SOMATIC SIGNALS REGULATING MRNA TRANSLATION IN MAMMALIAN OOCYTE.

In oocytes, resumption and completion of meiosis I are driven by post-transcriptional mechanisms on a background of repressed mRNA transcription. However, a comprehensive understanding of this regulation in higher-order mammals has been limited by challenges in isolating polysome-associated mRNAs. This study aims to shed light on these processes and how they can be affected by in vitro maturation (IVM). This objective has been pursued through three sequential steps that addressed the specific aims. First, we employed a bioinformatics approach to gain information on mRNA polysome association (GSE56603 and GSE196484) and polyadenylation (GSE61717) in immature and in vitro matured bovine oocytes. The GEO-retrieved datasets were re-analyzed to determine differentially polyadenylated/polysome-associated genes using edgeR. While the overlap of differentially polyadenylated/polysome-associated genes between datasets was limited when comparing mature oocytes (27 genes), all comparisons conducted in immature oocytes were substantial (372 genes p<0.05). These findings indicate that the experimental approaches potentially yield comparable results, but that this homogeneity is lost with IVM, possibly suggesting that culture conditions contribute to changes in mRNA translation patterns. Next, we tested whether hormones/growth factors supplementation may participate in the regulation of the translation of some maternal transcripts by activating the oocyte PI3K/AKT/mTOR axis in bovine oocytes, as demonstrated for mice. For this, the previously mentioned datasets were compared with polysome-associated transcripts of mouse oocytes exposed to the EGF-like growth factor, amphiregulin (GSE46640). Notably, one bovine dataset showed a significant correlation with EGF network activation, supporting, at least in part, the observation that IVM conditions might affect mRNA translation. Secondly, to experimentally confirm the role of the EGF network, we monitored AKT phosphorylation during IVM using western blots in bovine oocytes and immunofluorescence in bovine and human oocytes. We observed significant phosphorylation in response to amphiregulin, indicating activation of the pathway. Furthermore, the activation of the PI3K/AKT/mTOR pathway required the physical presence of cumulus cells (CCs). AKT phosphorylation during rescue IVM of denuded human immature oocytes, as predicted by our model, failed to activate, providing a putative explanation for the suboptimal outcomes of rescue IVM in humans. Third, to understand if AKT activation affected translation, we investigated polyadenylation in target transcripts using two approaches. The first method attempted to indirectly catch differences in the reverse transcription efficiency in association with short or long poly(A) tail. The mRNA was split in half and reverse transcribed using random hexamers or oligo(dT). This assay did not highlight differences between immature and mature oocytes or IVM conditions. The second method consisted in directly assaying the poly(A) tail length in target transcripts and revealed significant polyadenylation in genes related to cell cycle progression and spindle stabilization such as AURKA and NEDD1, in amphiregulin-supplemented IVM. Furthermore, this assay allowed to assess that the poly(A) tail in immature oocytes was 100 bp-long, providing an explanation for the inability of observing differences when using random hexamers and oligo(dT). Since the extension of the poly(A) tail is necessary for polysome association and translation, these results confirm that, despite differences in timing and initial tail length, also in higher-order mammals, translation of genes crucial for proper oocyte maturation are regulated by the EGF network, likely through PI3K/AKT/mTOR. This research provides a mechanistical explanation of the empirical observations that hormones/growth factors supplementation and structural integrity of the cumulus-oocyte complex affect the quality of the in vitro matured bovine oocytes.