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Growth differentiation factor-9 (GDF-9), an oocyte-derived member of the TGF-ß superfamily, plays an essential role in regulation of follicular development. This study aimed to determine the cyclic changes in serum GDF-9 concentration, compare its levels before and after ovariohysterectomy (OHE), and investigate its potential as a tool in ovarian remnant syndrome (ORS) diagnosis in cats. GDF-9 measurements were performed on 50 cats referred for routine OHE. The stage of the estrous cycle was determined by vaginal cytology and measurement of serum estradiol and progesterone levels was carried out to detect the cyclic changes in circulating GDF-9. One week after OHE, serum samples were collected again from 30 cats to reveal differences in GDF-9 levels. GDF-9 levels in the follicular phase were significantly higher than those in the interestrus (p<0.05). The postoperative analysis could be performed. gDf-9 levels slightly decreased one week after OHE (p=0.053). In conclusion, blood GDF-9 levels change during the estrous cycle, and may decrease with age in cats. However, further studies are needed to reveal the efficiency of GDF-9 in ORS diagnosis.

Growth differentiation factor 9 (GDF9) was the first oocyte-specific growth factor identified; however, most information about GDF9 functions comes from studies in the mouse model. In this study, we created a mutant for Gdf9 gene ( gdf9-/- ) in zebrafish using TALEN approach. The loss of Gdf9 caused a complete arrest of follicle development at primary growth (PG) stage. These follicles eventually degenerated, and all mutant females gradually changed to males through sex reversal, which could be prevented by mutation of the male-promoting gene dmrt1 . Interestingly, the phenotypes of gdf9-/- could be rescued by simultaneous mutation of inhibin α ( inha -/-) but not estradiol treatment, suggesting a potential role for the activin-inhibin system or its signaling pathway in Gdf9 actions. In gdf9 -null follicles, the expression of activin βAa ( inhbaa ), but not βAb ( inhbab ) and βB ( inhbb ), decreased dramatically; however, its expression rebounded in the double mutant ( gdf9 -/- ;inha-/- ). These results indicate clearly that the activation of PG follicles to enter the secondary growth (SG) requires intrinsic factors from the oocyte, such as Gdf9, which in turn works on the neighboring follicle cells to trigger follicle activation, probably involving activins. In addition, our data also support the view that estrogens are not involved in follicle activation as recently reported.

Growth differentiation factor 9 (GDF9) is a secreted protein belonging to the transforming growth factor beta superfamily and has been well characterized for its role during folliculogenesis in the ovary. Although previous studies in mice and sheep have shown that mutations in GDF9 disrupt follicular progression, the exact role of GDF9 in pigs has yet to be elucidated. The objective of this study was to understand the role of GDF9 in ovarian function by rapidly generating GDF9 knockout (GDF9–/–) pigs by using the CRISPR/Cas9 system. Three single-guide RNAs designed to disrupt porcine GDF9 were injected with Cas9 mRNA into zygotes, and blastocyst-stage embryos were transferred into surrogates. One pregnancy was sacrificed on day 100 of gestation to investigate the role of GDF9 during oogenesis. Four female fetuses were recovered with one predicted to be GDF9–/– and the others with in-frame mutations. All four had fully formed oocytes within primordial follicles, confirming that knockout of GDF9 does not disrupt oogenesis. Four GDF9 mutant gilts were generated and were grown past puberty. One gilt was predicted to completely lack functional GDF9 (GDF9–/–), and the gilt never demonstrated standing estrus and had a severely underdeveloped reproductive tract with large ovarian cysts. Further examination revealed that the follicles from the GDF9–/– gilt did not progress past preantral stages, and the uterine vasculature was less extensive than the control pigs. By using the CRISPR/Cas9 system, we demonstrated that GDF9 is a critical growth factor for proper ovarian development and function in pigs. Summary Sentence Inactivation of growth differentiation factor 9 revealed its critical role for proper ovarian development and function in pigs.

