Production of matrix metalloproteinases by cultured bovine theca and granulosa cells

Abstract

Matrix metalloproteinases (MMPs) degrade the proteinaceous components of the extracellular matrix and are presumably essential for follicular growth culminating in ovulation or atresia. The objectives of this study were to characterize the gelatinolytic and caseinolytic MMPs secreted by cultured bovine thecal and granulosal cells and to determine the effect of luteinizing hormone (LH) on MMP secretion. Thecal and granulosal cells were collected from small bovine follicles (<5 mm) on day 2 or 5 of the estrous cycle (day 0=estrus). A serum-free culture system was utilized in which bovine thecal and granulosal cells do not spontaneously luteinize, but produce androstenedione and estradiol in response to physiological concentrations of LH and follicle-stimulating hormone (FSH) respectively. The effect of LH (0, 1 or 100 ng/ml) on MMP production was determined in conditioned media collected every 48 h for 144 h. MMPs were detected by gelatin and casein zymography and MMP activity was quantified by image analysis. Thecal and granulosal cell conditioned media contained MMPs that had a relative molecular size (Mr) ranging from 53,000 to 200,000 and addition of 1,10 phenanthroline (MMP inhibitor) blocked gelatinolytic and caseinolytic activity. Patterns of gelatinolytic activity in thecal and granulosal cell conditioned media differed over time with the Mr 62,000 and 83,000 MMPs being increased (P <0.05) and the Mr 53,000 MMP being decreased (P <0.05) at 96 h of culture. LH (1 or 100 ng/ml) increased (P <0.05) gelatinolytic activity of the Mr 53,000 and 62,000 gelatinases within thecal cell conditioned media but not granulosal cell conditioned media. The Mr 62,000 and 83,000 gelatinolytic activities corresponded to the active forms of gelatinase A (Mr 62,000) and B (Mr, 83,000) and gelatinase A was detected in thecal cell conditioned media by Western blot analysis. Caseinolytic activity (Mr 83,000) was detected in both thecal and granulosal cell conditioned media and increased from 48 to 96 h. In summary, thecal and granulosal cells secrete gelatinolytic and caseinolytic MMPs and thecal cell production of gelatinase A was stimulated by LH.