ABSTRACT
BACKGROUND: The KITL-KIT interaction is known as an important initiator in oocyte activation through the downstream pathway of PI3K-AKT-FOXO3 signalling. Previous studies utilising germ cell-specific Kit mutant knockin and kinase domain knockout models with Vasa-Cre suggested the crucial role of KIT in oocyte activation at the primordial follicle stage. METHODS: We utilised mice with complete postnatal deletion of KIT expression in oocytes via Gdf9-iCre and conducted analyses on ovarian follicle development, specific markers, hormone assays, and fertility outcomes. FINDINGS: Our findings reveal contrasting phenotypes compared to previous mouse models with prenatal deletion of Kit. Specifically, postnatal deletion of Kit exhibit no defects in germ cell nest breakdown, follicle activation, and folliculogenesis during development. Remarkably, upon reaching full maturity, mice with postnatal deletion of Kit experience a complete loss of ovarian reserve, growing follicles, and ovarian function. Furthermore, mice display smaller ovarian size and weight, delayed folliculogenesis, and phenotypes indicative of primary ovarian insufficiency (POI), including elevated serum levels of FSH, reduced AMH, and absence of ovarian follicles, ultimately resulting in infertility. Additionally, the ovaries exhibit randomly distributed expression of granulosa and theca cell markers such as Inhibin α, ACVR2B, and LHR. Notably, there is the uncontrolled expression of p-SMAD3 and Ki67 throughout the ovarian sections, along with the widespread presence of luteinised stroma cells and cleaved Caspase-3-positive dying cells. INTERPRETATION: These genetic studies underscore the indispensable role of KIT in oocytes for maintaining the survival of ovarian follicles and ensuring the reproductive lifespan. FUNDING: This work was supported by National Institutes of Health grant R01HD096042 and startup funds from UNMC (S.Y.K.).
Subject(s)
Oocytes , Ovarian Follicle , Proto-Oncogene Proteins c-kit , Animals , Female , Oocytes/metabolism , Mice , Proto-Oncogene Proteins c-kit/metabolism , Proto-Oncogene Proteins c-kit/genetics , Ovarian Follicle/metabolism , Cell Survival/genetics , Reproduction , Mice, Knockout , Biomarkers , Growth Differentiation Factor 9/metabolism , Growth Differentiation Factor 9/genetics , Primary Ovarian Insufficiency/metabolism , Primary Ovarian Insufficiency/geneticsABSTRACT
BACKGROUND: Primary ovarian insufficiency (POI) affects around 2-4% of women before the age of 40. Genetic factors play an important role in POI. The GDF9 gene has been identified as a significant genetic contributor of POI. However, the pathogenicity and penetrance of GDF9 variants remain uncertain. METHODS: A next-generation sequencing approach was employed to investigate the entire coding region of the GDF9 gene in a cohort of 1281 patients with POI or diminished ovarian reserve (DOR). The frequency of each identified GDF9 variant was then compared with that of the general population, taking into account the ethnicity of each individual. RESULTS: By screening the entire coding region of the GDF9 gene, we identified 19 different variants, including 1 pathogenic frameshift variant. In total, 36 patients with POI/DOR (2.8%) carried at least one GDF9 variant. With regard to missense variants, no significant overrepresentation of the most common variants was observed in our POI/DOR cohort in comparison to the general or specific ethnic subgroups. Only one homozygous subject had a frameshift loss of function variant. CONCLUSION: This epidemiological study suggests that the vast majority of heterozygous missense variants could be considered as variants of uncertain significance and the homozygous loss-of-function variant could be considered as a pathogenic variant. The identification of a novel case of a homozygous POI patient with a heterozygous mother carrying the same variant with normal ovarian function strongly suggests that GDF9 syndrome is an autosomal recessive disorder.
