubated at 38°C for 60 min under dark and moist conditions. Positive control

blastocysts were treated with 50 IU/mL of DNase I (Promega, Madison, WI, USA) for 20 min

at 37°C before being incubated with TUNEL reagent. Negative control blastocysts were

incubated with TUNEL reagent in the absence of the enzyme terminal deoxynucleotidyl

transferase. Counter staining was carried out by incubating blastocysts in the PBS solution

containing 50 µg/mL propidium iodide, and 50 µg/mL RNase A for 40 min at 25°C and then

followed by 10 µg/mL bisbenzemide for 1 min. Blastocysts were washed and mounted with 10

µL drops of anti-fading solution prepared with 0.2 M diazabicyclo octane in 50% (v/v)

glycerol-PBS solution. Blastocysts were observed under an epifluorescence microscope

(Nikon Eclipse E 600) using a FITC filter (excitation 450-490, dichoric mirror 505, and barrier

160

filter 520 nm) to examine numbers of TUNEL positive nuclei and a DAPI filter for

determining numbers of total nuclei. Not every yellowish-green fluorescence staining was

recorded as TUNEL positive. Nuclei were recorded as positive for TUNEL labeling only

when they showed yellowish-green fluorescence on the red fluorescence background of

propidium iodide and that particular nucleus must be stained with bisbenzemide as well.

Experimental design

Beginning at 18 hours post insemination presumptive IVF zygotes were randomly

cultured in either control (with no added growth factor) or treatment groups, i.e. with 50 ng/ml

IGF-I (experiment1) or 5 ng/ml EGF (experiment 2). These concentrations were selected

based on our previous results (Sirisathien et al., 2001). Data were recorded at 72 hours post

insemination as proportions of 4-cell stage embryos that developed from inseminated oocytes.

Proportions of 4-cell stage embryos reaching blastocyst stages were recorded at 168 hours and

192 hours post insemination for day 7 and day 8 blastocysts, respectively.

Statistical analyses

Proportions of oocytes reaching the 4-cell stage and, of oocytes and 4-cell embryos

reaching the blastocyst stage according to treatments were analyzed by Chi-square. Numbers

of blastocyst nuclei, TUNEL stained nuclei, as well as percentages of TUNEL stained nuclei

were analyzed by t-test. When P< 0.05, the differences were taken as significant.

RESULTS

Inclusion of IGF-I to embryo culture media had no effect on proportions of

presumptive zygotes developed to 4-cell stage embryos compared to the untreated control

(67.7% vs. 65.8%, respectively, Table 6.1) but significantly promoted blastocyst development

from 4-cell stage embryos compared to the controls (56.1% vs. 43.4%, respectively, P<0.01).

161

Thus, resulted in significantly higher proportions of blastocysts from oocytes inseminated

(38.0% vs. 28.5%, respectively, P<0.01).

No significant differences were found between the percentages of TUNEL positive

nuclei of day 7 and day 8 blastocysts from the control group (2.4 % vs. 3.3 %, respectively,

Table 6.2). IGF-I treatment did not significantly increase cell numbers of day 7 blastocysts

compared to those of the controls (145 vs. 139, respectively). Similarly, the percentages of

TUNEL positive nuclei were not different between day 7 IGF-I treated blastocysts and day 7

control blastocysts (1.9% vs. 2.4%, respectively). However, day 8 blastocysts resulting from

the IGF-I treated group had higher cell numbers than those from the control group (162 vs.

141, P<0.05). The percentages of TUNEL positive nuclei were significantly lower in IGF-I

treated day 8 blastocysts than those in controls (2.1% vs. 3.3%, respectively, P<0.05). The

majority of blastocysts contained at least one TUNEL positive nucleus, 62 out of 68 for

controls and 65 out of 76 for IGF-I treated.

Inclusion of EGF to embryo culture media significantly improved blastocyst

development from 4-cell stage embryos compared to the control (51.3% vs. 41.5%, P<0.01,

Table 6.3) without having any effect on zygotic development to the 4-cell stage (75.0% vs.

74.0%). Development of blastocysts from oocytes was significantly improved by EGF

supplementation compared to untreated controls (38.5% vs. 30.7%, P<0.01).

EGF treatment produced no effect on cell numbers of day 7 or day 8 blastocysts (140 vs.

137 for day 7 blastocysts and 144 vs.143 for day 8 blastocysts, Table 6.4). The percentages of

TUNEL positive nuclei of day 8 control blastocysts were significantly higher than those of day 7

control blastocysts (2.4% vs. 1.5%, respectively, P<0.05). Percentages of TUNEL positive nuclei

were also higher in day 8 EGF-treated blastocysts than those in day 7 EGF-treated blastocysts

(2.2% vs. 1.2%, respectively, P<0.05). However, no significant differences in percentages of

TUNEL positive nuclei were found when compared between control and EGF-treated groups

(1.5% vs. 1.2% for day 7 blastocysts and 2.4% vs. 2.2% for day 8 blastocysts, respectively, Table

162

6.4). Again, the majority of blastocysts in this experiment contained at least one TUNEL positive

nucleus, i.e. 59 out of 68 for controls and 66 out of 74 for EGF treated.

Four-cell stage and blastocyst developmental rates of parthenogenetically activated oocytes

were similar to those obtained by IVF (Table 6.5). However, parthenogenetic blastocysts

contained fewer cell numbers and higher percentages of TUNEL positive nuclei than those found

in IVF blastocysts.

