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Gene Expression in Granulosal Cells From Persistent Follicles

ABSTRACT: Granulosa cells form highly specialized membrane connections with the oocyte and each other, allowing the passage of regulatory molecules and metabolites between cells. These connections must be maintained for oocyte growth in vitro and have been implicated in oocyte aging both in vivo and in vitro. Changes in gene expression in granulosa cells from persistent follicles may adversely affect oocyte development. Previous studies have reported changes in gene expression in oocytes and high embryonic loss associated with persistent follicles. Cows were assigned randomly to two groups; growing follicles on day 8 (young cells) and persistent follicles on day 15 (aged cells) of the estrous cycle (estrus = d 0). Cows were super-stimulated with Folltropin-V® on day 1 to 4 and received 25 mg PGF2α on day 6. Cows in P15 group received progesterone from CIDR-B devices on day 4 through 13. Follicles were aspirated immediately after colpotomy. Granulosa cells were collected from each aspirate, pelleted, and stored at -80° until total RNA isolation. Three biological replicates, after linear amplification, were used in a direct comparison microarray experiment using a bovine oligo array. Microarray data were analyzed using GenePix AutoProcessor (GPAP 3.2; http://darwin.biochem.okstate.edu/gpap32/). The analysis revealed up regulation of 272 genes (M-value ≥ 0.9, q ≤ 0.05) and down regulation of 203 genes (M-value ≤ -0.9, q ≤ 0.05) in granulosa cells from persistent follicles (aged cells) in comparison to growing follicles (young cells). Grouping of these genes into themes revealed down regulation of many genes involved in protein metabolism, amino acid transport, and mitosis regulation and an up-regulation of apoptotic genes. However, there was no change in the expression of gap junction genes. These data suggest that aged cells have a reduced capacity to regulate mitosis and provide amino acids to the oocyte. Supported by project Hatch 427 (NE 1007) of the WV Agricultural and Forestry Experiment Station. Keywords: direct comparison, cell type comparison

ORGANISM(S): Bos taurus

PROVIDER: GSE4930 | GEO | 2006/06/21

SECONDARY ACCESSION(S): PRJNA95879

REPOSITORIES: GEO

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Project description:Granulosa cells form highly specialized membrane connections with the oocyte and each other, allowing the passage of regulatory molecules and metabolites between cells. These connections must be maintained for oocyte growth in vitro and have been implicated in oocyte aging both in vivo and in vitro. Changes in gene expression in granulosa cells from persistent follicles may adversely affect oocyte development. Previous studies have reported changes in gene expression in oocytes and high embryonic loss associated with persistent follicles. Cows were assigned randomly to two groups; growing follicles on day 8 (young cells) and persistent follicles on day 15 (aged cells) of the estrous cycle (estrus = d 0). Cows were super-stimulated with Folltropin-VÂ® on day 1 to 4 and received 25 mg PGF2Î± on day 6. Cows in P15 group received progesterone from CIDR-B devices on day 4 through 13. Follicles were aspirated immediately after colpotomy. Granulosa cells were collected from each aspirate, pelleted, and stored at -80Â° until total RNA isolation. Three biological replicates, after linear amplification, were used in a direct comparison microarray experiment using a bovine oligo array. Microarray data were analyzed using GenePix AutoProcessor (GPAP 3.2; http://darwin.biochem.okstate.edu/gpap32/). The analysis revealed up regulation of 272 genes (M-value â¥ 0.9, q â¤ 0.05) and down regulation of 203 genes (M-value â¤ -0.9, q â¤ 0.05) in granulosa cells from persistent follicles (aged cells) in comparison to growing follicles (young cells). Grouping of these genes into themes revealed down regulation of many genes involved in protein metabolism, amino acid transport, and mitosis regulation and an up-regulation of apoptotic genes. However, there was no change in the expression of gap junction genes. These data suggest that aged cells have a reduced capacity to regulate mitosis and provide amino acids to the oocyte. Supported by project Hatch 427 (NE 1007) of the WV Agricultural and Forestry Experiment Station. This experiment employed a direct comparison between day 8 and day 15 granulosa cells. There are two technical replicates per slide and a total of 5 biological replicates were used for each sample (5 from day 8 and 5 from day 15). Three day 8 samples were labeled with Cy3 and two with Cy5. Two day 15 samples were labeled with Cy3 and three samples with Cy5.

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. In this project, we proposed to study the genetic determinism that underlies the difference of ovulation rate between species (cattle and pigs), by studying two processes that exist in ovary: follicular development and follicular atresia. Towards this purpose, we made a comparative transcriptomics study on granulosa cells. Pig and cattle comparison was achieved by a transcriptome analysis with a 9K nylon pig microarray (GPL3729) on granulosa cells from either small healthy antral follicles (SHF), small atretic follicles (SAF) or large healthy antral follicles (LHF). The images were quantified using AGscan software and the data were managed with BASE software. Statistical analysis was performed using R software. Transcriptomic analysis on pig and cattle evidenced 997 differentially expressed genes (FDR1%) between the three follicle classes and/or the two species. 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 34 RNA samples: 10 small healthy follicles samples (6 for sows and 4 for cows), 13 small atretic follicles samples (7 for sows and 6 for cows) and 11 samples for large healthy follicles (6 for sows and 5 for cows). They were hybridized on a 9K pig nylon microarray (GPL3729).

Project description:The main objective of superovulatory treatment is to achieve multiple ovulations and produce multiple embryos. The technique is intended to rescue a cohort of follicles within a follicular wave that would otherwise regress. Superstimulory treatments change the intra-follicular and systemic hormonal milieu, but it is not clear how and to what extent treatment alters gene expression of follicular cells. In this study, a genome-wide bovine oligo-microarray was used to compare the gene expression of granulosa cells from post-LH preovulatory dominant follicle with those from follicles of the same status after superstimulatory treatment. Cows were allocated randomly to two groups (superstimulation group and control group, n=6 cows per group). A new follicular wave was induced by transvaginal ultrasound-guided ablation of follicles ?5 mm in diameter, and a progesterone-releasing device (CIDR) was placed in vagina. The superstimulation group was given eight doses of 25 mg FSH im at 12-h intervals starting from the day of wave emergence (Day 0), whereas the control group was not given any FSH treatment. Both groups were given prostaglandin F2? im twice, 12 h apart, on Day 3 and the CIDR was removed at the second injection; 25 mg pLH was given im 24 h after CIDR removal, and cows were ovariectomized 24 h later. Granulosa cells were collected for RNA extraction, amplification and microarray hybridization. To translate microarray results to a physiological context, a list of differentially expressed transcripts were biologically annotated. A total of 190 genes were down-regulated and 280 genes were up-regulated in superstimulated group when compared with the reference (non-superstimulated control) group. straight comparison of superstimulation group (the treatment; group 1) versus non-superstimulated ( the reference; group 4) using 3 different aninals (biological replicates) in each group and performed dye swap. For example on array 1: group1 cow 1 versus group 4 cow 1 (cy3 vs cy5) and on array 4 is the dey swap group 4 cow 1 versus group 1 cow 1 (cy3 vs cy5).

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Gene Expression in Granulosal Cells From Persistent Follicles

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