MOLECULAR REPRODUCTION AND DEVELOPMENT 72:88–97 (2005)

Embryotropic Effect of Insulin-Like Growth Factor(IGF)-I and Its Receptor on Development of PorcinePreimplantation Embryos Produced by In VitroFertilization and Somatic Cell Nuclear TransferSUE KIM,1 GAB SANG LEE,1 SO HYUN LEE,1 HYE SOO KIM,1 YEON WOO JEONG,1 JI HYE KIM,1

SUNG KEUN KANG,1,2* BYUNG CHUN LEE,1,2 AND WOO SUK HWANG1,2,3

1Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University,Seoul, Korea2The Xenotransplantation Research Center, Seoul National University Hospital, Seoul, Korea3School of Agricultural Biotechnology, Seoul National University, Seoul, Korea

ABSTRACT Insulin-like growth factor (IGF)-I isa receptor-mediated autocrine/paracrine growth/survi-val factor for mammalian embryo development. Thepresent study investigated the temporal expression andregulation of porcine IGF-I receptor (IGF-IR) mRNA andthe role of IGF-I on development of porcine in vitrofertilized (IVF) and somatic cell nuclear transfer (SCNT)embryos. As assessed by semi-quantitative reversetranscription-polymerase chain reaction (RT-PCR), thelevel of IGF-IR mRNA expression was high in unferti-lized oocytes, 2-cell and 4-cell embryos and graduallydecreased in 8-cell embryos, morulae, and blastocystsin both IVF and SCNT series. The IVF or SCNT embryoswere cultured with 0, 1, 10, 50, or 100 ng/ml IGF-I for168 hr. Supplementing with 50 ng/ml IGF-I increasedblastocyst formation and the number of cells in innercell masses (ICMs) in both IVF and SCNT embryos. In asecond experiment, more blastocysts were obtainedwhen IVF or SCNT embryos were cultured for the first48 hr or for the entire 168 hr with 50 ng/ml IGF-Icompared to culturing without IGF-I for 48 hr or withIGF-I for the last 120 hr or without IGF-I for the entire168 hr. Treating IVF or SCNT embryos with 50 ng/mlIGF-I significantly up-regulated IGF-IR mRNA com-pared to untreated control embryos. In conclusion, thepresent study demonstrated that IGF-IR mRNA isexpressed in porcine IVF and SCNT embryos, and thatIGF-I improved the developmental competence of IVFand SCNT embryos through its specific receptors. Mol.Reprod. Dev. 72: 88–97, 2005.� 2005Wiley-Liss, Inc.

Key Words: IGF-I; IGF-I receptor; porcine; IVF;SCNT

INTRODUCTION

Somatic cell nuclear transfer (SCNT) technology inpigs has limitless potential in biomedical research andits applications, such asmaking a bioreactor for produc-ing therapeutic proteins or to provide organ-donationsfor humans (Lai et al., 2002). Because both in vitro

manipulation and gene targeting embryo technologiesare indispensable for the realization of such achieve-ments, many research groups have attempted to pro-duce large numbers of viable reconstructed SCNTembryos. Among innumerable components, the in vitroembryo culture protocol is the most critical factorfor successful production of cloned transgenic pigs(Betthauser et al., 2000; Onishi et al., 2000; Polejaevaet al., 2000), as well as for supporting many biologicalevents such as embryonic genomic activation (De Sousaet al., 1998) and inner cell mass (ICM) apposition (De laFuente and King, 1997; Machaty et al., 1998).

Membrane receptors are key proteins that mediatebiological effects in an autocrine and/or paracrinemanner (Eckert and Niemann, 1998; Kawamura et al.,2003; Lee et al., 2004). Numerous growth factors thatfunction via these receptors are secreted from theuterine wall adjacent to developing embryos (Kaye andHarvey, 1995). In particular, the insulin-like growthfactor (IGF) family is known to affect mammalianembryo development. In mice, IGF-I and IGF-I receptor(IGF-IR) mutations resulted in developmental re-tardation and neonatal lethality (Baker et al., 1993).The IGF-I stimulates oocyte maturation and embryodevelopment in various species (Narula et al., 1996;Herrler et al., 1998;Moreira et al., 2002; Lin et al., 2003)including humans (Lighten et al., 1998). In addition,ICM cell number was increased in buffalo and humanblastocysts cultured in medium supplemented withIGF-I (Narula et al., 1996; Lighten et al., 1998). In con-trast to humans,mice, and cattle, few studieswith IGF-I

� 2005 WILEY-LISS, INC.

Grant sponsor: Korean Ministry of Science and Technology (Biodis-covery).

*Correspondence to: Sung Keun Kang, DVM, PhD, Department ofTheriogenology and Biotechnology, College of Veterinary Medicine,Seoul National University, Seoul 151-742, Korea.E-mail: kangsnu@snu.ac.kr

Received 26 February 2005; Accepted 20 April 2005Published online 12 June 2005 in Wiley InterScience(www.interscience.wiley.com).DOI 10.1002/mrd.20327

have been reported on pig embryo development, and noattempt has beenmade to examine the effect of IGF-I onthe cell number of porcine blastocysts, which is a criticalparameter for assessing the likelihood of successfulpregnancy after embryo transfer (Enright et al., 2000).Accordingly, the present study investigated (1) the

temporal expression pattern of IGF-IR mRNA, (2) theeffect of IGF-I on development of porcine preimplanta-tionembryosderived frominvitro fertilization (IVF) andSCNT, and (3) the regulation of IGF-IR mRNA expres-sion by IGF-I treatment in IVF and SCNT blastocysts.

