226. Expression of matrix metalloproteinases in bovine thecal cells

Abstract

As follicles grow the thecal layers expand. It is likely that extracellular matrix is remodelled in this process and possibly by matrix metalloproteinases (MMPs). A promising candidate to regulate MMPs is insulin-like factor 3 (INSL3). It is produced by thecal cells, its receptor, LGR8, is expressed in the theca interna (unpublished) and a related molecule, relaxin, regulates turnover of matrix in a number of tissues. For this reason we sort to examine the role of INSL3 in matrix turnover. However, in all thecal cell culture systems examined LGR8 receptors appear to be down regulated within 24 h. We therefore examined the effects of second messenger pathway activators. Thecal and granulosa cells were isolated and cultured and the levels of RNA for MMP2 and 9 quantitated by RT-PCR. MMP2 mRNA levels in thecal tissues were >10 fold higher than in granulosa cells (n = 19 follicles >10 mm). MMP2 levels were substantially greater than MMP9. At 12 h phorbol ester (100 nM phorbol 12,13-didecanoate) increased thecal expression of MMP 9 mRNA levels 11.5 fold (P < 0.001) and at 48 h MMP2 mRNA was increased 5 fold (P < 0.01). Pieces of whole follicle wall [follicles <5 mm in diameter, classified as healthy (n = 12) or atretic (n = 6)] were cultured in serum free media. Expression of the steroidogenic enzymes 17β HSD and P450scc but not 3β HSD were detected by immunohistochemistry even after 10 days. MMP activity on day 2 was analysed by gelatin zymography. Treatment with phorbol ester increased active MMP9 19 fold (P < 0.001). Treatment of thecal cells or follicle walls with 1 mM dibutyryl cAMP induced additional MMP activities at sizes of 110 and 122kDa. No effects on MMP2 activity were observed. In conclusion whilst we do not know the ligand inducers of the synthesis and activator of MMPs in thecal cells they can be regulated. Hence MMPs are candidates for remodelling the extracellular matrix of thecal layers.