Department of Obstetrics and Gynecology, Showa University School of Medicine, 1-5-8, Hatanodai, Shinagawa-ku, Tokyo, 142-8666, Japan
1 To whom correspondence should be addressed. Email: hasejun{at}oak.dti.ne.jp
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Abstract |
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Key words: cumulus cells/follicular fluid progesterone/human embryo morphology/IVF/progesterone receptors
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Introduction |
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Zhang and Armstrong reported that only about 20% of oocytes from untreated rats were fertilized in vitro, in contrast to 70% of oocytes fertilized from FSH-treated rats, whereas 80% of oocytes underwent germinal vesicle breakdown, regardless of the treatments to donor rat. The addition of progesterone during in vitro maturation period duplicated the beneficial effect of FSH on fertilization rate. Although having no apparent effect on nuclear maturation of the oocyte, it is possible that gonadotrophin and ovarian steroids are important regulators, either directly and indirectly, of some of the cytoplasmic changes in oocytes that lead to the normal fertilization of the oocyte. Moreover, Mori et al. suggested that gap junction communications between the oocyte and cumulus cells might play an important role in regulating cytoplasmic maturation (Mori et al., 2000).
The recognition of oocyte maturation and regulation of cumulus cells, and the harvesting and culturing of numerous good-quality oocytes has clinical significance for improving fertility treatment. The aforementioned studies led to the objective of this study: developmental competence of human oocytes in IVF can be predicted from analysis of PR in cumulus cells surrounding mature oocytes. We examined the localization and expression of PR in human cumulus cells at the time of oocyte collection during IVF cycles via immunohistochemistry and real-time quantitative polymerase chain reaction (RTPCR).
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Materials and methods |
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IVF procedure
The ovaries were stimulated with clomiphene citrate (Clomid®; Shionogi, Osaka, Japan), human menopausal gonadotrophin (HMG; HMG Injection TEIZO®, Teikoku-zouki, Tokyo, Japan), pure FSH (Fertinorm P®; Serono, Tokyo, Japan), or a combination of the three; the stimulation was done after pituitary desensitization with gonadotrophin-releasing hormone agonist (GnRH-a; Suprecure; Aventis Pharma, Tokyo, Japan) according to the long protocol. Ovarian follicle diameter was assessed by transvaginal sonography, and gonadotropins were administered daily until the second largest follicle reached a diameter of 18 mm. When the follicle grew beyond that diameter, (HCG; Gonatropin®; Teikoku-zouki, Tokyo, Japan) 10000 IU or GnRH-agonist 600 µg was administered; 35 h later, oocytes were retrieved under ultrasonographic guidance. Prior to oocyte retrieval, the follicular diameter was measured by ultrasonography and follicular fluid was carefully collected from each mature follicle without any contamination with flushing medium. Serum and follicular fluid samples were stored at 20°C prior to hormone measurements. Cumulus cells were manually separated from COC under a microscope after oocyte retrieval.
Oocyte and embryo culture
Oocytes were cultured in dishes with 500 µl P-1TM medium (preimplantation-1 medium; Irvine Scientific) under mineral oil with 10% human serum at 37°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2. Oocytes underwent conventional IVF or ICSI, depending on the patient's specific needs.
Embryo quality assessment
Three days after insemination, embryo quality (EQ) was investigated under a microscope with a micro-manipulator. EQ was categorized as good morphology group if the number of blastomeres was greater than seven cells with less than 5% fragmentation.
Serum and follicular fluid analysis
Follicular fluid concentrations of estradiol (E2), progesterone and LH were determined by a commercial enzyme-linked immunosorbent assay kit (AIA-600II, TOSOH, Tokyo, Japan). After determining the follicular fluid steroid concentration, the follicular fluid was diluted 1:1000 in steroid-free serum before the measurements.
