Dynamic mRNA expression during chicken ovarian follicle development.

Ovarian follicle development is a complex and well-orchestrated biological process of great economic significance for poultry production. Specifically, understanding the molecular mechanisms underlying follicular development is essential for high-efficiency follicular development can benefit the entire industry. In addition, domestic egg-laying hens often spontaneously develop ovarian cancer, providing an opportunity to study the genetic, biochemical, and environmental risk factors associated with the development of this cancer. Here, we provide high-quality RNA sequencing data for chicken follicular granulosa cells across 10 developmental stages, which resulted in a total of 204.57 Gb of clean sequencing data (6.82 Gb on average per sample). We also performed gene expression, time-series, and functional enrichment analyses across the 10 developmental stages. Our study revealed that SWF (small while follicle), F1 (F1 hierarchical follicles), and POFs (postovulatory follicles) best represent the transcriptional changes associated with the prehierarchical, preovulatory, and postovulatory stages, respectively. We found that the preovulatory stage F1 showed the greatest divergence in gene expression from the POF stage. Our research lays a foundation for further elucidation of egg-laying performance of chicken and human ovarian disease.

Comparative analyses in transcriptome of human granulosa cells and follicular fluid micro-environment between poor ovarian responders with conventional controlled ovarian or mild ovarian stimulations.

BACKGROUND: Both mild and conventional controlled ovarian stimulation are the frequently used protocols for poor ovarian responders. However, there are some debates about which treatment is better. Moreover, little is known about the follicular physiology after the two ovarian stimulation protocols. This study was intended to investigate the features in granulosa cells and follicular fluid micro-environment after the two different ovarian stimulation protocols in poor responders. METHODS: Granulosa cells RNA were sequenced using Illumina Hiseq technology. Specific differently expressed genes and proteins were verified by real-time quantitative PCR and Western blot analysis. Moreover, hormone and cytokine concentrations in the follicular fluid were measured by electrochemiluminescence immunoassay and enzyme-linked immunoabsorbent assay. The correlation between the results of molecular experiments and the laboratory outcomes were analyzed by Spearman correlation analysis. RESULTS: The differentially expressed genes between the two groups were involved in 4 signaling pathways related to the follicular development; three proteins pertinent to the TGF-ß signaling pathway were expressed differently in granulosa cells between the two, and the constituents in the follicular fluid were also different. Further, a correlation between the TGF-ß signaling pathway and the good-quality embryo was observed. CONCLUSIONS: The present study made a comparison for the first time in the transcriptome of human granulosa cells and the follicular fluid micro-environment between poor responders with the conventional controlled ovarian stimulation or the mild ovarian stimulation, showing that the TGF-ß signaling pathway may correlate with the good-quality of embryos in the mild group, which may be instrumental to the choice of optimal management for IVF patients.

circSLC41A1 Resists Porcine Granulosa Cell Apoptosis and Follicular Atresia by Promoting SRSF1 through miR-9820-5p Sponging.

Ovarian granulosa cell (GC) apoptosis is the major cause of follicular atresia. Regulation of non-coding RNAs (ncRNAs) was proved to be involved in regulatory mechanisms of GC apoptosis. circRNAs have been recognized to play important roles in cellular activity. However, the regulatory network of circRNAs in follicular atresia has not been fully validated. In this study, we report a new circRNA, circSLC41A1, which has higher expression in healthy follicles compared to atretic follicles, and confirm its circular structure using RNase R treatment. The resistant function of circSLC41A1 during GC apoptosis was detected by si-RNA transfection and the competitive binding of miR-9820-5p by circSLC41A1 and SRSF1 was detected with a dual-luciferase reporter assay and co-transfection of their inhibitors or siRNA. Additionally, we predicted the protein-coding potential of circSLC41A1 and analyzed the structure of circSLC41A1-134aa. Our study revealed that circSLC41A1 enhanced SRSF1 expression through competitive binding of miR-9820-5p and demonstrated a circSLC41A1-miR-9820-5p-SRSF1 regulatory axis in follicular GC apoptosis. The study adds to knowledge of the post-transcriptional regulation of follicular atresia and provides insight into the protein-coding function of circRNA.

Protein Palmitoylation in Bovine Ovarian Follicle.

