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.

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.

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.

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.

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.

Expression of atresia biomarkers in granulosa cells after ovarian stimulation in heifers.

The use of younger gamete donors in dairy cattle genetic selection programs significantly accelerates genetic gains by decreasing the interval between generations. Ovarian stimulation (OS) and the practice of follicle-stimulating hormone (FSH) withdrawal, also known as coasting, are intensively used in pre-pubertal heifers without detrimental effects on subsequent reproductive performance but generally with lower embryo yields. However, recent data from embryo transfer programs showed similar embryo yields in younger and sexually mature animals but with a significant difference in the coasting period. The aim of the present study was to identify a set of granulosa cell biomarkers capable of distinguishing optimal follicle differentiation from late differentiation and atresia in order to assess the differences in coasting dynamics between pre- and post-pubertal donors. We integrated transcriptomic data sets from a public depository and used vote counting meta-analysis in order to elucidate the molecular changes occurring in granulosa cells during late follicle differentiation and atresia. The meta-analysis revealed the gene expression associated with follicle demise, and most importantly, identified potential biomarkers of that status in bovine granulosa cells. The comparison of the expression of six biomarkers between pre- and post-pubertal donors revealed that younger donors had more signs of atresia after the same period of coasting. We found different follicular dynamics following coasting in younger donors. It is possible that younger donors are less capable to sustain follicular survival most likely due to insufficient luteinizing hormone signaling. In summary, the pre-pubertal status influences follicular dynamics and reduces the oocyte developmental competence curve following OS and FSH withdrawal in heifers.

Regulation by FSH of the dynamic expression of retinol-binding protein 4 in the mouse ovary.

BACKGROUND: Ovarian retinoid homeostasis plays an important role in the physiological function of the ovary. Retinol-binding protein 4 (RBP4) acts as the mediator for the systemic and intercellular transport of retinol and is heavily involved in cellular retinol influx, efflux, and exchange. However, the expression patterns and regulatory mechanisms of Rbp4 in the ovary remain unclear. METHODS: The expression pattern of ovarian Rbp4 was examined in immature mice during different developmental stages and in adult mice during different stages of the estrous cycle. The potential regulation and mechanisms of ovarian Rbp4 expression by estrogen and related gonadotropins in mouse ovaries were also investigated. RESULTS: The present study demonstrated that the ovarian expression of Rbp4 remained constant before puberty and increased significantly in the peripubertal period. In adult female mice, the expression of Rbp4 increased at proestrus and peaked at estrus at both the mRNA and protein levels. The protein distribution of RBP4 was mainly localized in the granulosa cell and theca cell layer in follicles. In addition, the expression of Rbp4 was significantly induced by follicle-stimulating hormone (FSH) or FSH + luteinizing hormone (LH) in combination in immature mouse (3 weeks old) ovaries in vivo and in granulosa cells cultured in vitro, both at the mRNA and protein levels. In contrast, treatment with LH or 17ß-estradiol did not exhibit any observable effects on ovarian Rbp4 expression. Transcription factors high-mobility group AT-hook 1 (HMGA1), steroidogenic factor 1 (SF-1), and liver receptor homolog 1 (LRH-1) (which have been previously shown to be involved in activation of Rbp4 transcription), also responded to FSH stimulation. In addition, H-89, an inhibitor of protein kinase A (PKA), and the depletion of HMGA1, SF-1, and LRH-1 by small interfering RNAs (siRNAs), resulted in a dramatic loss of the induction of Rbp4 expression by FSH at both the mRNA and protein levels. CONCLUSIONS: These data indicate that the dynamic expression of Rbp4 is mainly regulated by FSH through the cAMP-PKA pathway, involving transcriptional factors HMGA1, SF-1, and LRH-1, in the mouse ovary during different stages of development and the estrous cycle.

Histological and immunohistochemical evaluation of granulosa cells during different stages of folliculogenesis in bovine ovaries.

Bovine granulosa cells (GC) vary in their morphological aspect during different stages of folliculogenesis. In this study, 10 morphologically normal bovine ovaries were collected to study the structural aspects of different stages of GC using intermediate filament protein antibodies including cytokeratin AE1/AE3 (AE1/AE3), vimentin, nectin-4 and desmin. Hormonal immunolocalization was assessed using the immunomarkers anti-Müllerian hormone (AMH) and inhibin alpha. In addition, tumour markers and proliferation markers using c-erbB-2 oncoprotein and proliferating cell nuclear antigen, respectively, were investigated. The immunolabelling of AE1/AE3 in GC was strongest in the early follicle stage and gradually decreased when reaching the Graafian follicle stage. Its immunolabelling increased again as the stage progressed from stage I to stage III. The immunolabelling of inhibin alpha was inversely proportional to that of AE1/AE3 in the developing ovarian follicles as their immunolabelling is opposite to each other during folliculogenesis. AMH was immunopositive in almost all GC stages in different intensities and percentages, except for some negative staining in the atretic IV follicles. The atretic IV follicle is a unique type of atretic follicle that shows Call-Exner body formation, which was mainly found in older cows in this study. The distinct patterns of immunoreactivity for various types of immunomarkers in the different GC stages will play an important role in diagnostic assistance of various follicle conditions, including cystic ovaries and GC tumours.

