Growth Differentiation Factor 9 Expression Research Articles

Vitrification alters growth differentiation factor 9 and follicle-stimulating hormone receptor expression in human cumulus-mural granulosa cells.

Ovarian tissue vitrification is widely utilized for fertility preservation in prepubertal and adolescent female patients with cancer. The current literature includes reports of successful pregnancy and live birth following autografting. However, the effects of the vitrification process on cumulus-mural granulosa cells (C-mGCs)-somatic cells in ovarian tissue crucial for oocyte maturation and early embryonic development-remain unclear. This study was conducted to explore the impact of vitrification on the cellular function of C-mGCs by quantifying the expression of growth differentiation factor 9 (GDF-9), bone morphogenetic protein 15 (BMP-15), follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), connexin 37, survivin, and caspase 3. Mature and immature C-mGCs were obtained from 38 women with polycystic ovary syndrome who participated in an in vitro fertilization program. The C-mGCs were then divided into two groups: fresh and vitrified. The expression levels of target genes were assessed using real-time quantitative polymerase chain reaction. After vitrification, GDF-9 expression was significantly decreased among both mature and immature C-mGCs, with 0.2- and 0.1-fold changes, respectively (p<0.01). Similarly, FSHR expression in the mature and immature groups was reduced by 0.1- and 0.02-fold, respectively, following vitrification (p<0.01). The expression levels of the other genes, including BMP-15, LHR, connexin 37, survivin, and caspase 3, remained similar across the examined groups (p>0.05). Vitrification may compromise oocyte maturation through reduced GDF-9 and FSHR expression in C-mGCs after warming.

Comparing GDF9 in mature follicles and clinical outcomes across different PCOS phenotype

BackgroundPolycystic ovary syndrome (PCOS) is main cause of anovulatory infertility in women with gestational age. There are currently four distinct phenotypes associated with individualized endocrinology and metabolism. Growth differentiation factor 9 (GDF9) is a candidate as potential biomarker for the assessment of oocyte competence. The effect on oocyte capacity has not been evaluated and analyzed in PCOS phenotypes. ObjectiveWe aimed to screen the expression levels of GDF9 in mature follicles of women with controlled ovarian hyperstimulation (COS) with different PCOS phenotypes. To determine the correlation between the expression level of GDF9 and oocyte development ability. MethodsIn Part 1, we conducted a retrospective study comparing the clinical outcomes and endocrine characteristics of patients with PCOS according to different subgroups (depending on the presence or absence of the main features of polycystic ovarian morphology (PCOM), hyperandrogenism (HA), and oligo-anovulation (OA)) and non-PCOS control group. We stratified PCOS as phenotype A (n = 29), phenotype B (n = 18) and phenotype D (n = 24). In Part 2, the expression of GDF9 in follicular fluid (FF) and cumulus cells (CCs) were detected by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry, respectively. ResultsIn Part 1, the baseline clinical, hormonal, and ultrasonographic characteristics of the study population were matched with the presence or absence of the cardinal features of each PCOS phenotypes showed a clear difference. Phenotypes A and D had statistically significant associations with blastocyst formation and clinical pregnancy compared with phenotypes B (p < 0.001). In Part 2, the levels of GDF9 in FF and CCs for phenotype A and B were significantly were higher than those of phenotype D (P = 0.019, P = 0.0015, respectively). Multivariate logistic regression analysis showed that GDF9 was an important independent predictor of blastocyst formation (P＜0.001). The blastocyst formation rate of phenotype A was higher than that of phenotype B and D (P＜0.001). Combining the results of the two parts, GDF9 appears to play a powerful role in the development of embryos into blastocysts. ConclusionsGDF9 expression varies with different PCOS phenotypes. Phenotype A had higher GDF9 levels and blastocyst formation ability.

