In Vitro Culture of Bovine Uterine and Oviduct Epithelial

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1991

In Vitro Culture of Bovine Uterine and Oviduct Epithelial Cells. In Vitro Culture of Bovine Uterine and Oviduct Epithelial Cells.

John Kevin Thibodeaux Louisiana State University and Agricultural & Mechanical College

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In vitro cu ltu re of bovine u terine and oviduct epithelial cells

Thibodeaux, John Kevin, Ph.D.

The Louisiana State University and Agricultural and Mechanical Col., 1991

U M I300 N. Zeeb Rd.Ann Aibor, MI 48106

IN VITRO CULTURE OF BOVINE UTERINE AND OVIDUCT EPITHELIAL CELLS

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and

Agricultural and Mechanical College in partial fulfillment of the

requirements for the degree of

Doctor of Philosophy

in the

Department of Dairy Science

by

John Kevin Thibodeaux

B.S., University of Southwestern Louisiana, 1986 M.S., Louisiana State University, 1989

December, 1991

ACKNOWLEDGEMENTS

The author wishes to thank his major professor, Dr. J.D. Roussel for his

guidance, understanding and support during his graduate study. Thanks are also

extended to Dr. R.A. Godke, minor professor, for his helpful discussions in preparing

this dissertation. Both have provided the confidence and training necessary for me to

develop as a scientist.

Appreciation is also extended to members of the graduate committee, Dr. R.W.

Adkinson, Dr. G.F. Amborski, Dr. L.L. Goodeaux and Dr. R.H. Gough for their

assistance and critical review of this manuscript. Appreciation is also extended to Dr.

Y. Menezo for demonstrating various cell culture techniques used in these experiments

and for the excellent discussions over the past few years.

A very special thanks are extended to fellow graduate students, James R.

Broussard and Mike W. Myers for their assistance with animals and conducting

experiments. The author is also grateful to the following Research Associates, John

Moreau, Department of Dairy Science and Richard Denniston, Department of Animal

Science, for laboratory assistance, Marylin Dietrich, Department of Veterinary

Microbiology and Parasitology, for flow cytometry analysis and Lora Younger,

Department of Veterinary Anatomy, for help with electron microscopy studies.

The author is deeply grateful to his wife, Holly Thibodeaux, for being my

biggest supporter and demonstrating unlimited patience during the long hours of

conducting experiments during the past few years.

Finally, this dissertation is dedicated to my recently bom daughter, Sydney

Paige Thibodeaux, who has provided an enormous joy in my life and may one day

achieve an accomplishment such as this.

TABLE OF CONTENTSPage

ACKNOWLEDGEMENTS ................................................................... ii

LIST OF TABLES ............................................................................... via

LIST OF FIGURES ............................................................................ ix

ABSTRACT .......................................................................................... xi

INTRODUCTION ....................... 1

LITERATURE REVIEW ....................................................................... 3

In Vitro Co-Culture of Mammalian Embryos ................................. 3

Trophoblastic Vesicle Co-Culture ........................................... 3

Uterine Cell Co-Culture ......................................................... 5

Oviductal Cell Co-Culture ..................................................... 8

Granulosa Cell Co-Culture ..................................................... 13

Chick Embryo Co-Culture ..................................................... 16

Other Co-Culture Systems ..................................................... 17

In Vitro Culture of Epithelial Cells ................................................. 18

Uterine Epithelial Cells ......................................................... 18

Oviduct Epithelial Cells ........................................................ 23

Summary............................................................................................. 27

EXPERIMENT I : A METHOD FOR IN VITRO CELLCULTURE OF SUPERFICIAL BOVINE UTERINEENDOMETRIAL EPITHELIUM ........................................................ 29

Introduction ...................................................................................... 29

Materials and Methods ..................................................................... 30

Materials ................................................................................. 30

Procedure ................................................................................ 31

iii

TABLE OF CONTENTS (cont’d)Page

Results ............................................................................................. 35

Discussion ....................................................................... 38

Suppliers .......................................................................................... 39

EXPERIMENT I I : EFFECTS OF STAGE OF THE BOVINE ESTROUS CYCLE ON IN VITRO CHARACTERISTICS OF UTERINE AND OVIDUCT EPITHELIAL CELLS ......................... 40

Introduction ..................................................................................... 40

Materials and Methods .................................................................... 41

Experimental Animals and Treatment Groups........................ 41

Isolation of Epithelial Cells ................................................... 42

In Vitro Characteristics........................................................... 43

Freezing Epithelial Cells ....................................................... 44

Electron Microscopy............................................................... 44

Immunocytochemistry............................................................ 44

Cell Cycle Analysis ............................................................... 45

Progesterone Levels ............................................................... 45

Statistical Analysis ................................................................ 45

Results .............................................................................................. 46

In Vitro Characteristics .......................................................... 46

Cell Freezing ......................................................................... 47

Electron Microscopy............................................................... 47

Immunocytochemistry............................................................ 48

Cell Cycle Analysis ............................................................... 48

Progesterone Levels ............................................................... 48

iv


Discussion . . .......................................................................................... S3

EXPERIMENT m : THE EFFECT OF CULTURE MEDIUM AND TEMPERATURE ON IN VITRO GROWTH AND PROLIFERATION OF UTERINE AND OVIDUCT EPITHELIAL CELLS ............................................................................. 57

Introduction ......................................................................................... 57

Materials and M ethods......................................................................... 58

Cell Source................................................................................. 58

Isolation of Epithelial Cells ...................................................... 58

Experiment 1 ............................................................................. 59

Growth Curve and Cell Cycle Analysis ............... 60

Experiment 2 ............................................................................. 61

Statistical A nalysis..................................................................... 61

Results .................................................................................................. 62

Experiment 1 ............................................................................. 62

Experiment 2 ............................................................................. 69

Discussion............................................................................................. 73

EXPERIMENT IV : MORPHOLOGICAL EVALUATION OFBOVINE UTERINE AND OVIDUCT EPITHELIAL CELLSUSING IMAGE ANALYSIS .................................................................. 76

Introduction .......................................................................................... 76

Materials and M ethods......................................................................... 77

Experimental Anim als............................................................... 77

Isolation of Epithelial Cells ...................................................... 77

Morphological Assessments and Optical Density ................... 79

Electron Microscopy.................................................................. 79

v


Statistical Analysis................................................................... 80

R esults................................................................................................. 80

Optical D ensity......................................................................... 80

Electron Microscopy................................................................. 81

Discussion............................................................................................ 87

EXPERIMENT V : IN W7R0-MATURATTON, IN VITRO- FERTILIZATION AND CLEAVAGE OF BOVINE OOCYTES INCUBATED IN DIFFERENT CULTURE M EDIA........................... 89

Introduction ......................................................................................... 89

Materials and M ethods........................................................................ 90

Recovery of Oocytes ................................................................ 90

Oocyte Maturation and Fertilization......................................... 90

Fixation of Oocytes.................................................................. 91

Oviductal Cell Culture.............................................................. 91

Embryo Co-culture................................................................... 92

Statistical Analysis................................................................... 92

R esults................................................................................................. 92

Discussion............................................................................................ 98

EXPERIMENT VI : CO-CULTURING IVF-DERTVED BOVINE EMBRYOS WITH OVIDUCT EPITHELIAL CELLS ORIGINATING FROM DIFFERENT DAYS OF THE ESTROUS CYCLE.................. 101

Introduction......................................................................................... 101

Materials and M ethods........................................................................ 103

Experimental Embryos.............................................................. 103

Isolation and Culture of Oviductal Cells ............................... 103

vi


Experiment 1 ............................................................................. 104

Experiment 2 ............................................................................. 105

Statistical A nalysis..................................................................... 106

Results ................................................................................................... 106

Experiment 1 ............................................................................. 106

Experiment 2 ............................................................................. 107

Discussion............................................................................................. 113

CONCLUSIONS ...................................................................................... 116

REFERENCES.......................................................................................... 119

VITA ..................................................................................................... 133

LIST OF TABLES

Table Page

1. Effects of the estrous cycle on in vitro evaluations of uterine and oviduct epithelial cells(least squares means±SE) ........................................................ 49

2. Mean percent of cells proliferating for uterine and oviduct epithelial cells during a 8-day growth curvein different m edia...................................................................... 68

3. Mean percent of cells proliferating for uterine and oviduct epithelial cells during a 8-day growth curve atdifferent temperatures .............................................................. 72

4. Fertilization and cleavage rates of bovine oocytescultured in different media ...................................................... 94

5. Development rates of cleaved oocytes 48 h afterfertilization ............................................................................... 95

6. Developmental rates and viability of cleavedembryos following in vitro culture for 6 days ........................ 96

7. Development of 2- to 4-cell bovine embryos with oviduct epithelial cells isolated between d 4 to 6or d 14 to 16 of the bovine estrous cycle................................. 110

LIST OF FIGURES

Figure Page

1. A growth curve of uterine epithelial cells during thethird subculture conducted for an 8-day interval ................... 37

2. Scanning electron micrograph of oviductal (a) and uterine (b) epithelial cells during primary culture isolated from the same animal on d 4 to 6 of the estrous cycle. Notethe differences in cilia across the cell surface (2,700 x ) ...... SO

3. Scanning electron micrograph of oviductal (a) and uterinep>) epithelial cells at the third subpassage. These are cells isolated from the same animal as Figure 2 (20,000 x) ........ 51

4. Mean percent of cells proliferating isolated from different stages of the estrous cycle. Values representuterine and oviductal cells combined ..................................... 52

5. A growth curve on uterine epithelial cells conductedfor 8 days in different culture media ..................................... 64

6. A growth curve on oviduct epithelial cells conductedfor 8 days in different culture media ..................................... 65

7. An example of flow cytometry analysis of the cell cycle.Total events are the number of cells analyzed with individual quadrants gated based on distinct cell populations. Quadrants III indicates the percent ofthe cell population in a resting state whereas Quadrant IV indicates a proliferative state of the cell cycle ....................... 66

8. Growth rates of uterine and oviduct epithelial cells for 8 days of incubation. All culture media treatment groups were combined and presented as mean values foruterine and oviductal ce lls...................................................... 67

9. Growth rates for uterine epithelial cells culturedfor 8 days at 37°C or 39°C ................................................... 70

10. Growth rates for oviduct epithelial cells culturedfor 8 days at 37°C or 39°C ................................................... 71

11. An example of procedures used for harvesting oviduct epithelial cells. The same procedurewas used to isolate uterine epithelial cells ............................. 78

ix

LIST OF FIGURES (coal’d)

Figure Page

12. Mean optical density readings for uterine and oviductal cells combined for the five samplereplicates .............................................................. :............... 82

13. Mean optical density readings for uterine and oviductal cells combined during primary cultureand following the first and third subpassages.......................... 83

14. Mean optical density readings of good, flair and poor quality ceils for uterine andoviductal cells combined........................................................ 84

15. Examples of good (a) and poor (b) quality bovine oviductal cells stained with Giemsa stain followingin vitro culture (20 x ) ............................................................ 85

16. Scanning electron micrograph of oviductal (a) and uterine (b) epithelial cells during primary culture(oviductal cells=20,000 x and uterine cells=20,000 X ) 86

17. Percent embryo viability following in vitro culture in medium alone or co-culture withoviduct epithelial cells............................................................ 97

18. An example of free-floating clusters of oviductepithelial cells used during embryo co-culture ...................... 109

19. Proteins secreted by oviduct epithelial cells isolated between d 4 to 6 or d 14 to 16 of the estrous cycle.Evaluations were conducted 24 and 48 h following in vitro culture. Data are mean countsfrom three replicates............................................................... I l l

20. Cellular proteins in oviduct epithelial cells isolated between d 4 to 6 or d 14 to 16 of the estrous cycle.Evaluations were conducted 24 and 48 h following in vitro culture. Data are mean countsfrom three replicates............................................................... 112

x

ABSTRACT

A reliable procedure was developed to harvest purified populations of bovine

uterine epithelial cells from the luminal endometrium that allowed cells to be

maintained in vitro for multiple subpassages. This method allowed adequate numbers

of cells for in vitro culture studies to evaluate uterine secretion rates, uterine-embryo

interactions and a new embryo co-culture system.

In a second study, cells were isolated from the uterus and oviducts collected

from cattle on day of estrus, on d 4 to 6, d 8 to 10 and d 14 to 16 of the cycle to

monitor growth and development following incubation in vitro. The highest percent

cell viability and attachment during primary culture was noted for cells isolated on the

day of estrus or between d 4 to 6 for both uterine and oviductal cell populations.

The effects of different culture media and incubation temperatures on growth

and proliferation of uterine and oviduct epithelial cells in vitro were evaluated. Cells

incubated in CMRL and MB2 had the highest growth rates, however, there were no

differences in proliferation rates among media evaluated. There were no differences

in growth and proliferation rates between different incubation temperatures (37°C vs

39°C).

Morphological observations of cell integrity and quality were made using image

analysis during primary culture and following the first and third subpassages. Optical

density readings were influenced by cell subpassage and quality. Cell quality

following in vitro culture could be effectively monitored using image analysis.

Bovine oocytes were matured and fertilized in vitro in different culture media.

Culture medium did not influence fertilization or cleavage rates. Early embryo

development was enhanced following incubation in CMRL and TCM media. Embryo

viability percent improved when morulae were maintained on oviductal cells compared

with those cultured in medium alone.

Bovine embryos derived from in W/ro-fertilization procedures were co-cultured

with oviductal cells isolated between cycle d 4 to 6 or 14 to 16 and proteins secreted

by oviductal cells monitored. Cycle stage oviductal cells were isolated did not

influence developmental rates or proteins secreted. However, co-culture with oviduct

epithelial cells were superior to culture in medium alone.

IN T R O D U C T IO N

The use of embryo transfer methodologies in research has evolved from simple

embryo collection and transfer to recipient females in early years to the more technical

and complex embryo biotechnologies. These new embryo biotechnology procedures

encompass a wide range of laboratory techniques such as cryopreservation, micro

manipulation, in vitro fertilization (TVF), gene insertion and embryonic cloning. The

development of IVF procedures for hum animals in the late 1980’s (48,94) has

provided increased numbers of laboratory-derived embryos available for research

purposes. The access to large numbers of early-stage embryos has increased research

efforts in the areas of embryo bisection methodology (63), DNA insertion (69) and

nuclear transfer techniques (120,130,163) for farm animals.

For many of these new embryo biotechnologies to become successful, embryos

will have to be maintained and develop in vitro prior to recipient transfer. In addition,

the chances of successful pregnancies increase in farm animals if embryos develop to

the morulae or blastocyst-stages prior to their transfer. In most instances this requires

maintenance of early-stage embryos in vitro for up to 7 days. However, the specific

requirements for in vitro development of early-stage embryos of domestic animals are

still not fully understood (see review by 167). In 1965, Cole and Paul (32) first

reported that a high percentage of mouse embryos developed and hatched from their

zona pellucida when cultured in vitro with a feeder layer of irradiated HeLa cells.

This initial study provided a basis for embryo co-culture methodology currently in use

today. The development of embryo co-culture systems using various types of "helper

cells" has provided more efficient means of maintaining embryos in vitro for extended

periods with minimal reductions in viability (see review by 124). In addition, embryo

2

co-culture systems may offer the unique advantage of improving pregnancy rates of

marginal-quality embryos prior to recipient transfer.

The development of embryo co-culture systems has resulted in the emergence

of three different types of co-culture systems (trophoblastic vesicles, cellular

monolayers and chick embryo co-culture). In virtually all instances, co-culture

systems have improved embryo development over that of incubating similar embryos

in culture medium alone. The present hypotheses explaining the beneficial effects of

embryos co-culture systems are that monolayers remove embryotoxic substances from

the medium surrounding the developing embryo, secrete embryotropic factor(s) to

enhance embryo development and/or a combination of both (see review by 8,124).

Although the beneficial effects of co-culture systems have been demonstrated

by many different laboratories in recent years, there are still many biochemical

components and interactions of cell monolayers that are not fully understood.

Developmental capacity of individual embryos in vitro can be enhanced with co

culture, however, most scientists agree that optimum success rates have likely not been

achieved. The variability of success rates using similar co-culture systems across

studies and among different laboratories can be attributed to the cells of the monolayer

or the environmental conditions that the cells are maintained during incubation. It

would seem logical that conditions altering monolayer cell growth or characteristics

while maintained in vitro may ultimately affect embryotropic factor(s) produced during

co-culture. This review will discuss current in vitro co-culture systems used for

mammalian embryos and in vitro culture studies conducted on bovine epithelial

monolayers of uterine and oviductal origin.

L IT E R A T U R E R E V IE W

In Vitro Co-Culture of Mammalian Embryos

Trophoblastic Vesicle Co-Culture. In the mid-1980’s French scientists (27,72)

developed a unique embryo co-culture system using trophoblastic tissue segments of

d 12 to d 14 elongating bovine concentuses. The trophectoderm of elongating cattle

and sheep blastocyst were sectioned and incubated in vitro. These trophoblastic

segments subsequently formed spherical vesicles (trophoblastic vesicles), and were

suggested to secrete luteotropic and embryo development promoting factors (72).

Camous et al. (27) investigated the ability of one- to eight-cell bovine embryos

to overcome the eight- to 16-cell block stage in vitro by co-culturing with d 13 or d

14 bovine trophoblastic vesicles. In this study, 46% of the one- to eight-cell embryos

developed to the morula stage when cultured with trophoblastic vesicles for 3 to 4 days

in Menezo’s B2 medium (MB2). In contrast, <20% of similar stage embryos reached

the morula-stage when embryos were cultured in the control MB2 medium alone. In

a later study, Heyman et al. (73) co-cultured one- to eight-cell bovine embryos with

d 14 bovine trophoblastic vesicles and 46% reached the morula stage compared with

only 18% reaching the morula stage in control medium. In a second experiment, 55

one-cell embryos were co-cultured with trophoblastic vesicles and 44 % cleaved beyond

the eight-cell stage compared with only 13% of the one-cell embryos cultured in

medium alone. In addition, when one-cell ovine embryos were co-cultured with d 12

sheep trophoblastic vesicles 75% of the embryos reached the morula stage compared

with 35% of the embryos cultured in control medium alone.

Camous et al. (27) and Heyman and Menezo (71) have suggested that tropho

blastic vesicles may provide important metabolic component(s) (e.g. lipids), that are

3

required for normal embryo cleavage in utero. In addition, it was proposed that these

embryotropic factors are secreted into the culture medium, and that direct contact

between the developing embryo and trophoblastic cells during the co-culture was not

necessary to elicit this response. Heyman et al. (73) further verified that direct

embryo-trophoblastic vesicle contact was not necessary by showing that one- to two

cell bovine embryos could develop to the 16-cell stage by culturing in conditioned

medium harvested from bovine trophoblastic vesicles.

In a more recent study, Pool et al. (119) attempted to improve the efficiency

of trophoblastic vesicle co-culture for farm animals by, individually placing morula-

stage bovine embryos into the lumen cavity of bovine trophoblastic vesicles during

incubation. It was noted that embryos placed inside trophoblastic vesicles during

culture resulted in fewer grade 1 and 2 embryos (36%) following 60 h of culture

compared with that of similar embryos individually co-cultured outside of the

trophoblastic vesicles (69%). The authors suggested that the decreased viability of

embryos placed inside trophoblastic vesicles was possibly due to the level of

embryotropic factors being too concentrated inside the vesicle during incubation and/or

the metabolic by-products of the confined embryo accumulated to a toxic level in the

lumen of the vesicle during co-culture. It was concluded that placement of embryos

inside trophoblastic vesicles was less effective when compared with the conventional

co-culture method using trophoblastic vesicles (27,72).

Although initial reports gave positive results with trophoblastic vesicle co

culture in cattle, Rexroad and Powell (127) reported no beneficial effects with

trophoblastic vesicle co-culture when evaluated with ovine embryos. These conflicting

results may be due to the day the conceptus was harvested for trophoblastic vesicle

production. In the latter study, d 14 elongated ovine embryos for producing

5

trophoblastic vesicles resulted in lower cleavage rates for co-culture, similar to the

one-cell embryos cultured in medium alone. This study differed from the reports by

French scientists (72) when d 12 ovine blastocyst were used for trophoblastic vesicle

production for the in vitro co-culture of ovine embryos. Correspondingly, no embryo

tropic effect from trophoblastic vesicle co-culture was noted when d IS caprine

blastocyst were used for trophoblastic vesicle production for co-culture with early-stage

caprine embryos (16). The latter authors suggested that trophoblastic vesicles from

d 15 caprine blastocyst were too moiphologically advanced during incubation to

enhance development of blastocyst from two- to eight-cell goat embryos.

