Development of a short-term fluorescence-based assay to assess the toxicity of anticancer drugs on rat stem/progenitor spermatogonia in vitro.

In vitro culture of rodent spermatogonial stem cells (SSCs) has become an important asset in the study of mammalian SSC biology. Supported by added growth factors, SSCs divide in culture and form aggregates of stem/progenitor spermatogonia, termed clusters. Recent studies have shown that serial passaging of clusters results in long-term maintenance and amplification of the SSC pool and that this culture system can also be used for short-term semiquantification of SSC activity. Here, we report the development of an automated assay to assess the activity of rat stem/progenitor spermatogonia in vitro and its application for investigating the cytotoxicity of chemotherapeutic drugs on these cells. Cultures of EGFP-expressing rat spermatogenic cells allowed us to determine the number and two-dimensional surface area of clusters using an automated fluorescence imaging system, thereby providing quantitative data of SSC activity. Using this assay, we examined the germ cell toxicity of three drugs that are routinely used in testicular cancer therapy, namely, bleomycin, cisplatin, and etoposide, alone and in combination. All three drugs showed a significant and dose-dependent reduction of cluster number and surface area, indicating their adverse effects specific to spermatogonia. The inhibitory concentration at which cluster number and surface area are inhibited by 50% (IC(50)) was the lowest with etoposide and the highest with cisplatin, implying that etoposide was most toxic to spermatogonia in vitro. These results suggest that the SSC culture should provide an effective and efficient system to assess the germ cell toxicity of various drugs and chemical compounds.

Effects of chemotherapeutic agents for testicular cancer on the male rat reproductive system, spermatozoa, and fertility.

Testicular cancer is the most common cancer affecting men of reproductive age. Advances in treatment of the disease, which include the coadministration of bleomycin, etoposide, and cis-platinum (BEP), have brought the cure rate to over 90%. This high cure rate, coupled with the young age of patients, makes elucidation of the impact of the treatment on reproductive function, fertility, and progeny outcome increasingly important. The goal of this study was to determine the effects of BEP, in doses analogous to those given to humans, on the male reproductive system, spermatozoa, fertility, and progeny outcome in an animal model. Male Sprague-Dawley rats were treated daily with BEP for 3 cycles of 3 weeks each, for a total of 9 weeks. After 6 and 9 weeks, males were mated to 2 groups of untreated females. BEP treatment resulted in decreases in testicular and epididymal weights of 52% and 28%, respectively, when compared to control. Decreased testis and epididymis weights were accompanied by impairment of spermatogenesis and by a decrease in spermatozoal count of nearly 90% (11.9 x 10(7) spermatozoa per caput epididymidis in control vs 1.65 x 10(7) in BEP-treated rats). The percent of motile spermatozoa in the treated rats was more than 30% lower than in controls. Defects in the flagella of spermatozoa increased by more than twofold in the midpiece, and by more than sixfold in the principal piece. Paternal BEP treatment, for either 6 or 9 weeks, did not affect fertility, pre- or postimplantation loss, litter size, or sex ratio among progeny on gestation day 21. In contrast, among the pregnancies allowed to proceed to delivery, a significant number of pups sired by males treated with BEP for 9 weeks died between birth and postnatal day 2; this was not observed in pups sired by males treated for 6 weeks. Markers of postnatal development were not affected in the surviving offspring from either group. Thus, despite the dramatic effects of the testicular cancer drug regimen on spermatogenesis, the numbers of spermatozoa, and their motility and morphology, male rats were fertile. While fetal development was apparently normal, early postnatal mortality, which may be associated with a delay in parturition, was elevated among the progeny sired by males exposed to BEP for 9 weeks.

Effects of chemotherapeutic agents for testicular cancer on rat spermatogonial stem/progenitor cells.

Spermatogonial stem cells (SSCs) are responsible for the production of spermatozoa throughout adulthood and for the recovery of spermatogenesis following exposure to cytotoxic agents. Previously, we have shown that the combined administration of bleomycin, etoposide, and cisplatin (BEP) used in the treatment of testicular cancer causes impaired spermatogenesis and reduced sperm production in the rat. However, definitive evidence about the potential impact of such chemotherapy on SSCs is still lacking. The objective of this study was to determine whether chronic exposure to BEP treatment causes adverse effects on rat SSC activity. We first investigated the effects of BEP treatment on the clonal organization of undifferentiated spermatogonia by staining whole-mount preparations of rat seminiferous tubules for GFRA1 and ZBTB16 (previously known as PLZF), 2 established markers of undifferentiated spermatogonia. We found that BEP treatment drastically reduced the number of A-aligned spermatogonia while sparing A-single and A-paired cells from the effect. Next, we determined the SSC activity following BEP exposure. Adult transgenic rats carrying EGFP expression in the germ line were treated with BEP for 9 weeks, and SSCs were quantified using spermatogonial transplantation. We found that BEP treatment significantly decreased SSC numbers, which were restored to the control level after a 9-week recovery period. These results demonstrate that BEP treatment transiently affects the activity of rat SSCs.

Reversibility of the effects of the chemotherapeutic regimen for non-Hodgkin lymphoma, cyclophosphamide, doxorubicin, vincristine, and prednisone, on the male rat reproductive system and progeny outcome.