DISCUSSION

In agreement with our previous study (Sirisathien et al., 2000), supplementation of

chemically defined embryo culture media with IGF-I and EGF was beneficial to the in vitro

production of bovine blastocysts by stimulating the advance of 4-cell stage embryos toward the

blastocyst stage without having any effect on proportions of presumptive zygotes reaching the 4-

cell stage. In agreement with other studies (Neuber et al., 2002, Van Soom et al., 2002) day 7 and

day 8 blastocysts from the control group had comparable blastocyst cell numbers indicating that

the ages at which blastocysts are formed had no significant effect on cell numbers. However, the

ages at which blastocysts were formed seemed to affect the incidences of TUNEL positive nuclei.

Day 8 blastocysts contained significantly higher numbers of TUNEL positive nuclei than did day

7 blastocysts (only in experiment 2); this tendency was also reported by Neuber et al (2002) but

was not observed by Van Soon et al (2002). EGF treatment had no effect on blastocyst cell

numbers of either day 7 or day 8 blastocysts confirming our previous results (Sirisathien et al.,

2000) and those of Lonergan et al (1996). IGF-I treatment significantly increased cell numbers of

day 8 blastocysts but did not increase cell numbers of day 7 blastocysts. In this study, we could

not distinguish between numbers of inner cell mass and trophoblast cells. However, from our

earlier work (unpublished data) with day 8 blastocysts, the same IGF-I treatment significantly

increased numbers of cells comprising the inner cell mass by comparison to controls.

163

In general, we observed very light impact of IGF-I and EGF supplementation on the

incidences of DNA fragmented nuclei determined by TUNEL staining. IGF-I indirectly reduced

the percentages of TUNEL positive nuclei by increasing blastocyst cell numbers rather than

reduce the actual numbers of TUNEL positive nuclei per blastocyst. This corresponds with

previous reports (Byrne et al., 2002a, Makarevich and Markkula, 2002). Since EGF had no effect

on blastocyst cell numbers, no significant effect on the percentages of TUNEL positive nuclei

were found. Nonetheless, it would wrong to conclude that IGF-I and EGF do not have any anti-

apoptotic effect during early bovine blastocyst development. The purpose of this study was not to

address the anti-apoptotic effect of IGF-I or EGF. Therefore, neither stressful conditions (Herrler

et al., 1998, Chi et al., 2000) nor induction of apoptosis (Matwee et al., 2000, Kolle et al., 2002,

Byrne et al., 2002b) were applied to the embryos.

In the present work it was not determined whether those TUNEL positive nuclei were

apoptotic or necrotic nuclei. However, the TUNEL labeling pattern observed in this study

corresponded to that of apoptotic nuclei in which the staining was confined to nuclei while the

TUNEL staining of necrotic nuclei are accompanied by diffuse staining in the cytoplasm as well

(Charriaut-Marlangue and Ben-Ari, 1995, Jurisicova et al., 1996). Despite being cultured in

chemically defined media involving replacement of bovine serum albumin with PVA, our bovine

blastocysts had similar numbers of TUNEL positive nuclei to those in other studies (Byrne et al.,

2002a, Van Soom at al., 2002, Neuber et al., 2002, Kolle et al., 2002). We obtained similar

proportions of blastocyst development between parthenogenetically activated oocytes and

inseminated oocytes. Parthenogenetic blastocysts, however, contained lower blastocyst cell

numbers and higher numbers of TUNEL positive nuclei than IVF blastocysts as reported earlier

(Neuber et al., 2002). Curiously, a slight reduction in cell numbers and a slight increase in

TUNEL positive nuclei would not explain the lack of ability to establish pregnancy after transfer.

Additionally, parthenogenetic blastocysts are able to secrete interferon-tau, the maternal

recognition signal, as well as IVF blastocysts (Kubisch et al, 2002). Therefore, it can be

164

speculated that gene imprinting should be the major explanation for factors that prevent fetal

development of bovine parthenogenetic blastocysts after embryo transfer.

In conclusion, most of in vitro produced bovine blastocysts in this study contained at

least one TUNEL positive nucleus. IGF-I and EGF had no major impact on numbers of TUNEL

positive nuclei under our conditions but the percentage of TUNEL positive nuclei was lower in

IGF-I treated blastocysts. Both growth factors significantly improved blastocyst yields from

oocytes.

Acknowledgements


Veterinary Medicine Award for Service, Research, or Teaching; Royal Thai Embassy and The

University of Georgia Graduate School (Fellowships for S. Sirisathien); Ares Advanced

Technology, Inc., Randolph, MA; Brown Packing Co., Gaffney, SC; and Genex/CRI, Ithaca, NY.

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170

Table 6.1. Effect of in vitro culture with IGF-I (50 ng/mL) on bovine blastocyst development.

(Data from 5 replicates).

Treatment No. oocytes No. 4-cell stage embryos (%)

No. Blastocysts† from

4-cell stage embryos (%)

% Blastocysts†

from oocytes

Control 564 371 (65.8) 161 (43.4)a 28.5c

+ IGF-I 558 378 (67.7) 212 (56.1)b 38.0d

a,b,c,d Different superscripts within columns denote significant differences p < 0.01 (Chisquare).

† 8 days post-insemination

171

Table 6.2. TUNEL staining results for day 7 and day 8 bovine blastocysts after in vitro culture

with or without IGF-I (50 ng/mL). Data are shown as mean ± SEM.

Treatment

Blastocysts

(n)

Total nuclei

TUNEL stained nuclei

% TUNEL stained

nuclei

Control 35 139 ± 6.06 3.4 ± 0.48 2.4 ± 0.33

Day 7 + IGF-I 45 145 ± 6.12 2.4 ± 0.31 1.9 ± 0.31

Control 33 141 ± 6.59a 4.1 ± 0.52 3.3 ± 0.42c

Day 8 + IGF-I 31 162 ± 6.83b 3.5 ± 0.62 2.1 ± 0.28d

ab,cd Different superscripts within columns denote significant differences p < 0.05 compared to

control (t-test)

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Table 6.3. Effect of in vitro culture with EGF (5 ng/mL) on bovine blastocyst development.


