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

Abstract

-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)

Involvement of the metabolic hormones leptin, ghrelin, obestatin, IGF-I and of MAP kinase in control of porcine oocyte maturation

“…Requirement of MAPK/ ERK1-2 for promotion of meiosis is confirmed by the inhibitory effect of MAPK blocker PD98059 on both MAPK/ERK1-2 accumulation and oocyte maturation after culture observed in our experiments. Our observations are in line with maturationassociated accumulation or activation of MAPK and with the ability of MAPK blocker to prevent meiosis in porcine oocytes observed by other authors (Inoue et al, 1995 andMotlik et al, 1998;Lee et al, 2000;Sirotkin et al, 2000;Wehrend and Meinecke, 2001;Ye et al, 2007;Ebeling et al, 2007; …”

“…The nuclear maturation of porcine oocytes after isolation and culture, as well as the presence of MAPK/ERK1-2 in these oocytes, is in line with previous reports (Inoue et al, 1995 andMotlik et al, 1998;Lee et al, 2000;Sirotkin et al, 2000;Wehrend and Meinecke, 2001;Ye et al, 2007;Ebeling et al, 2007;Kimura et al, 2007;Li et al, 2008). It was shown that a part of the oocytes were mature already before long-term culture.…”

“…Oocyte-cumulus complexes were isolated and processed as described earlier (Sirotkin et al, 2000). Briefly, the oocyte-cumulus complexes were aspirated by syringe from medium size (3 to 8 mm in diameter) antral ovarian follicles.…”

“…Furthermore, these hormones could be useful for regulation of oocyte maturation (meiotic divisions of chromosomes and cytoplasmic maturation enabling oocyte fertilization and further development (Nebreda and Ferby, 2000) in assisted reproduction and biotechnology. IGF-I was reported to be a promoter of porcine oocyte nuclear maturation/meiosis (Xia et al, 1994;Illera et al, 1998;Sirotkin et al, 2000). Some authors (Craig et al, 2005;Kun et al, 2007) have reported the promotion of meiosis in porcine oocytes after leptin addition.…”

Unexpected detrimental effect of Insulin like growth factor-1 on bovine oocyte developmental competence under heat stress

“…Also, it has been established that IGF-1 has a beneficial effect on oocyte nuclear maturation [21,22], whereas, oocyte expresses IGF-1 receptor [23]. Forthermore, IGF-1 stimulates resumption of meiosis and completion of nuclear maturation of porcine oocytes [24]. It has been detected that exposure of bovine oocytes to EGF and IGF-1 accelerated progression of meiosis in COCs [25].…”