MATERIALS AND METHODS

Chemicals

All chemicals were purchased from Sigma-AldrichCorp. (St. Louis, MO) unless otherwise stated.

Oocyte Collection and Culture ofCumulus–Oocytes Complexes

Porcine ovaries were obtained from prepubertal giltsat a local slaughterhouse and transported to thelaboratory at 30–358C in 0.9% (w/v) NaCl containing75 mg/ml penicillin G and 50 mg/ml streptomycin sulfate.Follicular fluid and cumulus–oocyte complexes (COCs)from 3 to 6mm follicles were aspirated using a 18-gaugeneedle attached to a 5 ml disposable syringe. The COCswere then washed three times in HEPES-bufferedNorth Carolina State University (NCSU)-23 mediumbefore being transferred to tissue culture medium(TCM)-199 supplemented with 10 ng/ml epidermalgrowth factor (EGF), 4 IU/ml eCG (Intervet, Boxmeer,Holland), 4 IU/ml hCG (Intervet), and 10% (v/v) porcinefollicular fluid (pFF). The pFF was aspirated from 3 to7 mm follicles of the prepubertal gilt ovaries. Aftercentrifugation at 300g for 30 min, supernatants werecollected and filtered sequentially through 1.2 and0.45 mm syringe filters (Gelman Sciences, Ann Arbor,MI). Prepared pFFwas then stored at�208Cuntil used.Agroupof 50COCswas cultured in500ml of themodifiedTCM-199 medium in a 4-well dish (Nunc, Roskilde,Denmark) at 398C inahumidifiedatmosphere of 5%CO2

and 95% air. After culturing for 22 hr, COCs werewashed three times and cultured in eCG- and hCG-freeNCSU-23 medium at 398C under 5% CO2 in humidifiedair for another 22 hr. At the end of maturation culture,COCs were transferred to HEPES-buffered NCSU-23medium containing 0.5 mg/ml hyaluronidase for 1 minand the cumulus cells were subsequently removed bygentle pipetting.

In Vitro Fertilization

A straw of frozen boar semen was thawed at 398C for1 min in a water bath and washed in 10 ml PBS bycentrifuging twice at 300g for 3 min. After washing, thesperm pellet was resuspended in modified Tris-bufferedmedium(mTBM) containing113.1mMNaCl, 3mMKCl,7.5mMCaCl2 � 2H2O, 20mMTris, 11mMglucose, 5mMsodium pyruvate, and 0.2% (w/v) BSA. After 44 hr ofin vitro maturation (IVM), oocytes freed from cumulus

cells were washed three times in mTBM that was pre-equilibrated for 18 hr at 398C under 5% CO2. Afterwashing, groups of 20 oocyteswere randomly placed into45 ml droplets of mTBM medium covered with pre-warmed mineral oil. After appropriate dilution, 5 ml ofsperm suspension was added to a 45 ml drop of ferti-lization medium (mTBM) to give a final sperm concen-tration of 2�106 sperm/ml.Gameteswere culturedwithspermatozoa for 6hr at 398C inahumidified atmosphereof 5% CO2 and 95% air. After 6 hr, gametes wereremoved from the fertilization drops, washed three orfour times in HEPES-buffered NCSU-23 medium andcultured in 25 ml microdrops (10 oocytes per drop) ofNCSU-23 supplemented with 4 mg/ml fatty acid-freeBSA (A-6003, lot number: 102K7400) covered withmineral oil incubated under 5% O2, 5% CO2, and 90%N2 at 398C for 168hr. Cleavage andblastocyst formationwere evaluated under a stereomicroscope at 48 and168 hr after insemination, respectively.

Preparation of Porcine FetalFibroblasts for SCNT

Fetal fibroblasts were isolated from fetuses at Day 40of gestation. The head was removed using iris scissorsand soft tissues such as liver and intestine were dis-carded by scooping out with two watchmaker’s forceps.After washing twice with PBS (Life Technologies,Rockville, MD), the carcass was minced with a surgicalblade on a 100-mm culture dish (Becton Dickinson,Lincoln Park, NJ). The minced fetal tissues weredissociated in DMEM (Life Technologies) supplement-ed with 0.1% (w/v) trypsin and 1 mM EDTA (LifeTechnologies) for 1–2 hr. Trypsinized cells werewashedonce by centrifugation at 300g for 10 min and subse-quently seeded into 100 mm plastic culture dishes.Seeded cells were subsequently cultured for 6–8 daysin DMEM supplemented with 10% (v/v) FBS (LifeTechnologies), 1 mM sodium pyruvate, 1% (v/v) non-essential amino acids (Life Technologies), and 10 mg/mlpenicillin-streptomycin solution at 398C in a humidifiedatmosphere of 5% CO2 and 95% air. After removal ofunattached clumps of cells or explants, attached cellswere further cultured until confluent, subcultured atintervals of 5–7 days by trypsinization for 5 min using0.1% trypsin and 0.02% EDTA and stored after two pas-sages in freezing medium in liquid nitrogen at �1968C.The freezingmediumconsisted of 80% (v/v)DMEM,10%(v/v) DMSO, and 10% (v/v) FBS. Prior to SCNT, cellswere thawed and subsequently cultured for 3–4 daysuntil 80% confluence and then subjected to serum star-vation. For serum starvation, cells were cultured for3 days in DMEM supplemented with 0.5% FBS andindividual cells were retrieved from the monolayer bytrypsinization for 30 sec and subsequently used forSCNT.

