The role of gonadotropin-releasing hormone(GnRH) and its receptor in development ofporcine preimplantation embryos derived

from in vitro fertilization

Dong Hyun Nama, So Hyun Leea, Hye Soo Kima, Gab Sang Leea,Yeon Woo Jeonga, Sue Kima, Ji Hye Kima, Sung Keun Kanga,b,*,

Byeong Chun Leea,b, Woo Suk Hwanga,b,c

aDepartment of Theriogenology and Biotechnology, College of Veterinary Medicine,

Seoul National University, Seoul 151-742, South KoreabThe Xenotransplantation Research Center, Seoul National University Hospital, Seoul 110-744, South Korea

cSchool of Agricultural Biotechnology, Seoul National University,

Seoul National University, Seoul 151-742, South Korea

Received 14 December 2003; received in revised form 30 March 2004; accepted 15 April 2004

Abstract

This study was performed to investigate the expression of embryo-derived gonadotropin-releasing

hormone (GnRH) and its receptor, and to determine the role of GnRH in porcine preimplantation

embryos. In Experiment 1, porcine blastocysts derived from in vitro fertilization (IVF) and cultured in

North Carolina State University (NCSU)-23 medium were subjected to reverse transcription

polymerase chain reaction (RT-PCR) amplification with specific primers for GnRH and its receptor.

The results showed that GnRH and its receptor were expressed in porcine IVF blastocysts. In order to

investigate the role of GnRH in embryo development, porcine IVF embryos were cultured in NCSU-

23 supplemented with different concentrations (0, 0.1, 1, or 10 mM) of a GnRH agonist (leuprolide,

Experiment 2) or GnRH antagonist (antide, Experiment 3). Supplementing the culture medium with

0.1 or 1 mM leuprolide increased the rate of blastocyst formation (28.5 or 27.6% versus 20.2%) and

mean total cell number (129 versus 104) compared to the control group. In contrast, antide

significantly decreased the rate of blastocyst formation [12.6% (0.1 mM), 10.2% (1.0 mM), or

8.9% (10.0 mM) versus 22.8% (control)] and total cell number [69 (1 mM) or 68 (10 mM) versus

104 (control)]. In Experiment 4, porcine IVF embryos were cultured in NCSU-23 medium containing

1 mM antide plus 1 mM leuprolide. The embryotrophic effect of GnRH agonist was reversed by

co-supplementing with GnRH antagonist. In conclusion, the present study demonstrated that

Theriogenology 63 (2005) 190–201

* Corresponding author. Tel.:þ82 2 880 1247; fax: þ82 2 884 1902.

E-mail address: kangsnu@snu.ac.kr (S.K. Kang).

0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.theriogenology.2004.04.004

supplementing a culture medium with GnRH agonist can improve blastocyst formation and the

quality of porcine IVF embryos, and that this action was mediated through GnRH receptors.

# 2004 Elsevier Inc. All rights reserved.

Keywords: Porcine; GnRH analogues; In vitro culture; RT-PCR

1. Introduction

The establishment of a reliable culture system for porcine embryos derived from in vitro

fertilization (IVF) is important not only for use in basic research but also in embryo

manipulation procedures such as somatic cell nucleus transfer (SCNT). Although embryos

have been successfully produced in vitro using in vitro matured porcine oocytes and

subsequent IVF [1–5], their developmental potential is still lower than that of oocytes

matured and fertilized in vivo. Studies have been performed to improve in vitro culture

(IVC) of preimplantation embryos by supplementing culture medium with various growth

factors, oxygen, energy substrates, amino acids and albumin [6–11].

In addition to its well documented role in the pituitary resulting in stimulation of both

synthesis and release of FSH and LH, gonadotropin-releasing hormone (GnRH) is thought

be an autocrine regulator in the reproductive tissues of the ovary, uterus, testis and placenta

[12]. Recently, an effect of GnRH on embryo development was also demonstrated in

bovine [13], murine [14,15], and human [16]. In cattle, the cleavage rate of bovine oocytes

fertilized in vitro was significantly increased by incubation with GnRH or with GnRH

agonist. This effect was abolished by the addition of a GnRH antagonist. In mice,

development of preimplantation embryos was significantly enhanced by incubating them

with increasing concentrations of GnRH agonist, and was decreased by GnRH antagonist.

