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Endocrinology, doi:10.1210/en.2004-1646
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Endocrinology Vol. 146, No. 9 4105-4116
Copyright © 2005 by The Endocrine Society

Gonadotropin-Releasing Hormone I Analog Acts as an Antiapoptotic Factor in Mouse Blastocysts

Kazuhiro Kawamura, Jun Fukuda, Jin Kumagai, Yasushi Shimizu, Hideya Kodama, Akira Nakamura and Toshinobu Tanaka

Departments of Obstetrics and Gynecology (K.K, J.F., J.K., Y.S., T.T.) and Information Science (A.N.) and Faculty of Health Science (H.K.), Akita University School of Medicine, Akita 010-8543, Japan

Address all correspondence and requests for reprints to: Kazuhiro Kawamura, Department of Obstetrics and Gynecology, Akita University School of Medicine, Hondo 1-1-1, Akita 010-8543, Japan. E-mail: kawamura{at}yf7.so-net.ne.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Both GnRH-I and its receptor (GnRHR)-I have been shown to be expressed in the mammalian preimplantation embryo. In this study, we investigated the molecular mechanisms of GnRH-I in the regulation of early embryonic development in mouse. We found that GnRH-I and GnRHR-I mRNAs were detectable throughout early embryonic stages and that expression levels of both increased significantly after the early blastocyst stage. In blastocysts, GnRH-I and GnRHR-I expression was detected in both inner cell mass and trophectoderm cells. The pregnant uterus also expressed both genes, suggesting that preimplantation embryos could be affected by GnRH through both paracrine and autocrine signaling. Treatment with GnRH-I agonist, buserelin, promoted development of two-cell-stage embryos to the expanded and hatched blastocyst stages and inhibited apoptosis in a dose-dependent manner. In contrast, treatment with GnRH-I antagonist, ganirelix acetate, inhibited development of preimplantation embryos beyond the expanded blastocyst stage and induced apoptosis; both effects could be reversed by cotreatment with GnRH-I agonist. GnRH-I antagonist-induced cell death was mediated by disruption of mitochondrial function, release of cytochrome c, and activation of caspase-3. Furthermore, treatment with GnRH-I antagonist decreased expression of two antiapoptotic growth factors, epidermal growth factor and IGF-II, in blastocysts. These results indicate that GnRH-I, acting as an antiapoptotic factor, is an important growth factor in development of mouse blastocysts.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GnRH, ALSO CALLED LHRH, is a key hormone in the regulation of the mammalian reproduction system (1, 2, 3). GnRH, which is secreted from the hypothalamus into the hypophyseal portal blood system, binds to specific receptors on the anterior pituitary, inducing the stimulation of both synthesis and release of the pituitary gonadotropin hormones (4). GnRH receptor (GnRHR) is a member of the G protein-coupled receptor superfamily. In mammals, two forms of GnRH (GnRH-I and -II) and their cognate receptors (GnRHR-I and-II), which are encoded by separate genes, have been identified (5, 6, 7). However, GnRH-II and GnRHR-II are absent in mouse, and full-length human GnRHR-II is not likely translated because of a frameshift and a premature stop codon in the GnRHR-II gene, despite evidence of GnRH-II function in human tumor cells and decidual stromal cells (5, 6, 7). There is increasing evidence indicating that GnRHR-I is not limited to pituitary gonadotropes; the expressions of GnRHR-I or GnRH-binding sites have been identified in peripheral tissues, including placenta, granulosa cells, myometrium, and lymphoid cells as well as in breast, ovarian, and endometrial tumors (8). Therefore, in addition to its central action through the pituitary-gonadal axis, GnRH-I may also function as a modulator of the activity of diverse systems in many peripheral organs.

Both GnRH-I and GnRHR-I have been shown to be expressed in human and mouse preimplantation embryos (9, 10). In addition, human uterine endometrium expresses GnRH-I during the menstrual cycle (11, 12, 13). These findings suggest an autocrine and/or paracrine function of GnRH-I in the development of preimplantation embryos. A previous report showed that GnRH-I analog promotes the development of mouse preimplantation embryos from the two-cell to the hatching blastocyst stage through paracrine signaling (9). Development of mouse two-cell-stage embryos treated with GnRH-I antagonist was reported to be arrested at early stages and to not progress beyond the blastocyst stage in vitro; and this suppression of embryo development was reversed by cotreatment with GnRH-I agonist (9). These data support the idea that GnRH-I is involved in mammalian preimplantation embryogenesis, and in addition to paracrine effects, GnRH-I plays an important autocrine role in the development of preimplantation embryos. However, the molecular mechanisms underlying the effects of GnRH-I on the development of preimplantation embryos are completely unknown.

Apoptosis, or programmed cell death, is an essential physiological process in almost all tissues (14, 15). Recent studies have focused on the role of apoptosis in the degeneration of preimplantation embryos during in vitro culture (16). In in vitro culture, induction of apoptosis in both inner cell mass (ICM) and trophectoderm (TE) cells of blastocysts has been attributed to a lack of maternal factors, such as essential growth factors and cytokines released by maternal cells (16). In addition, blockading the autocrine effects of growth factors in the absence of maternal factors has been shown to induce embryonic apoptosis in vitro (17).

The objective of the present study was to investigate the molecular mechanisms of GnRH-I in the development of mouse preimplantation embryos. We sought to determine 1) whether GnRH-I acts as an antiapoptotic factor in preimplantation embryos and 2) the molecular signaling of GnRH-I in the regulation of apoptosis in embryos. Our results demonstrate that mouse blastocysts are sensitive to GnRH-I stimulation at various developmental stages, and GnRH-I analog acts as a paracrine factor to inhibit apoptosis in mouse blastocysts. Furthermore, we show that interruption of the effects of GnRH-I by GnRH-I antagonist, ganirelix acetate, induces apoptosis in blastocysts via the intrinsic mitochondrial pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and embryo culture media
To obtain mouse preimplantation embryos, female B6D2F1 mice at 25 d of age (Institute for Animal Reproduction, Ibaragi, Japan) were superovulated by a single ip injection of 7 IU pregnant mare serum gonadotropin (PMSG) (Sigma Chemical Co., St. Louis, MO) followed 48 h later by 10 IU of human chorionic gonadotropin (hCG) (Sigma). All procedures involving the care and use of animals were approved by the Animal Research Committee, Akita University School of Medicine (Akita, Japan). M2 medium (MR-015-D; Chemicon Inc., Temecula, CA) or modified M16 medium (MR-010-D; Chemicon) without serum was used in all experiments.