SCNT and Embryo Culture

After 42 hr of IVM, a cumulus-free oocyte was heldwith a holding micropipette and the zona pellucida (ZP)waspartially dissectedwithafine glass needle tomakea

EMBRYOTROPIC ROLE OF IGF-I ON PORCINE EMBRYOS 89

slit near the first polar body. The first polar body andadjacent cytoplasm presumably containing the meta-phase-II chromosomes were extruded by squeezingoocytes with the same needle. Oocytes were enucleat-ed in HEPES-buffered NCSU-23 supplemented with0.3% BSA and 7.5 mg/ml cytochalasin B. After enuclea-tion, oocytes were stained with 5 mg/ml bisbenzimide(Hoechst 33342) for 5 min and observed under aninverted microscope equipped with epifluorescence.Oocytes still containing DNA were excluded from theexperiments. Using a fine pipette, a trypsinized fetalfibroblast with smooth cell surface was transferred intothe perivitelline space of an enucleated oocyte. Thecouplets were equilibrated with 0.26Mmannitol solutioncontaining 0.5 mM HEPES, 0.1 mM CaCl2, and MgSO4

for 4 min and transferred to a chamber containing twoelectrodes overlaid with mannitol solution. Coupletswere fused and activated simultaneously with a singleDC pulse of 2.0 kV/cm for 30 msec using a BTX Electro-Cell Manipulator 2001 (BTX, Inc., San Diego, CA).Activated oocytes were washed three times with NCSU-23 supplemented with 4 mg/ml fatty acid-free BSA, andplaced in 25 mlmicrodrops (10 oocytes per drop) ofNCSU-23 undermineral oil and cultured at 398Cunder 5%CO2,5% O2, and 90% N2. The reconstructed embryos werecultured for 168 hr after activation, and cleavage andblastocyst formation were monitored under a stereomi-croscope at 48 and 168 hr, respectively.

Evaluation of Blastocyst Quality

The quality of blastocysts was assessed by differentialstaining of the ICM and the trophectoderm (TE) cellsaccording to a modified method of Hardy et al. (1989).Briefly, the zona pellucida (ZP) of blastocysts on Day 7wasremovedby treatingwith0.25%pronase for2–3minat room temperature followed by washing with NCSU-23medium at least three times. Zona free embryos wereplaced in 15mM trinitrobenzene sulfonic acid at 48C for30minand then exposed to goat anti-dinitrophenyl-BSA(ICN Biomedicals, Inc., Cleveland, OH) for 20 min at398C. After rinsing three times with NCSU-23, embryoswere incubated for 2 hr in a solution containing 0.01mg/ml of propidium iodide and 15% (v/v) guinea pigcomplement, transferred into absolute alcohol contain-ing bisbenzimide and stored overnight at 48C. Embryoswere then placed in absolute alcohol for at least 1 hr andmounted on a glass slide under a coverslip. The cellnumber was counted under an inverted microscope(Nikon Corp., Tokyo, Japan) equipped with epifluores-cence. ICM cell nuclei labeled with bisbenzimideappeared blue and trophectoderm (TE) labeledwith bothbisbenzimide and propidium iodide appeared pink.

RT-PCR Amplification andQuantification of IGF-IR mRNA

Identical numbers of cells (150 1-cell embryos, 752-cell embryos, 37 4-cell embryos, 9 16-cell embryos,5morulae, and3blastocysts)were collected based on ourtotal cell number counting and a previous report (Hardy

