Microarray analysis of gene expression in the primate ovulatory follicle: Selected gene transcripts involved in cumulus–oocyte expansion

Abstract

To identify, using a genome-wide approach, genes whose expression is regulated by LH in the primate ovulatory follicle. Particular attention was given to those genes implicated in cumulus-oocyte expansion. Longitudinal study of gene expression at specific timepoints during controlled ovulation (COv) of the naturally selected, dominant follicle in rhesus monkeys during the menstrual cycle. The preovulatory follicle (n = 4–8/time point) was collected before (0 hr) and 12, 24, and 36 hr after injection of an ovulatory bolus of hCG. Since ovulated follicles were observed at 36 hr, samples at this timepoint were divided into unruptured and ruptured follicles (n = 4/group). Total RNA was prepared from individual follicles. An aliquot used for DNA microarray analysis (Affymetrix GeneChip® Macaque Genome Array) and the remainder stored for validation of expression patterns for selected genes by real-time PCR. The latter data were analyzed by one-way ANOVA followed by S-N-K tests. Between timepoint comparisons revealed profound changes (P<0.05) in mRNA levels. Those for an enzyme catalyzing hyaluronan synthesis (HAS2) and for a component (tumor necrosis factor-alpha induced protein, TNFAIP6) linking hyaluronan in the cumulus, were low at 0 hr, increased markedly (5–10 fold) at 12 hr, then declined to baseline by 36 hr before rupture. Also, mRNA levels for the epidermal growth factor family members, amphiregulin and epiregulin (not betacellulin) also increased up to 100 fold by 12 hr, and decreased markedly before rupture. In contrast, mRNA levels for the rate-limiting enzyme in prostaglandin synthesis (PTGS2) increased modestly at 12 hr, followed by a second rise (>10-fold) at 36 hrs in the ruptured follicle. The four receptors for PGE (PTGER1–4) were expressed, but mRNA levels did not change markedly (2–3 fold increase, PTGER2 and 3, P<0.05) following hCG treatment. Notably, mRNA levels for several of these genes (e.g., HAS2, TNFAIP6, amphiregulin, epiregulin) also increased 5–10 fold at 36 hr in ruptured compared to unruptured follicles. Several genes encoding factors involved in oocyte maturation and cumulus-oocyte expansion in rodents are also expressed in a pattern that is consistent with similar activities in the primate ovulatory follicle. Their subsequent expression in the ruptured follicle suggests an additional role during development of the functional corpus luteum.