Project description:Mouse oocytes control cumulus cell metabolic processes that are deficient in the oocytes themselves and this delegation is necessary for oocyte development. Oocyte-derived bone morphogenetic factor 15 (BMP15) and growth differentiation factor 9 (GDF9) appear to be key regulators of follicular development. The effect of these factors on cumulus cell function before the preovulatory surge of luteinizing hormone (LH) was assessed by analysis of the transcriptomes of cumulus cells from wildtype (WT), Bmp15-/-, and Bmp15-/- Gdf9+/- double mutant (DM) mice using microarray analysis. The biological themes associated with the most highly-affected transcripts were identified using bioinformatic approaches, IPA and GenMAPP/MAPPFinder. There were 5,332, 7,640, and 2,651 transcripts identified to be significantly changed in the comparisons of Bmp15-/- vs. WT, DM vs. WT, and DM vs. Bmp15-/- respectively by the criteria of FC (fold change) p <0.01. Among theses changed transcripts, 744 were commonly changed in all three pair-wise comparisons, and hence were considered to be the most highly affected transcripts by mutation of Bmp15 and Gdf9. IPA Analyses revealed that metabolism was the major theme associated with the most highly-changed transcripts: glycolysis and sterol biosynthesis were the two most significantly affected pathways. Most of the transcripts encoding enzymes for sterol biosynthesis were down-regulated in both mutant cumulus cells and in WT cumulus cell after oocytectomy. Similarly, there was a reduction of de novo-synthesized cholesterol in these cumulus cells. This suggests that oocytes regulate cumulus cell metabolism, particularly sterol biosynthesis, by promoting the expression of corresponding transcripts. Furthermore, in WT-mice, Mvk, Pmvk, Fdps, Sqle, Cyp51, Sc4mol, and Ebp, which encode enzymes in the sterol biosynthetic pathway, were found to be expressed robustly in cumulus cells, but expression was barely detectable in oocytes. Levels of de novo-synthesized cholesterol were significantly higher in cumulusâenclosed oocytes than denuded oocytes. These results indicate that mouse oocytes are deficient in their ability to synthesize cholesterol and require cumulus cells to provide them with products of the sterol biosynthetic pathway. Oocyte-derived BMP15 and GDF9 may promote this metabolic pathway in cumulus cells as compensation for their own deficiencies. Experiment Overall Design: Three sets of independent cumulus cell samples were collected for each genotype (wild type, Bmp15-/-, and Bmp15-/-Gdf9+/-) of mice, and were used for the array study as shown below. Experiment Overall Design: Array Genotype Sample Experiment Overall Design: GC_430_2_GES05_0161_033105_1.CEL WT 1 Experiment Overall Design: GC_430_2_GES05_0162_033105_1.CEL WT 2 Experiment Overall Design: GC_430_2_GES05_0163_033105_1.CEL WT 3 Experiment Overall Design: GC_430_2_GES05_0164_033105_1.CEL Bmp15-/- 4 Experiment Overall Design: GC_430_2_GES05_0165_033105_1.CEL Bmp15-/- 5 Experiment Overall Design: GC_430_2_GES05_0166_033105_1.CEL Bmp15-/- 6 Experiment Overall Design: GC_430_2_GES05_0167_033105_1.CEL DM 7 Experiment Overall Design: GC_430_2_GES05_0168_033105_1.CEL DM 8 Experiment Overall Design: GC_430_2_GES05_0169_033105_1.CEL DM 9
Project description:Mouse oocytes control cumulus cell metabolic processes that are deficient in the oocytes themselves and this delegation is necessary for oocyte development. Oocyte-derived bone morphogenetic factor 15 (BMP15) and growth differentiation factor 9 (GDF9) appear to be key regulators of follicular development. The effect of these factors on cumulus cell function before the preovulatory surge of luteinizing hormone (LH) was assessed by analysis of the transcriptomes of cumulus cells from wildtype (WT), Bmp15-/-, and Bmp15-/- Gdf9+/- double mutant (DM) mice using microarray analysis. The biological themes associated with the most highly-affected transcripts were identified using bioinformatic approaches, IPA and GenMAPP/MAPPFinder. There were 5,332, 7,640, and 2,651 transcripts identified to be significantly changed in the comparisons of Bmp15-/- vs. WT, DM vs. WT, and DM vs. Bmp15-/- respectively by the criteria of FC (fold change) p <0.01. Among theses changed transcripts, 744 were commonly changed in all three pair-wise comparisons, and hence were considered to be the most highly affected transcripts by mutation of Bmp15 and Gdf9. IPA Analyses revealed that metabolism was the major theme associated with the most highly-changed transcripts: glycolysis and sterol biosynthesis were the two most significantly affected pathways. Most of the transcripts encoding enzymes for sterol biosynthesis were down-regulated in both mutant cumulus cells and in WT cumulus cell after oocytectomy. Similarly, there was a reduction of de novo-synthesized cholesterol in these cumulus cells. This suggests that oocytes regulate cumulus cell metabolism, particularly sterol biosynthesis, by promoting the expression of corresponding transcripts. Furthermore, in WT-mice, Mvk, Pmvk, Fdps, Sqle, Cyp51, Sc4mol, and Ebp, which encode enzymes in the sterol biosynthetic pathway, were found to be expressed robustly in cumulus cells, but expression was barely detectable in oocytes. Levels of de novo-synthesized cholesterol were significantly higher in cumulus–enclosed oocytes than denuded oocytes. These results indicate that mouse oocytes are deficient in their ability to synthesize cholesterol and require cumulus cells to provide them with products of the sterol biosynthetic pathway. Oocyte-derived BMP15 and GDF9 may promote this metabolic pathway in cumulus cells as compensation for their own deficiencies. Keywords: Gdf9, Bmp15, cumulus cells, transcriptome, metabolism, sterol biosynthesis, genotype comparison, microarray, ingenuity pathways analyses.