Subject(s)
Growth Differentiation Factor 9 , Primary Ovarian Insufficiency , Humans , Female , Primary Ovarian Insufficiency/genetics , Growth Differentiation Factor 9/genetics , Adult , Frameshift Mutation , Mutation, Missense , High-Throughput Nucleotide Sequencing , Genetic Predisposition to Disease , Cohort Studies , Genes, RecessiveABSTRACT
Acetaminophen is an emerging endocrine disrupting chemical and has been detected in various natural matrices. Numerous studies have documented developmental toxicity associated with prenatal acetaminophen exposure (PAcE). In this study, we established a PAcE Kunming mouse model at different time (middle pregnancy and third trimester), doses (low, middle, high) and courses (single or multi-) to systematically investigate their effects on fetal ovarian development. The findings indicated PAcE affected ovarian development, reduced fetal ovarian oocyte number and inhibited cell proliferation. A reduction in mRNA expression was observed for genes associated with oocyte markers (NOBOX and Figlα), follicular development markers (BMP15 and GDF9), and pre-granulosa cell steroid synthase (SF1 and StAR). Notably, exposure in middle pregnancy, high dose, multi-course resulted in the most pronounced inhibition of oocyte development; exposure in third trimester, high dose and multi-course led to the most pronounced inhibition of follicular development; and in third trimester, low dose and single course, the inhibition of pre-granulosa cell function was most pronounced. Mechanistic investigations revealed that PAcE had the most pronounced suppression of the ovarian Notch signaling pathway. Overall, PAcE caused fetal ovarian multicellular toxicity and inhibited follicular development with time, dose and course differences.
Subject(s)
Acetaminophen , Ovary , Female , Animals , Pregnancy , Mice , Acetaminophen/toxicity , Ovary/drug effects , Ovary/metabolism , Dose-Response Relationship, Drug , Oocytes/drug effects , Prenatal Exposure Delayed Effects/chemically induced , Bone Morphogenetic Protein 15/genetics , Growth Differentiation Factor 9/genetics , Growth Differentiation Factor 9/metabolism , Cell Proliferation/drug effectsABSTRACT
BACKGROUND: Antral follicles consist of an oocyte cumulus complex surrounding by somatic cells, including mural granulosa cells as the inner layer and theca cells as the outsider layer. The communications between oocytes and granulosa cells have been extensively explored in in vitro studies, however, the role of oocyte-derived factor GDF9 on in vivo antral follicle development remains elusive due to lack of an appropriate animal model. Clinically, the phenotype of GDF9 variants needs to be determined. METHODS: Whole-exome sequencing (WES) was performed on two unrelated infertile women characterized by an early rise of estradiol level and defect in follicle enlargement. Besides, WES data on 1,039 women undergoing ART treatment were collected. A Gdf9Q308X/S415T mouse model was generated based on the variant found in one of the patients. RESULTS: Two probands with bi-allelic GDF9 variants (GDF9His209GlnfsTer6/S428T, GDF9Q321X/S428T) and eight GDF9S428T heterozygotes with normal ovarian response were identified. In vitro experiments confirmed that these variants caused reduction of GDF9 secretion, and/or alleviation in BMP15 binding. Gdf9Q308X/S415T mouse model was constructed, which recapitulated the phenotypes in probands with abnormal estrogen secretion and defected follicle enlargement. Further experiments in mouse model showed an earlier expression of STAR in small antral follicles and decreased proliferative capacity in large antral follicles. In addition, RNA sequencing of granulosa cells revealed the transcriptomic profiles related to defective follicle enlargement in the Gdf9Q308X/S415T group. One of the downregulated genes, P4HA2 (a collagen related gene), was found to be stimulated by GDF9 protein, which partly explained the phenotype of defective follicle enlargement. CONCLUSIONS: GDF9 bi-allelic variants contributed to the defect in antral follicle development. Oocyte itself participated in the regulation of follicle development through GDF9 paracrine effect, highlighting the essential role of oocyte-derived factors on ovarian response.