Collection of mouse preimplantation embryos and uteri
Two-cell-stage embryos were obtained by flushing the oviducts of mated mice at 46–47 h after hCG injection. The embryos were washed three times with M2 medium. Subsequently, groups of 30 embryos were placed in 30-µl drops of modified M16 medium, covered by mineral oil, and cultured at 37 C in air with 5% CO2. For quantitative real-time RT-PCR analysis, embryos at the four-cell, eight-cell, morula, early blastocyst, and expanded blastocyst stages were collected from cultures in individual microdrops at 50–52, 59–60, 70–72, 94–96, and 119–120 h, respectively, after hCG injection. Mouse uteri were obtained from immature mice at 25 and 28 d of age, from mice at 24 and 48 h after treatment with PMSG (7 IU), from mice at 12 h after treatment with hCG (the PMSG primed mice followed 48 h later by 10 IU hCG), and from pregnant mice at 2 and 4 d after mating (corresponding to two-cell and expanded blastocyst stages, respectively).

Quantitative real-time RT-PCR
Quantifications of GnRH-I, GnRHR-I, epidermal growth factor (EGF), IGF-I, IGF-II and TNF-{alpha} transcript levels in mouse preimplantation embryos and GnRH-I and GnRHR-I transcript levels in uteri were performed using a SmartCycler (Takara, Tokyo, Japan) as suggested by the manufacturer. The mRNA extraction and RT was performed as described previously (18, 19, 20); 30 embryos were used in the initial poly (A)+ mRNA isolation step. Primers and hybridization probes are shown in Table 1Go and were synthesized and purified using reverse-phase HPLC (Nihon Gene Research Laboratories, Sendai, Japan).


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  		TABLE 1. Sequences of the oligonucleotide primers and hybridization probes

 
Quantitative PCR was performed in total reaction volumes of 25 µl per reaction tube using the SmartCycler format. The 25-µl reaction mixture comprised 2x QuantiTect Probe PCR Master Mix (QIAGEN, Tokyo, Japan), 0.5 µM primer pairs, 0.2 µM TaqMan probe, and suitable dilutions of template cDNA. In all reactions, HotStarTaq DNA polymerase was activated by an initial denaturation at 95 C for 15 min, followed by 60 cycles of denaturation at 94 C for 15 sec and annealing/elongation at 60 C for 60 sec. Fluorescent signals were monitored at the end of the combined annealing/elongation phase for each cycle for real-time RT-PCR.

To determine the absolute copy number of target transcripts, cloned plasmid cDNAs were used to generate a calibration curve. Briefly, total RNA was extracted from mouse placenta using the RNeasy Mini kit (QIAGEN), and RT was performed as described previously (18, 19, 20). Target transcripts were amplified using conventional RT-PCR with specific primers (Table 1Go). PCR cycling conditions were as follows: denaturation for 3 min at 95 C, followed by 30 cycles of denaturation at 95 C for 30 sec, annealing at 60 C for 30 sec, and extension at 72 C for 30 sec. PCR products were validated as described above. Purified plasmid cDNA templates were measured, and copy number was calculated using absorbance at 260 nm.

A calibration curve was created by plotting the threshold cycle against the known copy number for plasmid templates diluted in log increments from 105 to 100. Each run included diluted plasmid standards used to generate the calibration curve, a negative control without template, and samples with unknown mRNA concentrations. Copy numbers for all unknown samples were determined using SmartCycler software 2.0 (Takara). Although the amount of mRNA present throughout the preimplantation period is not constant for most genes, including common housekeeping genes such as glyceraldehyde-3-phosphate dehydrogenase and ß-actin, the expression levels of histone H2a are constant across the preimplantation period (21). Thus, to correct for differences in both RNA quality and quantity among embryonic samples, data were normalized by dividing the copy number of the target cDNA by the histone H2a copy number.

Isolation of ICM and TE cells
ICM and TE cells were isolated by immunosurgery and used for mRNA extraction (22). After zona removal and lysis, blastocysts were incubated sequentially at room temperature in the presence of an RNA stabilizing solution (RNAlater; Ambion, Austin, TX) first in rabbit antimouse serum (Sigma) diluted 1:5 in PBS-BSA (Sigma) for 30 min and then in guinea pig complement serum (Sigma) diluted 1:5 in PBS-BSA for 30 min. The lysed TE cell fraction was subjected to RNA extraction. To completely remove the remaining TE cells, ICM cells were washed through small-bore glass pipettes with PBS containing 8.0 mg/ml polyvinylpyrrolidone (Sigma) and then subjected to RNA extraction. Total RNA was extracted using the RNeasy Micro kit (QIAGEN), and RT was performed as described previously (18, 19, 20). To confirm the purity of ICM and TE cDNA preparations, RT-PCR was performed with primers specific for Oct-4 (23) and urokinase plasminogen activator (uPA) (24) mRNAs, respectively.

Embryo cultures
For embryo cultures, female B6D2F1 mice were superovulated as described above. Cumulus-zygote complexes were obtained by dissection of the oviducts of mated mice at 22–23 h after hCG injection. After treatment with hyaluronidase (Chemicon) for 1–2 min, zygotes were separated from cumulus cells using a small-bore pipette under Hoffman modulation contrast microscopy (Nikon Inc., Tokyo, Japan). After three washes in M2 medium, the zygotes were cultured overnight in modified M16 medium. The following morning (at 44–46 h after hCG injection), normally developed, two-cell-stage embryos were collected and washed twice with M2 medium. Randomly selected single embryos were placed in 20-µl drops of modified M16 medium and covered by mineral oil with or without GnRH-I agonist (buserelin; Sigma) or antagonist (ganirelix acetate; Organon, West Orange, NJ). In addition, groups of more than 15 embryos were also cultured with or without GnRH-I agonist or antagonist. The medium was changed at 24-h intervals. Embryos were cultured over 144 h up to the hatched blastocyst stage at 37 C in air with 5% CO2. To examine whether the effects of GnRH-I antagonist on preimplantation embryos is mediated through GnRHR-I, two-cell-stage embryos were cultured in modified M16 medium with 5 µM GnRH-I antagonist and 10 µM GnRH-I agonist, according to a previously described method (9). Embryonic development was monitored after 96, 108, 120, and 144 h of culture to determine the proportion of morula, early blastocyst, expanded blastocyst, and hatched blastocyst stage embryos, respectively. At the end of culture, all embryos were subjected to the caspase-3 assay to detect apoptosis. Expanded blastocyst-stage embryos cultured with or without 10 µM GnRH-I antagonist were also subjected to RNA extraction to measure the levels of EGF, IGF-I, IGF-II, and TNF-{alpha} by quantitative real-time RT-PCR.

