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.

Influence of BMP-2 on early follicular development and mRNA expression of oocyte specific genes in bovine preantral follicles cultured in vitro.

This study evaluates the effect of different concentrations (0, 10, 50 and 100ng/mL) of bone morphogenetic protein-2 (BMP-2) on primordial and secondary follicle development. It also investigates the effects of FSH and BMP-2 on the growth, morphology, ultrastructure and expression of mRNA for GDF9, NLRP5 and NPM2 genes in secondary follicles cultured for 18 days. The presence of BMP-2 at all tested concentrations increased the development of primordial follicles in vitro, but the highest concentration of BMP-2 (100 ng/mL) reduced the percentage of normal follicles when compared with tissues cultured with 10 ng/mL BMP-2. During culture of secondary follicles, in contrast to higher concentrations (50 or 100 ng/mL), 10 ng/mL BMP-2 kept the morphology of follicles during initial stages of in vitro culture. This concentration of BMP-2 also benefits maintenance of the ultrastructure of 18-day cultured follicles. The presence of both BMP-2 and FSH in culture medium resulted in a significant (P<0.05) increase in follicular diameter after 18 days of culture. However, both FSH and BMP-2 reduced follicular mRNA expression of GDF9 and NLRP5 when compared to follicles cultured in media containing only FSH. In combination with FSH, BMP-2 reduced the mRNA levels of NPM2, when compared to follicles cultured in control medium. It is concluded from these data that 10 ng/mL BMP-2 promotes the growth of primordial in vitro and it helps to maintain the ultrastructure of secondary follicles, while FSH is more important for better expression of follicular markers like GDF9 and NLRP5.

Effect of Human Ovarian Tissue Vitrification/Warming on the Expression of Genes Related to Folliculogenesis.

BACKGROUND: Ovarian tissue cryopreservation is an alternative strategy to preserve the fertility of women predicted to undergo premature ovarian failure. This study was designed to evaluate the expression of folliculogenesis-related genes, including factor in the germline alpha (FIGLA), growth differentiation factor-9 (GDF-9), follicle-stimulating hormone receptor (FSHR), and KIT LIGAND after vitrification/warming of human ovarian tissue. METHODS: Human ovarian tissue samples were collected from five transsexual women. In the laboratory, the ovarian medullary part was removed by a surgical blade, and the cortical tissue was cut into small pieces. Some pieces were vitrified and warmed and the others were considered as non-vitrified group (control). Follicular normality was assessed with morphological observation by a light microscope, and the expression of FIGLA, KIT LIGAND, GDF-9,, and FSHR genes was examined using real-time RT-PCR in both the vitrified and non-vitrified groups. RESULTS: Overall, 85% of the follicles preserved their normal morphologic feature after warming. The percentage of normal follicles and the expression of FIGLA, KIT LIGAND, GDF-9, and FSHR genes were similar in both vitrified and non-vitrified groups (P > 0.05). CONCLUSION: Vitrification/warming of human ovarian tissue had no remarkable effect on the expression of folliculogenesis-related genes.

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.

Retinoic acid induces mouse bone marrow-derived CD15⁺, Oct4⁺ and CXCR4⁺ stem cells into male germ-like cells in a two-dimensional cell culture system.

We have examined the effect of retinoic acid (RA) on differentiation of bone marrow-derived CD15(+) , Oct4(+) and CXCR4(+) cells into male germ cells. Bone marrow stem cells (BMSCs) were isolated from the femur of 3-4-week-old male C57BL/6 mice. Magnetic-activated cell sorting (MACS) system was used to sort CD15(+) , Oct4(+) and CXCR4(+) cells. RT-PCR was used to follow the expression of pluripotency markers. Sorted CD15(+) , Oct4(+) and CXCR4(+) cells were cultured in an undifferentiated condition on a feeder layer of mitomycin C-inactivated C2C12. The embryoid-like bodies were differentiated into male germ cells by retinoic acid. To identify the expression of male germ specific markers, differentiated cells were analysed by means of reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence staining. RT-PCR and immunofluorescence show that bone marrow-derived CD15(+) , Oct4(+) and CXCR4(+) cells express pluripotency markers, Oct4, Nanog, Rex-1, SOX-2 and AP. The purified CD15(+) , Oct4(+) and CXCR4(+) formed structures like embryoid bodies when plated over a feeder layer; these bodies were alkaline phosphatase positive. When cells were induced by RA, bone marrow-derived CD15(+) , Oct4(+) and CXCR4(+) were positive for Mvh, Dazl, Piwil2, Dppa3 and Stra8, that known molecular markers of male germ cells. Thus RA can induce differentiation of mouse bone marrow-derived CD15(+) , Oct4(+) and CXCR4(+) cells into male germ cells in vitro. Negative results for the gene expression analysis of female germ cells markers, GDF9 and ZP3, confirmed this conclusion.

