3-methyl-4-nitrophenol (PNMC), a well-known constituent of diesel exhaust particles and degradation products of insecticide fenitrothion, is a widely distributed environmental contaminant. PNMC is toxic to the female reproductive system; however, how it affects meiosis progression in oocytes is unknown. In this study, in vitro maturation of mouse oocytes was applied to investigate the deleterious effects of PNMC. We found that exposure to PNMC significantly compromised oocyte maturation. PNMC disturbed the spindle stability; specifically, it decreased the spindle density and increased the spindle length. The weakened spindle pole location of microtubule-severing enzyme Fignl1 may result in a defective spindle apparatus in PNMC-exposed oocytes. PNMC exposure induced significant mitochondrial dysfunction, including mitochondria distribution, ATP production, mitochondrial membrane potential, and ROS accumulation. The mRNA levels of the mitochondria-related genes were also significantly impaired. Finally, the above-mentioned alterations triggered early apoptosis in the oocytes. In conclusion, PNMC exposure affected oocyte maturation and quality through the regulation of spindle stability and mitochondrial function.

The ten-eleven translocation (TET) enzyme family, which includes TET1/2/3, participates in active DNA demethylation in the eukaryotic genome; moreover, TET1/2/3 are functionally redundant in mice embryos. However, the combined effect of TET1/2/3 triple-gene knockdown or knockout on the porcine oocytes or embryos is still unclear. In this study, using Bobcat339, a specific small-molecule inhibitor of the TET family, we explored the effects of TET enzymes on oocyte maturation and early embryogenesis in pigs. Our results revealed that Bobcat339 treatment blocked porcine oocyte maturation and triggered early apoptosis. Furthermore, in the Bobcat339-treated oocytes, spindle architecture and chromosome alignment were disrupted, probably due to the huge loss of 5-hydroxymethylcytosine (5hmC)and concurrent increase in 5-methylcytosine (5mC). After Bobcat339 treatment, early parthenogenetic embryos exhibited abnormal 5mC and 5hmC levels, which resulted in compromised cleavage and blastocyst rate. The mRNA levels of EIF1A and DPPA2 (ZGA marker genes) were significantly decreased, which may explain why the embryos were arrested at the 4-cell stage after Bobcat339 treatment. In addition, the mRNA levels of pluripotency-related genes OCT4 and NANOG were declined after Bobcat339 treatment. RNA sequencing analysis revealed differentially expressed genes in Bobcat339-treated embryos at the 4-cell stage, which were significantly enriched in cell proliferation, cell component related to mitochondrion, and cell adhesion molecule binding. Our results indicated that TET proteins are essential for porcine oocyte maturation and early embryogenesis, and they act by mediating 5mC/5hmC levels and gene transcription.

Fat deposition plays a key role in determining porcine meat quality traits, with lipid droplets serving as critical organelles for lipid storage in adipose tissue. Inhibiting lipid droplet biogenesis disrupts the lipid storage capacity of adipocytes. The Stearoyl-CoA Desaturase (SCD) family is crucial in regulating polyunsaturated fatty acid/monounsaturated fatty acid (PUFA/MUFA) composition, while its role in lipid droplet formation remains unclear. This study employed CRISPR/Cas9 to create SCD1-deficient porcine renal epithelial cells (PK15), enabling an investigation into SCD1’s role in fatty acid composition and lipid droplet regulation. RNA-seq analysis was conducted to elucidate the mechanisms underlying SCD1’s impact on lipid droplet numbers. Results showed that SCD1 deletion significantly decreased triacylglycerols (TAG) content, altered fatty acid composition, and decreased lipid droplet numbers. Conversely, SCD1 overexpression increased lipid droplet numbers, confirming SCD1’s role in regulating lipid droplet abundance. RNA-seq analysis revealed that SCD1 regulates lipid metabolism via Calsyntenin 3β (CLSTN3B). Experimental validation confirmed the SCD1-CLSTN3B regulation of lipid droplet numbers. In summary, we discovered the role of SCD1 in regulating the number of lipid droplets, highlighting its potential impact on lipid metabolism and adipocyte function in pigs.

N6-methyladenosine (m⁶A) is the most common modification in eukaryotic RNAs. Growing research indicates that m⁶A methylation is crucial for a multitude of biological processes. However, research on the m⁶A modifications in the regulation of porcine muscle growth is lacking. In this study, we identified differentially expressed genes in the neonatal period of muscle development between Large White (LW) and NingXiang (NX) pigs and further reported m⁶A methylation patterns via MeRIP-seq. We found that m⁶A modification regulates muscle cell development, myofibrils, cell cycle, and phosphatase regulator activity during the neonatal phase of muscle development. Interestingly, differentially expressed genes in LW and NX pigs were mainly enriched in pathways involved in protein synthesis. Furthermore, we performed a conjoint analysis of MeRIP-seq and RNA-seq data and identified 27 differentially expressed and m⁶A-modified genes. Notably, a typical muscle-specific envelope transmembrane protein, WFS1, was differentially regulated by m⁶A modifications in LW and NX pigs. We further revealed that the m⁶A modification accelerated the degradation of WFS1 in a YTHDF2-dependent manner. Noteworthy, we identified a single nucleotide polymorphism (C21551T) within the last exon of WFS1 that resulted in variable m⁶A methylation, contributing to the differing WFS1 expression levels observed in LW and NX pigs. Our study conducted a comprehensive analysis of the m⁶A modification on NX and LW pigs during the neonatal period of muscle development, and elucidated the mechanism by which m⁶A regulates the differential expression of WFS1 in the two breeds.