Protein palmitoylation is a reversible post-translational modification by fatty acids (FA), mainly a palmitate (C16:0). Palmitoylation allows protein shuttling between the plasma membrane and cytosol to regulate protein stability, sorting and signaling activity and its deficiency leads to diseases. We aimed to characterize the palmitoyl-proteome of ovarian follicular cells and molecular machinery regulating protein palmitoylation within the follicle. For the first time, 84 palmitoylated proteins were identified from bovine granulosa cells (GC), cumulus cells (CC) and oocytes by acyl-biotin exchange proteomics. Of these, 32 were transmembrane proteins and 27 proteins were detected in bovine follicular fluid extracellular vesicles (ffEVs). Expression of palmitoylation and depalmitoylation enzymes as palmitoyltransferases (ZDHHCs), acylthioesterases (LYPLA1 and LYPLA2) and palmitoylthioesterases (PPT1 and PPT2) were analysed using transcriptome and proteome data in oocytes, CC and GC. By immunofluorescence, ZDHHC16, PPT1, PPT2 and LYPLA2 proteins were localized in GC, CC and oocyte. In oocyte and CC, abundance of palmitoylation-related enzymes significantly varied during oocyte maturation. These variations and the involvement of identified palmitoyl-proteins in oxidation-reduction processes, energy metabolism, protein localization, vesicle-mediated transport, response to stress, G-protein mediated and other signaling pathways suggests that protein palmitoylation may play important roles in oocyte maturation and ffEV-mediated communications within the follicle.

Reproductive senescence impairs the energy metabolism of human luteinized granulosa cells.

RESEARCH QUESTION: Female age is the single greatest factor influencing reproductive performance and granulosa cells are considered as potential biomarkers of oocyte quality. Is there an age effect on the energy metabolism of human mural granulosa cells? DESIGN: Observational prospective cohort and experimental study including 127 women who had undergone IVF cycles. Women were allocated to two groups: a group of infertile patients aged over 38 years and a control group comprising oocyte donors aged less than 35 years. Individuals with pathologies that could impair fertility were excluded from both groups. Following oocyte retrieval, cumulus and granulosa cells were isolated and their bioenergetic properties (oxidative phosphorylation parameters, rate of aerobic glycolysis and adenine nucleotide concentrations) were analysed and compared. RESULTS: Human mural luteinized granulosa and cumulus cells present high rates of aerobic glycolysis that cannot be increased further when mitochondrial ATP synthesis is inhibited. Addition of follicular fluid to the experimental media is necessary to reach the full respiratory capacity of the cells. Granulosa cells from aged women present lower mitochondrial respiration (12.8 ± 1.6 versus 11.2 ± 1.6 pmol O2/min/mg; P = 0.046), although mitochondrial mass is not decreased, and lower aerobic glycolysis, than those from young donors (12.9 ± 1.3 versus 10.9 ± 0.5 mpH/min/mg; P = 0.009). The concurrent decrease in the two energy supply pathways leads to a decrease in the cellular energy charge (0.87 ± 0.01 versus 0.83 ± 0.02; P < 0.001). CONCLUSIONS: Human mural luteinized granulosa cells exhibit a reduction in their energy metabolism as women age that is likely to influence female reproductive potential.

Prediction of oocyte quality using mRNA transcripts screened by RNA sequencing of human granulosa cells.

RESEARCH QUESTION: Can RNA transcripts of granulosa cells be used to assess oocyte quality? The possibility of predicting the developmental competence of oocytes by RNA sequencing analysis of granulosa cells was evaluated. DESIGN: Granulosa cell samples were collected from 29 women undergoing assisted reproductive technology treatment and divided into two groups: 14 samples from the high blastocyst rate group and 15 from the low blastocyst rate group. Ten samples from each group were selected for RNA sequencing. RESULTS: A total of 129 differentially expressed genes associated with high developmental competence of oocytes were identified. COL1A2, renin and COL1A1 were selected and further examined by quantitative real-time polymerase chain reaction (qRT-PCR). Expression levels of COL1A2 and renin by qRT-PCR were consistent with the results of RNA sequencing. CONCLUSION: RNA sequencing data could provide novel marker genes for the non-invasive evaluation of oocyte quality and embryo developmental competence.

TMT-based proteomic and bioinformatic analyses of human granulosa cells from obese and normal-weight female subjects.