Treatment No. oocytes No. 4-cell stage embryos (%)

No. Blastocysts† from

4-cell stage embryos (%)

% Blastocysts† from

oocytes

Control 577 427 (74.0) 177 (41.5)a 30.7c

+ EGF 504 378 (75.0) 194 (51.3)b 38.5d

a,b,c,d Different superscripts within columns denote significant differences p < 0.01 (Chi-square).

† 8 days post-insemination

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Table 6.4. TUNEL staining results for day 7 and day 8 bovine blastocysts after in vitro culture

with or without EGF (5 ng/mL). Data are shown as mean ± SEM.

Treatment

Blastocysts

(n)

Total nuclei


% TUNEL stained

nuclei

Control 32 137 ± 4.2 2.1 ± 0.34a 1.5 ± 0.26c

Day 7 + EGF 35 140 ± 3.4 1.7 ± 0.25a 1.2 ± 0.18c

Control 36 143 ± 5.0 3.2 ± 0.35b 2.4 ± 0.26d

Day 8 + EGF 39 144 ± 4.9 3.0 ± 0.32b 2.2 ± 0.24d

ab,cd Different superscripts within columns denote significant differences p < 0.05 (t-test) between

day7 and day 8 blastocysts within the same treatment.

174

Table 6.5. Parthenogenetic blastocyst development and TUNEL staining results.


No. oocytes % 4-cell

embryos

% Blastocysts†

from oocytes

Total nuclei‡ ±

SEM (n)


mean ± SEM

% TUNEL stained nuclei

mean ± SEM

374 75.4 28.8 93 ± 5.7 (30) 5.0 ± 0.49 5.7 ± 0.69

† 8 days post activation

‡ 7 days post activation

CHAPTER 7

BULL SPERM UPTAKE OF EXOGENOUS DNA AND EFFORTS TO OBTAIN

TRANSGENIC EMBRYOS1

1S. Sirisathien and B. G. Brackett. Submitted to Transgenics, 10/15/2002.

176

ABSTRACT

The use of bull spermatozoa as an alternative, non-invasive gene transfer method for

production of transgenic embryos was investigated. Ejaculated, and epididymal spermatozoa

were incubated for increasing intervals with a fluorescence labeled plasmid. Ejaculated bull

sperm showed no signs of bound plasmid. In contrast, proportions of epididymal spermatozoa

that showed signs of bound plasmid at the end of 1, 2, and 4 h incubations were 86%, 98%, and

98%, respectively, and proportions of epididymal spermatozoa populations retaining bound

plasmid after DNase I treatment were 16%, 44%, and 40%, respectively. No transgene

expression was observed in bovine blastocysts resulting from in vitro fertilization with

epididymal spermatozoa preincubated with plasmid DNA for 2 h. However, 30% of those

blastocysts were positive for the presence of transgene after PCR analyses. Intracytoplasmic

spermatozoa injection (ICSI) of oocytes with DNA-treated epididymal spermatozoa, freeze-dried,

and membrane disrupted spermatozoa was also carried out. No transgene expression was

observed in blastocysts that developed in vitro after ICSI with DNA-treated epididymal

spermatozoa. Only 3 to 4 embryos (3%) showed evidence of transgene expression after ICSI

with either membrane disrupted, or freeze-dried spermatozoa that were pre-treated with plasmid

DNA. Results of this investigation emphasize the need for further study to develop a repeatable

bovine procedure for sperm mediated transgenesis.

Keywords: intracytoplasmic sperm injection, transgenic, blastocyst, green fluorescent protein

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INTRODUCTION

Pronuclear injection of mammalian zygotes was first described to produce genetically

transformed mouse embryos by Gordon et al [1]. To date, it has become a well established

technique for transgenic animal production, especially with murine species. However, successful

production of transgenic livestock animals after pronuclear injection is extremely low, compared

to that of murine species. Only 1 to 4 % transgenic offspring are expected. For instance,

Eyestone [2] reported 18 transgenic calves out of 226 calves born from 35,000 pronuclear

injected zygotes. As a result, several alternative methodologies have been under investigation.

Sperm mediated gene transfer is arguably the most attractive of these due to its simplicity and less

zygote manipulation. The sperm approach includes several methodologies including injection of

DNA solution alone into the vas deferens [3] or combined with liposome then injected into

semniferous tubules [4], isolation and culture of male germ cells, which can be transfected before

being reimplanted into testes depleted of germ cells [5,6,7], or a simple incubation of

spermatozoa and exogenous DNA. The latter methodology originated from the work of Brackett

et al [8] showing that rabbit spermatozoa were able to incorporate exogenous DNA (SV40 DNA)

and to transfer the foreign DNA into ova during fertilization to result in embryos from which viral

activity representing the expression of the novel DNA was recovered. Sperm mediated gene

transfer gained prominence when success with mouse spermatozoa was reported by Lavitrano et

al [9]. To date, spermatozoa from a variety of species including livestock are known to bind

DNA [for review 10,11]. Therefore, there are possibilities to utilize spermatozoa as vectors to

deliver exogenous DNA into oocytes during fertilization.

Although there have been numerous studies to define the biology of this phenomenon,

predominantly with mouse spermatozoa [12-17] this approach remains unreliable [18], especially with

other domestic animals [19-21]. Recently, sperm mediated gene transfer has incorporated

intracytoplasmic sperm injection (ICSI) to produce live transgenic mice [22], rhesus monkey embryos

178

[23], and porcine embryos [24]. Here, we report experimentation designed to obtain sperm mediated gene

transfer to produce transgenic bovine embryos through in vitro fertilization (IVF) and via ICSI.


