The role of the PI3K-Akt signaling pathway in the developmental competence of bovine oocytes

Abstract

The ovarian follicle encloses oocytes in a microenvironment throughout their growth and acquisition of competence. Evidence suggests a dynamic interplay among follicular cells and oocytes, since they are constantly exchanging "messages". We dissected bovine ovarian follicles and recovered follicular cells (FCs-granulosa and cumulus cells) and cumulus-oocyte complexes (COCs) to investigate whether the PI3K-Akt signaling pathway impacted oocyte quality. Following follicle rupture, COCs were individually selected for in vitro cultures to track the follicular cells based on oocyte competence to reach the blastocyst stage after parthenogenetic activation. Levels of PI3K-Akt signaling pathway components in FCs correlated with oocyte competence. This pathway is upregulated in FCs from follicles with high-quality oocytes that are able to reach the blastocyst stage, as indicated by decreased levels of PTEN and increased levels of the PTEN regulators bta-miR-494 and bta-miR-20a. Using PI3K-Akt responsive genes, we showed decreased FOXO3a levels and BAX levels in lower quality groups, indicating changes in cell cycle progression, oxidative response and apoptosis. Based on these results, the measurement of levels of PI3K-Akt pathway components in FCs from ovarian follicles carrying oocytes with distinct developmental competences is a useful tool to identify putative molecular pathways involved in the acquisition of oocyte competence.

Fig 1

Relative expression of (A) PTEN and (B) PI3K mRNAs in follicular cells according to oocyte competence. (C) Levels of the phosphorylated Akt protein (p-Akt) obtained from western blotting analyses and (D) representative protein bands for p-Akt (~ 60 kDa) and Actin B (ACTB; 42 kDa) from western blotting analyses. Non-cleaved (NC), cleaved non-blastocyst (CNB), blastocyst (BL). Bars depict means and error bars depict standard errors of the means. Differences were statistically significant when p < 0.05.

Fig 2. Relative levels of bta-miR-494 and bta-miR-20a in follicular cells.

(A) Relative levels of bta-miR-494 in non-cleaved (NC), cleaved non-blastocyst (CNB), and blastocyst (BL) groups. Bovine miRNA-494 and its validated target PTEN. A bioinformatics analysis of the 3’UTR of PTEN revealed two conserved target sites among mammals. (B) Relative levels of bta-miR-20a in non-cleaved (NC), cleaved non-blastocyst (CNB), and blastocyst (BL) groups. (C) Luciferase assay of the bovine PTEN 3’UTR in granulosa cells. Firefly luciferase activity was evaluated in cells transfected the empty pmirGLO vector, the vector with the wild type PTEN 3’UTR insert, and the vector with the mutated insert following the transfection of the bta-miR-494 mimic. Different letters indicate p<0.05. ** indicate p<0.01.

Fig 3. Relative expression of mRNAs for genes responsible for the downstream cellular effects of PI3K-Akt signaling on non-cleaved (NC), cleaved non-blastocyst (CNB) and blastocyst (BL) groups.

Bars depict means and error bars depict standard errors of the means. Different superscripts (a and b) indicate significant differences (p < 0.05).

Fig 4. Schematic representation of the PI3K-Akt signaling pathway model and cellular responses in ovarian follicular cells (FCs) associated with competent oocytes that were able to reach the blastocyst stage (BL) and incompetent oocytes that were unable to initiate embryo development after maturation (NC).

Pentagons indicate whether a given component of the pathway was upregulated (upward arrow) or downregulated (downward arrow) in FCs from the BL and NC groups. Lines ending in arrowheads indicate stimulation, whereas lines ending in bars indicate blockage.