et al., 1989). To amplify IGF-IRmRNA, embryos derivedfrom IVF or SCNT were washed in three changes ofPBS and transferred into 0.2 ml of 4M guanidiumisothiocyanate lysis solution containing 1% b-mercap-toethanol. For a positive control, porcine fetal fibroblastcells were transferred into 1 ml TRIzol Reagent (LifeTechnologies). Total RNA was extracted by thiocyanateextraction and dissolved in 12 ml RNase-free water asdescribed by Szafranska et al. (1995). The concentrationof total RNA was determined by absorbance at 260 nmusing a Beckman DU 600 spectrophotometer (BeckmanInstruments, Jan Ramon, CA). Total RNA was subject-ed to reverse transcription-polymerase chain reaction(RT-PCR).Reverse transcriptionwas carried out at378Cfor 60 min as follows. Individual RT reactions (15 mleach) consisted of 5 mM MgCl2, 1� RT buffer, 2.5 mMoligo(dT), 1 mM dNTP, 50 IU murine leukemia virusreverse transcriptase (Amersham Pharmacia Biotech.,Oakville, Canada) and 8 ml total RNA. In order toamplify the complete coding region of the IGF-IR gene,primers amplifying IGF-IR transcript were designedbased on the published porcine IGF-IR gene coding se-quence (accessionnumber:AB003362). The primers are:sense, 50-AGAAGACGACCATCAACAAC-30 and anti-sense 50-AGAGGAGTTTGATGCTGAGA-30. The cDNA(5 ml) was amplified in a 50 ml PCR reaction containing250 U Ex-Taq polymerase (Takara, Shiga, Japan) andits buffer, 2.5 mM deoxy-NTP, and 50 pmol specificprimers. The PCR amplification was carried out for1 cycle with denaturing at 958C for 15 min, and sub-sequently for 33 cycles with denaturing at 958C for30 sec, annealing at 638C for 30 sec, extension at 728C for60 sec, and a final extension at 728C for 15 min. Ampli-fied PCR products were subjected to Southern blotanalysis. Ten microliters of PCR products were fractio-nated on a 1.0% agarose gel, and stained with ethidiumbromide. The PCR products were transferred to a nylonmembrane and hybridized with a digoxigenin-labeled390 bp cDNA probe for porcine IGF-IR (accessionnumber: AB003362) following the manufacturer’s re-commended procedure (Roche Molecular Biochemicals,Mannheim, Germany). After washing, the membraneswere exposed to Kodak Omat X-ray film (EastmanKodak Co., Rochester, NY). The photographs werescanned and quantified using the Scion Image AnalysisProgram (Scion Corporation, beta 4.02, 2000). Eachtreatment was replicated five times. For sequence anal-ysis, the PCR product was purified from the gel with anagarose gel extraction kit (Qiagen, Hilden, Germany)and was cloned into a pCRTopo cloning vector (Invitro-gen, SanDiego,CA).Thenucleotide sequence of thePCRproducts was analyzed using an automated DNA se-quence analyzer (ABI 3100, Applied Biosystems, FosterCity, CA).

Treatment of Exogenous IGF-I

Human recombinant IGF-I was reconstituted in 0.1Msterile acetic acid to give a stock concentration of 100mg/ml and stored in 10 ml aliquots at �208C. The stocksolution was diluted in NCSU-23 medium to give dif-

90 S. KIM ET AL.

ferent concentrations of IGF-I (1, 10, 50, and 100 ng/ml).Embryos produced by IVF or SCNT were randomlyallocated to each experimental treatment (10 embryos/drop). In order to determine the physiological role ofIGF-I, IVF or SCNT embryoswere cultured inNCSU-23medium supplemented with different concentrationsof IGF-I (0, 1, 10, 50, or 100 ng/ml) for 7 days. Inanother experiment, IVF or SCNT embryos werecultured in NCSU-23 medium supplemented as follows:(1) no supplementation, (2) 50 ng/ml IGF-I for 168 hr,(3) with 50 ng/ml IGF-I for the first 48 hr only, or (4)without IGF-I for the first 48 hr andwith 50 ng/ml IGF-Ifor the last 120 hr. The rates of cleavage and blastocystformationweremonitored at 48and168hr after culture,respectively. The cell numbers and allocation (ICM orTE) in blastocysts were determined at 168 hr.

Regulation of IGF-IR mRNA byExogenous IGF-I Treatment

After culturing embryos for 168 hr with 50 ng/ml ofIGF-I, total RNA was extracted from 10 blastocysts andsubjected to semi-quantitativePCR.Porcine b-actinwasused an internal positive control for RT-PCR ampli-fication. Relative IGF-IR mRNA expression levels wererepresented as the ratio of IGF-IR to b-actin. Theprimers for b-actin were designed based on publishedsequences (accession number: U07786). The primersare: sense, 50-GAGACCTTCAACACGCCG-30 and anti-sense 50-GGAAGGT GGACAGCGAGG-30. The PCRamplification for IGF-IR was performed as describedabove (see ‘‘RT-PCRAmplification andQuantification ofIGF-IR mRNA’’). Ten microliters of PCR products werefractionated on a 1.0% agarose gel, and stained withethidium bromide. The cDNA (5 ml) for b-actin wasamplified under the same conditions as for IGF-IR. Theexperiment was repeated three times with differentbatches of samples. The photographs were scanned andquantified using the Scion Image Analysis Program(Scion Corporation, beta 4.02, 2000). Each treatmentwas replicated five times.

Statistical Analysis

Thedifferences in embryodevelopmentamongexperi-mental groups were analyzed using one-way ANOVAafter arcsine transformation to maintain homogeneityof variance. Post hoc analyses to identify between-groupdifferences were performed using the LSD test. Thesame test was used to determine statistical significancein the cell numbers of blastocysts among experimentalgroups without arcsine transformation. All analyseswere performed using SAS (SAS Institute, version 8.1).Significant differences among the treatments weredetermined where the P value was less than 0.05.