Subject(s)
Infertility, Female , Mice , Animals , Female , Humans , Infertility, Female/metabolism , Ovarian Follicle/metabolism , Oocytes/chemistry , Oocytes/metabolism , Granulosa Cells/metabolism , Estrogens/metabolism , Growth Differentiation Factor 9/genetics , Growth Differentiation Factor 9/analysis , Growth Differentiation Factor 9/metabolismABSTRACT
Both oocyte secretory factors (OSFs) and estrogen are essential for the development and function of mammalian ovarian follicles, playing synergistic role in regulating oocyte growth. OSFs can significantly affect the biological processes regulated by estrogen in cumulus cells (CCs). It is a scientific question worth investigating whether oocyte secretory factors can influence the expression of estrogen receptors in CCs. In our study, we observed a significant increase in the mRNA and protein expressions of estrogen receptor ß (Esr2/ERß) and G-protein-coupled estrogen receptor (GPER) in cumulus cells of goat cumulus-oocyte complexes (COCs) cultured in vitro for 6 h. Furthermore, the addition of 10 ng/mL growth-differentiation factor 9 (GDF9) and 5 ng/mL bone morphogenetic protein 15 (BMP15) to the culture medium of goat COCs resulted in a significant increase in the expressions of ERß and GPER in cumulus cells. To explore the mechanism further, we performed micromanipulation to remove oocyte contents and co-cultured the oocytectomized complexes (OOXs) with denuded oocytes (DOs) or GDF9/BMP15. The expressions of ERß and GPER in the co-culture groups were significantly higher than those in the OOXs group, but there was no difference compared to the COCs group. Mechanistically, we found that SB431542 (inhibitor of GDF9 bioactivity), but not LDN193189 (inhibitor of BMP15 bioactivity), abolished the upregulation of ERß and GPER in cumulus cells and the activation of Smad2/3 signaling. In conclusion, our results demonstrate that the oocyte secretory factor GDF9 promotes the activation of Smad2/3 signaling in cumulus cells during goat COCs culture in vitro, and the phosphorylation of Smad2/3 induces the expression of estrogen receptors ERß and GPER in cumulus cells.
Subject(s)
Cumulus Cells , Receptors, Estrogen , Female , Animals , Cumulus Cells/physiology , Receptors, Estrogen/metabolism , Growth Differentiation Factor 9/genetics , Growth Differentiation Factor 9/metabolism , Goats/metabolism , Estrogen Receptor beta/genetics , Estrogen Receptor beta/metabolism , Oocytes/physiology , Estrogens/metabolism , Bone Morphogenetic Protein 15/metabolismABSTRACT
Unexpected poor ovarian response (UPOR) occurs when nine or fewer oocytes are retrieved from a young patient with normal ovarian reserve. Bone morphogenetic protein15 (BMP15) and growth differentiation factor 9 (GDF9) are two oocyte-specific factors with pivotal role in folliculogenesis. The aim of this study was to assess the relation between BMP15 and GDF9 variants with UPOR. Hundred women aged ≤ 39 with AMH ≥ 1.27 IU/ml participated as UPOR and normal ovarian responders (NOR) based on their oocyte number. Each group consisted of 50 patients. After genomic DNA extraction, the entire exonic regions of BMP15 and GDF9 were amplified and examined by direct sequencing. Western blotting was performed to determine the expression levels of BMP15 and GDF9 in follicular fluid. Additionally, in silico analysis was applied to predict the effect of discovered mutations. From four novel variants of BMP15 and GDF9 genes, silent mutations (c.744 T > C) and (c.99G > A) occurred in both groups, whereas missense variants: c.967-968insA and c.296A > G were found exclusively in UPORs. The latter variants caused reduction in protein expression. Moreover, the mutant allele (T) in a GDF9 polymorphism (C447T) found to be more in NOR individuals (58% NOR vs. 37% UPOR (OR = 2.3, CI 1.32-4.11, p = 0.004).The novel missense mutations which were predicted as damaging, along with other mutations that happened in UPORs might result in ovarian resistance to stimulation. The mutant allele (T) in C447T polymorphism has a protective effect. It can be concluded that there is an association between BMP15 and GDF9 variants and follicular development and ovarian response.
Subject(s)
Bone Morphogenetic Protein 15 , Growth Differentiation Factor 9 , Humans , Female , Growth Differentiation Factor 9/genetics , Growth Differentiation Factor 9/metabolism , Bone Morphogenetic Protein 15/genetics , Bone Morphogenetic Protein 15/metabolism , Ovary/metabolism , Oocytes/metabolism , Follicular Fluid/metabolismABSTRACT
BACKGROUND: DNA methylation is an epigenetic mechanism that takes place at gene promoters and a potent epigenetic marker to regulate gene expression. OBJECTIVE: The study aimed to improve the milk production of Zaraibi goats by addressing the methylation pattern of two milk production-related genes: the growth hormone receptor (GHR) and the growth differentiation factor-9 (GDF-9). METHODS: 54 and 46 samples of low and high milk yield groups, respectively, were collected. Detection of methylation was assessed in two CpG islands in the GDF-9 promoter via methylation-specific primer assay (MSP) and in one CpG island across the GHR promoter using combined bisulfite restriction analysis (COBRA). RESULTS: A positive correlation between the methylation pattern of GDF-9 and GHR and their expression levels was reported. Breeding season was significantly effective on both peak milk yield (PMY) and total milk yield (TMY), where March reported a higher significant difference in PMY than November. Whereas single birth was highly significant on TMY than multiple births. The 3rd and 4th parities reported the highest significant difference in PMY, while the 4th parity was the most effective one on TMY. CONCLUSION: These results may help improve the farm animals' milk productive efficiency and develop prospective epigenetic markers to improve milk yield by epigenetic marker-assisted selection (eMAS) in goat breeding programs.