BACKGROUND: Increasing evidence supports a relationship between obesity and either infertility or subfertility in women. Most previous omics studies were focused on determining if the serum and follicular fluid expression profiles of subjects afflicted with both obesity-related infertility and polycystic ovary syndrome (PCOS) are different than those in normal healthy controls. As granulosa cells (GCs) are essential for oocyte development and fertility, we determined here if the protein expression profiles in the GCs from obese subjects are different than those in their normal-weight counterpart. METHODS: GC samples were collected from obese female subjects (n = 14) and normal-weight female subjects (n = 12) who were infertile and underwent in vitro fertilization (IVF) treatment due to tubal pathology. A quantitative approach including tandem mass tag labeling and liquid chromatography tandem mass spectrometry (TMT) was employed to identify differentially expressed proteins. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were then conducted to interrogate the functions and pathways of identified proteins. Clinical, hormonal, and biochemical parameters were also analyzed in both groups. RESULTS: A total of 228 differentially expressed proteins were noted, including 138 that were upregulated whereas 90 others were downregulated. Significant pathways and GO terms associated with protein expression changes were also identified, especially within the mitochondrial electron transport chain. The levels of free fatty acids in both the serum and follicular fluid of obese subjects were significantly higher than those in matched normal-weight subjects. CONCLUSIONS: In GCs obtained from obese subjects, their mitochondria were damaged and the endoplasmic reticulum stress response was accompanied by dysregulated hormonal synthesis whereas none of these changes occurred in normal-weight subjects. These alterations may be related to the high FFA and TG levels detected in human follicular fluid.

A high-androgen microenvironment inhibits granulosa cell proliferation and alters cell identity.

A naturally occurring bovine model with excess follicular fluid androstenedione (High A4), reduced fertility, and polycystic ovary syndrome (PCOS)-like characteristics has been identified. We hypothesized High A4 granulosa cells (GCs) would exhibit altered cell proliferation and/or steroidogenesis. Microarrays of Control and High A4 GCs combined with Ingenuity Pathway Analysis indicated that High A4 GCs had cell cycle inhibition and increased expression of microRNAs that inhibit cell cycle genes. Granulosa cell culture confirmed that A4 treatment decreased GC proliferation, increased anti-Müllerian hormone, and increased mRNA for CTNNBIP1. Increased CTNNBIP1 prevents CTNNB1 from interacting with members of the WNT signaling pathway thereby inhibiting the cell cycle. Expression of CYP17A1 was upregulated in High A4 GCs presumably due to reduced FOS mRNA expression compared to Control granulosa cells. Furthermore, comparisons of High A4 GC with thecal and luteal cell transcriptomes indicated an altered cellular identity and function contributing to a PCOS-like phenotype.

Granulosa cell genes that regulate ovarian follicle development beyond the antral stage: The role of estrogen receptor ß.

Follicle development beyond the preantral stage is dependent on gonadotropins. FSH signaling is crucial for the advancement of preantral follicles to the antral stage, and LH signaling is essential for further maturation of preovulatory follicles. Estrogen is intricately tied to gonadotropin signaling during the advanced stages of folliculogenesis. We observed that Erßnull ovarian follicles fail to develop beyond the antral stage, even after exogenous gonadotropin stimulation. As ERß is primarily expressed in the granulosa cells (GCs), we explored the gonadotropin-regulated GC genes that induce maturation of antral follicles. Synchronized follicle development was induced by administration of exogenous gonadotropins to wildtype 4-wk-old female rats. The GC transcriptome was analyzed via RNA-sequencing before and after gonadotropin stimulation. An Erßnull mutant model that fails to show follicle maturation was also included in order to identify the ERß-regulated genes involved at this step. We observed that specific groups of genes were differentially expressed in response to PMSG or hCG administration in wildtype rats. While some of the PMSG or hCG-induced genes showed a similar expression pattern in Erßnull GCs, a subset of PMSG- or hCG-induced genes showed a differential expression pattern in Erßnull GCs. These latter ERß-regulated genes included previously known FSH or LH target genes including Lhcgr, Cyp11a1, Cyp19a1, Pgr, Runx2, Egfr, Kiss1, and Ptgs2, which are involved in follicle development, oocyte maturation, and ovulation. We also identified novel ERß-regulated genes including Jaml, Galnt6, Znf750, Dusp9, Wnt16, and Mageb16 that failed to respond to gonadotropin stimulation in Erßnull GCs. Our findings indicate that the gonadotropin-induced spatiotemporal pattern of gene expression is essential for ovarian follicle maturation beyond the antral stage. However, expression of a subset of those gonadotropin-induced genes is dependent on transcriptional regulation by ERß.