Infertile woman undergoing in vitro fertilization (IVF) and embryo transfer had a

significantly higher pregnancy and implantation rate if the administration of GnRH agonist

was maintained during the early stages of embryonic development and implantation.

Because of its potential for improving porcine preimplantation embryo development,

this study was performed to investigate the expression of embryo-derived GnRH and its

receptor, and the effect of GnRH agonist and antagonist on cultured embryos.

2. Materials and methods

2.1. Oocyte collection and culture

Ovaries were retrieved from prepubertal gilts at a local abattoir and transported to the

laboratory in 0.9% (v/v) NaCl solution at 30–35 8C within 2 h. Follicular fluid and

cumulus–oocytes complexes (COCs) from follicles 3 to 6 mm in diameter were aspirated

using an 18-gauge needle attached to a 5 mL disposable syringe. Compact COCs were

selected and cultured in tissue culture medium (TCM)-199 (Life Technologies, Rockville,

MD) supplemented with 10 ng/mL epidermal growth factor (Sigma–Aldrich Corp., St.

Louis, MO), 4 IU/mL of equine chorionic gonadotropin (eCG, Intervet, Boxmeer,

D.H. Nam et al. / Theriogenology 63 (2005) 190–201 191

Netherlands), 4 IU/mL of human chorionic gonadotropin (hCG, Intervet), 10% (v/v)

porcine follicular fluid (pFF). The pFF was aspirated from 3 to 7 mm follicles from

the prepubertal gilt ovaries. After centrifugation at 1600 � g for 30 min, supernatants were

collected and filtered sequentially through 1.2 and 0.45 mM syringe filters (Gelman

Sciences, Ann Arbor, MI). Prepared pFF was then stored at �20 8C until use. Each group

of 50 COCs was cultured in 500 mL of TCM-199 placed in a CO2 incubator maintained at

39 8C in a humidified atmosphere of 5% CO2 and 95% air. After culturing for 22 h, COCs

were washed three times and cultured in PMSG- and hCG-free TCM-199 medium for

another 22 h.

2.2. In vitro fertilization and embryo culture

Frozen semen was thawed at 39 8C for 1 min in a water bath, diluted in 10 mL

Dulbecco’s PBS (Life Technologies) supplemented with 0.1% BSA (Sigma–Aldrich

Corp.), 75 mg/mL potassium penicillin G (Sigma–Aldrich Corp.), and 50 mg/mL strepto-

mycin sulfate (Sigma–Aldrich Corp.) and centrifuged twice at 350 � g for 2 min. The

sperm pellet was resuspended in modified Tris-buffered medium (mTBM) containing

113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl2�2H2O, 20 mM Tris, 11 mM glucose, 5 mM

sodium pyruvate and 0.1% (w/v) BSA (Sigma–Aldrich Corp.). Each group of 15 matured

oocytes was placed into 50 mL mTBM droplets and inseminated with 2 � 106 spermatozoa/

mL for 6 h at 39 8C. The IVF zygotes were cultured in 25 mL microdrops (10 per drop) of

NCSU-23 supplemented with 4 mg/mL fatty acid-free BSA (Sigma–Aldrich Corp.)

overlaid with mineral oil (Sigma–Aldrich Corp.) for 7 days at 39 8C in an atmosphere

of 5% O2, 5% CO2, and 90% N2. At day 4 of embryo culture, 2.5 mL of fetal bovine serum

(FBS, Life Technologies) was added to the microdrops to make final concentration of 10%

FBS in order to increase embryo viability and enhance hatching of blastocysts at day 7 [17].

Development of oocytes to the 2-cell, 4-cell, 8-cell, morula, and blastocyst stages was

checked under a stereomicroscope at 48, 72, 96, 144, and 168 h, respectively.

