Molecular cloning and induction of bovine prostaglandin E synthase by gonadotropins in ovarian follicles prior to ovulation in vivo

Prostaglandin E-2 (PGE(2)) is thought to be an ultimate prostaglandin effec tor during the ovulatory process, and the objectives of this study were to clone bovine PGE synthase (PGES) and to characterize its regulation by gona dotropins in preovulatory follicles in vivo. The bovine PGES complementary DNA (cDNA) was shown to contain a 5 ' -untranslated region of eight base pa irs (bp), an open reading frame of 462 bp and a 3 ' -untranslated region of 406 bp. The putative bovine PGES open reading frame encodes a 153-amino ac id protein that is 85, 78, and 78% identical to the human, rat, and mouse P GES homologs, respectively. The regulation of PGES during ovulation was stu died using three different models in vivo: 1) human chorionic gonadotropin (hCG)-induced ovulation during a normal estrous cycle; 2) hCG-induced ovula tion following ovarian hyperstimulation; and 3) spontaneous ovulation durin g natural estrus. Results from semi-quantitative reverse transcription-poly merase chain reaction/Southern blotting analyses showed that the hCG/lutein izing hormone surge caused a significant increase in PGES mRNA. Levels of P GES transcripts were low or undetectable prior to hCG/luteinizing hormone b ut increased markedly 18-24 h after hCG in models I and 2, and 18-24 h afte r the onset of natural estrus in model 3 (p < 0.05). Analyses on isolated p reparations of granulosa and theca interna cells indicated that the granulo sa cell layer was the predominant site of follicular PGES expression. The r egulation of the protein was studied in the same models using a specific an tibody raised against a fragment of bovine protein (Delta PGES; from Glu(49 ) to Val(146)). Results from immunoblots showed an induction of bovine PGES (M-r = 17,000) 18-24 h after hCG treatment or onset of estrus (p < 0.05). The protein was detected in extracts of granulosa cells but not in theca in terna. Collectively, these results demonstrate that the ovulatory process i s associated with a gonadotropin-dependent induction of PGES in granulosa c ells of ovarian follicles in vivo, thus establishing for the first time the regulation of the enzyme in a physiological context.