RESULTS

Detection of IGF-IR mRNA inPreimplantation Embryos

In order to quantify the expression level of IGF-IRmRNA during embryo development, semi-quantitative

RT-PCR and Southern blot analysis were performed. Todetermine the conditions under which PCR amplifica-tion for IGF-IRmRNAwas in the logarithmic phase, 5 mlcDNA were amplified using different numbers of PCRcycles (30, 35, and 40). A linear relationship betweenPCR products and amplification cycles was observed(data not shown). Thirty-three cycles were employed forquantification of IGF-IR mRNA. In both IVF and SCNTembryos, the abundance of IGF-IR transcripts wassignificantly higher in oocytes than following stages(Fig. 1). In IVF embryos, the expression level wasdecreased from oocytes to 4-cell in a stage-dependent

Fig. 1. Representative gel photographs showing expression ofinsulin-like growth factor-I receptor (IGF-IR) mRNA by reversetranscription-polymerase chain reaction (RT-PCR). First strandcDNAs from porcine in vitro fertilized (IVF,A) and somatic cell nucleartransfer (SCNT, B) embryos were amplified using one set of PCRprimers derived from the porcine IGF-IR sequence. After confirmingthe identity of PCR products by sequence analysis, amplified productswere scanned and quantified (IVF, A and SCNT, B) using Scion ImageSoftware. Data were represent the mean�SE of five individualexperiment (P< 0.05). M, 1k marker; O, MII oocytes; 2C, 2-cell; 4C,4-cell; 8C, 8-cell; Mo, morulae; Bl, blastocysts.

EMBRYOTROPIC ROLE OF IGF-I ON PORCINE EMBRYOS 91

manner,however, the expressionwas constantduring4-cell to blastocyst. A similar effect was found in SCNTembryos, except that the expression level in 4-cell wassimilar to that of 2-cell.

Effect of Different Concentrations of IGF-Ion Development of IVF and SCNT Embryos

In IVF embryos, as shown in Table 1, a highercleavage rate was observed in medium supplementedwith 50 ng/ml IGF-I than in the other two treatmentgroups (P< 0.05, 71.1% vs. 60.2%–62.5%). More blas-tocysts were obtained with 50 ng/ml IGF-I (30.7% vs.24.3%–28.1%) and more hatched blastocysts wereobtained at 10 (7.4%) or 50 ng/ml IGF-I (8.2%) comparedto the other treatment groups (3.3%–4.5%). Increasedtotal cell number (P<0.05) was obtained at 10 (74.0) or50 ng/ml IGF-I (76.4) compared to the other treatmentgroups (53.2–58.4) and more ICM cells (P< 0.01) wereobtained under the same conditions (9.3–9.4 vs. 5.8–6.6) for the other treatments. As shown in Table 2, thecleavage rate of SCNT embryos was higher (P< 0.05) inall IGF-I treated groups (89.6%–95.3%) compared to thecontrol (80.8%). A significant effect of IGF-I on blas-tocyst development was found in 50 ng/ml (21.8% vs.11.6%–13.0%) compared to other treatments and thecontrol group.More ICMcells were observed at 50 ng/mlIGF-I compared to other experimental groups. No signi-ficance changes in numbers of total and TE cells wereobserved.

Effect of Different Timing of SupplementationWith IGF-I on IVF and SCNT

Embryo Development

Because the optimal embryotrophic effect of IGF-Iwas observed at 50 ng/ml IGF-I, the subsequent ex-periment to examine supplement timing of IGF-I wasperformed with 50 ng/ml IGF-I in both IVF and SCNTembryos. In IVF embryos, as shown in Table 3, more 2-cell embryos and blastocysts (P<0.05) were producedwhen 50 ng/ml IGF-I was added for the first 48 hr only(75% and 29.4%, respectively) or for the entire 168 hr(78.3% and 31.4%, respectively) compared to culturingembryoswithout IGF-I for the first 48 hr andwith 50 ng/ml IGF-I for the last 120 hr (61.5% and 27.2%,respectively) or without IGF-I (61.8% and 25.8%,respectively). More hatched blastocysts were obtainedwhen embryoswere culturedwith 50ng/ml IGF-I for theentire culture period compared to other experimentalgroups. When counted, the numbers of total cells, ICMcells and TE cells were all increasedwhen embryoswerecultured with IGF-I for the first 48 hr only or for theentire culture period. Similarly, in SCNT embryos, asshown in Table 4, more blastocysts were obtained whenthe medium was supplemented with 50 ng/ml IGF-I forthe first 48 hr only (25.8%) or for the entire cultureperiod (24.8%) compared to culturing embryos, withoutIGF-I for the first 48 hr and with 50 ng/ml IGF-I for thelast 120 hr (13.6%) or without IGF-I (17.1%). Also, whenthemedium contained 50 ng/ml IGF-I either for the first48 hr only or the entire culture period, numbers of total TABLE

1.EffectofDifferentConcentrationsofIn

sulin-L

ikeGrowth

Facto

r(IGF)-IonDevelopmentofPorcin

eIn

VitroFertilized(IVF)Embryosand

Cell

Allocationsto

InnerCell

Mass

(ICM)andTrophecto

derm

(TE)in

Blastocysts

Con

centration

ofIG

F-I

(ng/m

l)No.

ofoo

cytes

cultured

No.