Preparation of oocytes

Bovine oocytes were harvested from ovarian follicles of surface diameters less than 6 mm using

10 ml syringes and 18G needles. Oocytes were cultured in groups of 20-22 in 100 µl drops of

maturation medium covered with light mineral oil. The maturation medium was TCM-199 (M-0650,

Sigma Chemical Co., St. Louis, MO, USA) supplemented with 50 µg/ml sodium pyruvate, 25 mM

NaHCO3, 1 mg/ml polyvinyl alcohol (PVA), 0.25 mM glutamine, 0.1 mM cystine, 0.1 mM cysteamine,

10 mM hydroxyethylpiperazine ethanesulfonic acid (HEPES), 50 µg/ml gentamicin sulfate plus 0.1

IU/ml of recombinant human FSH (1.7 IU/ µg, Ares Advanced Technology Inc., Randolph, MA, USA),

5 ng/ml of recombinant human IGF-I, and 5% fetal calf serum. Oocytes were incubated under moist

5% CO2, 5% O2 and 90% N2 in a modular incubator chamber (Billups-Rothenberg Inc, Del Mar, CA,

USA) for 24 hours. This atmosphere was also employed throughout the in vitro cultures for

fertilization (IVF) and early embryonic development.

Preparation of spermatozoa and in vitro fertilization

Frozen-thawed ejaculated bull spermatozoa were prepared by swim-up in low

bicarbonate-TALP [25] modified to contain 1 mg/ml bovine serum albumin (BSA), 5.15 mM

caffeine, and 3.35 mM D-penicillamine. Thus, two straws of frozen Holstein semen (108 sperm

per 0.5 ml straw) were thawed at 37°C for 30 sec; then 115 µl of semen was layered under 1.5 ml

of TALP in seven 12×75 mm tubes. The tubes were held at a 45° angle for 60 min at 39°C under

moist 5% CO2 in air. Then, 950 µL aliquots from each tube were pooled into a 15 ml tube which

179

was centrifuged at 500 × g for 10 min. The sperm pellet was resuspended and the concentration

was adjusted to 20×106 sperm per ml.

Bovine epididymides were obtained from a slaughterhouse on the same day that ovaries

were collected. Epididymides were held at 4° C under mineral oil for three to four days [26]. For

in vitro fertilization, they were kept overnight and used on the following day. Epididymal

spermatozoa were retrieved by flushing upon cutting the distal portion of the cauda epididymis

following its distension with media forced through the vas deferens via a 25 gauge needle

attached to a 5 ml syringe. The flushing medium was low bicarbonate-TALP containing 5 mM

aurintricarboxylic acid to preserve fertilizing ability of epididymal spermatozoa [27]. Contents

were expelled into a petri dish containing 5 ml of TALP. A period of 30 min was allowed for

epididymal spermatozoa to initiate motility. Motile spermatozoa were purified by using 45/67

percent percoll discontinuous gradient centrifugation [28]. Sperm concentrations were adjusted to

20×106 sperm per ml. For IVF, 10 µl of this sperm suspension was added to each 90 µl drop of

Fert-TALP [25] modified to contain 1 mg/ml BSA, 5 mM glucose, 5 mM HEPES, 3.35 mM D-

penicillamine and 11 µg/ml heparin. Matured oocytes were added to each drop 10 min later. This

marked the beginning (0 h insemination) of an 18 h insemination (IVF) interval.

In vitro embryo culture

Cultures were carried out in 3 successive media. Presumptive zygotes were cultured in

groups of 20 in 50 µl of synthetic oviductal fluid (SOF; 29) modified to contain 0.1 mM non-

essential amino acids (M-7145, Sigma), 0.5 mM glutamine, 0.4 mM threonine, 10 mM HEPES, 1

mg/ml BSA, and no glucose. From 72 to 144 hours post insemination, embryos were cultured in

citrate-supplemented SOF-based medium (c-SOF+NEA; 30) plus 0.4 mM threonine and 5% fetal

calf serum. After 144 hours post insemination, embryos were cultured in the maturation medium

without FSH or IGF-I.

180

Sperm treatments prior to ICSI

Freeze-drying of ejaculated spermatozoa

Sperm lyophilization was carried out as follow. A straw of frozen bull spermatozoa was

thawed by immersion in a 370C water bath for 15 sec. The thawed semen was layered over 1 mL

of both 45% and 90% Enhance-S-plus (Conception Technologies Inc., 6835 Flanders Drive San

Diego, CA 92121) in a 15 mL centrifuge tube. The tube was centrifuged at 1200 g for 15 min to

obtain the motile fraction of spermatozoa. The pellet containing the motile sperm fraction and

500 µL of 90% Enhance-S plus was carefully removed from the bottom of the tube by a 1 mL

pipette and transferred to another 15 mL centrifuge tube. The spermatozoa were then

resuspended in 2.0 mL of HEPES-TALP medium and centrifuged at 300 g for 4 min. The

supernatant was removed and 4 mL of modified DMEM medium (10315-026, Gibco) containing

10% FBS (Atlanta Biologicals, S1550, Lot# E01 10), 1M L-glutamine (25030-08 1, Gibco), 0.2

mM sodium pyruvate (1136-070, Gibco), 100 x non-essential amino acids (11140-050, Gibco),

