Research Centre for Reproductive Health, Department of Obstetrics and Gynecology, The University of Adelaide, The Queen Elizabeth Hospital, Woodville, South Australia, Australia
Submitted 26 August 2004 ; accepted in final form 6 December 2004
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ABSTRACT |
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blastocyst; blastomere allocation; mouse; ovarian stimulation; recombinant gonadotropin
Therefore, in the current study, we have designed a model of rhFSH-induced ovarian stimulation in the mouse and investigated the impact of this stimulation on preimplantation embryo development. The parameters used to assess preimplantation embryo development were in vitro development to blastocyst, blastocyst cell numbers, and cell allocation between the ICM and TE of the blastocyst. Furthermore, because growth factors and their receptors have been demonstrated to play an important role in cell proliferation and differentiation in preimplantation embryos (16, 26, 27), we have also determined the impact of ovarian stimulation on the expression of insulin-like growth factor 2 (IGF-II), the IGF-II receptor, and VEGF in blastocysts.
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MATERIALS AND METHODS |
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All procedures were approved by The University of Adelaide Animal Ethics Committee and The North Western Adelaide Health Service Animal Ethics Committee. Adult female F1 hybrid CBA x C57Bl/6 F1 mice (810 wk) and male C57Bl/6 F1 mice (315 mo) were obtained from the Central Animal House at the University of Adelaide. The animals were housed under a 14:10-h light-dark regimen, fed a standard pellet diet ad libitum, and had free access to water.
Gonadotropin Treatment
The gonadotropins used in this study were diluted in 0.9% saline, and an injection volume of 0.1 ml was used. The rhFSH treatments were given as two subcutaneous injections 24 h apart, since the activity of rhFSH has a relatively short duration.
rhFSH treatments. Female mice were stimulated with a total of 2.5, 5.0, 10.0, or 20.0 IU rhFSH (Gonal-F; Serono). Ovulation was induced after an intraperitoneal injection of 5 IU hCG (Pregnyl, Organon, Holland) 24 h after the second rhFSH injection, and mice were immediately caged overnight with fertile male mice.
eCG treatment. Female mice were stimulated with a subcutaneous injection of 5 IU eCG (Folligon serum gonadotropin; Intervet Australia, Victoria, Australia) followed 48 h later with an intraperitoneal injection of 5 IU hCG. Immediately after the hCG injection, mice were placed with fertile male mice.
An additional treatment group consisted of female mice injected with 5 IU hCG only before being caged overnight with fertile male mice.
A control group of mice consisted of naturally cycling females that were caged with fertile males at the same time as the experimental groups received their hCG injections.
The presence of a copulation plug the morning after females were caged with males indicated a successful mating, and only females with a copulation plug were included in the study.
Embryo Recovery and In Vitro Culture
Female mice were killed by cervical dislocation 2224 h post-hCG, and cumulus enclosed zygotes were flushed from the oviducts and collected in HEPES-buffered handling media. The embryos were denuded by a short incubation (i.e., <1 min) in ovine hyaluronidase (50 U/ml; Sigma Chemical, St. Louis, MO) in handling media and washed three times in the handling media before being placed in a culture drop. The culture media consisted of potassium simplex optimized media (KSOM) without amino acids (3) but containing L-alanyl-L-glutamine (1.0 mM) (GlutaMax; Invitrogen, Carlsbad, CA) instead of glutamine. Groups of 1015 morphologically normal presumptive zygotes were cultured in 20 µl drops of KSOM media under mineral oil (Sigma) in 35-mm Falcon 1008 dishes (Becton-Dickinson Labware, Franklin Lakes, NJ) at 37°C in 5% O2-6% CO2-89% N2. Presumptive zygotes with fragmentation and unfertilized or immature oocytes (exhibiting a germinal vesicle or metaphase I) were classified as abnormal and therefore were not included for culture. After 88 h of culture, the stages of embryonic development (compact morulas, early to fully expanded blastocysts, and hatching blastocysts) were recorded. Blastocysts were either used for cell staining or were frozen in groups of 2040 in 400 µl Tri Reagent and stored at 80°C for subsequent RNA extraction.