Subject(s)
DNA Methylation , Milk , Pregnancy , Female , Animals , Milk/metabolism , DNA Methylation/genetics , Goats/genetics , Growth Differentiation Factor 9/genetics , Growth Differentiation Factor 9/metabolism , Egypt , Prospective Studies , Epigenesis, GeneticABSTRACT
Bone morphogenetic protein (BMP15) and growth differentiation factor (GDF9) are critical for ovarian follicular development and fertility and are associated with litter size in mammals. These proteins initially exist as pre-pro-mature proteins, that are subsequently cleaved into biologically active forms. Thus, the molecular forms of GDF9 and BMP15 may provide the key to understanding the differences in litter size determination in mammals. Herein, we compared GDF9 and BMP15 forms in mammals with high (pigs) and low to moderate (sheep) and low (red deer) ovulation-rate. In all species, oocyte lysates and secretions contained both promature and mature forms of BMP15 and GDF9. Whilst promature and mature GDF9 levels were similar between species, deer produced more BMP15 and exhibited, together with sheep, a higher promature:mature BMP15 ratio. N-linked glycosylation was prominant in proregion and mature GDF9 and in proregion BMP15 of pigs, and present in proregion GDF9 of sheep. There was no evidence of secreted native homo- or hetero-dimers although a GDF9 dimer in red deer oocyte lysate was detected. In summary, GDF9 appeared to be equally important in all species regardless of litter size, whilst BMP15 levels were highest in strict monovulatory species.
Subject(s)
Bone Morphogenetic Protein 15 , Growth Differentiation Factor 9 , Litter Size , Animals , Female , Pregnancy , Bone Morphogenetic Protein 15/genetics , Deer , Fertility , Growth Differentiation Factor 9/genetics , Oocytes/metabolism , Ovulation , Sheep , SwineABSTRACT
Infertility is defined as the inability of couples to conceive after 1 year of regular unprotected intercourse, which affects 10-15% of couples. The present study aimed to investigate the influence of Interleukin-17 (IL-17) and growth differentiation factor 9 (GDF9) on three groups of infertile males, including control, azoospermia, and oligozoospermia. In total, this study was performed on 93 participants, consisting of 18, 65, and 10 subjects in the Azoospermia, oligozoospermia, and control groups, respectively. The mean plasma levels of IL-17 in the azoospermia and oligozoospermia groups were 21.317±3.605 and 15.101±2.416 ng/l, respectively, which were significantly higher than that in the control group (5.392±1.731 ng/l). Furthermore, the mean plasma levels of GDF9 in the azoospermia and oligozoospermia groups were 3.299±1.051 and 6.2603±2.621 ng/l, respectively, which was significantly higher than that in the control group (12.807±2.170 ng/l). One-way analysis of variance and least significant difference post-hoc test were performed to assess significant differences among means. R-squared measures how well the linear regression model fits the data. It can be interpreted as the proportion of variance of the outcome Y explained by the linear regression model. R-squared is a number between 0 and 1. In non-obstructive forms of severe oligozoospermia and azoospermia, like the case in the current study, intracytoplasmic sperm injection is suggested by using testicular biopsy for spermatozoa extraction, if viable spermatozoa are present.
