Unknown,Transcriptomics,Genomics,Proteomics

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Contribution of CBX4 to cumulus oophorus cell phenotype in mice, and attendant effects in cumulus cell cloned embryos

ABSTRACT: Cumulus oophorus cells play an essential role in oocyte development. CBX4 is a member of the Polycomb complex, which plays a role in regulating cellular differentiation. We used siRNA mediated knockdown of Cbx4 expression followed by Affymetrix array analysis to evaluate the role of CBX4 in cumulus cell differentiated state.

ORGANISM(S): Mus musculus

SUBMITTER: Uros Midic

PROVIDER: E-GEOD-46565 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Contribution of CBX4 to cumulus oophorus cell phenotype in mice and attendant effects in cumulus cell cloned embryos.

Hao Lanping L Midic Uros U Garriga Judith J Latham Keith E KE

Physiological genomics 20131126 2

Cumulus oophorus cells play an essential role in oocyte development. They are also widely employed as donor cells for cloning by somatic cell nuclear transfer. Our previous studies revealed that Cbx4 mRNA was overexpressed in cloned two-cell embryos. These data indicated that CBX4 may regulate normal cumulus cell differentiation and that its overexpression in clones could contribute to aberrant gene regulation. We used siRNA-mediated knockdown of Cbx4 to assess its role in determining cumulus ce ...[more]

PMID: 24280258

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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:The present study was undertaken to discover molecular markers in bovine cumulus cells predictive of oocyte competence and elucidate their functional significance. Differences in RNA transcript abundance in cumulus cells harvested from oocytes of adult versus prepubertal animals (model of poor oocyte quality) were identified by microarray analysis. Four genes of interest encoding for the lysosomal cysteine proteinases cathepsin B, S, K and Z and displaying greater transcript abundance in cumulus cells surrounding oocytes harvested from prepubertal animals were chosen for further investigation. Greater mRNA abundance for such genes in cumulus cells of prepubertal oocytes was confirmed by real time RT-PCR. Elevated transcript abundance for cathepsins B, S and Z was also observed in cumulus cells surrounding adult metaphase II oocytes that developed to the blastocyst stage at a low percentage following parthenogenetic activation, versus those that developed at a high percentage. Functional significance of cumulus cell cathepsin expression to oocyte competence was confirmed by treatment of cumulus oocyte complexes during in vitro oocyte maturation with a cell permeable cysteine proteinase (cathepsin) inhibitor. Inhibitor treatment decreased apoptotic nuclei in the cumulus layer and enhanced development of parthenogenetically activated and in vitro fertilized adult oocytes to the blastocyst stage. Stimulatory effects of inhibitor treatment during meiotic maturation on subsequent embryonic development were not observed when oocytes were matured in the absence of cumulus cells. Results support a functional role for cumulus cell cathepsins in compromised oocyte competence and suggest that cumulus cell cathepsin mRNA abundance may be predictive of oocyte quality. Keywords: Bovine, microarray, cDNA, cumulus cells Differences in RNA transcript abundance in cumulus cells harvested from oocytes of adult versus prepubertal animals (model of poor oocyte quality) were identified by microarray analysis. Total RNA from pools of cumulus cells (n = 4) collected from adult and prepubertal animals for microarray experiments was amplified. Two color microarray experiments were conducted using a bovine cDNA array containing expressed sequence tags (ESTs) representing approximately 15200 unique genes. Hybridizations were performed on duplicate slides (prepubertal versus adult) and incorporated a dye swap. The total number of slides used is eight.

Dataset Information

Contribution of CBX4 to cumulus oophorus cell phenotype in mice, and attendant effects in cumulus cell cloned embryos

Publications

Contribution of CBX4 to cumulus oophorus cell phenotype in mice and attendant effects in cumulus cell cloned embryos.

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