Testicular MTHFR deficiency may explain sperm DNA hypomethylation associated with high dose folic acid supplementation.

Supplementation with high doses of folic acid, an important mediator of one-carbon transfers for DNA methylation, is used clinically to improve sperm parameters in infertile men. We recently detected an unexpected loss of DNA methylation in the sperm of idiopathic infertile men after 6 months of daily supplementation with 5 mg folic acid (>10× the daily recommended intake-DRI), exacerbated in men homozygous for a common variant in the gene encoding an important enzyme in folate metabolism, methylenetetrahydrofolate reductase (MTHFR 677C>T). To investigate the epigenomic impact and mechanism underlying effects of folic acid on male germ cells, wild-type and heterozygote mice for a targeted inactivation of the Mthfr gene were fed high-dose folic acid (10× the DRI) or control diets (CDs) for 6 months. No changes were detected in general health, sperm counts or methylation of imprinted genes. Reduced representation bisulfite sequencing revealed sperm DNA hypomethylation in Mthfr+/- mice on the 10× diets. Wild-type mice demonstrated sperm hypomethylation only with a very high dose (20×) of folic acid for 12 months. Testicular MTHFR protein levels decreased significantly in wild-type mice on the 20× diet but not in those on the 10× diet, suggesting a possible role for MTHFR deficiency in sperm DNA hypomethylation. In-depth analysis of the folic acid-exposed sperm DNA methylome suggested mouse/human susceptibility of sequences with potential importance to germ cell and embryo development. Our data provide evidence for a similar cross-species response to high dose folic acid supplementation, of sperm DNA hypomethylation, and implicate MTHFR downregulation as a possible mechanism.

Developmental windows of susceptibility for epigenetic inheritance through the male germline.

Exposure of developing male germ cells to environmental insults has been linked to adverse effects in the offspring. One mechanism by which germ cell defects may be passed intergenerationally is through perturbations in the epigenome at the level(s) of DNA methylation, histone post-translational modifications and/or small non-coding RNAs. Epigenetic programs are particularly dynamic in germ cells undergoing erasure, re-establishment and maintenance of patterns, events potentially susceptible to prenatal and/or postnatal exposures. In this review, we focus on the epigenetic events occurring at each phase of male germ cell development including the prenatal period covering primordial germ cells and prospermatogonia and the postnatal period covering mitotic spermatogonia, meiotic spermatocytes and post-meiotic haploid spermatids and spermatozoa. Strong barriers to the passage of abnormal epigenetic patterns between generations are erected at two times of genome-wide epigenomic reprogramming, first in the germline in primordial germ cells and second, post-fertilization, during preimplantation development. Evidence from high resolution profiling studies that not all epigenetic marks are erased during germ cell and embryonic reprogramming provides a potential explanation for the intergenerational inheritance of abnormal epigenetic marks that may affect offspring health.

Intergenerational impact of paternal lifetime exposures to both folic acid deficiency and supplementation on reproductive outcomes and imprinted gene methylation.