(%)of

oocytesdev

elop

edto

No.

ofblastocysts

examined

Totalcell

number

ICM

TE

ICM:total

cells

2-cell(%

)aBlastocysts(%

)bHatched

blastocysts(%

)b

0527

324(61.5)c

89(27.5)c

14(4.3)c

43

56.5�15.4

c,d

6.6

�1.8

c50.0�13.7

c,d

0.13

1458

286(62.5)c

76(26.6)c

13(4.5)c

39

53.2

�13.4

d5.8

�1.7

c47.4�12.0

c0.10

10

498

310(62.3)c

87(28.1)c,d

23(7.4)d

40

74.0

�17.3

c9.3

�3.1

d64.8�16.6

c,d

0.12

50

481

342(71.1)d

105(30.7)d

28(8.2)d

45

76.4

�18.4

c9.4

�2.9

d67.7�16.3

d0.13

100

498

300(60.2)c

73(24.3)c

10(3.3)c

38

58.4�18.1

c,d

6.1

�2.7

c52.3�16.0

c,d

0.11

aPercentageof

thenumber

ofoo

cytescu

ltured.

bPercentageof

thenumber

ofclea

ved

oocytes.

c,dValues

withdifferentsu

perscripts

within

thesa

mecolumnare

significantlydifferent(P

<0.05).

Data

werederived

from

11times

replicate

experim

ents.

92 S. KIM ET AL.

TABLE

2.EffectofDifferentConcentrationsofIG

F-I

onDevelopmentofPorcin

eSomaticCellNuclearTransfer(S

CNT)EmbryosandCellAllocationsto

ICM

andTE

inBlastocysts

Con

centration

ofIG

F-I

(ng/m

l)

No.

ofoo

cytes

No.

(%)of

oocytesdev

elop

edto

No.of

blastocysts

examined

Totalcell

number

ICM

TE

ICM:totalcells

Injected

Recon

stru

cted

(%)a

2-cell(%

)bBlastocysts(%

)c

0150

99(66.0)

80(80.8)d

10(12.5)d

942.6

�8.5

3.9

�0.8

d38.8�8.5

0.11

1147

98(66.7)

89(90.8)e

11(12.4)d

10

46.0

�9.4

3.8

�1.0

d42.2�8.9

0.87

10

152

102(67.1)

92(92.2)d

12(13.0)d

,e11

51.5�10.2

5.6

�1.1

d,e

45.8�9.5

0.10

50

151

106(70.2)

101(95.3)d

22(21.8)e

15

57.7�12.0

6.6

�1.4

e51.0

�10.9

0.11

100

144

96(66.7)

86(89.6)d

10(11.6)d

10

43.0�11.5

4.8

�1.4

d,e

38.2

�10.5

0.12

aPercentageof

thenumber

ofoo

cytesinjected

withdon

orcells.

bPercentageof

thenumber

offusedcouplets.

c Percentageof

thenumber

ofclea

ved

embryos.

d,eValues

withdifferentsu

perscripts

within

thesa

mecolumnare

significantlydifferent(P

<0.05).

Data

werederived

from

fivetimes

replicate

experim

ents.

TABLE

3.EffectofDifferentTim

ingofSupplementa

tionWith50ng/m

lIG

F-I

onth

eDevelopmentandCellAllocationsofCellsto

ICM

andTEin

Porcin

eEmbryosDerivedFrom

InVitroFertilization

Presence

(þ)or

Absence

(�)of

IGF-I

No.

(%)oo

cytes

cultured

No.

(%)of

oocytesdev

elop

edto

No.

ofblastocysts

examined

Totalcell

number

ICM

TE

ICM:total

cells

First

48hr

Nex

t120hr

2-cell(%

)aBlastocysts(%

)bHatched

blastocysts(%

)b

(�)

(�)

207

128(61.8)c

33(25.8)c

6(4.7)c

30

52.7�15.0

c6.1

�2.1

c46.6�13.1

c0.12

(�)

(þ)

221

136(61.5)c

37(27.2)c

8(5.9)c

32

63.0�17.7

c,d

7.3

�2.2

c,d

55.7�16.8

c,d

0.12

(þ)

(�)

204

153(75.0)d

48(29.4)d

11(7.2)c,d

36

72.1�18.9

d9.1

�3.1

d62.9�18.0

d0.13

(þ)

(þ)

203

159(78.3)d

50(31.4)d

16(10.1)d

38

74.3�19.6

d8.7

�2.8

d65.6�18.8

d0.12

aPercentageof

thenumber

ofoo

cytescu

ltured.

bPercentageof

thenumber

ofclea

ved

oocytes.

c,dValues

withdifferentsu

perscripts

within

thesa

mecolumnare

significantlydifferent(P

<0.05).

Data

werederived

from

sixtimes

replicate

experim

ents.

cells, ICMcells, andTEcellswere increased compared tothe other treatment groups.

