Effect of sexual maturation and some biologically active substances on function of porcine ovarian cells

The aim of our experiments was to study the influence of sexual maturation on level of progesterone (P), insulin-like growth factor I (IGF-I), IGF-binding proteins-3 (IGFBP-3) in blood plasma and on release of these substances by porcine ovarian granulosa cells cultured in vitro. Besides the role of IGF-I, oxytocin (OT), cAMP and intracellular messengers in the control of ovarian function and sexual maturation was studied in pigs. 85 gilts were divided in two groups according to the sexual maturation (sexually immature gilts and sexually mature gilts). We determined the basal secretions of substances by granulosa cells, isolated from immature and preovulatory ovaries, together with the effect of treatment by IGF-I (10 ng/ml), OT (100 ng/ml and dbcAMP (10 mg/ml) during culture. The following products were assayed in plasma, cell culture medium and cells using RIA and immunocytochemistry: P, IGF-I, IGFBP-3, IGFBP-4, protein kinases A (PKA) and G (PKG), phosphotyrosine (Pht), ERK1,2 related MAP kinase, CREB1 and proliferation-associated (PCNA, P34/cdc2, cyclin B1), apoptotic (BAX) and antiapoptotic (BCL-2) peptides. It was observed, that sexual maturation of gilts is associated with an increase in the accumulation of P, IGF-I, IGFBP-3 in both blood plasma and cell culture medium. There were also increases in content of IGFBP-3, Pht, BCL-2, PCNA, PKA, BAX, P34/cdc2, CREB1 and decreases in the presence of IGFBP-4, ERK1,2, PKG and cyclin B1 in cultured granulosa cells. In both groups of animals, dbcAMP increased the secretion of P and IGF-I and the expression of ERK1,2 and PKG, and decreased the content of IGFBP-3, IGFBP-4 and PKA in cultured granulosa cells. In immature animals, dbcAMP stimulated PCNA, P34/cdc2 and inhibited Pht, BCL-2, BAX, cyclin B1 and CREB1, whilst in mature animals effect was opposite. In both groups, IGF-I increased P secretion and the expression of PCNA and IGFBP-3 but decreased the expression of Pht and P34/cdc2. IGF-I induced accumulation of IGFBP-4, PKA, BAX and CREB1 in immature gilts but had the opposite effect in mature animals. In both groups, OT increased IGF-I secretion and the content of IGFBP-3, IGFBP-4 and PCNA but decreased the content of Pht, BCL-2, ERK1,2 and PKA. In immature gilts, OT increased the expression of BAX, P34/cdc2, PKG, CREB1 and decreased P secretion. It had the opposite effect in mature animals. These data demonstrate that in pigs IGF-I, OT, cAMP, IGFBP-3 and IGFBP-4 control the proliferation and apoptosis of ovarian cell, the secretion of ovarian substances and sexual maturation. They also indicate differences in contents of P, IGF-I, IGFBP-3, IGFBP-4, PKA, PKG, Pht, ERK1,2, CREB1, PCNA, P34/cdc2, cyclin B1, BAX and BCL2 produced by granulosa cells during sexual maturation in gilts. These findings suggest the potential application of OT, IGF-I and cAMP preparation for regulation of porcine reproduction