Project description:Follicle stimulating hormone (FSH) and epidermal growth factor (EGF) are currently used on cumulus-oocyte complexes to mimic the luteinizing hormone surge in vitro and induce oocyte maturation and cumulus expansion. We have previously shown that addition of exogenous recombinant growth differentiation factor 9 (GDF9) during oocyte in vitro maturation led to an improvement of oocyte quality, as shown by an increased blastocyst percentage and fetal survival. Our objective was to characterize the effect of FSH/EGF and GDF9 treatments on mouse cumulus cells expression profile by microarray analysis. Cumulus-oocyte complexes (COCs) were recovered from 21 to 26 day old female 129/SV mice, 44 hours post equine chorionic gonadotropin treatment (eCG (5 IU)). For the microarray experiment whole COCs were treated with 293H control medium (0.125% v/v), with 20 ng/ml GDF9 or with a combination of 50 mIU/ml FSH and 10 ng/ml EGF. After 8 hours of in vitro maturation, COCs were denuded by gentle pipetting, the oocytes were removed and the cumulus cells centrifuged and extracted RNA analysed by microarray.
Project description:Follicle stimulating hormone (FSH) and epidermal growth factor (EGF) are currently used on cumulus-oocyte complexes to mimic the luteinizing hormone surge in vitro and induce oocyte maturation and cumulus expansion. We have previously shown that addition of exogenous recombinant growth differentiation factor 9 (GDF9) during oocyte in vitro maturation led to an improvement of oocyte quality, as shown by an increased blastocyst percentage and fetal survival. Our objective was to characterize the effect of FSH/EGF and GDF9 treatments on mouse cumulus cells expression profile by microarray analysis.
Project description:Purpose: The goal of this study is to compare oocyte transcriptome profiling in WT and Kat8 Gdf9 cKO mice Methods: Oocyte mRNA profiles of 2 weeks old WT and Kat8 Gdf9 cKO mice were generated by deep sequencing using Illumina HiSeq 2500. Results: Using an optimized data analysis workflow, we mapped about 54-63 million sequence reads per sample to the mouse genome (build mm10) and identified 18,468 transcripts in the oocytes of WT and Kat8 Gdf9 cKO mice. Approximately 5% of the transcripts showed differential expression between the WT and Kat8 Gdf9 cKO oocyte, with a fold change ≥1.5 and p value <0.05. Conclusions: Our study represents the first detailed analysis of oocyte transcriptomes after kat8 deletion, which is generated by RNA-seq technology.Our results show that Kat8 affects numerous gene expression in mouse oocytes.
Project description:Transcriptomes of mouse mural granulosa cells were sequenced to identify transcripts expressed in mural granulosa cells of ovaries. Moreover, transcriptomes of cumulus cells were compared between those of young (2 month-old) and old mice (10 month-old) to assess the effects of ageing on cumulus cells. In addition, transcriptomes of cumulus-oocyte complexes were compared between DBA/2 and (C57BL/6 x DBA/2)F1 mice to assess the strain differences.
Project description:Cumulus cells and mural granulosa cells (MGCs) are spatially and functionally distinct cell types in antral follicles: cumulus cells contact the oocyte and most MGCs contact the basal lamina. For transcriptomic analyses, both cell types were collected from small and large antral follicles, before and after stimulation of immature mice with eCG, respectively. Both cell types underwent dramatic transcriptomic changes and the differences between them became greater with follicular growth. Although cumulus cells of both stages of follicular development are competent to undergo expansion in vitro, they were otherwise remarkably dissimilar with transcriptomic changes quantitatively equivalent to those of MGCs. Gene Ontology (GO) analysis showed that cumulus cells of small follicles were enriched in transcripts generally associated with catalytic components of metabolic processes while those from large follicles were involved in regulation of metabolism, cell differentiation, and adhesion. Upon contrasting cumulus cells versus MGCs, cumulus cells were enriched in transcripts associated with metabolism and cell proliferation while MGCs were enriched for transcripts involved in cell signaling and differentiation.