关键词: 甜菜碱, 猪, 胚胎, 孤雌激活, m⁶A修饰

Abstract:

This study aimed to investigate the effect of betaine on the in vitro developmental ability of porcine parthenogenetic embryos. Porcine ovaries were collected from a local slaughterhouse. Cumulus-oocyte complexes were aspirated and selected. After in vitro maturation and parthenogenetic activation, the activated embryos were subjected to betaine treatment at a range concentrations (0, 0.25, 0.5, and 1.0 mmol·L^-1) during in vitro culture. The cleavage and blastocyst formation rates were recorded at 48 h and 168 h, respectively, in order to determine the optimal concentration of betaine. Hoechst 33342 staining was conducted to calculate the total cell number of blastocyst. The expression levels of pluripotency-related genes (OCT4, NANOG, and SOX2) in blastocysts were analyzed by RT-qPCR. Reactive oxygen species (ROS) and glutathione (GSH) levels in blastocysts were measured. m⁶A methylation levels in embryos at the 2-cell, 4-cell, 8-cell, morula, and blastocyst stages were measured by immunofluorescence staining. The expressions of genes encoding m⁶A methyltransferases and demethylases were also examined. All the experiments were repeated at least 3 times. The results showed that the blastocyst rates after parthenogenetic activation in the 0.25 and 0.5 mmol·L^-1 betaine groups were significantly higher than those of the control group and 1.0 mmol·L^-1 group (P < 0.05), with the highest blastocyst rate in the 0.5 mmol·L^-1 group. Compared with the control group, the 0.5 mmol·L^-1 treatment group showed a significant increase in the total blastocyst cell number (P < 0.01), upregulated expressions of OCT4, NANOG, and SOX2 associated with pluripotency (P < 0.05), decreased ROS levels (P < 0.01), and increased GSH levels (P < 0.000 1). Following 0.5 mmol·L^-1 betaine exposure, the m⁶A methylation levels and the expressions of m⁶A methyltransferase-related genes (WTAP, METTL3, and METTL14) in embryos at the 8-cell, morula, and blastocyst stages were significantly higher than those in the control group (P < 0.05). However, the expressions of m⁶A demethylase-related genes (ALKBH5 and FTO) were not significantly affected. In conclusion, enriching the in vitro culture medium with 0.5 mmol·L^-1 betaine improved the in vitro developmental competence and embryonic quality of porcine parthenogenetic embryos. The protective effects of betaine on embryonic development may be attained through the combine mechanisms of its anti-oxidative capacity and enhancement of m⁶A modification ability.

Key words: betaine, porcine, embryo, parthenogenetic, m⁶A modification