Age-related decline in the expression of GDF9 and BMP15 genes in follicle fluid and granulosa cells derived from poor ovarian responders.

BACKGROUND: Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) genes play important roles in folliculogenesis. Altered expression of the two have been found among patients with poor ovarian response (POR). In this prospective cohort study, we have determined the expression of the GDF9 and BMP15 genes in follicle fluid (FF) and granulosa cells (GCs) derived from poor ovarian responders grouped by age, and explored its correlation with the outcome of in vitro fertilization and embryo transfer (IVF-ET) treatment. METHODS: A total of 196 patients with POR were enrolled from a tertiary teaching hospital. The patients were diagnosed by the Bologna criteria and sub-divided into group A (< 35 year old), group B (35-40 year old), and group C (> 40 year old). A GnRH antagonist protocol was conducted for all patients, and FF and GCs were collected after oocyte retrieval. Expression of the GDF9 and BMP15 genes in the FF and GCs was determined with enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. RESULTS: Compared with group C, groups A and B had significantly more two pronuclei (2PN) oocytes and transplantable embryos, in addition with higher rates of implantation and clinical pregnancy (P <  0.05). The expression level of GDF9 and BMP15 genes in the FF and GCs differed significantly among the three groups (P <  0.05), showing a trend of decline along with age. The ratio of GDF9/BMP15 mRNA levels were similar among the three groups (P > 0.05). The relative levels of GDF9 and BMP15 proteins in GCs have correlated with the relative mRNA levels in GCs and protein concentrations in FF (P <  0.05). CONCLUSIONS: For poor ovarian responders, in particular those over 40, the expression of GDF9 and BMP15 is declined along with increased age and in accompany with poorer oocyte quality and IVF outcome, whilst the ratio of GDF9/BMP15 mRNA levels remained relatively constant. TRIAL REGISTRATION: Chinese Clinical Trial Registry Center ( ChiCTR1800016107 ). Registered on 11 May 2018.

Improved developmental competence in embryos treated with lycopene during in vitro culture system.

In vitro embryo development remains suboptimal compared to in vivo development due to the challenge from various stressors associated with in vitro culturing of oocytes. When 0.2 µM lycopene was added to oocyte in vitro maturation and embryo culture media, to assess its antioxidant effects on embryo development, we observed a significant (p < 0.05) increase in cleavage and blastocyst development rates compared to the corresponding controls (84.3 ± 0.6% vs. 73.1 ± 1.9% and 41.0 ± 1.4% vs. 33.4 ± 0.7%, respectively). Lycopene also significantly reduced (p < 0.05) intracellular reactive oxygen species concentrations in oocytes and blastocysts, whereas lipid peroxidation and mitochondrial activity increased compared to control conditions. The number of apoptotic nuclei was significantly reduced in the lycopene-treated compared to the control group (1.7 ± 0.1 vs. 4.7 ± 0.3), and the quantity of cells in the trophectoderm (207.1 ± 1.6 vs. 171.3 ± 1.0, respectively) and inner cell mass (41.9 ± 0.4 vs. 36.7 ± 0.4, respectively) was higher following treatment-although the inner cell mass-to-trophectoderm ratio was unchanged (1:3.3 vs. 1:3.4 for lycopene vs. control, respectively). Lycopene supplementation also significantly (p < 0.05) attenuated expression of IKBKB (Inhibitor of nuclear factor kappa B kinase, subunit beta) and reduced Caspase 9 and Caspase 3 protein abundance, while up-regulating GDF9 (Growth and differentiation factor 9), BMP15 (Bone morphogenetic protein 15), SOD2 (Superoxide dismutase 2), NDUFA2 (NADH dehydrogenase), ACADL (Acyl-CoA dehydrogenase, long chain), and ACSL3 (Acyl-CoA synthetase 3, long-chain membrane 3) transcription compared to control. Therefore, co-culturing with lycopene during oocyte maturation improved bovine embryo developmental potential during in vitro culture by improving embryonic resilience to stress.

Fibroblast growth factor 10 markedly improves in vitro maturation of porcine cumulus-oocyte complexes.