Differential Nuclear Staining
The number of cells within the ICM or TE of expanded and hatching blastocysts was assessed using methods based on Handyside and Hunter (13). Blastocysts were incubated in acidic Tyrode solution (Sigma) at room temperature for up to 3 min to remove the zona pellucida, before being washed in 4 mg/ml polyvinyl alcohol in PBS (PBS-PVA). This was followed by an incubation in ice-cold 10 mM 2,4,6-trinitrobenzenesulfonic acid (Sigma) in PBS-PVA for 10 min, followed by a wash in PBS-PVA. Blastocysts were subsequently incubated in 0.1 mg/ml anti-dinitrophenyl-BSA (Sigma) in protein-free KSOM for 10 min at 37°C. After a PBS-PVA wash, complement mediated lysis was induced by incubating the blastocysts in guinea pig complement (Sigma) diluted 1:5 in PBS with 5 µg/ml propidium iodide (Sigma) for 20 min at 37°C. Blastocysts were then briefly washed in PBS-PVA before being placed in 20 mg bisbenzimide/ml (Hoechst 33342; Sigma) in ethanol overnight at 4°C. The next morning, embryos were mounted in 50% glycerol on a glass slide and overlayed with a coverslip. The differential color of the nuclei was examined under ultraviolet light using an Olympus AH-3 microscope (Olympus Optical) whereby the ICM (stained with Hoechst) appeared blue and the dual-stained TE cells appeared red/pink. Numbers of ICM and TE cells were obtained for each embryo, and ratios of ICM to total number of cells was calculated for individual embryos.
RNA Extraction
Total RNA was extracted from blastocysts using Tri Reagent (Sigma) according to the manufacturer's instructions. The tubes containing Tri Reagent and blastocysts were thawed at room temperature and vortexed briefly before standing for a further 5 min at room temperature. Chloroform (100 µl; Sigma) was added, and samples were vigorously shaken and incubated at room temperature for 15 min. Samples were centrifuged at 13,000 rpm for 15 min at 4°C in an Eppendorf centrifuge 5145C (Eppendorf, Hamburg, Germany), and the upper aqueous phase was removed. Isopropanol (250 µl; Sigma) and glycogen (20 µg; Invitrogen) were added, and RNA was precipitated by overnight incubation at 80°C, followed by centrifugation at 13,000 rpm for 30 min at 4°C. RNA pellets were washed with 70% ethanol, air-dried, and redissolved in 10 µl sterile water. DNA contamination was excluded by treatment with 2 units DNase (2 U/µl, DNA-free; Ambion, Austin, TX) for 30 min at 37°C. DNase was inactivated by addition of 5 µl DNase Inactivation Reagent (DNA-free; Ambion), incubation at room temperature for 2 min, and centrifugation at 13,000 rpm for 1 min. The RNA solution was removed from the pelleted DNase Inactivation Reagent (DNA-free; Ambion) and used immediately for reverse transcription.
Placental tissue from day 18 pregnant CBA x C57 Bl6 mice was collected and snap-frozen. Total RNA was extracted using Tri Reagent, as described above. Final pellets were redissolved in 40 µl sterile water and treated with DNase as above. The concentration of RNA extracted from placental tissue was determined by spectrophotometric analysis at 260 nm.
Reverse Transcription
Total RNA was reverse transcribed to cDNA using Superscript II RNase H RT (Invitrogen). For embryos, total RNA from each pool (2055 blastocysts) was reverse transcribed. For placenta, ten 2-µg aliquots of RNA were reverse transcribed. RNA was initially incubated with random primers (250 ng) at 70°C for 10 min. Reverse transcription was then performed in a final volume of 25 µl containing First-Strand Buffer (50 mM Tris·HCl, pH 8.3, 75 mM KCl, and 3 mM MgCl2), 250 nM of each dNTP (Invitrogen), 0.01 M dithiothreitol, and 200 units Superscript II (Invitrogen). Before addition of the RT enzyme, reactions were incubated at 25°C for 10 min and then 42°C for 2 min. The RT was added, and reactions were incubated for 50 min at 42°C. The reaction was inactivated by incubation at 70°C for 15 min. Embryo cDNAs were stored at 20°C until use. Placental cDNAs were pooled, diluted to a concentration of cDNA equivalent to that generated from 25 ng starting RNA/µl, and stored in aliquots at 20°C.
