Ovary

Molecular Characterization of Equine P-Selectin (CD62P) and Its Regulation in Ovarian Follicles During the Ovulatory Process

Abstract
Ovulation is accompanied by a marked infiltration of leukocytes into thecal layers after the gonadotropin surge. P-selectin is known to play a critical role in the initial steps of leukocyte recruitment from the bloodstream during inflammation. Thus the objective was to investigate the potential regulation of P-selectin by gonadotropins in equine preovulatory follicles. The full-length equine P-selectin cDNA was cloned by a combination of RT-PCR and 5'- and 3'-rapid amplification of cDNA ends. Results showed that equine P-selectin cDNA encodes a 829-amino acid protein that is highly conserved when compared to the human protein (80% identity). Semiquantitative RT-PCR/Southern blot analyses were performed to study the regulation of P-selectin transcript in preovulatory follicles isolated during estrus at 0, 12, 24, 30, 33, 36 and 39 h after an ovulatory dose of hCG (ovulation occurs between 39 and 42 h post-hCG in this model). Results showed that levels of P-selectin mRNA remained very low or undetectable throughout the ovulatory process in extracts prepared from the granulosa cell layer. In contrast, a significant increase in P-selectin transcript was observed between 30 and 39 post-hCG in extracts obtained from thecal layers (P < 0.05). Likewise, immunohistochemistry revealed an increase of immunoreactive P-selectin protein in the vascular endothelium present in theca layers of follicles isolated 36 and 39 h post-hCG. Thus, this study describes for the first time the primary structure of equine P-selectin and the regulation of P-selectin transcript and protein in follicular thecal endothelial cells prior to ovulation.

Key words: Ovary • Follicle • Human chorionic gonadotropin • Ovulation

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