Growth factors synthesized by ovarian somatic cells affect cumulus cell expansion and oocyte maturation in vitro. Fibroblast growth factor 10 (FGF10), for example, is a known regulator of mammalian cumulus-oocyte complex maturation. In this study, we investigated the effects of 0, 5, 10, 50, and 100 ng/mL FGF10 (5F, 10F, 50F, and 100F, respectively) on in vitro cumulus cell expansion, oocyte maturation, and embryo development. The percentage of fully expanded cumulus cells at the oocyte's metaphase-II (MII) stage was significantly higher in the 10F-treated group than in the control. Transcript abundance of the cumulus cell expansion-related gene encoding hyaluronian synthase 2 (HAS2) in cumulus cells at oocyte germinal vesicle breakdown (GVBD) was significantly higher in the 10F- and 50F-treated groups compared to untreated controls, whereas the mRNA abundance of the protease cathepsin B (CTSB) at the oocyte MII stage was remarkably decreased in the 10F-treated group. The percentage of oocytes with normal spindles was greater in the 10F- and 50F-treated group at GVBD than in the other groups; the 5F-, 10F-, and 100F-treated groups were higher than the control; and the 50F-treated group was highest at MII. The abundance of GDF9 and BMP15 transcript at GVBD and BMP15 and CCNB1 transcripts at MII increased in the 10F-treated group. Cleavage rate, blastocyst formation rate, and total cell number were significantly higher in the 5F- to 50F-treated groups. These results demonstrate that FGF10 markedly improves cumulus cell expansion, oocyte maturation, and subsequent embryo development. Mol. Reprod. Dev. 84: 67-75, 2017. © 2016 Wiley Periodicals, Inc.

bmp15l, figla, smc1bl, and larp6l are preferentially expressed in germ cells in Atlantic salmon (Salmo salar L.).

Atlantic salmon is a valuable commercial aquaculture species that would benefit economically and environmentally by controlling precocious puberty and preventing escapees from reproducing with wild populations. One solution to both these challenges is the production of sterile individuals by inhibiting the formation of germ cells, but achieving this requires more information on the specific factors that control germ cell formation. Here, we identified and characterized novel factors that are preferentially expressed in Atlantic salmon germ cells by screening for gonad-specific genes using available adult multi-tissue transcriptomes. We excluded genes with expression in tissues other than gonads based on quantity of reads, and then a subset of genes was selected for verification in a multi-tissue PCR screen. Four gonad-specific genes (bmp15l, figla, smc1bl, and larp6l) were chosen for further characterization, namely: germ cell specificity, investigated by comparing mRNA abundance in wild-type and germ cell-free gonads by quantitative real-time PCR, and cellular location, visualized by in situ hybridization. All four genes were expressed in both testis and ovary, and preferentially within the germ cells of both sexes. These genes may be essential players in salmon germ cell development, and could be important for future studies aiming to understand and control reproduction. Mol. Reprod. Dev. 84: 76-87, 2017. © 2016 Wiley Periodicals, Inc.

Effect of anti-Mullerian hormone in culture medium on quality of mouse oocytes matured in vitro.

Anti-mullerian hormone (AMH) is thought to reflect the growth of follicles and the ovarian function. However, the role of AMH in culture medium during in vitro maturation (IVM) on oocyte quality and subsequent development potential is unclear. The objective of this study is to investigate the effect of recombinant human AMH (rh-AMH) supplemented into IVM medium on oocyte quality. Cumulus-oocyte complexes (COCs) were obtained from ICR mice and cultured in vitro with the different concentrations (0-1,000 ng/ml) of rh-AMH. Following 16-18 h of culture, quantitative PCR and ELISA were performed to analyze GDF9 and BMP15 mRNA expression and protein production from the oocytes. Subsequently, in vitro fertilization (IVF) and early embryonic development were employed to further evaluate the quality of in vitro matured oocytes. The results showed that AMH was only expressed in cumulus cells but not in the oocytes. However, AMH most specific receptor, AMHR-II, was expressed in both oocytes and cumulus cells. The levels of GDF9 and BMP15 expression and blastocyst formation rate were significantly increased (p<0.05) when the IVM medium was supplemented with 100 ng/ml of rh-AMH. With AdH1-SiRNA/AMH for knocking down of AMH expression during IVM significantly reduced (p<0.05) the levels of GDF9 and BMP15 expression and blastocysts formation rate. These results suggest that AHM improves oocytes quality by up-regulating GDF9 and BMP15 expressions during IVM.

