Recent purification and amino-terminal analysis of the isoform of prostaglandin endoperoxide H synthase (PGS) induced in rat preovulatory follicles by gonadotropins identified it as a novel distinct isoform of PGS (rPGSi) which exhibited a high degree of homology to the deduced sequence of PGS-related cDNAs recently cloned in chicken and mice. To further verify the similarities of these novel gene products and to examine the hormonal regulation of rPGSi mRNA in ovarian cells, three different in vivo and in vitro models were used. Northern blots using a cDNA encoding the mouse homologue of rPGSi detected a 4.4-kilobase transcript which was rapidly but transiently induced in granulosa cells of preovulatory follicles exposed in vivo to an ovulatory dose of human chorionic gonadotropin. The rPGSi mRNA was undetectable at 0 h, peaked 4 h after human chorionic gonadotropin, and had almost disappeared by 6 h. Increases in rPGSi protein (immunoblots) lagged by about 1 h, peaked at 5 h, and remained present at 11 h. PGSi mRNA and protein were also induced in a time- and dose-dependent manner when preovulatory follicles were isolated and incubated with elevated levels of follicle-stimulating hormone (500 ng/ml) or luteinizing hormone (500 ng/ml), or when differentiated granulosa cell cultures were stimulated with follicle-stimulating hormone, luteinizing hormone, or with gonadotropin-releasing hormone (10(-6) M). In both in vitro systems, rPGSi mRNA peaked at 4-5 h. When the same RNA samples were probed with the mouse cDNA encoding the other PGS isoform, no mRNA transcripts (2.8 kilobases) were observed. These results show for the first time that a rapid and transient induction of mRNA encoding a novel PGS enzyme occurs in granulosa cells of preovulatory follicles prior to ovulation and that results in vitro closely mimicked those in vivo and thereby provide models for studying the molecular mechanisms of rPGSi gene expression.