Vaspin, a novel adipokine in woman granulosa cells physiology and PCOS pathogenesis?

Vaspin is a novel adipokine mainly expressed in visceral adipose tissue and closely related to obesity and insulin-resistance. Currently, data about its ovarian expression are limited to animal models and its role in human reproduction is largely unexplored. Our study's aims were then to characterise vaspin expression in the human ovary and to study in vitro its effects on granulosa cells physiology. Secondly, we assessed vaspin and its receptor GRP78 variations in granulosa cells and follicular fluid of a cohort of 112 infertile women undergoing an in vitro fertilisation procedure and allocated to three groups, each including normal-weight and obese subjects: 34 PCOS patients, 33 women with isolated polycystic ovary morphology (ECHO group) and 45 controls. Vaspin and GRP78 expression in the ovary was assessed by immunohistochemistry, RT-qPCR and Western blot. Granulosa cells and follicular fluid were analysed by RT-qPCR and ELISA, respectively. In vitro, granulosa cells metabolism was studied after stimulation with recombinant human vaspin, with and without a siRNA directed against GRP78. Vaspin was highly expressed in the human ovary and concentration-dependently enhanced granulosa cells steroidogenesis, proliferation and viability through GRP78 (P < 0.0001). Vaspin levels in both granulosa cells and follicular fluid were significantly higher in obese women (P < 0.0001) and in the normal-weight ECHO group (P < 0.001), which also had the highest expression rates of GRP78 (P < 0.05). Although further investigation is needed, vaspin appears as a novel modulator of human granulosa cells physiology and possibly plays a role in PCOS pathogenesis, notably protecting from insulin-resistance induced complications.

Effects of α-lipoic acid and myo-inositol supplementation on the oocyte environment of infertile obese women: A preliminary study.

Obesity is becoming pandemic and is associated with impaired reproductive potential. Oxidative stress, low-grade chronic inflammation and mitochondrial dysfunctions, which characterize obesity, strongly affect oocyte environment and function. Supplementation with antioxidant and anti-inflammatory compounds has been suggested to improve fertility. Here we evaluated the effect of α-lipoic acid and myo-inositol supplementation on the oocyte environment of infertile obese women. Nineteen normal-weight and twenty-three obese women, infertile for non-ovarian reasons, were recruited. For two months before ovarian stimulation, all women received 400 µg/die folic acid, whereas 15 obese were additionally supplemented with 800 mg α-lipoic acid, 2 g myo-inositol/die. Antioxidant capacity was measured in follicular fluid by enzymatic assay; mitochondrial DNA (mtDNA) content and mRNA levels of two respiratory chain subunits were analyzed in granulosa cells by Real-time PCR. Pregnancy rate was similar between normal-weight and treated obese, and lower in untreated obese patients. Supplemented women showed significantly higher antioxidant levels in follicular fluid compared to the two groups taking only folic acid. Conversely, granulosa cells mtDNA content was decreased in treated and higher in untreated obese patients compared to normal-weight women, suggesting mtDNA increases to compensate for oxidative-stress damages. Reduced expression of respiratory subunits in untreated obese may confirm mitochondria impairment. Interestingly, mtDNA levels inversely correlated to both total and metaphase II oocyte number. In this preliminary study, combined supplementation of α-lipoic acid and myo-inositol in infertile obese women was associated with amelioration in the oxidative status of the oocyte environment, possibly contributing to a higher pregnancy rate.

Granulosa cell-conditioned medium enhances steroidogenic competence of buffalo (Bubalus bubalis) theca cells.