0.25 mL of l0 x nucleosides {160 mg adenosine (A-4036), 170 mg guanosine (G-6264), 146 mg

cytidine (C-4684), 146 mg uridine (U-3003), 48 mg thymidine (T-1895)}, and 0.01 mL

penicillin-streptomycin (P-3539)/mL (S11550, Lot# E0110, Atlanta Biologicals, Atlanta GA) was

added. Sperm concentration was adjusted to 0.5x105/ml; 100 µL sperm suspension was aliquoted

into 1.0 mL Eppendorf tubes and the tubes were plunged into a liquid nitrogen bath. Then, the

tubes were placed in a precooled (-470C) freeze-flask attached to a Flexi-Dry freeze dryer (FTS

Systems, Inc. Stone Ridge, NY 12484). Inlet pressure was 190x103 mBar. About 12-18 h later,

the flask was removed from the system and the tubes were closed and stored at 40C for 1-3

months before use.To observe DNA binding ability, rhodamine-labeled plasmid (pGeneGrip,

Gene Therapy Systems Inc, San Diego, CA, USA) was mixed with nuclear isolation medium

(NIM, 31) for rehydrating freeze-dried spermatozoa.

181

Triton X-100 treatment

Triton X-100 was used to remove sperm plasma membranes. Swim-up selected frozen-

thawed ejaculated spermatozoa were incubated in 0.05% Triton X-100 in ice cold NIM for 1 min.

Then, the sperm cells were washed twice with ice cold NIM by centrifugation (600 x g for 5 min).

Intracytoplasmic sperm injection and oocyte activation

Oocyte holding medium for ICSI was TCM-199 (M-0650, Sigma) supplemented with 50 µg/ml

sodium pyruvate, 5 mM NaHCO3, 20 mM HEPES, and 5% fetal calf serum. Mature oocytes were striped

of cumulus cells by pipetting. Two drops were made on the cover of a 60 mm diameter petri dish and

covered with mineral oil; the drops consisted of 75 µL of holding medium containing 12 to 15 oocytes

and a small drop of diluted spermatozoa. Spermatozoa were diluted in 10% PVP in TALP or NIM (when

freeze-dried spermatozoa or Triton X-100-treated spermatozoa were used). ICSI was carried out at 400

X magnification on the stage of an inverted microscope (Ziess Axiophot) equipped with Hoffman

modulation contrast and an electronic micromanipulator (TransferMan NK, Eppendorf, Brinkmann

Instruments, Inc. Wesbury, NY, USA). The injection pipette with an inner diameter at the tip of 7-8 µm

(Humagen Fertility Diagnostic, Charlottesville, VA, USA) was connected to CellTram Oil (Eppendorf),

and the holding pipette (100-120 µm outer diameters, Humagen) was connected to CellTram Air

(Eppendorf). A selected spermatozoon was aspirated tail-first into the injection pipette and then moved to

the drop containing the oocytes to be injected. Oocytes were positioned with the holding pipette to have

polar bodies located at 6 or 12 o’clock and the injection pipette, to approach from 3 o’clock. A small

volume of ooplasm was drawn into the pipette tip and by observing the movement of ooplasm the

position for sperm injection into the ooplasm was verified. The spermatozoon and the aspirated ooplasm

were expelled into the oocyte with minimal PVP solution.

After injection, oocytes were incubated with 5 µM ionomycin for 5 min followed by 5

min washing to stop the activation. When oocytes were injected with freeze-dried or TritonX-

182

100 treated spermatozoa they were incubated for 1.5 h further incubated with 2.5 mM dimethyl

aminopurine (DMAP) for 1.5 h [32, 33]. All steps of the activation process were carried out in

the maturation medium prepared without FSH and IGF-I. Subsequent cultures were carried out

as for presumptive zygotes and embryos after IVF.

PCR analysis

Hatched blastocysts (devoid of zonae pellucidae) nine days after in vitro insemination were

studied to avoid any amplification of DNA from contaminating plasmid attached with or without

sperm cells to zonae pellucidae. Embryos were incubated with 25 IU/ml DNase I for 30 min at

38.5°C, then washed twice and transferred into the digestion buffer (50 mM KCl, 10 mM Tris-

HCl, 1.5 mM MgCl2) prepared with 200 µg/ml of proteinase K. Embryos were individually

transferred into PCR tubes in 10 µl volume covered with oil and incubated for 2 h at 37°C for

digestion. Proteinase K was denatured by heating samples at 98°C for 10 min. Lysate (5 µl) was

used for PCR analysis in 50 µl final volume. The PCR reaction mixture included 50 mM KCl, 10

mM Tris-HCl, 1.5 mM MgCl2, 10mg/ml BSA, 0.4% DMSO, and 3 ng of each primer. Reactions

were started with 95°C for 10 min followed by amplification for 45 cycles of 94°C for 1 min

denaturation, 56°C for 30 sec annealing, 72°C for 1 min extension and completed with 72°C for 5

min for final extension. Primers were 5’GAGCGCCACCATCTTCT TCAAGGAC-3’ and 5’-

AACTCCAGGAGGACCATGTGATCG-3’ with a predicted PCR product of 386 base pairs [34].

Plasmid pEGFP N-1 (0.5 pg) was used as a positive control and lysates from pooled (3 to 5,

depending on availability) regular hatched blastocysts (fertilized by control epididymal

spermatozoa) were used as negative controls.

183

Experimental studies

Experiment 1. Following preliminary work with ejaculated and cryopreserved

spermatozoa, trials were conducted to study the ability of epididymal spermatozoa to bind

exogenous DNA and to take up DNA. Spermatozoa were prepared as described above.

Rhodamine-labeled plasmid DNA (pGeneGrip) was incubated with motile spermatozoa in a

plasmid DNA to sperm cell ratio of 100 ng DNA per 106 spermatozoa. Spermatozoa (20×106

sperm per ml, 300 µl) were incubated with plasmid DNA at 38.5°C for 1, 2, and 4 h intervals.