Abstract Context The role of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) on aromatase regulation is poorly understood in humans. Objective Determine GDF9 and BMP15 effects on FSH stimulation of estradiol production in primary human cumulus granulosa cells (GCs). We hypothesized that the combination of GDF9 and BMP15 potentiates FSH-induced aromatase expression. Design Primary human cumulus GCs in culture. Setting University infertility center. Patients or Other Participants GCs of 60 women undergoing in vitro fertilization were collected. Interventions Cells were treated with GDF9 and/or BMP15 (GB) in the presence or absence of FSH, dibutyryl cAMP, or SMAD inhibitors. Main Outcome Measures Promoter activity, mRNA, protein, and estradiol levels were quantified. Results FSH and GB treatment increased CYP19A1 promoter activity, mRNA, and protein levels as well as estradiol when compared with cells treated with FSH only. GB treatment potentiated cAMP stimulation of aromatase and IGF2 stimulation by FSH. GB effects were inhibited by SMAD3 inhibitors and IGF1 receptor inhibitors. GB, but not FSH, stimulates SMAD3 phosphorylation. Conclusion The combination of GDF9 and BMP15 potently stimulates the effect of FSH and cAMP on CYP19a1 promoter activity and mRNA/protein levels. These effects translate into an increase in estradiol production. This potentiation seems to occur through activation of the SMAD2/3 and SMAD3 signaling pathway and involves, at least in part, the effect of the IGF system. Oocyte-secreted factors synergize with FSH to promote CYP19A1 mRNA and protein expression and estradiol production in primary human granulosa cells via SMAD2/3, SMAD3, and IGF1R signaling.

Fetal ovarian germ cells show characteristic energy metabolism status, such as enhanced mitochondrial metabolism as well as glycolysis, but their roles in early folliculogenesis are unclear. We show here that inhibition of pyruvate uptake to mitochondria by UK5099 in organ cultures of fetal mouse ovaries resulted in repressed early folliculogenesis without affecting energy production, survival of oocytes, or meiosis. In addition, the abnormal folliculogenesis by UK5099 was partially rescued by α-ketoglutarate and succinate, intermediate metabolites in the TCA cycle, suggesting the importance of those metabolites. The expression of TGFβ-related genes Gdf9 and Bmp15 in ovarian germ cells, which are crucial for folliculogenesis, was downregulated by UK5099, and the addition of recombinant GDF9 partially rescued the abnormal folliculogenesis induced by UK5099. We also found that early folliculogenesis was similarly repressed, as in the culture, in the ovaries of a germ cell-specific knockout of Mpc2, which encodes a mitochondria pyruvate carrier that is targeted by UK5099. These results suggest that insufficient Gdf9 expression induced by abnormal pyruvate metabolism in oocytes results in early follicular dysgenesis, which is a possible cause of defective folliculogenesis in humans. Summary sentence Summary Sentence Pyruvate uptake to mitochondria is crucial for early folliculogenesis via regulation of a TGF-β-related factor in perinatal mouse ovary. Graphical Abstract

Growth differentiation Factor 9 ( GDF9 ) has been confirmed to be closely related to the reproductive capacity of sheep. This study systematically investigated the genetic variation of the GDF9 gene across 75 global sheep breeds ( n  = 2,409) and explored its association with gestation days in Australian White sheep (AUW, n  = 120). Through whole-genome sequencing and SNP analysis, 49 SNPs (26 located in introns, 22 in exons, and 1 in the 3′ UTR region) and 20 InDel loci were identified within GDF9 gene. Haplotype analysis revealed six major haplotypes strongly correlated with geographical population distribution. Association studies in Australian White sheep demonstrated a significant difference between three SNPs loci (g.42115010T > C, g.42115254T > C, and g.42114509T > C) and gestation days: primiparous ewes with the CC genotype at g.42,115,010 exhibited the shortest gestation days (146.42 ± 2.57 days, P  = 0.030), while fourth-parity ewes with the AG genotype at g.42,114,509 showed an abnormally prolonged gestation (155.00 ± 12.81 days, P  = 0.030). Key missense mutations (e.g., E241K, R87H) were predicted to alter protein 3D structure, suggesting functional impacts on reproductive regulation. Despite limited sample sizes in certain parity groups, suggests that GDF9 may serve a potential genetic marker for optimizing reproductive efficiency, offering molecular strategies to shorten primiparous gestation and uncovering its evolutionary role in sheep domestication.