Molecular characterization of gdf9 and bmp15 genes in rare minnow Gobiocypris rarus and their expression upon bisphenol A exposure in adult females.

Growth differentiation factor 9 (Gdf9) and bone morphogenetic protein 15 (Bmp15) are members of transforming growth factor ß (TGFß) superfamily that plays important roles in regulating ovarian functions. We cloned the cDNAs of gdf9 and bmp15 in rare minnow Gobiocypris rarus. The full length cDNAs of gdf9 and bmp15 were 1999 and 1721 bp, encoding 431 and 384 amino acids respectively. They both contained conserved TGFß superfamily domain, with six conserved cysteine residues. Tissue distribution showed that both gdf9 and bmp15 are highly expressed in the G. rarus ovary. Following bisphenol A (BPA) treatment, ovarian transcripts of gdf9 and bmp15 together with the gonadosomatic index and the ovarian histology were altered. It suggests that the altered gdf9 and bmp15 expression may play roles in the weight gain and abnormal development of the ovary following BPA exposure.

Growth and antrum formation of bovine primary follicles in long-term culture in vitro.

Successful antral formation in vitro from bovine preantral follicles (145-170 µm) has been described previously, but antrum formation from the primary follicle (50-70 µm) has not yet been achieved in vitro. The aim of the study was to establish an optimal culture system supporting the growth and maturation of bovine primary follicles (50-70 µm) in vitro. Bovine primary follicles were cultured in a three-dimensional culture system for 13 or 21 days in alpha-minimum essential medium. Various treatments including follicle stimulating hormone (FSH), luteinizing hormone (LH), 17ß-estradiol (E2), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were tested. The follicular diameter and antrum formation rate were recorded, and follicular maturation markers (P450 aromatase, CYP19A1; anti-Mullerian hormone, AMH; growth differentiation factor-9, GDF9; bone morphogenetic protein-15, BMP15; and type III transforming growth factor ß receptor, TGFßR3) were analyzed by real-time RT-PCR. After 21 days of culture under each treatment condition, the follicular diameter was significantly enlarged in the presence of FSH + LH + E2 + bFGF or FSH + LH + E2 + bFGF + EGF (p<0.05). An addition of 50 ng/ml bFGF or bFGF +25 ng/ml EGF initiated antrum formation by day 19 and day 17 of culture, and the antral cavity formation rate was 16.7% and 33.3% by 21 days of culture, respectively. The expression of follicular maturation markers (CYP19A1, AMH, GDF9, BMP15 and TGFßR3) was significantly altered. We conclude that addition of 50 ng/ml bFGF +25 ng/ml EGF to media containing FSH + LH + E2 turned out to be the most effective optimized culture conditions to support the growth and maturation of bovine primary follicles in vitro.

Inhibitory effects of controlled ovarian stimulation on the expression of GDF9 and BMP15 in oocytes from women with PCOS.

PURPOSE: To explore the effects of controlled ovarian stimulation (COS) on the expression of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in oocytes and granulosa cells from patients with or without polycystic ovary syndrome (PCOS). METHODS: This case-control study was conducted in the university affiliated hospital. The study comprised four groups of patients: eighteen PCOS patients with COS (stimulated-PCOS) and twenty-two PCOS patients without COS (unstimulated-PCOS), twenty-nine normal ovulatory women with COS (stimulated-control) and twenty-eight normal ovulatory women without COS (unstimulated-control). The oocytes and granulosa cells were collected and the abundance of GDF9 and BMP15 mRNA in the cells were detected by nested quantitative real-time PCR. RESULTS: The abundance of GDF9 and BMP15 mRNA was significantly higher both in oocytes (P < 0.01, P < 0.001, respectively) and GCs (P < 0.01, P < 0.05, respectively) from stimulated-control group than in unstimulated-control group. However, there was no significant difference for GDF9 or BMP15 mRNA in oocytes from stimulated-PCOS goup compared with unstimulated-PCOS group (P > 0.05, P > 0.05, respectively). The abundance of GDF9 mRNA was significantly lower (P < 0.01) while the abundance of BMP15 mRNA was significantly higher (P < 0.001) in GCs from stimulated-PCOS group than in unstimulated-PCOS group. CONCLUSIONS: The controlled ovarian stimulation can promote the expression of GDF9 and BMP15 both in oocytes and GCs from normal ovulatory women. However, the stimulating effects may be inhibited in oocytes from PCOS patients, which subsequently impair cytoplasm maturation and lead to poor oocyte quality.