Regulation of IGF-IR mRNA Expression byIGF-I Treatment in IVF and SCNT Blastocysts

In order to investigate whether exogenous IGF-Iregulates its own receptor, the expression level of IGF-IRmRNA in IGF-I-treated IVF or SCNTblastocystswasdetermined and compared with each non-treated con-trol. As shown in Figure 2, IGF-IR mRNA expressionwas significantly up-regulated in presence of 50 ng/ml ofIGF-I in both IVF and SCNT embryos (P<0.05).

DISCUSSION

In order to investigate the role of IGF-I in porcinepreimplantation embryos, the present study investi-gated the temporal expression and regulation of IGF-IRmRNA and the effect of IGF-I on embryo development.The results demonstrate that exogenous IGF-I stimu-lates the development of porcine embryos derived fromboth IVF and SCNT, and that this effect is mediated bypre-existing IGF-IR in oocytes and by pre-existing ornewly produced IGF-IR in developing embryos after

Fig. 2. Representative gel photographs of RT-PCR analysis of IGF-IR mRNA in IGF-I-treated in vitro fertilized (IVF) blastocysts andSCNT blastocysts. Total RNA was extracted from 15 blastocysts andIGF-IRmRNA (1,295 bp) level were normalized against b-actin mRNA(685 bp). Data were expressed relative to untreated in vitro fertilized(IVF) blastocysts and SCNT blastocysts and represent the mean�SEof three different experiments (P< 0.05).T

ABLE

4.EffectofDifferentTim

ingofSupplementa

tionWith50ng/m

lIG

F-I

onth

eDevelopmentandAllocationsofCellsto

ICM

andTE

inPorcin

eEmbryosDerivedFrom

SCNT

Presence

(þ)or

Absence

(�)

ofIG

F-I

Nex

t120hr

No.

ofoo

cytes

cultured

No.

offused

oocytes(%

)a

No.

(%)of

oocytesdev

elop

edto

No.

ofblastocysts

examined

Totalcell

number

ICM

TE

ICM:total

cells

First

48hr

2-cell(%

)bBlastocysts(%

)c

(�)

(�)

168

141(83.9)

123(87.2)

21(17.1)d

,e20

43.7�10.3

d3.9

�0.9

d39.8�9.5

d0.09

(�)

(þ)

174

146(83.9)

125(85.6)

17(13.6)d

17

49.7

�9.7

d,e

4.8

�1.3

d44.9

�10.1

d,e

0.10

(þ)

(�)

180

153(85.0)

128(83.7)

33(25.8)e

31

54.1�13.7

e6.2

�1.5

e47.9�12.4

e0.11

(þ)

(þ)

174

150(86.2)

129(86.0)

32(24.8)e

30

56.4�14.4

e6.7

�1.9

e49.7�13.9

e0.12

aPercentageof

thenumber

ofem

bryos

cultured.

bPercentageof

thenumber

offusedcouplets.

c Percentageof

thenumber

ofclea

ved

embryos.

d,eValues

withdifferentsu

perscripts

within

thesa

mecolumnare

significantlydifferent(P

<0.05).

Data

werederived

from

fivetimes

replicate

experim

ents.

94 S. KIM ET AL.

embryonic genomic activation. Furthermore, it is de-monstrated that IGF-I exerted its embryotropic effect onporcine IVF and SCNT embryos during the first 48 hr(Tables 3 and 4).Recent studies have demonstrated the presence of

several growth factors in the oviduct epithelium duringdevelopment of embryos in various species includinghuman (Pfeifer and Chegini, 1994), cattle (Schmidtet al., 1994), and pigs (Chang et al., 2000). The repro-ductive tract produces growth factors including IGFfamilies, colony stimulating factor-I, transforminggrowth factor-b, leukaemia inhibitory factor, granulo-cyte-macrophage colony- stimulating factor, EGF, andplatelet activating factor (Schell et al., 1994; Lonerganet al., 1996; Senturk and Arici, 1998; De Moraes et al.,1999; Tiemann et al., 2001; Pushpakumara et al., 2002;Davies et al., 2004). These growth factors play animportant role in blastocyst development, in regulationof cellular events and in maternal-embryonic dialogue.A wide range of growth factor ligands and theirreceptors are expressed during preimplantation devel-opment and exogenous growth factors seem to affectblastocyst formation (Collier et al., 1990; Chia et al.,1995; Sharkey et al., 1995; Lighten et al., 1997, 1998;Osterlund and Fried, 2000; Lee et al., 2005). In thepresent study, the expression of IGF-IR mRNA inporcine IVF and SCNT embryos was detected by RT-PCR amplification and confirmed by Southern blot andsequence analysis. In both IVF and SCNT embryos, theexpression of IGF-IRmRNA reached its maximum levelin oocytes, however, it gradually decreased as embryosdevelop, and was maintained constantly from 8-cellto blastocyst. Similarly, using a microarray analysis,Whitworth et al. (2005) demonstrated that IGF-IRtranscript decreased between germinal vesicle stageand the blastocyst stage; no differences were foundbetween the 4-cell and blastocyst stage. Such patterns ofIGF-IR mRNA expression in porcine IVF and SCNTembryos were somewhat different from reports in otherspecies. Smith et al. (1993) reported that IGF-IR trans-cripts inmouseembryosweredetectable onlyafter the8-cell stage. Moreover, only negligible amounts of IGF-IRwere found in oocytes and the maximum level inblastocysts (Doherty et al., 1994; Kowalik et al., 1999).The differences in amounts of IGF-IR reported betweenmouse (Smith et al., 1993; Kowalik et al., 1999) and pigoocytes in the present study may be due to differentculture systems used for IVM. Generally, no specificIVM procedure is required for murine oocytes and thisprocess takes only about 12 hr, whereas severalchemical mediators (eCG, hCG, and pFF) are neededto support IVM of porcine oocytes, as well as prolongedculture (44 hr). It has been reported that gonadotropins(eCG or hCG) stimulate secretion of estrogen and IGF-Ifrom cumulus cells into bovine (Nuttinck et al., 2004)and buffalo oocytes (Pawshe et al., 1998). Thus, thesehormones could be responsible for the accumulation ofIGF-IR that we observed in porcine oocytes duringprolonged IVMculture, thereby helping to support earlycleavage stage embryo development.