Real-time PCR
Real-time PCR was performed in an Applied Biosystems 5700 GeneAmp Sequence Detection System (Applied Biosystems, Foster City, CA). Oligonucleotide primers were designed using Primer Express (Applied Biosystems) and were synthesized by GeneWorks (Adelaide, Australia). Primer sequence details are described in Table 1. PCR was performed in 20-µl volumes containing 1x SyBr Green Master Mix (Applied Biosystems) and 500 nM forward and reverse primers. cDNAs were diluted in water to a concentration per microliter equivalent to cDNA generated from 0.25 embryo. For IGF-II and the IGF-II receptor, 4 µl diluted cDNA were analyzed (1 embryo equivalent), for VEGF, 8 µl diluted cDNA were analyzed (2 embryo equivalents), and for 18S, 2 µl diluted cDNA were analyzed (0.5 embryo equivalent). The thermal cycling program consisted of 2 min at 50°C and 10 min at 95°C followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. A cycle threshold (Ct) was calculated for each sample using the GeneAmp 5700 software. Dissociation curves, to detect nonspecific amplification, were generated for all reactions, and the presence of a single amplified product was also confirmed by gel electrophoresis.
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Statistics
All statistical analyses were carried out using SPSS for Windows (SPSS). Data are presented as means ± SE. A minimum of five replicates for each experiment was used. All percentage data were subjected to arcsine transformation. Gene expression data in the rhFSH treatment groups only were subject to linear trend analysis. If no significant trend was found, the rhFSH groups were combined for subsequent analysis. All data were analyzed using a one-way ANOVA and a Bonferroni's or a least-significant difference test for multiple comparisons. A probability level of 5% (P < 0.05) was taken as significant for all analyses.
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RESULTS |
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There was a significantly greater total number of embryos recovered from mice treated with of 5.0, 10.0, or 20 IU rhFSH compared with the nontreated control group and the hCG only treated group (Table 2). The number of embryos recovered from the 5 IU eCG-treated mice was also significantly greater than the control and hCG only groups (Table 2). Furthermore, the number of embryos recovered from the 5 IU eCG-treated mice was the same as the 2.5 and 5.0 IU rhFSH groups but was significantly less than the 10.0 and 20.0 IU rhFSH treatment groups (Table 2). Of the total embryos recovered, those with fragmentation were classified as abnormal. There was a greater proportion of abnormal embryos collected from mice treated with 10.0 IU rhFSH, 20 IU rhFSH, and 5 IU eCG compared with the control group (Table 2).
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The in vitro development of morphologically normal presumptive zygotes to blastocysts was not affected by ovarian stimulation with 2.5 or 5.0 IU rhFSH or by ovulation induction by hCG compared with the development of embryos in the control group (Table 2). There was a significant reduction in the proportion of embryos to reach the blastocyst stage in embryos from mice treated with 10.0 or 20.0 IU rhFSH compared with embryos recovered from nontreated control mice (Table 2).
Differential Nuclear Staining
There was no effect of hCG and 2.5 or 10 IU rhFSH on total blastocyst cell number (Fig. 1A). The total cell number in blastocysts derived from 5.0 and 20.0 IU rhFSH- and eCG-treated mice was significantly reduced compared with blastocysts derived from nontreated control mice (Fig. 1A). The number of TE cells within blastocysts was also significantly reduced in the 20.0 IU rhFSH group (Fig. 1B); however, TE cell number was not altered in the 2.5 IU rhFSH, 5.0 IU rhFSH, 10.0 IU rhFSH eCG and hCG groups (Fig. 1B). There was a significant reduction in the number of ICM cells in the 5.0, 10.0, and 20.0 IU rhFSH groups and the eCG group compared with the untreated control group (Fig. 1C). There was, however, no significant effect of 2.5 IU rhFSH or hCG treatment on the number of ICM cells (Fig. 1C). There was a significant decrease in the ICM-to-total cell ratio in the 10.0 IU rhFSH and eCG groups compared with the control group (Fig. 1D). The ICM-to-total cell ratio was not altered in response to 2.5, 5.0, and 20.0 IU rhFSH or hCG (Fig. 1D).
