170 Effect of Folic Acid Supplementation on In Vitro Maturation of Oocytes and Folate Cycle

Abstract

Folic acid (vitamin B₉) is the principal component of one-carbon (C1) metabolism in mammalian cells. It acts as a coenzyme in C1 unit transfer during nucleotide and amino acid metabolism. Therefore, it is unquestionably essential for the formation of thymidylate (TMP) for DNA synthesis and methylation, which is in turn crucial for post-implantation embryonic brain and nerve chord development. However, the role of folic acid in maturation of oocytes and subsequent blastocyst production is largely ambiguous in different mammalian species. The aim of this study was to investigate the effect of folic acid supplementation on expression of oocyte maturation markers and folate cycle enzymes in cumulus cells in addition to blastocyst development rate in goat. Immature oocytes were isolated from ovaries, and 1600 oocytes were matured in maturation medium (medium-199) supplemented with 0 (control), 50, 100, or 150 µM folic acid (Sigma Aldrich, India), comprising total 400 oocytes in each group in 5 replicates. The oocytes were in vitro fertilized and embryos were cultured for 7 days. Blastocyst rate was calculated in all 4 groups. Cumulus cells from matured oocytes from each group were analysed for the differential expression of oocyte maturation markers: PTX3, PTGS2, CTSB, and CTSS, and folate cycle enzyme transcripts MTR, MAT2A, ACHY, DHFR, and SLC19A1, by qRT-PCR. For statistical analysis, one-way ANOVA was used. The results of present study show that blastocyst rate was significantly higher (P ≤ 0.05) at 50 µM folic acid (21.4 ± 1.2%) compared with control (13.6 ± 0.4%), 100 µM (10.4 ± 0.34%), and 150 µM (7.4 ± 0.54%). However, there was no effect on the number of cleaved embryos among control (72 ± 0.21%), 50 µM (70.6 ± 0.58%), 100 µM (73.75 ± 0.31%), and 150 µM (73.02 ± 0.04%). The expression of the oocyte maturation markers PTX3 and PTGS2 was up-regulated, whereas that of CTSS and CTSSB was down-regulated (P ≤ 0.05), in the 50 µM group, but did not change (P ≤ 0.05) among the other groups. The expression of these genes did not change significantly among the other groups. The expression of genes of the folate cycle, MTR, MAT2A, ACHY, and DHFR, increased (P ≤ 0.05) by 5.7-, 3.6-, 2.2-, and 1.7-fold, respectively, at 50 µM, but did not change (P ≤ 0.05) among the other groups. The expression of SLC19A1 did not change significantly among all the groups. The results of present study show that the supplementation of 50 µM folic acid to the maturation medium results in increased maturation of oocytes, making them more competent for the blastocyst development. Up-regulation of transcripts of folate cycle enzymes at 50 µM might explain the increase in the blastocyst production rate, which further needs to be validated.