Gene Expression Profiling of Differentially Expressed Genes in Granulosa Cells of Bovine Dominant Follicles Using Suppression Subtractive Hybridization1

Abstract

Development of antral follicles beyond 3 to 4 mm in cattle appears as a wave pattern that occurs two to three times during the estrous cycle. Each wave presents a cyclic recruitment of multiple follicles at the 3- to 4-mm stage, followed by the selection of a single follicle that becomes the dominant follicle (DF). The molecular determinants involved in the follicular dominance process remain poorly understood. The objective of the current study was to compare gene expression in granulosa cells (GCs) between growing dominant follicles from Day 5 of the estrous cycle and nonselected small follicles (<or=4 mm) using the suppression subtractive hybridization (SSH) approach to identify candidate genes differentially expressed in GCs of the DF. Small follicle cDNAs were subtracted from DF cDNAs (DF-SF) and used to establish a DF GC-subtracted cDNA library. A total of 42 nonredundant cDNAs were identified. Detection of previously identified genes such as CX43, CYP19, INHBA, and SERPINE2 supported the validity of our experimental model and the use of SSH as the method of analysis. For selected genes such as ApoER2, CPD, CSPG2, 14-3-3 epsilon, NR5A2/SF2, RGN/SMP30, and SERPINE2, gene expression profiles were compared by virtual Northern blot or reverse transcriptase-polymerase chain reaction, and results confirmed an increase or induction of their mRNA in GCs of dominant follicles compared with that of small follicles. We conclude that we have identified novel genes (known and unknown) that are up-regulated in bovine GCs that may affect follicular growth, dominance, or both.

A pilot study on transcriptome data analysis of folliculogenesis in pigs

“…Among them, three had already been selected by other experiments on cellular models: VIIC2 had been found in a differential hybridisation screening between control and FSH-treated pig granulosa cells (Tosser-Klopp et al, 1997), GPX and calpaïn had been found in suppression subtractive experiments between control and FSH-treated pig granulosa cells (Bonnet et al, 2006) and may play a role during folliculogenesis. NR5A2 transcript has been shown to be up-regulated in bovine dominant follicles, compared to small follicles (Fayad et al, 2004), which is consistent with our findings but has not been described yet in pig ovary. NPTX2 transcript regulation has never been described in granulosa cells.…”

Differentiation of the bovine dominant follicle from the cohort upregulates mRNA expression for new tissue development genes

“…These two transcripts encoded a novel 3 0 truncated aromatase variant (deposited under NCBI accession number DQ004742 and recently matched to Bt.4447), and an untranslated mRNA sequence recently matched to the UniGene cluster Bt.33875 (deposited under NCBI accession number AY999166). In addition, SAGE analysis of transcripts upregulated in the DF granulosa cells confirmed four known genes encoding cytochrome P450 aromatase (CYP19A1), glutathione S-transferase A2 (GSTA2), inhibin a-subunit (INHA) and glia-derived nexin (SERPINE2), which regulate follicular E synthesis, antioxidant activity and extracellular tissue remodelling in the selected DF (Mihm et al 1997, Sisco et al 2003, Evans et al 2004, Fayad et al 2004.…”

“…These proteins have diverse cellular functions in signalling (calreticulin (CALR)), metabolism (cytochrome c oxidase subunit VIIb (COX7B), glutathione peroxidase 1 (GPX1)), DNA binding (high-mobility group nucleosomal binding domain 2 (HMGN2), poly(rC) binding protein 1 (PCBP1)), RNA synthesis (non-POU domain containing, octamer binding (NONO)), protein synthesis (Tu translation elongation factor, mitochondrial (TUFM)) and metabolism (heat shock 70 kDa protein 8 (HSPA8), heat shock protein 90 kDa b (Grp94), member 1 (HSP90B1)) ( Table 2). The remaining three transcripts encoded genes (inhibin, b A (INHBA), hydroxy-d-5-steroid dehydrogenase, 3b-and steroid d-isomerase 1 (HSD3B1), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, 3 polypeptide (YWHAE)) previously found to show higher mRNA expression in the newly selected DF , Sisco et al 2003, Fayad et al 2004; Table 2). …”

“…While this candidate gene approach has merit, it is clear that approaches that utilise global gene and protein expression profiling are required to identify all key factors and pathways which regulate cell differentiation during DF development. Genomic approaches so far used (suppression subtractive hybridisation, custom cDNA microarray) have focussed on the comparison between the highly oestrogenic selected DF at the beginning of the rapid growth phase with subordinate follicles undergoing or initiating atresia (Sisco et al 2003, Evans et al 2004, Fayad et al 2004). Such comparisons have identified genes that regulate E synthesis or apoptosis within the growing cohort (Sisco et al 2003) for example.…”

Signaling by the Extracellular Matrix Protein Reelin Promotes Granulosa Cell Proliferation in the Chicken Follicle

“…Recent attempts to identify genes that regulate the selection and recruitment of follicles to develop into mature oocytes during the bovine estrous cycle by expression profiling led to the identification of ApoER2 as a candidate for such a function (10,11). In another study, ApoER2 has been shown to be preferentially expressed in bovine dominant follicles when compared with subordinate follicles (12).…”