To assess the effect of GnRH-I antagonist together with a caspase inhibitor, groups of 15 embryos were placed in 20-µl drops of modified M16 medium alone or modified M16 medium containing GnRH-I antagonist at 5 µM with or without the following diverse caspase inhibitors (at 1 µM): z-DEVD-fmk (caspase-3 inhibitor), z-IETD-fmk (caspase-8 inhibitor), and z-LEHD-fmk (caspase-9 inhibitor) (MBL, Nagoya, Japan). Embryonic development was monitored daily over 144 h of culture, and at the end of culture, all embryos were assayed for caspase-3 activity.

To examine the effects of EGF and IGF-II on apoptosis in GnRH-I antagonist-treated embryos, groups of 15 two-cell-stage embryos were placed in 20-µl drops of modified M16 medium alone or with 5 µM GnRH-I antagonists in the presence or absence of EGF (at 10 ng/ml) (Invitrogen, Inc., Carlsbad, CA) and/or IGF-II (at 13 nM) (Sigma). Concentrations of each growth factor were selected following previous studies of early embryos (25, 26). After 144 h of culture, all embryos were assayed for caspase-3 activity.

The caspase-3 assay for detection of apoptosis
Activated caspase-3 in preimplantation embryos was detected using the PhiPhilux G1D2 kit (OncoImmunin Inc., College Park, MD) as described previously (20). Briefly, 10–15 embryos were incubated with 30 µl caspase-3 substrate for 1 h at 37 C in air with 5% CO2. After three washes in dilution buffer, embryos were fixed with 4% paraformaldehyde (PFA) in PBS for 15 min at 4 C in the dark. After three washes in PBS, embryos were transferred on a slide and analyzed under an epifluorescent microscope (Olympus Corp., Tokyo, Japan).

The mitochondrial function assay for detection of apoptosis
Changes in the membrane potential of mitochondria induced by apoptosis were examined using the MitoTracker Orange CMTMRos (Molecular Probes Inc., Eugene, OR) according to the manufacturer’s protocol, with slight modifications. Briefly, embryos were incubated in 250 nM MitoTracker probe for 30 min at 37 C in air with 5% CO2. After three washes in prewarmed modified M16 medium, embryos were fixed with 4% PFA in PBS for 15 min at 37 C in the dark. The embryonic nuclei were stained with 5.0 µg/ml Hoechst 33342 (Molecular Probes) at room temperature for 30 min. After three washes in PBS, embryos were transferred on a slide and analyzed under an epifluorescent microscope.

Immunohistochemistry
Embryos were fixed with 4% PFA in PBS for 15 min at room temperature after thorough washing in 1% PBS-BSA. Embryos were then permeabilized in PBS with 0.2% Triton X-100 (Sigma) for 5 min at room temperature. After blocking at room temperature with Image iT FX Signal Enhancer (Molecular Probes) for 30 min, embryos were incubated with 50 µg/ml mouse anti-cytochrome c monoclonal antibody conjugated with fluorescein isothiocyanate (FITC) (eBioscience, San Diego, CA) in 1% PBS-BSA with 0.05% Tween 20 (Sigma) overnight at 4 C. After three washes in Tris-buffered saline with 0.1% Tween 20, embryos were transferred to a drop of SlowFade Light antifade (Molecular Probes) on a slide and analyzed under an epifluorescent microscope. For negative controls, sections were subjected to the same method with the exception that antibodies were replaced by the same concentration of mouse nonimmunized IgG1 (Dako Corp., Kyoto, Japan) conjugated with FITC using an EZ-label FITC protein labeling kit (Pierce Biotechnology, Inc., Rockford, IL).

Statistical analysis
The Mann-Whitney U test was used to compare the expression levels of EGF, IGF-I, IGF-II, and TNF-{alpha} in blastocysts. One-way ANOVA, followed by Fisher’s protected least-significant difference test, was used to evaluate differences in other experiments. Results are presented as means ± SEM of at least three separate experiments. To analyze the dose-dependent effects of GnRH-I and GnRH-I antagonist on embryonic development and apoptosis, trend analysis ({chi}2 test) was performed using Excel 2001 (Microsoft Corp., Redmond, WA), according to published procedures (27).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Temporal and spatial expression of GnRH-I and GnRHR-I in mouse preimplantation embryos
To examine the temporal changes in the expression levels of GnRH-I and GnRHR-I in mouse preimplantation embryos during development, quantitative real-time PCR was performed to measure the levels of GnRH-I (Fig. 1AGo) and GnRHR-I (Fig. 1BGo) mRNAs in different stages of the early embryos (two-cell, four-cell, eight-cell, morula, early blastocyst, and expanded blastocyst stages). Levels of GnRH-I mRNA were significantly high at the two-cell stage (P < 0.005), decreased as the embryos developed into the eight-cell stage, and then significantly increased after the early blastocyst stage (P < 0.05), and reached their highest levels at the expanded blastocyst stage (Fig. 1AGo). In contrast, levels of GnRHR-I mRNA were low from the two-cell stage up to the morula stage and then were significantly increased at the early blastocyst stage (P < 0.005) and reached their highest levels at the expanded blastocyst stage (Fig. 1bGo).



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  		FIG. 1. Temporal and spatial expression of GnRH-I and GnRHR-I mRNAs in mouse preimplantation embryos. The GnRH-I (A) and GnRHR-I (B) mRNAs levels in preimplantation embryos during development were measured by quantitative real-time RT-PCR. Two-cell-stage embryos were obtained by flushing the oviducts of the mated mice at 46–47 h after hCG injection. Embryos at the four-cell, eight-cell, morula, early blastocyst, and expanded blastocyst stages were collected from cultures in individual microdrops at 50–52, 59–60, 70–72, 94–96, and 119–120 h, respectively, after hCG injection. In each developmental stage, at least three samples prepared from 30 embryos were used for real-time RT-PCR, and levels of GnRH-I and GnRHR-I mRNAs were normalized against levels of histone H2a mRNA in the same sample. Expression levels were independently determined three times and are shown as means ± SEM. The data were analyzed by one-way ANOVA followed by Fisher’s protected least-significant difference test. *, P < 0.05; {dagger}, P < 0.005 vs. eight-cell stage. C, Expression of GnRH-I and GnRHR-I mRNAs in ICM and TE cells. After isolation of ICM and TE cells from blastocysts by immunosurgery, RT-PCR was performed to detect the expression of GnRH-I and GnRHR-I mRNAs. Oct-4 and uPA mRNAs were amplified simultaneously as markers for purity of ICM and TE cDNA preparations, respectively. Levels of ß-actin served as loading controls. Blastocyst cDNA (Blast) was used in positive control tests, whereas no-template cDNA was included for negative controls (NC).

