Project description:We used MethylCap-seq and RRBS to profile methylomes of purified human ovarian granulosa cells. Genomic DNA methylation patterns in ovarian granulosa cells were compared between two groups of women: i) oocyte donors (n=20) who were young (age 26 ± 2.2 years) and had robust response to ovarian stimulation during assisted reproductive technology (ART) (mean number of oocytes retrieved = 25); versus ii) poor responders (n=20) who were older (age 40 ± 2.3 years) and responded poorly to ovarian stimulation during ART (oocytes retrieved ≤4 and peak estradiol level ≤ 1000 pg/ml). The first group served as healthy control. The second group represented the majority of women in their early 40s who have the natural age-related decline of ovarian functions and therefore respond poorly to ovarian stimulation during ART. We compared DNA methylomes in ovarian granulosa cells from oocyte donors versus poor responders using two approaches: MethylCap-seq for broader genomic coverage, and RRBS for absolute quantification. Due to very limited amount of materials available from each poor responder, samples containing equal amounts of granulosa cell DNA were pooled from 10 individuals in each group. A second set of experiments pooling granulosa cell DNA samples from independent donor and poor responder groups (ten individuals each) was then performed.
Project description:RNA was extracted from normal human granulosa cells from IVF patients (hGC1 and hGC2 samples) and from adult-type ovarian granulosa cell tumor samples (H1, H8, H20, H23, H24, H28, H30, H33, H4, H18) as described in Jamieson et al, 2010. RNA from all samples was linearly amplified using the Whole Transcriptome Amplification kit (Sigma), starting from 300ng of RNA, and with 12 amplifications cycles. cDNA was purified on columns and sent to the Nimblegen platform for hybridization and transcriptional profiling. The FOXL2 locus was gentoyped in tumor samples, and all samples were found positive for the recurrent somatic mutation p.Cys134Trp which is present in >95% of adult-type ovarian granulosa cell tumors (Shah et al, 2009).
Project description:The objective of this study was to investigate the differences in the gene expression profile of the granulosa cells from preovulatory follicles obtained after controlled ovarian hyperstimulation (COH) with either recombinant FSH (rFSH) or urine derived human menopausal gonadotropins (hMG).
Project description:An in vitro model of human ovarian follicles would greatly benefit the study of female reproduction. Ovarian development requires the combination of germ cells and their supporting somatic cells, known as granulosa cells. Whereas efficient protocols exist for generating human primordial germ cell-like cells (hPGCLCs) from human iPSCs, a method of generating granulosa cells has been elusive. Here we report that simultaneous overexpression of two transcription factors (TFs) can direct the differentiation of human iPSCs to granulosa-like cells. We elucidate the regulatory effects of several granulosa-related TFs, and establish that overexpression of NR5A1 and either RUNX1 or RUNX2 is necessary and sufficient to generate granulosa-like cells. Our granulosa-like cells have transcriptomes similar to human fetal ovarian cells, and recapitulate key ovarian phenotypes including follicle formation and steroidogenesis. When aggregated with hPGCLCs, our cells form ovary-like organoids (ovaroids) and support hPGCLC development from the premigratory to the gonadal stage as measured by induction of DAZL expression. This model system will provide unique opportunities for studying human ovarian biology, and may enable the development of therapies for female reproductive health.
Project description:An in vitro model of human ovarian follicles would greatly benefit the study of female reproduction. Ovarian development requires the combination of germ cells and their supporting somatic cells, known as granulosa cells. Whereas efficient protocols exist for generating human primordial germ cell-like cells (hPGCLCs) from human iPSCs, a method of generating granulosa cells has been elusive. Here we report that simultaneous overexpression of two transcription factors (TFs) can direct the differentiation of human iPSCs to granulosa-like cells. We elucidate the regulatory effects of several granulosa-related TFs, and establish that overexpression of NR5A1 and either RUNX1 or RUNX2 is necessary and sufficient to generate granulosa-like cells. Our granulosa-like cells have transcriptomes similar to human fetal ovarian cells, and recapitulate key ovarian phenotypes including follicle formation and steroidogenesis. When aggregated with hPGCLCs, our cells form ovary-like organoids (ovaroids) and support hPGCLC development from the premigratory to the gonadal stage as measured by induction of DAZL expression. This model system will provide unique opportunities for studying human ovarian biology, and may enable the development of therapies for female reproductive health.