The chemotherapeutic agents used to treat non-Hodgkin lymphoma, cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), have adverse effects on male reproductive function and progeny outcome. To determine the reversibility of these effects, male rats received a CHOP treatment mimicking human exposure. CHOP reduced testicular and epididymal weights; these remained decreased after 9 weeks recovery. This treatment also decreased testicular sperm number and increased spermatozoal DNA damage. Although sperm production returned to control values after 9 weeks recovery, DNA damage persisted. Decreased litter size, and increased pre- and post-implantation losses were observed among litters sired by CHOP-exposed males. Litter size and pre-implantation loss returned to control within 3 weeks post-treatment and post-implantation loss by 6 weeks. Thus, effects of CHOP on progeny outcome were reversed 9 weeks post-treatment, although germ cell DNA breaks remained elevated. These data suggest that the ability to sire viable progeny may not be a sensitive measure of spermatozoal quality in rats.

Reversibility of the effects of subchronic exposure to the cancer chemotherapeutics bleomycin, etoposide, and cisplatin on spermatogenesis, fertility, and progeny outcome in the male rat.

Testicular cancer is the most common cancer among young men of reproductive age. A regimen of bleomycin, etoposide, and cisplatin (BEP regimen) is the standard chemotherapy for testicular cancer. BEP has adverse effects on spermatogenic function that pose a long-term reproductive health risk to cancer survivors and their progeny. Using a rat model, we investigated the persistence of the effects of BEP on male reproductive function, fertility, and progeny outcome. Adult male Sprague-Dawley rats received a BEP regimen mimicking human clinical exposure (three 21-day cycles of etoposide and cisplatin on days 1-5 and bleomycin on days 2, 9, and 16, or vehicle). Reproductive and progeny outcome parameters were assessed at the end of BEP treatment and up to 9 weeks post-treatment, at 3-week intervals. BEP treatment reduced testicular weights and impaired spermatogenesis, characterized by abnormal testis histology and germ cell depletion. Germ cell apoptosis increased at least 3-fold in BEP-treated rats compared with controls at the end of treatment; 9 weeks posttreatment, germ cell apoptosis in BEP-treated rats did not differ from controls. BEP-exposed males were fertile; a decrease in litter size and an increase in preimplantation and postimplantation losses were observed. Preimplantation loss remained elevated in litters sired by BEP-treated males up to 9 weeks posttreatment; however, neither postimplantation loss nor litter sizes differed from controls. Thus, both germ cell apoptosis and the postimplantation loss induced by BEP treatment were reversible. The persistence of the elevation in preimplantation loss 9 weeks after BEP treatment suggests that spermatogonia are affected.

Transient DNA strand breaks during mouse and human spermiogenesis new insights in stage specificity and link to chromatin remodeling.

In the course of mammalian spermiogenesis, a unique chromatin remodeling process takes place within elongating and condensing spermatid nuclei. The histone-to-protamine exchange results in efficient packaging and increased stability of the paternal genome. Although not fully understood, this change in chromatin architecture must require a global but transient appearance of endogenous DNA strand breaks because most of the DNA supercoiling is eliminated in the mature sperm. To establish the extent of DNA strand breakage and the stage specificity at which these breaks are created and repaired, we performed a sensitive terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay to detect in situ DNA strand breaks on both mice and human testis cross sections. In the mouse, we established that DNA strand breaks are indeed detected in the whole population of elongating spermatids between stages IX and XI of the seminiferous epithelium cycle perfectly coincident with the chromatin remodeling as revealed by histone H4 hyperacetylation. Similarly, TUNEL analyses performed on human testis sections revealed an elevated and global increase in the levels of DNA strand breaks present in nuclei of round-shaped spermatids also coincident with chromatin remodeling. The demonstration of the global character of the transient DNA strand breaks in mammalian spermiogenesis suggests that deleterious consequences on genetic integrity of the male gamete may arise from any disturbance in the process. In addition, this investigation may shed some light on the origin of the low success rate that has been encountered so far with intracytoplasmic injection procedures making use of round spermatids in humans.

Transition nuclear proteins are required for normal chromatin condensation and functional sperm development.

The histone-to-protamine transition is important in the formation of spermatozoa. In mammals this involves two steps: replacement of histones by transition nuclear proteins (TPs) and replacement of TPs by protamines. To determine the functions of the TPs and their importance for sperm development, we generated mice lacking both TPs, since mice lacking only TP1 or TP2 were fertile. Our results indicated that TP1 and TP2 had partially complemented each other. In mice lacking both TPs, nuclear shaping, transcriptional repression, histone displacement, and protamine deposition proceeded relatively normally, but chromatin condensation was irregular in all spermatids, many late spermatids showed DNA breaks, and protamine 2 was not posttranslationally processed. Nevertheless, genomic integrity was maintained in mature spermatids, since efficient fertilization and production of offspring were achieved by intracytoplasmic sperm injection. However, many mature spermatids were retained in the testis, epididymal spermatozoa were drastically reduced in number and were highly abnormal, and the mice were sterile. Most epididymal spermatozoa were incapable of fertilization even using intracytoplasmic sperm injection. Thus, in mammals TPs are required for normal chromatin condensation, for reducing the number of DNA breaks, and for preventing the formation of secondary defects in spermatozoa, eventual loss of genomic integrity, and sterility.