Five azacytidine, a DNA methyltransferase inhibitor, specifically inhibits testicular cord formation and Sertoli cell differentiation in vitro.
In mammals, Sry, Sox9, and M33 act as regulators at a chromatin level and promote the maturation of embryonic gonads into testes. Recently, it was shown that transcriptional regulation by DNA methylation plays crucial roles in gene expression during the differentiation and development of various cell types. To determine the involvement of DNA methylation in sex determination of the gonad, we developed and performed organ culture of gonad with the DNA methyltransferase inhibitor 5-azacytidine to induce global DNA methylation status changes. In vitro treatment with 5-azacytidine specifically inhibited testicular cord formation in a dose-dependent manner; however, no appreciable defect was observed in ovarian explants. Inhibition of testicular cord was observed only in gonads from 11.5 days post-coitus embryos. These effects were not observed in 5-azacytidine-treated gonads from 12.0 days post-coitus embryos. To determine the effect of 5-azacytidine on Sertoli and Leydig cell differentiation in the testis, we performed whole mount in situ hybridization analysis. The Leydig and stromal cell marker genes Lhx9, Mfge8, and 3beta-Hsd were normally induced in 5-azaytidine-treated testicular explants. Sertoli cell marker genes, Sox9 and MIS were normally induced, but Col9a3, encoding an extracellular matrix component, was inhibited in 5-azacytidine-treated testicular explants. Thus, our data show that DNA methylation is involved in testicular cord formation and Sertoli cell differentiation, acting directly on the gonad at 11.5 days post-coitus.
Mizukami
Takuo
T
Department of Veterinary Anatomy, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan. tmiz@nih.go.jp
Kanai
Yoshiakira
Y
Fujisawa
Masahiko
M
Kanai-Azuma
Masami
M
Kurohmaru
Masamichi
M
Hayashi
Yoshihiro
Y
D015096:3-Hydroxysteroid Dehydrogenases / Q000235:genetics; D000818:Animals; D000954:Antigens, Surface / Q000235:genetics; D001374:Azacitidine / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects; D019175:DNA Methylation / Q000187:drug effects; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D006609:High Mobility Group Proteins / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics; D060850:LIM-Homeodomain Proteins; D007985:Leydig Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D008894:Milk Proteins / Q000235:genetics; D009924:Organ Culture Techniques; D010053:Ovary / Q000166:cytology / Q000187:drug effects / Q000196:embryology / Q000378:metabolism; D055758:SOX9 Transcription Factor; D012708:Sertoli Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D017154:Stromal Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D013737:Testis / Q000166:cytology / Q000187:drug effects* / Q000196:embryology* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics
D000954:Antigens, Surface; D004791:Enzyme Inhibitors; D006609:High Mobility Group Proteins; D018398:Homeodomain Proteins; D060850:LIM-Homeodomain Proteins; C117058:Lhx9 protein, mouse; C490919:Mfge8 protein, mouse; D008894:Milk Proteins; D055758:SOX9 Transcription Factor; C527933:Sox9 protein, mouse; D014157:Transcription Factors; D015096:3-Hydroxysteroid Dehydrogenases; D015254:DNA Modification Methylases; D001374:Azacitidine