Granulosa cells (GCs) and theca cells (TCs) are the main components of follicles, and the interactions between GCs and TCs play a significant role in steroidogenesis, follicular growth, and atresia. However, the effects of GCs in the form of conditioned medium on steroidogenesis in buffalo TCs remain unclear. In the present study, the impacts of GC-conditioned medium (GCCM) on androgen synthesis in buffalo TCs were examined. The results showed that GCCM collected at 48 h promoted both the expression levels of androgen synthesis-related genes (CYP11A1, CYP17A1, 3ß-HSD, and Star) and the secretion levels of testosterone in TCs. The treatment time of 48 h in GCCM improved both the expression levels of androgen synthesis-related genes (CYP11A1, CYP17A1, 3ß-HSD, and Star) and the secretion levels of testosterone in TCs. Furthermore, GCCM that was collected at 48 h and applied to TCs for 48 h (48 h and 48 h) promoted the sensitivity of buffalo TCs to LH. This study indicated that GCCM (48 h and 48 h) enhanced the steroidogenic competence of TCs mainly through facilitating the responsiveness of TCs to LH in buffalo. This study provides a basis for further exploration of interactions between GCs and TCs for steroidogenesis in the ovary.

Type 1 diabetes affects zona pellucida and genome methylation in oocytes and granulosa cells.

Diabetes affects oocyte nuclear and cytoplasmic quality. In this study, we generated a type 1 diabetes (T1D) mouse model by STZ injection to study the effects of T1D on zona pellucida and genomic DNA methylation of oocytes and granulosa cells. T1D mice showed fewer ovulated oocytes, reduced ovarian reserve, disrupted estrus cycle, and significantly ruptured zona pellucida in 2-cell in vivo embryos compared to controls. Notably, diabetic oocytes displayed thinner zona pellucida and treatment of oocytes with high concentration glucose reduced the zona pellucida thickness. Differential methylation genes in oocytes and granulosa cells were analyzed by methylation sequencing. These genes were significantly enriched in GO terms by GO analysis, and these GO terms were involved in multiple aspects of growth and development. Most notably, the abnormal methylation genes in oocytes may be related to oocyte zona pellucida changes in diabetic mice. These findings provide novel basic data for further understanding and elucidating dysgenesis and epigenetic changes in type 1 diabetes mellitus.

MicroRNA-486-5p inhibits ovarian granulosa cell proliferation and participates in the development of PCOS via targeting MST4.

OBJECTIVE: To explore whether microRNA-486-5p affected the proliferation of ovarian granulosa cells by targeting MST4 (silk/threonine protein kinase 4), thereby promoting the development of polycystic ovary syndrome (PCOS). MATERIALS AND METHODS: The level of microRNA-486-5p in PCOS tissues and adjacent normal tissues was detected by quantitative real-time polymerase chain reaction (qRT-PCR). After microRNA-486-5p up-regulation in KNG cells, the mRNA and protein level of related genes was examined using qRT-PCR and western blot assay, respectively. Meanwhile, cell proliferation and cell cycle were analyzed by cell counting kit-8 (CCK-8) assay and flow cytometry. After insulin treatment of KNG cells, expressions of microRNA-486-5p and MST4, cell proliferation as well as cell cycle, were detected by qRT-PCR, CCK-8 and flow cytometry, respectively. Furthermore, cell proliferation and cycle situation were examined after simultaneous up-regulation of MST4 and microRNA-486-5p in vitro. RESULTS: MicroRNA-486-5p expression in PCOS tissues was significantly lower than that of normal tissues. In KNG cells, up-regulation of microRNA-486-5p significantly inhibited cell proliferation and cell cycle. The levels of cycle-associated proteins including CDK2 and CCNB1 decreased significantly. The results of dual-luciferase reporter gene assay showed that microRNA-486-5p could bind to MST4. After up-regulating microRNA-486-5p, both the mRNA and protein levels of MST4 decreased remarkably. MST4 expression was found significantly elevated in PCOS tissues as well. After overexpression of MST4, cell proliferation was enhanced, cell cycle was promoted, and expressions of cycle-related proteins increased. After treatment with different concentrations of insulin in KNG cells, the expression level of microRNA-486-5p decreased in a concentration-dependent manner. However, opposite results were observed in MST4 level. Meanwhile, the proliferation ability and cell cycle of insulin-treated cells were significantly enhanced. In addition, the inhibitory effect of microRNA-486-5p on cell proliferation and cell cycle could be partially reversed by simultaneous up-regulation of MST4 and microRNA-486-5p. CONCLUSIONS: MicroRNA-486-5p can bind to MST4 in a targeted manner and inhibit the proliferation of ovarian granulosa cells, thereby inhibiting the development of PCOS.

Thermotolerance of camel (Camelus dromedarius) somatic cells affected by the cell type and the dissociation method.

