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This Article Full Text (PDF) All Versions of this Article: jas.2009-2088v1 87/12/3955    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Sessions, D. R. Articles by Fitzgerald, B. P. PubMed PubMed Citation Articles by Sessions, D. R. Articles by Fitzgerald, B. P.

Characterization of Matrix Metalloproteinase-2 and -9 and their inhibitors in equine granulosa cells in vivo and in vitro

Department of Veterinary Science, Maxwell H. Gluck Equine Research Center, University of Kentucky, Lexington, KY 40546-0099

bfitz{at}uky.edu

Abstract

Matrix metalloproteinases (MMP) and tissue inhibitors of MMP (TIMP) regulate tissue remodeling events necessary for ovulation. Thus, changes in MMP and TIMP expression and protein enzyme activity were examined in vivo and in vitro during follicular development and atresia in the horse. Equine granulosa cells and follicular fluid from medium (15 to 29 mm) healthy and atretic follicles and from large (>30 mm) healthy and preovulatory follicles were collected by transvaginal aspiration. The cells were either snap frozen (in vivo study) or cultured for 48 h (in vitro study) to determine gene expression and protein enzyme activity of MMP-2 and -9 and TIMP-1 and -2. Concentrations of progesterone and estradiol were determined by RIA in follicular fluid and conditioned media and were used along with follicle dynamics to classify follicles. In vivo, expression of MMP-2 and TIMP-2 was increased (P < 0.05) in large preovulatory follicles, while TIMP-1 was decreased. The ratio of MMP-2:TIMP-2 expression was decreased (P < 0.05) in medium healthy and large preovulatory follicles, while MMP-9:TIMP-1 ratio was increased only in large preovulatory compared to large healthy follicles. Estradiol was greatest (P < 0.05) in fluid of large healthy and large preovulatory follicles. However, medium atretic follicles were associated with the lowest estradiol concentrations, both in vivo and in vitro. Progesterone concentrations were greatest (P < 0.05) in large preovulatory follicles both in vivo and in vitro. In healthy follicles in vivo, the diameter correlated to estradiol concentration, estradiol:progesterone ratio, MMP-9 and TIMP-1 expression, and MMP-2 and -9 protein activity. In contrast to in vivo studies, the ratio of MMP-9:TIMP-1 expression was increased (P < 0.05) in medium healthy follicles; TIMP-2 expression decreased in large preovulatory follicles in vitro. In addition, MMP-9 protein activity was decreased (P < 0.05) in the media samples of cells from large healthy compared to medium healthy follicles. These results indicate changes in MMP-2 and -9 activities may be essential to the tissue reorganization necessary for ovulation in the equine ovary.

Key Words: atresia • equine • follicle growth • granulosa cells • matrix metalloproteinases • tissue inhibitors