Assessing the effect of adipose-tissue-derived stem cell conditioned medium on follicles and stromal cells in bovine ovarian tissue culture

Research questionDoes adipose tissue-derived stem cell-conditioned medium (ASC-CM) supplementation enhance follicle and stromal cell outcomes in vitro? DesignBovine ovaries (n=8) were sectioned and in vitro-cultured for 8 days in two different groups: (i) standard culture (OT Ctrl D8); and (ii) culture with ASC-CM supplementation (OT+CM D8). Half of the culture medium was replaced every other day, and stored for estradiol (E2) production measurement. Follicle classification was established using hematoxylin and eosin (H&E) staining. Follicle and stromal cell DNA fragmentation was assessed by TUNEL assays, while growth differentiation factor-9 (GDF-9) staining served as a follicle quality marker. Additionally, three factors, namely vascular endothelial growth factor (VEGF), interleukin 6 (IL-6), and transforming growth factor beta 1 (TGF-β1), were evaluated in ASC-CM in order to appraise potential underlying ASC mechanisms of action. ResultsOT+CM D8 showed significantly higher secondary follicle proportions (p=0.02) than OT Ctrl D8. OT+CM D8 also demonstrated significantly lower follicle (p=0.014) and stromal cell (p=0.001) TUNEL-positive percentages than OT Ctrl D8. Furthermore, follicles in OT+CM D8 exhibited a significant increase (p=0.002) in GDF-9 expression compared to those in OT Ctrl D8, and E2 production was significantly higher (p=0.04) after 8 days in OT+CM. All studied factors were found to be present in the ASC-CM. VEGF and IL-6 were the most widely expressed, while TGF-β1 showed the lowest concentrations. ConclusionsAddition of ASC-CM to culture medium enhances follicle survival, development and E2 secretion, and promotes stromal cell viability. VEGF, IL-6 and TGF-β1 could be possible paracrine mediators underlying the beneficial effects.

Effects of cortisol on granulosa cell function during follicle maturation in the horse

Follicle maturation and acquisition of developmental competence by the oocyte require an interplay between granulosa cells and the oocyte, involving Growth Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15) mediated modulation of follicular steroidogenesis. In the horse, dynamic intrafollicular concentrations of cortisol were associated with follicle growth and oocyte maturation, suggesting a functional relationship between cortisol and oocyte-derived factors. The aim of this study was to analyze effects of cortisol on steroidogenic activity, apoptosis and proliferative capacity of equine granulosa cells, as well as the relationship with the oocyte-derived factor GDF9. For this purpose, granulosa cells were collected three times in four mares each during the breeding season by transvaginal aspiration in the absence of a dominant follicle. Cells collected on the same day were pooled and then seeded on 12-well plates to grow to 70-80% confluence before being stimulated with 100 ng/ml IGF1 for 24h, in the presence or absence of cortisol in increasing concentrations: 50, 100 and 150 ng/ml. These concentrations were previously observed in antral follicles before deviation (50 ng/ml) and after deviation (120 ng/ml). In blocking experiments, cells were preincubated with 20 uM H89 (protein kinase A inhibitor). All experiments were repeated three times. GraphPad software was used for statistical analysis with one-way analysis of variance (ANOVA), followed by non-parametric multiple comparison post-hoc test.Expression of Star was similar in all groups (P>0.05), independent of cortisol concentration and was decreased (P<0.05) after preincubation with H89. Transcriptional levels of HSD3B2 were higher (P<0.001) after adding IGF1 and cortisol simultaneously, but not separately. The rise in HSD3B2 levels was independent of cortisol dose used (P>0.05). Blocking of Star with H89 increased GDF9 expression (P<0.01), both in the presence and absence of cortisol. Interestingly, addition of cortisol alone in all analyzed dosages raised BCL2 levels (P<0.05) and this effect was inhibited by H89 (P<0.05). However, there was no effect on CASP3 levels (P>0.05), regardless of treatment used. A higher proliferating capability of equine granulosa cells, as assessed by PCNA levels, was observed in response to IGF1 treatment (P<0.05), but only in the presence of 150 ng/ml cortisol. Despite apparently elevated, the expression of Ki67 marker in response to IGF1 in the presence of cortisol proved to be not significant (P>0.05). Taken together, our results demonstrate enhanced steroidogenesis as well as reduced apoptosis in equine granulosa cells in response to cortisol. Moreover, reduced steroidogenic activity (i.e. decreased expression of genes involved in progesterone synthesis) of granulosa cells increased GDF9 expression, which has previously been shown to be detrimental for the acquisition of developmental competence by the oocyte.

Decreased GDF9 and BMP15 in follicle fluid and granulosa cells and outcomes of IVF-ET among young patients with low prognosis.

To 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. A 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. The 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). The 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. ChiCTR1800016107 (Chinese Clinical Trial Registry), May 11, 2018. ( http://www.chictr.org.cn/edit.aspx?pid=27216&htm=4 ).