Project description:The granulosa cells in the mammalian ovarian follicle respond to gonadotropin signalling and are involved in the processes of folliculogenesis and oocyte maturation. Studies on gene expression and regulation in human granulosa cells are of interest due to their potential for estimating the oocyte viability and IVF success. The current study determined the mRNA profile by deep sequencing of the two intrafollicular somatic cell types: mural and cumulus granulosa cells isolated from women undergoing controlled ovarian stimulation and in vitro fertilization.
Project description:Granulosa cells (GCs) have many endocrine functions. However, in long-term in vitro culture GCs can change their properties. GCs were collected from hyper-stimulated ovarian follicles from woman during IVF procedures. They were cultured in in vitro long-term culture. RNA was collected after 1, 7, 15 and 30 days of culture. Expression microarrays were used for analysis, which allowed to identify groups of genes characteristic for particular cellular processes. In this study, we demonstrated the gene expression profile of long time primary cultured human ovarian granulosa cells.
Project description:The Forkhead Box, FOXO1 and FOXO3, transcription factors regulate multiple functions in mammalian cells. Selective inactivation of the Foxo1 and Foxo3 genes in murine ovarian granulosa cells severely impairs follicular development and apoptosis causing infertility, and as shown herein, granulosa cell tumor (GCT) formation. Coordinate depletion of the tumor suppressor Pten gene in the Foxo1/3 strain enhanced the penetrance and onset of GCT formation A direct comparison of ovarian granulosa cells from wild type d25 and FOXO/PTEN knockout granulosa cell tumors.
Project description:The granulosa cells in the mammalian ovarian follicle respond to gonadotropin signalling and are involved in the processes of folliculogenesis and oocyte maturation. Studies on gene expression and regulation in human granulosa cells are of interest due to their potential for estimating the oocyte viability and IVF success. The current study determined the mRNA profile by deep sequencing of the two intrafollicular somatic cell types: mural and cumulus granulosa cells isolated from women undergoing controlled ovarian stimulation and in vitro fertilization. Paired cumulus and mural granulosa samples were analysed from 3 women participating in IVF procedure. Differential gene expression study was performed. The identified gene expression profile was also used for predicting targets for miRNAs that were also identified from the same samples (GSE46489).
Project description:FOXL2 is a transcription factor essential for female fertility, expressed in somatic cells of the ovary, notably granulosa cells. In the mouse, Foxl2 deletion leads to partial sex reversal postnatally. However, deletion of the gene in 8-week-old females leads to granulosa to Sertoli cell transdifferentiation. We hypothesised that different outcomes of Foxl2 deletion in embryonic versus adult ovary may depend on a different role played across ovarian development. Therefore, we characterised the dynamics of gene expression and chromatin accessibility changes in purified murine granulosa cells across key developmental stages (E14.5, 1 and 8 weeks). We then performed genome-wide identification of FOXL2 target genes and on-chromatin interacting partners by ChIP-SICAP. We found that FOXL2 regulates more genes at postnatal stages, through the interaction with factors regulating primordial follicle activation (PFA), such as NR5A2, and others regulating steroidogenesis including AR and ESR2. As a proof of principle experiment, we chose one FOXL2 interactor, Ubiquitin specific protease 7 (USP7) and showed that deletion of this gene in granulosa cells leads to a blockage of PFA, impaired ovary development and sterility. Our study constitutes a comprehensive resource for exploration of the molecular mechanisms of ovarian development and causes of female infertility.