Identification of a 30 000 Mr Polypeptide Secreted by Cultured Ovine Granulosa Cells and Luteal Tissue as a Tissue Inhibitor of Metalloproteinases1

Previous studies have revealed that the gonadotropin surge initiates, via transcriptional mechanisms, synthesis and secretion of a 30,000 M(r) polypeptide by the granulosa cells of ovine preovulatory follicles. This polypeptide also appears to be secreted by luteal tissue and has been tentatively identified as a tissue inhibitor of metalloproteinases (TIMP; 68% NH2-terminal amino acid sequence identity to a human TIMP). Therefore, the objective of the present study was to confirm that TIMP is produced by granulosa cells and luteal tissue in the ewe. A series of experiments was conducted to determine whether the 30,000 M(r) polypeptide secreted by granulosa cells and luteal cells is similar to TIMP in biochemical properties (degree of N-linked glycosylation), in biological activity (as ascertained by gelatin zymographic analysis), and in immunoreactivity (as ascertained by Western blot analysis). Incubation of granulosa cells or luteal cells with tunicamycin revealed that the 30,000 M(r) polypeptide is glycosylated. The form lacking N-linked chains had an M(r) (approximately 20,000) similar to that of the unglycosylated form of TIMP in other species. Gelatin zymographic analysis detected significant metalloproteinase inhibitor activity associated with polypeptides of M(r) approximately 21,000 and 30,000 secreted by granulosa cells and luteal cells. Northern hybridization of granulosa cell RNA and Day 10 luteal RNA with an anti-sense TIMP oligonucleotide probe detected an approximately 900-base transcript, which is similar in size to that reported for TIMP mRNA in other species. Finally, Western blot analysis with a rabbit anti-human TIMP antiserum detected immunoreactive polypeptides of M(r) 30,000 secreted by granulosa cells and luteal cells.(ABSTRACT TRUNCATED AT 250 WORDS)