Resolving the challenge of insoluble production of mature human growth differentiation factor 9 protein (GDF9) in E. coli using bicistronic expression with thioredoxin

Growth differentiation factor 9 (GDF9) is an oocyte-derived protein with fundamental functions in folliculogenesis. While the crucial contributions of GDF9 in follicular survival have been revealed, crystallographic studies of GDF9 structure have not yet been carried out, essentially due to the insoluble expression of GDF9 in E. coli and lack of appropriate source for structural studies. Therefore, in this study, we investigated the impact of different expression rate of bacterial thioredoxin (TrxA) using bicistronic expression constructs to induce the soluble expression of mature human GDF9 (hGDF9) driven by T7 promoter in E. coli. Our findings revealed that in BL21(DE3), the high rate of TrxA co-expression at 30 °C was sufficiently potent for the soluble expression of hGDF9 and reduction of inclusion body formation by 4 fold. We also successfully confirmed the bioactivity of the purified soluble hGDF9 protein by evaluation of follicle-stimulating hormone receptor gene expression in bovine cumulus cells derived from small follicles. This study is the first to present an effective approach for expression of bioactive form of hGDF9 using TrxA co-expression in E. coli, which may unravel the current issues regarding structural analysis of hGDF9 protein and consequently provide a better insight into hGDF9 functions and interactions.

Expression of caspase-3, caspase-9, GDF-9 and IGF-1 genes in ovine oocytes cultured with optimum and elevated doses of Amphiregulin, Neuregulin-1 and Tumour necrosis factor-α in-vitro

The present study was conducted to evaluate the expression of caspase-3, caspase-9, growth differentiation factor-9 (GDF-9) and insulin growth factor-1 (IGF-1) genes in oocytes cultured in vitro with optimum and elevated doses of amphiregulin (50 ng and 150 ng), neuregulin-1 (25 ng and 150 ng) and tumor necrosis factor-α (25 ng and 150 ng) during in vitro maturation of oocytes based on the results of effects of AREG or NRG-1 or TNF-α concentration on maturation of oocytes which caused significant effect, were selected for gene expression studies along with the basal/control level. Total RNA was extracted from in vitro matured oocytes using Trizol method and Real-time reverse transcription polymerase chain reaction was used to evaluate the expression of genes. There was an upregulation of caspase-3 at 150 ng of TNF-α, caspase-9 at 50 ng of AREG, GDF-9 at 150 ng of AREG and IGF-1 at 150 ng of AREG and 25 ng of TNF-α. These results suggested that AREG at elevated dose and TNF-α at optimum dose enhanced the expression of GDF-9 and IGF-1, while the presence of elevated dose of TNF-α and optimum dose of AREG activated caspase-3 and caspase-9, respectively in oocytes cultured in vitro.

Altered Expression of GDF9 and BMP15 Genes in Granulosa Cells of Diminished Ovarian Reserve Patients: A Case-Control Study.

Objective Diminished ovarian reserve (DOR) is a challenging issue encountered during assisted reproductive technology. Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) belong to the transforming growth factor-beta (TGF-β) superfamily which are essential for folliculogenesis. We aimed to the evaluation of the GDF9 and BMP15 expression in the granulosa cells (GCs) of DOR patients. Materials and Methods This case-control study included 14 women with DOR and 12 controls, who were between 28- 40 years of age undergoing controlled ovarian stimulation with a gonadotropin releasing hormone (GnRH) antagonist protocol. DOR patients were selected by the Bologna criteria. The GCs were extracted from the aspirated follicular fluids and RNA isolated from this. The fold change of gene expressions was assessed by real-time polymerase chain reaction (PCR). Results GDF9 expression in patients was 0.23 times lower than the control group, which was significant (P<0.0001). BMP15 expression in patients was 0.32 times lower than the control group, which was significant (P<0.0001). The number of archived oocytes, MII, and two pronuclei (PN) embryos was higher in the control group and these differences were statistically significant (P<0.05). ConclusionGiven that GDF9 and BMP15 are specifically involved during follicular recruitmen., we expect expression of these two genes in DOR patients which is greatly reduced by reducing follicular reserve.

High expression of CFTR in cumulus cells from mature oocytes is associated with high-quality of oocyte and subsequent embryonic development.