Granulosa cell biomarkers to predict pregnancy in ART: pieces to solve the puzzle.

Cumulus and mural granulosa cells of the ovarian follicle surround and interact with the developing oocyte. These follicular cells reflect the oocyte's overall health and may indicate subsequent developmental competence of embryos. Biomarkers of granulosa cells associated with individual oocytes could potentially be used in assisted reproduction to indicate which embryos have the best chance of implanting in the uterus and completing gestation. In this review, we have performed a comprehensive assessment of the recent literature for human cumulus and mural granulosa cell mRNA biomarkers as they relate to pregnancy and live birth. A critical discussion of variables affecting granulosa gene expression profiles for in vitro fertilization patients, including patient demographics and ovarian stimulation regimens, is presented. Although studies with microarray data were evaluated, this synopsis focuses on expressed genes that have been validated by quantitative RT-PCR. Furthermore, we summarize the current published data that support or refute identified granulosa expressed genes as potential biomarkers of embryos that give rise to ongoing pregnancy and live birth. Finally, we review studies that offer predictive models for embryo selection for uterine transfer based on biomarkers that show differential gene expression.

Serine threonine kinase receptor associated protein regulates early follicle development in the mouse ovary.

The molecular mechanisms involved in regulating the development of small, gonadotrophin-independent follicles are poorly understood; however, many studies have highlighted an essential role for TGFB ligands. Canonical TGFB signalling is dependent upon intracellular SMAD proteins that regulate transcription. STRAP has been identified in other tissues as an inhibitor of the TGFB-SMAD signalling pathway. Therefore, in this study we aimed to determine the expression and role of STRAP in the context of early follicle development. Using qPCR, Strap, Smad3 and Smad7 revealed similar expression profiles in immature ovaries from mice aged 4-16 days containing different populations of early growing follicles. STRAP and SMAD2/3 proteins co-localised in granulosa cells of small follicles using immunofluorescence. Using an established culture model, neonatal mouse ovary fragments with a high density of small non-growing follicles were used to examine the effects of Strap knockdown using siRNA and STRAP protein inhibition by immuno-neutralisation. Both interventions caused a reduction in the proportion of small, non-growing follicles and an increase in the proportion and size of growing follicles in comparison to untreated controls, suggesting inhibition of STRAP facilitates follicle activation. Recombinant STRAP protein had no effect on small, non-growing follicles, but increased the mean oocyte size of growing follicles in the neonatal ovary model and also promoted the growth of isolated preantral follicles in vitro Overall findings indicate STRAP is expressed in the mouse ovary and is capable of regulating development of small follicles in a stage-dependent manner.

Local effect of bisphenol A on the estradiol synthesis of ovarian granulosa cells from PCOS.

Close relationship between polycystic ovary syndrome (PCOS) and bisphenol A (BPA) has drawn much attention in recent years, while the underlying mechanisms are poorly understood. In our study, we aim to detect BPA concentration in the follicular fluid and investigate its effect on estradiol synthesis in human granulosa cells from PCOS and non-PCOS patients. Follicular fluid and granulosa cells were collected from women who underwent controlled ovarian stimulation for in vitro fertilization or intracytoplasmic sperm injection. BPA concentration in the follicular fluid from PCOS patients (440.50 ± 63.70 pg/ml) was significantly higher than that from non-PCOS patients (338.00 ± 57.88 pg/ml). Expression of aromatase and estradiol synthesis in cultured granulosa cells was examined after treatment with BPA from 0.01 to 1 µM for 24 h. Expression of aromatase and estradiol synthesis was downregulated by BPA in a dose-dependent manner in PCOS, but no effect was observed in granulosa cells from non-PCOS patients. These findings provide evidence that increased BPA concentration in the follicular fluid of PCOS patients may play an important role in its pathogenesis by attenuating the expression of aromatase in granulosa cells.

Involvement of PAPP-A and IGFR1 in Cystic Ovarian Disease in Cattle.