Half of sperm samples were treated with DNase I (25 U/ml) for 30 min. Sperm suspensions were

repeatedly washed by centrifugation at 600×g for 5 min (3X) to free the cells of unbound

plasmid. Spermatozoa in suspension were fixed by adding an equal volume of 4%

paraformaldehyde for 15 min. Spermatozoa were also counterstained with 10 µg/ml of

bisbenzemide. Sperm-bound plasmid DNA was observed under an epifluorescence microscope

(Nikon Eclipse E 600) with a rhodamine filter at 100X magnification. At least 200 spermatozoa

were counted for each time point. Experiments were replicated 4 times.

Experiment 2. IVF with epididymal spermatozoa incubated with plasmid DNA was

carried out to determine whether bound or incorporated plasmid DNA could be introduced into

bovine oocytes during fertilization. Plasmid pEGFP N-1 (Clonetech Inc, Palo Alto, CA, USA)

encoding a variant (replacement of Ser 65 by Thr) of the green fluorescent protein (GFP) was

used. This S65T GFP could be observed with a conventional fluorescein isothiocyanate (FITC)

filter set. GFP expression was under the control of cytomegalovirus (CMV) immediate-early

promoter/enhancer. Epididymal bull spermatozoa were incubated with plasmid pEGFP N-1

(circular form) for 2 h before IVF. Resulting embryos were observed under an inverted,

fluorescence microscope (Ziess Axiophot) with a FITC filterset for signs of GFP expression.

Some of the resulting embryos were cultured until they reached the hatched blastocyst stage for

PCR analysis.

184

Experiment 3. Intracytoplasmic sperm injection (ICSI) was carried out with epididymal

spermatozoa incubated with plasmid DNA (pEGFP N-1) at a ratio of 100 ng DNA per 106

spermatozoa for at least 2 h prior to ICSI. Two other sperm treatments to improve sperm and

plasmid DNA interaction were also carried out, i.e. membrane disruption with Triton X-100 and

use of freeze-dried ejaculated spermatozoa. Plasmid pEGFP N-1 was mixed with NIM upon

rehydrating the freeze-dried spermatozoa. Membrane disrupted spermatozoa were mixed with

plasmid pEGFP N-1 (100 ng DNA per 106 spermatozoa) just prior to ICSI. No washing steps

were applied to all of the above sperm-plasmid DNA treatments for ICSI.

RESULTS

Association of plasmid DNA with bull spermatozoa (Experiment 1)

Initial experimentation involved studies to assess binding of DNA by cryopreserved

ejaculated (frozen-thawed) bull spermatozoa. There were no signs of bound plasmid DNA to

ejaculated bull spermatozoa. In contrast to ejaculated spermatozoa, epididymal spermatozoa

avidly bound plasmid DNA. Almost all of the bound DNA was observed as multiple dots of red

fluorescence on the sperm head. Some spermatozoa (less than 5%) showed uniform red

fluorescence over the post acrosomal region. Proportions of epididymal spermatozoa found

associated with plasmid DNA after 1, 2, and 4 h incubation were 86%, 98%, and 98%,

respectively (Figure 7.1). Proportions of epididymal spermatozoa retaining fluorescence signal

after DNase I treatment following incubation with plasmid DNA for 1, 2, and 4 h were 16%, 44%

and 40%, respectively (Figure 7.1).

In vitro fertilization with epididymal spermatozoa (Experiment 2)

Incubation with plasmid DNA had no effect on fertilization by epididymal spermatozoa,

or on development of resulting embryos to the blastocyst stage (Figure 7.2). The proportion of 4-

cell embryos resulting from fertilization with DNA-treated spermatozoa was 33.8% (154/455)

185

while 39.2% (102/255) of oocytes fertilized by control epididymal spermatozoa reached the 4-cell

stage. Proportions of 4-cell embryos reaching the blastocyst stage were 29.9% (46/154) for

DNA-treated spermatozoa and 31.4% (32/102) for controls. No sign of GFP expression was

detected by fluorescence microscopy in resulting embryos. However, 11 out of 29 (30%) of

embryos recovered at the hatched blastocyst stage after fertilization by DNA-treated epididymal

spermatozoa were positive for the presence of GFP gene by PCR analysis. None of pooled

hatched blastocysts fertilized by control epididymal spermatozoa (negative controls) were

positive by PCR analysis

Intracytoplasmic injection of spermatozoa incubated with plasmid DNA (Experiment 3)

One hundred percent of freeze-dried spermatozoa showed signs of bound plasmid DNA

as multiple red fluorescent dots over the sperm head and all of them retained red fluorescence

after DNase I treatment. Results of blastocyst development after ICSI according to different

sperm treatments are shown in Table 7.1. The percentage of oocytes developing to 4-cell

embryos after ICSI: with epididymal spermatozoa incubated with plasmid DNA for 2 h was

58.3%; 61.8%, after the use of freeze-dried ejaculated spermatozoa; and 70.9%, after use of

Triton X-100 treated spermatozoa. For the same groups, percentages of 4-cell embryos reaching

the blastocyst stage were 20.4%, 25.5%, and 14.1%, respectively. No sign of GFP expression

was detected by fluorescence microscopy in embryos injected with epididymal spermatozoa after

simple plasmid DNA incubation. However, three and four embryos at 4- to 8- cell stages showed

green fluorescence in one to three blastomeres after being injected with Triton X-100 pretreated,

or freeze-dried ejaculated spermatozoa, respectively.