Researchers dealing with heat stress experiments use different cell kinds and use trypsin that has been reported to affect the cellular proteins of cultured cells. Therefore, we compared the effects of acute and chronic exposures to high temperature (45 °C) on camel skin fibroblast and granulosa cells. Primary culture of fibroblasts and granulosa cells tolerated the acute heat shock for 2 h; however, granulosa cells cultured for long duration (20 h) showed thermotolerance when compared with the fibroblasts. Moreover, the effect of cell dispersion method (trypsin and mechanical dissociation) on the thermotolerance of sub-cultured cells was examined. Trypsin altered the morphology of fibroblasts and granulosa cells exposed to 45 °C for 4 h. Moreover, trypsin significantly reduced the fibroblast and granulosa cell migration in the wound healing assay. The current results demonstrate that cell passaging and cell type can affect the thermotolerance of the cells; it also revealed that trypsin could alter the cellular response to the heat shock. We raise the demand for another alternative method for cell dispersion in experiments dealing with cellular responses to the heat shock.

Effective and economical column-based method for RNA isolation from animal cells.

OBJECTIVES: To develop an efficient, economical, and low-toxicity method for the extraction of RNA from animal cells to meet a basic requirement of biological research: the isolation of high-quality RNA. RESULTS: Guanidine hydrochloride was used as a lysis buffer and Na-acetate was used as a wash buffer to extract RNA fragments from TM3 Leydig cells and ovarian granulosa cells efficiently. The functionality of the extracted RNA samples was verified through polymerase chain reaction (PCR) and real-time fluorescence quantitative PCR (RT-PCR). PCR results showed that the normal DNA column-based method could guarantee RNA integrity and could be used to amplify gene fragments successfully. RT-PCR analysis showed that the RNA samples isolated through the proposed method could be used to detect the expression levels of steroidogenic acute regulatory protein and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 mRNA in TM3 Leydig cells under induction by luteinizing hormone. The proposed method could be used to isolate RNA from mammalian cells and provided RNA yields of > 120 ng/5 × 106 cells. This method provided RNA with purities and yields that are sufficient for cDNA synthesis and PCR amplification in gene expression studies. CONCLUSIONS: The proposed RNA extraction method has the advantages of low toxicity, safe handling, and low cost. Isolation can be completed in 20 min. The proposed method can be used to extract RNA from various animal cell samples and is worth promoting.

Characterization of mRNA profiles of the exosome-like vesicles in porcine follicular fluid.

Extracellular vesicles such as exosomes contain several types of transcripts, including mRNAs and micro RNAs (miRNAs), and have emerged as important mediators of cell-to-cell communication. Exosome-like vesicles were identified in the ovarian follicles of several mammalian species. Although the miRNA contents have been extensively characterized, the detailed investigation of their mRNA profiles is lacking. Here, we characterize the mRNA profiles of exosome-like vesicles in ovarian follicles in a pig model. The mRNA contents of the exosome-like vesicles isolated from porcine follicular fluid were analyzed and compared with those from mural granulosa cells (MGCs) using the Illumina HiSeq platform. Bioinformatics studies suggested that the exosomal mRNAs are enriched in those encoding proteins involved in metabolic, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) -protein kinase B (AKT), and mitogen-activated protein kinase (MAPK) pathways. While the mRNA profile of the exosome-like vesicles resembled that of MGCs, the vesicles contained mRNAs barely detectable in MGCs. Thus, while the majority of the vesicles are likely to be secreted from MGCs, some may originate from other cell types, including theca cells and oocytes, as well as the cells of non-ovarian organs/tissues. Therefore, the mRNA profiles unveiled several novel characteristics of the exosome-like vesicles in ovarian follicles.

DNA methylome profiling of granulosa cells reveals altered methylation in genes regulating vital ovarian functions in polycystic ovary syndrome.

