Molecular characterization of equine P-selectin (CD62P) and its regulation in ovarian follicles during the ovulatory process.
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.
Sayasith
Khampoune
K
Centre de recherche en reproduction animale and Département de biomédecine vétérinaire, Faculté de médicine vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, Canada.
Bouchard
Nadine
N
Boerboom
Derek
D
Brown
Kristy A
KA
Doré
Monique
M
Sirois
Jean
J
D000595:Amino Acid Sequence; D000818:Animals; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D030762:Estrous Cycle / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006107:Granulosa Cells / Q000378:metabolism; D006736:Horses / Q000235:genetics / Q000378:metabolism*; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D006080:Ovarian Follicle / Q000166:cytology / Q000378:metabolism*; D019007:P-Selectin / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D012016:Reference Values; D017386:Sequence Homology, Amino Acid; D013799:Theca Cells / Q000378:metabolism; D014018:Tissue Distribution
D019007:P-Selectin; D012333:RNA, Messenger