Molecular Mechanisms of Insulin-Like Growth Factor 1 Promoted Synthesis and Retention of Hyaluronic Acid in Porcine Oocyte-Cumulus Complexes1

The aim of this study was to investigate the involvement of the serine/threonine protein kinase AKT (also called protein kinase B) in the control of meiosis of porcine denuded oocytes (DOs) matured in vitro. Western blot analysis revealed that the two principal AKT phosphorylation sites, Ser473 and Thr308, are phosphorylated at different stages of meiosis. In freshly isolated germinal vesicle (GV)-stage DOs, Ser473 was already phosphorylated. After the onset of oocyte maturation, the intensity of the Ser473 phosphorylation increased, however, which declined sharply when DOs underwent GV breakdown (GVBD) and remained at low levels in metaphase I-and II-stage (MI-and MII-stage). In contrast, phosphorylation of Thr308 was increased by the time of GVBD and reached maximum at MI-stage. A peak of AKT activity was noticed around GVBD and activity of AKT declined at MI-stage. To assess the role of AKT during meiosis, porcine DOs were cultured in 50 mM SH-6, a specific inhibitor of AKT. In SH-6-treated DOs, GVBD was not inhibited; on the contrary, a significant acceleration of meiosis resumption was observed. The dynamics of the Ser473 phosphorylation was not affected; however, phosphorylation of Thr308 was reduced, AKT activity was diminished at the time of GVBD, and meiotic progression was arrested in early MI-stage. Moreover, the activity of the cyclin-dependent kinase 1 (CDK1) and MAP kinase declined when SH-6-treated DOs underwent GVBD, indicating that AKT activity is involved in the regulation of CDK1 and MAP kinase. These results suggest that activity of AKT is not essential for induction of GVBD in porcine oocytes but plays a substantial role during progression of meiosis to MI/MII-stage. Reproduction (2009) 138 645-654

Section: Discussionmentioning

confidence: 99%

AKT (protein kinase B) is implicated in meiotic maturation of porcine oocytes

Kalous¹,

Kubelka²,

Solc³

et al. 2009

“…An incentive for these studies was a finding that insulin-like growth factor 1, a known activator of PI3K/AKT signaling, is the component of serum that enables expansion of pig cumulus cells in vitro in response to FSH (Singh & Armstrong 1997). Further studies have revealed that PI3K/AKT signaling is involved in regulation of connexin 43 phosphorylation (Shimada et al 2001) and in promotion of FSH-stimulated synthesis and retention of hyaluronic acid in pig COCs (Nagyova et al 1999, Nemcova et al 2007). Inhibition of PI3K/AKT signaling in matured pig COCs by LY294002 showed interesting differences between FSH-and AREG-induced maturation in our study.…”

Section: Signaling In Pig Cumulus-oocyte Complexesmentioning

confidence: 99%

“…PI3K/AKT activity is required for gonadotropininduced expansion of pig cumulus cells in vitro (Shimada et al 1988, 2001, Nemcova et al 2007.…”

Section: Signaling In Pig Cumulus-oocyte Complexesmentioning

confidence: 99%

Signaling pathways regulating FSH- and amphiregulin-induced meiotic resumption and cumulus cell expansion in the pig

