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

• Published in: Biology of reproduction 2005-03, Vol.72 (3), p.736-744
• Main Authors: SAYASITH, Khampoune, BOUCHARD, Nadine, BOERBOOM, Derek, BROWN, Kristy A, DORE, Monique, SIROIS, Jean
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Biological and medical sciences; Endothelium, Vascular - cytology; Endothelium, Vascular - metabolism; Estrous Cycle - genetics; Estrous Cycle - metabolism; Female; Fundamental and applied biological sciences. Psychology; Granulosa Cells - metabolism; Hormone metabolism and regulation; Horses - genetics; Horses - metabolism; Immunohistochemistry; Mammalian female genital system; Molecular Sequence Data; Ovarian Follicle - cytology; Ovarian Follicle - metabolism; P-Selectin - genetics; P-Selectin - metabolism; Reference Values; RNA, Messenger - analysis; Sequence Homology, Amino Acid; Theca Cells - metabolism; Tissue Distribution; Vertebrates: reproduction
• ISSN: 0006-3363; 1529-7268
• DOI: 10.1095/biolreprod.104.034710

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 reverse transcription-polymerase chain reaction (RT-PCR) and 5′- and 3′-rapid amplification of cDNA ends. Results showed that equine P-selectin cDNA encodes an 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 h 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 thecal layers of follicles isolated 36 and 39 h post-hCG. Thus, the present study describes, to our knowledge 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 before ovulation. Abstract First report of P-selectin induction by hCG in preovulatory follicles