DISCUSSION

Pronuclear injection is a rather inefficient way to produce transgenic livestock because of

two related problems. First, exogenous DNA must be mixed with host pronuclei before the first

186

DNA replication takes place in order to have a successful genomic integration; otherwise a

mosaic pattern will occur if exogenous DNA integrates into the host genome during successive S-

phases [35, 36]. Second, pronuclei of bovine zygotes are not easily visualized (compared to those

of murine zygotes) and most of them cannot be seen until late, or near the end of, S-phase.

Around 60% of bovine zygote pronuclei could be visualized after centrifugation to displace

opaque cytoplasmic material [2, 37]. Alternatively, the concept of sperm-mediated gene transfer

should be able to avoid those problems. Exogenous DNA taken up and carried into oocytes by

spermatozoa could be concentrated within male pronuclei during sperm DNA decondensation,

long before pronuclei could be visualized for microinjection.

Fluorescence-labeled plasmid DNA is proving to be very useful in studying the DNA

binding ability of spermatozoa. Previously, radio-labeled plasmid DNA preparations were used

to demonstrate sperm DNA binding ability with measurement of the radio-isotope signal of

plasmid DNA retained in the sperm pellet after intensive washing [12,13,29,38]. Carballada &

Esponda [39] demonstrated that commercially available rhodamine-labeled plasmid DNA could

be easily observed when associated with individual sperm cells within whole spermatozoan

populations.

In contrast to other bovine studies [38,40], we saw no signs of plasmid DNA binding to

ejaculated frozen-thawed spermatozoa. Glycoproteins present in seminal fluid have been shown

to prevent ejaculated spermatozoa from binding to exogenous DNA [12,14]. In this study, we

could not rule out the possibility that some components in frozen semen extender might prevent

frozen-thawed ejaculated bull spermatozoa from binding to exogenous DNA. In addition, the

minimal numbers of plasmids needed to be detectable under fluorescence microscopy are not

known. It is possible that the fluorescence intensity of plasmids bound to ejaculated bull

spermatozoa were below the observable threshold of fluorescence microscopy. In contrast,

virtually all epididymal spermatozoa showed signs of bound DNA. Forty percent of epididymal

spermatozoa retained red fluorescence after DNase I treatment. Therefore, at least some of the

187

bound DNA was taken up, i.e. within the plasma membrane or tightly associated with some

sperm cell structure(s) hidden from the degradative enzyme. However, it is beyond our data to

speculate whether plasmid DNA became bound to submembrane structures or was taken into the

nuclear matrix as suggested by McCarthy & Ward [41]. When compared to the binding pattern

of mouse epididymal spermatozoa [39] in which all spermatozoa displayed a uniform red

fluorescence over the post acrosomal region, less than 5% of bull epididymal spermatozoa was

found to display that pattern. Murine spermatozoa are profoundly distinct from bovine

spermatozoa, especially in acrosomal morphology. However, with regard to association with

exogenous DNA murine and bovine epididymal spermatozoa have two things in common. First,

almost every spermatozoon showed signs of bound DNA. Second, less than half of the sperm

population retained red fluorescence after DNase I treatment.

In murine species, this phenomenon seems to be a regulated event. Binding of exogenous

DNA to murine epididymal spermatozoa had been shown to be mediated by a 30-35 kDa sperm

membrane protein identified by using Southwestern blot analysis [12]. This 30-35 kDa sperm

membrane protein was also found in human, bovine, and porcine spermatozoa [40]. Two other

molecules present on the sperm cell surface have been implicated to play a crucial role. MHC

type II molecules play a role in DNA binding and CD4 molecules play a role in DNA uptake

[17,42]. Sperm cells from CD4 knockout mice have failed to internalize exogenous DNA, even

though they had the identical ability to bind exogenous DNA, when compared to sperm cells from

wild type mice. However, those two molecules could not provide a definitive explanation. Both

molecules are also present on other cell types but no reports of DNA binding or of DNA uptake

are available. Nonetheless, exogenous DNA has been shown to be taken up and incorporated

within the nuclear matrix of murine spermatozoa [16,41].

The failure to observe green fluorescence of GFP albeit detectable by PCR analyses has

raised some concerns related to fertilization events, especially about applying this technique with

bull spermatozoa. Unlike those of murine spermatozoa, half of bull sperm heads are covered by

188

acrosomes. Epididymal spermatozoa that simply bind exogenous DNA externally, unable to

incorporate exogenous DNA with any submembrane structure, could fail to deliver exogenous

DNA into ooplasm. The majority of externally bound DNA might be lost at the time of the

acrosome reaction; some could be lost while spermatozoa are penetrating through zonae

pellucidae. Furthermore, spermatozoa leave their plasma membranes behind by intermingling

with plasma membranes of oocytes. Therefore, any exogenous DNA that failed to incorporate

within the sperm nuclear matrix could have remained in the ooplasm. Interestingly, 30% of

hatched blastocysts were positive for exogenous DNA by PCR analyses and this figure

corresponded to the proportions of epididymal spermatozoa that retained red fluorescence after

DNase I treatment. Then, why did these embryos fail to express GFP? The simplest explanation

is that exogenous DNA remained in the cytoplasm of those blastocysts. Another scenario is that

exogenous DNA integrated into the sperm genome but was subjected to DNA rearrangement

[16].

Failures to obtain transgenic embryos through IVF have been reported before [23,24,43].

However, those studies involved ejaculated spermatozoa which apparently have very low binding

capability for exogenous DNA. Despite showing a great ability for binding as well as uptake of

some bound plasmid DNA, epididymal bull spermatozoa were unable to be used to obtain

transgenic embryos via either IVF or ICSI. Interestingly, all of the freeze-dried ejaculated

spermatozoa retained red fluorescence signals of plasmid DNA even after DNase I treatment.