BACKGROUND: Women with polycystic ovary syndrome (PCOS) manifest a host of ovarian defects like impaired folliculogenesis, anovulation, and poor oocyte quality, which grossly affect their reproductive health. Addressing the putative epigenetic anomalies that tightly regulate these events is of foremost importance in this disorder. We therefore aimed to carry out DNA methylome profiling of cumulus granulosa cells and assess the methylation and transcript expression profiles of a few differentially methylated genes contributing to ovarian defects in PCOS. A total of 20 controls and 20 women with PCOS were selected from a larger cohort of women undergoing IVF, after carefully screening their sera and follicular fluids for hormonal and biochemical parameters. DNA extracted from cumulus granulosa cells of three women each, from control and PCOS groups was subjected to high-throughput, next generation bisulfite sequencing, using the Illumina HiSeq 2500® platform. Remaining samples were used for the validation of methylation status of some identified genes by pyrosequencing, and the transcript expression profiles of these genes were assessed by quantitative real-time PCR. RESULTS: In all, 6486 CpG sites representing 3840 genes associated with Wnt signaling, G protein receptor, endothelin/integrin signaling, angiogenesis, chemokine/cytokine-mediated inflammation, etc., showed differential methylation in PCOS. Hypomethylation was noted in 2977 CpGs representing 2063 genes while 2509 CpGs within 1777 genes showed hypermethylation. Methylation differences were also noted in noncoding RNAs regulating several ovarian functions that are dysregulated in PCOS. Few differentially methylated genes such as aldo-keto reductase family 1 member C3, calcium-sensing receptor, resistin, mastermind-like domain 1, growth hormone-releasing hormone receptor and tumor necrosis factor, which predominantly contribute to hyperandrogenism, premature luteolysis, and oocyte development defects, were explored as novel epigenetic candidates in mediating ovarian dysfunction. Methylation profiles of these genes matched with our NGS findings, and their transcript expression patterns correlated with the gene hypo- or hypermethylation status. CONCLUSION: Our findings suggest that the epigenetic dysregulation of genes involved in important processes associated with follicular development may contribute to ovarian defects observed in women with PCOS.

Transcriptomic Diversification of Granulosa Cells during Follicular Development in Chicken.

Granulosa cells play important roles in ovarian follicular development. To better understand the molecular mechanisms involved in this physiological process in chicken, high-throughput transcriptome analyses were performed to study the expression profiles of granulosa cells harvested from 6 mm white follicles, F5 follicles and F1 follicles. The analyses elucidated a clear tendency of granulosa cells in shifting its expression profile from proliferation to differentiation during follicular development. Transcripts down-regulated during this process were mainly associated with cell division, cell cycle and DNA replication while the up-regulated transcripts were related to ribosomal function, lipid metabolism and protein synthesis. Our study for the first time provides the complete gene expression profiles along follicular development supporting the active involvement of many genes characterized in cell signaling (AMH, Inhibins, Activins, BMPs) and transcription factors (SMAD3, SMAD5, ID1, ID2, ID3). Their temporal expression profiles support the notion of continual cross-talk between granulosa cells and its neighboring cells and shed light on the mechanisms behind avian follicular selection and pave the way to the better understanding of reproductive efficiency.

Identification and potential value of candidate microRNAs in granulosa cells of polycystic ovary syndrome.

BACKGROUND: Polycystic Ovary Syndrome (PCOS) is a major cause of anovulatory infertility. Some studies showed that miRNAs were used as diagnostic/prognostic biomarkers for various diseases. OBJECTIVE: To identify candidate miRNAs in Granulosa Cells (GCs) of PCOS and evaluate their potential values for PCOS diagnosis. METHODS: We screened differentially expressed miRNAs in GCs between PCOS and controls by the microarray data from the GEO database. GCs were collected from 21 controls and 24 PCOS. The candidate miRNAs were verified by qRT-PCR. The correlation was investigated between candidate miRNAs and clinical characteristics in participants. Diagnostic value of candidate miRNAs was analyzed by receiver operating characteristic (ROC) curve. RESULTS: Seven miRNAs were differentially expressed in PCOS compared with controls. Furthermore, the validation results demonstrated that hsa-miR-3188 and hsa-miR-3135b showed higher levels in GCs with PCOS patients (p< 0.05). In addition, the expressions of hsa-miR-3188 and hsa-miR-3135b were negative correlated with FSH and hsa-miR-3188 was positive correlated with BMI (p< 0.05). ROC analysis indicated that hsa-miR-3188 and hsa-miR-3135b could differentiate PCOS from controls, and the hsa-miR-3188/3135b improved the predictive accuracy for PCOS. CONCLUSIONS: The expressions of hsa-miR-3188 and hsa-miR-3135b in human GCs were significantly associated with PCOS. Moreover, the hsa-miR-3188/3135b has certain diagnostic value for distinguishing PCOS.