 
Based on RT-PCR performed using isolated ICM and TE cells, spatial variation in expression of GnRH-I and GnRHR-I in blastocysts (Fig. 1CGo) was confirmed. GnRH-I mRNA expression was detected in both ICM and TE cells, and GnRHR-I was also expressed in both cell lineages. The purity of cDNA preparations of ICM and TE cells was confirmed by the amplification of each cell-specific gene, such as Oct-4 for ICM and uPA for TE (Fig. 1CGo).

Expression of GnRH-I and GnRHR-I in mouse uteri during preimplantation period
To examine whether the expression of GnRH-I and GnRHR-I mRNAs in mouse uteri is regulated during the preimplantation period, we measured the levels of GnRH-I (Fig. 2AGo) and GnRHR-I (Fig. 2BGo) mRNAs in the uteri by quantitative real-time PCR. The levels of GnRH-I and GnRHR-I mRNAs were significantly high in immature mice at both 25 and 28 d of age (P < 0.005; Fig. 2Go, A and B). After PMSG treatment, both GnRH-I and GnRHR-I transcript levels were decreased and maintained at a low level up until 12 h after hCG treatment, which corresponds to the ovulation stage (Fig. 2Go, A and B). Although the GnRH-I and GnRHR-I mRNA levels were increased in pregnant mice on both d 2 and 4 as compared with those in uteri treated with gonadotropin, expanded blastocysts expressed 2.5 times higher levels of GnRH-I mRNA and 1.5 times higher levels of GnRHR-I mRNA than those in d 4 pregnant uteri (the levels of GnRH-I and GnRHR-I mRNAs in expanded blastocysts and pregnant uteri on d 4 were as follows: GnRH-I, 19.8 ± 0.7 x 10–3 and 7.9 ± 0.3 x 10–3; GnRHR-I, 12.7 ± 0.3 x 10–3 and 8.2 ± 0.1 x 10–3, respectively; Fig. 2Go, A and B).



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  		FIG. 2. Quantification of GnRH-I (A) and GnRHR-I (B) mRNAs levels in mouse uteri using real-time RT-PCR. Uteri were obtained from immature mice at 25 and 28 d of age, from mice at 24 and 48 h after treatment with PMSG, and from PMSG-primed mice after 12 h of treatment with hCG, which corresponded to the ovulation stage. After mating, uteri were obtained from the pregnant mice at d 2 and 4, which corresponded to the two-cell and expanded blastocyst stages. In each of the reproductive states, three samples prepared from three individual animals were used for real-time RT-PCR, and levels of GnRH-I and GnRHR-I mRNAs were normalized against levels of histone H2a mRNA in the same sample. To compare the expression levels of GnRH-I and GnRHR-I mRNAs between uteri and embryos, the results of expanded blastocysts are shown in the same figure. Expression levels were determined independently and are shown as means ± SEM. The data were analyzed by one-way ANOVA followed by Fisher’s protected least-significant difference test. *, P < 0.05; {dagger}, P < 0.005 vs. uteri treated with PMSG for 48 h.

 
Effects of GnRH-I agonist and antagonist on the development of preimplantation embryos in vitro
The expression of GnRH-I and GnRHR-I in mouse preimplantation embryos and uteri suggests that the GnRH-I signaling system plays a role in early embryonic development. To examine the effect of endogenous GnRH-I on the in vitro development of preimplantation embryos, two-cell-stage embryos were cultured singly with or without GnRH-I antagonist (Fig. 3Go, A and B). Up until 108 h of culture, GnRH-I antagonist treatment showed no significant effect on the development of preimplantation embryos into morula and early blastocyst stages (data not shown). After 120 h of culture, 5 and 10 µM GnRH-I antagonist significantly inhibited the development of embryos from the early blastocyst into the expanded blastocyst stage (P < 0.05 vs. control for both 5 and 10 µM; Fig. 3AGo). After 144 h of culture, the formation of hatched blastocysts from expanded blastocysts was significantly inhibited by GnRH-I antagonist treatment in a dose-dependent manner (P < 0.01, control vs. 2.5 µM, and P < 0.005, control vs. both 5 and 10 µM; Fig. 3BGo).



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  		FIG. 3. The dose-dependent effects of GnRH-I antagonist on the in vitro development of mouse preimplantation embryos. Two-cell-stage embryos in groups at a density of more than 15 embryos were cultured with modified M16 medium alone (control); 2.5, 5, or 10 µM GnRH-I antagonist (GnRH AN); or 5 µM GnRH-I antagonist and 10 µM GnRH-I agonist (GnRH AN + GnRH). Embryonic development was monitored after 120 and 144 h of culture to evaluate the proportion of expanded blastocyst (A) and hatched blastocyst stage embryos (B), respectively. Results of examination of a total of 47–192 embryos in each group were summarized. Values are means ± SEM. The data were analyzed by one-way ANOVA followed by Fisher’s protected least-significant difference test as well as trend analysis. *, P < 0.05; **, P < 0.01; {dagger}, P < 0.005 vs. control.

 
To confirm the specificity of the inhibitory effects of GnRH-I antagonist on the preimplantation embryos, we examined whether the inhibitory effects could be overcome by treatment with GnRH-I agonist. As expected, the inhibitory effects of GnRH-I antagonist on the development of embryos from the early blastocyst into the expanded blastocyst stage and the expanded blastocyst into the hatched blastocyst stage were blocked by cotreatment with GnRH-I agonist (Fig. 3Go, A and B).