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Although there was no significant effect of rhFSH treatment on the expression of the IGF-II gene in blastocysts, 5 IU eCG treatment significantly increased the expression of IGF-II in blastocysts compared with the control group (P < 0.05, Fig. 2A). Furthermore, there was no significant effect of ovarian stimulation on the expression of the IGF-II receptor compared with the nontreated control group (Fig. 2B). There was also no significant effect of ovarian stimulation on VEGF mRNA abundance in blastocysts derived from any ovarian stimulation treatment (Fig. 2C).
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DISCUSSION |
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Because rhFSH is not routinely used to induce superovulation in the mouse, we investigated the ovarian response to a range of rhFSH doses. We found that 2.5 IU rhFSH resulted in a basal level of stimulation, that the response to 5.0 IU rhFSH was three times greater than basal stimulation levels, and that a maximum ovarian response resulted from stimulation with 10 and 20 IU rhFSH.
In the present study, although high levels of rhFSH induced ovarian stimulation and produced large numbers of embryos, this was associated with an increased proportion of abnormal zygotes; however, the mechanisms responsible for this are not yet clear. Interestingly, the proportion of abnormal zygotes was not altered in response to 2.5 or 5.0 IU rhFSH, suggesting that there is a threshold in follicle stimulation required to produce this adverse effect. This suggests that the number of embryos produced by the higher levels of ovarian stimulation treatments has an impact on the developmental outcome of embryos, which probably reflects oocytes being recruited that would normally become atretic, as well as oocytes that are forced to mature ahead of time. We also found an increase in the proportion of abnormal zygotes recovered from eCG-stimulated mice compared with nontreated control mice, which is in agreement with previous studies that have recovered one-cell (24) or two-cell embryos (10) after eCG treatment. This has mainly been attributed to the long half-life of eCG and its high level of luteinizing hormone activity, which are thought to prematurely induce meiotic maturation of oocytes, resulting in the fertilization of meiotically aged oocytes (5, 30) that are often fragmented or abnormal in appearance.
The proportion of embryos developing in vitro was also reduced in embryos recovered from mice treated with 10 or 20 IU rhFSH, suggesting that development competence has been compromised after high levels of follicle stimulation. It has previously been reported that ovarian stimulation with eCG resulted in delayed and/or impaired (10, 11, 32) in vitro development of mouse embryos. Interestingly, we did not find any adverse effects of eCG treatment on in vitro blastocyst development, which conflicts with previous reports. The present study, however, is the first study to our knowledge to report that the in vitro development of mouse embryos is impaired after ovarian stimulation with recombinant FSH. It has been reported that exogenous gonadotropin treatment increases the frequency of chromosomal abnormalities (6, 7, 33), and it is possible that this underlies the impaired in vitro embryo development after rhFSH and eCG treatments.
Embryos recovered from mice treated with 5.0 and 20 IU rhFSH and eCG developed into blastocysts with significantly reduced cell numbers. The reduction in total cell number in the 20 IU rhFSH group appeared to be because of a decrease in both the number of ICM cells and TE cells, since the proportion of ICM cells to total cell number was not altered. In the 5.0 IU rhFSH and eCG groups, there was a reduction in the number of ICM cells only. These results indicate that different mechanisms may be responsible for the observed decrease in blastocyst cell numbers in the 20.0 IU rhFSH group compared with the 5.0 IU rhFSH and eCG groups, which may also explain why there was no observed decrease in blastocyst cell numbers in the 10 IU rhFSH group. It has previously been reported that the total blastocyst cell number and the number of ICM cells are positively correlated with fetal development after embryo transfer (18). The results of the present study suggest, therefore, that treatment with 5.0, 10.0, and 20.0 IU rhFSH, or eCG, results in embryos that may have a reduced developmental capacity posttransfer.
