Coordinate Induction of Transcripts Encoding Endothelin-2 and Endothelin Receptor B, but Not Endothelin Receptor A, in Equine Follicles Prior to Ovulation.

Abstract

The release of the oocyte from the follicle during the final moments of the ovulatory process is thought to involve a follicular contraction responsible for the physical expulsion of its content. Such a mechanism appears even more needed in a species like the horse that has a relatively large preovulatory follicle (40-45 mm in diameter). The objective of the study was to characterize the expression and regulation of endothelin-2 (EDN-2), a potent smooth muscle contractor, as well as of endothelin receptor A and B (EDNRA and EDNRB) in equine follicles during ovulation. Preovulatory follicles were isolated during estrus 0, 12, 24, 30, 33, 36 and 39 h after an ovulatory dose of hCG (ovulation occurred 39-42 h post-hCG), and corpora lutea were obtained on day 8 of the estrous cycle. Preparations of follicle wall (theca interna with attached granulosa cells), as well as isolated preparations of granulosa cells and theca interna, were obtained and total RNA extracts were prepared and analyzed by semi-quantitative RT-PCR/Southern blot analyses. Results showed a marked and transient induction of EDN-2 in equine preovulatory follicles after gonadotropin treatment. Levels of EDN-2 transcript were low or undetectable prior to hCG (0 h), significantly increased from 30 to 39 h post-hCG in both granulosa and theca cells (P < 0.05), and returned to basal levels in day-8 corpora lutea. The regulation of EDNRA and EDNRB followed distinct patterns. EDNRA mRNA decreased significantly in granulosa cells between 24-39 h and in theca cells 30-39 post-hCG (P< 0.05). In contrast, a marked and significant induction in EDNRB transcript was observed in granulosa cells 30-39 h post-hCG, which paralleled the induction of END-2 in these cells. Levels of EDNRB remained high and unchanged throughout the ovulatory process in theca cells. High levels of both receptors were observed in corpora lutea. Thus, this study characterizes for the first time the pattern of regulation of EDN-2 and its receptors in follicle cells of a monoovulatory species. The marked induction of EDN-2 in both granulosa and theca interna cells of equine follicles supports its putative role in follicular contraction at the time of ovulation, and the concomitant elevated levels of EDNRB, but not of EDNRA, suggest that the former has a predominant role in this species. (This study was funded by NSERC of Canada) (poster)