Leptin and nonessential amino acids enhance porcine preimplantation embryo development in vitro by intracytoplasmic sperm injection

Abstract

Intracytoplasmic sperm injection (ICSI) has been considered one of the strong assisted reproductive technologies for producing transgenic animals as well as treating infertility in animals and humans. However, in porcine ICSI, embryos produced by in vitro methods show low pregnancy rates with high abnormal offspring and blastocyst formation rate as well as quality are poor compared with those in other species. For these reasons, developing a protocol for porcine ICSI is essential to efficiently generate transgenic pigs. Since amino acids were introduced to embryo development because of their beneficial effects, many embryologists have been using nonessential amino acid (NEAA) in culture medium for embryonic development in pig and other species. Leptin also has been shown to be beneficial in embryonic development for increasing rate of cleavage and blastocyst development. However, the effects of NEAA and leptin were not fully understood in the development of porcine ICSI-derived embryos. Here we investigated the optimization of NEAA and leptin supplementation in culture medium to improve developmental competence and quality of preimplantation embryos after ICSI in pig. The proportion of embryos that developed to the blastocyst stage was significantly greater when 1% vol/vol NEAA (24.6%) or 100 ng/mL leptin (27.1%) was supplemented in the culture medium compared with other concentrations or no supplement. When NEAA and leptin (24.8%) were supplemented together, blastocyst formation was significantly higher than other single supplementation groups. We also evaluated the effects of different supplementation periods of NEAA or leptin on the preimplantation embryonic development after ICSI. Both NEAA and leptin showed that supplementation for the entire 7 days significantly increased the blastocyst formation rate compared with the other groups of supplementation for the first 4 days and for the subsequent 3 days. A second goal of our research was to evaluate the quality of developed blastocysts after ICSI. The supplementation of 100 ng/mL leptin in culture medium made blastocysts express less of the proapoptosis genes BAX and BAK and more of the antiapoptosis genes BCL-XL and BCL-2 after the ICSI procedure. Furthermore, terminal deoxynucleotidyl transferase dUTP nick end labeling index, fragmentation, and total apoptosis were significantly decreased and the total cell number was significantly increased when the ICSI-derived embryos were cultured to blastocyst stage in the presence of the combination of NEAA and leptin. These results suggest that NEAA and leptin could improve not only the quantity but also quality of ICSI-derived porcine embryos during in vitro culture with the optimal concentration of each reagent.