Molecular characterization of equine prostaglandin G/H synthase-2 and regulation of its messenger ribonucleic acid in preovulatory follicles.

Abstract

To increase our understanding of the molecular control of PG synthesis in equine preovulatory follicles, the specific objectives of this study were to clone and determine the primary structure of equine prostaglandin G/H synthase-2 (PGHS-2) and to characterize the regulation of PGHS-2 messenger RNA (mRNA) in follicles before ovulation. A complementary DNA (cDNA) library prepared from follicular mRNA and a genomic library were screened with a mouse PGHS-2 cDNA probe to isolate the equine PGHS-2 cDNA and gene, respectively. The expression library yielded three nearly full-length clones that differed only in their 5'-ends; clones 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 transcription initiation site, as determined by primer extension analysis, and to contain 120 bp of 5'-untranslated region (UTR), 1812 bp of open reading frame, and 1466 bp of 3'-UTR. The open reading frame encodes a 604-amino acid protein that is more than 80% identical to PGHS-2 homologs in other species. Numerous repeats (n = 11) of the Shaw-Kamen's sequence (ATTTA) are present in the 3'-UTR, a motif typically indicative of mRNAs with a short half-life. The complete equine PGHS-2 gene was isolated and sequenced from a approximately 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 except 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 isolated 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 blots 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 (approximately 4 kilobases) only at 30 h, and reached maximal levels 33 h post-hCG. PGHS-2 mRNA was selectively induced in granulosa cells and not in theca interna. Thus, this study provides for the first time the primary structure of the equine PGHS-2 gene, transcript, and protein. It also demonstrates that the induction of PGHS-2 gene expression in equine granulosa cells is a long molecular process (30 h post-hCG), thereby providing a model to study the molecular basis for the late transcriptional activation of PGHS-2 in species with a long ovulatory process.