Induction of prostaglandin endoperoxide synthase-2 by human chorionic gonadotropin in bovine preovulatory follicles in vivo

Abstract

Gonadotropins have recently been shown to induce prostaglandin endoperoxide synthase-2 (PGHS-2) in rat preovulatory follicles before ovulation, but hormonal induction of this enzyme has not yet been reported in preovulatory follicles of any other species. To determine whether the selective induction of PGHS-2 by the ovulatory gonadotropin surge is a molecular process that has been conserved in other mammalian species with different reproductive cycles, the regulation of PGHS isoforms was studied in bovine preovulatory follicles isolated 0, 6, 12, 18, 20, 22, 24, and 26 h after an ovulatory dose of hCG. Each follicle was dissected into three preparations: the intact follicle wall (i.e. theca interna with attached granulosa cells), theca interna, and isolated granulosa cells. Solubilized cell extracts were obtained from each preparation and analyzed by Western blots, using polyclonal antibodies that selectively recognize PGHS-2 and PGHS-1 isoforms. RNA extracts were prepared and analyzed by Northern blots, using a mouse PGHS-2 complementary DNA. The results indicated that the expression of PGHS-2 messenger RNA (mRNA) and protein, but not that of PGHS-1, was regulated by hCG in bovine preovulatory follicles before ovulation. Levels of PGHS-2 protein (mol wt, 74,000) in the follicle wall were undetectable at 0, 6, and 12 h, but increased dramatically after 18 h to reach maximal levels 24 h post-hCG treatment. Northern blots revealed a similar temporal induction of PGHS-2 mRNA (4.0 kilobases). Analyses of isolated preparations of theca interna and granulosa cells clearly showed that PGHS-2 was expressed exclusively in granulosa cells and not in theca interna. Significant increases in follicular fluid concentrations of prostaglandins E2 and F were associated with the induction of follicular PGHS-2 protein and mRNA. Thus, this study establishes that the induction by gonadotropin of PGHS-2 in granulosa cells before ovulation appears to be a conserved mechanism by which the synthesis of prostaglandins necessary for the ovulation process is regulated in different species. However, the striking difference between the time course of PGHS-2 induction previously reported in rat follicles (4 h post-hCG treatment) and that observed in bovine follicles (18 h post-hCG treatment) suggests that the control of PGHS-2 expression could be one of the determinants involved in dictating the species-specific progression (length) of the ovulation process.