2.3. Total RNA isolation and reverse transcription-polymerase chain reaction

(RT-PCR) amplification

Blastocysts were washed three times in PBS and transferred into 0.2 mL of 4 M

guanidium isothiocyanate (Sigma–Aldrich Corp.) lysis solution containing 1% b-mercap-

toethanol. The blastocysts were frozen-stored in liquid nitrogen until use. Total RNA (from

20 blastocysts) was extracted by acid phenol–guanidium thiocyanate–chloroform extrac-

tion [18] and dissolved in 15 mL RNase-free water. Reverse transcription was carried out

using total RNA (15 mL) at 37 8C using the First-strand cDNA Synthesis Kit (Amersham

Biotech, Piscataway, NJ) in 33 mL reaction volume according to the manufacturer’s

suggested procedure. The primers for GnRH, GnRH receptor (GnRHR) and b-actin, as

listed in Table 1, were designed to span the exon–intron region to rule out the amplification

of genomic DNA. The PCR for b-actin was performed to rule out the possibility of RNA

degradation and was used to control the variation in mRNA concentration in the RT

reaction. The cDNA (5 mL) was amplified in a 50 mL PCR reaction containing 1.25 units

hot start Taq polymerase (Qiagen, Hilden, Germany) and its buffer, 1.5 mM MgCl2, 2 mM

192 D.H. Nam et al. / Theriogenology 63 (2005) 190–201

deoxy-NTP, and 25 pmol specific primers. The PCR amplification was carried out for 1

cycle with denaturing at 95 8C for 15 min, and subsequently for 35 cycles with denaturing

at 95 8C for 30 s, annealing at 55 8C for 30 s, extension at 72 8C for 90 s, and a final

extension at 72 8C for 15 min. Amplified PCR products (10 mL) were fractionated on a

1.5% agarose gel, stained with 0.2 mg/mL ethidium bromide and visualized with a Gel

Documentation system (Gel-DocTM 2000, BioRad, Hercules, CA). For GnRHR, nested

PCR with outer primers was performed using 2 mL of PCR product of the first amplification

as a template. The PCR products were purified from the gel with an agarose gel extraction

kit (Qiagen) and cloned into pCRTopo cloning vector (Invitrogen, San Diego, CA).

Sequence analysis was performed to confirm the identity of amplified PCR products

using an automated DNA sequence analyzer (ABI 3100, Applied Biosystems, Foster

City, CA).

2.4. Differential staining

The quality of blastocysts was assessed by differential staining of the inner cell mass

(ICM) and the trophectoderm (TE) cells according to a modified staining procedure [19].

Briefly, TE cells of blastocysts at 7 days were stained with 100 mg/mL fluorochrome

propidium iodide (Sigma–Aldrich Corp.) after treatment with permeabilizing solution

containing 1% (v/v) Triton X-100 ionic detergent (Sigma–Aldrich corp.). Blastocysts were

then incubated in a second solution containing 100% ethanol (for fixation) at 4 8C and

bisbenzimide (Sigma–Aldrich Corp.). Fixed and stained whole blastocysts were mounted

and assessed for cell number using epifluorescence microscopy.

2.5. Experimental design

In Experiment 1, the expression of GnRH and its receptor mRNA in porcine IVF derived

blastocysts were investigated by RT-PCR amplification. Separately, IVF procedures were

done to obtain embryos for analyzing effects of GnRH agonist/antagonist, and the

experiment was repeated at least three times with different sets of embryos. The porcine