Species differences in the expression and activity of bone morphogenetic protein 15.

Oocyte-derived bone morphogenetic protein 15 (BMP15) regulates ovulation rate and female fertility in a species-specific manner, being important in humans and sheep and largely superfluous in mice. To understand these species differences, we have compared the expression and activity of human, murine, and ovine BMP15. In HEK293F cells, human BMP15 is highly expressed (120 ng/ml), ovine BMP15 is poorly expressed (15 ng/ml), and murine BMP15 is undetectable. Because BMP15 synthesis is dependent upon interactions between the N-terminal prodomain and the C-terminal mature domain, we used site-directed mutagenesis to identify four prodomain residues (Glu(46), Glu(47), Leu(49), and Glu(50)) that mediate the high expression of human BMP15. Substituting these residues into the prodomains of murine and ovine BMP15 led to significant increases in growth factor expression; however, maximal expression was achieved only when the entire human prodomain was linked to the mature domains of the other species. Using these chimeric constructs, we produced and purified murine and ovine BMP15 and showed that in a COV434 granulosa cell bioassay, these molecules displayed little activity relative to human BMP15 (EC(50) 0.2nM). Sequence analysis suggested that the disparity in activity could be due to species differences at the type I receptor binding interface. Indeed, murine BMP15 activity was restored when specific residues through this region (Pro(329)/Tyr(330)) were replaced with the corresponding residues (Arg(329)/Asp(330)) from human BMP15. The identified differences in the expression and activity of BMP15 likely underlie the relative importance of this growth factor between species.

Regulatory role of kit ligand-c-kit interaction and oocyte factors in steroidogenesis by rat granulosa cells.

Although kit ligand (KL)-c-kit interaction is known to be critical for oogenesis and folliculogenesis, its role in ovarian steroidogenesis has yet to be elucidated. We studied the impact of KL-c-kit interaction in regulation of steroidogenesis using rat oocyte/granulosa cell co-culture. In the presence of oocytes, soluble KL suppressed FSH-induced estradiol production and aromatase mRNA expression without affecting FSH-induced progesterone production. The KL effect on steroidogenesis was interrupted by an anti-c-kit neutralizing antibody, suggesting that KL-c-kit interaction is involved in suppression of estrogen by granulosa cells through oocyte c-kit action. The cAMP-PKA pathway activity was not directly involved in the estrogen regulation by KL-c-kit action. It was of note that KL treatment increased the expression levels of oocyte-derived FGF-8, GDF-9 and BMP-6, while it reduced the expression levels of oocyte-derived BMP-15 in the oocyte-granulosa cell co-culture. Given the findings that FGF-8, but not GDF-9, BMP-6 or -15, suppressed FSH-induced estrogen production by granulosa cells, oocyte-derived FGF-8 is linked to suppression of FSH-induced estrogen production through the KL-c-kit interaction. Furthermore, the suppression of FSH-induced estrogen production by KL in the co-culture was reversed by a FGF receptor kinase inhibitor and the effect of the inhibitor was enhanced in combination with extracellular-domain protein of BMPRII, which interferes with BMP-15 and GDF-9 activities. Thus, the actions of endogenous oocyte factors including FGF-8 and BMP-15/GDF-9 were involved in the KL activity that inhibited FSH-induced estradiol production. Collectively, the results indicate that KL-c-kit interaction plays a role in estrogenic regulation through oocyte-granulosa cell communication.

Quantitative analysis of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) gene expression in calf and adult bovine ovaries.

