Evidence that growth factors IGF-I, IGF-II and EGF can stimulate nuclear maturation of porcine oocytes via intracellular protein kinase A

The aim of our in vitro experiments was to study the role of growth factors and protein kinase A (PKA)-dependent intracellular mechanisms in the contr ol of nuclear maturation of porcine oocytes. Oocytes were cultured with or without growth factors (IGF-I, IGF-II, EGF; 10 ng.mL(-1) medium) and inhibi tors of PKA (Rp-cAMPS or KT5720; 100 ng.mL(-1)). Stages of meiosis were det ermined from the structure of chromosomes after staining with Giemza. Intra cellular levels of PKA were evaluated immunocytochemically using primary an tisera against the PKA regulatory and catalytic subunits and by Western imm unoblotting using primary antiserum against the PKA catalytic subunit. It w as found that after 24 h culture the majority of oocytes had resumed nuclea r maturation (they were at a stage of meiosis after diplotene) and that aft er 48 h culture the majority of cells had completed maturation (they had re ached metaphase II of meiosis). Addition of IGF-I, IGF-II or EGF, or a comb ination of IGF-I and EGF, significantly increased the proportion of oocytes which resumed and completed meiosis. Immunocytochemistry demonstrated a si gnificant increase in the proportion of cells containing catalytic and, in some cases, the regulatory subunits of PKA after addition of IGF-I, IGF-II and EGF. Immunoblotting showed the presence of 2 forms of the PKA catalytic subunit within the oocytes (MW approximately 52 and 40 kD). EGF, but not I GF-I or IGF-II, increased the content of both isoforms. Inhibitors of PKA, when given alone, did not substantially influence the proportion of oocytes which resumed or completed meiosis. However, Rp-cAMPS and KT5720 both prev ented the stimulatory effects of IGF-I, IGF-II and EGF on the resumption an d completion of oocyte maturation. The present observations suggest (1) tha t IGF-I, IGF-II and EGF are potent stimulators of both resumption and compl etion of porcine oocyte nuclear maturation, (2) that PKA is present in oocy tes, and (3) that PKA-dependent intracellular mechanisms can mediate the ac tion of growth factors on porcine oocytes.