Subject(s)
Azoospermia , Infertility, Male , Oligospermia , Humans , Male , Growth Differentiation Factor 9 , Interleukin-17 , Iraq , SemenABSTRACT
In brief: The regulatory role of BMP15 on porcine ovarian follicular development still remains unclear. This study reveals that biallelic editing of BMP15 impairs SMAD signaling and inhibits granulosa cell proliferation, resulting in porcine follicular development arrest and ovarian hypoplasia. Abstract: Bone morphogenetic protein 15 (BMP15) is a member of the transforming growth factor beta (TGF-ß) superfamily, which is critical for facilitating ovarian folliculogenesis in mono-ovulatory mammalian species but is not essential in polyovulatory mice. Our previously established BMP15-edited pigs presented varied female reproductive phenotypes, suggesting the important role of BMP15 in ovarian folliculogenesis in polyovulatory pigs. To understand the regulatory mechanism underlying the effect of BMP15 on porcine ovarian follicular development, we molecularly characterized infertile biallelic-BMP15-edited gilts with ovarian hypoplasia. We found that an absence of BMP15 proteins in biallelic-BMP15-edited gilts can lead to premature activation of primordial follicles, possibly through the upregulation of KITLG-KIT-PI3K-AKT signaling pathways. However, this absence severely impaired SMAD (Sma and Mad proteins from Caenorhabditis elegans and Drosophila, respectively) signaling, causing severely reduced granulosa cell proliferation, leading to the arrest of follicular development during the preantral stage and ovarian hypoplasia, resulting in complete infertility. Our study expands the understanding of the molecular functions of BMP15 in nonrodent polyovulatory mammals.
Subject(s)
Bone Morphogenetic Protein 15 , Phosphatidylinositol 3-Kinases , Female , Swine , Animals , Mice , Bone Morphogenetic Protein 15/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Ovarian Follicle/metabolism , Ovary/metabolism , Transforming Growth Factor beta/metabolism , Growth Differentiation Factor 9/genetics , Mammals/metabolismABSTRACT
OBJECTIVE: Folliculogenesis is a complex process involving various ovarian paracrine factors. During folliculogenesis, vitamin D3 and progesterone are significant for the proper development of follicles. This study aimed to investigate the effects of vitamin D3 and selective progesterone receptor modulator ulipristal acetate on ovarian paracrine factors. METHODS: In the study, 18 female Wistar-albino rats were randomly divided into three groups: control group (saline administration, n=6), vitamin D3 group (300 ng/day vitamin D3 oral administration, n=6), and UPA group (3 mg/kg/day ulipristal acetate oral administration, n=6). Ovarian tissue was analyzed by histochemistry and immunohistochemistry. For quantification of immunohistochemistry, the mean intensities of growth differentiation factor 9, bone morphogenetic protein 15, and forkhead box O3a expressions were measured by Image J and MATLAB. Blood samples were collected for the analysis of serum anti-Müllerian hormone levels by ELISA. RESULTS: Atretic follicles and hemorrhagic cystic structures were observed in the UPA group. After immunohistochemistry via folliculogenesis assessment markers, growth differentiation factor 9, bone morphogenetic protein 15, and cytoplasmic forkhead box O3a expressions decreased in the UPA group (p<0.05). Anti-Müllerian hormone level did not differ significantly between the experimental groups (p>0.05). CONCLUSION: Ulipristal acetate negatively affects folliculogenesis via ovarian paracrine factors. The recommended dietary vitamin D3 supplementation in healthy cases did not cause a significant change.
