192 The effect of in vitro maturation on the PI3K-Akt pathway in bovine cumulus cells
G. Andrade A , M. Del Collado A , R. Nociti A , W. J. Da Silva B , F. Meirelles A , J. Da Silveira A and F. Perecin AA Veterinary Medicine Department, Faculty of Animal Sciences and Food Engineering, University of Sao Paulo, Pirassununga, São Paulo State, Brazil;
B Department of Genetics, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo State, Brazil
Reproduction, Fertility and Development 32(2) 224-224 https://doi.org/10.1071/RDv32n2Ab192
Published: 2 December 2019
Abstract
Oocyte quality is influenced by in vitro oocyte maturation (IVM) because the culture conditions can alter the metabolism and gene expression of cumulus cells. Proper oocyte development requires fine regulation of signalling pathways involved with cell proliferation and survival, such as the PI3K-Akt (phosphatidylinositol-3-kinase/protein kinase B) signalling pathway. The aim of this study was to determine the effect of IVM on the expression of PI3K-Akt-related genes in bovine cumulus cells. To this aim, cumulus cells associated with immature oocytes, associated with oocytes in vitro-matured for 24 h, or associated with oocytes in vivo-matured were compared in terms of gene expression. Pools (n = 4) of cumulus cells from 20 cumulus-oocyte complexes (COCs) per group were submitted to total RNA extraction using the TRizol protocol, libraries were prepared with TruSeq stranded mRNA sample prep kit (Illumina Inc.), and the sequencing was performed in the HisEqn 2500 V4 (Illumina Inc.). After quality check with FastQC and filtering with Trim Galore, read alignment was performed with STAR and analysis of differential gene expression was done using DESEqn 2 in R considering the Benjamini-Hochberg method for adjusted P-values < 0.10, and absolute value of log2-fold change >0.5. Principal component analysis was able to separate, with 94% cumulative variance (81% and 13% for PC1 and PC2, respectively), the cumulus cells groups, especially the immature from the matured counterparts. Gene ontology and enrichment analysis showed that the PI3K-Akt signalling pathway was affected in immature cumulus cells compared with cumulus cells from in vitro- or in vivo-matured oocytes, with 77 and 88 genes from PI3K-Akt pathway being differentially expressed, respectively. A total of 51 genes were common in in vivo- and in vitro-matured oocytes cumulus cells groups compared with immature group. Regarding cumulus cells after the maturation process, 48 genes from the PI3K-Akt pathway were differentially expressed; of those, 26 genes were upregulated in cumulus cells from in vitro-matured oocytes and 22 genes were upregulated in cumulus cells from in vivo-matured oocytes. Comparing the in vitro and in vivo cumulus cells, the main genes of the pathway (AKT, PI3K, and PTEN) were not differentially expressed. The differences in expression between in vitro and in vivo cumulus cells were in genes responsible for different cellular functions controlled by the PI3K-Akt pathway, such as apoptosis, protein synthesis, and DNA repair, among others, which, in general, were increased in cumulus cells from in vitro-matured oocytes. These results demonstrated the effect of culture conditions on cumulus cell gene expression modulating important pathways involved in oocyte competence acquisition, such as PI3K-Akt signalling.
Financial support was provided by FAPESP grants 2014/22887-0, 2018/01431-9, and 2018/13155-6.