Cystic ovarian disease (COD) is one of the main causes of infertility in dairy cattle. It has been shown that intra-ovarian factors, such as members of the insulin-like growth factor (IGF) system, may contribute to follicular persistence. The bioavailability of IGF to initiate its response by binding to specific receptors (IGFRs) depends on interactions with related compounds, such as pregnancy-associated plasma protein A (PAPP-A). The aim of this study was to determine IGFR1 and PAPP-A expression both in follicles at different stages of development and in cysts, to evaluate the roles in the etiopathogenesis of COD in cattle. The mRNA expression of PAPP-A was higher in granulosa cells of large tertiary follicles than in cysts, whereas the protein PAPP-A present in the follicular fluid from these follicles showed no differences. Although no PAPP-A mRNA expression was detected in smaller tertiary follicles, in their follicular fluid, this protease was detected in lesser concentration than in cysts. The mRNA expression of IGFR1 was lower in granulosa cells from cystic follicles than in those from tertiary ones. However, the protein expression of this receptor presented the highest levels in cystic structures, probably to increase the possibility of IGF response. The data obtained would indicate that animals with COD have an altered regulation of the IGF system in the ovary, which could be involved in the pathogenesis of this disease in cattle.

Involvement of hyaluronan synthesis in ovarian follicle growth in rats.

Most of the previous studies on ovarian hyaluronan (HA) have focused on mature antral follicles or corpora lutea, but scarcely on small preantral follicles. Moreover, the origin of follicular HA is unknown. To clarify the localization of HA and its synthases in small growing follicles, involvement of HA in follicle growth, and gonadotropin regulation of HA synthase (Has) gene expression, in this study, perinatal, immature, and adult ovaries of Wistar-Imamichi rats were examined histologically and biochemically and by in vitro follicle culture. HA was detected in the extracellular matrix of granulosa and theca cell layers of primary follicles and more advanced follicles. Ovarian HA accumulation ontogenetically started in the sex cords of perinatal rats, and its primary site shifted to the intrafollicular region of primary follicles within 5 days of birth. The Has1-3 mRNAs were expressed in the ovaries of perinatal, prepubertal, and adult rats, and the expression levels of Has1 and Has2 genes were modulated during the estrous cycle in adult rats and following administration of exogenous gonadotropins in immature acyclic rats. The Has1 and Has2 mRNAs were predominantly localized in the theca and granulosa cell layers of growing follicles respectively. Treatments with chemicals known to reduce ovarian HA synthesis induced follicular atresia. More directly, the addition of Streptomyces hyaluronidase, which specifically degrades HA, induced the arrest of follicle growth in an in vitro culture system. These results indicate that gonadotropin-regulated HA synthesis is involved in normal follicle growth.

Effects of progesterone and its metabolites on human granulosa cells.

The corpus luteum (CL) is under control of gonadotrophic hormones and produces progesterone, which is necessary for endometrial receptivity. Recent studies have shown that progesterone and its metabolites are involved in cell proliferation and apoptosis of cancer cells. Here weanalyzed the role of progesterone and its meta-bolites on luteinized granulosa cells (LGC) by FACS analysis and quantitative Real-Time PCR. We detected the mRNA of the progesterone metabolizing genes SRD5A1, AKR1C1, and AKR1C2 in LGC. The stimulation of LGC with progesterone or progesterone metabolites did not show any effect on the mRNA expression of these genes. However, a downregulation of Fas expression was found to be accomplished by progesterone and human chorionic gonadotropin. Our findings do not support the concept of an effect of progesterone metabolites on LGCs. However, it suggests an antiapoptotic effect of hCG and progesterone during corpus luteum development by downregulation of Fas.

Study on connexin gene and protein expression and cellular distribution in relation to real-time proliferation of porcine granulosa cells.

Granulosa cells (GCs) play an important role during follicle growth and development in preovulatory stage. Moreover, the proteins such as connexins are responsible for formation of protein channel between follicular-cumulus cells and oocyte. This study was aimed to investigate the role of connexin expression in porcine GCs in relation to their cellular distribution and real-time cell proliferation. In the present study, porcine GCs were isolated from the follicles of puberal gilts and then cultured in a real-time cellular analyzer (RTCA) system for 168 h. The expression levels of connexins (Cxs) Cx36, Cx37, Cx40 and Cx43 mRNA were measured by RQ-PCR analysis, and differences in the expression and distribution of Cx30, Cx31, Cx37, Cx43 and Cx45 proteins were analyzed by confocal microscopic visualization. We found higher level of Cx36, Cx37, and Cx43 mRNA expression in GCs at recovery (at 0 h of in vitro culture, IVC) compared to all analyzed time periods of IVC (24, 48, 72, 96, 120, 144 and 168 h; P<0.001). On the other hand, the expression level of Cx40 transcripts was higher after 24 h of IVC compared to 0 h and the other times of IVC (P<0.001). Similarly to mRNAs, the expression levels of Cx31, Cx37 and Cx45 proteins were higher before (0 h) compared to after 168 h of IVC. The expression of Cx30 and Cx43, however, did not vary between the groups. In all, the proteins were distributed throughout the cell membrane rather than in the cytoplasm both before and after IVC. After 24 h of IVC, we observed a significant increase in the proliferation of GCs (log phase). We found differences in the proliferation index between 72-96 and 96- 140 h within the same population of GCs. In conclusion, the decrease in the expression of Cx mRNAs and proteins following IVC could be associated with a breakdown in gap-junction connections (GJCs), and leads to the decreased of their activity, which may be a reason of non-functional existence of connexon in follicular granulosa cells. These data indicated that the differentiation and proliferation of GCs and lutein cells are regulated by distinct mechanisms in pigs.