Background In horses, the mechanisms behind ovarian follicle growth and oocyte maturation remain largely unknown. In other species, oocyte-secreted factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) have been related to the acquisition of developmental competence and to interaction with granulosa cells for the regulation of follicle development. This study assessed the expression and localization of GDF9 in the equine ovary, and its possible relationship with granulosa cell function. Results Using custom-made antibodies, GDF9 protein was localized in oocytes from the primary follicle stage onwards. Together with BMP15, its intrafollicular concentration was higher in small antral follicles compared to larger ones ( P  < 0.05). Negative correlations were observed between intrafollicular BMP15 concentration and estradiol sulfate (E2S) ( r = -0.36, P  = 0.048), as well as between BMP15 and E2S/P4 ratio ( r = -0.37, P  = 0.046). In vivo, equine granulosa cells showed increasing mRNA expression of genes involved in steroidogenesis ( STAR and HSD3B2 ) and cell proliferation ( KI67 ) with increasing follicle size, while expression of GDF9 and of apoptosis-related genes ( BCL2 and CASP3 ) were not affected by follicle size. Simultaneous stimulation of granulosa cells in vitro with IGF1 and cortisol significantly increased HSD3B2 and CYP19A1 transcriptional levels, as well as E2 concentration in culture media, while IGF1-induced P4 secretion was suppressed in the presence of cortisol. Blocking the stimulatory effect of IGF1 on E2, E2S and P4 by H89 was associated with increased GDF9 mRNA levels and reduced STAR , PCNA , KI67 and BCL2 mRNA expression. Significant negative correlations of GDF9 with STAR and PCNA mRNA, respectively, were seen in vivo and in vitro. Conclusions Together, our results show GDF9 localization and expression in the equine ovary and a temporal relationship with steroidogenesis and cell proliferation within the surrounding granulosa cells. Moreover, results of the in vitro study suggest a supporting role of cortisol during follicle maturation. Our study sheds light on possible mechanisms for the regulation of ovarian function in horses using GDF9.

Growth differentiation factor 9 ( GDF9) is an oocyte-specific paracrine factor involved in bidirectional communication, which plays an important role in oocyte developmental competence. In spite of its vital role in reproduction, there is insufficient information about exact transcriptional control mechanism of GDF9. Hence, present study was undertaken with the aim to study the expression of basic helix-loop-helix (bHLH) transcription factors (TFs) such as the factor in the germline alpha (FIGLA), twist-related protein 1 (TWIST1) and upstream stimulating factor 1 and 2 (USF1 and USF2), and nuclear receptor (NR) superfamily TFs like germ cell nuclear factor (GCNF) and oestrogen receptor 2 (ESR2) under three different in vitro maturation (IVM) groups [follicle-stimulating hormone (FSH), insulin-like growth factor-1 (IGF1) and oestradiol)] along with all supplementation group as positive control, to understand their role in regulation of GDF9 expression. Buffalo cumulus–oocyte complexes were aspirated from abattoir-derived ovaries and matured in different IVM groups. Following maturation, TFs expression was studied at 8 h of maturation in all four different IVM groups and correlated with GDF9 expression. USF1 displayed positive whereas GCNF, TWIST1 and ESR2 revealed negative correlation with GDF9 expression. TWIST1 & ESR2 revealing negative correlation with GDF9 expression were found to be positively correlated amongst themselves also. GCNF & USF1 revealing highly significant correlation with GDF9 expression in an opposite manner were found to be negatively correlated. The present study concludes that the expression of GDF9 in buffalo oocytes remains under control through the involvement of NR and bHLH TFs.