Based on the results reported with trophoblastic vesicle co-culture, it appears

that trophoblastic vesicles should be prepared following d 10 and prior to d 15 of

development in the cow, sheep and goat for maximum embryotropic activity during

in vitro embryo co-culture (16,27,72,73,119,127). The potential for biochemical

involvement of trophoblast cells and the developing embryo has recently been

reviewed (71).

Uterine Cell Co-Culture. Many different co-culture systems are now being

developed to enhance embryonic growth and development of mammalian embryos in

vitro. Initial sources of cells for co-culture were of reproductive origin, used in hopes

of mimicking the in vivo uterine environment. Early co-culture systems incorporated

monolayers of fibroblasts derived from reproductive tissues of different animal

sources. Fibroblast monolayers for embryo co-culture were prepared following three

to five subpassages of cells after their initial outgrowth from endometrial explants

(86,155). In one of the earliest reports co-culturing bovine embryos, Kuzan and

Wright (86) incubated morula-stage embryos in vitro and reported a higher percentage

of embryos developed to hatched blastocyst on either uterine or testicular fibroblast

monolayers compared with similar stage embryos cultured in medium alone.

In a later study, Allen and Wright (3) reported that early-stage porcine

embryos (four-cell to morula) had greater developmental rates following co-culture on

porcine endometrial monolayers compared with embryos cultured in medium alone.

Bovine uterine fibroblast co-culture monolayers have also been shown to be capable

of enhancing in vitro development of porcine embryos (85,87).

Voelkel et al. (155) further reported beneficial effects of uterine fibroblast co

culture with bovine demi-embryos. Improved viability of demi-embryos was evident

on uterine fibroblast monolayers after 72 h of co-culture compared with corresponding

demi-embryos cultured in medium alone. Also, beneficial effects of the fibroblast

monolayer were thought to be necessary during the in vitro repair process of the

micromanipulated demi-embryos. It was suggested by Kuzan and Wright (86) that

fibroblast monolayers could be releasing embryo growth factor(s) into the culture

medium or possibly removing toxic substances from the medium, thus resulting in

enhanced in vitro development.

With renewed interest in fibroblast co-cultures, Wiemer and co-workers

(158,159,163) developed a fetal uterine cell monolayer culture system that incorpo

rated uterine fibroblasts derived from near-term («270 days) bovine fetuses. This fetal

bovine uterine fibroblast co-culture system has been shown to enhance in vitro devel

opment of bovine (160), equine (163) and human (161,162) embryos over that of

culturing similar embryos in medium alone.

A majority of the studies reported on co-culturing embryos in uterine cell

monolayer systems have used primarily fibroblast cells originating from reproductive

tissue. In more recent studies, epithelial or epithelial-like cells for embryo co-culture

have been isolated from the uterine endometrial lining of the cow (148), goat (121)

and rhesus monkey (65). Prichard et al. (121) first used uterine and oviductal cells

to co-culture two- to four-cell goat embryos through the in vitro developmental block.

Although adequate hatching rates were noted on uterine cell monolayers (63%),

significantly greater hatching rates were noted with oviductal cell co-culture (87%).

Also, sequentially transferring another group of embryos from oviductal cell mono

layers to uterine cell monolayers (to mimic the in vivo environment) during the

incubation interval was not found to be beneficial over that of oviductal cell co-culture

(73 %). Blakewood et al. (14) have also reported caprine uterine epithelial monolayers

enhanced post-thaw development of bovine embryos when co-culturing prior to

freezing over that of medium alone.

Recently, a larger scale study was conducted evaluating uterine, oviductal and

granulosa cell co-culture systems on IVF-derived bovine embryos (77). These three

different cell co-culture systems were also evaluated in different combinations to

evaluate enhancing effects between cell types during embryo co-culture. The

percentage of hatched blastocyst obtained when cultured, with granulosa cell, oviductal

cell, uterine cells, granulosa and oviductal cells, granulosa and uterine cells, oviductal

and uterine cells and a combination of granulosa, oviductal and uterine cells ranged

from 58 to 73%. The addition of granulosa cells to uterine or oviductal cell co-culture

systems provided the highest hatching rates for IVF-derived bovine embryos. The

bovine uterine cell co-culture system resulted in the lowest percentage of hatched

blastocyst (58%) following incubation. Unexpectedly, these findings suggested other

cell types were better for culturing bovine embryos in vitro than cells derived from

uterine tissues.

The recent development of a nonsurgical embryo recovery technique in rhesus

monkeys has allowed access to early-stage embryos for subsequent culture research

(64). Goodeaux et al. (65) reported increased development and hatching rates of

rhesus monkey embryos co-cultured on rhesus uterine epithelial cells compared with

culture in medium alone (63% vs 25%, respectively). Furthermore, co-culture of

rhesus embryos on rhesus uterine epithelial cell monolayers followed by nonsurgical

transfer has resulted in the birth of a live transplant offspring. It was also shown in

this study that rhesus uterine epithelial monolayers were also capable of supporting

development of morula-stage cattle embryos.

Oviductal Cell Co-Culture. Although positive results have been reported with

the use of uterine cells as a co-culture system for later-stage embryos, oviductal cell

monolayers have been shown to be more effective in enhancing the development of

early-stage mammalian embryos in vitro. Over two decades ago, Biggers et al. (10)

noted that two-cell mouse embryos would develop through the in vitro developmental

block stage when embryos were cultured in explanted mouse oviducts maintained in

culture medium. Whittingham (157) later suggested that only the ampullar region of

the murine oviduct was capable of maintaining embryo development in vitro.

It has also been reported that oviductal cells of various mammalian species

were capable of stimulating embryonic development in different animal species.

Earlier studies have demonstrated the capabilities of the rabbit oviduct to maintain or

enhance in vivo embryonic development in mouse, sheep, cow, pig, goat and horse

embryos (see reviews by 8,18).

Rexroad and Powell (125) were among the first to report the use of oviduct

cell monolayers for short-term culture of early-stage ovine embryos. In addition,

Gandolfi and Moor (57) evaluated the developmental potential of pronuclear-stage

9

ovine embryos co-cultured on both oviductal epithelial and uterine fibroblast

monolayers. Both of these monolayer systems were capable of supporting embryo

development to the early blastocyst stage. After 6 days of incubation, 42% of the

ovine embryos developed into expanded blastocyst using oviductal cell co-culture

compared with only 5% co-cultured on uterine fibroblast monolayers. The transfer

of embryos co-cultured on oviductal cell monolayers resulted in higher pregnancy rates

in recipient sheep compared with those co-cultured on fibroblast monolayers.

Oviductal cell monolayers have also been used successfully for in vitro co

culture of early-stage bovine embryos (37,39,41). In an initial study, Eyestone et al.

(43) reported higher developmental rates of five- to eight-cell bovine embryos cultured

for 4 to 5 days in an oviductal cell co-culture system compared with culture medium

alone. In a subsequent study, Eyestone and First (41) evaluated the effects of ovi

ductal cell co-culture on embryonic growth by comparing this culture system with that

of conditioned medium from oviductal cells. A higher percentage of morula- and

blastocyst-stage embryos resulted when IVF-derived one-cell bovine embryos were co-

cultured on oviductal cell monolayers compared with that of culture in medium alone

(22% and 3%, respectively). In contrast, when embryo development on oviduct cell

co-culture was compared with that of oviductal-cell conditioned medium, in vitro

developmental rates for bovine embryos were similar (22% and 22%, respectively).

Furthermore, embryos transferred to recipient animals following co-culture in

conditioned medium resulted in a 67% pregnancy rate.

McCaffrey et al. (97) evaluated the requirements of oviductal monolayer-

embryo contact and age of monolayer during co-culture using one- to four-cell bovine

embryos. Oviductal monolayers were prepared for co-culture 3 days prior to embryo

recovery. These monolayer systems were used with or without a microporous (.4 /im)

10

membrane inserted between the monolayer cells and embryo during culture. Morulae

or blastocyst resulting were similar following co-culture on monolayers prepared either

on the day of embryo recovery (69%) or 3 days earlier (62%). As was indicated in

a previous report (41), direct contact between the embryo and monolayer co-culture

system did not improve developmental rates in this study.

Recently, Ellington et al. (39) compared in vitro embryo development rates of

early-stage bovine embryos co-cultured on oviductal monolayers in a simple medium

(CZB) with that of in vivo development within the reproductive tract of the cow. In

this study, embryos obtained 40 to 48 h post-estrus were co-cultured in vitro for S

days, however, the developmental rate and number of nuclei per embryo did not differ

from embryos developed in vivo. This finding suggests that the oviductal cell co

culture system with a simple serum-free medium can function to enhance development

of bovine embryos. In addition, Ellington et al. (38) compared in vitro development

of one- to two-cell bovine embryos cultured in either a simple (CZB) or complex

(CMRL-1066 and Ham’s F-10) medium with bovine oviductal cells. Higher develop

ment of early-stage embryos in CZB with oviductal cells occurred compared with

similar embryos co-cultured in either of the complex media. Frozen-thawed oviductal

cells used for co-culture also produced similar rates of blastocyst development resulted

compared with fresh monolayers cultured in CZB medium (32 and 39%, respectively).

Significantly fewer blastocyst were obtained when the CZB oviductal-cell conditioned

medium (24%) was used in place of oviductal cells. It was concluded that a simple

serum-free medium would adequately support in vitro development of one-cell bovine

embryos to the blastocyst stage in the presence of oviductal cells.

In a recent study, Ellington et al. (37) co-cultured one- to two-cell bovine

embryos on oviductal qrithelial cells in a simple medium (CZB) or in the oviducts of

an intermediate host rabbit prior to transferring the cultured morulae and blastocyst to

recipient animals. The pregnancy rate for embryos co-cultured on oviductal cells was

57% compared with 58% for similar embryos cultured in rabbit oviducts. It was

concluded that oviductal epithelial cell co-culture systems supported embryo

development in vitro to obtain an adequate pregnancy rate, even when a simple culture

medium (without protein supplementation) was used for in vitro culture of bovine

embryos.

Recently, extensive studies have been conducted with oviductal cell monolayers

to promote in vitro development of IVF-derived bovine embryos (17,40,52,84,133)

and IVF-derived chimeric embryos (82). In each case, oviductal cell monolayers were

more effective than culturing embryos in medium alone. In addition, Aoyagi et al.

(4) evaluated the ability of six different culture systems to promote blastocyst develop

ment for IVF-derived bovine embryos. The co-culture systems included cumulus

cells, oviductal epithelial cells, trophoblastic vesicles, amniodc sac cells and in vivo

culture in rabbit oviducts. No difference was reported in blastocyst development

among oviductal cells, trophoblastic vesicles and amniotic sac cells, with develop

mental rates ranging between 39 and 51%. These culture systems were more effective

for stimulating blastocyst development in vitro than when bovine cumulus cell co

culture, culture in rabbit oviducts and culture medium alone were used to evaluate

embryo development in vitro.

Oviductal cell monolayers are also capable of stimulating in vitro development

of early-stage pig embryos. White et al. (156) reported enhanced development of two-

to 16-cell pig embryos co-cultured on porcine oviductal cell monolayers. In this

study, in vitro development was evaluated during culture with either medium alone,

porcine oviductal cells, porcine endometrial fibroblasts or a bilayer of porcine

12

endometrial fibroblast and oviductal cells. The highest percentage of hatched

blastocyst were noted with co-culture of porcine oviductal cells and a bilayer of

oviductal and fibroblasts cells (54% and 61 %, respectively). It was concluded that

oviductal cell co-culture provided necessary factors to overcome the four-cell block in

the pig.

Oviductal cell monolayers capable of supporting development of sheep

embryos to the blastocyst stage in vitro have been found to secrete proteins similar to

those found in ovine oviductal fluid (58). Two main classes of proteins were found

to be secreted by sheep oviductal cells; one secreted throughout the cycle (MW range

of 10,000 to 100,000) and those exhibiting cyclic variations in secretion. The latter

class of proteins is further divided into a 92,000 MW protein and a 46,000 MW

protein. Both proteins were secreted during the first 4 days of the cycle but the

96,000 MW protein then declined sharply, thereafter. In addition, the proteins

produced during the first 4 days of the ovine estrous cycle have the ability to bind to

the zona pellucida (59).

A present hypothesis suggests that oviduct epithelial cell feeder-layers enhance

the development of preimplantation embryos by one or more mechanisms. First,

oviductal monolayers may secrete specific growth factors (e.g. protein, peptides) that

are required by early-stage embryos to maintain normal rate of development (58,59).

Another possibility is that monolayers have the ability to remove toxic components that

are detrimental to the embryo from the surrounding medium during co-culture (for

review see 8). In addition to secreting embryotropic components and/or removing

embryotoxic substances from the medium, Bavister (8) has proposed that both cell

types may lower oxygen tension in the immediate vicinity of the embryo to enhance

development. It was suggested that the major difference between uterine fibroblast

13

and oviduct epithelial cell co-culture systems is that fibroblast feeder-layers are capable

of removing embryotoxic components, reducing oxygen tension, thereby, improving

embryo development over that of culture medium alone but are unable to secrete

embiyotropic factors. In contrast, it was concluded that oviductal cell feeder layers

apparently have the ability to remove embryotoxic components, lower oxygen tension

in addition to having embryotropic capabilities.

Results thus far suggest that oviductal cell monolayers are the most effective

co-culture system for early-stage farm animal embryos while fibroblast monolayers are

adequate for development of later-stage embryos (morulae to blastocyst) but less

effective for developing earlier stage embryos through the hatched blastocyst stage

(124). Due to the limited number of studies available evaluating embryo culture on

uterine epithelial cells, it is unclear whether development of early-stage embryos is

dependent upon the source of cells (uterus vs oviduct) for co-culture.

Granulosa Cell Co-Culture. Research efforts have now demonstrated the

importance of bovine granulosa/cumulus cells for the in vitro maturation of bovine

oocytes obtained from abattoir ovaries. Critser and First (34) suggested that

granulosa/cumulus cells are not only beneficial for in vitro maturation of oocytes but

maybe important for the development of IVF-derived embryos to the morula and

blastocyst stages. Faundez et al. (46) have reported that in vitro fertilization rates of

bovine oocytes were higher with granulosa cells than when similar oocytes were

exposed to IVF procedures without the aid of granulosa cells. In addition, the highest

fertilization rates resulted when cumulus-intact bovine oocytes were incubated on

granulosa cell monolayers prior to and during the IVF procedure.

In an initial study, Goto et al. (66) reported successful bovine transplant preg

nancies were obtained following in vitro co-culture of IVF-derived embryos with

14

bovine cumulus cells for 6 or 7 days. In a similar study using granulosa cell co

culture, Goto et al. (67) reported that 25% of in vz'rro-matured and fertilized bovine

oocytes reaching the eight-cell stage following 3 to 4 days of incubation, with 21%

reaching the morula and blastocyst stages following IVF procedures.

Fukuda et al. (55) have also used cumulus cell monolayers to maintain IVF-

derived cattle embryos in vitro. Following co-culture on cumulus monolayers, six

frozen-thawed blastocyst were transferred into three recipients, with one recipient

producing twin offsprings. In addition, seven fresh IVF-derived blastocyst were

transferred to six recipients and two recipients delivered live calves. This study

demonstrated the ability of cumulus cell monolayers to produce live offspring from

both fresh and frozen-thawed IVF-derived embryos.

More recently, Zhang et al. (172) used a different procedure for in vitro

fertilization of bovine oocytes and reported 54% cleavage rates and 41% of the total

oocytes placed into culture reaching the morula stage of development using a cumulus

cell co-culture system. In addition, a bovine cumulus cell co-culture system was

reported to be effective for in vitro development of porcine IVF-derived embryos

(171). Younis and Brackett (170) reported similar results using a bovine cumulus cell

co-culture system on IVF-derived bovine embryos. In addition, Berg and Brem (9)

noted significantly higher rates of development of IVF-derived bovine embryos to

morulae and blastocyst (32%) using granulosa cell co-culture compared with oviductal

epithelial cell co-culture (17%).

In 1990, Japanese researchers evaluated the proportion of inner cell mass

(ICM) cells of bovine blastocyst fertilized in vitro and in vivo (76). In this study,

IVF-derived bovine embryos were co-cultured on cumulus cell monolayers or placed

in rabbit oviducts for 8 to 12 days following fertilization. The proportion of ICM and

15

trophectoderm cells of IVF-derived embryos was compared between an in vitro and

two in vivo culture systems by a differential fluorochrome staining technique after 7

days. In this study, the highest proportion of ICM was noted with in vivo culture for

early (25%), expanded (21%) and hatched (27%) blastocyst in the rabbit oviducts.

There was a significantly lower proportion of ICM noted with IVF-derived in vitro-

cultured (cumulus cells) embryos for early (16%), expanded (15%) and hatched (13%)

blastocyst when compared with those cultured in an in vivo system. In addition, there

was a significantly lower proportion of ICM noted with IVF-derived in vivo cultured

(rabbit oviducts) embryos for early (23 %) and expanded (21 %) blastocyst but not with

hatched blastocyst (17%) compared with the in vitro system. It was concluded that

the differential staining of ICM and trophectoderm nuclei provided an effective method

for evaluating embryo development in an in vitro co-culture system.

Nakao and Nakatsuki (109) recently compared bovine trophoblastic vesicle and

cumulus cell co-culture systems for the in vitro culture of IVF-derived bovine

embryos. Embryos developing to the morula stage were similar when co-cultured on

either trophoblastic vesicles (17%), cumulus cells (19%) or co-cultured with both

vesicles and cumulus cells (16%). However, in this study there was no enhanced

embryotropic effect evident when both cell types were combined in a single culture

unit.

Although most results indicate that granulosa cell co-culture systems are

similar to other co-culture systems, the major advantage of the granulosa cell culture

system is that the cells are available for harvesting from the follicle at the time of

oocyte collection. This provides a simple and cost efficient co-culture system without

lowering success rates of in vitro culture systems.

16

Chick Embryo Co-Culture. The first report of using the amniotic cavity of

developing chick embryos to culture mammalian embryos was reported by Blakewood

et al. (12). Blakewood and Godke (11) embedded pronuclear-stage mouse embryos

in agarose and injected embryos into the amniotic cavity of a 96 h-old chick embryo

incubated at 37eC. In the initial experiment (13), pronuclear mouse embryos from

two different lines were placed in the amniotic cavity of chick embryos for 72 to 96

h of incubation. Following incubation, significantly more embryos developed into

hatching blastocyst within both strains of mice compared with those in culture medium

alone. Blakewood et al. (14) additionally reported success using the chick embryo co-

culture system to enhance post-thaw development of precompaction-stage bovine

morulae. Further studies have demonstrated that the chick embryo amnion was

capable of supporting development of bovine morulae (IS) and two- to eight-cell goat

embryos (16).

Blakewood et al. (16) demonstrated the ability of the chick embryo co-culture

system to promote development of two- to eight-cell goat embryos through the in vitro

block stage. Both the fetal bovine uterine fibroblast monolayer and chick embryo co

culture systems resulted in significantly more expanded and hatched blastocyst

compared with embryos incubated in medium alone following 72 h of incubation. In

a second experiment, two- to eight-cell caprine embryos were maintained on uterine

fibroblast monolayers or in the amniotic cavity of the chick embryo for 96 h. It was

reported that no early-stage goat embryos developed to the expanded or the hatched

blastocyst stages when co-cultured on the fetal uterine monolayer or with the control

medium. However, 86% of the embryos co-cultured in the chick embryo amnion

reached the expanded blastocyst stage and 82% of the embryos developed to the

hatched blastocyst stage in vitro. In addition, four recipient goat females received

17

surgically transplanted chick-embryo co-culture morulae, two maintained pregnancies

to term and a total of six live-transplant offspring (50% of all embryos transferred)

were bom. These findings further indicate that the developing chick embryo has

potent embryotropic properties that are evident with mammalian embryos across

species.

Other Co-Culture Systems. Glass et al. (61) evaluated different types of

helper cells (L-cells, liver, JLS-V11 and teratocarcinoma cells) for culturing mouse

embryos to the hatching stage and reported no differences among cell types. More

recently, Overskei and Cincotta (86) noted adequate success rates (83%) when two-cell

mouse embryos were co-cultured in vitro on hamster hepatocyte monolayers. In

addition, Hu et al. (75) reported excellent development of two-cell mouse embryos co-

cultured on a established cell line of Buffalo Rat liver cells. It was noted that Buffalo

Rat liver cells produced better results during in vitro culture than did similar embryos

incubated on mouse oviductal cells.

