The developmental potential of porcine oocytes cultured in vitro was remarkably enhanced in a medium containing FGF2, LIF and IGF1 (FLI) when compared to a medium supplemented with gonadotropins and EGF (control). We analyzed the molecular background of the enhanced oocyte quality by comparing the time course of MAPK3/1 and AKT activation, and the expression of genes controlled by these kinases in cumulus-oocyte complexes (COCs) cultured in FLI and the control medium. The pattern of MAPK3/1 activation in COCs was very similar in both media, except for a robust increase in MAPK3/1 phosphorylation during the first hour of culture in the FLI medium. The COCs cultured in the FLI medium exhibited significantly higher activity of AKT than in the control medium from the beginning up to 16 h of culture; afterwards a deregulation of AKT activity occurred in the FLI medium, which was not observed in the control medium. The expression of cumulus cell genes controlled by both kinases was also modulated in the FLI medium, and in particular the genes related to cumulus-expansion, signaling, apoptosis, antioxidants, cell-to-cell communication, proliferation, and translation were significantly overexpressed. Collectively, these data indicate that both MAPK3/1 and AKT are implicated in the enhanced quality of oocytes cultured in FLI medium.

Background: Ovarian follicular fluids (FFs) contain several kinds of regulatory factors that maintain a suitable microenvironment for oocyte development. Extracellular vesicles (EVs) are among the factors that play essential roles in regulating follicle and oocyte development through their cargo molecules that include microRNAs (miRNAs). This study aimed to investigate small-EV (s-EV) miRNAs in porcine FFs and their potential association with oocyte quality. Methods: Individual aspirated oocytes were stained with lissamine green B stain (LB), a vital stain for oocyte quality, and each oocyte was classified as high-quality (unstained; HQ) or low-quality (stained; LQ). FFs corresponding to oocytes were pooled together into HQ and LQ groups. Small-EVs were isolated from FFs, characterized, and their miRNA cargo was identified using the Illumina NovaSeq sequencing platform. Additionally, s-EVs from the HQ and LQ groups were utilized to investigate their effect on oocyte development after co-incubation during in vitro maturation. Results: A total of 19 miRNAs (including miR-125b, miR-193a-5p, and miR-320) were significantly upregulated, while 23 (including miR-9, miR-206, and miR-6516) were downregulated in the HQ compared to the LQ group. Apoptosis, p53 signaling, and cAMP signaling were among the top pathways targeted by the elevated miRNAs in the HQ group while oocyte meiosis, gap junction, and TGF-beta signaling were among the top pathways targeted by the elevated miRNAs in the LQ group. The supplementation of small-EVs during maturation does not affect the oocyte developmental rates. However, LQ s-EVs increase the proportion of oocytes with homogeneous mitochondrial distribution and decrease the proportion of heterogeneous distribution. Conclusion: Our findings indicated that FF-EVs contain different miRNA cargos associated with oocyte quality and could affect the mitochondrial distribution patterns during oocyte maturation.

Summary: Brilliant cresyl blue (BCB) vital labelling is a powerful method for analyzing the quality of porcine cumulus–oocyte complexes. Our aim was to investigate the correlation between the selection of porcine oocytes using BCB labelling and selected intranuclear characteristics of porcine oocytes and parthenotes. Moreover, BCB labelling was correlated with the diameter of the oocyte and the developmental potential of the parthenotes. The following methods were used: BCB labelling, measurement of the diameter of the oocyte, parthenogenetic activation, immunocytochemistry, transmission electron microscopy, enucleation and relative protein concentration (RPC) analysis. We determined that the diameter of the oocytes in the BCB-positive (BCB+) group was significantly larger than in the BCB-negative (BCB−) group. Immediately after oocyte selection according to BCB labelling, we found significant difference in chromatin configuration between the analyzed groups. BCB+ oocytes were significantly better at maturation than BCB− oocytes. BCB+ embryos were significantly more competent at cleaving and in their ability to reach the blastocyst stage than BCB− embryos. Ultrastructural analyses showed that the formation of active nucleoli in the BCB+ group started at the 8-cell stage. Conversely, most BCB− embryos at the 8-cell and 16-cell stages were fragmented. No statistically significant difference in RPC in nucleolus precursor bodies (NPBs) between BCB+ and BCB− oocytes was found. We can conclude that BCB labelling could be suitable for assessing the quality of porcine oocytes. Moreover, the evaluation of RPC indicates that the quantitative content of proteins in NPB is already established in growing oocytes.