PARENT SESSION
Corpus Luteum

TISSUE INHIBITOR OF METALLOPROTEINASES–3 LOCALIZATION AND mRNA EXPRESSION IN THE OVINE OVARY.

Willis, Erin¹, Ricke, William¹, Smith, George², Smith, Michael¹, ^{1 2}

ABSTRACT- Tissue inhibitors of metalloproteinases (TIMPs)–1, 2, 3, and 4 are multifunctional proteins that play an important regulatory role(s) in tissue remodeling. We have previously reported that TIMP–1 and 2 are differentially expressed in ovine periovulatory follicles and corpora lutea. However, the localization and ontogeny of TIMP–3 expression in the ruminant ovary is not known. Unlike TIMP–1 and 2, TIMP–3 binds to the extracellular matrix (ECM) and thus may play a unique role in maintenance of an ECM microenvironment conducive to follicular or luteal function. Thus, our objectives were to characterize TIMP–3 mRNA and protein expression in ovine follicles and corpora lutea. Ovine periovulatory and adjacent small antral follicles were collected 30 hr following injection of PGF₂ on day 14 post–estrus (day 0 = estrus; presurge; n = 4) or 12–14 h after an LHRH–induced gonadotropin surge at 36 hr following PGF₂ injection (post-surge; n = 5). Corpora lutea were collected on days 2, 4, 10, and 15 post–estrus (day 0 = estrus; n = 5 to 10 per group) for Northern, Western blot, and reverse zymographic analysis. Northern blot analysis confirmed a major transcript of 4.5 kB (similar to other species) and Western blot analysis and reverse zymography demonstrated two distinct bands representing TIMP–3 at 24,000 and 50,000 Mr. Low levels of TIMP–3 mRNA were localized to thecal and granulosa cells of ovine pre and post surge follicles by in situ hybridization. However, an intense hybridization signal for TIMP–3 mRNA was detected within the granulosa layer(s) of adjacent smaller follicles in both groups. Within luteal tissue, relative levels of TIMP–3 mRNA were greater (P < 0.05) during the mid–late luteal phase (days 10 and 15) versus the early luteal phase (days 2 and 4). In summary, TIMP–3 mRNA was detected in ovine follicles and TIMP–3 mRNA and protein were detected in corpora lutea. Although regulation of follicular TIMP–3 mRNA expression during the periovulatory period was not detected, TIMP–3 mRNA expression was increased during the mid to late luteal phase. Supported by USDA 98–35203–6282 (MFS) and USDA 98–35203–6226 (GWS).

KEY WORDS: Corpus Luteum, Follicle, TIMP, Extracellular Matrix