The purpose of this study was to explore the association of expression of cystic fibrosis transmembrane conductance regulator (CFTR) in cumulus cells (CCs) from mature oocytes with oocyte quality and embryonic development. A total of 338 infertile women who underwent ovarian stimulation cycle of oocyte retrieval in Zhejiang University School of Medicine were retrospectively enrolled in this study. The relative mRNA expression levels of CFTR, bone morphogenetic protein 15 (BMP15), and growth differentiation factor 9 (GDF9) in CCs were detected by qPCR technology. ROC curve was applied for the diagnosis of oocyte maturation. The serum levels of anti-Müllerian hormone (AMH), E2, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and androstenedione were measured. Oocyte maturation rate, fertilization rate, cleavage rate, high-quality embryo formation rate, and implantation rate after embryo transfer were also determined. The mRNA expression levels of CFTR in CCs were significantly increased in metaphase II (MII) oocytes compared to that in metaphase I (MI) or germinal vesicle (GV) oocytes. The ROC curve analysis illustrated that CFTR mRNA expression could efficiently discriminate MII oocytes from MI or GV oocytes (AUC = 0.954), and revealed that 0.695 RQU is the optimal cut-off value for diagnosis. So the cut-off value of 2-ΔΔCT = 0.70 was used to divide the patients into two groups: low- (n = 114) and high-CFTR group (n = 224). The mRNA expression of CFTR in CCs was positively correlated with the antral follicular count (AFC), number of oocytes retrieved, number of MII oocytes, serum E2 level on hCG day, and BMP15 and GDF9 expression in CCs. Under continuous stimulation with the same dose of recombinant follicle-stimulating hormone (rFSH), the number of follicles, average recovered oocytes, recovered oocytes, MII oocytes, as well as the oocyte recovery rate, fertilization rate, oocyte cleavage rate, high-quality embryo formation rate, and implantation rate were decreased in patients with lower CFTR. This study suggests that CFTR expression in CCs is associated with the developmental potential of human oocytes.

Effect of vitrification on the expression of genes in porcine blastocysts derived from in vitro matured oocytes

This study aimed to examine the effect of vitrification on the expression of genes that are crucial for porcine early embryo development; cathepsin B (CTSB), growth differentiation factor 9 (GDF9), caudal type homeobox 2 (CDX2), and OCT-4, which play an important role in the maintenance of embryonic cell pluripotency. Their gene expression was investigated in expanded blastocysts (day 6–7) derived from in vitro matured oocytes. The quantitative real-time PCR method was used to assess the amount of relative specific transcripts in 20 vitrified (treatment group) and 32 fresh non-vitrified (control group) blastocysts. Vitrification was performed using 7.5% dimethyl sulfoxide (DMSO) plus 7.5% ethylene glycol (EG), and in the final step, 15% DMSO plus 15% EG and a 0.5 M sucrose solution and cryotop as a vitrification device. The blastocysts were warmed in 1 M, 0.5 M, and 0.25 M sucrose solution and kept in a culture medium for six hours before their fixation and further qPCR analysis. A significant upregulation in the targeted genes CTSB (p<.006), GDF9 (p<.04), and CDX2 (p<.003) was observed in the vitrified embryos compared to the fresh control group. Interestingly, the OCT-4 mRNA expression level was not affected by vitrification and remained comparable to that of the fresh non-vitrified embryos. In summary, the results of this pilot study showed, that vitrification induced substantial alteration in the expression of CTSB, GDF9, and CDX2 genes but did not influence the expression of OCT-4 gene in porcine in vitro derived blastocysts. Our data on the expression of developmentally important genes in vitrified porcine blastocyst may facilitate: (1) future improvements in culture conditions and/or cryopreservation protocol and (2) understanding the mechanism(s) of cryoinjuries inducing compromised post-thaw embryo development followed by the poor pregnancy outcome after blastocyst transfer.

Paclitaxel is evidence to reduce growing ovarian follicle growth in mice model study