Immunohistochemistry
Formalin-fixed, paraffin-embedded tissue was cut into 3 µm sections. The sections were then deparaffinized in xylene three times for 5 min each, and fixed in 100% ethanol three times for 5 min each, and then washed in water for 5 min. Endogenous peroxidase activity was blocked with a methanol solution, which included 3% H2O2, for 20 min at room temperature. To activate antigens, the sections were autoclaved in 10 mM sodium citrate buffer (pH 6.0) at 121°C for 15 min. The sections were rinsed in phosphate-buffered saline (PBS) at room temperature and incubated with primary antibodies at 4°C overnight. We used primary antibodies as follows: anti-human PR-A mouse monoclonal antibody (PR Ab7; Neo Markers, Foremont, CA); and anti-human PR-B mouse monoclonal antibody (PR Ab2; Neo Markers). The following day, the slides were washed in PBS for three times for 5 min each and treated for 30 min at room temperature with Histofine Simple StainPO(M) (Nichirei Corporation, Tokyo, Japan), which is a labelled polymer prepared by combining amino acid polymers with peroxidase and goat anti-mouse immunoglobulin reduced to Fab' fragment. The slides were washed in PBS for three times for 5 min each, and AEC (3-amino-9-ethyl carbazole) was used for colour development. Nuclei were counterstained with Mayer's hematoxylin. Normal proliferative endometrium was used as a positive control for PR-A and PR-B. Negative control sections, which had not been exposed to primary antibody, were included in all staining runs. Staining was assessed using a consulting microscope (Model BX51; Olympus, Tokyo, Japan).
RNA preparation and reverse transcription, and real-time quantitative TaqMan PCR
Total RNA was isolated from cumulus cells by phenolchloroform extraction. The cDNAs were synthesized from 5 µg of total RNA using random hexamer and Superscript II reverse transcriptase at 55°C for 30 min and 99°C for 5 min. Five microlitres of this reaction were analysed by RTPCR.
RTPCR was performed using TaqMan PCR® (ABI PRISM 7700 Sequence Detection System; PerkinElmer Applied Biosystems, CA, USA). Because PR-A has no specific sequence to distinguish it from PR-B mRNA, PR-A mRNA cannot be distinguished from PR-B by RTPCR. Therefore, we used an intersectional primer (Hs00172183_m1, Assay on demand Gene Expression probes; Applied Biosystems, CA) of PR-A and PR-B. glyceraldehyde-3-phosphatedehydrogenase (GAPDH) cDNA fragments were amplified as positive controls. Two RTPCR reactions were performed with each sample.
Statistics
Comparisons between the groups were made by MannWhitney's U-test, and relationships between PR/GAPDH and follicular fluid concentrations were analysed by linear regression. For all analyses, P<0.05 was considered statistically significant.
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Results |
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Immunohistochemistry
In cross-section, immunoreactive PR-A was mainly localized in the cytoplasm of 58 (75%) of 77 human cumulus cells (Figure 1a). Immunoreactive PR-B was localized in the nuclei of 44 (62%) of 71 human cumulus cells in cross-section. No cytoplasmic staining was found for PR-B (Figure 1b).
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Discussion |
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It is known that premature luteinization is related to low ovarian reserve (Younis et al., 2001). Premature luteinization is defined as an early rising of the serum concentration of progesterone (progesterone/E2>1) before the LH surge. In our study, because follicular fluid concentrations of progesterone at the time of oocyte collection were fully affected by LH, we were unable to determine a correlation between premature luteinization and follicular fluid progesterone levels.
