RNAseq of whole ovarian follicles from young and old mice.
ABSTRACT: Compare gene expression in whole ovarian follicles from young (6-12 weeks) and old (14-17 months) mice. Whole follicles,consist of the oocyte and granulosa cells. As oocytes are very difficult to extract intact from old follicles due to fibrosis, whole follicles were sequenced instead, so that old and young could be properly compared. 2-3 follicles each were sequenced from 4 old mice and 5 young mice. Each sample has both single- and paired-end runs.
Project description:Multilayer murine follicles were grown in 3% alginate and follicles were isolated for transcriptomics analysis at day 0, 2, 4,5,6,and 8 after encapsulation. This data complements to the GSE42795, where follicles were grown in vitro in 0.5% alginate gels A total of 8 samples were analyzed, each of them repeted in triplicate
Project description:The relevance of immune-endocrine interactions to the regulation of ovarian function in teleosts is virtually unexplored. As part of the innate immune response during infection, a number of cytokines such as tumor necrosis factor α (TNFα) and other immune factors, are produced and act on the reproductive system. However, TNFα is also an important physiological player in the ovulatory process in mammals. In the present study, we have examined for the first time the effects of TNFα in vitro in preovulatory ovarian follicles of a teleost fish, the brown trout (Salmo trutta). In control and recombinant trout TNFα (rtTNFα)-treated granulosa cells, we examined the percentage of apoptosis by flow cytometry analysis and cell viability by propidium iodide (PI) staining. Furthermore, we determined the in vitro effects of rtTNFα on follicle contraction and testosterone production in preovulatory trout ovarian follicles. In addition, we analyzed the gene expression profiles of control and rtTNFα-treated ovarian tissue by microarray and real-time PCR (qPCR) analyses.Treatment with rtTNFα induces ovarian cell apoptosis, decreases granulosa cell viability and stimulates the expression of genes known to be involved in the normal ovulatory process in trout. In addition, rtTNFα causes a significant increase in follicle contraction and testosterone production. Also, using a salmonid-specific microarray platform (SFA2.0 immunochip) we observed that rtTNFα induces the expression of genes known to be involved in inflammation, proteolysis and tissue remodeling. In view of these results, we propose that TNFα could have an important role in the biomechanics of follicle weakening, ovarian rupture and oocyte expulsion during ovulation in trout, primarily through its stimulation of follicular cell apoptosis and the expression of genes involved in follicle wall proteolysis and contraction. Reproductively mature female brown trout (Salmo trutta) from a cultured stock at the Piscifactoria de Bagà (Generalitat de Catalunya, Bagà, Spain) were kept under natural conditions of temperature and photoperiod. Fish at the preovulatory stage (according to the position of the germinal vesicle (GV) in oocytes that were cleared using a solution previously described), were anesthesized in 3-aminobenzoic acid ethyl ester (0.1 g/l; Sigma, Alcobendas, Spain) dissolved in fresh water, and the fish were sacrificed by concussion prior to the collection of the ovaries. The dissected ovaries were immediately used for the various in vitro assays. After dissection, brown trout preovulatory ovaries were placed in Hank´s balanced salt solution (HBSS) and individual ovarian follicles were manually separated with forceps from each ovary on ice, as previously described. To collect ovarian tissue for RNA extraction, preovulatory follicles from each of a total of three females were incubated (400 follicles/50 ml) in HBSS-BSA in the absence or presence of rtTNFα (100 ng/ ml, dissolved directly in HBSS-BSA), at 15ºC for 24 h with gentle shaking (100 rpm). At the end of the incubation follicles (previously de-yolked by gentle pressure) were removed, flash frozen in liquid nitrogen and stored at -80ºC until assayed.