Subject(s)
Anti-Mullerian Hormone , Bone Morphogenetic Protein 15 , Forkhead Box Protein O3 , Growth Differentiation Factor 9 , Ovary , Animals , Female , Rats , Anti-Mullerian Hormone/metabolism , Bone Morphogenetic Protein 15/metabolism , Cholecalciferol/pharmacology , Growth Differentiation Factor 9/metabolism , Rats, Wistar , Forkhead Box Protein O3/metabolismABSTRACT
Induced molting (IM) can restore the laying rate of aged laying hens to the peak level of laying and rejuvenate ovarian function for the second laying cycle. To explore the mechanism of ovarian function remodeling during IM in laying hens, in this study, ninety 71-wk-old laying lady hens with 60% laying rate and uniform weight were selected for molting induction by fasting. Samples (serum and fresh ovarian tissue) were collected on the day before fasting (F0), the 3rd and 16th days of fasting (F3, F16), and the 6th, 15th, 32nd days of refeeding (R6, R15, and R32), and the number of follicles in each period was counted. Then, the reproductive hormone levels in serum and antioxidant levels in ovarian tissues were detected at different stages, and the gene expression of the KIT-PI3K-PTEN-AKT pathway and GDF-9 in ovaries was measured by qRT-PCR. The results showed that the laying rate increased rapidly after refeeding and returned to the prefasting level by R32. At F16 and R6, the number of mature follicles significantly decreased; the number of primary and secondary follicles significantly increased; the contents of follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), and progesterone (P4) in serum decreased; the relative expression of KIT, PI3K, AKT, and GDF-9 significantly increased; and that of PTEN significantly decreased. At R15 and R32, except for GDF-9, which maintained a high expression state, other indicators showed opposing trends to those observed at F16 and R6. In conclusion, IM activated the KIT-PI3K-PTEN-AKT signaling pathway and promoted the activation of primordial follicles during the fasting period and early resumption of feeding; gonadotropin secretion increased gradually, which promoted the rapid development of primary and secondary follicles to mature follicles and ovulation. This study explained the mechanism of ovarian function remodeling in the process of IM and provided a theoretical basis for improving the ovarian function of laying hens and optimizing the IM program.
Subject(s)
Chickens , Growth Differentiation Factor 9 , Female , Animals , Chickens/physiology , Molting , Proto-Oncogene Proteins c-akt , Luteinizing Hormone , Follicle Stimulating Hormone , Progesterone , Phosphatidylinositol 3-KinasesABSTRACT
The Tibetan cashmere goat is a prolific goat breed in China. In sheep breeds, natural mutations have demonstrated that the transforming growth factor beta (TGF-ß) super family ligands, such as growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) and their type I receptor (bone morphogenetic protein receptor (BMPR1B), are essential for ovulation and increasing litter size. In this study, 216 female Tibetan cashmere goats were sampled, and candidate genes with fecundity traits were detected via restriction fragment length polymorphism (RFLP) and sequenced. Four polymorphic loci were found in specific amplification fragments of BMP15 and GDF9. Two SNP sites of the BMP15 gene were discovered, namely G732A and C805G. The G732A mutation did not cause the change in amino acids, and the frequencies of each genotype were 0.695 for the GG type, 0.282 for the GA type and 0.023 for the AA type. The C805G mutation caused amino acids to change from glutamine to glutamate. The genotype frequencies were 0.620 for the CC type, 0.320 for the CG type and 0.320 for the CG type. For the GG type 0.060, the G3 and G4 mutations of the GDF9 gene were all homozygous mutations. Two known SNP sites, C719T and G1189A, were detected in the Tibetan cashmere goat GDF9 gene, of which the C719T mutation caused a change of alanine to valine, with a genotype frequency of 0.944 for the CC type and 0.056 for the CT type, whereas no TT type was found. The G1189A mutation caused valine to become isoleucine, and the frequencies of each genotype were 0.579 for the GG type, 0.305 for the GA type and 0.116 for the AA type; G1, B2, B3, B4, FecXH, FecXI, FecXL, G2, G5, G6, G7, G8, FecGE, FecTT and FecB mutations were not found in Tibetan cashmere goats. The results of this study provide a data basis for future studies of BMP15, GDF9 and BMPR1B gene mutations in goats.
Subject(s)
Bone Morphogenetic Protein 15 , Growth Differentiation Factor 9 , Animals , Sheep/genetics , Female , Bone Morphogenetic Protein 15/genetics , Growth Differentiation Factor 9/genetics , Goats/genetics , Tibet , Amino AcidsABSTRACT
AIMS: Phytoestrogens can act as natural estrogens owing to their structural similarity to human estrogens. Biochanin-A (BCA) is a well-studied phytoestrogen with a wide variety of pharmacological activities, whereas not reported in the most frequently encountered endocrinopathy called polycystic ovary syndrome (PCOS) in women. PURPOSE: This study aimed to investigate the therapeutic effect of BCA on dehydroepiandrosterone (DHEA) induced PCOS in mice. MAIN METHODS: Thirty-six female C57BL6/J mice were divided into six groups: sesame oil, DHEA-induced PCOS, DHEA + BCA (10 mg/kg/day), DHEA + BCA (20 mg/kg/day), DHEA + BCA (40 mg/kg/day), and metformin (50 mg/kg/day). KEY FINDINGS: The results showed a decrease in obesity, elevated lipid parameters, restoration of hormonal imbalances (testosterone, progesterone, estradiol, adiponectin, insulin, luteinizing hormone, and follicle-stimulating hormone), estrus irregular cyclicity, and pathological changes in the ovary, fat pad, and liver. SIGNIFICANCE: In conclusion, BCA supplementation inhibited the over secretion of inflammatory cytokines (TNF-α, IL-6, and IL-1ß) and upregulated TGFß superfamily markers such as GDF9, BMP15, TGFßR1, and BMPR2 in the ovarian milieu of PCOS mice. Furthermore, BCA reversed insulin resistance by increasing circulating adiponectin levels through a negative correlation with insulin levels. Our results indicate that BCA attenuated DHEA-induced PCOS ovarian derangements, which could be mediated by the TGFß superfamily signaling pathway via GDF9 and BMP15 and associated receptors as first evidenced in this study.
