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 G_sα–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.

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.

This study aimed to determine the underlying mechanism of ramelteon on the competence of oocyte and subsequent embryo development in pigs during in vitro maturation (IVM). Our results showed that the cumulus expansion index was significantly lower in the control group compared to the ramelteon groups (p < 0.05). Moreover, supplementation of 10⁻¹¹ and 10⁻⁹ M ramelteon significantly increased the cumulus expansion and development-related genes expression, and reduced apoptosis in cumulus cells (p < 0.05). In oocytes, the nuclear maturation rate was significantly improved in 10⁻¹¹, 10⁻⁹, and 10⁻⁷ M ramelteon groups compared to the control (p < 0.05). Additionally, the level of intracellular GSH was significantly increased and ROS was significantly decreased in ramelteon-supplemented groups, and the gene expression of oocyte development and apoptosis were significantly up- and down-regulated by 10⁻¹¹ and 10⁻⁹ M ramelteon (p < 0.05), respectively. The immunofluorescence results showed that the protein levels of GDF9, BMP15, SOD1, CDK1, and PGC1α were significantly increased by 10⁻¹¹ M ramelteon compared to the control (p < 0.05). Although there was no significant difference in cleavage rate, the blastocyst formation rate, total cell numbers, and hatching/-ed rate were significantly improved in 10⁻¹¹ M ramelteon group compared to the control (p < 0.05). Furthermore, embryo development, hatching, and mitochondrial biogenesis-related genes were dramatically up-regulated by 10⁻¹¹ M ramelteon (p < 0.05). In addition, the activities of lipogenesis and lipolysis in oocytes were dramatically increased by 10⁻¹¹ M ramelteon compared to the control (p < 0.05). In conclusion, supplementation of 10⁻¹¹ M ramelteon during IVM improved the oocyte maturation and subsequent embryo development by reducing oxidative stress and maintenance of lipid homeostasis.

Tannin (TA) improves porcine oocyte cytoplasmic maturation and subsequent embryonic development after in vitro fertilization (IVF). However, the mechanism through which TA blocks polyspermy after IVF remains unclear. Hence, the biological function of organelles (cortical granule [CG], Golgi apparatus, endoplasmic reticulum [ER], and mitochondria) and the incidence of polyspermic penetration were examined. We found no significant difference in oocyte nuclear maturation among the 1 µg/mL, 10 µg/mL TA, and control groups. Moreover, 100 μg/mL TA significantly reduced 1st polar body formation rate compared to the other groups. Additionally, 1 and 10 μg/mL TA significantly increased the protein levels of GDF9, BMP15, and CDK1 compared to the control and 100 μg/mL TA groups. Interestingly, 1 and 10 μg/mL TA improved the normal distribution of CGs, Golgi, ER, and mitochondria by upregulating organelle-related gene expression and downregulating ER stress (CHOP) gene expression. Simultaneously, 1 and 10 μg/mL TA significantly increased the proportion of normal fertilized oocytes (2 pronuclei; 2 PN) and blastocyst formation rate compared to the control, as well as that of 100 μg/mL TA after IVF by upregulating polyspermy-related genes. In conclusion, TA during IVM enhances 2PN and blastocyst formation rates by regulating organelles’ functions and activities.

Zygotic genome activation (ZGA) is a pivotal event during early mammalian embryonic development and is regulated by multiple molecular factors. In mice, previous studies have demonstrated that long non-coding RNAs (lncRNAs) and transposable elements (TEs) participate in the regulation of this process. However, the functional roles of TEs-associated lncRNAs in porcine ZGA remain largely unknown. In this study, we identified a previously unannotated lncRNA associated with a SINE transposable element sequence in porcine parthenogenetically activated embryos, which we named LincSAKP. LincSAKP exhibited peak expression at the 8-cell stage, coinciding with ZGA, was predominantly localized in the nucleus, and lacked protein-coding potential. Functional analyses demonstrated that depletion of LincSAKP resulted in developmental arrest at the 8-cell stage, accompanied by reduced chromatin accessibility, impaired nascent transcription, and aberrant expression of multiple ZGA marker genes and transcription factors. Transcriptomic analysis combined with rescue experiments identified EP300 as a critical downstream target of LincSAKP. Notably, Depletion of either LincSAKP or EP300 impairs histone H3K27ac modification. EP300 overexpression effectively alleviated the developmental defects caused by LincSAKP depletion. Collectively, our findings identify EP300 as a downstream mediator of LincSAKP in regulating zygotic genome activation, providing insights into the functional network of SINE-associated lncRNAs during parthenogenetically activated early embryonic development. This study expands our understanding of the functional roles of TE-associated non-coding RNAs in early embryonic development and provides a potential theoretical basis for improving the efficiency of in vitro culture of porcine embryos.