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.

During spermiogenesis, histones are replaced first by transition proteins and then by protamines, resulting in a very condensed sperm DNA structure that is absolutely critical for normal sperm function. We have demonstrated previously that, despite a 9-wk recovery period, mature sperm from rats treated for 9 wk with bleomycin, etoposide, and cis-platinum (BEP), the drugs used to treat testicular cancer, have reduced levels of protamine 1 and a concomitant upregulation of specific histones, highlighting a problem in histone eviction. Here, we demonstrate that regulators of histone removal are increased in elongating spermatids following recovery; however, Ac-H4 and gamma H2AX histones remain elevated in elongating spermatids or caudal epididymal spermatozoa 9 wk post-BEP treatment. This indicates that chromatin remodelers and effector proteins that respond to histone removal cues may be a target of BEP treatment. A decrease in the expression of SMARCE1 in elongating spermatids may explain the persistent retention of histones in cauda epididymal sperm 9 wk after the cessation of BEP treatment. Remarkably, proteins implicated in the translational control and posttranslational processing of protamine 1 are also significantly elevated 9 wk post-BEP treatment, suggesting that histone eviction may dictate the DNA availability for protamine binding. Males mated to control females 9 wk after BEP treatment have reduced litter sizes; moreover, the profile of gene expression in the developing testes of their pups is altered. Altering the proportion of histones to protamine in mature spermatozoa has an adverse impact on male fecundity, with modifications to epigenetic marks potentially threatening normal progeny development.

SUMMARYCo-administration of bleomycin, etoposide, and cis-platinum (BEP) has increased the 5-year survival rate of testis cancer patients to over 90%; however, this treatment induces chemotoxic effects on male germ cells. Treatment of male rats with BEP, using a similar schedule to that used in man, affects reproductive organ weights and sperm count, motility, and DNA integrity, as well as pup survival rates. Telomeres, specialized structures at the termini of chromosomes, play an important role in the maintenance of genetic stability. In previous studies, we demonstrated, using a spermatogonial cell line, that cis-platinum and bleomycin damage telomeres and that cis-platinum also inhibits telomerase activity. Our objective here was to test the hypothesis that in vivo exposure to the BEP regimen used to treat testis cancer targets telomeres in the male germ line. Adult male Brown Norway rats received chronic treatment with a BEP regimen. DNA double strand breaks were increased significantly in zygotene germ cells, as assessed by c-H2AX immunofluorescence. Interestingly, treatment with this BEP regimen increased c-H2AX foci in the telomere region of zygotene spermatocytes, but not in other germ cell types, such as pachytene cells, round spermatids, or elongating spermatids. Mean telomere lengths were reduced in zygotene, pachytene, round spermatid, elongating spermatid and cauda epididymal spermatozoa compared with the saline control group. Thus, telomere lengths did not recover during germ cell development. These studies demonstrate that BEP treatment is associated with an effect on telomeres.

Treatment of testicular cancer includes the coadministration of bleomycin, etoposide and cis-platinum (BEP); however, along with its therapeutic benefit, BEP exposure results in extensive reproductive chemotoxic effects, including alterations to sperm chromatin integrity. As an intact paternal genome is essential for successful fertilization and embryogenesis, we assessed the effect of paternal exposure to BEP on sperm fertilization capacity and the resulting consequences on early embryonic gene expression. Adult male Brown Norway rats received a 9-week treatment with BEP or saline and then were sacrificed immediately or subject to a 9-week recovery period. HSP90AA1, HSP90B1 and PDIA3, involved in spermatozoa-egg interactions, were overexpressed in BEP-exposed spermatozoa after the 9-week treatment period; overexpression was also observed in spermatozoa from BEP-treated rats after 9 weeks of recovery. These proteins were localized to the plasma membrane of the sperm head; this localization may facilitate their role in spermatozoa-egg interactions as the highest staining intensities were observed in capacitated spermatozoa. The fertilization potential of spermatozoa was determined by in vitro fertilization with oocytes from unexposed naturally cycling female rats. Interestingly, the fertilization potential of spermatozoa following a 9-week recovery period from BEP treatment was significantly enhanced compared with controls. Moreover, stem cell transcription factors, involved in the regulation of a plethora of early embryonic events, were upregulated by more than twofold in eight-cell stage embryos sired by BEP recovery males compared with controls; this suggests that there are potential deleterious effects on embryo development well after termination of BEP exposure.

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