Effect of growth factors on proliferation, apoptosis and protein kinase A expression in cultured porcine cumulus oophorus cells

Abstract

-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

Epidermal growth factor activates cytosolic [Ca2+] elevations and subsequent membrane permeabilization in mouse cumulus–oocyte complexes

“…In both freshly isolated and established cultures of granulosa cells from pigs, tyrosine phosphorylation and activation of MAP kinase is induced by EGF in a concentration-dependent manner that requires phosphatidylinositol 3-kinase (Keel et al 1995, Keel & Davis 1999. Granulosa cells in culture are stimulated by EGF to proliferate (Gospodarowicz & Bialecki 1979, Sirotkin et al 2002. These results demonstrate that EGF directly activates phosphorylation cascades in granulosa cells.…”

Adipose-derived stem cells improved mouse ovary function after chemotherapy-induced ovary failure

“…Zcchc11 deficiency significantly decreased IGF-1 mRNA in the liver and insulin-like growth factor (IGF)-1 protein in the blood during the neonatal period [24]. IGF-1, a growth-hormone mediator, can stimulate GC proliferation, inhibit apoptosis, and promote follicular antrum formation [25,26]. IGF-1 may play a key role in sensitizing ovarian GCs to follicle-stimulating hormone (FSH) during terminal follicular growth [27] and also plays an endocrine and/or paracrine role in ovarian follicular development.…”

Effects of Basal Media and Supplements on Diethylstilbestrol‐Treated Immature Mouse Primary Granulosa Cell Growth and Regulation of Steroidogenesis In Vitro

“…2008). And the significant rise of StAR and P450scc resulted in the progesterone increase; 10 ng/ml of EGF was chosen based on previous studies (Sirotkin et al. 2002) and was thought to mediate its biological actions through the activation of mitogen‐activated protein kinases (Mao et al.…”

“…2005) and EGF (Broussard et al. 1995; Sirotkin et al. 2002) were supplied, respectively, to maintain cellular viability or support proliferation in former studies.…”

“…A commercial product containing insulin, transferrin and selenium (Irusta et al 2010), BSA (Broussard et al 1995), PNa (McNatty et al 2005) and EGF (Broussard et al 1995;Sirotkin et al 2002) were supplied, respectively, to maintain cellular viability or support proliferation in former studies. Yet, they were not compared with serum to study the growth of GCs before.…”