Project description:In mammals, ovarian folliculogenesis leading to the ovulation of completely mature oocytes is a long and complex process that is regulated at different levels. The mechanisms that underlie the selection of one or several dominant follicles as well as the regulation of the number of ovulating follicles are largely unknown. Atresia is a phenomenon which affects the majority of developing follicles. In this project, we proposed to study the gene regulation of small antral follicles that are either healthy or undergoing atresia in pigs). Towards this purpose, we made a comparative transcriptomics study on granulosa cells, using a 9K nylon pig microarray (GPL3729) on granulosa cells from either small healthy antral follicles (SHF) or small antretic follicles (SAF). The images were quantified using AGscan software and the data were managed with BASE software. Statistical analysis was performed using R software. Transcriptomic analysis evidenced 1682 (912) differentially expressed genes with a 5% (1%) FDR between the two follicle classes. This research project which implicated three laboratories from INRA: "Laboratoire de Genetique Cellulaire" (UMR444-LGC), "Station d'Amelioration Genetique des Animaux" (UR 631-SAGA) and "Physiologie des Comportements et de la reproduction" (UMR 85-PRC) benefited from both European funding through SABRE project and French ANR funding through GenOvul project. Keywords: transcriptome analysis, pig, cattle, ovary, folliculogenesis, gene expression, cDNA microarray The data were obtained from 13 RNA samples: 6 small healthy follicles samples and 7 small atretic follicles. They were hybridized on a 9K pig nylon microarray (GPL3729).
Project description:Coordinated interactions between ovarian granulosa and theca cells are required for female endocrine function and fertility. To elucidate these interactions the regulation of the granulosa and theca cell transcriptomes during bovine antral follicle development were investigated. Granulosa cells and theca cells were isolated from small (<5 mm), medium (5-10 mm), and large (>10 mm) antral bovine follicles. A microarray analysis of 24,000 bovine genes revealed that granulosa cells and theca cells each had gene sets specific to small, medium and large follicle cells. Transcripts regulated (i.e., minimally changed 1.5-fold) during antral follicle development for the granulosa cells involved 446 genes and for theca cells 248 genes. Only 28 regulated genes were common to both granulosa and theca cells. Regulated genes were functionally categorized with a focus on growth factors and cytokines expressed and regulated by the two cell types. Candidate regulatory growth factor proteins mediating both paracrine and autocrine cell-cell interactions include macrophage inflammatory protein (MIP1 beta), teratocarcinoma-derived growth factor 1 (TDGF1), stromal derived growth factor 1 (SDF1; i.e., CXCL12), growth differentiation factor 8 (GDF8), glia maturation factor gamma (GMFG), osteopontin (SPP1), angiopoietin 4 (ANGPT4), and chemokine ligands (CCL 2, 3, 5, and 8). The current study examined granulosa cell and theca cell regulated genes associated with bovine antral follicle development and identified candidate growth factors potentially involved in the regulation of cell-cell interactions required for ovarian function. Experiment Overall Design: Granulosacell RNA samples from three groups of follicles different in size - small, medium, and large (pooled untreated ovaries) are compared between each other. Each group has 2 separate biological replicas; each replica contained pooled RNA from 20-40 ovaries from 6-10 different animals.
Project description:Gene expression analysis of yw follicles at S9/10a, S10B, S12, and S14; Gene expression analysis of pxt mutant follicles (f01000 and EY03052) at S10B, S12, S14 Drosophila ovarian follicles complete development using a spatially and temporally controlled maturation process in which they resume meiosis and secrete a multi-layered, protective eggshell before undergoing arrest and/or ovulation. Microarray analysis revealed more than 150 genes that are expressed in a stage-specific manner during the last 24 hours of follicle development. These include all 30 previously known eggshell genes, as well as 19 new candidate chorion genes and 100 other genes likely to participate in maturation. Mutations in pxt, encoding a putative Drosophila cyclooxygenase, cause many transcripts to begin expression prematurely, and are associated with eggshell defects. Somatic activity of Pxt is required, as RNAi knockdown of pxt in the follicle cells recapitulates both the temporal expression and eggshell defects. One of the temporally regulated genes, cyp18a1, which encodes a cytochromome P450 protein mediating ecdysone turnover, is downregulated in pxt mutant follicles, and cyp18a1 mutation itself alters eggshell gene expression. These studies further define the molecular program of Drosophila follicle maturation and support the idea that it is coordinated by lipid and steroid hormonal signals. minimum of 2 replicates per stage and genotype. yw=control follicles. pxt=mutant follicles.