Table 1

Primers used for polymerase chain reaction for amplification of gonadotropin-releasing hormone (GnRH) and its

receptor

mRNA Direction Primer sequence Fragment

size (bp)

b-Actin Forward 50-CACCACTGGCATTGTCATGGACTCT-30 429

Reverse 50-TGTCCACGTCGCACTTCATGATCG-30

GnRH Forward 50-ATGGAGCCAATTCCGAAACTTCTAGC-30 400

Reverse 50-GCAAACAGGTGCAACTTGGCATAAGA-30

GnRH receptor Forward of outer pair 50-CTACATCAGTTGGGGAAGGATGGCA-30 1088

Reverse of outer pair 50-ATGCTTTGTGCTTGTCATTCCCCA-30

Forward of inner pair 50-CGGAGAGTTCCTCTGCAAAGTCCT-30 480

Reverse of inner pair 50-GTCATCTTCAGAGTCCTCAACCGA-30

D.H. Nam et al. / Theriogenology 63 (2005) 190–201 193

IVF embryos were cultured in NCSU-23 medium supplemented with different concentra-

tions (0, 0.1, 1.0, or 10 mM) of a GnRH agonist (leuprolide acetate; Sigma–Aldrich Corp.,

Experiment 2) or GnRH antagonist (antide; Sigma–Aldrich Corp., Experiment 3). In

Experiment 4, the IVF embryos were cultured in NCSU-23 medium supplemented with

vehicle control, leuprolide (1 mM), antide (1 mM) or 1 mM leuprolide þ 1 mM antide.

Embryo development to 2-cell, 4-cell, 8-cell, morula, and blastocyst, and the quality of

blastocysts were monitored as experimental parameters. Each experiment was replicated at

least 10 times.

2.6. Statistical analysis

Oocytes were randomly distributed in each experimental group. The differences in

embryo development among experimental groups were analyzed using one-way ANOVA

after arcsine transformation to maintain homogeneity of variance. Post hoc analyses to

identify between-group differences were performed using the LSD test. The same test was

used to determine to statistical significance in the cell number of blastocysts among

experimental groups without arcsine transformation. All analyses were performed using

SAS (SAS Institute, version 8.1). Significant difference among the treatment was deter-

mined where the P value was less than 0.05.

3. Results

3.1. Expression of GnRH and its receptor in porcine IVF blastocysts: Experiment 1

The GnRH and its receptor mRNA were amplified by RT-PCR using sets of primers

shown in Table 1. As shown in Fig. 1, the expected size of PCR products were observed for

GnRH (400 bp) and GnRHR (480 bp, observed after nested PCR) and confirmed by

sequence analysis. The possibility of cross-contamination was ruled out, because no PCR

products were observed and detected in the negative control [Tm (�), without template in

the RT reaction]. Sequence analysis revealed that GnRH and its receptor have sequences

Fig. 1. Detection of gonadotropin-eleasing hormone (GnRH) mRNA and gonadotropin-eleasing hormone

receptor (GnRHR) mRNA by reverse transcription-polymerase chain reaction (RT-PCR) amplification. First

strand cDNAs from porcine IVF blastocysts were amplified using sets of PCR primers for porcine GnRH,

GnRHR and b-actin. The expected products of GnRH (400 bp), GnRHR (480 bp) and b-actin (429 bp) were

observed on an ethidium bromide-stained gel. For GnRHR, nested PCR with inner primers was performed using

a 2 mL of PCR product of first amplification (amplified with the outer primers) as a template.

194 D.H. Nam et al. / Theriogenology 63 (2005) 190–201

Table 2

Effect of a GnRH agonist (leuprolide; 0, 0.1, 1 or 10 mM) on the development of porcine in vitro fertilized embryos in Experiment 2

Leuprolide

(mM)

No. of oocytes

cultured

No. (%) of oocytes developed to No. of cells (mean � S.E.)1 ICM:TE (%)

2-cell

(48 h)

4- to 8-cell

(96 h)

Morula

(144 h)

Blastocyst

(168 h)

ICM TE Total

0 258 181 (70.2) 169 (65.5) 105 (40.7) 52 (20.2)a 27.2 � 3.1 77.0 � 5.1a 104.2 � 5.6a 39.0 � 4.6a

0.1 274 193 (70.4) 185 (67.5) 129 (47.1) 78 (28.5)b 26.8 � 2.4 72.8 � 3.5a 99.6 � 5.0a 37.1 � 2.8a

