Transcriptomic Analysis of Bovine Cumulus Cells Investment of Oocytes at Different Stage of Differentiation Based on the Degree of Their Chromatin Configuration: Preliminary Study

The identification of biomarkers that can predict oocyte viability and developmental potential has important implications in treating human infertility as well as managing breeding schemes of domestic mammals. During ovarian follicle development, interactions between somatic and germ cells are critical for normal follicular and oocyte development as well as for the acquisition of oocyte competence. Moreover, companion granulosa cells serve a critical role in the regulation of global transcriptional silencing and in the modulation of chromatin configuration changes that occur before meiotic resumption. Importantly, chromatin configurations within the oocyte germinal vesicle (GV) are indicative of oocytes metabolic state as well as in vitro developmental potential. However, the mechanisms acting in the somatic compartment that regulate oocyte differentiation and competence are far from being fully understood. Starting from these observations, the aim of this study was to assess gene expression profile by microarray platform in cumulus cells (CC) isolated from ovarian follicles, which may be linked to the oocyte developmental competence. To this aim we have determined the expression profile of CC investment of oocytes at different stage of differentiation, and selected according to their chromatin configuration by DNA staining. Oocytes were classified as GV0, GV1, GV2 and GV3 according to their chromatin pattern. CC belonging to each group were collected and processed for further analysis. Four pools of CC from 10 oocytes per group were used. RNA was extracted, amplified and hybridized on a bovine embryo-specific 44K Agilent slide (EmbryoGene). The GV1, GV2 and GV3 classes were each hybridized against the GV0, which represents a stage of early oocyte differentiation with poor development competence. Data were normalized with Loess and Fold changes (FC) of differentially expressed genes were determined with Limma procedure. Finally, the regulation pattern genes and function predicted was studied with Ingenuity Pathway Analysis software. Transcriptome analysis (FC >1.5; p<0.05) reveals 1441 genes differentially expressed in GV1 vs GV0 (684 down and 757 upregulated), 1474 in GV2 vs GV0 (699 down and 775 upregulated) and 2047 in GV3 vs GV0 (981 down and 1066 upregulated), with 347 genes that where common among groups. Interestingly, the majority of these genes appeared to change either in one direction or in other among groups. Preliminary analysis showed an up-regulation of several transcripts associated to extracellular matrix formation and stabilization: Hyaluronan synthase 2 (HAS2), Progesterone receptor (PGR) and Sprouty homolog 2 (SPRY2). Another class of differentially regulated genes was represented by apoptosis-related transcripts: SH3 domain containing ring finger 2 (SH3RF2), disabled homolog 2, mitogen-responsive phosphoprotein (DAB2), Ras association (RalGDS/AF-6) domain family (N-terminal) member 7 (RASSF7) and caspase 3, apoptosis-related cysteine peptidase (CASP3). This study provides multiple candidate genes to be further analysed to elucidate their role in the process of oocyte developmental competence acquisition. Moreover, it would help in the identification of the signal(s) through which the somatic compartment controls the function of the oocyte genome. Finally, these findings will facilitate identification of non-invasive biomarkers of oocyte health status. Work supported by the NSERC Strategic Network EmbryoGENE, Can