Follicular fluid exosome-derived miR-339-5p enhances in vitro maturation of porcine oocytes via targeting SFPQ, a regulator of the ERK1/2 pathway.

In this study, we aimed to investigate cytoplasmic maturation and miRNA expression of mature oocytes cultured in porcine follicular fluid exosomes. We also examined the effect of miR-339-5p on oocyte maturation. Twenty eight differentially expressed miRNAs were detected using miRNA-seq. We then transfected cumulus oocyte complexes with miR-339-5p mimics and inhibitor during culture. The results showed that exosomes increased endoplasmic reticulum levels and the amount of lipid droplets, and decreased ROS levels, lipid droplet size, and percentage of oocytes with abnormal cortical granule distribution. Overexpressing miR-339-5p significantly decreased cumulus expansion genes, oocyte maturation-related genes, target gene proline/glutamine-rich splicing factor (SFPQ), ERK1/2 phosphorylation levels, oocyte maturation rate, blastocyst rate, and lipid droplet number, but increased lipid droplet size and the ratio of oocytes with abnormal cortical granule distribution. Inhibiting miR-339-5p reversed the decrease observed during overexpression. Mitochondrial membrane potential and ROS levels did not differ significantly between groups. In summary, exosomes promote oocyte cytoplasmic maturation and miR-339-5p regulating ERK1/2 activity through SFPQ expression, thereby elevating oocyte maturation and blastocyst formation rate in vitro.

Effects of follicular fluid exosomes on in vitro maturation of porcine oocytes.

Exosomes are related to effective communication between cells. In this study we aimed to investigate the effect of porcine follicular fluid exosomes (FF-Exo) on cumulus expansion, oocyte mitochondrial membrane potential, and maturation in in vitro culture. We used different concentrations of FF-Exo (Exo-0, Exo-1, Exo-10, Exo-20, and Exo-40) and added them to an oocyte maturation medium. Transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blot (WB) showed that the isolated samples were exosomes. Immunofluorescence showed that exosomes could be taken up by cumulus cells. Compared with the Exo-0 group, there was no significant difference in oocyte maturation rate in the Exo-1 group (p > 0.05), while the Exo-10 group (p < 0.05), Exo-20 group (p < 0.01) and Exo-40 group (p < 0.01) significantly increased. The maturation rate of the Exo-20 and Exo-40 groups was the highest, and there was no significant difference between the two groups (p > 0.05). However, different concentrations of treatment could not effectively induce cumulus expansion and the results of JC1 showed that it had no significant effect on mitochondrial membrane potential (p > 0.05). In conclusion, the results suggest that porcine FF-Exo are involved in oocyte nuclear maturation.

Inhibition of 26S proteasome enhances AKAP3-mediated cAMP-PKA signaling during boar sperm capacitation.

This study investigates the effects of the ubiquitin-proteasome pathway (UPP) on porcine sperm capacitation and its interactions with the cAMP-PKA pathway. The semen of adult Landrace boars was divided into four groups: non-capacitated, capacitated, 10 µM/mL MG132, and 10 µM/mL DMSO groups. We characterized the parameters related to sperm dynamics using a computer-assisted sperm analysis system. The level of sperm protein tyrosine phosphorylation was detected using Western blotting, and the change of zinc ion signal was detected via flow cytometry. The relationship between A-kinase-anchor protein 3 (AKAP3), ubiquitin (Ub), and protein kinase A (PKA) was assessed by co-precipitation assays; to evaluate the interactions between the UPP and cAMP-PKA pathway, threonine, serine, and tyrosine phosphorylation were detected using Western blotting to evaluate the interaction between the UPP and cAMP-PKA pathway; Hoechst staining was used to detect the sperm-egg binding state and evaluate the effects of UPP inhibition. During capacitation, the levels of protein tyrosine, serine, and threonine phosphorylation and ubiquitination of porcine sperm increased, and sperm-egg binding was inhibited (P < 0.05). AKAP3 was degraded by UPP, and after inhibiting the 26 S proteasome, ubiquitinated AKAP3 accumulated in large quantities. Our findings indicate that, after the 26 S proteasome was inhibited, PKA was uncoupled from AKAP3 and degraded by UPP; the level of tyrosine phosphorylation induced by PKA-AKAP3 was reduced, the level of serine threonine phosphorylation increased, and the ubiquitination pathway interacted with the phosphorylation pathway and was involved in sperm capacitation.

MicroRNA-101 regulates oocyte maturation in vitro via targeting HAS2 in porcine cumulus cells.

RNA-seq technology can be used for the detection of miRNA transcripts in tissues and cells at specific periods and under specific treatment conditions, which can easily and effectively screen out differential transcripts. The purpose of this study was to compare miRNA expression in porcine cumulus cells before and after oocyte maturation, and to investigate the mechanism whereby cumulus cells may influence oocyte maturation. To that aim, cumulus cells surrounding GV- and MII-stage oocytes were isolated, and their differences in miRNA expression were examined using miRNA-seq. 143 differentially expressed miRNAs were identified, among which miR-101 was selected and further verified by qPCR. Moreover, miR-101 was found to target HAS2 through target gene prediction, luciferase-based co-transfection and cumulus cells transfection. Transfection of COCs with miR-101 mimics or inhibitor revealed that the miR-101 could inhibit oocyte IVM by regulating the expanding of CCSs, but had no effect on the embryo development competence. These findings demonstrated that miR-101 regulated oocyte maturation in vitro via targeting HAS2 in porcine cumulus cells.

Comparative proteomic identification of capacitated and non-capacitated sperm of Yanbian Yellow Cattle.

To investigate the proteomic differences between the capacitated and non-capacitated sperm of the Yanbian yellow cattle, semen was collected from three Yanbian yellow cattle, aged 3-5 years, pooled, and divided into capacitated and non-capacitated groups (n = 9) to be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-mass spectrometry. Gene ontology enrichment, Kyoto Encyclopedia of Genes and Genomes pathway enrichment, and protein-protein interactions were used for bioinformatics analysis. The results showed that 598 were described as differentially expressed proteins during sperm capacitation. In the capacitated sperm, 89 proteins were up-regulated, and 509 proteins were downregulated, compared to the non-capacitated sperm. Western blot analysis confirmed that the expression of the Heat Shock 70-kDa Protein 5 (HSPA5) proteins was down-regulated. After HSPA5 inhibition, the level of tyrosine phosphorylation decreased, and the cleaved caspase 3 increased. The results showed that HSPA5 could regulate sperm capacitation and slow down apoptosis. Thus, providing a basis for studying the changes in protein expression during sperm capacitation, which can help identify the marker proteins potentially related to sperm capacitation. Hence, this study can deepen our understanding of the molecular processes involved in the sperm capacitation in Yanbian yellow cattle and provide a framework for future research.