Involvement of EGFR and Smad2/3 Activation in FSH-Induced Porcine Oocytes-Cumulus Cell Complex Maturation.

Abstract

It has been shown that the activation of both EGF receptor and SMAD2 and SMAD3 in mouse cumulus cells is essential for their preovulatory maturation. In the present study we investigated whether these changes are also required for the final differentiation of pig cumulus cells. Porcine oocyte-cumulus complexes (OCCs) were stimulated in vitro with FSH in the absence or in the presence of the following inhibitors: AG1478 (an EGFR tyrosine kinase inhibitor), SB431542 (a specific inhibitor of SMAD2 and SMAD3 activation) and SIS3 (a specific inhibitor of SMAD3 activation). We found that EGFR inhibitor AG1478 completely blocked FSH-induced cumulus expansion by preventing hyaluronic acid production and its incorporation within the extracellular matrix. In addition, in the presence of AG1478, most of FSH-stimulated OCCs were in germinal vesicle stage (GV) and did not mature. Conversely, AG1478 did not affect the stimulation of progesterone synthesis by FSH. In agreement, EGF was unable to stimulate progesterone synthesis by OCCs cultured in vitro. The SB431542, that inhibits the activation of SMAD2 and SMAD3, only partially inhibited cumulus expansion by slightly decreasing both total production and matrix accumulation of hyaluronic acid. Surprisingly, SIS, that inhibits only SMAD3, produced an inhibitory effect higher than SB431542. A divergent effect of the two inhibitors was observed on steroid synthesis: FSH-induced progesterone synthesis increased in the presence of SB431542 and decreased in the presence of SIS. Both inhibitors did not affect the induction of oocyte meiotic resumption promoted by FSH. These results suggest that in pig, like in mice, FSH stimulates cumulus cells to produce EGF that acting in an autocrine way induces the formation of the expanded matrix and the signaling for oocyte meiotic resumption. The results also suggest that SMAD2 and SMAD3 activation is required for optimal stimulation of hyaluronic acid and proteins involved in the organization of this polymer in the pig OCC matrix. Finally, FSH stimulation of progesterone synthesis seems to be independent from EGF, while it appears to be counteracted by SMAD2, but not SMAD3 activation, thereby suggesting that specific SMAD2 signaling pathway is probably indispensable to prevent luteinization of cumulus cells. Supported by grant GACR 523/08/0111 and VEGA 2/0153/08. (poster)