The presence of IGF-IR mRNA in porcine preimplan-tation embryos derived by IVF and SCNT proceduresindicate an autocrine and/or paracrine role of IGF-I inembryo development. It was demonstrated that exces-sive amounts of IGF-I lower the viability of embryos(Pinto et al., 2002) and lead to down-regulation of thereceptor (Chi et al., 2000). Several studies were per-formed to optimize the dosage of IGF-I in embryo culturemedia in cattle (50–100 ng/ml) (Byrne et al., 2002;Sirisathien et al., 2003) andmice (30–100 ng/ml) (Desaiet al., 2000; Fabian et al., 2004). Within this range,addition of IGF-I enhanced development and the cellnumber of blastocysts in mice (Kurzawa et al., 2002),cattle (Sirisathien et al., 2003), and humans (Spanoset al., 2000). Immunocytochemistry using an alphaIR3antibody confirmed the presence of IGF-IR in humanand mouse blastocysts and the effect of IGF-I wasinhibited by addition of alphaIR3 antibody (Chi et al.,2000). Addition of IGF-I to the culture medium signi-ficantly increased the proportion of embryos developingto theblastocyst stage (Lightenetal., 1998;Spanosetal.,2000) and these blastocysts had significantly increasednumbers of ICM cells (Lighten et al., 1998). In thepresent study, exogenous IGF-I improved porcineembryo development and increased cell numbers inboth IVF and SCNT embryos. Consistent with otherreports, IGF-I at 50 ng/ml increased blastocyst forma-tion and cell numbers of blastocysts in both IVF andSCNT embryos. In addition to the concentration of IGF-I, the timing of supplementation with IGF-I affected thedevelopment of IVF and SCNT embryos. Culturingembryos with IGF-I for the first 48 hr only or for theentire 168 hr of culture significantly improved develop-ment to blastocysts, while culturing without IGF-I forthe first 48 hr or with IGF-I only for the last 120 hr didnot increase blastocyst formation. Along with theprevious studies (Chi et al., 2000; Desai et al., 2000;Spanos et al., 2000; Byrne et al., 2002; Kurzawaet al., 2002; Pinto et al., 2002; Sirisathien and Brackett,2003; Sirisathien et al., 2003; Fabian et al., 2004)our results indicate that using an optimal concentra-tion of IGF-I and correct timing of its addition im-prove porcine embryo development and the quality ofblastocysts.

Mouse and bovine embryos are known to respond ina specific manner to exogenous IGF-I during differentdevelopmental stages via the expression of functionalIGF-IRmolecules. Inmouse embryos, confocal immuno-fluorescent microscopy and Western blot analysis re-vealed that IGF-IR expressionwas reduced to half whenthe embryos were exposed to a high dose of IGF-I(130 nM), and this effect was mediated by blockingdownstream insulin-stimulated glucose uptake, leadingto embryonic apoptosis due to lack of substrates forglycolysis (Chi et al., 2000). In bovine embryos showedthat treatment of embryos with IGF-I (100 ng/ml) de-creased IGF-IR mRNA levels by 20% compared with anegative control (Prelle et al., 2001). In the presentstudy, expression of IGF-IR mRNA was upregulated inIVF and SCNT blastocysts cultured with IGF-I (Fig. 2),

EMBRYOTROPIC ROLE OF IGF-I ON PORCINE EMBRYOS 95

suggesting that the embryotrophic effect of IGF-I wasmediated through its specific receptor.

In conclusion, the present study demonstrated thatIGF-IR mRNAwas expressed in porcine IVF and SCNTembryos during preimplantation embryo development,and that IGF-I improved in vitro development of theseembryos through its specific receptor. The improvementof in vitro culture system with IGF-I for in vitro pro-duction (IVP) of porcine SCNT embryos will leads tomore successful birth of cloned piglets.

ACKNOWLEDGMENTS

We thank Dr. Barry D. Bavister (University of NewOrleans) for his valuable editing of the article. Theauthors acknowledge a graduate fellowship provided bythe Ministry of Education through BK21 program.

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EMBRYOTROPIC ROLE OF IGF-I ON PORCINE EMBRYOS 97