Ovary

Identification of Genes Involved in Apoptosis and Dominant Follicle Development During Follicular Waves in Cattle

Abstract
We hypothesize that granulosa and theca cells from growing dominant follicles, with relatively high intrafollicular concentrations of estradiol, have a greater expression of genes involved in inhibiting apoptosis pathways and lower expression of genes involved in apoptosis pathways than growing subordinate follicles with lower estradiol concentrations. Using the well-characterized bovine dominant follicle model, we collected granulosa and theca cells from individual dominant and the largest subordinate follicle 3 days after initiation of a follicular wave in four animals. Based on ultrasound analysis, both follicle types were in the growth phase at the time of ovariectomy. However, dominant follicles were larger (9.8±1.0 versus 7.6±0.6 mm in diameter, P<0.05) and had greater intrafollicular concentrations of estradiol (132.2& [plusmn]38.5 versus 24.1±12.1 ng/ml, P<0.05) compared with the largest subordinate follicles. We used bovine cDNA microarrays, which contained a total of 1400 genes including a subset of 53 genes known to be involved in apoptosis pathways, to determine which apoptosis and marker genes from each of the four dominant versus subordinate follicles were potentially differentially expressed. Using a low stringency-screening criterion, 22 genes were identified. Quantitative real-time PCR confirmed that 16 of these genes were differentially expressed. Our novel results demonstrate that the high intrafollicular concentrations of estradiol in growing dominant follicles were positively associated with enhanced expression of mRNAs in granulosa cells for aromatase, LH receptor, estradiol receptor , DICE-1 and MCL-1 compared with granulosa cells from subordinate follicles (all survival-associated genes). In contrast, the relatively low intrafollicular concentrations of estradiol in growing subordinate follicles were positively associated with enhanced expression of mRNAs in granulosa cells for glycan, COX-1, TNF, CAD and DRAK-2, and in theca cells for glycan, caspase 13, P58(IPK), Apaf-1, BTG-3, and TS-BCLL compared with granulosa or theca cells from dominant follicles (genes that are all associated with cell death and/or apoptosis). We suggest that that these genes may be candidate estradiol target genes and that they may be early markers for the final stages of follicle differentiation and initiation of apoptosis and thus selection of dominant follicles during follicular waves.

Key words: Apoptosis • Estradiol • Follicle • Follicular development

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