This indicated that plasmid DNA was tightly associated with the spermatozoa, possibly being

absorbed through osmotic suction when spermatozoa were rehydrated. The failure to obtain

transgenic embryos via ICSI was unexpected, especially after use of Triton X-100 treated and

freeze-dried spermatozoa. Clearly, further research is warranted.

189

ACKNOWLEDGEMENTS


Veterinary Medicine Experiment Station (VMES 02-001); Royal Thai Embassy and UGA

Graduate School Fellowships ( S. Sirisathien); and generous gifts provided by Ares Advanced

Technology, Inc., Randolph, MA; Brown Packing Co., Gaffney, SC; and Genex/CRI, Ithaca, NY.

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Cell. Res. 2001;262:104-13.

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exogenous DNA by mammalian spermatozoa: specific localization of DNA on sperm heads.

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194

Table 7.1 In vitro development of bovine oocytes after ICSI

Sperm preparation No.

Oocytes

No. 4-cell

embryos (%)

No. blastocysts from

4-cell embryos (%)

Freeze-dried 215 133 (61.8) 34 (25.5)

Epididymal 168 98 (58.3) 20 (20.4)

TritonX-100 110 78 (70.9) 11 (14.1)

195

0

20

40

60

80

100

+ DNase I

1h 4h2 h

Sperm incubation intervals with rhodamine-labeled DNA

% sp

erm

atoz

oa sh

owin

g re

d flu

ores

cenc

e

Figure 7.1 Association of exogenous DNA with epididymal spermatozoa. Percentages of

epididymal spermatozoa showing red fluorescence after incubation with rhodamine-labeled

plasmid DNA for increasing intervals are shown without (upper line) or after treatment with

DNase I (+DNase I).

196

0

10

20

30

40

50Control

+ DNA

4-cell stage Blastocyst

% o

f ooc

ytes

inse

min

ated

102/255

154/455

32/25546/455

Figure 7.2 Results of bovine embryo development after IVF with epididymal spermatozoa pre-

incubated without (control) or with (+DNA) plasmid DNA

(pEGFP N-1).

197

CHAPTER 8

CONCLUSIONS

Inclusion of leukemia inhibitory factor (LIF) at a concentration of 100 ng/mL to protein-

free embryo culture media begining at the morula stage stimulated blastocyst development

proceeding to blastocyst completely hatched from zonae pellucidae. LIF treatment also

predominantly stimulated the proliferation of inner cell mass cells rather than trophoblast cells.

However, the same concentration of LIF when supplemented to culture media at the 4-cell stage

was found to reduce embryonic development to the blastocyst stage. This led to conclusions that

either LIF at the level of 100 ng/mL was detrimental for early embryonic stages or premature

exposure to LIF is harmful for subsequent embryonic development to the blastocyst stage. LIF

treatment produced no improvement to the in vitro survival after cryopreservation of resulting

blastocysts. Therefore, inclusion of LIF to protein-free embryo culture media had no major

impact on in vitro production of bovine blastocysts.

In this study, it was demonstrated that in vitro developmental competence of bovine

embryos is not only affected by culture conditions starting with the in vitro oocyte maturation

conditions but developmental competence is also regulated by changes within the intrafollicular

environment prior to oocyte aspiration. Data demonstrated that two hours postmortem delay prior

to oocyte aspiration increased in vitro developmental competence of preimplantation stage

embryos. Higher proportions of 4-cell stage embryos derived from oocytes recovered after two

hours of postmortem delay developed to the blastocyst stage. It is of interest for further

198

experiments to examine whether factors needed in the intrafollicular environment could be

supplied by adjustment of culture conditions.

Inclusion of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) to

protein-free embryo culture media proved to benefit in vitro production of bovine blastocysts.

Higher proportions of blastocyst development were obtained when either EGF at a concentration

of 5 ng/mL or IGF-I at a concentration of 50 ng/mL were added to embryo culture media.

However, a significant additive improvement of EGF combined with IGF-I was not detected. For

practical purposes, it may be wise to supplement culture medium with both EGF and IGF-I as this

combined treatment resulted in the highest proportions of blastocyst obtained (although not

significant in present work). Data suggest to the author that appropriate adjustment of both

growth factor might produce less variation in responses to each individual growth factor. The

IGF-I treatment increased numbers of inner cell mass cells of the resulting blastocysts whereas

the EGF treatment had no effect on blastocyst cell numbers. Additionally, the IGF-I treatment

also reduced the percentages of DNA-fragmented nuclei of bovine blastocysts while the EGF

treatment showed no effect in this regard.

In the last part of this study, it was demonstrated that bovine epididymal

spermatozoa possess an ability to bind to exogenous DNA and take up some of the bound

DNA while ejaculated spermatozoa have been shown to be unable to bind to exogenous

DNA. Attempts were made to use bovine spermatozoa as an alternative gene transfer

method for production of transgenic embryos either in conjunction with in vitro

fertilization or intracytoplasmic sperm injection (ICSI). No transgene expression was

observed in bovine embryos resulting from either in vitro fertilization or ICSI with

epididymal spermatozoa preincubated with plasmid DNA. Three per cent of bovine

embryos showed evidence of transgene expression after ICSI with membrane disrupted,

or freeze-dried spermatozoa that were pre-treated with plasmid DNA. Therefore, in

199

contrast to recent findings with murine spermatozoa, there remains a barrier in efforts to

adopt this approach for bovine spermatozoa as gene transfer vectors.

200

APPENDICES

A representative bovine blastocyst after TUNEL staining. Five nuclei (yellow) are TUNEL-

positive.

A representative picture of blastocyst differential staining. Inner cell mass nuclei are blue and

trophoblast cell nuclei are red.

A bovine embryo after ICSI expressing green fluorescent protein

201

202

203

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