In this study, follicular fluid concentrations of progesterone were higher in the good morphology group than the other group (Table III). Chian et al. reported that during in-vitro meiotic maturation of COC in humans, progesterone was produced by the cumulus cells, and that the level of progesterone was increased by stimulation with LH, FSH, or forskolin (Chian et al., 1999). Moreover, it has been demonstrated that significantly increased concentrations of progesterone were observed in COCs cultured with LH and FSH for 28 h (Shimada and Terada, 2002a
). Other researchers have compared the follicular fluid steroid concentrations during the maturation of the morphological COCs and reported that the follicular fluid concentrations of progesterone in the mature COCs were significantly higher than they were in the immature stage (Botero-Ruiz et al., 1984
; De Sutter et al., 1991
). Furthermore, it has been reported that progesterone/E2 ratios were higher in follicles whose oocytes were fertilized (Enien et al., 1995
), that higher progesterone values associated with oocyte capable of fertilization by ICSI (Mendoza et al., 1999
), and that two pronuclei zygotes that failed to undergo cleavage developed from oocytes that were harvested from follicles with lower concentrations of progesterone (Mendoza et al., 2002
). However, it has been reported that there was no significant difference between the follicular fluid concentration of progesterone and either EQ at day 3 of an IVF (Hasegawa et al., 2003
) or the pregnancy rate (Andersen, 1993
). It is questionable whether higher follicular fluid concentrations of progesterone accurately reflect either oocyte quality or the capacity for embryo development. Differences in ovarian stimulation protocols also may introduce increased variability (Mendoza et al., 1999
). However, regardless of progesterone concentration, good embryos were retrieved from COCs whose PR expressions were lower than the others (Table IV). It has been reported that the luteal phase of the cycle is marked by a sharp decrease in PR-B expression that precedes the decrease in PR-A by several days (Brandon et al., 1993
). PR-A mRNA appears to persist longer than PR-B mRNA for 24 h in LH-stimulated rat granulosa cells (Natraj and Richards, 1993
). A recent study also reported that the level of PR B m-RNA in cumulus cells was up-regulated by FSH and LH during the first 8 h of cultivation and then significantly decreased at 12 h; furthermore, PR-A predominated at 20 h, and high level of total PR mRNA was maintained up to a cultivation period of 20 h in porcine oocytes (Shimada et al., 2004b
). In the present study, PR expression was neither significantly different between the fertilized group and the unfertilized group nor between the group which cleaved and group which did not cleave. However, PR expression was lower in the good morphology group than in the other group. In view of these results and previous studies, we suggest that full reduction of PR expression in cumulus cells at the time of oocyte collection (which may be transiently stimulated by LH) may be essential to acquire developmental competence of the human oocyte, and that oocyte maturation is controlled by the expression and the reduction of PRs in humans just as it is in other mammalian oocytes.
It has been reported that progesterone-induced Xenopus oocyte maturation is mediated via an extranuclear receptor, and is independent of gene transcription (Bayaa et al., 2000). Other investigators have reported that PRs were increased mainly in the cytoplasm during the proliferative phase. In the early luteal phase, PRs decreased in the cytosol; however, they remained high in the nuclei (Bayard et al., 1978
; Pollow et al., 1981
). In the present study, we found for the first time that PR-A was localized in cytoplasm and PR-B was localized in nuclei of human cumulus cells by the technique of immunohistochemistry in IVF cycles. Shimada et al. suggested that the induction of PR isoforms in cumulus cells and their binding to progesterone appear to have an impact on proliferation and differentiation in a time-dependent manner, and a shift from PR-B to PR-A may help mediate certain events (Shimada et al., 2004b
). These previous studies and our results support the theory that time-dependent expression of PR isoforms and the distinction of localization between PR-A and PR-B affect follicular growth, oocyte maturation, and early embryo development.
In conclusion, regardless of follicular fluid steroid or LH concentration at the time of oocyte collection, good morphology embryos were retrieved from COCs whose PR expressions were lower than the others. Our findings indicate that the full reduction of total PR expression may be essential to acquire developmental competence in the human. Although it could not be specifically demonstrated in this study, selective contribution of the two PR isoforms to progesterone action might affect oocyte maturation. It is possible that expression and reduction of PRs and localization of PR isoforms transiently stimulated by LH might regulate oocyte maturity and embryo development.
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Acknowledgements |
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References |
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Submitted on November 18, 2004; resubmitted on February 23, 2005; accepted on February 25, 2005.
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