STUDY QUESTION: Do paternal exposures to folic acid deficient (FD), and/or folic acid supplemented (FS) diets, throughout germ cell development adversely affect male germ cells and consequently offspring health outcomes? SUMMARY ANSWER: Male mice exposed over their lifetimes to both FD and FS diets showed decreased sperm counts and altered imprinted gene methylation with evidence of transmission of adverse effects to the offspring, including increased postnatal-preweaning mortality and variability in imprinted gene methylation. WHAT IS KNOWN ALREADY: There is increasing evidence that disruptions in male germ cell epigenetic reprogramming are associated with offspring abnormalities and intergenerational disease. The fetal period is the critical time of DNA methylation pattern acquisition for developing male germ cells and an adequate supply of methyl donors is required. In addition, DNA methylation patterns continue to be remodeled during postnatal spermatogenesis. Previous studies have shown that lifetime (prenatal and postnatal) folic acid deficiency can alter the sperm epigenome and increase the incidence of fetal morphological abnormalities. STUDY DESIGN, SIZE, DURATION: Female BALB/c mice (F0) were placed on one of four amino-acid defined diets for 4 weeks before pregnancy and throughout pregnancy and lactation: folic acid control (Ctrl; 2 mg/kg), 7-fold folic acid deficient (7FD; 0.3 mg/kg), 10-fold high FS (10FS, 20 mg/kg) or 20-fold high FS (20FS, 40 mg/kg) diets. F1 males were weaned to their respective prenatal diets to allow for diet exposure during all windows of germline epigenetic reprogramming: the erasure, re-establishment and maintenance phases. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: F0 females were mated with chow-fed males to produce F1 litters whose germ cells were exposed to the diets throughout embryonic development. F1 males were subsequently mated with chow-fed female mice. Two F2 litters, unexposed to the experimental diets, were generated from each F1 male; one litter was collected at embryonic day (E)18.5 and one delivered and followed postnatally. DNA methylation at a global level and at the differentially methylated regions of imprinted genes (H19, Imprinted Maternally Expressed Transcript (Non-Protein Coding)-H19, Small Nuclear Ribonucleoprotein Polypeptide N-Snrpn, KCNQ1 Opposite Strand/Antisense Transcript 1 (Non-Protein Coding)-Kcnq1ot1, Paternally Expressed Gene 1-Peg1 and Paternally Expressed Gene 3-Peg3) was assessed by luminometric methylation analysis and bisulfite pyrosequencing, respectively, in F1 sperm, F2 E18.5 placenta and F2 E18.5 brain cortex. MAIN RESULTS AND THE ROLE OF CHANCE: F1 males exhibited lower sperm counts following lifetime exposure to both folic acid deficiency and the highest dose of folic acid supplementation (20FS), (both P < 0.05). Post-implantation losses were increased amongst F2 E18.5 day litters from 20FS exposed F1 males (P < 0.05). F2 litters derived from both 7FD and 20FS exposed F1 males had significantly higher postnatal-preweaning pup death (both P < 0.05). Sperm from 10FS exposed males had increased variance in methylation across imprinted gene H19, P < 0.05; increased variance at a few sites within H19 was also found for the 7FD and 20FS groups (P < 0.05). While the 20FS diet resulted in inter-individual alterations in methylation across the imprinted genes Snrpn and Peg3 in F2 E18.5 placenta, ≥50% of individual sites tested in Peg1 and/or Peg3 were affected in the 7FD and 10FS groups. Inter-individual alterations in Peg1 methylation were found in F2 E18.5 day 10FS group brain cortex (P < 0.05). LARGE SCALE DATA: Not applicable. LIMITATIONS REASONS FOR CAUTION: The cause of the increase in postnatal-preweaning mortality was not investigated post-mortem. Further studies are required to understand the mechanisms underlying the adverse effects of folic acid deficiency and supplementation on developing male germ cells. Genome-wide DNA and histone methylome studies as well as gene expression studies are required to better understand the links between folic acid exposures, an altered germ cell epigenome and offspring outcomes. WIDER IMPLICATIONS OF THE FINDINGS: The findings of this study provide further support for paternally transmitted environmental effects. The results indicate that both folic acid deficiency and high dose supplementation can be detrimental to germ cell development and reproductive fitness, in part by altering DNA methylation in sperm. STUDY FUNDING AND COMPETING INTERESTS: This study was supported by a grant to J.M.T. from the Canadian Institutes of Health Research (CIHR #89944). The authors declare they have no conflicts of interest.

Maraba MG1 virus enhances natural killer cell function via conventional dendritic cells to reduce postoperative metastatic disease.

This study characterizes the ability of novel oncolytic rhabdoviruses (Maraba MG1) to boost natural killer (NK) cell activity. Our results demonstrate that MG1 activates NK cells via direct infection and maturation of conventional dendritic cells. Using NK depletion and conventional dendritic cells ablation studies in vivo, we established that both are required for MG1 efficacy. We further explored the efficacy of attenuated MG1 (nonreplicating MG1-UV(2min) and single-cycle replicating MG1-Gless) and demonstrated that these viruses activate conventional dendritic cells, although to a lesser extent than live MG1. This translates to equivalent abilities to remove tumor metastases only at the highest viral doses of attenuated MG1. In tandem, we characterized the antitumor ability of NK cells following preoperative administration of live and attenuated MG1. Our results demonstrates that a similar level of NK activation and reduction in postoperative tumor metastases was achieved with equivalent high viral doses concluding that viral replication is important, but not necessary for NK activation. Biochemical characterization of a panel of UV-inactivated MG1 (2-120 minutes) revealed that intact viral particle and target cell recognition are essential for NK cell-mediated antitumor responses. These findings provide mechanistic insight and preclinical rationale for safe perioperative virotherapy to effectively reduce metastatic disease following cancer surgery.

Transgenerational impact of grand-paternal lifetime exposures to both folic acid deficiency and supplementation on genome-wide DNA methylation in male germ cells.

