RESUMO
The anticancer agent, 5-aza-2'-deoxycytidine (5-azaCdR, decitabine), causes DNA hypomethylation and a robust, dose-dependent disruption of spermatogenesis. Previously, we have shown that altered testicular histology and reduced sperm production in 5-azaCdR-treated animals is associated with decreased global sperm DNA methylation and an increase in infertility and/or a decreased ability to support preimplantation embryonic development. The goal of this study was to determine potential contributors to 5-azaCdR-mediated infertility including alterations in sperm motility, fertilization ability, early embryo development, and sequence-specific DNA methylation. We find that although 5-azaCdR-treatment adversely affected sperm motility and the survival of sired embryos to the blastocyst stage, the major contributor to infertility was a marked (56-70%) decrease in fertilization ability. Sperm DNA methylation was investigated using Southern blot, restriction landmark genomic scanning, and quantitative analysis of DNA methylation by real-time polymerase chain reaction. Interestingly, hypomethylation was restricted to genomic loci that have been previously determined to acquire methylation during spermatogenesis, demonstrating that 5-azaCdR selectively inhibits de novo methylation activity. Similar to previous studies, we show that mice that are heterozygous for a nonfunctional Dnmt1 gene are partially protected against the deleterious effects of 5-azaCdR; however, methylation levels are not restored in these mice, suggesting that adverse effects are due to another mechanism(s) in addition to DNA hypomethylation. These results demonstrate that clinically relevant doses of 5-azaCdR specifically impair de novo methylation activity in male germ cells; however, genotype-specific differences in drug responses suggest that adverse reproductive outcomes are mainly mediated by the cytotoxic properties of the drug.
Assuntos
Azacitidina/análogos & derivados , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Espermatozoides/fisiologia , Animais , Azacitidina/efeitos adversos , DNA (Citosina-5-)-Metiltransferase 1 , Decitabina , Genótipo , Infertilidade Masculina/induzido quimicamente , Masculino , Camundongos , Espermatozoides/efeitos dos fármacosRESUMO
Metabolism of folate is essential for proper cellular function. Within the folate pathway, methylenetetrahydrofolate reductase (MTHFR) reduces 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a methyl donor for remethylation of homocysteine to methionine, the precursor of S-adenosylmethionine. S-adenosylmethionine is the methyl donor for numerous cellular reactions. In adult male mice, MTHFR levels are highest in the testis; this finding, in conjunction with recent clinical evidence, suggest an important role for MTHFR in spermatogenesis. Indeed, we show here that severe MTHFR deficiency in male mice results in abnormal spermatogenesis and infertility. Maternal oral administration of betaine, an alternative methyl donor, throughout pregnancy and nursing, resulted in improved testicular histology in Mthfr-/- offspring at Postnatal Day 6, but not at 8 mo of age. However, when betaine supplementation was maintained postweaning, testicular histology improved, and sperm numbers and fertility increased significantly. We postulate that the adverse effects of MTHFR deficiency on spermatogenesis, may, in part, be mediated by alterations in the transmethylation pathway and suggest that betaine supplementation may provide a means to bypass MTHFR deficiency and its adverse effects on spermatogenesis by maintaining normal methylation levels within male germ cells.
Assuntos
5,10-Metilenotetra-Hidrofolato Redutase (FADH2)/deficiência , 5,10-Metilenotetra-Hidrofolato Redutase (FADH2)/metabolismo , Betaína/administração & dosagem , Infertilidade Masculina/dietoterapia , Infertilidade Masculina/enzimologia , Espermatogênese/efeitos dos fármacos , Animais , Suplementos Nutricionais , Esquema de Medicação , Fertilidade/efeitos dos fármacos , Fertilidade/fisiologia , Fármacos para a Fertilidade Masculina/administração & dosagem , Ácido Fólico/metabolismo , Células Germinativas/citologia , Células Germinativas/efeitos dos fármacos , Células Germinativas/enzimologia , Infertilidade Masculina/tratamento farmacológico , Masculino , Metilação/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Contagem de Espermatozoides , Espermatogênese/fisiologia , Estatísticas não Paramétricas , Testículo/citologia , Testículo/efeitos dos fármacos , Testículo/enzimologiaRESUMO
Because of the ability of cytidine analogues, such as 5-aza-2'-deoxycytidine, to incorporate into DNA and lead to decreases in DNA methylation, there has recently been renewed interest in using these drugs in anticancer therapy. To determine the effects of paternal 5-aza-2'-deoxycytidine treatment on spermatogenesis and progeny outcome in the mouse and whether effects are modulated by decreased levels of the predominant DNA methyltransferase, DNMT1, adult Dnmt1(+/+) and Dnmt1-deficient (Dnmt1(c/+)) male mice were treated with 5-aza-2'-deoxycytidine for 7 weeks, which resulted in dose-dependent decreases in testicular weight, an increase in histological abnormalities, and a decline in sperm counts, with no apparent effect on androgen status. Testes of Dnmt1(c/+) mice, however, were less severely affected by 5-aza-2'-deoxycytidine than were those of wild-type mice. The exposure of Dnmt1(+/+) male mice to even low doses of 5-aza-2'-deoxycytidine followed by mating elicited significantly reduced pregnancy rates and elevated preimplantation loss in females. Dnmt1 deficiency, however, protected against such drug-induced decreases in pregnancy rate but not preimplantation loss. Altered DNA methylation or DNMT1 activity may explain such adverse effects, because treatment resulted in dose-dependent decreases in the global methylation of sperm DNA. Thus, in the mouse, paternal administration of 5-aza-2'-deoxycytidine interferes with normal male germ cell development and results in reduced fertility, whereas lowering DNMT1 levels appears to partially protect the seminiferous epithelium from deleterious drug effects.