MOLECULAR CHARACTERIZATION OF EQUINE PROSTAGLANDIN G H SYNTHASE-2 ANDREGULATION OF ITS MESSENGER-RIBONUCLEIC-ACID IN PREOVULATORY FOLLICLES/

To increase our understanding of the molecular control of PG synthesis in equine preovulatory follicles, the specific objectives of this stu dy were to clone and determine the primary structure of equine prostag landin G/H synthase-2 (PGHS-2) and to characterize the regulation of P GHS-2 messenger RNA (mRNA) in follicles before ovulation. A complement ary DNA (cDNA) Library prepared from follicular mRNA and a genomic lib rary mere screened with a mouse PGHS-2 cDNA probe to isolate the equin e PGHS-2 cDNA and gene, respectively. The expression library yielded t hree nearly full-length clones that differed only in their 5'-ends; cl ones 3, 5, and 6 were 2946, 3138, and 3398 bp in length, respectively. The longest clone was shown to start 9 bp downstream of the transcrip tion initiation site, as determined by primer extension analysis, and to contain 120 bp of 5'-untranslated region (UTR), 1812 bp of open rea ding frame, and 1466 bp of 3'-UTR. The open reading frame encodes a 60 4-amino acid protein that is more than 80% identical to PGHS-2 homolog s in other species. Numerous repeats (n = 11) of the Shaw-Kamen's sequ ence (ATTTA) are present in the 3'-UTR, a motif typically indicative o f mRNAs with a short half-life. The complete equine PGHS-2 gene was is olated and sequenced from a similar to 17-kilobase clone obtained from the genomic library. The equine PGHS-2 gene structure (10 exons and 9 introns; total length of 6991 bp) is similar to its human homolog exc ept for lacking sequence elements in introns 4, 8, and 9 and in the 3' -UTR region of exon 10. To characterize the regulation of PGHS-2 mRNA in equine follicles before ovulation, preovulatory follicles were isol ated during estrus, 0, 12, 24, 30, 33, 36, and 39 h (n = 4-5 follicles /time point) after an ovulatory dose of hCG. Results from Northern blo ts showed significant changes in steady state levels of PGHS-2 mRNA in preovulatory follicles after hCG treatment (P < 0.05). The transcript remained undetectable between 0-24 h post-hCG, first appeared (simila r to 4 kilobases) only at 30 h, and reached maximal levels 33 h post-h CG. PGHS-8 mRNA was selectively induced in granulosa cells and not in theca interna. Thus, this study provides for the first time the primar y structure of the equine PGHS-2 gene, transcript, and protein. It als o demonstrates that the induction of PGHS-2 gene expression in equine granulosa cells is a long molecular process (30 h post-hCG), thereby p roviding a model to study the molecular basis for the late transcripti onal activation of PGHS-2 in species with a long ovulatory process.