To examine the paracrine effects of GnRH-I on the in vitro development of preimplantation embryos, two-cell-stage embryos were cultured with or without GnRH-I agonist. However, because the effect of GnRH-I agonist on the development of embryos was not obvious when embryos were cultured in groups (data not shown), we performed single-embryo culture to eliminate the effects of embryonically derived factors (including GnRH-I), which are endogenously produced and secreted into the medium by the embryos (Fig. 4Go, A and B). Up until 108 h of culture, GnRH-I agonist treatment showed no significant effect on the development of preimplantation embryos into the morula and early blastocyst stages (data not shown). After 120 h of culture, GnRH-I agonist treatment significantly promoted the development of embryos from the early blastocyst into the expanded blastocyst stage in a dose-dependent manner (P < 0.05, control vs. 2.5 µM, and P < 0.01, control vs. both 5 and 10 µM; Fig. 4AGo). After 144 h of culture, the frequency of formation of hatched blastocysts from expanded blastocysts showed a dose-dependent increase in response to GnRH-I agonist treatment (P < 0.01, control vs. 2.5 µM, and P < 0.005, control vs. both 5 and 10 µM; Fig. 4BGo).



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  		FIG. 4. The dose-dependent effects of GnRH-I agonist on the in vitro development of mouse preimplantation embryos under single-embryo culture. To eliminate the effects of embryo-derived factors, including GnRH, two-cell-stage embryos were singly cultured with modified M16 medium alone [C (1)] or 2.5, 5, or 10 µM GnRH-I agonist (GnRH). Embryonic development was monitored after 120 and 144 h of culture to evaluate the proportion of expanded blastocyst (A) and hatched blastocyst (B) stage embryos, respectively. Results of examination of a total of 57–102 embryos in each group were summarized. Values are means ± SEM. The data were analyzed by one-way ANOVA followed by Fisher’s protected least-significant difference test as well as trend analysis. *, P < 0.05; **, P < 0.01; {dagger}, P < 0.005 vs. C (1).

 
These statistical observations were also confirmed by the trend analysis ({chi}2 test, 2 x k contingency table; P < 0.00001 for all the data of Figs. 3Go and 4Go).

Effects of GnRH-I agonist and antagonist on apoptosis in cultured mouse blastocysts
To examine whether the detrimental effects of GnRH-I antagonist on embryonic development resulted from induction of apoptosis, we evaluated apoptosis in embryos treated with or without GnRH-I antagonist and in embryos treated with both GnRH-I antagonist and GnRH-I agonist (Fig. 5Go). In these experiments, apoptosis was evaluated by caspase-3 assay, and representative fluorescence images of caspase-3-positive and -negative embryos are shown in Fig. 5BGo. In blastocysts, both ICM and TE cells were affected by apoptosis induced by GnRH-I antagonist. The groups treated with GnRH-I antagonist for 144 h showed a significant increase in the proportion of caspase-3-positive embryos relative to the control group in a dose-dependent manner (P < 0.05, 2.5 µM vs. control, and P < 0.001, both 5 and 10 µM vs. control; Fig. 5AGo). The effect of GnRH-I antagonist on the induction of apoptosis in embryos was reversed by cotreatment with GnRH-I agonist, suggesting a specific effect of GnRH-I antagonist (Fig. 5AGo).



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  		FIG. 5. The dose-dependent effects of GnRH-I antagonist on the induction of apoptosis in mouse blastocysts. a, Two-cell-stage embryos were cultured in groups at a density of more than 15 embryos with modified M16 medium alone (control); 2.5, 5, or 10 µM GnRH-I antagonist (GnRH AN); or 5 µM GnRH-I antagonist and 10 µM GnRH-I agonist (GnRH AN + GnRH). After the end of 144-h cultures, all embryos were used a in caspase-3 assay to detect apoptosis. Apoptosis was evaluated in more than three independent experiments in which 67–222 embryos were used for each treatment group. Values are means ± SEM. Data were analyzed by one-way ANOVA followed by Fisher’s protected least-significant difference test. *, P < 0.05; **, P < 0.005 vs. control. b, Detection of activated caspase-3 in mouse preimplantation embryos. A, Caspase-3-positive embryo; B, caspase-3-negative embryo. Caspase-3-positive cells were observed in ICM (arrow) and TE (arrowhead). Original magnification, x400.

 
To determine whether GnRH-I acts as a survival factor through a paracrine manner in mouse preimplantation embryos, two-cell-stage embryos were cultured in the presence or absence of GnRH-I agonist and then assayed for apoptosis using caspase-3 activity. Similar to the results of embryo development assays, the effect of GnRH-I agonist on the occurrence of apoptosis in blastocysts was not clear when embryos were cultured in groups (data not shown), and thus, we performed single-embryo culture (Fig. 6Go). Treatment with GnRH-I agonist for 144 h significantly decreased the proportion of caspase-3-positive embryos, as compared with the control group, in a dose-dependent manner [P < 0.005 vs. C (1), a control group containing embryos cultured singly]. The results of statistical tests were also confirmed by the trend analysis ({chi}2 test; P < 0.00001 for all the data shown in Figs. 5Go and 6Go).



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  		FIG. 6. The dose-dependent effects of GnRH-I agonist on the inhibition of apoptosis in mouse blastocysts under single-embryo culture. To eliminate the effects of embryo-derived factors, including GnRH, two-cell-stage embryos were cultured singly with modified M16 medium alone [C (1)] or 2.5, 5, or 10 µM GnRH-I agonist (GnRH). After the end of 144-h cultures, all embryos were used in a caspase-3 assay to detect apoptosis. Apoptosis was evaluated in more than three independent experiments in which 45–92 embryos were used for each treatment group. Values are means ± SEM. Data were analyzed by one-way ANOVA followed by Fisher’s protected least-significant difference test. **, P < 0.005 vs. C (1).

 
Effects of caspase inhibitors on apoptosis in blastocysts treated with GnRH-I antagonist
Induction of apoptosis results in the activation of a caspase cascade from either the extrinsic pathway, which is initiated by activation of membrane-bound death receptors leading to cleavage of caspase-8, or the intrinsic pathway, which is characterized by mitochondrial dysfunction, release of cytochrome c, and subsequent activation of caspase-9 and -3 (28, 29, 30, 31, 32, 33). To determine the molecular pathways underlying GnRH-I antagonist-induced apoptosis in blastocysts, two-cell-stage embryos were cultured in modified M16 medium containing GnRH-I antagonist in combination with diverse caspase inhibitors. The groups treated with GnRH-I antagonist in combination with caspase-3 and -9 inhibitors showed a significant decrease in the proportion of caspase-3-positive embryos compared with those of the group treated with GnRH-I antagonist alone (both P < 0.001), whereas there was no significant difference in the group treated with GnRH-I antagonist in combination with caspase-8 inhibitor (Fig. 7Go). Thus, the GnRH-I antagonist-induced apoptosis in blastocysts is mediated through the intrinsic pathway.



