Under endoplasmic reticulum (ER)‐stress conditions, the unfolded protein response (UPR) generates a defense mechanism in mammalian cells. The regulation of UPR signaling is important in oocyte maturation, embryo development, and female reproduction of pigs. Recent studies have shown that melatonin plays an important role as an antioxidant to improve pig oocyte maturation. However, there is no report on the role of melatonin in the regulation of UPR signaling and ER‐stress during in vitro maturation (IVM) of porcine oocytes. Therefore, the objective of this study was to investigate the antioxidative effects of melatonin on porcine oocyte maturation through the regulation of ER‐stress and UPR signaling. We investigated the changes in the mRNA/protein expression levels of three UPR signal genes (Bip/Grp78, ATF4, P90/50ATF6, sXbp1, and CHOP) on oocytes, cumulus cells, and cumulus‐oocyte complexes (COCs) during IVM (metaphase I; 22 hours and metaphase II; 44 hours) by Western blot and reverse transcription‐polymerase chain reaction analysis. Treatment with the ER‐stress inducer, tunicamycin (Tm), significantly increased expression of UPR markers. Additionally, cumulus cell expansion and meiotic maturation of oocytes were reduced in COCs of Tm‐treated groups (1, 5, and 10 μg/mL). We confirmed the reducing effects of melatonin (0.1 μmol/L) on ER‐stress after pretreatment with Tm (5 μg/mL; 22 hours) in maturing COCs. Addition of melatonin (0.1 μmol/L) to Tm‐pretreated COCs recovered meiotic maturation rates and expression of most UPR markers. In conclusion, we confirmed a role for melatonin in the modulation of UPR signal pathways and reducing ER‐stress during IVM of porcine oocytes.
Mito-TEMPO is a well-known mitochondria-specific superoxide scavenger. However, the effect of Mito-TEMPO on porcine embryo development, to our knowledge, has not been studied yet. In the present study, porcine embryos were classified into two groups (G1 and G2) based on the cytoplasm lipid contents at the zygote stage. The development of blastocysts derived from G2 zygotes was reduced (G2:16.2 ± 7.9% vs G1: 26.5 ± 5.9%; 1.6-fold, p < 0.05) compared to those from G1 zygotes. In G2 embryos, the proportion of TUNEL-positive cells was also higher than that of G1 embryos. Superoxide in G2 embryos was significantly increased compared to that in G1 embryos. Mitochondrial membrane potential and ATP production were lower in G2 embryos than in G1 embryos. Phosphorylation of Drp1 at Ser 616 increased in G1 embryos during the cleavage stages compared to that in the zygote but was not significantly different in G2 embryos. Then, the effects of Mito-TEMPO were investigated in G2 embryos. Blastocyst formation rate (G2: 19.1 ± 5.1% vs G2 + Mito-TEMPO: 28.8 ± 4.0%; 1.5-fold, p < 0.05) and mitochondrial aggregation were recovered after superoxide reduction by Mito-TEMPO treatment. Thus, we showed that Mito-TEMPO improves blastocyst development by superoxide reduction in porcine embryos in vitro.
Bisphenol A (BPA) is synthetic organic compound that exhibits estrogen-like properties and it induces mitochondrial superoxide production. Melatonin (Mela) protects against BPA-mediated cell damage and apoptosis. However, the antioxidative effects of Mela against BPA-induced superoxide production in porcine oocytes are still not known. In this study, we investigated the antioxidative effects of Mela against BPA-derived superoxide on oocyte maturation in pigs. To investigate the effects of the superoxide specific scavenger, Mito-TEMPO, on porcine oocyte maturation in response to BPA exposure apoptosis proteins, we treated the oocytes with Mito-TEMPO (0.1 µM) after pre-treating them with BPA (75 µM) for 22 h. As expected, the reduction in meiotic maturation and cumulus cell expansion of cumulus-oocyte-complexes (COCs) in the BPA (75 µM) treated group was recovered (p < 0.01) by treatment with Mito-TEMPO (0.1 µM). An increase in the levels of mitochondrial apoptotic proteins (AIF, cleaved Cas 3 and cleaved Parp1) in response to BPA-induced damage was also reduced by Mito-TEMPO treatment in porcine COCs. Interestingly, we confirmed the positive effects of Mela with respect to superoxide production upon BPA exposure during oocyte maturation and also confirmed the reduction in mitochondrial apoptosis in Mela (0.1 µM)-treated porcine COCs. These results provide evidence for the first time that antioxidative effects of Mela on BPA-derived superoxide improve porcine oocyte maturation.
Gangliosides are components of the mammalian plasma membrane that help regulate receptor signaling. Ganglioside GM3, for example, plays an important role in initiating apoptosis in cancer cells; however, physiological roles for GM3 in normal processes, such as during pig oocyte maturation, are not clear. The aim of this study was to investigate the functional link between GM3 and cellular apoptosis in porcine cumulus-oocyte-complexes (COCs) during in vitro maturation. Our results indicated that denuded oocytes possess less ST3GAL5, a GM3-synthesizing enzyme, than cumulus cells or COCs after 44 hr of in vitro maturation. GM3 also affected the meiotic maturation of cultured pig oocytes, as evaluated by orcein staining. In vitro treatment of COCs with exogenous GM3 also reduced cumulus cell expansion, the proportion of meiotic maturation, and increased cumulus cell transcription of PTX3, TNFAIP6, and HAS2. Interestingly, GM3 treatment reduced the expression of Epidermal growth factor receptor (EGFR)-mediated Phosphoinositide 3-kinase/AKT signaling proteins in COCs in a concentration-dependent manner, instead increasing the abundance of pro-apoptotic factors such as AIF, activated Caspase 9, cleaved PARP1, and Caspase 3 were. Thus, GM3 might affect porcine oocyte maturation via suppression of EGFR-mediated PI3K/AKT signaling and/or induction of apoptosis during in vitro maturation.
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