Study of Gene Expression in Bovine Granulosa Cells from Different Follicle Growth Phases.

Abstract

It is known that 1 in 3 follicles contains a good a competent oocyte, but to this day the features of those particular follicles have not been characterized. The objective of this study was to build the transcriptomic profile of granulosa cells (GC's) originating from pre-dominance follicle (6 to 9mm diameter) in dairy cattle using microarrays and validating by Q-PCR. Granulosas originating from three different phases of antral follicle growth were compared: growing (G), plateau (P) and atresia (A), as categorized by flow cytometry on DNA content. The growing and atretic conditions we're each hybridized against the plateau condition as a reference in order to understand the specific biological mechanisms underway in this class of follicles, which is believed to hold oocytes with the best capacities to produce an offspring following in-vitro assisted reproduction technologies such as maturation (MIV) and fertilisation (FIV). 2,942 genes were differentially expressed (P<0.05) in G vs P and 1,974 in P vs A. Ingenuity Pathway Analysis (IPA) software was used to study the regulation pattern of genes and functions predicted to be enriched in the dataset. Cytometry was efficient in categorizing the follicles in the different phases, as determined by the down-regulation of steroidogenesis genes: cytochrome P450, subfamily XIX (CYP19A1, i.e. aromatase), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1) and cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1) in the granulosa cells of P follicles relative to G and probably in response to the decreasing FSH level. On the other hand, the P vs A contrast showed up-regulation of multiple transcripts associated to apoptosis: tissue transglutaminase (TGM2; transglutaminase 2 (C polypeptide, protein-glutamine-gamma-glutamyltransferase)), SH3 domain containing ring finger 1(SH3RF1, also known as POSH), disabled-2 (DAB2), desmoglein-2 (DSG2), death receptor 5 (DR5), caspase-4 (CASP4), Ras association (RalGDS/AF-6) domain family member 1 (RASSF1), chaperonin containing TCP1, subunit 2 (beta) (CCT2) and interferon regulatory factor 5 (IRF5). Functions such as cell death, secretion of lipids and ploidy of cells, were predicted to be increased in P-follicle-granulosa compared to G-follicles, while functions such as growth of cells were predicted to be decreased in P relative to G. Although the genes more expressed in the A condition did not correspond to common atretic markers, they are likely to be indicators differentiating between the late atresia status of the follicle and early atresia, a hallmark characteristic of plateau follicles. This study offers multiple candidate genes to be further studied at a biochemical level in order to elucidate their role in the modulation of oocyte competence. This work was supported by NSERC of Canada.