Procházka¹,

Blaha²,

Němcová³

2012

To define signaling pathways that drive FSH-and epidermal growth factor (EGF)-like peptide-induced cumulus expansion and oocyte meiotic resumption, in vitro cultured pig cumulus-oocyte complexes were treated with specific protein kinase inhibitors. We found that FSH-induced maturation of oocytes was blocked in germinal vesicle (GV) stage by protein kinase A (PKA), MAPK14, MAPK3/1, and EGF receptor (EGFR) tyrosine kinase inhibitors (H89, SB203580, U0126, and AG1478 respectively) whereas phosphoinositide-3-kinase/v-akt murine thymoma viral oncogene homolog (PI3K/AKT) inhibitor (LY294002) blocked maturation of oocytes in metaphase I (MI). Amphiregulin (AREG)-induced maturation of oocytes was efficiently blocked in GV by U0126, AG1478, and low concentrations of LY294002; H89, SB203580, and high concentrations of LY294002 allowed the oocytes to undergo breakdown of GV and blocked maturation in MI. Both FSH-and AREG-induced cumulus expansion was incompletely inhibited by H89 and completely inhibited by SB203580, U0126, AG1478, and LY294002. The inhibitors partially or completely inhibited expression of expansion-related genes (HAS2, PTGS2, and TNFAIP6) with two exceptions: H89 inhibited only TNFAIP6 expression and LY294002 increased expression of PTGS2. The results of this study are consistent with the idea that PKA and MAPK14 pathways are essential for FSH-induced transactivation of the EGFR, and synthesis of EGF-like peptides in cumulus cells and MAPK3/1 is involved in regulation of transcriptional and posttranscriptional events in cumulus cells required for meiotic resumption and cumulus expansion. PI3K/AKT signaling is important for regulation of cumulus expansion, AREG-induced meiotic resumption, and oocyte MI/MII transition. The present data also indicate the existence of an FSH-activated and PKA-independent pathway involved in regulation of HAS2 and PTGS2 expression in cumulus cells.

“…3). Activity of protein kinase A (PKA) and AKT kinase (also known as protein kinase B) was assessed in the COCs 60 min after stimulation and activity of MAPK3/1 at 2 h after stimulation; the intervals were chosen on the basis of our previous results (Nemcova et al 2007). FSH, AREG, and EREG promptly induced phosphorylation of MAPK3/1 and AKT kinase ( Fig.…”

Section: Stimulation Of Oocyte Maturation and Cumulus Cell Expansionmentioning

confidence: 99%

“…In the pig, both phosphatidylinositol 3-kinase (PI3K) and MAPK activation in cumulus is required for gonadotropin-stimulated resumption of meiosis (Shimada et al 2001, Meinecke & Krischek 2003, Liang et al 2005. PI3K/AKT-dependent pathway was also involved in the promotion of FSH-stimulated production of hyaluronic acid in pig COCs by insulinlike growth factor 1 (Nemcova et al 2007). Therefore, in this study, we assessed the activation of PKA, MAPK, and AKT in the cumulus cells by FSH and EGF-like peptides and also the expression of the expansion-related genes.…”

Section: Effect Of Egf-like Peptides On Pig Cocsmentioning

confidence: 99%

Effect of epidermal growth factor-like peptides on pig cumulus cell expansion, oocyte maturation, and acquisition of developmental competence in vitro: comparison with gonadotropins

Procházka

Petlach²,

Nagyová³

et al. 2011

Self Cite

The aim of this work was to assess the FSH-stimulated expression of epidermal growth factor (EGF)-like peptides in cultured cumulusoocyte complexes (COCs) and to find out the effect of the peptides on cumulus expansion, oocyte maturation, and acquisition of developmental competence in vitro. FSH promptly stimulated expression of amphiregulin (AREG) and epiregulin (EREG), but not betacellulin (BTC) in the cultured COCs. Expression of AREG and EREG reached maximum at 2 or 4 h after FSH addition respectively. FSH also significantly stimulated expression of expansion-related genes (PTGS2, TNFAIP6, and HAS2) in the COCs at 4 and 8 h of culture, with a significant decrease at 20 h of culture. Both AREG and EREG also increased expression of the expansion-related genes; however, the relative abundance of mRNA for each gene was much lower than in the FSH-stimulated COCs. In contrast to FSH, AREG and EREG neither stimulated expression of CYP11A1 in the COCs nor an increase in progesterone production by cumulus cells. AREG and EREG stimulated maturation of oocytes and expansion of cumulus cells, although the percentage of oocytes that had reached metaphase II was significantly lower when compared to FSH-induced maturation. Nevertheless, significantly more oocytes stimulated with AREG and/or EREG developed to blastocyst stage after parthenogenetic activation when compared to oocytes stimulated with FSH alone or combinations of FSH/LH or pregnant mares serum gonadotrophin/human chorionic gonadotrophin. We conclude that EGF-like peptides do not mimic all effects of FSH on the cultured COCs; nevertheless, they yield oocytes with superior developmental competence.