Inhibition of proteolysis in histiotrophic nutrition pathways alters DNA methylation and one-carbon metabolism in the organogenesis-stage rat conceptus

Epigenetic modifications, including DNA methylation, contribute to the transcriptional regulation of developmental genes that control growth and differentiation during embryogenesis. The methyl donor, S-adenosylmethionine (SAM), is biosynthesized from methionine and adenosine triphosphate by methionine adenosyltransferase 2a (Mat2a) in the one-carbon (C1) metabolism pathway. SAM biosynthesis requires a steady supply of nutrients, vitamins and cofactors obtained by the developing conceptus through histiotrophic nutrition pathways (HNPs). The visceral yolk sac (VYS) captures proteins and their substrate cargos by receptor-mediated endocytosis and degrades them using lysosomal proteases. We hypothesize that leupeptin, a protease inhibitor, reduces the availability of methionine and C1 substrates, restricting SAM biosynthesis and altering patterns of DNA methylation. Rat conceptuses were exposed to 50 and 100 μM leupeptin in whole embryo culture for periods of 26 h from gestational day (GD) 10 or 6 h on GD11. After 6 h on GD11, the 100-μM leupeptin treatment significantly decreased methionine in embryo (EMB) and VYS, reduced Mat2a protein levels and inhibited Mat2a specific activity, all of which produced a significant 52% reduction of SAM in the VYS. The 50- and 100-μM leupeptin treatments significantly decreased global methylation levels by 6%-9% in EMB and by 11%-15% in VYS following both 6- and 26-h exposure periods. This study demonstrates that HNP disruption alters C1 activity and significantly reduces global DNA methylation during organogenesis. Because epigenetic reprogramming is crucial for normal differentiation and growth, these findings suggest a possible mechanism through which nutrients and environmental factors may alter early developmental regulation.