Project description:Somatic cells surrounding the oocyte were sampled at the following stages: developmentally incompetent or poorly competent prophase I oocytes (NC1 oocytes), developmentally competent prophase I oocytes (C1 oocytes), and developmentally competent metaphase II oocytes (C2 oocytes). NC1 samples were collected from immature stage IV follicles, C1 samples from immature stage VI follicles, and C2 samples from in vitro matured stage VI follicles. Global transcriptional profiling was performed using somatic cells collected from xenopus ovarian follicles during in vivo oocyte developmental competence acquisition. Somatic cells were collected at 3 stages of oogenesis: early stage follicles (stage IV, vitellogenic, prophase I arrested oocytes, meiotically competent but developmentally incompetent, n=5), late stage follicles (stage VI, post-vitellogenic, prophase I arrested oocytes, meiotically competent and developmentally competent, n=5) and ovulatory follicles collected after in vitro maturation induction with hCG of post-vitellogenic follicles (metaphase II arrested oocytes, developmentally fully competent, n=5).
Project description:Fgf18 gene is strongly expressed in hair follicles of mouse dorsal skin during regressing (catagen) and resting (telogen) phases of hair cycle, but not in growth (anagen) phase. This study aims at identifying the function of Fgf18 in the regulation of hair cycle. To define target genes of Fgf18 during telogen phase of hair cycle, we generated mice in which Fgf18 gene is conditionally knocked out in keratin 5-positive epithelial cells (referred to as Fgf18 cKO below). We carried out microarray experiments with mouse back skin samples harboring telogen hair follicles obtained from three 42-d-old Fgf18 cKO male mice, or from three 42-d-old C57BL/6 male mice as control. Total RNA was isolated from each mouse and further purified to polyA RNA using oligo dT30 columns. The RNA samples were pooled for each group. Gene expression was analyzed by one-color analysis using duplicate arrays for each group.
Project description:Cumulus cells surrounding the oocyte were sampled at the following stages: developmentally incompetent or poorly competent prophase I oocytes (NC1 oocytes), developmentally competent prophase I oocytes (C1 oocytes), and developmentally competent metaphase II oocytes (C2 oocytes). NC1 samples were collected from immature, unexpanded cumulus-oocytes complexes (COC) from prepubertal (3-week-old) mice, C1 samples from immature, unexpanded cumulus-oocytes complexes (COC) from adult (8-week-old) and C2 samples from mature, expanded COCs obtained from the oviduct from 8-week-old mice after standard superovulation protocol. Global transcriptional profiling was performed using cumulus cells collected from murine ovarian follicles during in vivo oocyte developmental competence acquisition. Cumulus cells were collected at 3 stages: early stage follicles (prophase I arrested oocytes, meiotically competent but developmentally incompetent, n=5), late stage follicles (prophase I arrested oocytes, meiotically competent and developmentally competent, n=5) and ovulatory follicles collected in vivo (metaphase II arrested oocytes, developmentally fully competent, n=5).
Project description:A microarray study to compare gene expression during ovarian follicle development to better understand transcriptional changes during follicular growth. Multi-layered secondary follicles (150-180 µm in diameter) were mechanically isolated from ovaries of 16-day old CD-1 mice and individually encapsulated in 0.5% alginate. Follicles were culture for up to 8 days. Day 0 samples correspond to follicles that were isolated and immediately frozen. Samples were collected after 0, 2, 4, 5, 6 and 8 days in culture in triplicate and day 0 was collected 5 times.
Project description:Granulosa cells mature and die as ovarian follicles enlarge and die (undergo atresia) under the influence of hormones and intrafollicular factors. Later in follicular development, a fluid-filled antrum is formed, a process which is accompanied by a high rate of atresia. These small antral follicles (5 mm or less in diameter in the cow) contain granulosa of 2 different phenotypes, rounded or columnar, whereas follicles larger than 5 mm have the rounded phenotype only. Prior to ovulation, in larger follicles greater than 10 mm in size, the granulosa begin to migrate and differentiate in preparation for oocyte release and formation of the corpus luteum. These two key phases of follicular development were studied by gene expression microarray analysis using a bovine model to dissect the molecular mechanisms underlying these processes. Four groups of bovine ovarian follicles were selected for analysis. Follicle size, type and array number for each group are as follows: small (3-5 mm) healthy rounded (n=5), small healthy columnar (n=5), atretic (n=5) and large healthy (>10 mm; n=4). For each group, the RNA from a single follicle was used to hybridise an array, except for 3 small healthy samples which were pooled from 2 follicles each due to low RNA.