LH-Induced Steroidogenesis in the Mouse Ovary, but Not Testis, Requires Matrix Metalloproteinase 2- and 9-Mediated Cleavage of Upregulated EGF Receptor Ligands1

Abstract

Oocyte maturation and cumulus cell expansion depend on luteinizing hormone (LH)-mediated upregulation of membrane-bound epidermal growth factor (EGF)-like ligands, including amphiregulin, epiregulin, and betacellulin. These ligands then transactivate the EGF receptor (EGFR) after release by matrix metalloproteinases (MMPs). However, direct measurement of released EGF-like ligands or MMPs from granulosa cells has not been formally evaluated, nor has direct identification of responsible MMPs. Here we address these issues by analyzing LH-induced steroidogenesis, which is also MMP and EGFR dependent, in freshly isolated mouse primary granulosa cells. We demonstrate a correlation between amphiregulin and epiregulin mRNA induction and steroid production in LH-treated granulosa cells as well as in ovaries of human chorionic gonadotropin-treated mice. In contrast, LH does not alter Mmp1, Mmp2, Mmp3, Mmp8, Mmp9, or Adam17 mRNA expression. We demonstrate that, in primary mouse granulosa cells, LH triggers release of soluble amphiregulin that correlates with steroid production, both of which are blocked by MMP2/9 inhibition, confirming that MMP2/9 likely regulates LH-induced amphiregulin release and downstream processes. Notably, LH does not alter secretion of MMP2/9 from primary granulosa cells, nor does it modulate MMP activity. These findings indicate that, in the ovary, LH dictates EGFR-mediated processes not by regulating MMPs, but instead by increasing EGF-like ligand availability. In contrast, LH stimulation of primary mouse Leydig cells does not induce EGF-like ligand expression or require MMP2/9 for steroidogenesis, confirming marked differences in LH receptor-induced processes in the testes. Our results suggest that MMP inhibition may be a means of attenuating excess ovarian steroid production in diseases like polycystic ovary syndrome.

Developmental consequences of supplementing with matrix metallopeptidase-9 during <i>in vitro</i> maturation of heat-stressed bovine oocytes

“…This inverse relationship is divergent from reports derived by others suggesting that MMP2/9 may be required for progesterone biosynthesis [30,31,32,33]. These studies, performed on mural granulosa cells or intact follicles, showed that MMP2/9 activation of EGF-like ligands was required for LH-induced steroidogenesis [30,31,32,33]. Our studies, however, examined the responsiveness of the cumulus-oocyte complex after removal from an antral follicle.…”

“…This cumulus-oocyte complex has low expression of LH receptors [34, 35] and reduced amounts of EGF-like ligands [36]. Bovine cumulus-oocyte complexes may be similar to cultured Leydig cells which do not appear to require EGF-like ligands or MMP2/9 for steroidogenesis [30]. In support of this notion, when bovine cumulus-oocytes were matured in the presence of 300 ng/ml hMMP9 for the entire maturation period at a thermoneutral temperature, no impact on progesterone levels were observed by our laboratory [37].…”

Ovarian Folliculogenesis

“…Downstream to the AC/cAMP/PKA pathway, the CG express the EGF-like factors amphiregulin (AREG), beta-cellulin (BTC), and epiregulin (EREG) in GC. These EGF-like factors undergo proteolytic cleavage-by ADAM17/TACE in porcine (Yamashita and Shimada 2012) or by matrix metalloproteinases MMP2 and MMP9 in mice (Light and Hammes 2015). Subsequently, the EGFR is activated and initiates the ERK1/2 pathway, thus inducing expression of downstream target genes responsible for cumulus expansion, steroidogenesis, oocyte maturation, and ovulation.…”

Expression pattern of matrix metalloproteinases changes during folliculogenesis in the cat ovary

“…MMP7 mRNA has also been observed in pre‐ovulatory granulosa cells of the macaque (Chaffin & Stouffer, ), but is undetectable in the mouse ovary (Hägglund et al., ). MMP3 immunolocalization has been detected in the hamster (Whited, Shahed, McMichael, & Young, ), so as the mRNA expression in the human (McCord, Li, Rosewell, Brännström, & Curry, ) rat (McCord et al., ) and mouse ovary (Light & Hammes, ).…”