BACKGROUND: It has been reported that calf oocytes are less developmentally competent than oocytes obtained from adult cows. Bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) play critical roles in folliculogenesis, follicular development and ovulation in mammalian ovaries. In the present study, we attempted to compare the expression patterns of BMP15 and GDF9 in the cells of calf and cow ovaries to determine a relationship between the level of these genes and the low developmental competence of calf oocytes. METHODS: Bovine tissues were collected from 9-11 months-old calves and from 4-6 years-old cows. We characterized the gene expression of BMP15 and GDF9 in calf and adult bovine oocytes and cumulus cells using quantitative real-time reverse transcriptase polymerase chain reaction (QPCR) and in situ hybridization. Immunohistochemical analysis was also performed. RESULTS: The expression of BMP15 and GDF9 in cumulus cells of adult ovaries was significantly higher than that in calf ovaries, as revealed by QPCR. GDF9 expression in the oocytes of calf ovaries was significantly higher than in those of the adult ovaries. In contrast, BMP15 expression in the oocytes of calf and adult ovaries was not significantly different. The localization of gene expression and protein were ascertained by histochemistry. CONCLUSIONS: Our result showed for the first time BMP15 and GDF9 expression in bovine cumulus cells. BMP15 and GDF9 mRNA expression in oocytes and cumulus cells was different in calves and cows.

Different follicle-stimulating hormone exposure regimens during antral follicle growth alter gene expression in the cumulus-oocyte complex in mice.

Follicle-stimulating hormone (FSH) and oocyte-secreted factors influence granulosa cell differentiation and follicle development. Whereas FSH stimulates the expression of mural cell transcripts, oocyte-secreted factors regulate specific cumulus cell genes and suppress the appearance of mural cell transcripts. This study addresses the extent to which clinically relevant changes in FSH doses applied during antral follicle development in vitro could alter the expression of oocyte and cumulus cell transcripts. A 12-day culture system in which mouse ovarian preantral follicles can grow to preovulatory follicles was used. The following three FSH regimens were considered: 1) continuous exposure to an FSH level of 10 mIU/ml (control), 2) decreasing concentrations of FSH (low FSH), and 3) an FSH level of 25 mIU/ml (high FSH) as soon as the antrum is formed. Transcripts in oocytes (Gdf9, Bmp15, and Fgf8) and in cumulus cells (Amh, Lhcgr, Ar, and Pfkp) were quantified by real-time PCR. Under high FSH, the three oocyte transcripts were upregulated, while in cumulus cells a shutdown of the Amh signal and substantial increases in Lhcgr and Ar expression were measured. In contrast, low FSH tended to reduce Lhcgr to levels comparable to those in vivo. Levels of Pfkp were not affected by FSH doses. These results demonstrate that a 2.5-fold increase in FSH changes both oocyte and cumulus cell transcript levels. Conversely, a decrease in FSH does not affect transcript levels but seems to limit inappropriate Lhcgr expression. Modulating FSH within physiological ranges during the antral phase of culture alters cumulus cell differentiation.

Expression of growth differentiation factor-9 and bone morphogenetic protein-15 in oocytes and cumulus granulosa cells of patients with polycystic ovary syndrome.

OBJECTIVE: To evaluate the effect of growth differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15) on the development of follicles among patients with polycystic ovary syndrome (PCOS). DESIGN: Case-control study. SETTING: University Hospital. PATIENT(S): Twenty-two oocytes were obtained from 15 patients with PCOS and 67 oocytes from 58 controls. Cumulus granulosa cells (GC) were obtained from 16 patients with PCOS and controls treated with intracytoplasmic sperm injection. INTERVENTION(S): Immunofluorescence combined with laser scanning confocal microscopy and immunocytochemistry were used to analyze the expression of GDF-9 and BMP-15 in oocytes and cumulus GCs. MAIN OUTCOME MEASURE(S): To detect the protein expression levels. RESULT(S): No significant difference was found in the expression of GDF-9 and BMP-15 in the oocytes and BMP-15 in the cumulus GCs of patients with PCOS and controls. However, the expression of GDF-9 in cumulus GCs of patients with PCOS was decreased significantly compared with controls (8.88 +/- 1.52 vs. 5.01 +/- 0.83). CONCLUSION(S): The expression of GDF-9 and BMP-15 in the oocytes of patients with PCOS who received ovulation induction treatment was in the normal range, but the GDF-9 expression in cumulus GCs from patients with PCOS was significantly lower than the normal. Reduced GDF-9 expression in cumulus GCs of patients with PCOS appears to be associated with decreased long-term developmental potential of the oocytes of patients with PCOS.

