The objective was to analyze gene expression in bovine granulosa cells of the dominant follicle by mRNA differential display. Total RNA was extracted from granulosa cells of <or=4 mm follicles, day 5 (D5) dominant follicles, and hCG-induced preovulatory follicles. A differentially expressed cDNA observed in the dominant follicle group was used to screen a granulosa cell cDNA library, which resulted in the cloning of a 2,096 bp cDNA. Amino acid comparison showed identity level of 91.4, 83.9, and 83.1% when compared to human, rat, and mouse serine protease inhibitor E2, SERPINE2, also called Glia-derived nexin or protease Nexin-1. A single transcript of 2.4 kb was shown to be differentially expressed in different bovine tissues. Immunoblotting with a specific antibody raised against a fragment of SERPINE2 (S(12)-R(196)) showed that SERPINE2 migrated at 47.5 kDa in support of glycosylation. Primordial, primary, and secondary pre-antral follicles showed immunostaining associated with granulosa cells and oocytes, and strong labeling in large antral follicles was located with granulosa cells and follicular fluid. Heterogeneity of SERPINE2 labeling was observed in CL. Semi-quantitative real-time fluorescent RT-PCR showed a six-fold increase (P = 0.0002) in mRNA level of SERPINE2 in granulosa cells of D5 dominant follicle compared to granulosa cells collected from the <or=4 mm or preovulatory hCG-induced follicles. This report demonstrates that SERPINE2 mRNA is regulated in a spatio-temporal pattern with highest levels in granulosa cells of growing dominant bovine follicles, and support the hypothesis that a high expression of SERPINE2 may contribute to follicular growth whereas a decrease following hCG injection may contribute to ovulation.
Copyright 2003 Wiley-Liss, Inc.