We aimed to investigate the effects of paclitaxel on ovarian follicle development and oocyte meiotic competence. Early secondary follicles were cultured individually with or without paclitaxel 2.5 × 10−10, 2.5 × 10−9, and 2.5 × 10−8 M for 12 days. Thereafter, the follicles were treated with human chorionic gonadotropin (hCG) and epidermal growth factor (EGF). Follicle morphology, oocytes meiotic maturation, immunofluorescence for α-tubulin of the oocytes, mRNA expression of growth differentiation factor 9 (GDF9), B-cell lymphoma 2 (Bcl2), and Bcl2-associated X (Bax) were examined every 4, 8, and 12 days of the culture period. We demonstrated that high dose paclitaxel treatment decreased follicle survival (p ≤ 0.05), while lower dose (2.5 × 10−9 M) reduced the survival compared to control after 12 days of culture. The number of oocytes at MII stage was not significantly different between control and paclitaxel groups (p > 0.05). Paclitaxel increased GDF9 expression in oocytes after 4 days of the culture (p ≤ 0.05). Bcl2 declined significantly compared to control after 8 days (p ≤ 0.05 for all groups), while Bax expression tended to be consistent (p ≥ 0.05 for all groups). To conclude, high concentration paclitaxel reduces follicle preantral follicle growth, while in lower concentration it decreases more growing follicle growth and survival.

2-Hydroxy-4-Methylselenobutanoic Acid Promotes Follicle Development by Antioxidant Pathway.

Selenium (Se) is assumed to promote the follicle development by attenuating oxidative stress. The current study was developed to evaluate the effects of dietary 2-hydroxy-4-methylselenobutanoic acid (HMSeBA) supplementation on the follicle development in vivo and on the function of ovarian granulosa cells (GCs) in vitro. Thirty-six gilts were randomly assigned to fed control diet (CON), Na2SeO3 diet (0.3 mg Se/kg) or HMSeBA diet (0.3 mg Se/kg). The results showed that HMSeBA and Na2SeO3 supplementation both increased the total selenium content in liver and serum compared with control, while HMSeBA increased the total selenium content in liver compared with Na2SeO3 group. HMSeBA tended to increase the total selenium content in ovary compared with control. HMSeBA and Na2SeO3 supplementation both increased the weight of uteri in gilts at the third estrus. Moreover, HMSeBA supplementation down-regulated the gene expression of growth differentiation factor-9 (GDF-9) and bone morpho-genetic protein-15 (BMP-15) in cumulus-oocyte complexes (COCs). HMSeBA supplementation decreased malondialdehyde (MDA) content in serum, liver and ovary, increased activity of T-AOC in liver, TXNRD in ovary and GPX in serum, liver and ovary, while up-regulated the liver GPX2, SOD1 and TXNRD1, ovarian GPX1 gene expression. In vitro, HMSeBA treatment promoted GCs' proliferation and secretion of estradiol (E2). HMSeBA treatment increased the activity of T-AOC, T-SOD, GPX, TXNRD and decreased MDA content in GCs in vitro. Meanwhile, HMSeBA treatment up-regulated SOD2 and GPX1 gene expression in GCs in vitro. In conclusion, HMSeBA supplementation is more conducive to promoting follicle development by antioxidant pathway.

Effect of conditioned medium of umbilical cord-derived mesenchymal stem cells as a culture medium for human granulosa cells: An experimental study.

Background The umbilical cord-derived mesenchymal stem cells conditioned medium (UC-MSCs-CM) produces secretomes with anti-apoptotic properties, and has the potential to prevent apoptosis of granulosa cells (GC) during controlled ovarian hyperstimulation.ObjectiveTo observe the effect of UC-MSCs-CM on the interaction between pro- and anti-apoptotic proteins and the influence of growth differentiation factor 9 (GDF9) production in GC.Materials and MethodsUC-MSCs-CM was collected from umbilical cord stem cell culture on passage 4. GC from 23 women who underwent in vitro fertilization were cultured and exposed to UC-MSCs-CM for 24 hr. Then RNA of the GC was extracted and the mRNA expression of BCL-2 associated X (BAX), survivin and GDF9 were analysed using quantitative real-time PCR. The spent culture media of the GC were collected for measurement of insulin growth factor 1 using ELISA.ResultsThe expression of BAX was significantly different after UC-MSCs-CM exposure (4.09E-7 vs. 3.74E-7, p = 0.02). No significant changes occurred in survivin, BAX/survivin ratio, and GDF9 expression after UC-MSCs-CM exposure (p 0.05). The IGF-1 level of the CM was significantly higher after the CM was used as a culture medium for GC (2.28 vs. 3.07 1.72, p 0.001). A significant positive correlation was found between survivin and GDF9 (r = 0.966, p 0.001).Conclusion IGF-1 produced by UC-MSCs-CM can work in paracrine fashion through the IGF receptor, which can inhibit BAX and maintain GDF9 production. Moreover, under the influence of UC-MSCs-CM, GC are also capable of producing IGF-1, which can impact GC through autocrine processes.