Recently, bovine fetal spleen (BFS) cell and chicken embryo fibroblast (CEF)

monolayers were used for the in vitro co-culture of morula-stage mouse (79) and

bovine (80) embryos. The BFS and CEF monolayers produced more hatched bovine

embryos (75 and 83%, respectively) than did culture in medium alone (45%). In

addition, Kim et al. (81) compared these two monolayer co-culture systems with a

bovine cumulus cell co-culture system using IVF-derived bovine embryos. The best

success with development and hatching occurred when using bovine cumulus cells

compared with BFS and CEF monolayers.

Ouhibi et al. (113) evaluated development of one-cell mouse embryos co-

cultured on reproductive tract cells (oviduct epithelium) or established cell lines

derived from cells other than of reproductive origin (kidney cells). The oviductal cell

18

co-culture treatments consisted of mouse oviductal organ cultures, mouse oviductal

cells and bovine oviductal monolayers consisting of both polarized and unpolarized

cells. The highest percentage of morulae and blastocyst were obtained from embryos

placed in mouse oviductal organ cultures (77%). The percentage of morula- and

blastocyst-stage embryos resulting from mouse oviductal, bovine unpolarized and

polarized monolayers were 67%, 48% and 14%, respectively. Also, Vero unpolarized

and polarized monolayers had lower development rates (8% and 4%, respectively)

compared with MDBK unpolarized and polarized cells (74% and 21 %, respectively).

Overall, maintenance of cell polarization resulted in lower development during mono

layer co-culture. From this study, it appears that the established Vero cell line was

not suitable for development of mouse embryos.

These studies indicate that various cell lines and cell monolayers developed

from cells other than from adult and fetal reproductive tissue are capable of supporting

development of early-stage embryos for up to 72 h during in vitro culture. The major

advantage of established cell line co-culture systems is a pathogen-free cell line with

embryotropic capabilities and high viability and growth of cells following repeated

subpassages.

Id Vitro Culture of Epithelial Cells

Uterine Epithelial Cells. The development of an effective uterine epithelial

cell culture system may improve embryo co-culture systems for IVF-derived embryos.

In addition, this in vitro culture system would allow researchers to investigate

prostaglandin release, protein synthesis, early embryo development, embryo/uterine

interactions and implantation using uterine cell monolayers or explanted tissue. The

majority of research efforts have concentrated on isolation and culture of epithelial or

19

stromal (fibroblast) cells from laboratory and farm animal species. It has been

suggested that vital communications occur in vivo between stromal and epithelial cells

to construct the complexity of the uterus (62). These communications support the

integrity or continued secretion of embryotropic factor(s) by cells. Secretion of

embryotropic factor(s) by monolayers is continued even though an in vitro culture

system results in some degree of transformation or removal of the in vivo system.

Research efforts have utilized membrane inserts for in vitro culture of epithelial cells

to retain polarity of the cells and create a more in vivo type co-culture system

(113,131,133,156). However, these systems have not proven beneficial over standard

culture conditions for enhancing embryonic development.

Several studies have been conducted to evaluate the action of progesterone and

estrogen on protein synthesis of uterine epithelial cells in vitro. In one study (30),

endometrial sections obtained from guinea-pigs were digested and isolated cells

incubated in CMRL-1066 medium in vitro. Following 3 days of incubation, cells were

incubated with estradiol- 17B and progesterone for 48 h and the confluent monolayers

radiolabelled with 3SS-methionine. It was reported that progesterone supplemented in

the culture medium modified the patterns of proteins compared with those proteins

from monolayers incubated with estradiol-17B. It was further suggested that a single

protein (MW 19,000) was the major contributor to the antagonist effects of prog

esterone on estradiol-17B action, but the protein disappeared when both progesterone

and estradiol-17B was supplemented in the incubation medium. From this study, it

was concluded that the action of steroid hormones on uterine epithelial cells does not

require the presence of stromal cells. In addition, progesterone altered the synthesis

of proteins when combined with estradiol-17B and affected both cellular and secreted

proteins.

20

In the guinea-pig, progesterone induced secretory morphological changes in

epithelial cells maintained in vitro (1,2). In addition, the effects of estradiol-17B and

progesterone on progesterone receptors have been previously reported for guinea-pigs

(100,101,102,142,143). In ovariectomized guinea-pigs, estrone sulphate induced an

increase in progesterone receptors (104). In a later study, estrone sulphate was

reported to induce modifications in the shape and formation of microvilli (1). Further

investigations revealed that estrone sulphate induces the sulphation of proteins and

more specifically increased the sulphate incorporation into the cellular proteins (143).

The authors concluded that estrone sulphate acts with progesterone to preferentially

enhance the secretion of sulphated proteins.

Rickets et al. (129) digested portions of rabbit uterine mucosa in attempts to

characterize cell types used during in vitro culture systems. Both epithelial and

stromal cells were isolated and provided evidence that the source of uteroglobulin

proteins are derived from epithelial cells in rabbits. The roles of estrogens in

endometrial cell proliferation have also been previously investigated in the rabbit (33).

A possible role of estrogen in regulation of uterine cell function using in vitro culture

systems was suggested. Estrogen administered to ovariectomized rabbits resulted in

increased proliferation rates of quiescent epithelial cells. In addition, estrogens

increased cell migration towards the uterine lumen whereas progesterone was suggested

to promote development of new uterine glands.

As in rabbits, estrogen stimulates uterine epithelial cell proliferation in adult

ovariectomized mice (123,141). Estrogen also regulates the secretion of uterine

proteins along with morphological and functional differentiation of the uterine

epithelium (96). The roles of estrogen in regulating mouse uterine cell activity

appears to be mediated via epidermal growth factor (EGF) receptors and increased

21

levels of EGF peptides (106). Tomooka et al. (151) also reported stimulatory effects

of EGF on mouse uterine epithelial cells maintained in vitro. An increase in cell

number was associated with supplementation of EGF to culture medium. However,

this increase did not occur when nerve growth factor (NGF), multiplication-stimulating

activity (MSA), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF)

or somatomedian-C was added to incubation medium. Tomooka et al. (151) also

demonstrated the ability of mouse uterine cells to maintain EGF receptors in vitro and

EGF had stimulatory roles in cell proliferation. The presence of EGF receptors were

also detected in rat uterine cells (105) and EGF levels increased under the influence

of estrogens (106).

As in laboratory animals, protein synthesis by the endometrium is influenced

by ovarian steroids in the ewe. Estrogen treatment of ovariectomized ewes resulted

in increased uptake of 35S-methionine into both cellular and secreted proteins and

progesterone increased the proportion of newly synthesized proteins in both luminal

and glandular epithelial cells (136).

Using an in vitro culture system, Salamonsen et al. (136) suggested possible

roles of ovarian steroids on uterine protein secretion in the ewe. Both estrogen and

progesterone act synergisdcally, with estrogen stimulating overall synthesis of proteins

and progesterone promoting the increased synthesis of secretory proteins. In a

subsequent study, Salamonsen et al. (137) provided evidence that ovine blastocyst

influence uterine epithelial cells on d 13 of pregnancy by increasing the overall protein

synthesis and secretion of specific proteins by uterine cells.

lim ited studies have been reported on the in vitro culture of uterine epithelial

cells in the cattle. Recently, Munson et al. (108) reported methods for establishing

endometrial epithelial cell lines derived from both adult and fetal bovine uteri. For

adult uteri, layers of intercarunclar endometrium were removed and minced into small

fragments. Tissue fragments were separated from small clusters of cells by filtration

and centrifugation. In contrast, the fetal uteri were filled with collagenase and

incubated for 1 h at room temperature. Attempts to produce cell lines by digestion

with trypsin were unsuccessful. However, three adult and one fetal cell line were

established using collagenase digestion techniques. It was reported that fetal cell lines

were more viable than adult lines following subpassages and cry opreservation. In

addition, fetal lines survived longer in culture than adult cell lines (10 and four

subpassages, respectively). These initial experiments provided the basis for the in

vitro culture of bovine endometrial epithelial cells derived from adult and fetal uteri.

The in vitro culture of human endometrial cells has recently received attention

due to the increased efforts in human IVF procedures. The human endometrium

undergoes morphological and biochemical changes during the menstrual cycle and

pregnancy (35,168). These changes have prompted many researchers to develop in

vitro culture systems for endometrial tissue to study interactions between epithelial and

stromal cells.

Several studies have provided culture techniques for maintaining endometrial

tissue in vitro with varied success (47,83,152). Varma et al. (154) cultured human

endometrial tissue in vitro in an attempt to characterize and study growth properties

of various cell types. Different cell types were obtained following digestion of

endometrial tissue with glandular tissue and individual cells separated and placed into

culture. In addition, explants of endometrial tissue were placed in culture dishes to

promote fibroblast outgrowth. Extensive electron microscopy studies revealed three

cell types proliferating during primary culture: epithelial cells, stromal cells (epithelial-

like) and fibroblasts. Both stromal and fibroblast cells could be subcultured up to 20

23

passages in vitro, however, epithelial cells only proliferated during primary culture and

could not be subpassaged.

In a subsequent study by the same laboratory, Dorman et al. (36) indicated that

in vitro cultures of endometrial stromal cells exhibited aggregation following estrogen

and progesterone treatment. In addition, the plating efficiency of subcultured cells was

highly variable (32% to 75%). The addition of estrogen and progesterone to culture

medium did not enhance plating efficiency. Varma et al. (154) and Dorma et al. (36)

concluded that extensive characterization of endometrial cell lines used in in vitro

culture systems is required to understand basic morphological changes the uterus

exhibits throughout the menstrual cycle and pregnancy.

In an earlier study, Liszczak et al. (93) obtained epithelial monolayers from

human endometrial samples by separation of stromal cells by cloning cylinders.

Cultures of epithelial cells were incubated in steroid-free medium in the presence of

10"6 M estradiol-17B, 10"6 M progesterone or a combination of both for 7 days. No

alterations in cell morphology were noted from any steroid treatments. Furthermore,

criteria for classification of epithelial cells were conducted using electron microscopy.

These criteria were the presence of structures such as junctional complexes,

perinuclear microfilaments and microvilli surrounded by glycocalyx. In addition,

structures of secretory cells were abundant rough endoplasmic reticulum, golgi

apparatus and the presence of membrane-bound electron-dense bodies in the cytoplasm.

Oviduct Epithelial Cells. Recently, Ouhibi et al. (112) reported procedures

for isolation and in vitro culture of oviductal epithelial cells derived from the mouse,

rabbit, cow and humans. Epithelial cells were isolated by filling oviducts with .25%

trypsin solution and incubating at 37°C for 45 min. Following the incubation period,

cell clusters were disrupted by repeated pipetting and layered on Percoll gradients.

24

Harvested cells were seeded in culture dishes and incubated in MB2 medium with the

addition of 10 ng/ml serotonin. Serotonin added to the culture medium was suggested

to provide necessary components for cell attachment and differentiation and has

selective properties against fibroblast proliferation.

Quhibi et al. (112) reported that bovine oviductal monolayers were easily

obtained and could be subcultured for 14 to 18 passages before the appearance of an

in vitro crisis phase. Similarly, rabbit oviductal monolayers could be maintained for

10 passages before the appearance of altered growth and morphology. However,

limited numbers and slower growth rates of human oviductal cells were noted

compared with other species. In addition, monolayers could be maintained up to the

sixth passage. It was reported that establishment of mouse monolayers were pH

dependent and growth was only initiated in MEM in place of MB2 medium. In

addition, all attempts to obtain subpassaged monolayers were unsuccessful in this

study.

French scientists have also used radiolabelled methionine to evaluate protein

secretions by monolayers during in vitro culture as an indication of cell quality.

Ouhibi et al. (113) evaluated protein secretions by subpassaged oviductal monolayers

of different species using radiolabelled methionine incorporation and noted that

monolayers maintained high protein secretion rates following 96 h in culture.

Evaluation of radiolabelled protein secretion by monolayers during in vitro culture will

likely provide vital information on embryotropic factors produced by monolayers

during embryo co-culture. Identifying the factors that alter or directly influence cell

monolayers of embryo co-culture systems would provide the key to establishing the

most efficient co-culture systems for mammalian embryos.

Joshi (78) conducted extensive transmission electron microscopy and immuno-

cytochemical studies on oviduct epithelial cells isolated from the cow. Epithelial cells

were isolated by inflating oviducts with .1% collagenase and incubating at 37°C for

90 min. Harvested cells were incubated in DMEM and Ham’s F-10 medium and they

reported isolating both ciliated and nonciliated secretory cells. Using electron

microscopy, ciliated cells were characterized by presence of fewer endoplasmic

reticulum that secretory cells, many mitochondria, free ribosomes, polyribosomes and

microtubules. Attached ciliated cells lost their cilia following 4 to 5 days during in

vitro culture. However, free floating ciliated cells maintained cilia for 10 to 12 days.

These results provided specific criteria for evaluation of two specific cell types

obtained from the bovine oviduct and further verified epithelial origin of cells by

immunocytochemical studies.

Using similar culture techniques as previously reported (78), Hishinuma et al.

(74) evaluated growth and monolayer culture of bovine oviduct epithelial cells and

similar characteristics were noted. In this study, the addition of lO*5 and 10*9 M

estradiol-17B did not affect ciliary activity or growth of oviductal cells. Furthermore,

the authors noted different types of secretory cells maintained in vitro, those with

homogenous matrix and those possessing lamellar structures within the matrix.

Limited studies have been reported in the scientific literature on the in vitro

culture of human epithelial cells. In a recent study, Henriksen et al. (70) described

methods for the in vitro culture of human oviduct epithelial cells. Epithelial cells were

harvested by dissection of mucosa pieces obtained from the ampullary region of

oviducts. Tissue pieces were minced thoroughly with scissors the resuspended in

RPMI 1640 medium supplemented with 20% bovine calf serum. Following a 48 h

incubation period, the levels of calf serum in medium were lowered to 10%.

26

In this study, extensive election microscopy and immunocytochemical studies

were conducted using monoclonal antibodies to cytoskeleton proteins for character

ization of cell types. The plating efficiency of cultures ranged between 10 to 20%

within 48 h after initial seeding. Growth curves of cells during primary culture

revealed increases in the number of cells for 7 days followed by stationary periods of

cell growth. However, cultures could be maintained for 6 to 8 wks before

degeneration of cells. It was noted that cells isolated from human oviducts were of

epithelial origin as judged by presence of microvilli, typical desmosomes, cilia and

positive staining for cytokeratin antigens. It was further suggested that the low plating

efficiency noted may be attributed to the ciliary activity of cells. Furthermore,

pretreatment of culture vessels with fibronectin did not improve plating efficiency. It

was concluded that the specific origin of cells during in vitro culture must be based

on electron microscopy and presence of cytokeratin antigens to fully distinguish

between epithelial and fibroblast cell types.

Bongso et al. (20) successfully established epithelial monolayers isolated from

ampullary regions of human oviducts. Monolayers could be established regardless of

donor age or phase of the menstrual cycle, and it was reported that epithelial

monolayers were maintained for four to six subpassages. However, monolayers were

transformed to fibroblast-like cells following repeated subpassages. Although scanning

microscopy was used, extensive characterization of cell types using immunocyto-

chemistry was not performed.

Oki et al. ( I ll) evaluated the effects of age and stage of menstrual cycle of

donor subjects on performance of human oviduct epithelial cells in vitro. Oviductal

cells were obtained by treatment with collagenase then incubated in TCM-199 with

10% fetal calf serum. Survival of oviductal monolayers were determined following

27

7 days of incubation. Monolayer survival rates of 78%, 50% and 100% was reported

for subject age groups between 20-39 yr, 40-59 yr and over 59 yr, respectively. In

addition, cell survival rates for menstruating and post-menopausal women were 63%

and 83%, respectively. Although there were no differences in survival rates of

monolayers, the highest percentage of confluent cultures were obtained in the younger

age groups (44%) and for menstruating women (25%). It was further stated that there

were no differences in monolayer survival for cells obtained from women in follicular

and luteal stages of the menstrual cycle. However, the highest percentage of confluent

monolayers were obtained in the follicular phase opposed to luteal phase of the

menstrual cycle (40% and 17%, respectively). In addition, the authors reported

growth of human oviductal cells in vitro was only successful up to the third

subpassage.

Summary

Repeatable techniques for producing in v?Y/t>-fertiIized farm animal embryos

have made the early-stage embryo more accessible for biochemical and morphological

study (67,95,172). This accessibility to laboratory-derived embryos has illustrated the

need for culture systems that have the ability to promote normal in vitro development

of early-stage farm animal embryos. For farm animals, the ability to produce larger

numbers of early-stage embryos in the laboratory would offer little benefit without

developing an in vitro culture system that would allow the embryos to progress

through the in vitro block stage. Since acceptable pregnancy rates in cattle are not

obtained unless embryos of the morulae and blastocyst stages of development are

transferred, the need for a simple, efficient culture systems becomes increasingly more

important.

28

With recent advances made in embryo culture systems for farm animals, it

appears that in vitro incubation problems may have been partially alleviated with the

helper cell co-culture systems, that permit embryonic growth and development through

the in vitro block stage to morulae and blastocyst while in culture.

The use of oviductal cell co-culture systems have improved in vitro develop

ment of embryos in nearly all studies conducted. However, there still remains

variability in results between studies. Based on in vitro studies of uterine and oviduct

epithelial cells, culture conditions and cell source needs to be investigated in an effort

to explain differences in co-culture systems. Furthermore, specific characterization

of cell types used in co-culture systems are needed to evaluate results based on

different cell types.

The evaluation of factors that directly affect epithelial monolayers themselves

may provide the ultimate embryo co-culture system. The best possible monolayer

becomes increasingly more important due to the fact that the epithelial cell co-culture

system appears to be the source of embryotropic secretions during co-culture.

E X P E R IM E N T I

A METHOD FOR M VITRO CELL CULTURE O F SUPERFICIAL BOVINE UTERINE ENDOMETRIAL EPITHELIUM

Introduction

Efforts to culture early stage bovine embryos in vitro have met with limited

success (167). However, the use of various co-culture systems, including fetal uterine

(161,163) and adult uterine (86,118,155) fibroblasts, oviduct epithelial cells

(41,57,122), trophoblastic vesicles (27,73,119,127), and shell-less chick embryos (13)

have been shown to enhance early development of mammalian embryos in vitro.

Although embryo development benefits from co-culture, the establishment of an

adequate uterine epithelial cell in vitro culture system provides an attractive method

to evaluate uterine epithelial cell secretory capacity during the estrous cycle. In

addition, this model could be used to study early embryonic signals produced by the

uterus and the conceptus during early embryonic development in farm animals.

Isolation and culture of uterine endometrial epithelial cells has been previously

reported in mice (89,151), rats (62), guinea-pigs (1,28,29,30), rabbits (33,107), pigs

(3), sheep (136,137), rhesus monkeys (65), humans (31) and the cow (108). Although

epithelial cells have been isolated from the uterine endometrium of the cow and other

species, these procedures often rely on dissecting endometrial tissue samples for

placement directly in culture or digesting sections of endometrial tissue to obtain cells

for culture. A major drawback of utilizing these techniques is the possible contamin

ation of the culture with uterine stromal cells.

Uterine stromal cells may, themselves, have a secretory roll in the functional

uterus (62). However, to effectively conduct studies on epithelial cell secretory

capacity and/or interactions with embryos, a pure cell population is needed. The

29

30

objective of this study was to develop a rapid and efficient procedure for recovering

and maintaining a monolayer of uterine lumen epithelial cells to study uterine secretory

activity and uterine cell to embryo interactions. This procedure was developed based

on methods previously reported for recovery of oviductal cells from various species

(112). Using the procedure reported herein, only the superficial cell layers of the

uterine endometrium are removed without requiring digestion of excised endometrial

tissue samples.

