MICROARRAY ANALYSIS OF GENE EXPRESSION IN THE PRIMATE OVULATORY FOLLICLE: VALIDATION OF SELECTED GENE TRANSCRIPTS BY REAL-TIME PCR

Abstract

The recent development of genomic approaches permits, for the first time, a complete evaluation of LH-regulated genes in the ovulatory, luteinizing follicle. Therefore, experiments were designed to evaluate gene expression, using the Affymetrix rhesus macaque cDNA microarray, in a controlled ovulation (COv) model which permits evaluation of the naturally selected, dominant follicle at specific timepoints after exposure to an ovulatory (exogenous hCG) stimulus during the menstrual cycle in rhesus monkeys. The preovulatory follicle (n = 4–8/timepoint) was collected before (0 hr) and 12, 24, and 36 hr post-hCG injection. Since ovulated follicles were observed at 36 hr, samples at this timepoint were divided (n = 4/group) into unruptured and ruptured follicles. Total RNA was prepared from individual follicles, with an aliquot used for microarray analysis (Affymetrix Rhesus Macaque Genome Array) and the remainder stored for real-time PCR assays. Initial results indicate that: (a) RNA preparation, labeling and hybridization met high quality standards, (b) microarray data from individual samples distinctly clustered according to timepoint, and ovulated follicles displayed markedly different expression patterns from unruptured follicles at 36 hr, and (c) between timepoint comparisons revealed profound changes in mRNA expression profiles. In every instance, the dynamic pattern of mRNA expression for steroidogenic enzymes (CYP17A, CYP19A, HSD3B, HSD11B1, HSD11B2), StAR, and receptors for gonadotropin receptors (LHCGR, FSHR) determined by microarray analysis correlated precisely with those from blinded real-time PCR assays. Notably, mRNA levels for gonadotropin receptors declined (p<0.05) markedly to low levels within 12 hr post-hCG injection; those for CYP17A1 and CYP19A also declined (p<0.05) but did not reach low levels until 24 hr post-hCG. However, levels for LHCGR and CYP19A later increased (p<0.05) in ruptured, compared to unruptured, follicles at 36 hr post-hCG. Levels of HSD11B1 mRNA displayed a different pattern, increasing (p<0.05) initially between 0 and 12 hrs post-hCG and again in the ruptured follicle at 36 hr post-hCG. In contrast, mRNA levels for HSD11B2 declined (p<0.05) after hCG injection and remained low after follicle rupture. Thus, several mRNAs displayed the expected expression pattern for purported theca-cell specific (e.g., CYP17A), granulosa-cell specific (CYP19A, FSHR), and surface epithelium-related (HSD11B) genes in the rodent/primate ovulatory follicle. This genomic database is of great value in analyzing molecular and cellular pathways associated with follicle rupture and other periovulatory events in the primate follicle (e.g. luteinization, neuronal differentiation, inflammatory response, hypoxia, and angiogenesis), and for identifying novel gene products controlling mammalian fertility. Supported by Schering Female AMPPA, U54 SCCPIR HD18185, RR00163 (platform)