MTHFR-DEFICIENT MICE AS A MODEL FOR EPIGENETIC DISTURBANCE OF SPERMATOGENESIS THROUGH FOLATE PATHWAY DISRUPTION

Abstract

MTHFR (5,10-methylenetetrahydrofolate reductase) controls the flow of methyl groups from folate to S-adenosylmethionine production and therefore their availability for cytosine methylation, an important and heritable DNA modification that regulates gene expression. Human males homozygous for a common MTHFR (677C->T) polymorphism, which creates a thermolabile enzyme with reduced activity, are over-represented in the infertile population, linking folate availability and effective sperm production. Gene-targeted male Mthfr−/− mice are infertile on a BALB/c background but are fertile on a C57BL/6 background. This differential phenotype provides a strategy for studying the mechanism of folateassociated disruption of spermatogenesis. We examined testes from BALB/c mice at 1, 2, and 4 days post partum (dpp) and found similar germ cell numbers in Mthfr−/−, Mthfr+/−, and Mthfr+/+ animals, but significantly fewer at day 6 in Mthfr−/− testes. Day 4 BALB/c mice were given i.p. injections of 5-bromo-2′-deoxyuridine (BrdU), a thymidine analogue, and immunohistochemistry revealed significantly fewer proliferating germ cells in Mthfr−/− than those in Mthfr+/− or Mthfr+/+ testes. Thus, spermatogenic disruption manifests around 4dpp in these mice. In contrast, when mice on a C57BL/6 background were injected i.p. with BrdU and their testes examined at 0, 2, 4, and 6 dpp, total and proliferating germ cells were found to be similar among all genotypes at each sampling day. We used Restriction Landmark Genomic Scanning (RLGS), a technique using methylation-sensitive restriction enzymes and two-dimensional electrophoresis to assess methylation sites across the genome, in C57BL/6 epididymal spermatozoa to assess if DNA methylation changes were associated with MTHFR deficiency. After elimination of known polymorphic sites, methylation changes at 30 different loci were observed between Mthfr−/− and Mthfr+/+ mice, with 17 occurring in more then one animal. Both hyper- and hypo-methylation occurred at approximately equal frequencies. Thus, we have identified, in a murine model, the time-frame for spermatogenic disruption by MTHFR deficiency and demonstrated related germ cell methylation changes even among fertile individuals. Supported by NSERC and CIHR. (platform)