Materials and Methods

I. MATERIALS

A. Equipment

1. C02 incubator, model 3158, Forma Scientific1

2. Laminar-flow hood, Nuaire2

3. Inverted microscope, phase contrast, Diaphot, Nikon3

4. Centrifuge, table top, model TJ-6, Beckman4

5. Programmable cell freezer, Planer Biomed Kryo 10 Series5

6. Peristaltic pump, no. XX80 000 00, Millipore6

7. Temperature-controlled water bath, 50°C

B. Culture media and chemicals

1. Tissue culture medium-199 (TCM-199), no. M0393, Sigma7

2. Dulbecco’s phosphate-buffered saline (PBS), no. D5773, Sigma7

3. Dulbecco’s phosphate-buffered saline (PBS), without Ca2+ and Mg2+, no.

D5773, Sigma7

4. Heat-treated fetal bovine serum (FBS), Gibco8

5. Penicillin-streptomycin (100 X ), Gibco®

6. Fungizone (100 X ), E.R. Squibb9

7. Trypsin (10 X ), .25%, no. T5650, Sigma7

8. Sodium bicarbonate, no. S5761, Sigma7

9. Serotonin, no. H4511, Sigma7

10. Dimethyl sulfoxide (DMSO), no. D2650, Sigma7

11. Ultrapure water, MilliQ system6

12. Red blood cell lysing agent, no. RT7757, Sigma7

13. EDTA, no. E8008, Sigma7

C. Supplies

1. Tissue culture flasks, 25 cm2 vented, Costar 305610

2. Tissue culture flasks, 75 cm2 vented, Costar 337610

3. Centrifuge tubes, 15 ml, Costar 321510

4. Pasteur pipettes, borosilicate glass, no. 033-76111

5. Cryovials, no. 368632, Nunc12

6. Sterile plastic petri dish, 100 x 15 mm, no. 1012, Falcon13

7. Hemacytometer, Brightline, Reichert14

8. Syringes plastic, 10 ml, no. 88291, Air-Tite15

9. Nalgene sterilization filter units, no. 125-002016

10. Glass slides, 7.5 X 2.5 cm, no. 166-645, Coming11

11. Hypodermic needles, 18 gauge x 3.75 cm length

12. Stainless steel forceps, small

13. Stainless steel scissors, medium

14. Stainless steel hemostats, medium

H. PROCEDURE

A. Preparation of media and solutions

32

1. Prepare TCM-199 and PBS according to the directions of the manu

facturer using ultrapure water.

2. Adjust the pH to 7.3 and filter medium (.2 jxm filter).

3. Store medium at 4°C until use.

4. Prepare serotonin by diluting in 10 ml of sterile PBS and freezing in

1 ml aliquots at -20°C.

5. Combine TCM-199 (450 ml), FBS (50 ml), serotonin (.5 ml) and

penicillin-streptomycin (.5 ml).

6. This results in a final concentration of 10 ng serotonin, 100 units

penicillin and 100 jig streptomycin/ml of culture medium (TCM-199).

7. Combine 500 ml PBS (without Ca2+ and Mg2+) with 1.0 ml penicillin,

1.0 ml streptomycin and .5 ml fungizone.

8. This results in a final concentration of 200 units penicillin, 200 jig

streptomycin and .25 jig fungizone/ml of PBS which is used to transport

uterine cornua to the laboratory.

9. Add .2 mg/ml EDTA to .25% trypsin solution.

10. Dilute .25% trypsin solution with sterile PBS (without Ca2+ and Mg2+)

to achieve .025% trypsin solution.

B. Isolation of epithelial cells

1. Uterine cornua are aseptically collected from the abattoir and placed on

ice in sterile PBS (without Ca2+ and Mg2+) containing 200 units

penicillin, 200 ng streptomycin and .25 pg fungizone/ml PBS.

2. Intact uterus and cornua are placed under a laminar flow hood and the

connective tissue removed.

3. The trypsin solution (.25%) is placed in a 37°C water bath prior to use.

33

4. Separate uterine homs from uterus and flush each horn with 5 ml of

warm trypsin solution (.25%) to remove debris with 10 ml syringe and

18 gauge needle.

5. Clamp one end of the uterine horn with a pair of hemostats and fill with

warm trypsin (.25%) solution until inflated and then clamp the proximal

end.

6. Repeat procedure for second uterine horn.

7. Place the fluid-filled uterine horn in sterile petri dish with a small volume

of PBS (without Ca2+ and Mg2+) and incubate for 45 min at 37°C.

8. Remove hemostats and recover the trypsin solution from each uterine

horn into a second sterile petri dish.

9. Flush both uterine horn with fresh trypsin solution (.25%).

10. Open uterine homs longitudinally with scissors and gently scrape the

lumen surface (once) with a sterile glass slide.

11. Separate epithelial clusters in the petri dish by repeated pipetting with a

pasteur pipette.

12. Place trypsin solution containing the harvested cells into 15 ml centrifuge

tubes and add equal volumes of culture medium (TCM-199) to inactivate

trypsin.

13. Centrifuge harvested uterine epithelial cells at 200 X g for 10 min.

14. Remove supernatant and resuspended pellet in 1 ml of red blood cell

lysing agent for 1 min.

15. Add 10 ml of fresh culture medium (TCM-199) to red blood cell lysing

agent and centrifuge at 200 x g for 10 min.

16. Wash the epithelial cells three times with fresh TCM-199.

34

17. Assess cell viability by trypan blue exclusion as reported by Freshney

(51).

18. Resuspend epithelial cells in 5 ml of TCM-199 and place in 25 cm2

flasks.

19. Culture the cells at 37°C in a atmosphere of 95% air and 5% C02.

20. Replace TCM-199 at 48-h intervals during the incubation period.

C. Subculture and cryqpreservation of uterine epithelial cells

1. After cells reach 90% confluency remove culture medium, add 3 ml of

.025% trypsin solution.

2. After 2 min remove trypsin solution, add fresh trypsin solution (.025%)

and incubate at 37°C and 5% C02.

3. Observe for cell rounding and for cells loosening from the bottom of the

culture flask 5 min).

4. Shake the flask gently to remove loosely adhered cells and then pipette

solution into 15 ml centrifuge tubes with pasteur pipette.

5. An equal volume of TCM-199 is added to the centrifuge tube to

inactivate trypsin.

6. Pellet cells by centrifugation at 200 X g for 10 min, and seed new 25

cm2 flasks at a density of 1 x 106 viable cells/flask with 5 ml of TCM-

199.

7. At the third subculture, prepare the cell suspension as described for

subculturing above.

8. Prepare the freezing solution consisting of TCM-199 supplemented with

30% FBS, 10% DMSO, 100 units penicillin and 100 Mg streptomycin/ml.

9. Dilute cells to 1 X 106 viable cells/ml in the freezing solution.

35

10. Place 1 ml aliquots of cells into cryovials and place in a programmable

freezer.

11. Cool cells in programmable freezer with a curve of 5°C/min to

-20°C, hold for 5 min, then decrease l°C/min to -30°C, then decrease

2°C/min to -60°C.

12. Transfer subpassaged epithelial cells to liquid nitrogen for storage.

D. Developing a growth curve

1. At the third subculture, trypsinize the epithelial cells as previously

outlined.

2. Place 2.5 x 10s cells/flask in 15 sterile 25 cm2 flasks.

3. On d 1, 2, 4, 6 and 8 of culture, triplicate 25 cm2 flasks are trypsinized

using the same procedures, except use .25% trypsin solution to ensure

removal of all cells.

4. Under 10 x power determine number of cells with a hemacytometer.

Evaluate cell viability (51).

Results

Uterine cornua were recovered from 10 adult, mixed-breed cows in various

stages of the estrous cycle. Using this cell harvesting method, the mean number of

cells isolated per uterine horn was 3.3 x 106 cells. When placed in culture flasks,

uterine epithelial cells adhered to the plastic surface and began to form monolayers

within 48 h. Ciliated and nonciliated epithelial cells were noted in the culture flasks

during primary culture. Confluent monolayers were obtained within 5 to 7 days after

initial seeding. Cilia were not evident in cultures after the first subculture. Viability

of uterine cells at different subcultures ranged from 87 to 92%. Uterine epithelial

36

cells were subcultured for seven to eight subcultures before exhibiting a decrease in

cell viability, attachment efficiency and vital cell characteristics. Decreased growth

rate, enlarged cells with a flattened appearance and vacuolization of cytoplasm were

associated with epithelial cell death.

Cells thawed rapidly at 37°C (after 60 days of storage) were placed in fresh

TCM-199. Good post-thaw cell viability and cell attachment were noted during in

vitro culture. The mean (±SE) post-thaw viability and attachment efficiency were

88.8±.8% and 88.5±2.2%, respectively. Cells could be cultured for four

subpassages following thawing before becoming flattened in appearance and losing

viable cell characteristics.

A growth curve for these uterine epithelial cells is presented in Figure 1. The

mean values presented include the number of cells for five replicates. Uterine

epithelial cells exhibited a slight decrease in cell number during the first day in culture

but exhibited a log increase in cell growth for 7 days. A stationary phase did not

appear during the culture period examined. On d 8 of culture there was still an

increase in the number of cells. The mean (±SE) number of cells were: 2 .7 ± .l,

3.7±.2, 8.3±.6, 11.7±1.7 and 13.6±.5 x 10s for d 1, 2, 4, 6 and 8 of culture,

respectively.

37

Num ber o f Days in Culture

Figure 1. A growth curve of uterine epithelial cells during the third subculture conducted for an 8-day interval.

38

Discussion

Munson et al. (108) previously established endometrial epithelial cells derived

from adult pregnant cows (d SO to 120 of gestation). In this study, epithelial cells

were successfully established by both explants and collagenase digestion techniques.

Establishment of epithelial monolayers during primary culture was not possible, in this

case, most likely due to stromal cell contamination. Efforts were made to remove

stromal cells contaminants after the first subpassage with difficulty as noted by these

researchers (108).

The major advantage of the procedure reported herein is the ability to obtain

epithelial monolayers during the primary culture free from stromal cell contamination.

This is possible by harvesting and trypsinizing superficial lumen of the uterus, instead

of obtaining cells from explants or collagenase digestion.

Munson et al. (108) reported procedures for removal of fibroblast cells from

epithelial monolayers at the first subpassage. It was noted that trypsinized fibroblasts

detached from the culture surface more readily than epithelial cells. We also found

that warm (37°C) trypsin solution (.025%) placed on cultured cells for 1 min

effectively removes any fibroblasts without removing epithelial cells. Gently shaking

the flasks also helps to remove any loosely adhered fibroblast cells. This can be

incorporated into the epithelial cell procedures at each subpassage. The use of

serotonin in the culture medium also helps maintain monolayers of epithelial cells. It

has been previously reported that serotonin has selective properties against fibroblast

proliferation in vitro (112).

Uterine epithelial monolayers have been used to enhance embryo development

of mammalian embryos in vitro. However, the identification of embryotropic factor(s)

produced by endometrial cell monolayers during co-culture is still unknown (124). The

39

ability to establish and maintain uterine epithelial cells in vitro may be a potential

model to study their secretory capacity and biochemical interactions between the

embryo and uterine endometrium.

With the procedure reported herein, uterine epithelial cells isolated from the

adult bovine uterine endometrium developed and proliferated in culture. Uterine

epithelial cells harvested by this method provided excellent monolayers virtually free

of fibroblast cell contamination. The findings while developing this method for our

laboratory indicate that uterine epithelial cells may be isolated and in vitro cultured

with good success rates. Since this system is simple and efficient it may be used in

the future to provide a model to study uterine secretory capacity, embryo interactions

with the uterus and a new embryo co-culture method.

Suppliers

1 Forma Scientific, Inc., Marietta, OH2 Nuaire Inc., Plymouth, MN3 Advanced Scientific, Inc., Chalmette, LA4 Beckman Instruments, Inc., Palo Alto, CA5 T.S. Scientific, Peakasie, PA6 Millipore, Bedford, MA7 Sigma Chemical Co., St. Louis, MO8 GIBCO Laboratories, Grand Island, NY9 Pfizer, Inc., New York, NY

10 E.R. Squibb, Princton, NJ11 Costar, Cambridge, MA12 Curtin Matheson Scientific, Inc., Houston, TX13 Vangard International, Inc., Neptune, NJ14 Falcon, Oxnard, CA13 Reichert Scientific Instruments, Buffalo, NY16 Air-Tite of Virginia, Inc., Virginia Beach, VA17 VWR Scientific, Rochester, NY

E X PE R IM E N T H

EFFECTS OF STAGE OF THE BOVINE ESTROUS CYCLE ON IN VITRO CHARACTERISTICS OF UTERINE AND

OVIDUCT EPITHELIAL CELLS

Introduction

Oviductal epithelial cells of bovine and ovine origin have also been used for

in vitro co-culture of early-stage cow (41), goat (121) and sheep (126) embryos. In

each instance, the co-culture system used has proven to be effective in supporting

development of embryos through the characteristic in vitro block stages. These

findings suggest the existence of unidentified growth factor(s) produced by the cells

of monolayers that have embryotropic properties, perhaps via the secretion of specific

peptides and/or proteins (124). Studies have shown that ovarian hormones influenced

proliferation, ciliation and secretory processes of the oviduct epithelium in the rabbit

(50), monkey (25,110) and cow (98). Although various studies have used epithelial

monolayers for co-culture of early stage embryos (41,57,126), these studies have not

addressed possible differences in cells obtained for culture during different stages of

the estrous cycle and their effects on developing a successful embryo co-culture

system.

Salamonsen et al. (136) reported differences in appearance of endometrial

epithelial cells during primary culture when ovariectomized ewes were pre-treated with

different levels of progesterone and estrogen prior to cell harvest. Also, the hormonal

environment of the endometrium at the time of harvest resulted in a difference in

growth characteristics of human stromal endometrial cells during primary culture (49).

Changes in uterine epithelial glands were also reported following the administration

of progesterone to estradiol-primed ovariectomized animals (21,22). Progesterone has

40

41

also been reported to induce morphological changes in cultured uterine epithelial cells

obtained from the guinea pig (1,2).

Steroid treatment in sheep influenced in vivo incorporation of radiolabelled

methionine into cellular and secreted proteins of epithelial cells (136). In addition to

changes in cell morphology, cyclic changes in hormonal levels during the estrous cycle

may result in differences in cell characteristics. Differences in cell characteristics due

to the stage of the estrous cycle may ultimately have an effect on embryonic growth

in vitro when these cells are used in an in vitro co-culture system. The objective of

this study was to compare in vitro morphology, growth and monolayer foundation of

bovine uterine and oviduct epithelial cells when collected at different days of the

estrous cycle.

M aterials and Methods

Experimental Animals and Treatment Groups. A group of cyclic Holstein

heifers (range in estrous cycle length of 17 to 23 days), all in good body condition and

ranging in body weight from 350 to 400 kg, were used in this study. Heifers were

maintained on native grass pasture and provided daily with hay and com silage.

During the spring months heifers were synchronized with prostaglandin

(Lutalyse®; Upjohn Co., Kalamazoo, MI) then equally and randomly assigned within

day of the estrous cycle to one of four treatment (Trt) groups (n = 5/treatment). Only

heifers exhibiting standing estrus were used in the experiment. Treatment groups were

assigned by days of the estrous cycle (estrus — d 0): d 0 (Trt A), d 4 to 6 (Trt B), d

8 to 10 (Trt C) and d 14 to 16 (Trt D). Heifers in Trt A had one or more medium-

size follicles present on ovaries, plasma progesterone levels below 1 ng/ml and often

had visible mucus on the vulva. Females in Trt B, C and D had either a developing

42

corpus luteum or palpable corpus luteum and £ 2.0 ng/ml plasma progesterone at the

time the reproductive tracts were harvested.

Reproductive tracts were collected from animals at slaughter on respective

estrous cycle treatment days. Tracts were placed on ice immediately after collection

in PBS (without Ca2+ and Mg2+) containing 200 units penicillin, 200 Mg streptomycin

and .25 Mg Amphotericin-B/ml and transported to the tissue culture laboratory. The

time from the slaughter of animals until reaching the laboratory was «1 h.

Isolation of Epithelial Cells. Tissue Culture Medium-199 (Sigma) with 10%

heat-treated fetal bovine serum (FBS), 100 units penicillin, 100 fig streptomycin

(Gibco), 10 Mg serotonin (Sigma), .1 ml L-glutamine and .1 ml Na pyruvate/ml

(Gibco) of culture medium (TCM-199) was used as the base medium throughout the

study.

Uterine and oviductal cells were isolated and cultured with modifications of the

procedure previously reported for uterine (148) and oviduct (112) epithelial cell

cultures in cattle. With this procedure, a 10 cm section was removed from each

uterine hom 3 cm posterior to the utero-tubal junction and 3 cm posterior of the

ampulla for oviducts, then trimmed free of mesentery. Uterine horn and oviduct

sections were flushed with 5 ml of warm .25% trypsin supplemented with .2 mg/ml

EDTA to remove debris then filled with fresh trypsin solution, clamped and incubated

at 37°C for 45 min. The trypsin solution was recovered and the tissue flushed a

second time with fresh .25% trypsin. Uterine horns and oviducts were dissected

longitudinally and the lumen surface gently scraped once with a sterile glass slide.

Cells isolated from each section were combined and epithelial clusters separated by

repeated pipetting. The trypsin solution was inactivated by the addition of the base

culture medium (1:2 v/v). The entire procedure was carried out under a laminar flow

43

hood at room temperature.

Harvested cells were centrifuged at 200 x g for 10 min, the supernatant

removed and the cellular sediment resuspended in 1 ml of red blood cell lysing agent

(Sigma) for 1 min. Fresh TCM-199 was added (10 ml) and the suspension centrifuged

at 200 x g for 10 min. The supernatant fluid was removed and the cellular sediment

resuspended in TCM-199 (1 ml). A small portion of cells were assigned to electron

microscopy and the remaining cells placed in 25 cm2 vented tissue culture flasks

(Costar) with 5 ml of TCM-199. All cultures were maintained at 37°C in an

atmosphere of 95% air and 5% C02 and culture medium changed at 48-h intervals.

Subculturing confluent monolayers were conducted by trypsinization with .025%

trypsin supplemented with .2 mg/ml EDTA. Flasks were incubated at 37°C for 5 to

10 min until cells became rounded in shape and detached from the bottom of the flask.

The trypsin was inactivated by the addition of TCM-199 (1:2 v/v). Cells were

centrifuged, the supernatant removed then resuspended in fresh TCM-199 and seeded

at 1:2 ratios for each treatment group.

In Vitro Characteristics. Prior to initial seeding, the number of cells recovered

was determined on a hemacytometer and viability assessed by the trypan blue dye

exclusion method (51). Percent cell attachment was determined by removing the

culture medium and counting the number of cells that did not attach during the initial

48 h (51). The number of cells, percent viability and attachment were also determined

at the first and third subpassages. Histological evaluations of cells stained with

Giemsa stain (51) were made during primary culture and first and third subpassages.

During primary culture and after the first and third subpassage, samples of cells were

fixed for scanning and transmission electron microscopy. After the third subpassage,

cells were frozen in liquid nitrogen.

44

Freezing Epithelial Cells. Cells from the third subpassage were placed in 2 ml

cryovials (Nunc, Naperville, EL) at a concentration of 1 x 106 viable cells/vial and

frozen in TCM-199 adjusted for a final concentration of 30% FBS and 10% DMSO.

Cells were frozen in a Planer Biomed Kryo 10 Series programmable freezer using a

curve of 5°C/min to -20°C (held for 5 min), l°C/min to -30°C then 2°C/min to

-60°C and then transferred and stored in liquid nitrogen.

Electron Microscopy. For scanning electron microscopy, uterine and oviductal

cells were seeded in one well of a 24-well Transwell9 tissue culture plate (Costar).

At confluency, monolayer cultures were rinsed with PBS, fixed in 3% glutaraldehyde

in sodium-cacodylate buffer (. 1 M, pH 7.4) for 1 h, washed twice in buffer, post

fixed in 1% osmium tetroxide in sodium-cacodylate buffer (.1 M, pH 7.4) and

dehydrated in a dilution gradient of ethanol:water (30, SO, 70, 80, 90 and 100%

ethanol). Cells were critical-point dried then coated with gold-palladium and evaluated

with a Cambridge Stereoscan 150 scanning electron microscope.

For transmission electron microscopy, uterine and oviductal cells were seeded

in two-well plastic chamber slides (Lab-Tek). At confluency, monolayer cultures were

rinsed with PBS, fixed in 3% glutaraldehyde in sodium-cacodylate buffer (.1 M, pH

7.4) for 1 h, washed twice in buffer, post-fixed in 1 % osmium tetroxide in sodium-

cacodylate buffer (.1 M, pH 7.4). Samples were then dehydrated in a dilution

gradient of ethanol:water (30, 50, 70, 80, 90 and 100% ethanol), infiltrated in an

alcohol-Epon mixture and embedded in Epon and evaluated with a Zeiss-10

transmission electron microscope.