Background Oocyte secreted factors (OSFs), including growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), play an important role in the process of follicular development and oocyte maturation. Since OSFs are expressed in oocytes and cumulus granulosa cells, the aim of the present study was to explore whether the expression levels of GDF9 and BMP15 mRNAs in cumulus granulosa cells can be used as molecular markers for predicting oocyte developmental potential. Methods Cumulus cells of 2426 cumulus-oocyte complexes were collected from 196 female patients who underwent intracytoplasmic sperm injection (ICSI) and were used for mRNA detection on the egg retrieval day. Pearson correlation analysis was used to analyze the correlation between OSF expression and general physiological parameters. Partial correlation analysis was used to analyze the correlation between OSF expression and oocyte developmental potential. Covariance analysis was used to compare OSF expression among different groups. Receiver operating characteristic curves were used to examine the diagnostic value of GDF9 and BMP15 mRNA for predicting pregnancy. Results The expression levels of GDF9 and BMP15 mRNAs were significantly associated with age, body mass index (BMI), oocyte maturation, normal fertilization, and cleavage rate ( P  < 0.05). The expression levels of GDF9 and BMP15 mRNAs in the group with high-quality embryos were significantly higher than those in the group without high-quality embryos ( P  < 0.05). The expression levels of GDF9 and BMP15 mRNAs in the pregnancy group were significantly higher than those in the nonpregnancy group ( P  < 0.05). The cut-off value of GDF9 mRNA for predicting pregnancy was 4.82, with a sensitivity of 82% and a specificity of 64%. The cut-off value of BMP15 mRNA for predicting pregnancy was 2.60, with a sensitivity of 78% and a specificity of 52%. Conclusions The expression levels of GDF9 and BMP15 mRNAs were closely associated with oocyte maturation, fertilization, embryo quality, and pregnancy outcome; therefore, GDF9 and BMP15 mRNAs in cumulus granulosa cells may be considered as new molecular markers for predicting oocyte developmental potential.

PurposeTo analyze the level of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in follicle fluid (FF) and granulosa cells (GCs) derived from young patients with low prognosis for in vitro fertilization and embryo transfer (IVF-ET) treatment.MethodsA prospective cohort study was carried out by enrolling 52 young patients with low prognosis according to the POSEIDON classification group 3 (low prognosis group) and 51 young patients with normal ovarian reserve (control group). The concentration of the GDF9 and BMP15 proteins in FF was determined by enzyme-linked immunosorbent assay. The mRNA level of the GDF9 and BMP15 in the GCs was measured by quantitative real-time PCR.ResultsThe concentration of GDF9 (1026.72 ± 159.12 pg/mL vs. 1298.06 ± 185.41 pg/mL) and BMP15 (685.23 ± 143.91 pg/mL vs. 794.37 ± 81.79 pg/mL) in FF and the mRNA level of GDF9 and BMP15 in the GCs and the live birth rate per treatment cycle started (30.77% vs. 50.98%) and oocytes retrieved (4.25 ± 1.91 vs.12.04 ± 4.24) were significantly lower, whereas the canceled cycle rate was significantly higher (9.62% vs. 0) in the low prognosis group compared with the control group (P < 0.05). The expression of GDF9 and BMP15 in the ovary was positively correlated with live birth (P < 0.05).ConclusionThe expression of GDF9 and BMP15 in the ovary was decreased in young patients with low prognosis accompanied by a poorer outcome of IVF-ET treatment.Trial registrationChiCTR1800016107 (Chinese Clinical Trial Registry), May 11, 2018. (http://www.chictr.org.cn/edit.aspx?pid=27216&htm=4).