Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq of granulosa cells from IVF patients at different ages

ABSTRACT: Two major factors contributing to reduced fertility is use of exogenous hormones and old age. In previous experiments, we used mouse model to study transcriptional and cell-cell communication changes upon superovulation and ageing in female reproductive cells - oocytes (OC) - and somatic cells - granulosa (GC) - surrounding them. Here, we use granulosa cell samples from human patients undergoing in vitro fertilization to test how aged hormonally stimulated cell transcriptional profiles correspond to ones in mice. Total RNA was extracted from granulosa cell samples, collected during IVF procedures, using TRIzol. Further, RNA was cleaned from DNA with DNA-free™ DNA Removal Kit. ~1 µg of RNA was used to construct libraries with Truseq Stranded mRNA kit and barcoded with IDT for Illumina-TruSeq DNA and RNA UD Indexes. Samples were sequenced on Nextseq200 (Illumina) with 100bp paired-end sequencing.

INSTRUMENT(S): NextSeq 2000

ORGANISM(S): Homo sapiens

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PROVIDER: E-MTAB-13496 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The LH surge triggers dramatic transcriptional changes in genes associated with ovulation and luteinization. The present study investigated the spatiotemporal expression of nuclear factor interleukin-3 (NFIL3), a transcriptional regulator of the bZIP transcription factor superfamily, and its potential role in the ovary during the periovulatory period. NFIL3, also known as E4-binding protein 4 or NFIL3/E4BP4, was originally identified as a transcriptional repressor based on its DNA-binding activity at the promoter of the gene encoding the adenovirus E4 protein. Immature female rats were injected with PMSG, treated with hCG, and ovaries or granulosa cells were collected at various times after hCG. Nfil3 mRNA was highly induced both in intact ovaries and granulosa cells after hCG treatment. In situ hybridization demonstrated that Nfil3 mRNA was highly induced in theca-interstitial cells at 4-8 h after hCG, localized to granulosa cells at 12 h, and decreased at 24 h. Over-expression of NFIL3 in granulosa cells inhibited the induction of prostaglandin-endoperoxide synthase 2 (Ptgs2), progesterone receptor (Pgr), epiregulin (Ereg), and amphiregulin (Areg) and down regulated levels of prostaglandin E2. The inhibitory effect on Ptgs2 induction was reversed by NFIL3 siRNA treatment. In theca-interstitial cells the expression of hydroxyprostaglandin dehydrogenase 15-(NAD) (Hpgd) was also inhibited by NFIL3 over-expression. Data from luciferase assays demonstrated that NFIL3 over-expression decreased the induction of the Ptgs2 and Areg promoter activity. EMSA and ChIP analyses indicated that NFIL3 binds to the promoter region containing the DNA binding sites of CREB and C/EBP?. In summary, hCG induction of NFIL3 expression may modulate the process of ovulation and theca-interstitial and granulosa cell differentiation by regulating expression of PTGS2, PGR, AREG, EREG, and HPGD, potentially through interactions with CREB and C/EBP? on their target gene promoters. The granulosa cells were exposed to Ad- NFIL3 or Ad-GFP at a multiplicity of infection (MOI) of 50 pfu/cell. Two hours later, Medium was replaced with fresh Opti-MEM medium. At 24 h after adenovirus exposure, granulosa cells were treated with FSK + PMA for 6 h before collection for RNA isolation using a RNeasy kit according to the manufacturer’s instructions (Qiagen Inc., Valencia, CA). Five micrograms of total RNA were used as a template for cDNA synthesis by the University of Kentucky Microarray Core facility as described previously (47). The Affymetrix Rat 230-2.0 oligonucleotide array sets were hybridized, washed, and scanned using Affymetrix equipment and protocols (Affymetrix, Santa Clara, CA). The DNA microarray assays were performed on total RNA pooled from granulosa cells obtained from 3 separate experiments. The changes observed by DNA microarray analysis were confirmed by real-time PCR for a select subset of genes. Affymetrix GCS 3000 7G scanner was used.