Immunocvtochemistrv. Verification of uterine and oviductal cell origin using

immunofluorescence staining was performed as previously described (113). Cells were

fixed in paraformaldehyde, permeabilized with Triton X-100 then incubated with

45

bovine monoclonal anti-cytokeratin (clone K 8.13; Sigma) diluted 1:20 and finally

incubated with FITC-conjugated mouse and-IgG (1:50). Negative controls consisted

of a fibroblast cell line and positive controls were a MDBK cell line. Culture

preparations were examined using a Zeiss microscope equipped for FTTC excitation.

Cell Cycle Analysis. Immediately after cell isolation, 1 x 106 cells from the

remaining portion of the reproductive tract were obtained from animals in each

treatment. Cell cycle analysis was conducted using flow cytometry as described by

Lakhanpal et al. (88). Samples were fixed in 2% paraformaldehyde, then in 95%

ethanol, treated with 100 Mg RNase (Boehringer Mannheim Biochemicals, Indinapolis,

IN) and DNA stained with 5 ng/ml of propidium iodide (Sigma). Flow cytometry

was performed on a FASC 440 (Becton-Dickinson, San Jose, CA) fluorescence-

activated cell sorter that was configured for red fluorescence analysis that was linearly

amplified. Cells were exposed to 488 nm argon line and fluorescence emission was

detected using a 630/22 bandpass filter for propidium iodide detection. Data from

flow cytometry was analyzed using Consent-40 software (Becton-Dickinson) and the

percent of a cell population in a resting (Gap 1 or Gap 0) or proliferative (S, Gap 2

and mitosis phase) state of the cell cycle recorded (68).

Progesterone Levels. Bovine blood samples were collected via venipuncture

48 h prior to and immediately before transport to the abattoir. Blood samples were

collected in EDTA coated vacuum tubes, plasma harvested and stored at -20°C until

later assayed for progesterone according to procedures previously reported (150).

Statistical Analysis. The number of cells isolated, percent viability and

attachment were analyzed using a completely randomized split plot design with

repeated measures in time (60). Main effects within the model included treatment,

heifer within treatment, tissue type (uterine or oviduct), subpassage and important

46

interactions. Significant differences between least square means were determined by

preplanned comparisons of means.

Results

In Vitro Characteristics. The mean number (±SE) of cells isolated for uterine

and oviductal tissue were 3.3±.8 and 4 .4 ± .8 x 106, respectively. The mean number

of cells recovered from.uterine tract was not significantly different (P>.05) across

days of the estrous cycle or between uterine or oviductal cells. The percent viability

and attachment for each treatment group during primary culture are summarized in

Table 1. There was a reduction (P< .005) in percent viability of uterine cells in Trt

C and D compared with Trt A and B. For oviductal cells, Trt B had higher (P < .01)

percent viability than those of other treatments. There was a marked (PC .0001)

reduction in percent attachment in both uterine and oviductal cells noted for Trt C and

D during primary culture compared with that of Trt A and B.

Uterine and oviductal cells in Trt A and B adhered to the culture surface and

proliferated rapidly within 24 h after initial seeding. Confluent monolayers were most

often obtained within 5 to 6 days following the start of incubation. However, since

there were significantly fewer cells that attached in Trt C and D, confluent monolayers

were obtained in 10 to 14 days. The cells that attached to the culture surface began

to curl around each other and form colonies within a few h after seeding. Cells grew

in swirls composed of cohesive groups of polygonal cells. These characteristics were

observed for all treatment groups. Based on daily observations and Giemsa staining,

there were no noted differences in morphology between uterine and oviductal cells or

between treatment groups for day of the estrous cycle. Populations of ciliated and

nonciliated cells were isolated from both the uterus and oviducts. Ciliated cells could

47

be detected in cultures until 4 to 5 days after seeding but were not noted after the first

trypsinization (subpassage).

The cells that attached and proliferated in culture exhibited increases in percent

cell viability (PC.0001) and percent cell attachment (PC .0001) after the first and

third subpassages. There were ho significant differences in viability among treatment

groups for both uterine or oviductal cells following the first subpassage. Although

there were significant improvements in attachment efficiency following the first

subpassage, Trt C had a significantly lower percent attachment for oviductal cells than

did Trt B (PC .01) and Trt D (PC .05). However, after the third subpassage, there

were no significant differences in percent cell viability and cell attachment between

treatment groups for either uterine or oviductal cells.

Cell Freezing. After a minimum of 60 days, cells were thawed at 37°C and

washed three times in TCM-199. Percent post-thaw viability and attachment for

uterine and oviductal cell were not significantly different. Therefore, all data for

uterine and oviductal cells across all treatment groups were combined and expressed

a single percent. The mean (±SE) post-thaw percent viability and attachment were

88.8±.8 and 88.5±2.2, respectively.

Electron Microscopy. Scanning electron microscopy revealed numerous

bundles of cilia present on the cell surface of uterine and oviduct cells during primary

culture. Oviductal cells contained more dense patches of cilia present on cell surface

compared with uterine cells obtained from the same experimental animal (Figure 2).

Only microvilli were present on the culture surface at the first subpassage, indicating

a loss of cilia from the primary cultures (Figure 3). In many cells evaluated,

microvilli could be detected on the apical surface of the cell for all subpassages inves

tigated along with numerous mitochondria, desmosomes and microfilaments. These

48

characteristics were noted for both uterine and oviduct cells. Secretory granules could

be detected in specific cells at the first and third subpassages along with small clusters

of rough endoplasmic reticulum and some lipid inclusions.

Immunocvtochemistry. The presence of keratin filaments were detected on

both uterine and oviductal cultures during primary culture and the third subpassage.

The immunofluorescent detection of cytokeratin was easily observed in the MDBK

established cell line but absent for chick embryo fibroblast cell lines. These results

along with electron microscopy data confirm both uterine and oviductal cell cultures

to be of epithelial cell origin.

Cell Cvcle Analysis. The mean percent cell proliferation rates for treatments

A, B, C and D for both uterine and oviductal cells combined were 22.7, 7.6,22.1 and

17.7%, respectively (Figure 4). The coefficient of variation for each sample obtained

from cell cycle analysis ranged from 5.6 to 11.4 for all experimental animals

combined. The lowest percent cell proliferation observed in Trt B corresponds with

the highest percent cell attachment during primary cultures.

Progesterone Levels. The mean progesterone levels 48 h prior to and at

slaughter were .14 and .38 for heifers in (Trt A), .05 and 1.0 (Trt B), 9.91 and 10.34

(Trt C) and 7.59 and 5.26 (Trt D) ng/ml, respectively. Estrus detection records and

the progesterone values verified that the reproductive tract cells were sampled from

estrual, early, middle and late luteal phases of the estrous cycle.

49

Table 1. Effects of the estrous cycle on in vitro evaluations of uterine and oviduct epithelial cells (least squares means ±SE)

Treatment

ItemA

dOB

d 4-6C

d 8-10D

d 14-16

Primarv Culture Percent Cell Viability

Uterine Oviductal

Percent Attachment Uterine Oviductal

80.6±2.5*79.2±2.5*

84.6±3.9d79.7±3.9d

87.7±2.5b88.4±2.5b

87.5±3.9d87.2±3.9d

76.5 ±2.5*77.5 ±2.5*

10.8±3.9*27.3±3.9*

68.8±2.5* 74.1 ±2.5*

10.5±3.9C18.4±3.9®

Third Subpassaee Percent Cell Viability

Uterine Oviductal

Percent Attachment Uterine Oviductal

89.0±4.087.8±4.0

88.1 ±6.2 81.9±6.2

91.3±4.092.8±4.0

91.5±6.2 90.1 ±6.2

95.3±4.093.2±3.2

82.7±6.285.8±6.2

89.3±4.083.9±4.0

91.7±6.282.4±6.2

■*b,eSuperscripts with different letters within rows and tissue type differ (P< .05). ^'Superscripts with different letters within rows and tissue type differ (P< .0001).

Figure 2. Scanning electron micrograph of oviductal (a) and uterine (b) epithelial cells during primary culture isolated from the same animal on d 4 to 6 of the estrous cycle. Note the differences in cilia across the cell surface (2,700 x).

Figure 3. Scanning electron micrograph of oviductal (a) and uterine (b) epithelial cells at the third subpassage. These are cells isolated from the same animal as Figure 2 (20,000 x).

52

26 r2 4 -

T rea tm en t Groups

Figure 4. Mean percent of cells proliferating isolated from different stages of the estrous cycle. Values represent uterine and oviductal cells combined.

53

Discussion

There were no noted differences in morphology of uterine and oviduct epithelial

cells isolated from different stages of the estrous cycle. Stage of the estrous cycle

dramatically affected epithelial cell monolayer foundation in this study. Other

researchers (30,136) have suggested that progesterone may also act by interfering with

patterns of proteins synthesized and/or released by the reproductive tract epithelial

cells in vivo or in vitro. The marked reductions in percent attachment of uterine and

oviductal cells from animals in the mid to late luteal phases of the estrous cycle, may

be attributed to several factors such as physiological state of the cell (68), cell mor

phology and/or unknown factors.

Differences noted in cells harvested during mid to late luteal stages of the

estrous cycle may be attributed to fewer attachment factors present on the cell surface

during the late luteal phase of the estrous cycle. Another possible explanation for

reduced attachment may be cells during the late luteal phase have attachment factors

on the cell surface that are more sensitive to trypsinization. Removal of attachment

factors such as E-cadherin (S), present on the cell surface, would account for reduced

attachment and this sensitivity to trypsin may only be detected after removal in vivo.

Also, the highest proportion of cells in a proliferative state of the cell cycle (Trt C and

D) corresponded with the lowest percent cell attachment. These suggestions are

strengthened by the fact that after the first subpassage the percent attachment

increased, and this was similar for all treatment groups. There appears to be a

selection process occurring during primary culture with only cells that have the

greatest capacity for in vitro growth being maintained following subpassages.

Previous reports (74,78) indicate that three different cell types are routinely

isolated from oviducts, consisting of ciliated, nonciliated and columnar cells. Only

54

the nonciliated cells are generally classified as secretory cells. All three cell types

could be detected in the present study with both ciliated and nonciliated cells attaching

to the culture surface and rapidly proliferating. Although experimental animals in the

mid to late luteal stages of the estrous cycle had low percent attachments both ciliated

and nonciliated cells were detected in the culture dish. This suggests that the low

percent attachment is not specific to ciliated or nonciliated cells. These findings

suggest that epithelial cells examined in this experiment had similar morphology and

growth with observations previously reported (74,78). The results of the present study

also showed that most of the cilia activity was lost for both uterine and oviductal cells

during the first 4 to 5 days of incubation.

Loss of ciliation in cultures may be attributed to one or more factors. First,

changes in pH of culture medium may result in loss of cilia during primary cultures

or ciliated cells are not capable of dividing and there is a selection against these cells

during subpassages. Possibly, the ciliation process is the end point of differentiation

and cannot be easily induced in an in vitro system. Therefore, only secretory cells and

uncompleted ciliated cells existed during subpassages.

In this study it was noted that cells isolated from «10% of the animals became

vacuolated during incubation and appeared to enter a "crisis” phase. This was not

associated with any particular subpassage, hormonal status of animals at collection or

for cell type (uterine cells or oviductal cells). It was noted that if oviductal cells

began to enter a "crisis" phase then uterine cells from that same animal exhibited

similar patterns in vitro. This occurrence suggests individual animal variation that may

be due to the previous endocrinological history and/or present status of the animal.

There may be a limited number of cell cycles that are highly dependent upon each

animals response to circulating hormones. This model of programmed cell death may

55

be directly modulated by the previous endocrinological pattern of the animal. This

suggestion is further strengthened with electron microscopy revealing normal cell

morphology during subpassages and little damage or deteoriation of cells due to

trypsinization. Also, viable cell morphology and function were evident in the high

viability and percent attachment following the third subpassage.

Several preliminary trials conducted at this laboratory have suggested that once

cells begin vacuolation, this process associated with degeneration could be reversed by

increasing the serum content of the culture medium from 10 to 20% and replacing half

of the culture medium at 48-h intervals. Perhaps increasing the serum content

provides more growth factors to help reduce the cell degeneration process. The

vacuoles observed with degeneration in this study appeared to differ from lipid

containing vacuoles observed in healthy cells by Witkowska (166) and healthy cells in

this study (TEM evaluations).

Epithelial cell monolayers are now being used to enhance development of early

stage embryos in vitro. However, identification of embryotropic factors produced by

helper cell monolayers during co-culture are still unknown (124). When using primary

cultures of uterine or oviduct epithelial cells in an embryo co-culture system the source

of cells needs consideration. It has been reported, however, that subpassaged cells are

equally as effective or even more effective in some cases than primary cultures for co

culture (113,121). In this study, subpassaged cells exhibited the same characteristics

regardless of stage of estrous cycle uterine and oviductal cells were harvested. There

appears to be a selection process occurring for subpassaged cells harvested from the

mid to late luteal stage of the estrous cycle. This selection process may ultimately

affect embryonic development in an embryo co-culture system. Finally, it appears that

individual animal to animal variation may be reduced by the use of subpassed

monolayers to provide more uniform co-culture systems producing embryotrqpic

factors. Further experiments are needed to identify specific factors produced by cell

monolayers during embryo co-culture that result in enhanced in vitro development of

embryos.

E X PE R IM E N T m

THE EFFECT OF CULTURE MEDIUM AND TEMPERATURE ON IN VITRO GROWTH AND PROLIFERATION OF UTERINE

AND OVIDUCT EPITHELIAL CELLS

Introduction

Many studies have been conducted on uterine and oviduct epithelial cells using

different culture media to maintain in vitro growth and development. Epithelial cells

from uterine or oviductal origin have been isolated from the sheep (136), mouse (151),

rabbit (129) and guinea pig (28) and maintained in vitro in a variety of media such as

Dulbecco’s Modified Eagles medium (DMEM), a mixture of DMEM and Ham’s F-12

(HF12), DMEM and CMRL-1066 (CMRL), respectively. Also, a variety of media

has been used to study epithelial cell-embryo interactions with bovine uterine and

oviduct epithelial cells in in vitro culture systems. Excellent growth of bovine

epithelial cells has been obtained with Menezo’s B2 medium (MB2) (112), TCM-199

(148), DMEM (74) and a mixture of DMEM and HF12 (78).

In addition to establishing monolayer characteristics, oviductal cells are also

used for co-culturing embryos using various media. Oviduct epithelial cells have been

used for in vitro co-culture of early-stage cattle (40) and sheep (126) embryos. In

addition, uterine epithelial cells have been used for in vitro co-culture of

preimplantation embryos of rhesus monkeys (65). In each instance, the co-culture

system has proven effective in supporting some degree of development through the

characteristic in vitro block stages (for review see 124). A variety of culture media

have been used to establish oviductal cells prior to embryo co-culture experiments.

In the cow, culture medium used to maintain monolayers prior to and during embryo

co-culture consisted of TCM-199 (41,57,117), Ham’s F-10 (37), HF12 (14), CMRL

57

58

(37) and MB2 (116,138).

Recently, in vitro fertilization studies have used oviductal cell monolayers to

enhance embryonic growth following fertilization (40,82,84,133). In vitro fertilization

procedures are routinely conducted at 39 °C to more closely resemble body temperature

of the cow (38.9°C). The possibility exists that altering standard incubation

temperature of cell culture from 37°C to 39 °C may affect growth and embryotropic

factor production by epithelial cells used in embryo co-culture experiments. Although

various experiments have adjusted culture conditions in maintaining epithelial

monolayers for embryo co-culture, the authors are aware of no studies that have been

conducted to address if culture media and incubation temperature affect growth and

longevity of epithelial cells maintained in vitro. The optimum culture conditions for

growth of epithelial cells used in embryo co-culture systems could ultimately enhance

the embryotropic factor(s) secreted by monolayers during co-culture.

The objective of this study was to evaluate cell growth patterns and percent of

cells proliferating for uterine and oviduct epithelial cells incubated in different media

and temperatures.


Cell Source. Reproductive tracts from 10 mature cyclic cattle were obtained

from a local abattoir. Reproductive tracts were placed on ice immediately after

collection in phosphate-buffered saline without Ca2+ and Mg2+ (PBS) containing 200

units penicillin, 200 Mg streptomycin and .25 fig/ml Amphotericin B. The time from

slaughter until reaching the laboratory was »1 h.

Isolation of Epithelial Cells. The culture medium used to initiate cell growth

was Tissue Culture Medium-199 (Gibco) supplemented with 10% heat-treated fetal

59

bovine serum (FBS), 100 units penicillin, 100 /Jg streptomycin, 10 Mg serotonin

(Sigma) and . 1 ml L-glutamine/ml of medium (TCM).

Uterine and oviductal cells were isolated and cultured with modifications to

procedures previously reported by this laboratory (146,148). In summary, uterine

horn and oviduct sections were flushed with 5 ml of warm .25% trypsin supplemented

with .2 mg/ml ETDA to remove debris then filled with the trypsin solution, clamped

and incubated at 37°C for 45 min. The trypsin solution was recovered and the tissue

flushed a second time with warm flesh trypsin solution. Uterine horns and oviducts

were dissected longitudinally and the lumen surface gently scraped once with a sterile

glass slide. Epithelial clusters were then separated by repeated pipetting and the

trypsin solution inactivated by the addition of culture medium (1:2 v/v). Harvested

cells were then washed three times in TCM.

Cells were seeded in 25 cm2 tissue culture flasks with 5 ml of TCM. All

cultures were maintained at 37°C in an atmosphere of 95% air and 5% C02 and

culture medium replaced at 48-h intervals. Subculturing confluent monolayers were

conducted by trypsinization with .05 % trypsin supplemented with .2 mg/ml EDTA.

Flasks were incubated at 37°C for 5 to 10 min until cells became rounded in shape

and detached from the bottom of the flask. The trypsin was inactivated by the

addition of TCM (1:2 v/v). Cells were resuspended in flesh TCM and seeded at ratios

of 1:2.

Experiment 1.

The objective of this experiment was to evaluate the effects of five different

culture media on growth and percent of proliferating cells for uterine and oviduct

epithelial cells maintained in vitro. Reproductive tracts from five cyclic cows were

60

used in this experiment. At the second or third subpassage, uterine and oviductal cells

were trypsinized as previously described. Cells originating from each animal were

allotted within animals to five treatment groups. Treatment groups consisted of cells

incubated in TCM (Trt A), CMRL (Trt B), MEM (Trt C), MB2 medium (Trt D) or

HF12 (Trt E). All culture medium except MB2 (Biomerieux, France) was purchased

from Gibco Laboratories and supplemented with 10% FBS, 100 units penicillin and

100 Mg streptomycin/ml of medium. All cultures were maintained at 37°C in an

atmosphere of 95% air and 5% C02 and culture medium replaced at 48-h intervals.

Growth Curve and Cell Cvcle Analysis. Growth characteristics for uterine and

oviductal cells were determined by initially plating cells at a density of 2.5 X 10s

cells/flasks in ten 25 cm2 flasks/treatment. Cells were allowed to attach and initiate

growth for 24 h before the first cell count. At designated intervals duplicate flasks

from each treatment group were trypsinized with .25% trypsin solution and the

number of cells/flask counted on a hemacytometer (51). The number of cells from

each treatment and examinations of the cell cycle were determined on d 1, 2, 4, 6 and

8 of incubation.

Immediately following cell counts, the percent of the cell population in a

proliferative state of the cell cycle was determined using flow cytometry (88). Cells

from each treatment group were washed twice in PBS then fixed in 2% para

formaldehyde (Fisher Scientific Co., Fair Lawn, NJ) for 3 min at room temperature.

Cells were washed with PBS, centrifuged, the supernatant fluid removed, and the

pellet resuspended in PBS then incubated in 95% ethanol for 3 min. Samples were

washed twice in PBS then incubated for 30 min in 100 fil of PBS-EDTA and 100 Mg

of RNase (Boehringer Mannheim Biochemicals, Indianapolis, IN) in a 37°C water

bath. Finally, samples were resuspended in 200 m! PBS-EDTA with 5 Mg/ml of

61

propidium iodide for DNA staining. Flow cytometry was performed on a FASC 440

(Becton-Dickinson, San Jose, CA) fluorescence-activated cell sorter. Cells were

excited with the 488 nm argon line and fluorescence emission detected using a 630/22

bandpass filter for propidium iodide detection.

