Growth factors improve mouse oocyte developmental potential via increased MAPK and MTOR signaling activities in cumulus cells during in vitro maturation

Abstract

Oocyte in vitro maturation (IVM) is an emerging technology in human ART that has several potential advantages over traditional ovarian stimulation. Our previous work demonstrated a combination of human growth factors (GFs) resulted in better oocyte quality in pig and mouse, yielding increased blastocyst formation as well as improved fetal development after embryo transfer. The objective of this study is to understand how metabolic pathways were affected by these GFs in mouse cumulus cells (CCs) and oocytes. Research Study. Cumulus-oocyte complexes (COCs) were obtained from outbred CF1 mice and randomly allocated into groups either with or without the combination of GFs (FGF2, 40 ng/ml, LIF, 20 ng/ml, and IGF1, 20 ng/ml). Oocytes were matured for 18h in 6.5% O2/7.5% CO2 at 37.0 oC and then completely denuded from their surrounding CCs. CCs from 10 COCs and oocytes were frozen separately for subsequent analysis. The concentrations of phosphorylated MAPK (p-MAPK), p-mTOR, and p-AKT, and total MAPK (t-MAPK), t-MTOR and t-AKT were examined by Western blotting and the ratio of phosphorylated form relative to total protein concentrations was used to determine the activity of each signaling pathway (three replicates). In addition, genes correlated with changes in MAPK activation in CCs, including AREG, EREG, BTC, HAS2, TNFAIP6, were examined by quantitative PCR (three replicates). Protein data were analyzed by one-way ANOVA, and qPCR data were analyzed by REST 2009 software to determine relative expression levels between treatments (significance, p < 0.05). The ratios of p-MAPK/MAPK, p-mTOR/mTOR, and p-AKT/AKT remained unchanged in oocytes between the two treatment groups. On the other hand, both p-MAPK/MAPK and p-mTOR/mTOR were elevated in the GF treated CCs compared to control CCs, while p-AKT/t-AKT was not different. The EGF-like factor, EREG, and the cumulus expansion factor, TNFAI6, were significantly upgregulated in CCs treated by GFs. COCs treated with GFs during IVM exhibited greater MAPK and mTOR signaling activities in CCs, as well as upregulation of genes involved in cumulus-oocyte signaling and cumulus expansion. This work suggests that the improved oocyte quality previously observed following the addition of these growth factors may be mediated by enhanced metabolism and cell communication in CCs.