Adriamycin induces H2AX phosphorylation in human spermatozoa.
OBJECTIVE
To investigate whether adriamycin induces DNA damage and the formation of gammaH2AX (the phosphorylated form of histone H2AX) foci in mature spermatozoa.


METHODS
Human spermatozoa were treated with adriamycin at different concentrations. gammaH2AX was analyzed by immunofluorescent staining and flow cytometry and double-strand breaks (DSB) were detected by the comet assay.


RESULTS
The neutral comet assay revealed that the treatment with adriamycin at 2 microg/mL for different times (0.5, 2, 8 and 24 h), or for 8 h at different concentrations (0.4, 2 and 10 microg/mL), induced significant DSB in spermatozoa. Immunofluorent staining and flow cytometry showed that the expression of gH2AX was increased in a dose-dependent and time-dependant manner after the treatment of adriamycin. Adriamycin also induced the concurrent appearance of DNA maintenance/repair proteins RAD50 and 53BP1 with gammaH2AX in spermatozoa. Wortmannin, an inhibitor of the phosphatidylinositol 3-kinase (PI3K) family, abolished the co-appearance of these two proteins with gammaH2AX.


CONCLUSIONS
Human mature spermatozoa have the same response to DSB-induced H2AX phosphorylation and subsequent recruitment of DNA maintenance/repair proteins as somatic cells.
Li
Zhong-Xiang
ZX
Department of Reproductive Endocrinology, Women's Hospital, Zhejiang University School of Medicine, Hangzhou 310006, China.
Wang
Ting-Ting
TT
Wu
Yan-Ting
YT
Xu
Chen-Ming
CM
Dong
Min-Yue
MY
Sheng
Jian-Zhong
JZ
Huang
He-Feng
HF
D017766:Acid Anhydride Hydrolases; D000730:Androstadienes / Q000494:pharmacology; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D002478:Cells, Cultured; D020552:Comet Assay; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D004249:DNA Damage; D045643:DNA Repair Enzymes / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology*; D004347:Drug Interactions; D005434:Flow Cytometry; D006657:Histones / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008297:Male; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D013094:Spermatozoa / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D000071857:Tumor Suppressor p53-Binding Protein 1; D000077191:Wortmannin
D000730:Androstadienes; D000903:Antibiotics, Antineoplastic; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; D047428:Protein Kinase Inhibitors; C498301:TP53BP1 protein, human; D000071857:Tumor Suppressor p53-Binding Protein 1; D004317:Doxorubicin; D017766:Acid Anhydride Hydrolases; C112158:RAD50 protein, human; D045643:DNA Repair Enzymes; D000077191:Wortmannin