ABSTRACT
Porcine embryos are used for a variety of applications. However, the maturation rate in vitro remains low, and novel in vitro maturation (IVM) techniques that facilitate the collection of mature oocytes are necessary. C-C motif chemokine ligand 2 (CCL2) is a key periovulatory chemokine present in cumulus-oocyte complexes (COCs). We aimed to examine the effects of CCL2 supplementation during IVM on oocyte maturation and embryonic development. The CCL2 concentration was significantly higher in porcine follicular fluid (pFF) derived from follicles >8 mm in size than in pFF derived from smaller follicles. There was a significant increase in CCL2 mRNA levels in all follicular cells after IVM compared with that before IVM. We analyzed the localization of CCL2 and its receptor, the CCL2 receptor, in follicular cells. During IVM, different concentrations of CCL2 were added to COCs cultured in a maturation medium. After IVM, the group treated with 100 ng/mL CCL2 showed significantly higher metaphase II rates than the control group. All CCL2-treatment groups showed a significant increase in intracellular glutathione levels and a significant decrease in reactive oxygen species levels, compared to the control. In CCs treated with 100 ng/mL CCL2, the mRNA levels of BAX, CASP3, and NPR2 were significantly decreased. Furthermore, the mRNA levels of SOD1, SOD2, and CD44 were significantly increased. In oocytes treated with 10 ng/mL CCL2, mRNA levels of BAX and CASP3 were significantly decreased, whereas, NRF2 and NPM2 were significantly increased. ERK1 exhibited significantly increased mRNA expression in both CCs and oocytes treated with 10 ng/mL CCL2. The protein expression ratio of phosphorylated ERK1/2 to total ERK1/2 was significantly increased in CCs treated with 10 ng/mL CCL2. After parthenogenetic activation, cleavage rates were significantly improved in the 100 ng/mL CCL2 treatment group, and blastocyst formation rates were significantly enhanced in the 10 ng/mL CCL2 treatment group. Overall, our results suggest that IVM medium along with CCL2 improves porcine oocyte maturation and the development of parthenogenetically-activated embryos.
ABSTRACT
Background@#Ergothioneine (EGT) is a natural amino acid derivative in various animal organs and is a bioactive compound recognized as a food and medicine. @*Objectives@#This study examined the effects of EGT supplementation during the in vitro maturation (IVM) period on porcine oocyte maturation and subsequent embryonic development competence after in vitro fertilization (IVF). @*Methods@#Each EGT concentration (0, 10, 50, and 100 µM) was supplemented in the maturation medium during IVM. After IVM, nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels of oocytes were investigated. In addition, the genes related to cumulus function and antioxidant pathways in oocytes or cumulus cells were investigated. Finally, this study examined whether EGT could affect embryonic development after IVF. @*Results@#After IVM, the EGT supplementation group showed significantly higher intracellular GSH levels and significantly lower intracellular ROS levels than the control group. Moreover, the expression levels of hyaluronan synthase 2 and Connexin 43 were significantly higher in the 10 µM EGT group than in the control group. The expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and NAD(P)H quinone dehydrogenase 1 (NQO1) were significantly higher in the oocytes of the 10 µM EGT group than in the control group. In the assessment of subsequent embryonic development after IVF, the 10 µM EGT treatment group improved the cleavage and blastocyst rate significantly than the control group. @*Conclusions@#Supplementation of EGT improved oocyte maturation and embryonic development by reducing oxidative stress in IVM oocytes.