Subject(s)
Polycystic Ovary Syndrome , Animals , Female , Mice , Adiponectin/metabolism , Bone Morphogenetic Protein 15/genetics , Bone Morphogenetic Protein 15/metabolism , Dehydroepiandrosterone/therapeutic use , Estrogens/therapeutic use , Growth Differentiation Factor 9/genetics , Growth Differentiation Factor 9/metabolism , Insulin/metabolism , Polycystic Ovary Syndrome/chemically induced , Polycystic Ovary Syndrome/drug therapy , Polycystic Ovary Syndrome/metabolism , Signal Transduction , Transforming Growth Factor beta/metabolism , Up-RegulationABSTRACT
PURPOSE: The foremost drawback of ovarian tissue cryopreservation and re-transplantation (OTCT) technique is the rapid loss of the primordial follicle (PF) pool. In recent studies, we have demonstrated that post-transplantation burnout of the PFs occurs due to the altered expression of the activatory and inhibitory proteins that control PF reserve, and rapamycin prevented it. METHODS: Here, we investigated whether anti-Mullerian hormone administration in the bilateral oophorectomy and transplantation group and internal AMH in the unilateral oophorectomy and transplantation group protect follicle reserve by regulating the expression of the molecules that control follicle growth after OTCT in mice. RESULTS: After 14 days of OTCT, PF reserve is significantly reduced in both unilateral oophorectomy and transplantation and bilateral oophorectomy and transplantation groups, while anti-Mullerian hormone treatment attenuates PF loss after bilateral oophorectomy and transplantation. The expression of KitL, Bmp-15, and p27 decreased after unilateral oophorectomy and transplantation and bilateral oophorectomy and transplantation, yet recombinant anti-Mullerian hormone treatment did not restore the expression of these proteins in the BLO-T group. CONCLUSION: Exogenous recombinant anti-Mullerian hormone administration in the BLO-T group preserved the expressions of Tsc1 and Gdf-9 in PF and p-s6k and Gdf-9 in growing follicles after OTCT. Nonetheless, recombinant anti-Mullerian hormone administration did not affect granulosa cell proliferation and death rates in the growing follicles. These findings suggest a novel hormonal replacement strategy for fertility preservation by restoring anti-Mullerian hormone to regulate Tsc1 and p-s6k, thereby linking this hormone with the mTOR pathway and Gdf-9 signaling.
Subject(s)
Anti-Mullerian Hormone , Growth Differentiation Factor 9 , Female , Mice , Animals , Anti-Mullerian Hormone/metabolism , Growth Differentiation Factor 9/genetics , Growth Differentiation Factor 9/metabolism , Ovarian Follicle , Ovary/metabolism , CryopreservationABSTRACT
AIMS: Polycystic ovary syndrome (PCOS) is a hyper-androgenic endocrinopathy prevalent in premenopausal women with no cure available. The current study aimed to investigate the therapeutic effect of recombinant GDF-9 and Cetrorelix on the gestational origin of dehydroepiandrosterone (DHEA) induced PCOS in postnatal pups' delivered to rat dams. MAIN METHODS: The body weight measurement, blood and serum analysis for glucose tolerance, lipid profile, liver enzymes, sex hormones (Testosterone, Estradiol, and Progesterone), estrus cyclicity assessment, histological staining of ovary and liver, molecular markers expressions of pro-inflammatory by qRT-PCR and immuno-histochemistry technique for folliculogenesis genes and histological staining studies of liver and ovary were done. KEY FINDINGS: The combinational treatment was found to normalize the biochemical parameters and reduction in the estrus irregularity by altering the sex hormones as well as the glucose metabolism and insulin resistance via HOMA-IR value. Further, molecular markers expression confirmed the pro-inflammatory (IL-1ß, TNF-α, and IL-6) and folliculogenesis (GDF-9, BMPR2, and TGF-ßR1) genes associated with PCOS were improved by combinational therapy. SIGNIFICANCE: In conclusion, rGDF-9 could be a potential therapeutic agent in combination with Cetrorelix as a better treatment regime for metabolic and reproductive phenotypes in PCOS. However, the effect of rGDF-9 on infertility-associated phenotypes in PCOS needs further evaluation.