In the present study, we did not find any effects of rhFSH treatment on the expression of the IGF-II, IGF-II receptor, or VEGF genes in blastocysts compared with the control group. Therefore, it appears that, although some markers of embryo quality, such as blastocyst cell number, are altered in response to ovarian stimulation with rhFSH, other outcome measures, such as the expression of genes involved in developmental signaling pathways, such as IGF-II and its receptor, are largely unaltered. In contrast, ovarian stimulation induced by eCG increased IGF-II gene expression in blastocysts. It has previously been demonstrated, using antisense IGF-II oligonucleotides, that IGF-II plays an important role in cell number determination and the rate of progression to blastocyst in vitro (27). Because there is a reduction in cell number and a decreased in vitro blastocyst development in the eCG treatment group, the increased IGF-II expression in these blastocysts may be a compensatory mechanism by which embryos of suboptimal quality attempt to maintain appropriate levels of development. It has previously been reported that the gene expression of insulin-like growth factors in human embryos may be an indicator of embryonic growth potential and embryo quality (19). Furthermore, it has been demonstrated that in vitro culture of mouse embryos in different culture media affects the expression of IGF-II in blastocysts (14, 15). Interestingly, these studies found that suboptimal conditions were associated with a decrease in IGF-II expression, which contrasts with the present study, where an increase in IGF-II expression was found in the eCG treatment group. It is interesting to note that, in the studies by Ho et al. (14, 15), the in vivo control embryos were obtained from ovarian-stimulated mice treated with a combination of eCG and hCG. The decreased IGF-II expression in these studies was therefore in response to in vitro culture conditions. In contrast, in the present study, an increase in IGF-II expression was found in the eCG treatment group compared with control embryos. In this study, all embryos were exposed to the same in vitro culture conditions from the one-cell stage; however, the control embryos were obtained from natural ovulations in untreated control mice. Therefore, it appears that ovarian stimulation does alter embryo development, whereby treatment with eCG increases the expression of IGF-II in blastocysts. The relevance of these differences in gene expression in response to different stimuli, in terms of subsequent development, however, requires further investigation. It is interesting to speculate that, since IGF-II is an imprinted gene, eCG treatment may alter the methylation status or epigenetic regulation of imprinted genes in the oocyte, leading to alterations in subsequent embryo development. Because the parent of origin methylation marks are expressed in the genome of the mature oocyte, they may be susceptible to changes within the ovarian environment, such as ovarian stimulation. It has been demonstrated that manipulations of preimplantation embryos, such as in vitro culture, can alter the methylation status of imprinted genes and subsequently impact on fetal growth and development (8, 17, 20, 35). In future studies, therefore, it is important to determine if the alteration of imprinted genes by ovarian stimulation is a pathway by which oocyte and embryo quality is adversely affected.
It has previously been reported that ovarian stimulation with urinary human FSH decreased the mRNA levels of a specific VEGF isoform, VEGF120, at embryo implantation sites on embryonic days 5 and 6 and caused a delay in implantation (29). Although in the present study we measured total VEGF expression, we did not find any effect of the ovarian stimulation treatments on VEGF expression in blastocysts. These data suggest that ovarian stimulation may have a greater impact on VEGF expression in the endometrium and that the specific isoform VEGF120 may be more susceptible to ovarian stimulation compared with the total VEGF expression.
hCG was used to induce ovulation in the rhFSH and eCG groups, since it mimics the human situation where ovulation is routinely stimulated by hCG during IVF treatment. In the present study, we found that the proportion of abnormal zygotes in response to hCG was not different from the nontreated control group; therefore, it appears that the hCG treatment did not contribute to the increased proportion of abnormal embryos in the 10.0 and 20.0 IU rhFSH and eCG groups. There was also no difference in the in vitro development rates and blastocyst cell numbers between the hCG group and the control group; however, the hCG treatment group was also not different from the other gonadotropin treatment groups. The contribution of hCG to the adverse effects of rhFSH and eCG on preimplantation embryo development therefore cannot be ruled out.
In conclusion, ovarian stimulation with rhFSH and eCG impairs the in vitro development of preimplantation mouse embryos. We have found that both the developmental capacity and blastocyst cell numbers and allocation are altered by gonadotropin treatment. Ovarian stimulation induced by rhFSH did not significantly alter the expression of the genes for IGF-II, IGF-II receptor, or VEGF in blastocysts, indicating that the adverse effects of ovarian stimulation on embryo development were not as a result of differences in gene expression of the selected genes. IGF-II gene expression was increased, however, in the eCG treatment group. Further studies are required to determine if recombinant FSH has similar detrimental effects on embryo quality in species other than the mouse.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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