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.

Loss of expression of growth differentiation factor-9 (GDF9) in human kidney cancer and regulation of growth and migration of kidney cancer cells by GDF9.

BACKGROUND: Growth differentiation factor-9 (GDF9), a member of the bone morphogenetic protein (BMP) family and the transforming growth factor (TGF)-beta superfamily, has recently been implicated in the biological control of cancer cell behaviour. It has also been implicated in the development and spread of solid cancer. However, the role of GDF9 in kidney cancer remains to be investigated. In the present study, the expression of GDF9 in normal and malignant human kidney tissues and its molecular and cellular impact on human kidney cancer cells were investigated. MATERIALS AND METHODS: The expression of GDF9 in human kidney tissues and kidney cancer cell lines (UMRC-2 and CAKI-2) was assessed at both the mRNA and protein levels using reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively. GDF9 overexpression was induced by a mammalian GDF9 expression construct. The effect of GDF9 expression on cellular functions was examined in kidney cancer cells overexpressing GDF9 using a variety of in vitro assays. RESULTS: In normal kidney tissues, stronger staining of GDF9 was seen in renal tubular epithelial cells, both in the cytoplasm and in the nucleus. In contrast, the staining of GDF9 was notably weak or absent in cells of tumour tissues. Human kidney cancer cell lines UMRC-2 and CAKI-2 had lost their GDG-9 expression. Overexpression of GDF9 reduced in vitro invasion and cellular growth and migration of kidney cell lines in vitro. Using the electric cell-substrate sensing (ECIS) method, it was further revealed that overexpression of GDF9 in these cells markedly reduced cellular migration and adhesion. CONCLUSION: Human kidney tumours have a reduced or loss of expression of GDF9. In vitro, GDF9 overexpression suppresses the invasiveness, growth and migration of kidney cancer cells. This suggests that GDF9 is a potential tumour suppressor and may have prognostic and therapeutic implications in human kidney cancer.

Expression of CDC6 and GDF-9 and their clinicopathological significances in benign and malignant lesions of the gallbladder.

PURPOSE: Gallbladder cancers are cancers with high disease-specific mortality rates due to the lack of early diagnosis and effective therapy. In this study, we evaluated whether CDC6 and GDF-9 could be a marker for early diagnosis and target therapy. METHODS: CDC6 and GDF-9 expressions in 108 gallbladder adenocarcinomas, 15 gallbladder polyps, 35 chronic cholecystitis tissues, and 46 peritumoral tissues were detected using immunohistochemistry (IHC). RESULTS: We demonstrated that positive CDC6 and GDF-9 expressions were significantly higher in adenocarcinomas than that in peritumoral tissues, polyps, and chronic cholecystitis (p < 0.01). Benign lesions with positive CDC6 and negative GDF-9 expression showed moderately- or severely-atypical hyperplasia. The positive rates of CDC6 were significantly lower in cases with well-differentiated adenocarcinoma, small tumor mass, no metastasis of the lymph node, and no invasion of regional tissues (p < 0.05 or p < 0.01). In contrast, GDF-9 expression was significantly lower in the cases with poorly-differentiated adenocaarcinoma, lymph node metastasis, and invasion (p < 0.05 or p < 0.01). Univariate Kaplan-Meier analysis showed that CDC6 (p=0.046) or GDF-9 (p=0.032) expression was associated with decreased overall survival. Multivariate Cox regression analysis showed that increased expression of CDC6 (p=0.042) or decreased expression of GDF-9 (p=0.031) was an independent poor-prognostic predictor in gallbladder adenocarcinoma. CONCLUSION: CDC6 and GDF-9 might be closely related to the carcinogenesis, clinical biological behaviors, and prognosis of gallbladder adenocarcinoma. The positive expression of CDC6 and negative expression of GDF-9 have poor-prognosis in gallbladder carcinoma.

