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 effector during the ovulatory process, and the objectives of this study were to clone bovine PGE synthase (PGES) and to characterize its regulation by gonadotropins in preovulatory follicles in vivo. The bovine PGES complementary DNA (cDNA) was shown to contain a 5'-untranslated region of eight base pairs (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 acid protein that is 85, 78, and 78% identical to the human, rat, and mouse PGES homologs, respectively. The regulation of PGES during ovulation was studied using three different models in vivo: 1) human chorionic gonadotropin (hCG)-induced ovulation during a normal estrous cycle; 2) hCG-induced ovulation following ovarian hyperstimulation; and 3) spontaneous ovulation during natural estrus. Results from semi-quantitative reverse transcription-polymerase chain reaction/Southern blotting analyses showed that the hCG/luteinizing hormone surge caused a significant increase in PGES mRNA. Levels of PGES transcripts were low or undetectable prior to hCG/luteinizing hormone but increased markedly 18-24 h after hCG in models 1 and 2, and 18-24 h after the onset of natural estrus in model 3 (p < 0.05). Analyses on isolated preparations of granulosa and theca interna cells indicated that the granulosa cell layer was the predominant site of follicular PGES expression. The regulation of the protein was studied in the same models using a specific antibody 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 interna. Collectively, these results demonstrate that the ovulatory process is associated with a gonadotropin-dependent induction of PGES in granulosa cells of ovarian follicles in vivo, thus establishing for the first time the regulation of the enzyme in a physiological context.
Filion
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F
Centre de recherche en reproduction animale and the Département de biomédecine vétérinaire, Faculté de médecine vétérinaire, Université de Montréal, C.P. 5000, Saint-Hyacinthe, Québec J2S 7C6, Canada.
Bouchard
N
N
Goff
A K
AK
Lussier
J G
JG
Sirois
J
J
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001483:Base Sequence; D015139:Blotting, Southern; D002417:Cattle; D006063:Chorionic Gonadotropin / Q000737:chemistry; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D006062:Gonadotropins / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D019746:Intramolecular Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D006080:Ovarian Follicle / Q000378:metabolism*; D010060:Ovulation; D000071876:Prostaglandin-E Synthases; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014018:Tissue Distribution; D015854:Up-Regulation
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D000906:Antibodies; D006063:Chorionic Gonadotropin; D018076:DNA, Complementary; D006062:Gonadotropins; D012333:RNA, Messenger; D019746:Intramolecular Oxidoreductases; D000071876:Prostaglandin-E Synthases