The percent of the cell population in a resting state or proliferating state

recorded and data analyzed using Consent 40 software (Becton-Dickinson). Cells in

a resting state were composed of Gap 1 or Gap 0 while cells in a proliferating state

of the cell cycle were composed of S, Gap 2 and mitosis phases (68).

Experiment 2.

The objective of this experiment was to evaluate the effects of two incubation

temperatures on growth and percent of cells proliferating for uterine and oviduct

epithelial cells maintained in vitro. Based on the success of the culture medium in the

first experiment, CMRL supplemented with 10% FBS, 100 units penicillin and 100

tig streptomycin/ml of medium was used throughout the second experiment. Data for

fitting growth curves and cell cycle analysis were collected on the same experimental

days as in Experiment 1. However, treatment groups consisted of cells incubated at

37®C (Trt A) or 39°C (Trt B).

Statistical Analysis. A total of three replicates of duplicate flasks from each

animal were used to construct the growth curve for each experiment. The number of

cells and percent of cells proliferating were analyzed using a RBD split plot design

with repeated measures in time (60). Statistical comparisons of least squares means

were conducted using preplanned comparisons. For the analysis, cells originating

from each animal served as blocks to account for variation between animals.

62

Results

Experiment 1.

Uterine and oviductal epithelial cells attached and initiated growth in all culture

media evaluated. A growth curve of uterine epithelial cells is shown in Figure 5.

There was a slight decrease in cell numbers on the first day of incubation followed by

a log increase in growth for 5 days. On d 6 of incubation, Trt B had significantly

(P< .01) more cells than all other treatment groups. However, on d 8 of incubation,

Trt B and D had significantly (P < .0001) more cells than Trt A, C and E. There were

no significant differences between Trt A, C or E on d 6 or 8 of incubation. Cells

incubated in Trt D exhibited slow growth rates during the first 6 days of culture but

had a high growth rate at the end of the culture period. Cells incubated in Trt A, C

and E reached a stationary phase of growth by d 8 of incubation. However, cells

incubated in Trt B and did had not reach a stationary phase at the end of the culture

period. All treatment groups reached confluency by the end of the culture period.

There were no significant differences in cell growth between treatment groups

during the first 2 days of incubation for oviductal cells (Figure 6). However, Trt B

and C had significantly (PC .0001) more cells than Trt A, D and E on d 4 of

incubation. Cells incubated in Trt B had significantly (PC .0001) more cells than

other treatment groups on d 6 and 8 of incubation. Oviductal cells incubated in Trt

A, C and E exhibited stationary phases on d 6 of culture. However, cells in Trt B and

D were still exhibiting increases in cell growth at the end of the incubation period.

Oviductal cells cultured in Trt B reached confluency by d 6 of culture however, there

were still increases in cell numbers. All other treatments reached confluency by d 8

of incubation.

The percent of proliferating cells for each treatment were determined using

63

flow cytometry. All samples were collected, analyzed and results stored in the

computer prior to data processing. This allowed samples to be analyzed under similar

conditions and parameters. Gating of cell populations for analysis was conducted

based on histograms illustrating two distinct populations of cells (Figure 7). The

coefficient of variation for each peak observed during analysis was between 5 and 10%

for all samples. The mean percent of cells proliferating for uterine and oviduct

epithelial cells prior to the initiation of culture (d 0) were 50.2 and 49.6%,

respectively.

There was a decrease in percent of cells proliferating for uterine and oviduct

epithelial cells as the number of cells increased (Table 2). There was also an inverse

relationship between the number of cells and percent of cells proliferating. There were

no significant differences in the percent of cells proliferating between treatment groups

for any of the days in culture examined for both uterine and oviduct epithelial cells.

Those treatments with fewer cells at the end of the incubation period had slightly

higher percent of cells proliferating (Trt A, C and E). Epithelial cells incubated in Trt

B continued to increase in cell numbers even though percent of cells proliferating

indicated a resting state of development.

Oviduct epithelial cells gave evidence of growing more rapidly than uterine

epithelial cells. Therefore, all treatment groups were combined and growth of uterine

cells were compared to oviduct epithelial cells (Figure 8). There were no significant

differences between uterine and oviductal cells during the first 2 days of incubation.

However, there was a significant increase (PC .005) in the number of oviductal cells

compared with uterine cells for d 4, 6 and 8 of incubation.

64

inOx

jn*q3O

cuXIE3

2 5 r

20

15

10

CMRLMB2

V

TCM

MEM8 HF12

J. X X X81 2 3 4 5 6 7

Num ber of Days in Culture

Figure 5. A growth curve of uterine epithelial cells conductedfor 8 days in different culture media.

65

inO

x*w'm"55

a>x>E=3

5 0

4 5

4 0

3 5

3 0

2 5

2015

10

5

lT I | I I I I

0 1 2 3 4 5 6 7 80

V CMRL

MB2

MEM

HF12TCM

Number o f Days in Culture

Figure 6. A growth curve of oviduct epithelial cells conductedfor 8 days in different culture media.

66

64

48

32

Side SC (4)

16

Total E vents = 9835

Q uad ran ts a t [19,50]

1) 0 = 0.0%II) 0 = 0.0%

III) 5876 = 59.7%IV) 3959 = 40.3%

16 32Red Flu (3)

48 64

Figure 7. An example of flow cytometiy analysis of the cell cycle. Total events are the number of cells analyzed with individual quadrants gated based on distinct cell populations. Quadrant m indicates the percent of the cell population in a resting state whereas Quadrant IV indicates a proliferative state of the cell cycle.

67

Oviduct2 5

20o

U terusx

tn"a>O«*-o1_a)s iE3

2 3 4 - 5 6 7 810Number o f Days in Culture

Figure 8. Growth rates of uterine and oviduct epithelial cells for 8 days of incubation. All culture media treatment groups were combined and presented as mean values for uterine and oviductal cells.

68

Table 2. Mean percent of cells proliferating for uterine and oviduct epithelial cells during a 8-day growth curve in different media

Davs in CultureItem d 1 d 2 d 4 d 6 d 8

Trt A (TCM) Uterine Cells Oviductal Cells

37±344±6

36±4 31 ±4

48±4 25 ±4

23±3 23 ±4

24±3*22±4

Trt B (CMRL) Uterine Cells Oviductal Cells

37±340±4

42 ±4 37±3

37±324±4

21 ±3 14±3

15±3,,b15±3

Trt C (MEM) Uterine Cells Oviductal Cells

36±4 34 ±4

33±4 41 ±4

33 ±4 30±4

17±417±2

16±4'*b17±2

Trt D (MB2) Uterine Cells Oviductal Cells

37±336±4

32±530±4

36±531±4

22 ±3 20±4

19±3,,b17±3

Trt E (HF12) Uterine Cells Oviductal Cells

33±5 35 ±6

33 ±5 35 ±6

22±5 28 ±5

16±3 22 ±4

15±3b21±4

*,b Mean number of cells within columns with different superscripts differ (PC .05).

69

Experiment 2.

In this experiment, cells were cultured in CMRL medium to determine

maximum effects of incubation temperature on growth and percent of cells

proliferating for uterine and oviduct epithelial cells. There were no significant

differences between treatment groups for growth of uterine (Figure 9) or oviduct

(Figure 10) epithelial cells. Similar growth patterns were noted for uterine and

oviductal cells in this experiment as obtained for Trt B in Experiment 1. Also, there

were no significant differences in the percent of cells proliferating among temperature

treatment groups (Table 3). Oviductal cells incubated under these conditions exhibited

rapid cell growth and could be maintained for several subpassages without

degeneration of cell quality associated with cell death.

70

■nOx

m"oO

0)XIE3

2 5

20

15

10

39 C

37 C

O

O

1 2 3 4 5 6 7Number o f Days in Culture

8

Figure 9. Growth rates for uterine epithelial cells incubatedfor 8 days at 37°C or 39°C.

71

6 039 C

5 5

5 0 37 C4-5

4 0

3 5

3 0

2 5

20

B_l_

0 1 2 3 4 5 6 7 8Number o f Days in Culture

Figure 10. Growth curves for oviduct epithelial cells incubatedfor 8 days at 37°C or 39°C.

72

Table 3. Mean percent of cells proliferating for uterine and oviduct epithelial cells during a 8-day growth curve at different temperatures

Davs in CultureItem d 1 d 2 d 4 d 6 d 8

Trt A (37 °C) Uterine Cells Oviductal Cells

39±338±6

33 ±4 36±3

19±4 15±3 36±3 21±3

15±315±3

Trt B (39°C) Uterine Cells Oviductal Cells

40±338±3

29±337±3

18±3 16±3 32±3 22±3

16±317±3

73

Discussion

All culture media evaluated in this study adequately supported growth of

uterine and oviduct epithelial cells. Several cell populations were maintained for three

or four subpassages on each medium tested. Also, uterine and oviductal cells were

maintained at 39 °C for three or four subpassages. There were no adverse effects

detected due to culture medium or temperature.

Uterine and oviduct epithelial cells exhibited characteristic growth patterns.

Freshney (51) described cell growth as having a short lag period after seeding,

followed by a log increase in cell numbers for several days followed by a stationary

phase and subsequently a decline phase. Epithelial cells cultured in CMRL (Trt B)

and MB2 (Trt D) medium did not reach a stationary or decline phase in cell numbers

during the culture period examined. Other culture media had reached a stationary

phase of growth following 8 days in incubation. These results indicate that CMRL

and MB2 medium were still maintaining growth after other media were staring to enter

a decline phase. But for uterine cells, MB2 medium seems to combine a selection of

subpopulations as observed by a two step growth. However, the initial seeding density

may have affected subsequent cell growth during the culture period. Those cells

incubated in MB2 medium exhibited low growth rates during the initial days of

incubation. Ouhibi et al. (112) suggested that optimal seeding densities for bovine

oviduct epithelial cells to be 1 x 106 cells/25 cm2 flask when maintained in MB2

medium.

All cells in culture maintained the same general appearance and characteristics

regardless of medium used for culture. However, as oviductal cells incubated in

CMRL (Trt B) reached confluency, growth of fibroblast-like cells were noted in

culture flasks. After 8 days of incubation, the swirl-like morphology of these cells did

not overgrow epithelial cells. Cells originated from the same source cultured in other

media for extended periods of time did not have the same characteristics. Ouhibi et

al. (112) have reported that serotonin added to the culture medium was selective

against fibroblast proliferation. However, in this experiment oviductal cells cultured

in CMRL supplemented with serotonin still resulted in these growth patterns.

Extensive immunofluorescence studies were conducted using cytokeratin antibodies to

determine the true origin of fibroblast-like cells. Immunofluorescence staining

revealed these fibroblast-like cells to be true epithelial cells. Merchant (99) reported

similar characteristics for prostate epithelial cells maintained in vitro. The specific

alterations in cell morphology was termed dysplastic and Merchant (99) further stated

that the morphological alterations in cell characteristics was not a transformation form

epithelial to fibroblastic cell types. Perhaps the rapid cell growth noted with CMRL

medium may induce a more accelerated dysplastic cell state.

Under normal conditions cells enter a proliferative state of the cell cycle and

continue proliferating until a signal is received that growth conditions are sub-optimal.

These sub-optimal conditions result in cells entering an arrested state of growth and

may re-enter the cycle (proliferative) if conditions become favorable (68). In the

present study, the proliferative state consisted of those cells in a mitosis phase, gap 2

phase and a period in which DNA synthesis occurs (S phase). Our original hypothesis

for examining the cell cycle was that different culture conditions may provide a

optimum rate of DNA synthesis (and subsequently protein synthesis) after transcription

and translation at selected times during the culture period that may be beneficial during

embryo co-culture. However, there were no major differences in proliferation rates

during maximal increases in growth of uterine and oviductal epithelial cells.

In summary, all culture conditions examined adequately supported growth of

bovine uterine and oviduct epithelial cells. The maximum growth rates were obtained

when cells were cultured in CMRL and MB2 medium. Higher incubation temperature

routinely used for culture of IVF-derived embryos had no adverse affects on uterine

and oviduct epithelial cell growth and proliferation. Finally, flow cytometry provided

an efficient method to monitor the cell cycle of uterine and oviduct epithelial cells

during in vitro culture. The results provided here show the versatility of these cells

used for co-culture indicating that election of the best medium for embryo co-culture

should be based on the embryos metabolism.

E X PE R IM E N T IV

MORPHOLOGICAL EVALUATION OF BOVINE UTERINE AND OVIDUCT EPITHELIAL CELLS USING

IMAGE ANALYSIS

Introduction

The in vitro culture of early cleavage-stage mammalian embryos has met with

limited success and inconsistent results. Various embryo co-culture systems have been

used to enhance embryo development during in vitro culture. Cell types used to

increase embryonic development during co-culture include uterine fibroblasts

(86,87,163), trophoblastic vesicles (71,73,119) and uterine epithelial cells (65). In

addition, oviduct epithelial cells have been used for the in vitro co-culture of early-

stage cattle (37,38,41), goat (121), pig (156) and sheep embryos (126,127,128). In

each instance, the co-culture system used has proven to be effective in supporting

development of embryos through the characteristic in vitro developmental block stages

(124). These findings suggest the existence of unidentified factor(s) produced by these

cells that have embryotropic properties, perhaps via the secretion of specific peptides

and/or proteins (124). Furthermore, researchers now suggest that metabolism of

transport epithelium (113) and maintenance of cell polarity (116) are important during

embryo development in vitro.

Although various experiments have used epithelial monolayers for co-culture

of early stage embryos, these studies have not addressed possible differences in

embryotropic factor(s) produced by cells of different morphological qualities. The

objective of this study was to establish selection criteria for morphological assessment

of cell quality using a computer image analysis system. The development of a reliable

system to monitor cell quality in vitro may allow selection of the most competent

76

monolayers for embryo co-culture experiments.

77


Experimental Animals. Reproductive tracts were collected from five cyclic

cows immediately following slaughter. Tracts were placed in phosphate-buffered

saline without Ca2+ and Mg2+ (PBS) containing 200 units penicillin, 200 ng

streptomycin and .25 ng Amphotericin-B/ml on ice and transported to the laboratory.

Isolation of Epithelial Cells. Tissue Culture Medium-199 supplemented with

10% fetal bovine serum, 100 units penicillin, 100 ng streptomycin and 10 /zg

serotonin/ml of culture medium (TCM-199) was used throughout the study.

Uterine and oviductal cells were isolated and cultured with modifications of the

procedures previously reported by this laboratory (146,148). Briefly, uterine horns

and oviducts were removed, then trimmed free of mesentery tissue. Uterine horn and

oviduct sections were flushed with 5 ml of warm .25% trypsin then filled with fresh

trypsin solution, clamped and incubated at 37°C for 45 min (Figure 4.1). The trypsin

solution was recovered and tissues flushed a second time with fresh .25 % trypsin. The

luminal surface of uterine horns and oviducts were gently scraped once with a sterile

glass slide. Epithelial clusters were separated by repeated pipetting and the trypsin

solution inactivated by the addition of TCM-199 (1:2 v/v).

Harvested cells were then layered on Percoll gradients as previously described

(112). Following centrifugation, a small portion of harvested cells was assigned to

electron microscopy and remaining cells seeded in 25 cm2 tissue culture flasks with

5 ml of TCM-199. All cultures were maintained at 37°C in an atmosphere of 95%

air and 5% C02 and culture medium replaced at 48-h intervals. Subculture of

confluent monolayers were conducted by trypsinization with .05% trypsin. Culture

Epithelial Cell Preparation

Oviduct Inflated with .25% trypsin and Incubated 45 mln.

Cellscollected

Cells allowed to monolayerOviduct flushed with .25% trypsin C>

6

25 cm 1 flask

m onolayer trypslnlzed

W ashed 3 x resuspended in TCM-199

Figure 11. An example of procedures used for harvesting oviduct epithelial cells. The same procedure was used to isolate uterine epithelial cells.

79

flasks were incubated at 37°C for 5 to 10 min until cells became rounded in shape and

detached from the bottom of the culture vessel. Cells were then centrifuged, the

supernatant removed, and the cellular pellet resuspended in fresh TCM-199 and seeded

in new culture flasks at a 1:2 ratio.

Morphological Assessments and Optical Density. At designated subpassages,

confluent 25 cm2 flasks of uterine (n = 8) and oviduct (n = 13) epithelial cells were

fixed and stained with Giemsa stain to assess cell quality (51). Morphological

assessment of cell integrity and quality and optical density readings were made on cells

stained with Giemsa during primary culture (n = 6) and following the first (n — 6)

and third (n = 9) passages at confluency. For visual morphological assessment, cells

were assigned quality scores ranging from 1 to 3 (1 = good to 3 = poor cell quality).

Morphological scores were based on the presence of vacuoles, with a score of 1

having 10% or less of the culture surface containing visible vacuoles, 2 with 10% to

50% of the culture surface containing vacuoles and 3 with greater than 50% of the

culture surface containing vacuoles. All morphological evaluations were carried out

blind without regard to tissue type or passage. The formation of vacuoles within a

culture vessel has been associated with the onset of cell degeneration, which differed

from the presence of lipid inclusions (166).

Optical density readings were determined using a silicon-intensified target

camera and a computer based image analysis system (Micromeasure Version 3.0;

Analytical Imaging Concepts). All evaluations were conducted concurrently with light

source settings of 74 (lower threshold) and 255 (upper threshold).

Election Microscopy. For scanning electron microscopy, uterine and oviductal

cells were seeded on microporous membrane inserts in a 24-well Transwell9 tissue

culture plate (Costar). At confluency, monolayers were rinsed with PBS, fixed in 3%

80

glutaraldehyde in sodium-cacodylate buffer (.1 M, pH 7.4) for 1 h, washed twice in

buffer, post-fixed in 1 % osmium tetroxide in sodium-cacodylate buffer (. 1M, pH 7.4)

and dehydrated in a dilution gradient of ethanol: water (30, 50, 70, 80, 90 and 100%

ethanol). Cells were critical-point dried, then coated with gold-palladium and

evaluated with a scanning electron microscope (Cambridge Steieoscan 150).

Statistical Analysis. A total of five optical density readings for 100 by 100 pm

regions within each flask of cells w oe determined and analyzed using analysis of

variance. Main effects in the model included cell type (uterine or oviductal), passage

and cell quality score. Significant differences between least squares means were tested

using predicted differences.

Results

Optical Density. Cell type (uterine or oviductal) did not significantly (P > .05)

influence optical density readings. Therefore, for further comparisons, mean values

represent combined uterine and oviductal cell populations. In addition, there were no

significant (P> .05) differences between sample replicates, indicating that repeatable

measurements were obtained (Figure 12).

Comparisons were made between cells during primary culture and following

the first and third subpassages to determine optical density readings following repeated

subpassage. There was a significant increase (PC .05) in optical density following

each passage (Figure 13). Mean (±SE) optical density readings for combined uterine

and oviductal cell populations during primary culture and following the first and third

passage were 139.7±4.1, 154.5±3.6and 173.3±3.2, respectively.

Optical density readings were then compared with morphological evaluations

of cell quality. In this case, optical density was significantly (PC .01) influenced by

81

cell quality. There was an increase in optical density as cell quality scores increased.

The mean (±SE) optical density for good (1), fair (2) and poor (3) quality cells were

137.0±3.6, 159.7±3.4 and 170.8±3.9, respectively (Figure 14). An example of

good, fair and poor quality cells are shown in Figure IS.

Electron Microscopy. Jn this study, there were no noted differences in

morphology of uterine and oviductal epithelial cells when micrographs were evaluated.

Scanning electron microscopy revealed numerous bundles of cilia present on the cell

surface of uterine and oviduct cells during primary culture. Oviductal cells also

contained more dense patches of cilia present on the cell surface when compared with

uterine cells (Figure 16). However, only microvilli were observed on the uterine and

oviductal cell surface following the first passage. Furthermore, results from electron

microscopy in this study did verify the visual classification of cell quality.

Opt

ical

D

ensi

ty

82

170 r

160

150

140

130

120

1 1 0

1002 3 4

S a m p le R ep l ica tes

Figure 12. Mean optical density readings for uterine and oviductal cells combined for the five sample replicates.