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.

Altered gonadal expression of TGF-ß superfamily signaling factors in environmental contaminant-exposed juvenile alligators.

Environmental contaminant exposure can influence gonadal steroid signaling milieus; however, little research has investigated the vulnerability of non-steroidal signaling pathways in the gonads. Here we use American alligators (Alligator mississippiensis) hatched from field-collected eggs to analyze gonadal mRNA transcript levels of the activin-inhibin-follistatin gene expression network and growth differentiation factor 9. The eggs were collected from Lake Woodruff National Wildlife Refuge, a site with minimal anthropogenic influence, and Lake Apopka, a highly contaminated lake adjacent to a former EPA Superfund site. The hatchling alligators were raised for 13 months under controlled conditions, thus limiting differences to embryonic origins. Our data reveal sexually dimorphic mRNA expression in 13-month-old alligator gonads similar to patterns established in vertebrates with genetic sex determination. In addition, we observed a relationship between lake of origin and mRNA expression of activin/inhibin subunits α and ßB, follistatin, and growth differentiation factor 9. Our study suggests that embryonic exposure to environmental contaminants can affect future non-steroidal signaling patterns in the gonads of a long-lived species.

Expression of growth differentiation factor 9 (GDF9) and its receptor in adult cat testis.

Oocyte-secreted growth differentiation factor (GDF) 9 plays an essential role during follicle maturation through actions on granulosa cells. Despite its critical role in female reproduction, GDF9 expression, signalling and function are less well characterized during spermatogenesis. The purpose of this study was to investigate temporal and spatial expression and potential cellular targets of GDF9 in the adult cat testis. Our result confirmed that GDF9 is stage-specifically localized in the cytoplasm of round spermatids and pachytene spermatocytes of the cat seminiferous epithelium. In particular, activin receptor-like kinase (ALK) 5, the type I receptor of GDF9, is principally localized in the cytoplasm of round spermatids. Smad2/3, signal transducers for GDF9 signalling pathway, is mainly immunolocalized in the cytoplasm of germ cells, Sertoli cells and Leydig cells, but the expression in germ cells are weaker than in Sertoli cells. The expression pattern of ALK5 and Smad2/3 show that GDF9-ALK5-Smad2/3 may not be the only signalling pathway for testicular cell to respond to GDF9. Overall, our results demonstrate that GDF9 is a germ cell-specific factor in the adult cat testis, and that GDF9 regulates the tight junctions of Sertoli cells by paracrine secretion, and regulates the germ cells by autocrine secretion.

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-differentiating factor 9 and its type 1 receptor in human ovaries.

The expression of growth-differentiating factor 9 (GDF9) has not been studied in ovaries from girls and human fetuses nor has its receptor transforming growth factor-beta1 receptor (TGFbetaR1) been investigated in ovaries of girls/women. The aim of this study was to fill these gaps. Ovarian samples were obtained from 16 human fetuses at 21-35 gestational weeks and from 34 girls/women aged 5-39years. These specimens were prepared for immunohistochemical staining of the GDF9 and TGFbetaR1 proteins. Reverse transcription polymerase chain reaction was used to detect GDF9 mRNA transcripts and in-situ hybridization to localize TGFbetaR1 mRNA transcripts. Positive staining for the GDF9 protein was identified in oocytes and granulosa cells in all samples tested. GDF9 mRNA transcripts were present in all samples. Protein expression of TGFbetaR1 was identified in granulosa cells in all samples. Oocyte staining was identified in samples from girls/women but in only one fetal sample. TGFbetaR1 mRNA transcripts were identified in granulosa cells and oocytes in 50% of the samples from fetuses aged over 22 gestational weeks and in samples from girls/women. The detection of GDF9 and TGFbetaR1 at both at the protein and mRNA levels suggests that GDF9 may have functions in human preantral follicles.

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.

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.