To investigate the effects of tannins (TA) on porcine oocyte in vitro maturation (IVM), different concentrations of TA (0, 1, 10 and 100 μg/mL) were supplemented with a maturation medium and the COCs and subsequent embryonic development were examined. The results showed that 10 µg/mL TA significantly improved the cumulus expansion index (CEI), cumulus-expansion-related genes (PTGS1, PTGS2, PTX-3, TNFAIP6 and HAS2) expression and blastocyst formation rates after parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) compared to the control groups, but not oocyte nuclear maturation. Nevertheless, 10 µg/mL TA dramatically enhanced the mRNA expression of oocyte-development-related genes (BMP15, GDF9, CDC2 and CYCLIN B1), GSH, ATP, SOD1, PGC1α, BMP15, GDF9 and CDC2 levels and reduced intracellular ROS level in porcine oocytes. These results indicated that porcine oocyte cytoplasmic maturation was improved by 10 µg/mL TA treatment during IVM. In contrast, a high concentration of TA (100 μg/mL) significantly decreased the CEI and PTGS1, PTGS2, PTX-3 and HAS2 mRNA expressions in cumulus cells, and reduced oocyte nuclear maturation and the total cell numbers/blastocyst. In general, these data showed that 10 μg/mL TA supplementation has beneficial effects on oocyte cytoplasmic maturation and subsequent embryonic development in pigs.

Previous studies suggest that the inclusion of melatonin (MTn) in in vitro maturation protocols improves the developmental competence of oocytes by scavenging reactive oxygen species (ROS). However, the molecular mechanisms integrating melatonin receptor (MT)-mediated lipid metabolism and redox signaling during in vitro cumulus–oocyte complex (COC) development still remain unclear. Here, we aimed to elucidate the potential role of MTn receptors in lipid metabolic adjustments during in vitro porcine COC development. We observed that MTn-mediated Gsα–cAMP/PKA signaling facilitated lipolysis primarily through the MT2 receptor and subsequently increased fatty acid (FA) release by hydrolyzing intracellular triglycerides (TGs) in cumulus cells. Furthermore, CD36 was a critical FA transporter that transported available FAs from cumulus cells to oocytes and promoted de novo TG synthesis in the latter. In addition, MTn regulated lipogenesis and intracellular lipolysis to maintain lipid homeostasis and limit ROS production, thereby supporting oocyte cytoplasmic maturation and the subsequent embryo development. Taken together, these findings provide insight into the possible mechanism integrating MT2-mediated lipid homeostasis and redox signaling, which limits ROS production during in vitro COC development. Therefore, understanding the dynamics of the interactions between lipid homeostasis and redox signaling driven by MT2 is necessary in order to predict drug targets and the effects of therapeutics used to improve female reproductive health.

Pluripotent stem cells (PSCs) have unlimited self-renewal and multifunctional development potential in vitro. Porcine PSCs are highly desirable due to the conserved characteristics between pigs and humans. Extended PSCs (EPSCs) are additionally capable of differentiating into embryonic (Em) and extraembryonic (E×Em) parts. Here, we employed the LCDM culture system (consisting of human LIF, CHIR99021, (S) - (+) -dimethindene maleate, and minocycline hydrochloride), which can establish EPSCs from humans and mice, to derive and maintain stable porcine PSCs (pLCDM) from in vivo blastocysts. Transcriptome analysis revealed the unique molecular characteristics of pLCDMs compared with early-stage embryos. Meanwhile, the parallels and differences in the transcriptome features among pLCDMs, human EPSCs, and mouse EPSCs were carefully analyzed and evaluated. Most noteworthy, the trophoblast lineage differentiation tendency of pLCDMs was clarified by inducing trophoblast-like cells and trophoblast stem cells (TSCs) in vitro. Further research found that two of the small molecules in LCDM culture system, (S) - (+) -dimethindene maleate (DiM) and minocycline hydrochloride (MiH), probably play a crucial role in promoting trophoblast lineage differentiation potential of pLCDMs.