83

180 r

■S''tocoo"ooQ .O

_ a ,b ,c (P < .0 1 )

100 LPrim ary First

Cell P a s s a g e

c

Third

Figure 13. Mean optical density readings for uterine and oviductal cells combined during primary culture and following the first and third subpassages.

84

2*coca>ooa

CLo

180 i-

-| 7 Q _ a ,b ,c (P < .0 5 )

160 -

150

140 -

130 -

120 -

110

100 LGood Fair Poor

M orphologica l Cell Quality

Figure 14. Mean optical density readings of good, fair and poor quality cells for uterine and oviductal cells combined.

85

Figure 15. Examples of good (a) and poor (b) quality bovine oviductal cells stained with Giemsa stain following in vitro culture (20 X ).

Figure 16. Scanning electron micrograph of oviductal (a) and uterine (b) epithelial cells during primary culture (oviductal cells=20,000 X and uterine cells =20,000 X ).

87

Discussion

The morphological characteristics of uterine and oviduct epithelial cells noted

visually and with electron microscopy in this study were similar to those described in

the reports of others (74,78,112). Furthermore, basic uterine and oviductal epithelial

cell characteristics were maintained throughout the culture period.

A increase in optical density readings became evident following repeated

subpassages and was noted in both uterine and oviductal cells. Changes in epithelial

cells from a tight polygonal monolayer to a more flattened appearance may account

for the increasing density readings following subsequent subpassages. Although

bovine uterine and oviduct cells can be maintained in vitro for nine to 13 subpassages,

there are changes in cell shape noted following three subpassages (112,146). The

increasing optical density readings following subpassage may only be a reflection of

cell shape. This suggestion is further supported by reports of subpassaged oviductal

monolayers effectively supporting embryonic development compared with monolayers

of primary cultures (121). In addition, oviduct epithelial monolayers maintained high

protein secretion rates during extended in vitro culture following repeated passages

(113).

The increased optical density readings noted for poor quality cells were

associated with the high degree of vacuolation that resulted in a lower incorporation

of Giemsa stain. In contrast, good quality cells had increased quantities of stain

incorporated into the cellular material resulting in lower optical density readings. The

formation of vacuoles that coincides with cell degeneration may not be a result of

trypsinization (112) but rather a reflection of individual animal variation (146).

The results reported in this study provide a basis for continued investigations

on cell quality using image analysis. The use of a computer-based method of cell

evaluation may allow researchers to fully investigate changes in cellular structure

during in vitro culture. Although oviduct epithelial cell cultures are proven to

stimulate early embryonic development of farm animal embryos in vitro, there are

limited studies investigating sources of variation in cells used for embryo co-culture.

The investigation of factors that influence monolayers during co-culture may reveal an

optimal epithelial cell embryo co-culture system.

E X PER IM EN T V

IN VTTRO-MATURATION. IN VITRO-FERTILIZATION AND CLEAVAGE OF BOVINE OOCYTES INCUBATED

IN DIFFERENT CULTURE MEDIA

Introduction

During the last decade, there has been increased research interest in new

embryo biotechnology procedures (e.g. in vitro fertilization, nuclear transfer, gene

insertion). In recent years, research has focused on in vitro fertilization of oocytes to

supply supplemental embryos for research at low cost. This increased interest in in

vitro fertilization of bovine oocytes has prompted investigations aimed at enhancing

in vitro maturation, fertilization and subsequent development (6,7,19,24,54,90,92).

Aspects of in vitro fertilization for mammalian oocytes can be evaluated in several

excellent review articles (7,91,167).

Various in vitro fertilization techniques for bovine oocytes have been previously

reported by different laboratories for in vivo matured (24,139) and in vitro matured

oocytes (94,95,115,140,169). Although in vitro fertilization methodologies have been

improved in recent years, development beyond the bovine eight- to 16-cell in vitro

developmental block stage (41) has met with limited success. The use of oviductal

cells (41,53,95) and cumulus cell (67,172) co-culture systems have provided adequate

development of in vfrro-fertilized bovine oocytes to morulae and blastocyst stages.

Efforts are now being made to evaluate various culture media on the in vitro

fertilization efficiency of bovine oocytes. Most reports to date have focused on the

addition of hormones to the medium or using granulosa/cumulus cells for maturation,

fertilization and early cleavage of cattle oocytes. In a recent report, van de Sandt et

al. (153) evaluated eight different culture media for maturation, fertilization and

89

90

subsequent development of mouse oocytes. In this study, differences were reported

among treatment media for pre- and post-implantation development. Furthermore, it

was proposed that improved embryonic development following oocyte maturation

could be influenced by oocyte maturation medium.

Recently, researchers in this laboratory have reported that in vitro incubation

of bovine uterine and oviduct epithelial cells in different culture media and

temperatures resulted in varied responses in cell growth rates (147,149). In addition,

cells incubated in CMRL medium resulted in cell morphological alterations following

several subpassages. Monolayers of epithelial cells would begin to exhibit fibroblastic-

like characteristics. However, cytokeratin staining revealed the origin to be true

epithelial cells.

The objective of this study was to evaluate culture media previously used in

bovine uterine and oviduct epithelial cell culture experiments and to compare these

media for in vitro fertilization and development of cattle follicular oocytes.


Recovery of Oocytes. Ovaries were collected from heifers at a local abattoir

and transported to the laboratory in Dulbecco’s phosphate-buffered saline (DPBS)

(Gibco) at £25 °C. Oocytes were aspirated from 3 to 8 mm diameter follicles using

a 20 gauge needle and a 5 ml syringe. Recovered cumulus-oocyte complexes were

transferred into 15 ml conical centrifuge tubes and allowed to settle («5 min).

Supernatant fluid was then aspirated from tubes and remaining oocytes were washed

three times with DPBS with 10% fetal bovine serum (FBS).

Oocyte Maturation and Fertilization. Standard procedures for in vitro

maturation and fertilization of bovine oocytes are those developed by this laboratory

91

(172). Oocytes with intact, unexpanded cumulus oophorous (90) were randomly

distributed into four-well plates (Nunc) containing culture treatment medium. Oocytes

were incubated for 24 to 26 h at 39°C and 5% C02 in Tissue Culture Medium-199

(TCM) (Gibco), CMRL-1066 (CMRL) (Gibco) or Menezo’s B2 medium (MB2; API

Biomerioux) all supplemented with 10% FBS. Following the maturation period,

oocytes were washed with B-O medium (23) supplemented with 20 mg/ml bovine

serum albumin (BSA; Sigma) and transferred to 30 /d drops. Two units of frozen-

thawed semen was washed twice in B-O medium with 10 mM caffeine sodium

benzoate. Sperm cells were exposed to .1 /iM Ca2+ ionophore A23187 (Sigma) for

1 min to aid in capacitation then placed in microdrops of B-O medium containing

oocytes. Oocytes (20 to 25) and capacitated sperm cells were incubated for 5 to 6 h

in B-O medium with BSA containing l.S X 106 motile sperm/ml. Following the

fertilization interval, oocytes were washed in treatment medium and transferred into

four-well plates then incubated for an additional 48 h. Following the 48-h post

insemination interval, cumulus cells attached to oocytes were removed by gently

teasing with a 27 gauge needle. Oocytes were evaluated with a Nikon inverted

microscope (40-100 X ) to assess cleavage rates. Cleavage was defined as those

oocytes completing one or more cell divisions.

Fixation of Oocvtes. Oocytes were fixed and stained to determine fertilization

rates 18 h following insemination. Oocytes were mounted on glass slides and fixed

in acetic acid:ethanol (1:3) for 48 h, stained with 1% orcein in acetic acid and

examined for fertilization using a phase-contrast and Nomarski interface-contrast

microscope.

Oviductal Cell Culture. Bovine oviduct epithelial cells were isolated from

oviducts of heifers obtained at slaughter and maintained in vitro as previously reported

92

(146,148). Isolated epithelial cells were incubated in treatment media (TCM, CMRL

and MB2) and were used for co-culture following the first subpassage. Oviductal cells

were isolated, pooled together then cultured in respective treatment medium until

initiation of co-culture experiments. All media were supplemented with 10% FBS,

100 units penicillin and 100 /ig streptomycin/ml of treatment medium. Cells were

seeded in 25 cm2 tissue culture flasks (Costar) with 5 ml of TCM. All cultures were

maintained at 37°C in an atmosphere of 95% air and 5% C02 and culture medium

changed at 48-h intervals. Cells were seeded in 24-well tissue culture plates (Costar)

at a concentration of 1.0 x 10s viable cells/ml 48 h prior to co-culture. All wells had

reached ®90% confluency prior to initiation of co-culture experiments.

Embryo Co-culture. Two- to six-cell embryos (n = 180) obtained 48 h

following insemination from each treatment media (n = 60/medium) were randomly

allotted to respective treatment medium alone (n = 30/treatment) or co-cultured with

oviductal epithelial cells (n = 30/treatment). All cultures were maintained at 39°C

and 5% C02 with medium replaced at 48-h intervals. Embryos were co-cultured for

6 days (morulae stage of development) following in vitro fertilization.

Statistical Analysis. The proportion of oocytes that were fertilized and

completed at least one cell division 48 h following insemination were statistical

evaluated by the Chi-square test. In addition, treatment comparisons among groups

of embryos incubated in medium alone or with oviduct epithelial cells were compared

by the Chi-square test.

Results

The total number of fertilized oocytes consisted of those with two or more

pronuclei and/or with penetrated sperm. Those oocytes with only two pronuclei

93

present were further characterized (Table 4). There were no significant differences

(P> .05) between culture medium treatment groups for the total number of fertilized

oocytes. In addition, oocytes incubated in MB2 had significantly (P < .05) more

oocytes with only two pronuclei present compared with TCM medium. However,

there were no significant (P>.05) differences between treatment groups for the

number of oocytes completing at least one cell division 48 h following fertilization.

When oocytes were evaluated to determine cleavage rates, development stages*

of cleaved embryos were noted to evaluate if there were medium treatment differences

during early cell divisions (Table 5). There were significantly (P < .05) more two-cell

embryos in the MB2 treatment compared with TCM and CMKL treatments. Also,

there were more (P < .05) embryos £8 cells in the CMRL treatment compared with

MB2 medium.

The number of two- to six-cell embryos available for culture experiments were

75, 66 and 81 for TCM, CMRL and MB2 media, respectively. A total of 60

embryos/medium were randomly selected and further divided into culture in respective

treatment media alone (n = 30) or with oviduct epithelial cells (n = 30). Early-stage

embryos (n = 180) were cultured in vitro in respective treatment media or with

oviductal epithelial cells for 6 days following fertilization in hopes of enhancing

medium treatment effects (Table 6). However, no significant differences (P>.05)

were noted between treatment groups for embryos developing to the morula stage in

vitro. When culture medium treatment groups were combined, embryos co-cultured

on oviduct epithelial cells had more viable appearing embryos compared with similar

stage embryos maintained in media alone (Figure 17). There were 32% of morula-

stage embryos judged viable following in vitro culture in medium alone. In contrast,

70% of morula-stage embryos co-cultured on oviductal cells were judged viable.

94

Table 4. Fertilization and cleavage rates of bovine oocytes incubated in different media

TreatmentN o./group

Fertilization CleavageNo.

oocytesexamined

No. (%) oocytes_______ fertilized________

Total 2 Pronuclei

No.oocytescultured

No. (56)oocytescleaved

TCM 248 48 37(77%) 22(45 %)b 2 0 0 102(51%)CMRL 248 48 31(65%) 28(58%)b,c 2 0 0 104(52%)MB2 248 44* 33(75%) 30(68%)c 2 0 0 92(46%)

* Four oocytes were lost during fixation procedures.b,c Means within columns with different superscripts differ (P< .05).

95

Table 5. Developmental rates of cleaved oocytes 48 h after fertilization

Cleaved 2 cell 3 cell 4 cell 6 cell £8 cellTrt n n(% ) n (%) n (%) n(% ) n (%)

TCM 102 19(19%)* 8(8% ) 33(32%) 15(15%) 27(27%)*,bCMRL 104 18(17%)* 12(12%) 20(19%) 16(15%) 38(37%)bMB2 92 33(36%)b 15(16%) 24(26%) 9(10%) 11(12%)*

*,b Means within columns with different superscripts differ (PC .05).

96

Table 6. Developmental rates of cleaved embryos following in vitro culture for 6 days

Maturationmedium

No.cleaved Treatment

No.cultured

Morula n (%)

Viable embryos n (%)

TCM 102 TCM 30 12(40%) 3(10%)TCM+BOC* 30 13(43%) 10(33%)

CMRL 104 CMRL 30 14(47%) 5(17%)CMRL+BOC 30 17(57%) 11(37%)

MB2 92 MB2 30 12(40%) 4(13%)MB2+BOC 30 13(43%) 9(30%)

•BOC = bovine oviduct cells

Perc

ent

Viab

le

Em

bryo

s

97

100

90

SO

70

60

50

40

30

20

10

0

a ,b (P < .0 1 )

ControlMedium

b

OviductalCells

Figure 17. Percent embryo viability following in vitro culture in medium alone or co-culture with oviduct epithelial cells.

98

Discussion

Oocytes incubated in MB2 medium had more with two pronuclei present on

evaluation compared with TCM medium, however, this did hot affect the number of

oocytes completing one or more cell divisions. Oocytes maintained in CMRL medium

had greater level of morphological development at 48 h following fertilization

compared with IVF-derived embryos in MB2 medium. However, when embryos were

maintained in vitro for 6 days, development rates to the morula stage for embryos

incubated in CMRL or MB2 medium were similar. Similar in vitro fertilization and

cleavage rates of bovine oocytes have been recently reported (56,134).

Several studies have used various culture media to maintain oocytes or early-

stage bovine embryos in vitro for extended periods of time with adequate success rates.

Adequate in vitro development of early-stage bovine embryos have been reported

following incubation with MB2 (117) or CMRL (38) medium. In a early study, Fukui

et al. (54) evaluated different combinations of culture media, gonadotropins and

steroids supplemented in medium in an attempt to improve success rates for in vitro

maturation of bovine oocytes. The authors noted that the addition of steroids

(estradiol) was more critical for oocyte maturation than culture medium or

gonadotropins. However, the effects of steroids on oocyte maturation differed

depending on the culture medium selected.

More recently, Ellington et al. (38) compared in vitro development of one- to

two-cell bovine embryos incubated in either a simple serum-free (CZB) or complex

(CMRL or Ham’s F-10) medium with bovine oviduct epithelial cells. Similar

development rates (morula or blastocyst stages) were noted between three different

media following co-culture for 5 days. However, embryos incubated in Ham’s F-10

medium had lower embryo viability scores compared with those of CMRL and CZB

99

medium. In addition, CZB supplemented with insulin, transferrin and selenium (ITS)

was compared with CMRL containing serum and ITS. A higher percentage of morula

or blastocyst stage embryos were noted following incubation in CZB for S days in

vitro. Ellington et al. (38) concluded that a simple serum-free medium would support

in vitro development of early-stage bovine embryos following co-culture with oviduct

epithelial cells.

Differences in blastocyst development in mouse (153) and cow (56) embryos

incubated in vitro under different conditions suggest there are still mechanisms in early

embryonic development not fully understood. Although oviduct epithelial cell co

culture improved viability of morula-stage embryos in this study, the mode of action

of co-culture is still debatable. Researchers suggest somatic cell/embryo contact

necessitates beneficial effects (3) while others noted adequate success when using

conditioned medium (41,71). Although some researchers suggest somatic cells secrete

embryotropic factors that stimulate embryo development in vitro (57) others indicate

that a combination of embryotropic factor production and/or removal of toxic

substances occur (see review by 8).

The failure to detect differences between medium treatment groups may be

related to the length of in vitro incubation period. There were no developmental

differences noted between treatment groups even when embryos were co-cultured on

oviduct epithelial cells. However, this may be directly related to the reduction in

number of embryos per group by expanding treatment groups. Although there were

no differences in the number of embryos reaching the morula stage of development,

co-culture with oviductal cells improved the percent viability of embryos.

The present study demonstrated the ability of cell culture media evaluated to

stimulate development of in vzrro-matured, in vzVro-fertilized bovine oocytes to the

morula stage of development. In addition, co-culture with oviduct epithelial cells was

superior to culture in medium alone. Further studies are needed to substantiate

interactions between early embryonic development and in vitro culture conditions.

E X PER IM EN T V I

CO-CULTURING IVF-DERIVED BOVINE EMBRYOS WITH OVIDUCT EPITHELIAL CELLS ORIGINATING FROM DIFFERENT

STAGES OF THE ESTROUS CYCLE

Introduction

Bovine embryos of early cleavage stages do not readily develop beyond the

eight- to 16-cell developmental block under in vitro conditions (167). However,

researchers have recently been able to overcome the in vitro developmental block (42)

with the use of somatic cell co-culture with trophoblastic vesicles (27,71,73,119) and

oviductal tissue (38,41,57,128). Oviductal cells have been reported to enhance

embryo development in sheq> (57,125,126,127,128), goat (121,122,135), pig (156)

and cattle (38,39,97) embryos. In addition, medium harvested from oviductal cells

following 48-h of incubation will also support development of bovine embryos beyond

the eight- to 16-cell stage to the blastocyst and hatched blastocyst stages (41). The use

of oviductal tissue in embryo co-culture systems has received increased attention in an

effort to understand the mechanisms by which "helper" cells interact with embryos to

enhance in vitro development. Such studies have evaluated the use of fresh or

subpassaged monolayers (121,122). The use of subpassaged monolayers may provide

a more uniform and consistent co-culture system for mammalian embryos (113,121).

Other researchers have investigated using fresh and frozen-thawed monolayers for

embryo co-culture (38,127). In addition, researchers have demonstrated that oviductal

cell co-culture enhanced embryonic development when incubated in serum-free

medium. These studies (38,126,128) have provided the basis for noting that

embryotropic effects are directed by oviductal cells themselves and are not in part due

to serum effects.

101

102

Evaluation of specific factors that influence oviductal cells prior to and during

embryo co-culture may provide experimental evidence on the mechanism by which

embryotropic factors regulate embryonic development. These criteria become

increasingly important since the embryotropic properties of oviductal cells are

mediated perhaps via the secretion of specific proteins and/or peptides (see review by

124). The suggestion that the embryotropic effect of oviductal and uterine cells may

be hormonal dependent (128) has been further strengthened by a recent report from

this laboratory (146).

The latter report noted significant decreases in percent viability and attachment

of both uterine and oviduct epithelial cells in vitro following their daily exposure to

raised circulating progesterone in vivo. In this case, it suggested that the stage of

estrous cycle influenced the secretory capacity of both uterine and oviduct epithelial

cells while maintained in vitro. The possible secretion of specific proteins by

oviductal cells has been noted by reports of a estrus-specific protein present in the

ovine oviduct in vivo (144). In addition, Gandolfi et al. (58,59) detected a similar

molecular weight protein from in vitro cultured oviductal cells. The latter authors

further characterized in vitro secreted proteins into two groups: one secreted

throughout the estrous cycle and the second exhibiting cyclic patterns of secretion.

Based on the existing reports in the scientific literature, the objectives of the

present study was two fold. The first was to evaluate the ability of oviduct epithelial

cells isolated between d 4 to 6 or d 14 to 16 of the estrous cycle to support early in

vitro development of IVF-derived bovine embryos. The second objective was to

monitor proteins secreted by oviductal cells used for co-culture of embryos as an

indicator of cell secretory capacity. The evaluation of proteins secreted by oviductal

cell monolayers may provide an indication of the potential embryotropic effect during

103

embryo co-culture.

M aterials and Methods

Experimental Embrvos. Embryos were produced from in vzm?-matured and

in vz/ro-fertilized bovine oocytes with slight modifications of the procedure in use in

this laboratory (172). Briefly, cumulus intact oocytes were matured for 24 h in 500

Ml of Tissue Culture Medium-199 (TCM-199) supplemented with 10% fetal bovine

serum (FBS) (Hyclone Laboratories, Logan, UT) under paraffin oil at 39°C and 5%

C 02. Following maturation, oocytes were transferred to 50 /i 1 drops of B-O medium

(23) supplemented with 20 mg/ml BSA (Sigma). Two units of frozen-thawed semen

from a single dairy bull were washed twice in B-O medium with 10 mM caffeine

sodium benzoate (Sigma) and exposed to .1 pM Ca2+ ionophore A23187 (Sigma) for

1 min. A 50 jil portion of sperm cells containing 1.5 X 106 motile sperm cells/ml

were added to oocytes and incubated for an additional 5 h at 39°C and 5% C02.