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  		FIG. 7. The effect of caspase inhibitors on the occurrence of apoptosis in blastocysts cultured with GnRH-I antagonist. Two-cell-stage embryos in groups at a density of 15 embryos were cultured with modified M16 medium alone (control) or modified M16 medium containing GnRH-I antagonist at 5 µM with or without a 1 µM concentration of various caspase inhibitors: z-DEVD-fmk (caspase-3 inhibitor), z-IETD-fmk (caspase-8 inhibitor), or z-LEHD-fmk (caspase-9 inhibitor). Apoptosis was evaluated by caspase-3 assay in three independent experiments in which 90 embryos were used for each treatment group. Values are means ± SEM. Data were analyzed by one-way ANOVA followed by Fisher’s protected least-significant difference test. *, P < 0.001 vs. control.

 
GnRH-I antagonist induces loss of mitochondria function and release of cytochrome c in blastocysts
To further characterize the apoptotic effect of the GnRH-I antagonist in blastocysts, we examined loss of mitochondrial function using a mitochondrial membrane potential-sensitive dye, MitoTracker Orange CMTMRos, to assess the status of mitochondrial membrane potential during apoptosis (34). Functional mitochondria take up the dye and show a punctate staining pattern with orange fluorescence, whereas mitochondria in apoptotic cells are not stained. We found that the Mitotracker dye staining in the expanded blastocysts appeared punctate, as expected for their mitochondrial localization (Fig 8AGo). Treatment with the GnRH-I antagonist reduced Mitotracker dye staining dramatically in both ICM and TE cells of expanded blastocysts (Fig. 8BGo). Morphological changes in the nuclei were examined by Hoechst 33342 staining in the same sample sets. The number of nuclei in the expanded blastocysts with GnRH-I antagonist treatment were lower than in blastocysts cultured in modified M16 medium alone, whereas treatment with the GnRH-I antagonist showed no significant effect on the proportion of nuclear fragmentation in the blastocysts (Fig. 8Go, C and D).



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  		FIG. 8. Detection of functional mitochondria by MitoTracker Orange CMTMRos. Shown are epifluorescence images of optical sections of the following: control (A, C, and E) and GnRH-I antagonist (B, D, and F). Two-cell-stage embryos were cultured with modified M16 medium alone (control) or 10 µM GnRH-I antagonist. At the end of culture, embryos were incubated with MitoTracker probe and fixed. The embryonic nuclei were stained with Hoechst 33342. Control group embryos show a punctate staining pattern, as expected for their mitochondrial localization (A), whereas embryos treated with GnRH-I antagonist show a faint staining pattern in both ICM and TE cells (B). Nuclear morphology was examined by Hoechst 33342 staining. Although GnRH-I antagonist treatment shows no significant effect on the nuclear fragmentation (D), the numbers of nuclei in blastocysts with the GnRH-I antagonist treatment (D) were lower than those in blastocysts cultured in modified M16 medium alone (C). Original magnification, x400. Consistent results were obtained in three experiments in which a total of at least 15 embryos of each group were surveyed, and representative results are shown here.

 
The distribution of cytochrome c was visualized by immunofluorescence microscopy. Without GnRH-I antagonist treatment, cytochrome c was localized exclusively to the mitochondria (Fig. 9AGo). After GnRH-I antagonist-induced apoptosis, however, cytochrome c was discharged from the mitochondria and produced a diffused distribution pattern over the entire cytoplasm of both ICM and TE cells (Fig. 9BGo). No specific staining of cytochrome c was found in the negative controls containing nonimmunized IgG1 conjugated with FITC (Fig. 9CGo).



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  		FIG. 9. Immunofluorescence detection of cytochrome c in mouse blastocysts treated with the GnRH-I antagonist. Shown are epifluorescence images of optical sections of the following: modified M16 medium alone (A), GnRH-I antagonist (B), and negative control (C). Two-cell-stage embryos were cultured with modified M16 medium alone or 10 µM GnRH-I antagonist, and each group of embryos was fixed and stained with mouse anti-cytochrome c monoclonal antibody conjugated with FITC. The modified M16 medium alone control group shows a punctate staining pattern, as expected for their mitochondrial localization, whereas embryos treated with GnRH-I antagonist show a diffused distribution pattern over the entire cytoplasm in both ICM and TE cells. For negative controls, nonimmunized IgG1 conjugated with FITC at the same concentration of anti-cytochrome c monoclonal antibody was used, and no specific signal was observed (C) Original magnification, x400. Consistent results were obtained in three experiments in which a total of at least 15 embryos of each group were surveyed, and representative results are shown here.

 
Effect of GnRH-I antagonist on regulation of growth factors in cultured blastocysts
GnRH-I antagonist has been shown to affect the production of growth factors in other cell lines (35, 36, 37). We examined the effects of GnRH-I antagonist treatment on the levels of EGF, IGF-I, and IGF-II mRNAs in blastocysts using quantitative real-time PCR. The mRNA levels of the proapoptotic gene, TNF-{alpha}, were also measured as a control. Among the three growth factors, IGF-II mRNA showed both the highest basal level in expanded blastocysts and the most significant decrease upon GnRH-I antagonist treatment (P < 0.005; Fig. 10AGo). The levels of EGF mRNA also decreased in expanded blastocysts treated with the GnRH-I antagonist (P < 0.05), whereas GnRH-I antagonist treatment did not significantly decrease the levels of IGF-I mRNA in expanded blastocysts (Fig. 10AGo). Furthermore, there was no significant difference in the levels of TNF-{alpha} mRNA between control and GnRH-I antagonist treatment groups, confirming that the effects of GnRH-I antagonist were specific to EGF and IGF-II (Fig. 10BGo).