Project description:The molecular mechanisms that regulate the pivotal transformation processes observed in the follicular wall following the pre-ovulatory LH-surge, are still not established, particularly for cells of the thecal layer. To elucidate thecal and granulosa cell type-specific biological functions and signaling pathways, large dominant bovine follicles were collected before and 21 hrs after an exogenous GnRH induced LH surge. Because LH receptor density varies within the granulosa cell populations, antral granulosa (aGC; those aspirated by follicular puncture) and membrane associated granulosa (mGC; those scraped from the follicular wall) were compared to thecal cell expression profiles determined by mRNA microarrays. Thecal cell gene expression was less affected in the peri-ovulatory follicle when compared to granulosa cells, as evidenced by only 2% versus 25% of the ~11,000 genes expressed changing in response to the LH surge, respectively. The majority of the 203 LH-regulated thecal genes were also LH regulated in granulosa cells, leaving a total of 58 genes as LH-regulated theca cell specific genes. Most of the 58 genes (i.e., 74%) thecal specific genes including several known thecal markers (CYP17A1, NR5A1) were downregulated, while most genes identified are new to theca. Many of the newly identified upregulated thecal genes (e.g., PTX3, RND3, PPP4R4) were also upregulated in granulosa. Minimal expression differences were observed between aGC and mGC, however, transcripts encoding extracellular proteins (NID2) and matrix modulators (ADAMTS1, SASH1) predominated these differences. We also identified large numbers of unknown LH-regulated granulosa cell genes and discuss their putative roles in ovarian function. The single dominant ovarian follicle was collected from each cow before the LH surge or 22 hours after GnRH (used to induce LH surge). RNA was extracted from three independent cells within each follicle and there were hybridized on Affymetrix microarrays.

Project description:The forkhead box transcription factor FOXO1 is highly expressed in granulosa cells of growing follicles but is down-regulated by FSH in culture or by LH-induced luteinization in vivo. To analyze the function of FOXO1, we infected rat and mouse granulosa cells with adenoviral vectors expressing two FOXO1 mutants: a gain-of-function mutant FOXOA3 that has two serine residues and one threonine residue mutated to alanines rendering this protein constitutively active and nuclear, and a FOXOA3-mutant DNA-binding domain (mDBD) in which the DBD is mutated. The infected cells were then treated with vehicle or FSH for specific time intervals. Infection of the granulosa cells was highly efficient, caused only minimal apoptosis, and maintained FOXO1 protein at levels of the endogenous protein observed in cells before exposure to FSH. RNA was prepared from control and adenoviral infected cells exposed to vehicle or FSH for 12 and 24 h. Affymetrix microarray and database analyses identified, and real time RT-PCR verified, that genes within the lipid, sterol, and steroidogenic biosynthetic pathways (Hmgcs1, Hmgcr, Mvk, Sqle, Lss, Cyp51, Tm7sf2, Dhcr24 and Star, Cyp11a1, and Cyp19), including two key transcriptional regulators Srebf1 and Srebf2 of cholesterol biosynthesis and steroidogenesis (Nr5a1, Nr5a2), were major targets induced by FSH and suppressed by FOXOA3 and FOXOA3-mDBD in the cultured granulosa cells. By contrast, FOXOA3 and FOXOA3- mDBD induced expression of Cyp27a1 mRNA that encodes an enzyme involved in cholesterol catabolism to oxysterols. The genes up-regulated by FSH in cultured granulosa cells were also induced in granulosa cells of preovulatory follicles and corpora lutea collected from immature mice primed with FSH (equine choriogonadotropin) and LH (human choriogonadotropin), respectively. Conversely, Foxo1 and Cyp27a1 mRNAs were reduced by these same treatments. Collectively, these data provide novel evidence that FOXO1 may play a key role in granulosa cells to modulate lipid and sterol biosynthesis, thereby preventing elevated steroidogenesis during early stages of follicle development. Experiment Overall Design: Immature rat granulosa cells were affected with either control (GFP) or FOXO1 mutant adenovirus (FOXOA3 and FOXOA3-mDBD) for 4 hours. Then the cells were treated with either FSH or vehicle for 24 hours. The gene expression profiles for the four treatment samples (control, control +FSH, FOXOA3 + FSH, FOXOA3-mDBD + FSH) were compared using Rat Genome 230.2 Arrays with one array per sample.

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RNA-seq of granulosa cells from IVF patients at different ages

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