Subject(s)
Insulin Resistance , Polycystic Ovary Syndrome , Humans , Rats , Female , Animals , Polycystic Ovary Syndrome/metabolism , Growth Differentiation Factor 9/pharmacology , Gonadal Steroid HormonesABSTRACT
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.
Subject(s)
Growth Differentiation Factor 9 , Ovarian Follicle , Animals , Female , Mice , Bone Morphogenetic Protein 15/genetics , Bone Morphogenetic Protein 15/metabolism , Growth Differentiation Factor 9/genetics , Growth Differentiation Factor 9/metabolism , Oocytes/metabolism , Ovarian Follicle/metabolism , Ovary/metabolism , Sexual Maturation , Sheep , SwineABSTRACT
PURPOSE: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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. TRIAL REGISTRATION: ChiCTR1800016107 (Chinese Clinical Trial Registry), May 11, 2018. ( http://www.chictr.org.cn/edit.aspx?pid=27216&htm=4 ).
Subject(s)
Bone Morphogenetic Protein 15 , Growth Differentiation Factor 9 , Animals , Female , Bone Morphogenetic Protein 15/genetics , Fertilization in Vitro , Granulosa Cells/metabolism , Growth Differentiation Factor 9/genetics , Growth Differentiation Factor 9/metabolism , Oocytes/metabolism , Prognosis , Prospective Studies , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
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.
Subject(s)
Escherichia coli , Growth Differentiation Factor 9 , Humans , Animals , Cattle , Escherichia coli/genetics , Escherichia coli/metabolism , Growth Differentiation Factor 9/genetics , Growth Differentiation Factor 9/metabolism , Oocytes/metabolism , Thioredoxins/genetics , Thioredoxins/metabolism , Promoter Regions, Genetic/geneticsABSTRACT
Oocyte-secreted growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are critical paracrine regulators of female fertility. Recent studies demonstrated that serum concentrations are associated with the number of oocytes retrieved during IVF, and therefore potential clinical use as biomarkers. However, it is unknown if the presence of endometriosis affects serum GDF9 or BMP15. An exploratory case-control study was prospectively performed on 60 women who underwent laparoscopy between April 2017 and August 2018 at two hospitals. GDF9 and BMP15 were measured by validated immunoassays in pre-operative serum samples. Data were analysed relative to laparoscopic assessment of endometriosis and staging. There were 35 women with confirmed laparoscopic diagnosis of endometriosis and 25 controls with no evidence of endometriosis at laparoscopy. GDF9 was detectable in 40% of controls and 48% of cases. There was no difference in median GDF9 concentrations between controls (20.0 pg/ml, range 20.0-2504 pg/ml) and cases (20.0 pg/ml, range 20.0-2963 pg/ml). BMP15 was detectable in 48% of controls and 58% of cases, with no difference in median concentrations between controls (26.5 pg/ml, range 24.0-1499 pg/ml) and cases (24.0 pg/ml, range 24.0-796 pg/ml). Furthermore, there were no significant differences in the proportion of detectable samples or concentrations of GDF9 or BMP15 with differing severities of endometriosis. In conclusion, serum concentrations of oocyte-secreted factors, GDF9 and BMP15 did not differ between control patients and patients with endometriosis. For clinical application in reproductive medicine, GDF9 and BMP15 serum biomarker quantitation is unlikely to be aberrant in the presence of endometriosis.