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  		FIG. 10. The effects of GnRH-I antagonist on the expression of growth factors in mouse blastocysts. Two-cell-stage embryos were cultured with modified M16 medium alone (control) or 10 µM GnRH-I antagonist (GnRH AN). Thirty blastocysts were used for mRNA extraction, and levels of EGF, IGF-I, and IGF-II (A) and TNF-{alpha} (B) mRNAs were measured by real-time RT-PCR. In each gene, at least three samples prepared from 30 embryos were used for real-time RT-PCR. The mRNA levels of these growth factors were normalized against levels of histone H2a mRNA in the same sample. Expression levels were determined independently and are shown as means ± SEM. Data were analyzed by Mann-Whitney U test. *, P < 0.05; **, P < 0.005. C, The effect of cotreatment with GnRH antagonist and EGF and/or IGF-II on the occurrence of apoptosis in blastocysts. Two-cell-stage embryos were cultured with modified M16 medium alone (control) and modified M16 medium containing GnRH-I antagonist at 5 µM with or without EGF and/or IGF-II at 10 ng/ml and 13 nM, respectively. Apoptosis was evaluated by caspase-3 assay in three independent experiments in which 90 embryos were used for each treatment group. Values are means ± SEM. Data were analyzed by one-way ANOVA followed by Fisher’s protected least-significant difference test.

 
To examine whether the down-regulation of EGF and IGF-II is responsible for the apoptosis induced by the GnRH-I antagonist, we examined the occurrence of apoptosis in blastocysts treated with growth factors in the presence of GnRH-I antagonist using caspase-3 assay. Neither treatment with EGF or IGF-II alone nor treatment with both growth factors significantly reversed the effect of GnRH-I antagonist on induction of apoptosis (Fig. 10CGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous reports have shown that mouse and human preimplantation embryos express both GnRH-I and GnRHR-I (9, 10) and that human uteri express GnRH-I during the menstrual cycle (11, 12, 13). In the present study, we determined the regulation of both GnRH-I and GnRHR-I mRNAs in mouse preimplantation embryos and uteri during early embryonic development using quantitative real-time RT-PCR. In preimplantation embryos, expression of both GnRH-I and GnRHR-I was up-regulated after the early blastocyst stage and reached the highest levels at the expanded blastocyst stage. Thus, expanded blastocysts might be the most sensitive to GnRH-I stimulation, along with being the most potent autocrine machinery for GnRH-I. Furthermore, we found that GnRH-I mRNA was detectable in the uteri of pregnant mice, which corresponded to the two-cell and expanded blastocyst-stage embryos, implying the existence of paracrine mechanisms for GnRH-I signaling in preimplantation embryos. However, the level of GnRH-I mRNA in pregnant uteri was almost half that found in expanded blastocyst-stage embryos. Thus, in addition to the paracrine role of GnRH-I, the autocrine effects of GnRH-I may also be important for the development of mouse preimplantation embryos. We also found that GnRHR-I was expressed in mouse uteri with patterns similar to GnRH-I, suggesting the paracrine and autocrine roles of GnRH-I in the regulation of uterine functions. Interestingly, the highest levels of both GnRH-I and GnRHR-I mRNA were observed in immature uteri, and these levels decreased dramatically after PMSG treatment; however, additional studies are required to understand the physiological role of GnRH-I in immature uteri.

In the present study, we have shown that the addition of a GnRH-I antagonist, ganirelix acetate, to mouse embryo culture media inhibited preimplantation embryo development and that this effect was reversed by GnRH-I agonist. These results suggest that blockade of endogenous GnRH-I signaling by GnRH-I antagonist inhibits the development of certain stages of preimplantation embryos. Immunoneutralization of GnRH-I would further confirm the role of endogenous GnRH-I in embryo development. The stage-specific effects of GnRH-I antagonist are consistent with our postulation that sensitivity to GnRH stimulus increases after the early blastocyst stage in embryos. However, a previous report showed developmental arrest at the eight-cell and morula stages in embryos treated with a GnRH-I antagonist, detirelix (9). This discrepancy may be a result of using a different mouse strain with a different genetic background and/or using a different GnRH-I antagonist. The stage-selective effect of GnRH-I antagonist on embryonic development may not be caused by long periods of exposure of the GnRH-I antagonist because inhibition of development at the blastocyst stage was also observed in embryos for which the treatment of the GnRH-I antagonist was delayed for 48 h (data not shown). Although treatment with GnRH-I agonist showed no obvious effect on development of embryos cultured in groups, GnRH-I agonist promoted preimplantation embryo development under single-embryo cultures. Because embryonically derived factors promote development of preimplantation embryos when cultured at high density (38), the effect of GnRH-I agonist may be masked by endogenous factors, including GnRH-I, in group culture. Thus, GnRH-I agonist acts as a potent paracrine factor for preimplantation embryo development in vitro under conditions of missing endogenous effects of embryonically derived factors.

In porcine embryos, a recent study has revealed that treatment with GnRH-I agonist promotes blastocyst formation but not the formation of two-, four-, eight-cell, and morula stages and increases ICM and TE cell number in blastocysts (39). These results further support our findings of stage-selective effects of GnRH-I agonist in mouse preimplantation embryo development and suggest an important role of endogenous GnRH-I in the quality of porcine blastocysts. The hypogonadal (hpg) mouse is deficient in GnRH-I by virtue of a mutation in the GnRH-I gene that prevents full-length transcription and ultimately translation (40). Thus, if GnRH-I is necessary to prevent blastocyst apoptosis in vivo, the embryos of the hpg mouse should fail to develop. However, the embryos can develop normally in vivo (41). Because growth factors secreted from the maternal reproductive tract also act as antiapoptotic factors for preimplantation embryos (16), these inconsistent results may be explained by in vivo compensation of growth factors for antiapoptotic functions of GnRH-I in the hpg mouse.

Accumulated evidence indicates that a number of growth factors and cytokines act in coordination in paracrine and/or autocrine fashions in several important developmental programs, including the rate of embryo development, the proportion of embryos that develop to the blastocyst stage, the total cell number in blastocysts, and energy metabolism and apoptosis (reviewed in Ref. 38). As judged by caspase-3 activation (the last step of the apoptotic cascade and the one that leads to the cleavage of cellular substrates important for cell survival) (32), the GnRH-I agonist inhibited apoptosis in single-blastocyst cultures. The single-embryo culture can be regarded as a suboptimal condition, lacking both paracrine and autocrine factors for cell survival, and is a condition that induces apoptosis in blastocysts (42). This condition allow us to study the paracrine roles of the factors exogenously added to the culture media, excluding the effects of embryonically derived factors, and thus, our results suggest that GnRH-I agonist acts through paracrine signaling as a survival factor in blastocysts to inhibit apoptosis under suboptimal conditions.