Background Insulin regulates glucose homeostasis and has important effects on metabolism, cell growth, and differentiation. Depending on the cell type and physiological context, insulin signal has specific pathways and biological outcomes in different tissues and cells. For studying the signal pathway of insulin on glycolipid metabolism in porcine embryonic fibroblast (PEF), we used high-throughput sequencing to monitor gene expression patterns regulated by insulin. Objectives The goal of our research was to see how insulin affected glucose and lipid metabolism in PEFs. Methods We cultured the PEFs with the addition of insulin and sampled them at 0, 48, and 72 h for RNA-Seq analysis in triplicate for each time point. Results At 48 and 72 h, 801 and 1,176 genes were differentially expressed, respectively. Of these, 272 up-regulated genes and 264 down-regulated genes were common to both time points. Gene Ontology analysis was used to annotate the functions of the differentially expressed genes (DEGs), the biological processes related to lipid metabolism and cell cycle were dominant. And the DEGs were significantly enriched in interleukin-17 signaling pathway, phosphatidylinositol-3-kinase-protein kinase B signaling pathway, pyruvate metabolism, and others pathways related to lipid metabolism by Kyoto Encyclopedia of Genes and Genomes enrichment analysis. Conclusions These results elucidate the transcriptomic response to insulin in PEF. The genes and pathways involved in the transcriptome mechanisms provide useful information for further research into the complicated molecular processes of insulin in PEF.

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Background: The importance of the processes of lipogenesis and lipolysis in providing an essential energy source during oocyte maturation is increasingly being recognized. Recent our studies have demonstrated that melatonin up-regulated lipid metabolism during oocyte maturation. Nevertheless, there is still limited information regarding the underlying molecular mechanisms of action of melatonin on lipid metabolism in porcine cumulus-oocyte complexes (COCs). Here, our aim was to investigate the effect of melatonin on COCs, and the melatonin receptor-mediated lipid metabolism signaling pathway.Materials/methods: To determine the melatonin-mediated lipolysis pathway in cumulus cells, COCs were treated with melatonin and the correlated metabolic responses were assessed using melatonin receptor-mediated signaling.Results: The results showed that exposure of COCs to melatonin during in vitro maturation significantly increased cumulus expansion index, blastocyst formation rate and total cell numbers/blastocyst, although nuclear maturation was no significant difference. The levels of proteins MT1, MT2, Gsα, PKA, and lipolysis-related factors (AGTL, HSL, PLIN A+B) were significantly increased by melatonin supplementation, and this effect was inhibited by simultaneous treatment with melatonin antagonists (luzindole or 4P-PDOT), although 4P-PDOT treatment did not completely block the effect of melatonin on MT1. Further, the gene expression patterns reflected their relevant protein levels in cumulus cells. Melatonin-mediated lipolysis could significantly reduce lipid droplets (LDs) numbers and increase fatty acid (FA) production and ATP levels by increasing the β-oxidation-related gene expression in cumulus cells. Simultaneously, melatonin significantly increased the amount of LDs, FAs, ATP, and enhanced the lipid metabolism-related gene expression in oocytes. Finally, the oocyte quality was improved by increasing GDF9, BMP15 and GSH and decreasing ROS levels.Conclusion: These findings revealed that the MT2-mediated cAMP/PKA signaling pathway promotes intracellular lipolysis and FA production in cumulus cells, which provided an essential energy source for COCs development.

Simultaneously, multiplexed genome engineering and targeting multiple genomic loci are valuable to elucidating gene interactions and characterizing genetic networks that affect phenotypes. Here, we developed a general CRISPR-based platform to perform four functions and target multiple genome loci encoded in a single transcript. To establish multiple functions for multiple loci targets, we fused four RNA hairpins, MS2, PP7, com and boxB, to stem–loops of gRNA (guide RNA) scaffolds, separately. The RNA-hairpin-binding domains MCP, PCP, Com and λN22 were fused with different functional effectors. These paired combinations of cognate-RNA hairpins and RNA-binding proteins generated the simultaneous, independent regulation of multiple target genes. To ensure that all proteins and RNAs are expressed in one transcript, multiple gRNAs were constructed in a tandemly arrayed tRNA (transfer RNA)-gRNA architecture, and the triplex sequence was cloned between the protein-coding sequences and the tRNA-gRNA array. By leveraging this system, we illustrate the transcriptional activation, transcriptional repression, DNA methylation and DNA demethylation of endogenous targets using up to 16 individual CRISPR gRNAs delivered on a single transcript. This system provides a powerful platform to investigate synthetic biology questions and engineer complex-phenotype medical applications.