BACKGROUND: DNA methylation (DNAme) erasure and reacquisition occur during prenatal male germ cell development; some further remodeling takes place after birth during spermatogenesis. Environmental insults during germline epigenetic reprogramming may affect DNAme, presenting a potential mechanism for transmission of environmental exposures across multiple generations. OBJECTIVES: We investigated how germ cell DNAme is impacted by lifetime exposures to diets containing either low or high, clinically relevant, levels of the methyl donor folic acid and whether resulting DNAme alterations were inherited in germ cells of male offspring of subsequent generations. MATERIALS AND METHODS: Female mice were placed on a control (FCD), 7-fold folic acid deficient (7FD) or 10- to 20-fold supplemented (10FS and 20FS) diet before and during pregnancy. Resulting F1 litters were weaned on the respective diets. F2 and F3 males received control diets. Genome-wide DNAme at cytosines (within CpG sites) was assessed in F1 spermatogonia, and in F1, F2 and F3 sperm. RESULTS: In F1 germ cells, a greater number of differentially methylated cytosines (DMCs) were observed in spermatogonia as compared with F1 sperm for all folic acid diets. DMCs were lower in number in F2 versus F1 sperm, while an unexpected increase was found in F3 sperm. DMCs were predominantly hypomethylated, with genes in neurodevelopmental pathways commonly affected in F1, F2 and F3 male germ cells. While no DMCs were found to be significantly inherited inter- or transgenerationally, we observed over-representation of repetitive elements, particularly young long interspersed nuclear elements (LINEs). DISCUSSION AND CONCLUSION: These results suggest that the prenatal window is the time most susceptible to folate-induced alterations in sperm DNAme in male germ cells. Altered methylation of specific sites in F1 germ cells was not present in later generations. However, the presence of DNAme perturbations in the sperm of males of the F2 and F3 generations suggests that epigenetic inheritance mechanisms other than DNAme may have been impacted by the folate diet exposure of F1 germ cells.

Impact of mothers' early life exposure to low or high folate on progeny outcome and DNA methylation patterns.

The dynamic patterning of DNA and histone methylation during oocyte development presents a potentially susceptible time for epigenetic disruption due to early life environmental exposure of future mothers. We investigated whether maternal exposure to folic acid deficient and supplemented diets starting in utero could affect oocytes and cause adverse developmental and epigenetic effects in next generation progeny. Female BALB/c mice (F0) were placed on one of four amino acid defined diets for 4 weeks before pregnancy and throughout gestation and lactation: folic acid control (rodent recommended daily intake; Ctrl), 7-fold folic acid deficient, 10-fold folic acid supplemented or 20-fold folic acid supplemented diets. F1 female pups were weaned onto Ctrl diets, mated to produce the F2 generation and the F2 offspring were examined at E18.5 for developmental and epigenetic abnormalities. Resorption rates were increased and litter sizes decreased amongst F2 E18.5-day litters in the 20-fold folic acid supplemented group. Increases in abnormal embryo outcomes were observed in all three folic acid deficient and supplemented groups. Subtle genome-wide DNA methylation alterations were found in the placentas and brains of F2 offspring in the 7-fold folic acid deficient , 10-fold folic acid supplemented and 20-fold folic acid supplemented groups; in contrast, global and imprinted gene methylation were not affected. The findings show that early life female environmental exposures to both low and high folate prior to oocyte maturation can compromise oocyte quality, adversely affecting offspring of the next generation, in part by altering DNA methylation patterns.

Preventing postoperative metastatic disease by inhibiting surgery-induced dysfunction in natural killer cells.

Natural killer (NK) cell clearance of tumor cell emboli following surgery is thought to be vital in preventing postoperative metastases. Using a mouse model of surgical stress, we transferred surgically stressed NK cells into NK-deficient mice and observed enhanced lung metastases in tumor-bearing mice as compared with mice that received untreated NK cells. These results establish that NK cells play a crucial role in mediating tumor clearance following surgery. Surgery markedly reduced NK cell total numbers in the spleen and affected NK cell migration. Ex vivo and in vivo tumor cell killing by NK cells were significantly reduced in surgically stressed mice. Furthermore, secreted tissue signals and myeloid-derived suppressor cell populations were altered in surgically stressed mice. Significantly, perioperative administration of oncolytic parapoxvirus ovis (ORFV) and vaccinia virus can reverse NK cell suppression, which correlates with a reduction in the postoperative formation of metastases. In human studies, postoperative cancer surgery patients had reduced NK cell cytotoxicity, and we show for the first time that oncolytic vaccinia virus markedly increases NK cell activity in patients with cancer. These data provide direct in vivo evidence that surgical stress impairs global NK cell function. Perioperative therapies aimed at enhancing NK cell function will reduce metastatic recurrence and improve survival in surgical cancer patients.