The effect of gold nanoparticles synthesized with Achillea biebersteinii on gene expression in Cultured preantral Follicles derived from NMRI mice ovary

Infertility is one of the global public health problems facing society today. Studies on the causes of infertility have greatly expanded with the advancement of new technologies. The increasing use of nanomaterials has raised concerns about nanoparticles' potential dangers. The use of plant extracts is an easy and cost-effective approach to synthesize metal nanoparticles. In this comparative study, the effects of green and chemically synthesized Gold nanoparticles (GNPs), as well as Achillea biebersteinii (AM) (medicinal plant) extract, were investigated on the maturity of preantral Follicles (PF) of mice in vitro. The expression of GDF-9, BMP-15, GPX, and SOD-1 genes was analyzed by real-time polymerase chain reaction (Real Time-PCR). Twenty PF in control and experimental groups were treated with 50 μg/ml of AM extract, green GNPs, and GNPs, and were then used to determine intracellular ROS (Reactive oxygen species) in the first and fifth days after culture. AM extract also increased the expression of genes that stimulate preantral follicle growth (PF), including Growth differentiation factor-9 (GDF-9)and Bone morphogenetic protein 15 (BMP-15). Expression of the glutathione peroxidase (GPX) gene was observed in the group of NPs synthesized by a green method. Due to its antioxidant properties, this gene expression was also higher in the plant extract group than commercially synthesized GNPs. Overall, this study's findings showed that AM extract, due to its antioxidant effects, improves PF development, and AM-GNPs did not significantly impact PF growth, but GNPs inhibited PF growth. Findings suggest that the AM extract that covered the surface of AM-GNPs may be responsible for reducing the toxic effects of GNPs.

Regulation of GDF9 and CDKN1B expression in Tibetan sheep testes during different stages of maturity

Normal spermatogenesis is heavily dependent on the balance of germ cell proliferation, differentiation and apoptosis. Growth differentiation factor 9 (GDF9) and cyclin-dependent kinase inhibitor 1 B (CDKN1B) are strongly associated with cell cycle transition from G0/G1 to S and G2/M phase and hence regulating the growth and development of testicular germ cells and somatic cells. The current study was aimed at seeking out scientific evidence to determine if GDF9 and CDKN1B gene expression functions in the development of Tibetan sheep testes. To this end, developmental testes were derived from three-month-old (pre-puberty), one-year-old (sexual maturity), and three-year-old (adult) Tibetan sheep and then the expression and localization patterns of GDF9 and CDKN1B in these testes were evaluated using quantitative real-time PCR (qRT-PCR), Western blot and immunofluorescence. qRT-PCR and Western blot results showed that GDF9 and CDKN1B were detected in the testes throughout the different developmental stages. The abundance of GDF9 mRNA and protein in the testes of one- and three-year-old Tibetan sheep were higher than that in the testes of three-month-old Tibetan sheep; the mRNA and protein abundance of the CDKN1B gene in three-month-old Tibetan sheep testes were higher than that in the testes of the one-and three-year-old sheep. Moreover, immunofluorescence results suggested that the GDF9 protein was expressed in spermatogonia and Leydig cells, and that the CDKN1B protein was localized mainly in Leydig cells with some in the seminiferous epithelium throughout developmental stages. This indicated a novel role of the GDF9 and CDKN1B genes in Leydig cell development over and above their known roles in germ cell development. These findings have significant implications for our understanding of the molecular mechanisms of GDF9 and CDKN1B genes in Tibetan sheep spermatogenesis.

MiR-23b-3p inhibits chicken granulosa cell proliferation and steroid hormone synthesis via targeting GDF9

MicroRNAs (miRNAs) are ∼22 nt RNAs that direct post-transcriptional repression of mRNA targets in diverse eukaryotic lineages. Granulosa cells (GCs) are the earliest differentiated follicular somatic cells. From the initiation of primordial follicles, their differentiation and growth are closely related to the development of follicles. The research on follicular development mostly focused on the granular layer, as well as the hormone synthesis induced by granulosa cell differentiation before and after follicular selection. In this study, we evaluated the effects of miR-23b-3p on chicken granulosa cells, including granulosa cell proliferation and steroid hormone synthesis. Elevated expression of miR-23b-3p significantly inhibited granulosa cell proliferation and steroid hormone synthesis, but did not affect apoptosis. Furthermore, it was observed that the forecast growth differentiation factor 9 (GDF9) is a target gene of miR-23b-3p and miR-23b-3p can down-regulate expression of GDF9. Overall, this study demonstrated that miR-23b-3p can regulate the proliferation and steroid hormone synthesis of chicken granulosa cells by inhibiting the expression of GDF9.

