Molecular characterization of equine P-selectin (CD62P) and its regulation in ovarian follicles during the ovulatory process.

This study focused on understanding how the protein P-selectin, important for facilitating inflammation by enabling the movement of leukocytes from the bloodstream, is controlled by gonadotropins in horse preovulatory follicles. Researchers found that P-selectin, both at the gene and protein level, increases in the blood vessel-rich thecal layer of the follicle close to ovulation, providing initial insights into its role in equine ovulation.

Molecular Characterization of Equine P-Selectin

• The researchers cloned the full-length equine P-selectin cDNA using a combination of reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends.
• The cloned P-selectin cDNA encoded an 829-amino acid protein which was found to be highly similar to the human P-selectin protein (80% similar).

Regulation of P-Selectin in Ovarian Follicles during Ovulation

• The authors investigated the regulation of P-selectin in horse preovulatory follicles. They isolated these follicles during estrus at several time points (0, 12, 24, 30, 33, 36, and 39 hours) after an ovulatory dose of human chorionic gonadotropin (hCG), a hormone that induces ovulation.
• They used semiquantitative RT-PCR/Southern blot analyses to study the presence of P-selectin transcripts in these follicles.
• Results showed that P-selectin mRNA levels were very low or undetectable throughout the ovulatory process in granulosa cells, a type of cell found in the ovary.
• In contrast, a significant increase in the transcript of P-selectin was observed between 30 and 39 hours post-hCG in the thecal cells, another type of ovarian cell.

Localization and Timing of P-Selectin Production

• Immunohistochemistry was used to visualize the location and amount of P-selectin protein in these tissues. This involves staining tissues with antibodies that can bind to the protein and can be seen under a microscope.
• Results showed that just prior to ovulation, at 36 and 39 hours post-hCG, there was an increase in P-selectin protein in the vascular endothelium present in the thecal layers. Vascular endothelium is the innermost layer of blood vessels.

Conclusions

• This study provided the first insights into the structure of equine P-selectin and its regulation before ovulation in the thecal endothelial cells of follicles.
• The pattern of P-selectin expression suggests that this protein may play a role in recruiting leukocytes into the follicles during the ovulatory process, similar to its role in inflammation.