84 Folate-methionine cycle and folate transport in developing buffalo embryos

Abstract

Periconceptional folic acid is known to have a major role in the prevention of neural tube defects, leading to global recommendations for folic acid supplementation before and in early pregnancy. Maternal folate throughout pregnancy may have other roles in offspring health, including neurodevelopment and cognitive performance in childhood. Folate and folic acid (vitamin B₉) act as a co-enzyme essential for single carbon metabolism, a network of pathways involved in several biological processes including nucleotide synthesis, DNA repair, and methylation reactions. In general, rapidly growing and multiplying cells require an adequate supply of folate. A primary deficiency of natural folate resulting in an increase of the total homocysteine concentration may be detrimental to the quality of the oocyte, subsequent fertilisation, embryogenesis, implantation, and fetal development. However, to date, folate-methionine metabolism and folate transport have not been studied in developing buffalo embryos. The present study details transcript expression for genes encoding key enzymes in the linked folate-methionine cycles in the ovary tissue, cumulus cells, immature oocytes, IVM oocytes, and pre-implantation embryos and also estimates the folate concentration in follicular fluid (FF) of buffalo. The FF was pooled and collected by aspiration of different sizes of surface follicles (2-8 mm diameter). The total number of analysed samples was three, with different dilutions and estimation of folate in FF of buffalo done by chemiluminescence assay. Total RNA was extracted from oocytes, cumulus cells, ovarian tissue, and embryos produced from IVF. RT-PCR was performed to analyse the expression of folate-methionine cycle enzymes and folate transporters. Transcripts for all the enzymes of the folate-methionine cycle (i.e. SHMT, MTR, MTRR, MAT1A, MAT2B, GNMT, AHCY, CBS, and DHFR) and folate transporters (FOLR1, FOLR2) and reduced folate carrier (SLC19A1) were expressed in ovarian tissue, cumulus cells, oocytes, and pre-implantation embryos. Immunocytochemical analysis revealed FOLR2 and SLC19A1 protein expression on the plasma membrane and/or cytoplasm of the oocytes and embryos, and FOLR1 in the nucleus of pre-implantation embryos. The folate concentration in FF was 24 ng mL⁻¹. This is the first report to examine the concentration of folate in FF and revealed the identification of transcripts in different samples of buffalo species. The presence of these enzymes could have a profound effect on single-carbon metabolism within the ovary and pre-implantation embryo, therefore indicating that folate from FF is being disseminated through folate receptors within oocytes and embryos to participate in the folate pathway. This study advocates the necessity for examination of the result of folate supplementation throughout in vitro embryo production for improving the quality and quantity of transferable blastocysts and subsequently live calf births in buffalo.