Melatonin Restores the Developmental Competence of Heat Stressed Porcine Oocytes, and Alters the Expression of Genes Related to Oocyte Maturation.

Simple SummaryMelatonin improves the quality and in vitro maturation (IVM) of oocytes under heat stress. Melatonin treatment counteracts the adverse effects induced by heat stress (HS), such as the poor survival rate and maturation rate, distribution of α-tubulin and F-actin, expression of NRF2 and GDF9 mRNA. However, HS and melatonin have similar effects on increasing expression of HSP70 and NRF2 mRNA. Furthermore, HS inhibits expression of GDF9 mRNA.Melatonin enhances the quality and in vitro maturation (IVM) of oocytes under heat stress (HS), but the mechanism of melatonin in reducing HS injury on oocytes is not fully understood. In this study, porcine cumulus-oocyte complexes (COCs) were randomly divided into three groups. The COCs of the control group were cultured at 38.5 °C for 42 h, and the COCs of the HS group were cultured at 41.5 °C for 4 h, and then transferred into 38.5 °C for 38 h. The COCs of the HS + melatonin group were cultured with 10−9 M melatonin under the same conditions as the HS group. The survival rate, maturation rate, distribution of α-tubulin and F-actin of the oocytes were assessed. In addition, the expression profiles for genes related to the oocyte maturation, including heat shock protein 70 (HSP70), nuclear factor erythroid 2-related factor 2 (NRF2), cyclin-dependent kinase 1 (CDK1), growth differentiation factor 9 (GDF9) were analyzed by real-time quantitative PCR. The results showed that HS decreased the survival rate and maturation rate, distribution of α-tubulin and F-actin, but melatonin treatment could partly counteract these adverse effects. In addition, HS increased expression of HSP70 and NRF2 mRNA, and melatonin treatment had a similar effect on HSP70 expression, but had a contrary effect on NRF2 expression. Furthermore, HS inhibited expression of CDK1 and GDF9 mRNA, but melatonin treatment could weaken the effect on GDF9 expression induced by HS. In summary, melatonin treatment could attenuate the unfavorable effects induced by HS to enhance developmental competence of porcine oocytes during IVM.

Activation-induced cytidine deaminase is a possible regulator of cross-talk between oocytes and granulosa cells through GDF-9 and SCF feedback system

Activation-induced cytidine deaminase (AID, Aicda) is a master gene regulating class switching of immunoglobulin genes. In this study, we investigated the significance of AID expression in the ovary. Immunohistological study and RT-PCR showed that AID was expressed in murine granulosa cells and oocytes. However, using the Aicda-Cre/Rosa-tdRFP reporter mouse, its transcriptional history in oocytes was not detected, suggesting that AID mRNA in oocytes has an exogenous origin. Microarray and qPCR validation revealed that mRNA expressions of growth differentiation factor-9 (GDF-9) in oocytes and stem cell factor (SCF) in granulosa cells were significantly decreased in AID-knockout mice compared with wild-type mice. A 6-h incubation of primary granuloma cells markedly reduced AID expression, whereas it was maintained by recombinant GDF-9. In contrast, SCF expression was induced by more than threefold, whereas GDF-9 completely inhibited its increase. In the presence of GDF-9, knockdown of AID by siRNA further decreased SCF expression. However, in AID-suppressed granulosa cells and ovarian tissues of AID-knockout mice, there were no differences in the methylation of SCF and GDF-9. These findings suggest that AID is a novel candidate that regulates cross-talk between oocytes and granulosa cells through a GDF-9 and SCF feedback system, probably in a methylation-independent manner.

Expression Profiles of the Progesterone Receptor, Cyclooxygenase-2, Growth Differentiation Factor 9, and Bone Morphogenetic Protein 15 Transcripts in the Canine Oviducts during the Oestrous Cycle.