Regulation of membrane progestin receptors in the zebrafish ovary by gonadotropin, activin, TGF-beta and BMP-15.

Progestin hormones are vital for inducing oocyte maturation in fish by binding to membrane progestin receptors (mPRs). The aim of this study was to examine the expression and regulation of mPRalpha and mPRbeta in zebrafish follicles. First, defolliculated fully grown oocytes were subjected to immunofluorescent staining using anti-mPRalpha and mPRbeta antibodies, and their expression on the oocyte membrane was confirmed. Second, total protein was collected from zebrafish follicles and Western blotting revealed that the level of mPRalpha and mPRbeta increased with follicle development. We have previously shown that several members of the transforming growth factor-beta (TGF-beta) superfamily, including TGF-beta1, activin-A, and bone morphogenetic protein (BMP)-15, regulate oocyte maturation in zebrafish. Therefore, the third major focus of this study was to test if these growth factors, as well as gonadotropins, regulate the expression of mPRs. Overexpression of BMP-15 significantly reduced, while knockdown of BMP-15 increased, mPRbeta levels. However, mPRalpha expression level remained unchanged with BMP-15 overexpression or knockdown. Treatment of follicles with human chorionic gonadotropin (hCG) resulted in an increased in mPRbeta, but not mPRalpha, expression levels. Activin-A induced the expression of mPRalpha and mPRbeta in a dose- and time-dependent manner. On the other hand, TGF-beta1 treatment suppressed the expression of mPRbeta, but not mPRalpha. Taken together, these findings further support the role of mPRs in oocyte maturation and suggest that gonadotropins, BMP-15, activin-A, and TGF-beta1 exert their regulatory effects on oocyte maturation in part by regulating mPR expression.

Improvement of in vitro oocyte maturation with lectin supplementation and expression analysis of Cx43, GDF-9, FGF-4 and Fibronectin mRNA transcripts in Buffalo (Bubalus bubalis).

PURPOSE: To optimize In vitro maturation (IVM) of quality oocytes for embryo production through IVF and SCNT. METHODS: Buffalo oocytes were in vitro matured in the presence of the pokeweed lectin (Phytolacca americana), a potent lymphocyte mitogen. Lectin was supplemented in TCM + 10% FBS at the doses of 0, 1, 5, 10, 15, 20 and 40 microg/ml and cumulus expansion and gene expression patterns were characterized. RESULTS: The degree of cumulus expansion in different lectin treatment levels improved from 1.1 at 1 Ag/ml level to 3.60 at 10 microg/ml level and then decreased in higher concentration 20 microg/ml (1.66) and 40 microg/ml (0.64). IVF embryos showed highest cleavage rate (88.8%) in 10 microg/ml lectin treatment. Expression of all mRNA transcript studied (Cx43, GDF 9, FGF-4 and Fibronectin) was positively correlated with cumulus expansion and polar body extrusion. CONCLUSIONS: Mitogenic lectin supplemented maturation media improves oocyte quality for in vitro embryo production.

Oogenesis specific genes (Nobox, Oct4, Bmp15, Gdf9, Oogenesin1 and Oogenesin2) are differentially expressed during natural and gonadotropin-induced mouse follicular development.