1 257 176 (68.5) 168 (69.5) 116 (45.1) 71 (27.6)b 30.6 � 1.0 98.3 � 5.3b 128.9 � 5.3b 33.4 � 2.6b

10 272 201 (73.9) 190 (69.8) 109 (40.1) 59 (21.7)a,b 28.9 � 2.6 77.6 � 3.3a 106.5 � 4.5a 38.3 � 3.9a

Number in parentheses indicates hours after insemination. Blastocysts were subjected to differential staining to evaluate quality at 168 h after insemination. Within the

same column, values with different superscripts (a, b) were significantly different (P < 0.05).1 Number of examined blastocysts at different concentration of leuprolide (0, 0.1, 1, or 10 mM) was 19, 21, 21, and 20, respectively.

D.H

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l./Th

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3(2

00

5)

19

0–

20

11

95

Table 3

Effect of a GnRH antagonist (antide; 0, 0.1, 1 or 10 mM) on the development of porcine in vitro fertilized embryos in Experiment 3

Antide

(mM)

No. of oocytes

cultured

No. (%) of embryos developed to No. of cells (mean � S.E.)1 ICM:TE (%)

2-cell

(48 h)

4- to 8-cell

(96 h)

Morula

(144 h)

Blastocyst

(168 h)

ICM TE Total

0 228 173 (75.9) 131 (57.5) 87 (38.2) 52 (22.8)a 30.6 � 4.3a 73.6 � 2.0a 104.2 � 3.6a 43.2 � 6.4

0.1 230 177 (77.0) 107 (46.5) 54 (23.5) 29 (12.6)b 30.7 � 2.8a 69.7 � 3.8a 100.4 � 5.8a 44.2 � 3.1

1 234 180 (76.9) 107 (45.7) 60 (25.6) 24 (10.2)b,c 21.4 � 1.7b 48.0 � 3.8b 69.4 � 5.0b 46.1 � 2.7

10 235 173 (73.6) 112 (51.9) 58 (24.7) 21 (8.9)c 19.1 � 2.5b 49.1 � 3.8b 68.2 � 5.1b 39.5 � 5.5

Numbers in of parentheses indicates hours after insemination. Blastocysts were subjected to differential staining to evaluate quality at 168 h after insemination. Within

the same column, values with different superscripts (a, b, c) were significantly different (P < 0.05).1 Number of examined blastocysts at different concentration of antide (0, 0.1, 1 or 10 mM) was 14, 18, 19 and 13, respectively.

19

6D

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Na

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al./T

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gy

63

(20

05

)1

90

–2

01

Table 4

Effect of co-supplementing with a GnRH agonist (leuprolide, 1 mM) and an antagonist (antide, 1 mM) on the development of porcine in vitro fertilized embryo in

Experiment 3

Treatment No. of oocytes

cultured

No. (%) of embryos developed to No. of cells (means � S.E.)1 ICM:TE (%)

2-cell

(48 h)

4- to 8-cell

(96 h)

Morula

(144 h)

Blastocyst

(168 h)

ICM TE Total

Control 283 212 (74.9)b 196 (69.3)b 83 (29.3)b,c 56 (19.8)b 24.9 � 1.1b 70.0 � 2.2b 94.9 � 2.5b 36.6 � 2.2a

1 mM 1euprolide 305 229 (75.1)b 212 (69.5)b 102 (33.4)b 82 (26.9)c 28.6 � 1.2b 90.1 � 3.6c 118.7 � 4.6c 32.3 � 1.2b

1 mM antide 375 240 (64.0)c 205 (54.7)c 85 (22.7)c 50 (13.3)d 18.6 � 1.3c 52.7 � 2.5d 71.3 � 3.5d 35.4 � 1.7a

1 mM leuprolide þ1 mM antidea

385 258 (67.0)c 235 (61.0)c 109 (28.3)b,c 78 (20.1)b 24.5 � 1.2b 67.2 � 2.6b 91.7 � 3.3b 37.2 � 1.7a

Numbers in parentheses indicates hours after insemination. Blastocysts were subjected to differential staining to evaluate quality at 168 h after insemination. Superscript

‘a’ for porcine in vitro fertilized embryos were cultured in the NCSU-23 medium containing GnRH agonist (1 mM leuprolide) plus antagonist (1 mM antide). Within the

same column, values with different superscripts (b, c, d) were significantly different (P < 0.05).1 Number of examined blastocysts at treatment was 25, 32, 34, and 26, respectively.

