title: 5-aza-2'-deoxycytidine impairs mouse spermatogenesis at multiple stages through different usage of DNA methyltransferases.
abstract: Mammalian spermatogenesis is a progressive process comprising spermatogonial proliferation, spermatocytic meiosis, and later spermiogenesis, which is considered to be under the regulation of epigenetic parameters. To gain insights into the significance of DNA methylation in early spermatogenesis, 5-azadC was used as a molecular biological tool to mimic the level of DNA methylation in vivo. Since the drug is incorporated into DNA during the S-phase, spermatogonia and spermatocytes would be affected primarily in mouse spermatogenesis. Adult male ICR mice were intraperitoneally injected with 5-azadC at a dose of 0.25mg/kg/day for 10 consecutive days, allowing us to examine its maximum effect on the kinetics of spermatogonia and spermatocytes. In this short-term protocol, 5-azadC induced significant histological abnormalities, such as a marked increase in apoptosis of spermatogonia and spermatocytes, followed by severe loss of spermatids, while after termination of 5-azadC treatment, normal histology was restored in the testis within 35days. Quantification of the methylation level of CCGG sites as well as whole DNA showed spermatogonial hypomethylation, which correlated with increased apoptosis of spermatogonia. Interestingly, the hypomethylated cells were simultaneously positive for tri-methylated histone H3 at K4. On the other hand, no changes in methylation level were found in spermatocytes, but PCNA staining clearly showed disordered accumulation of S-phase spermatocytes, which increased their apoptosis in stage XII. In addition, different immunohistochemical staining pattern was found for DNA methyltransferases (DNMTs); DNMT1was expressed in the majority of all germ cells, but DNMT3a and b were only expressed in spermatogonia. Our results indicate that 5-azadC caused DNA hypomethylation in spermatogonia, but induced prolongation of S-phase in spermatocytes, resulting in the induction of apoptosis in both cases. Thus, 5-azadC affects spermatogenesis at more than one differentiation stage with different mechanisms, probably due to the specific usage of DNMTs.
authors.affiliation: Department of Histology and Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan; Department of Anatomy, Histology and Embryology, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Rd., Shanghai 200025, PR China.
authors.forename: Ning
authors.initials: N
authors.lastname: Song
authors.affiliation: Department of Histology and Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Endo
authors.affiliation: Department of Histology and Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan; Department of Anatomy, Histology and Embryology, School of Basic Medical Sciences, Fujian Medical University, NO. 1, Xueyuan Rd., Fuzhou, Fujian 350108, PR China.
authors.forename: Bin
authors.initials: B
authors.lastname: Song
authors.affiliation: Department of Histology and Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan.
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Shibata
authors.affiliation: Department of Histology and Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. Electronic address: tkoji@nagasaki-u.ac.jp.
authors.forename: Takehiko
authors.initials: T
authors.lastname: Koji
mesh_terms: D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000633:toxicity*; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000633:toxicity; D002454:Cell Differentiation / Q000187:drug effects; D019175:DNA Methylation / Q000187:drug effects; D015254:DNA Modification Methylases / Q000187:drug effects / Q000378:metabolism*; D000077209:Decitabine; D005854:Germ Cells / Q000187:drug effects / Q000648:ultrastructure; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D012671:Seminiferous Tubules / Q000187:drug effects / Q000648:ultrastructure; D013090:Spermatocytes / Q000187:drug effects / Q000378:metabolism; D013091:Spermatogenesis / Q000187:drug effects*; D013093:Spermatogonia / Q000187:drug effects / Q000378:metabolism; D013737:Testis / Q000187:drug effects / Q000378:metabolism
chemical_list: D000964:Antimetabolites, Antineoplastic; D000077209:Decitabine; D015254:DNA Modification Methylases; D001374:Azacitidine
keywords: 5-aza-2′-deoxycytidine; Apoptosis; DNA methylation; DNMT; HELMET; Mouse spermatogenesis