Using a semi-quantitative, single-cell sensitive RT-PCR method, we studied the expression of oogenesis specific genes (Nobox, Oct4, Bmp15, Gdf9, Oogenesin1 and Oogenesin2) in single oocytes collected from primordial, primary, secondary, preantral and antral follicles during natural and gonadotropin-induced mouse follicular development. We compared the number of transcripts of these genes, showing that they are differentially expressed, both in natural conditions and under gonadotropin-induction throughout the assessed developmental stages. Our data show a clear increase in the number of transcripts between the primordial until the preantral stages, with the exception of the Oogenesin1 transcripts under gonadotropin-induction. The number of transcripts starts decreasing at the antral stage and proceeds until the metaphase II stage, with values very similar to those obtained for the primordial oocytes in both analyzed conditions. Under exogenous gonadotropin-induction, oocyte recruitment to ovulation at the preantral stage is marked by an increase in Nobox and Oogenesin2 gene expression that is concomitant with a decrease in Oogenesin1 gene expression. Oocytes that are able to proceed into whole embryo development show a tight regulation of Nobox and Oct4 expression at the antral stage. A parallel immunocytochemical study at the protein level corroborates these findings.

BMP15 mutations associated with primary ovarian insufficiency cause a defective production of bioactive protein.

Bone morphogenetic protein-15 (BMP15) is selectively synthesized by oocytes as a pre-proprotein and is considered an ovarian follicle organizer whose adequate function is critical for female fertility. Missense mutations were reported in primary ovarian insufficiency (POI) but their biological impact remained unexplored. Here, screening of 300 unrelated idiopathic overt POI women with primary or secondary amenorrhea (SA) led to the identification of six heterozygous BMP15 variations in 29 of them. All alterations are nonconservative and include one insertion of three nucleotides (p.L262_L263insL) and five missense substitutions. Except for the p.S5R located in the signal sequence, the other variants (p.R68W, p.R138H, p.L148P, and p.A180T) localize in the proregion, which is essential for the processing and secretion of bioactive dimers. The mutations p.R68W, p.L148P, and the novel p.R138H lead to marked reductions of mature protein production. Their biological effects, evaluated by a novel luciferase-reporter assay in a human granulosa cell (GC) line, were significantly reduced. Cotransfection experiments of defective mutants with equal amounts of wild-type BMP15 cDNA, thus reproducing the heterozygous state seen in patients, did not generate a complete recovery of wild-type activity. No or minor deleterious effects were detected for the variants p.L262_L263insL, p.A180T, or p.S5R. In conclusion, heterozygous BMP15 mutations associated with the early onset of overt POI lead to defective secretion of bioactive dimers. These findings support the concept that an adequate amount of BMP15 secreted in the follicular fluid is critical for female fertility. We propose to consider the screening of BMP15 mutations among the analyses for the prediction of POI risk.

Fibroblast growth factor 7 stimulates in vitro growth of oocytes originating from bovine early antral follicles.

Essential factors required for growing oocytes derived from bovine early antral follicles and their mechanisms of action are poorly understood. Fibroblast growth factor 7 (FGF7) is a member of the heparin-binding FGF family with a distinctive pattern of target-cell specificity. The effect of FGF7 on the stimulation of oocyte growth in a culture of cumulus-oocyte complexes with granulosa cells (COCGs, oocyte diameter; 90-100 microm) was investigated. The oocyte diameter of COCGs was increased significantly in the FGF7-containing medium (10 ng/ml; 117.2 +/- 3.2 microm, 50 ng/ml; 116.5 +/- 3.5 microm) compared to the control (0 ng/ml; 110.5 +/- 2.8 microm) after 16 days. However, there was no stimulatory effect of FGF7 on the proliferation of cumulus-granulosa cells. The FGF7 receptor, fibroblast growth factor receptor 2IIIb (FGFR2IIIb), was detected in cumulus-granulosa cells from COCGs. Messenger RNA expression of FGFR2IIIb was induced to cumulus-granulosa cells by FGF7. The mRNA expression levels of KIT ligand (KITLG), KIT (KIT), growth differentiation factor 9 (GDF9), and bone morphogenetic protein 15 (BMP15) in the cultured COCGs were determined in FGF7-treated (10 ng/ml) cultures using real time RT-PCR analysis. The levels of KITLG and KIT, but not GDF9 and BMP15 mRNA expression were stimulated by FGF7. Furthermore, neutralizing antibody for KIT attenuated the stimulatory action of FGF7 on the oocyte growth. These results strongly suggest that FGF7 may be an important regulator for oocyte growth and its action is mediated via the KIT/KITLG signaling pathway.