D.H

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3(2

00

5)

19

0–

20

11

97

identical to those found in the hypothalamus and the pituitary, respectively (data not

shown).

3.2. Effect of different concentrations of GnRH agonist on porcine embryo

development and cell number in blastocysts: Experiment 2

As shown in Table 2, supplementing 0.1 or 1 mM leuprolide in the culture medium

significantly increased the rate of blastocyst formation (28.5 or 27.6% versus 20.2 to

21.7%) compared to other groups. No significant differences were found in the rates of 2-,

4-, 8-cell, and morula formation among the experimental groups. Supplementing with 1 mM

leuprolide significantly increased the total cell number of blastocysts (128.9 versus 99.6 to

106.5), and decreased the ICM to TE ratio (33 versus 39) compared to other groups.

3.3. Effect of different concentrations of GnRH antagonist on porcine embryo

development and cell number in blastocysts: Experiment 3

As shown in Table 3, 0.1, 1, or 10 mM antide in the culture medium significantly

inhibited the rate of blastocyst formation (12.6, 10.2, or 8.9% versus 22.8%) compared

to control. No significant differences were found in the rates of 2-, 4-, 8-cell, and morula

formation among the experimental groups. Supplementing with 1 or 10 mM antide

significantly decreased the total cell number of blastocysts (69.4 or 68.2 versus 100.4 to

104.2) compared other groups, due to decreases in ICM and TE cells. No significant

differences were observed in the ratio of ICM to TE among the experimental groups.

3.4. Effect of co-supplementing with GnRH agonist (1 mM leuprolide) and antagonist

(1 mM antide) on porcine embryo development and on cell number in blastocysts:

Experiment 4

As shown in Table 4, antide together with leuprolide in the culture medium blocked the

embryotrophic effect of leuprolide: the rate of blastocyst formation was decreased from

26.9 to 20.1% and blastocyst total cell number in was not significantly different from the

control group. As expected, leuprolide increased (26.9%) while antide inhibited (13.3%)

blastocyst formation compared to the control group (19.8%).

4. Discussion

In order to investigate the role of GnRH in porcine preimplantation embryos, the present

study investigated the expression of embryo-derived GnRH and the GnRHR gene and the

effect of GnRH on embryo development. The RT-PCR amplification demonstrated the

expression of GnRH and its receptor in porcine embryos. The rate of blastocyst formation

and total cell numbers were increased by the GnRH agonist and decreased by

the GnRH antagonist. Using the GnRH agonist together with the antagonist blocked

the embryotrophic effect of the GnRH agonist. These results indicate that GnRH plays its

embryotrophic role in porcine embryos via its specific receptor.

198 D.H. Nam et al. / Theriogenology 63 (2005) 190–201

In addition to its well documented role in the pituitary, GnRH is thought be an autocrine

and paracrine regulator in the reproductive tissues. This concept is based on the detection of

Gn/RH gene transcripts, synthesis of GnRH, and the multitude of effects attributed to

GnRH receptor-mediated signaling, in extrapituitary tissues including gonads, placenta,

and endometrium. Furthermore, the presence of GnRH and its receptor in preimplantation

embryos has been demonstrated. Immunoreactive GnRH was produced and secreted in

vitro by cultured rhesus monkey [20], mouse [15], and human embryos [21]. Immuno-

histochemical localization of GnRH showed intense staining in all the blastomeres at the

morula stage as well as in the trophectoderm and inner cell mass of blastocysts [21].