Deprivation of growth factors appears to be involved in the initiation of apoptosis in diverse cells (43). In preimplantation embryos, embryonically derived growth factors have been reported to prevent embryos from undergoing apoptosis (reviewed in Refs. 16 , 38 , and 44). Thus, blockade of the effects of the endogenous antiapoptotic growth factors causes a deprivation of growth factors to in vitro embryos, and that is expected to induce apoptosis in the absence of antiapoptotic paracrine factors. In the present study, we have shown that treatment with GnRH-I antagonist induced apoptosis in cultured blastocysts in a dose-dependent manner and that cotreatment with GnRH-I agonist reversed the effect of GnRH-I antagonist. These results suggest that the effects of GnRH-I antagonist are mediated through its specific receptor rather than through a nonspecific or cytotoxic effect, and in addition to the paracrine role, endogenous GnRH-I also inhibits apoptosis in blastocysts.

Signaling cascades that culminate in apoptosis can be divided into two categories: those that act through a mitochondria-dependent, intrinsic pathway and those that act in a death receptor-dependent, extrinsic pathway. The extrinsic pathway is initiated through activation of the death-receptor family receptors, followed by the formation of the death-inducing signaling complex. Recruitment to the death-inducing signaling complex activates caspase-8, which in turn either directly activates caspase-3 or indirectly activates the downstream caspases, leading to engagement of the intrinsic pathway. The intrinsic pathway, by contrast, is triggered by stress stimuli, including growth factor deprivation, leading to the disruption of mitochondrial function and subsequent release of cytochrome c from the mitochondria into the cytoplasm. The release of mitochondrial cytochrome c facilitates the formation of the apoptosome complex composed of the adapter molecule Apaf-1 and caspase-9, which then activates caspase-3 (reviewed in Refs. 28, 29, 30, 31, 32, 33). Therefore, in the signaling pathway of apoptosis induced by growth factor deprivation, activation of caspases is expected to occur in caspase-9 and -3 but not in caspase-8. In this study, we found that apoptosis induced by GnRH-I antagonist was suppressed by both caspase-9 and -3 inhibitors, whereas the caspase-8 inhibitor did not show significant effect on the occurrence of apoptosis, and furthermore, we detected a loss of mitochondrial function and the release of cytochrome c into the cytoplasm in blastocysts treated with GnRH-I antagonist. These findings further support the idea that treatment with GnRH-I antagonist induces apoptosis via the intrinsic pathway as a consequence of the growth factor deprivation for in vitro blastocysts. However, although nuclear fragmentation is known to be a distinctive feature of apoptosis (45), no significant increase was observed in the incidence of nuclear fragmentation in GnRH-I antagonist-treated blastocysts. Still, the result is not inconsistent with the idea that antagonist-treated embryos undergo apoptosis, because a previous report showed that mouse blastocysts treated with TNF-{alpha} did not have increased nuclear fragmentation but did undergo apoptosis as assayed by terminal transferase-mediated dUTP nick end labeling (46).

It is still unclear whether the blockade of the effects of endogenous GnRH-I directly induces apoptosis in mouse blastocysts through the mechanism of growth factor deprivation. GnRH-I antagonist could serve to regulate other apoptosis-related factors in the blastocyst, and then such factors may participate in apoptosis via the intrinsic pathway. Several reports have described a decrease of the synthesis of EGF and IGF-II in endometrial, mammary, and prostatic tumor cell lines by GnRH antagonist treatment (35, 36, 37). EGF is known to stimulate development of mouse preimplantation embryos (reviewed in Refs. 38 and 44), and activation of EGF receptors lead to suppression of apoptosis in mouse blastocysts through both paracrine and autocrine pathways (17, 42). IGF-I and IGF-II are also reported to stimulate embryo development and inhibit apoptosis in blastocysts (reviewed in Refs. 16 , 38 , and 44). In this study, treatment with GnRH-I antagonist significantly decreased the levels of EGF and IGF-II mRNAs in mouse blastocysts. Therefore, GnRH-I plays a key role in the regulation of EGF and IGF-II expression in blastocysts, and GnRH-I antagonist treatment may induce apoptosis in blastocysts through the attenuation of the effects of embryonically derived EGF and IGF-II. However, supplementation of culture medium with these growth factors did not reverse the effect of GnRH-I antagonist on the induction of apoptosis in the blastocysts. In gynecological cancer cells, GnRH-I exerts mitogenic and antiapoptotic activities through multiple pathways downstream of GnRHR-I (47). Thus, additional mechanisms may also be responsible for the induction of apoptosis with GnRH-I antagonist treatment in mouse blastocysts.

In conclusion, we demonstrate the regulation of GnRH-I and GnRHR-I expression in mouse preimplantation embryos and in the uteri. Although our results of GnRH-I and GnRHR-I expression were based on mRNA expression, mRNA levels are expected to accurately reflect all events in the blastocysts. GnRH-I promoted the development of preimplantation embryos and inhibited apoptosis after the expanded blastocyst stage. In blastocysts, blockade of the endogenous GnRH-I effects inhibited embryo development, induced apoptosis via the intrinsic pathway, and decreased the expression of antiapoptotic growth factors. These observations suggest that GnRH-I is an important growth factor in mouse blastocysts, acting as a cell survival factor. Because human preimplantation embryos and uteri also expressed GnRH-I and GnRHR-I (10, 11, 12, 13), the present findings suggest caution for the use of GnRH-I antagonist for in vitro fertilization programs. However, in human conventional in vitro fertilization cycles, GnRH-I antagonist treatment is discontinued when hCG is applied, and the half-life of the GnRH-I antagonist (ganirelix) is less than 13 h after sc administration (48). Thus, the potential harmful effect of GnRH-I antagonist on human embryos can be excluded when cultured embryos are transferred to the uterus between 5 and 7 d after the last administration of GnRH-I antagonist.


   Footnotes
 
First Published Online June 2, 2005

Abbreviations: EGF, Epidermal growth factor; FITC, fluorescein isothiocyanate; GnRHR, GnRH receptor; hCG, human chorionic gonadotropin; hpg, hypogonadal; PFA, paraformaldehyde; PMSG, pregnant mare serum gonadotropin; ICM, inner cell mass; TE, trophectoderm; uPA, urokinase plasminogen activator.

Received December 21, 2004.

Accepted for publication May 24, 2005.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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