-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 control 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 inhibitors of PKA (Rp-cAMPS or KT5720; 100 ng . mL -1 ). Stages of meiosis were determined from the structure of chromosomes after staining with Giemza. Intracellular levels of PKA were evaluated immunocytochemically using primary antisera against the PKA regulatory and catalytic subunits and by Western immunoblotting using primary antiserum against the PKA catalytic subunit. It was found that after 24 h culture the majority of oocytes had resumed nuclear maturation (they were at a stage of meiosis after diplotene) and that after 48 h culture the majority of cells had completed maturation (they had reached metaphase II of meiosis). Addition of IGF-I, IGF-II or EGF, or a combination of IGF-I and EGF, significantly increased the proportion of oocytes which resumed and completed meiosis. Immunocytochemistry demonstrated a significant 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 IGF-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 prevented the stimulatory effects of IGF-I, IGF-II and EGF on the resumption and completion of oocyte maturation. The present observations suggest (1) that IGF-I, IGF-II and EGF are potent stimulators of both resumption and completion of porcine oocyte nuclear maturation, (2) that PKA is present in oocytes, and (3) that PKA-dependent intracellular mechanisms can mediate the action of growth factors on porcine oocytes.insulin-like growth factor / epidermal growth factor / protein kinase A / meiosis Reprod. Nutr. Dev. 40 (2000)

-The proliferation, apoptosis and protein kinase A (PKA) in porcine cumulus oophorus (CO) before and after 40 h of culture together with oocytes in the presence of IGF-I, IGF-II and EGF (all at 10 ng . mL -1 medium) were compared. Cellular proliferation, apoptosis and PKA contents were evaluated by immunocytochemistry using specific antibodies against PCNA, TUNEL and catalytic (C-alpha) and regulatory (RI) subunits of PKA. The in-vitro culture of oocyte-CO complexes in a basal medium was accompanied by a decrease in the proportion of PCNA-positive CO cells (from 51 to 36%, p < 0.05). The addition of either IGF-I or EGF to the culture medium prevented this process and increased the proliferation rate (64 and 67% respectively, p < 0.001). During culture, the percentage of apoptotic (TUNEL-positive) CO cells increased from 42 to 57% (p < 0.01). The addition of IGF-I or EGF resulted in the inhibition of apoptosis to 36 and 12% respectively (p < 0.001). IGF-II and EGF reduced the amount of PKA catalytic subunits in the CO (percentage of cells with immunoreactive PKA catalytic subunits (28%, p < 0.05 and 27%, p < 0.05 respectively; versus control -41%), whilst the effect of IGF-I on this index was insignificant (31%). The expression of the PKA regulatory subunit was increased by EGF (51% compared with 29% in the control, p < 0.05), but not by IGF-I or IGF-II (30 and 29%).Our observations demonstrate that 40 h of culture of porcine CO resulted in a decrease in the proliferation and development of apoptosis in CO cells. IGF-I or EGF can stimulate proliferation and inhibit apoptosis. The influence of growth factors on the PKA content of the CO suggests that cAMP/PKA may be a mediator of the action of growth factors on these cells. The differential effects of IGFs and EGF on the regulatory subunit of PKA may indicate differences between their mechanisms of action.insulin-like growth factor I / insulin-like growth factor II / epidermal growth factor / cumulus oophorus / proliferation / apoptosis / protein kinase A Reprod. Nutr. Dev. 42 (2002) 35-43 35