Trophoblastic GnRH has been implicated as one of the primary regulators of the synthesis

and secretion of hCG in periimplantation embryos [15]. In line with previous reports, in this

study we detected GnRH and GnRHR mRNA in porcine blastocysts. The expression level

of GnRHR was lower than that of GnRH because GnRHR mRNA was amplified after a

second round of PCR. In order to investigate the role of GnRH and its receptor in porcine

embryo development, IVF embryos were cultured in the presence of GnRH agonist or

antagonist and the developmental competence of embryos was monitored. Our results

demonstrated that the rate of blastocyst formation was significantly higher in NCSU-23

medium containing 0.1 or 1 mM GnRH agonist compared to other experimental groups.

However, a GnRH antagonist (0.1, 1, or 10 mM) significantly decreased the rate of

blastocyst formation compared to control. The positive effect of the GnRH agonist was

eliminated by the GnRH antagonist, suggesting that the embryotrophic effect of GnRH was

mediated via its specific receptors, rather than a nonspecific or toxic effect. Along with the

presence of GnRH and its receptor, these results demonstrated a functional role of GnRH in

porcine embryo development. Our results are in agreement with previous results from

mouse [15] and primate embryos [22]. In contrast, Dodson et al. [23] showed that

leuprolide had no measurable effect on human embryo growth rates. The exact reasons

for the differential effect of GnRH on IVF embryo development in different species are not

known. This may due to species difference, and/or different culture conditions, among

others.

Ideally, a marker is needed to evaluate the quality of embryos obtained in vitro. Embryo

morphology is not sufficient, given that parthenogenetic blastocysts look very similar to in

vitro fertilized blastocysts, but are ultimately not viable. One approach to evaluate viable

embryos is to count the total cell number of blastocysts, and the proportion of ICM to total

cell numbers after differential staining. It is well documented that a total cell number close

to that of in vivo-derived blastocysts can be regarded as a valuable indicator of IVP embryo

viability [24–27]. Timed from activation (a similar starting time point to IVF derived

embryos in culture), in vivo developed porcine embryos contain over a hundred cells by

day 5, and several hundred cells by day 6 [28]. Although in the present study the mean cell

number of blastocysts was lower than for in vivo produced embryos, we observed increased

total cell numbers (129 versus 104) in blastocysts cultured with a GnRH agonist, due to

increases in the numbers of TE cells (98 versus 77). It has been suggested that the

proportion of ICM cells in blastocysts is crucial for postimplantation development [29,30].

Rivera et al. [31] also suggested that a decrease in TE cell number during early porcine

embryogenesis may improve embryo survival and liter size by reduction in placenta size

and thus less space of placenta are occupied by each fetus in prolific breeds. The

D.H. Nam et al. / Theriogenology 63 (2005) 190–201 199

importance of cell differentiation in blastocysts was recently considered from a different

point of view. In cloned cattle, it is suggested that increased total blastomere or ICM cell

numbers in blastocysts might cause the ‘‘large offspring syndrome (LOS)’’, emphasizing

the importance of cell differentiation into TE [32,33]. In line with this idea, in this study,

GnRH promoted TE cell differentiation resulting in an increase in total numbers of

blastomeres in blastocysts without affecting ICM cell number. However, unlike in cattle,

the importance of TE cell differentiation may not be applicable to the pig, a litter bearing

animal which produces many offspring in a limited uterine space because LOS was not

reported in cloned pigs. Therefore, the functional significance of GnRH-induced increase

in total cell number and TE cells is remains unknown. Large numbers of embryo transfers

are needed to determine the functional significance of GnRH-induced responses for

embryo quality in porcine embryos.

In conclusion, the present study demonstrated that supplementing a culture medium with

a GnRH agonist can improve blastocyst development and the quality of porcine IVF

embryos as assessed by total cell number.

Acknowledgements

We thank Dr. Barry D. Bavister for his valuable editing of the manuscript. This study was

supported by a grant from the Korea Research Foundation (041-E00246). The authors

acknowledge a graduate fellowship provided by the Ministry of Education through BK21

program.

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