Exposure to bleomycin, etoposide, and cis-platinum alters rat sperm chromatin integrity and sperm head protein profile

Abstract

Testicular cancer, currently the most common cancer affecting men of reproductive age, is one of the most curable malignancies due to the progress made in the early diagnosis and effective treatment of this disease. The coadministration of bleomycin, etoposide, and cis-platinum (BEP) has brought the 5-yr survival rate of testis cancer patients to over 90%. However, this treatment results in reproductive chemotoxic effects. We assessed the effect of BEP treatment on sperm chromatin integrity and sperm head protein profiles of adult male Brown Norway rats following 9 wk of treatment with BEP and in animals treated for 9 wk and then subjected to a 9-wk recovery period. Both the susceptibility of DNA to denaturation and the number of strand breaks were significantly increased in mature sperm following 9 wk of treatment with BEP; proteomic analysis revealed that the expression of several proteins, including HSP90AA1 and HSP90B1, was markedly affected. Following a 9-wk recovery period, mature sperm did not show significant DNA damage, indicating that repair had potentially occurred. Interestingly, the protamination level of the sperm of these animals was significantly decreased, while histones HIST1H1D (H1.2), HIST1H4B (H4), HIST2H2AA3 (H2A1), and HIST1H2BA (H2B1A) were concomitantly up-regulated; this was not observed in the sperm immediately following 9 wk of treatment. Thus, there are persistent effects on proteins in sperm heads from the cauda epididymidis 9 wk posttreatment, in the absence of DNA strand breaks. We suggest that these effects on the sperm head proteome may contribute to long-lasting adverse effects in the progeny of BEP-exposed males.

FIG. 1.

Strand breaks, as evaluated by the comet assay, in cauda spermatozoa immediately following treatment with saline (control) or BEP for 9 wk or after a 9-wk recovery period. Data for % tail DNA (A), tail length (B), and tail extent moment (C) are represented as mean ± SEM. *(P < 0.01) and **(P < 0.001) n = 6.

FIG. 2.

Sperm chromatin structure assay (SCSA/AO assay) determined in mature cauda spermatozoa, illustrating % DNA fragmentation index (DFI) (A) and % high DNA stainability (HDS) (B), a marker of poorly condensed spermatozoa, at 9 wk and following a 9-wk recovery period in saline and BEP-treated animals. Bars represent means ± SEM, n = 6. The asterisk indicates P < 0.05.

FIG. 3.

Protamine content, assessed indirectly by measuring CMA3 fluorescence in spermatozoa of animals treated with saline or BEP for 9 wk versus animals subjected to a recovery period of 9 wk. Bars represent means ± SEM, n = 6. The asterisk indicates P < 0.01.

FIG. 4.

Relative mRNA expression of Prm1 (A) in testis of control and BEP-treated rats after 9 wk of treatment or following a recovery period (n = 6). Qualitative analysis of PRM1 (red) immunofluorescence (B) with original magnification ×63. Quantitative analysis of the PRM1 staining intensity (C) in cauda spermatozoa of control and BEP-treated rats after 9 wk of treatment or following a recovery period. Bars represent means ± SEM, n = 4. The asterisk indicates P < 0.01.

FIG. 5.

Effects of BEP treatment at 9 wk and following a 9-wk recovery period on histone protein levels: HIST1H1D (H1.2) (A), HIST2H2AA3 (H2A1) (B), HIST1H2BA (H2B1A) (C), and HIST1H4B (H4) (D) in cauda spermatozoa. Bars represent means ± SEM. *(P < 0.05), **(P < 0.01), and ***(P < 0.001), n = 5–6.