Simple SummaryThe oestrous cycle in canines is specifically more extended than that in other mammals. This implies that the oocytes do not reach maturity within the ovarian follicle but undergo final maturation in the oviducts. Besides oocyte maturation, the oviduct provides the necessary milieu for fertilization and preimplantation embryonic development. Consequently, the oviductal environment presumably changes in the postovulatory period and throughout the entire reproductive cycle to provide a suitable condition for supporting different functions. In this study, we evaluated the gene expression of different genes associated with oocyte-embryo development, such as progesterone receptor, cyclooxygenase-2, growth differentiation factor 9, and bone morphogenetic protein 15 in the canine oviductal cells at different phases of the oestrous cycle. Using quantitative PCR (qPCR) analysis in bitch oviductal cells, this study revealed the ovarian cycle’s influence on the oviductal essential transcripts in the bitch. It also assessed the influence of the ovulated cumulus-oocytes complexes on the expression of GDF-9 and BMP-15 genes. Thus, the oestrous-cycle-dependent gene expression pattern of PR, COX-2, GDF-9, BMP-15 in the canine oviduct was found to execute the oviductal cell interactions necessary for the development and function of the canine reproductive tract.The gene expression in the canine oviduct, where oocyte maturation, fertilization, and early embryonic development occur, is still elusive. This study determined the oviductal expression of (PR), cyclooxygenase-2 (COX-2), growth differentiation factor 9 (GDF-9), and bone morphogenetic protein 15 (BMP-15) during the canine oestrous cycle. Samples were collected from bitches at anoestrus (9), proestrus (7), oestrus (8), and dioestrus (11), after routine ovariohysterectomy and the ovarian surface structures and plasma progesterone concentration evaluated the physiological status of each donor. The oviductal cells were isolated and pooled. Total RNA was isolated, and gene expression was assessed by qPCR followed by analysis using the t-test and ANOVA. The PR mRNA increased (P < 0.05) from the anoestrus to dioestrus with the plasma progesterone concentration (r = 0.8). COX-2 mRNA expression was low in the anoestrus and proestrus, and negligible in the oestrus, while it was around 10-fold higher (P < 0.05) in the dioestrus. The GDF-9 mRNA was expressed during all phases of the oestrous cycle and was most abundant (P < 0.05) during oestrus phase. The BMP-15 mRNA decreased (P < 0.05) in the anoestrus and proestrus phases. Thus, the transcripts were differentially expressed in a stage-dependent manner, suggesting the importance of oestrous cycle regulation for successful reproduction in dogs.

The Effect of Bone Marrow Transplantation on Oocyte-Granulosa Cell Interaction and Follicular Development of Cisplatin-Induced Ovarian Failure in Rat

Introduction: Chemotherapy has cytotoxic effect that induces follicular damage and abnormal folliculogenesis leads to ovarian failure. Two crucial growth factors in abnormal folliculogenesis, Growth Differentiation Factor-9 (GDF-9) and Kit-Ligand, will be disrupted and affect follicular development. In this study, we evaluate whether bone marrow transplantation (BMT) has a role on oocyte-granulosa cell interaction by analyzing GDF-9 and Kit-Ligand expressions and also follicular development by analyzing primordial, primary, secondary and graafian follicles of cisplatin-induced ovarian failure in rat. Material and method: Forty eight rats were divided into three groups: control, cisplatin and cisplatin + BMT. Ovarian failure was induced by administration of intraperitoneal cisplatin 5 mg/kg body weight for 1 week. BMT 2 x107 cells were injected through rat tail vein after cisplatin administration. Bone marrow was isolated from rat femur and characterized by CD44(+), CD45(-), CD105(+). Immunohistochemistry examinations for ovarian GDF-9, Kit-Ligand and follicle development evaluation were performed after 2 weeks of BMT injection. Results: The expressions of Kit-ligand among three groups by ANOVA were significant different (p=0.00), whereas by Post Hoc: cisplatin group lower than control group (p=0.00); cisplatin + BMT group higher than cisplatin group (p=0.00); and no significant different between control group and cisplatin + BMT group (p=0.955). The expressions of GDF-9 by Kruskal Wallis showed significant different (p=0.00) among three groups whereas cisplatin + BMT group higher than cisplatin group and control group. In cisplatin + BMT group the number of primordial, primary, secondary and graafian follicles were higher than those in cisplatin group; but were lower than those in control group (p=0.000). Positive Paul Kart Horan (PKH) labeling was seen in cisplatin + BMT group only. Conclusion: In cisplatin-induced ovarian failure in rat, bone marrow transplantation may improve oocytegranulosa cell interaction and follicular development.