Following the insemination interval, oocytes were washed twice and incubated in

TCM-199 with 10% FBS for an additional 42 h.

Isolation and Culture of Oviductal Cells. Bovine oviduct epithelial cells were

isolated with procedures similar to that previously reported at this laboratory (146).

Oviducts were obtained from animals immediately following slaughter and transported

to the laboratory on ice in Ca2+ and Mg2+ free phosphate-buffered saline (Gibco) with

200 units penicillin, 200 /Jg streptomycin and .25 fig Amphotericin-B/ml (PBS-AB).

Oviducts were trimmed free of mesentery tissue and washed three times with fresh

PBS-AB. Epithelial cells from both oviducts were then stripped with a pair of fine

forceps from the isthmic end distally to the infundibulum. Small clusters of ciliated

epithelial cells were recovered and resuspended in TCM-199 with 10% FBS, 100 units

104

penicillin and 100 Mg streptomycin/ml of medium (TCM). Oviductal cells were

washed three times with fresh medium by centrifugation (200 x g) and the cellular

pellet resuspended in 48 ml of TCM. The cell suspension was then incubated in 24-

well plates (1 ml suspension/well) at 39°C and 5% C02. Culture medium was

replaced at 48-h intervals and oviductal cells were used for co-culture experiments

within 48 h of initial seeding.

Experiment 1.

The objective of this experiment was to evaluate the efficiency of bovine

oviduct epithelial cells isolated from different stages of the estrous cycle to support in

vitro development of IVF-derived bovine embryos. Oviduct epithelial cells were

isolated and cultured from cyclic Holstein heifers between d 4 to 6 (n = 3) and d 14

to 16 (n = 3) of the estrous cycle (estrus = d 0). Embryos (two- to four-cell) were

randomly allotted («20 embryos/treatment/replicate) in suspension co-culture with cells

originating from either cycle d 4 to 6 (Trt A) or cycle d 14 to 16 (Trt B) 48 h

following cell isolation. Immediately prior to embryo co-culture, fresh medium

(TCM) was replaced on oviductal cell suspensions. The free-floating clusters of

epithelial cells, in this case, were allowed to remain in the culture wells (Figure 18).

A portion of oviductal cells often attached and would form monolayers («20%) during

the 48 h pre-incubation period. The control culture (Trt C) consisted of similar stage

embryos cultured in TCM. All cultures were maintained at 39°C and 5% C02 in 1

ml of TCM and a portion of culture medium (750 (A) replaced at 48-h intervals.

Embryos were evaluated for development to the morula, blastocyst and hatched

blastocyst stage on d 6, 8 and 10 following in vitro insemination, respectively.

105

Experiment 2.

The objective of this experiment was to monitor proteins secreted by oviduct

epithelial cells isolated from different stages of the bovine estrous cycle as an indicator

of cell secretory capacity. The oviduct epithelial cells were obtained from oviduct

tissue harvested between d 4 to 6 or d 14 to 16 used in Experiment 1. The same

culture conditions were used as in Experiment 1 with the following changes. Oviduct

epithelial cells were allowed to form confluent monolayers and all free-floating cells

and cell clusters were removed prior to the radiolabelling procedure. Oviductal cells

harvested from each female were seeded in eight wells of a 24-well tissue culture

plate.

Confluent monolayers were radiolabelled according to previously reported

procedures (113). Monolayers were washed twice and subsequently incubated for 2

h in methionine-free Dulbecco’s modified Eagle medium (Gibco) (DMEM-MET)

followed by a 90 min incubation period with DMEM-MET containing SO pCi of

[35S]methionine/ml of DMEM-MET. Radiolabelled [35S]methionine (Amersham Ltd)

had a specific activity of >1,000 Ci/mmol. Radiolabelling of monolayers were

conducted in 24-well plates (750 pi volume). Following a 90-min radiolabelling

period, monolayers were washed three times in DMEM and further incubated in TCM-

199 containing 10% FBS for an additional 48 h.

At 24- and 48-h intervals, aliquots of culture medium (500 pi) were harvested,

precipitated in .5 N perchloric acid. Free-labelled methionine was separated from the

bound methionine by centrifugation and the protein pellet counted by liquid

scintillation to determine 35S-methionine incorporation into secreted proteins. In

addition, cells were removed from the culture well, precipitated in .5 N perchloric

acid, centrifuged and the cellular pellet counted for 3SS-methionine incorporation into

106

the cellular proteins by liquid scintillation counting. Control samples consisted of

TCM-199 with 10% FBS alone and TCM-199 with 10% FBS and cells without

radiolabelling. In addition, control wells were treated with radiolabelled methionine

and washed to determine nonspecific activity. The amount of radiolabelled protein

secreted and the cellular protein content of oviductal cells were determined from each

of four wells at 24 and 48 h for each experimental animal across treatment days of the

estrous cycle. The number of samples for secreted proteins was 24 duplicates by cells

isolated from d 4 to 6 and d 14 to 16 of the estrous cycle.

Statistical Analysis. In Experiment 1, the proportion of embryos developing

to morula, blastocysts and hatched blastocyst were compared by the Chi-square

analysis. In Experiment 2, cpm of activity in secreted and cellular proteins were

compared across days of the estrous cycle using a split-plot design with repeated

measures in time (60) and a mixed model with animal as a random effect in the

model. Statistical comparisons of least square means were conducted with predicted

differences.

Results

Experiment 1.

There was a higher percentage (PC .05) of two- to four-cell embryos that

developed to morula stage when co-cultured on oviductal cells compared with that of

culture in medium alone (52% vs 32%, respectively). The percent of embryos

developing to the morula stage were similar for cells isolated between d 4 to 6 or

between d 14 to 16 (Table 7). Furthermore, developmental rates to the blastocyst

stage were higher with oviductal cell co-culture compared with culture in medium

alone (37% vs 9%, respectively) (PC .01). Similar results between d 4 to 6 and d 14

107

to 16 treatments were noted at the hatched blastocyst stage (14% and 15%,

respectively) and were higher than culture in medium (P < .01). In this experiment,

14 to 15% of the embryos hatched from their zona pellucida during co-culture while

no embryos hatched when incubated in medium alone.

Experiment 2.

All control samples exhibited low cpm indicating no nonspecific activity.

Aliquots of radiolabelled medium were harvested following the radiolabelling period

and counted to determine incorporation by monolayers. The percentage of

[35S]methionine incorporation based on total cpm averaged 16% and was similar for

both monolayer treatment groups. There was no differences noted among individual

wells within treatments, subsequently all data were combined for final analysis. The

stage of the estrous cycle from which cells were isolated did not influence the amount

of proteins secreted following 24 or 48 h of incubation in vitro (Figure 19). The mean

cpm (±SE) of secreted proteins following 24 h of incubation from cells isolated

between d 4 to 6 and d 14 to 16 were 128.9±10.3 and 111.2±10.3 x lOVlO6 cells,

respectively. In addition, similar secreted proteins were noted following 48 h of

incubation for d 4 to 6 and d 14 to 16 (116.6±10.3 and 130.8±10.3 x ltfVlO6 cells,

respectively). Epithelial cells isolated from both stages of the estrous cycle maintained

an extraded level of protein secretion following 48 h of in vitro culture.

For radiolabelled methionine incorporation into cellular proteins, stage of the

estrous cycle cells were harvested did not influence the amount of cellular protein

following 24 h of in vitro culture (P>.G5) (Figure 20). There was a significantly

lower (P<.05) amount of cell protein observed for cells isolated from d 14 to 16

compared with d 4 to 6 cells following 24 h of culture. Cells isolated between d 14

to 16 had significantly higher (P< .OS) amounts of cellular protein following 48 h of

culture compared with 24 h of incubation. The lower amount of cellular protein from

cells isolated between d 14 to 16 following 24 h of incubation may be attributed to a

higher turnover rate of protein synthesis and not necessarily a decrease in cell quality.

Figure 18. An example of free-floating clusters of oviduct epithelial cells used during embryo co-culture.

110

Table 7. Development of 2- to 4-cell bovine embryos on oviduct epithelial cells isolated between d 4 to 6 or d 14 to 16

of the bovine estrous cycle (3 replicates) f

Treatment nMorula

n (%)BLST*n(% )

HBLST* n (%)

A (d 4-6) 65 35 (54%)* 25 (39%)c 9 (14%)cB (d 14-16) 65 32 (49%)' 23 (35%)c 10 (15%)cC (control) 65 21 (32%)b 6 (9% )d 0 ( 0%)d

-{-Evaluations on embryo development were conducted on d 6 (Morula), d 8 (BLST) and d 10 (HBLST) following insemination.*BLST= blastocyst; HBLST= hatched blastocysta,b Means within columns with different superscripts differ (Pc.05).c,d Means within columns with different superscripts differ (P< .01).

Ill

m"55o

ID

KJ

Eo.u

1 501 4 01 30120110100

9 08 07 0605 04 03 02010

0d 4 - 6 d 1 4 - 1 6

2 4 Hoursd 4 - 6 d 1 4 - 1 6

4 8 Hours

Figure 19. Proteins secreted by oviduct epithelial cells isolated between d 4 to 6 or d 14 to 16 of the estrous cycle. Evaluations were conducted 24 and 48 h following in vitro culture. Data are mean counts from three replicates.

112

m"5o

co

O

X

EDlO

120

110100

9 0

8 0

7 0

6 0

5 0

4 0

3 0

20

10

0

_ a ,b (P < .0 5 )

d 4 - 6 d 1 4 - 1 6 2 4 Hours

d 4 - 6 d 1 4 - 1 64 8 Hours

Figure 20. Cellular proteins in oviduct epithelial cells isolated between d 4 to 6 and d 14 to 16 of the estrous cycle. Evaluations were conducted 24 and 48 h following in vitro culture. Data are mean counts from three replicates.

113

Discussion

In this study, protein secretions were evaluated to assess secretory patterns of

oviductal cells isolated between d 4 to 6 and d 14 to 16 of the estrous cycle. Proteins

secreted by oviductal cells in vitro in the present study were similar between both

stages of the estrous cycle. Gandolfi et al. (58,59) monitored in vitro protein

secretions by sheep oviductal cells and noted a small group of proteins secreted

throughout the estrous cycle and a larger group exhibiting cyclic variations. It was

further noted that in vitro secreted proteins, at a lower level, were similar to in vivo

secretion patterns.

The level of proteins secreted in the present study may be at a lower level,

which would have prevented detection of differences between stages of the estrous

cycle. However, the level of proteins secreted by oviductal cells in the present study

were similar to those reported for subpassaged oviductal monolayers (113). Oviductal

cells in the present study were maintained in vitro for approximately 7 days to reach

confluency prior to radiolabelling. Gandolfi et al. (58,59) also noted a reduction in

secretion of specific proteins following 3 days of in vitro incubation. In an earlier

study, Salamonsen et al. (136) noted a higher incorporation of radiolabelled

methionine into secreted and cellular proteins in ovariectomized ewes treated with

exogenous estrogen. The higher methionine incorporation into proteins secreted and

in cellular proteins were more pronounced than control ewes and those treated with

progesterone. It was suggested that exogenous treatment with estrogen influenced

incorporation of radiolabelled methionine into proteins and progesterone increased the

secretion of newly synthesized proteins.

The results reported in this study indicate that stage of the estrous cycle that the

oviductal cells are harvested had no overt effect on embryo development in vitro. In

114

addition, co-cultures with oviduct epithelial cells harvested from either early- and late-

luteal stages were superior for culture of bovine embryos over that for medium alone.

These results are in agreement with those recently reported by Eyestone et al. (44)

using oviductal-cell conditioned medium for culturing IVF-derived bovine embryos.

In the latter report, no difference in development of embryos was noted using medium

harvested from oviduct cells of cows either at estrus or in the luteal phase of the

estrous cycle. Furthermore, similar embryo development results have been reported

following in vivo culture within oviducts of sheep in estrus or during the luteal phase

or in an estrous and ovariectomized animals (45,103,164).

Previously, Thibodeaux et al. (146) reported significant differences in viability

and percent cell attachment of both uterine and oviduct epithelial cells harvested from

cattle on different days of the estrous cycle. It was noted that following the first cell

passage, percent cell attachment was similar for cells collected between different stages

of the cycle. In the present study, it appears that once cells begin to attach and grow

along with free-floating cells, both stages of the cycle investigated are capable of

enhancing embryo development in vitro. Based on a previous study (45) and the

results reported herein, indicate oviduct epithelial cells harvested from different stages

of the estrous cycle will enhance early-stage embryo development when incubated in

vitro. Secondly, clusters of free-floating unattached oviductal cells are capable of

stimulating embryo development.

Recently, researchers have demonstrated that oviductal cell co-culture combined

with a simple serum-free culture medium enhanced in vitro development of early-stage

sheep (128) and cattle (37,38) embryos. These studies noted that a serum-free

medium combined with oviductal cell co-culture would stimulate embryonic

development and resulted in transplant pregnancies. These reports further indicate that

115

oviduct cells are secreting embryotropic factors not dependant on the presence of

bovine serum. It has been suggested the embryotropic effects of oviductal cell co

culture may reflect their ability to maintain a required minimal amount of protein

secretion (58). Based on the findings thus far it appears that once cells are placed in

in vitro conditions a minimal amount of protein secretion is maintained resulting in

adequate embryonic development and co-culture of embryos with serum-free medium

does not alter the minimal amounts of proteins secreted.

Although there were no differences noted in developmental rates between

oviductal cells isolated from early- and late-luteal stages, several suggestions can be

made. Due to the efficiency of growth, increased ciliary activity, viability and

attachment, cells isolated from early luteal stages of the estrous cycle would be more

suitable candidates for embryo co-culture experiments. In this study, confluent

monolayers may not be necessary to obtain good embryo development in vitro. Free-

floating clusters of oviduct epithelial cells in primary culture may provide a more in

vivo type culture system for embryo development in vitro. This suggestion was

previously demonstrated with sheep embryos co-cultured with free-floating oviduct

cells (26). Finally, the simultaneous evaluation of embryo development and protein

secretions by monolayers provides an adequate evaluation of embryo co-culture

systems. These criteria may be used in future studies to explain possible variation in

monolayers used for embryo co-culture studies.

CO N CLU SIO N S

For almost a decade there has been a growing controversy on which was the

best type of helper cells to use for optimal embryo co-culture. Recently, positive

success has been reported using epithelial cells of sheep and cattle for embryo co-

culture over that of traditional uterine cell (e.g. fibroblast) co-culture systems.

Furthermore, the possibility exists that uterine and oviduct epithelial cells harvested

from different stages of the bovine estrous cycle may vary in developmental patterns,

cell attachment and secretory ability when used in an embryo co-culture system.

In order to maintain and evaluate epithelial cell activity in an embryo co

culture system, methodology was developed to maintain uterine and oviduct cells in

an epithelial cell state during in vitro incubation. In the first experiment, procedures

were established to isolate and maintain bovine uterine epithelial cells based on

modifications of methodology previously reported for bovine oviductal cells by French

scientists (112). A new method to maintain and culture bovine uterine epithelial cells

was then developed and evaluated in this laboratory. Immunofluorescence staining for

cytokeratin filaments verified that these procedures did maintain both uterine and

oviduct cells in an epithelial cell state during culture experiments.

When uterine and oviduct epithelial cells were harvested from heifers on day

of estrus, between d 4 to 6, d 8 to 10 or d 14 to 16 of the estrous cycle, there was a

difference in patterns of percent cell viability and cell attachment. The highest percent

cell viability and cell attachment resulted when uterine and oviductal cells were

harvested prior to d 6 of the cycle and the lowest percent viability and attachment

resulted when cells were isolated following d 8 of the estrous cycle. The possibility

existed that the secretory capacity of cells during in vitro culture may differ from cells

116

117

isolated on different stages of the estrous cycle. It was proposed that changes in

secretory capacity of these cells may lead to alterations in embryotropic capabilities.

Therefore, IVF-derived bovine co-cultured on uterine and/or oviductal cells harvested

from different stages of the estrous cycle may result in different developmental rates.

In vitro incubation conditions (culture medium) for maintaining uterine and

oviduct epithelial cells in the most favorable in vitro environment was evaluated. In

an experiment, four of the more established culture media were compared with a

standard tissue culture medium (TCM-199) for effects of in vitro cell growth and

proliferation. In addition, in vitro incubation temperatures routinely used for cell

culture experiments (37°C) was compared with incubation temperatures used in IVF

culture experiments (39°C). In summary, CMRL-1066 medium resulted in more

accelerated growth rates for uterine and oviduct epithelial cells but did not influence

the cell cycle when compared with the base culture medium. Also, incubation

temperatures evaluated influenced neither cell growth or the cell cycle.

A method of evaluating uterine and oviduct epithelial cell populations using

image analysis may provide a way to evaluate cell populations used for embryo co

culture. Using photometric measurements, changes in cell quality following repeated

subpassages were consistently identified. Thus, photometric image analysis may be

used in future studies to characterize changes in cell populations before cell monolayer

are used for embryo co-culture.

In uterine and oviduct epithelial cell culture experiments, CMRL-1066provided

a more suitable in vitro environment for uterine and oviductal cell growth. There

fore, CMRL-1066 was compared with Menezo’s B2 medium and the base culture

medium TCM-199 in a bovine IVF culture system. In this study there were no

differences in the media alone used to maintain bovine embryos to the morula stage

118

of development. However, using the three media with oviduct epithelial cells, a

marked improvement in embryo viability resulted following 6 days of in vitro culture,

verifying that epithelial cells are needed in addition to the medium for maximum

development of early-stage bovine embryos.

In an effort to establish if bovine epithelial cells would serve as an efficient

embryo co-culture system, IVF-derived bovine embryos were co-cultured with

oviductal cells harvested between d 4 to 6 or d 14 to 16 of the estrous cycle. In

addition to embryo co-culture, radiolabelled proteins secreted by oviductal cells were

monitored as an indicator of cell secretory capacity. Co-culturing IVF-derived bovine

embryos with oviductal cells isolated between d 4 to 6 or d 14 to 16 resulted in similar

developmental rates to the morula, blastocyst and hatched blastocyst stages.

Furthermore, proteins secreted by oviductal cells were not different between d 4 to 6

or d 14 to 16. Possibly, variability in cows within days of the estrous cycle may

account for there being no difference in embryo developmental rates among different

days of the bovine estrous cycle. The least amounts of animal variability, however,

was noted in cows with epithelial cells isolated between d 4 to 6 of the estrous cycle.

The experiments reported herein suggest that subpassaged monolayer cells

provided a more uniform population of cells than primary cultures for embryo co

culture studies. In addition, the use of subpassaged cells reduced the cell variation

observed during primary cultures as a result of host animal variation. However, co

culture with primary cultures of free-floating clusters of cells provided a more in vivo

type co-culture system for early-stage bovine embryos.

Further studies are needed to fully define the mechanisms by which uterine and

oviduct epithelial cells interact with early-stage embryos to allow development beyond

the eight- to 16-cell development block to the blastocyst stage.

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V IT A

John Kevin Thibodeaux was bom on November 9, 1963 in Lafayette,

Louisiana, the son of Herbert and Peggy Thibodeaux. He was raised on a small farm

located near Church Point, Louisiana and later graduated from Church Point High

School in May of 1981. Following high school graduation he attended the University

of Southwestern Louisiana. He majored in Animal Science and received his Bachelor

of Science degree in December of 1986. Following graduation he worked at the

University of Southwestern Louisiana Beef Farm as manager.

In August of 1987, he enrolled as a graduate student in the Department of

Dairy Science at Louisiana State University and completed the requirements for a

Master of Science degree in August of 1989. On March 20, 1991, his wife Holly S.

Thibodeaux gave birth to a beautiful baby girl named Sydney Paige Thibodeaux.

Currently, he is a candidate for the degree of Doctor of Philosophy in

Reproductive Physiology in the Department of Dairy Science under the direction of

Dr. Joseph D. Roussel.

133

DOCTORAL EXAMINATION AND DISSERTATION REPORT

Candidate: John Kevin Thibodeaux

Major Field: Dairy Science - Reproductive Physiology

Title of Dissertation: In V itro C u ltu re o f Bovine U te r in e and O viduct E p i th e l i a lC e lls

Approved:

M ajor P rofessor and C hairm an

D ean of the G raduate School

EXAM INING CO M M ITTEE:

Date of Examination:

August 26, 1991

Documents

In Vitro Culture of Bovine Uterine and Oviduct Epithelial