The influence of ovarian stromal/theca cells during in vitro culture on steroidogenesis, proliferation and apoptosis of granulosa cells derived from the goat ovary.

Early follicular development is closely related to oocyte-granulosa cells-ovarian stromal cells/theca cells. The aim of the present study was to investigate the effects of ovarian cortical, medullary stromal and theca cells on oestradiol and progesterone biosynthesis, proliferation and apoptosis of goat ovary granulosa cells in vitro. Using Transwell coculture system, we evaluated steroidogenesis, cell proliferation and apoptosis, and some molecular expressions regarding steroidogenic enzyme, luteinizing hormone receptor and apoptosis-related genes in granulosa cells. The results indicated that ovarian stromal/theca cells were able to stimulate oestradiol and progesterone production, promote cell proliferation and inhibit apoptosis of granulosa cells. Among all the three kinds of cells, theca cells affected strongly on granulosa cell function, and ovarian medullary stromal cells had the weakest effect on granulosa cells. These findings would provide an important knowledge of cell interaction among follicular cells during follicular development.

A novel approach to quantifying ovarian cell lipid content and lipid accumulation in vitro by confocal microscopy in lean women undergoing ovarian stimulation for in vitro fertilization (IVF).

PURPOSE: To quantify intracellular lipid levels in cumulus cells (CCs) and mural granulosa cells (MGCs) of lean women undergoing gonadotropin therapy for in vitro fertilization (IVF), based upon different cell preparation methods. METHODS: CCs and MGCs from 16 lean women undergoing ovarian stimulation for IVF were studied. Cells were pooled by cell type, with each type of cell separated into two groups for determination of initial lipid content (Method 1) and subsequent lipid accumulation in vitro (Method 2). Cells for initial lipid content were immediately fixed at the time of the oocyte retrieval with 4% paraformaldehyde in suspension, while those for subsequent lipid accumulation in vitro were cultured for 4 h with 5% fetal calf serum and then fixed. Cells were treated with lipid fluorescent dye BODIPY® FL C16 and nuclear marker DAPI. Intracellular lipid was quantified by confocal microscopy, using ImageJ software analysis. RESULTS: There was no significant effect of cell type (P = 0.2) or cell type-cell preparation method interaction (P = 0.8) on cell area (Method 1: CC 99.7 ± 5.1, MGC 132.8 ± 5.8; Method 2: CC 221.9 ± 30.4, MGC 265.1 ± 48.5 µm(2)). The mean area of all cells combined was significantly less for cells prepared by Method 1 (116.2 ± 4.9 µm(2)) vs. Method 2 (243.5 ± 22.5 µm(2), P < 0.00005). Intracellular lipid level, however, was significantly altered by cell preparation method (P < 0.05; cell preparation method-cell type interaction, P < 0.00001). Initial lipid content was significantly lower in CC (74.5 ± 9.3) than MGC (136.3 ± 16.7 fluorescence/cell area, P < 0.00005), while subsequent lipid accumulation in vitro was significantly higher in CC (154.0 ± 9.1) than MGC (104.6 ± 9.9 fluorescence/cell area, P < 0.00001). The relatively diminished initial CC lipid content compared to subsequent CC lipid accumulation in vitro (P < 0.00001), and the opposite pattern for MGC (P < 0.05), significantly lowered the CC/MGC lipid ratio in Method 1 (0.55 ± 0.04) vs. Method 2 (1.58 ± 0.10, P < 0.00001). CONCLUSIONS: Differential uptake or utilization of lipid by CC and MGC occurs during oocyte maturation and steroidogenesis, respectively, with the amount of lipid present in ovarian cells a function of both the follicular microenvironment at the time of the oocyte retrieval and the capacity of these cells to accumulate lipid in vitro over time.

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.