Effects of endocrine disrupting chemicals on gonad development: Mechanistic insights from fish and mammals.

Over the past century, evidence has emerged that endocrine disrupting chemicals (EDCs) have an impact on reproductive health. An increased frequency of reproductive disorders has been observed worldwide in both wildlife and humans that is correlated with accidental exposures to EDCs and their increased production. Epidemiological and experimental studies have highlighted the consequences of early exposures and the existence of key windows of sensitivity during development. Such early in life exposures can have an immediate impact on gonadal and reproductive tract development, as well as on long-term reproductive health in both males and females. Traditionally, EDCs were thought to exert their effects by modifying the endocrine pathways controlling reproduction. Advances in knowledge of the mechanisms regulating sex determination, differentiation and gonadal development in fish and rodents have led to a better understanding of the molecular mechanisms underlying the effects of early exposure to EDCs on reproduction. In this manuscript, we review the key developmental stages sensitive to EDCs and the state of knowledge on the mechanisms by which model EDCs affect these processes, based on the roadmap of gonad development specific to fish and mammals.

The effects of chemotherapy with bleomycin, etoposide, and cis-platinum on telomeres in rat male germ cells.

Co-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 γ-H2AX immunofluorescence. Interestingly, treatment with this BEP regimen increased γ-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.

Paternal exposure to testis cancer chemotherapeutics alters sperm fertilizing capacity and affects gene expression in the eight-cell stage rat embryo.

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.

Characterization of sperm chromatin quality in testicular cancer and Hodgkin's lymphoma patients prior to chemotherapy.

BACKGROUND: Although the incidences of testicular cancer and Hodgkin's lymphoma have increased in young men over the past decade, combination chemotherapy has improved survival. As fertility is of importance to these patients, characterization of sperm chromatin structure is needed. We assessed sperm chromatin in testicular cancer and Hodgkin's lymphoma patients prior to chemotherapy, in comparison with control community and idiopathic infertile volunteers. METHODS: DNA damage was assessed with the sperm chromatin structure assay (SCSA), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and comet assays; reactive thiols (SH) and DNA compaction were determined with the monobromobimane (mBBr) and chromomycin A3 (CMA3) assays, respectively. RESULTS: Both testicular cancer (37%) and Hodgkin's lymphoma (81%) patients had normospermic samples with increased DNA damage, compared with controls. Cancer patients also had higher reactive thiols and CMA3 staining, indicating low DNA compaction. CONCLUSIONS: Sperm DNA integrity and compaction were affected in testicular cancer and Hodgkin's lymphoma patients prior to chemotherapy. Although SCSA, TUNEL and comet assays all detected DNA damage, the latter was optimal for use in cancer patients. A combination of the comet assay with tests that evaluate sperm DNA compaction, such as flow cytometry-based CMA3 and mBBr assays, is a reliable strategy to characterize sperm chromatin quality in cancer patients at the time of sperm banking.

Exposure of male rats to cyclophosphamide alters the chromatin structure and basic proteome in spermatozoa.

BACKGROUND: The formation of mature sperm involves the expression of numerous proteins during spermiogenesis and the replacement of histones with protamines to package the genome. Exposure to cyclophosphamide (CPA), an anticancer alkylating agent, during spermiogenesis may disrupt chromatin condensation with adverse consequences to the offspring. METHODS: Adult male rats were given CPA in one of two schedules: (i) subchronic, 4 days - day 1 (100 mg kg(-1)) and days 2-4 (50 mg kg(-1) per day) or (ii) chronic - daily (6.0 mg kg(-1) per day). Animals were euthanized on days 14, 21 or 28. RESULTS: The effects of CPA on epididymal sperm chromatin structure were germ-cell-phase specific; mid-spermiogenic spermatids were most sensitive. The acridine orange DNA denaturation assay showed significant increases in susceptibility to denaturation (P < 0.01). Chromatin packaging assessment revealed 1,4-dithiothreitol-dependent chromomycin A3 DNA binding and less condensed, protamine-deficient sperm; the total thiol (P < 0.001) and protamine contents (P < 0.01), measured using monobromobimane and the HUP1N protamine 1 antibody, respectively, were reduced. The sperm basic proteome was also altered; proteins that were identified are involved in events during spermiogenesis and fertilization. CONCLUSIONS: Paternal exposure to CPA alters sperm chromatin structure, as well as the composition of sperm head basic proteins. We speculate that these changes underlie effects on fertilization and embryo development.

Expression of base excision, mismatch, and recombination repair genes in the organogenesis-stage rat conceptus and effects of exposure to a genotoxic teratogen, 4-hydroperoxycyclophosphamide.

BACKGROUND: DNA repair capability may influence the outcome of genotoxic teratogen exposure. The goals of this study were to assess the expression of base excision repair (BER), mismatch repair (MMR), and recombination repair (RCR) genes in the mid-organogenesis rat conceptus and to determine the effects on expression of exposure to the genotoxic teratogen, 4-hydroperoxycyclophosphamide (4-OOHCPA). METHODS: The expression of 17 BER, MMR, and RCR genes was examined in gestational day (GD) 10-12 rat conceptuses using the antisense RNA (aRNA) technique. Embryos were cultured with 10 microM 4-OOHCPA to examine effects on gene expression. RESULTS: Yolk sacs and embryos had similar gene expression patterns for all three DNA repair pathways from GD10-12. Transcripts for APNG, PMS1, and RAD54 were present at high concentrations in both tissues. The remainder of the genes were expressed at low levels in yolk sac, with a few not detected on GD10 and 11. In the embryo, transcripts for most genes were low on GD10 and 11; several increased by GD12. After exposure to 4-OOHCPA for 24 hr, XRCC1 and RAD57 expression decreased in yolk sac, whereas only RAD51 transcripts decreased in the embryo. By 44 hr, transcripts for all BER genes decreased in yolk sac; in the embryo, most BER, MMR, and RCR genes decreased, many below the level of detection. CONCLUSIONS: The expression of DNA repair genes in the mid-organogenesis rat conceptus is varied and subject to down-regulation by 4-OOHCPA. DNA repair gene expression may determine the consequences of genotoxicant exposure during development.

Acute cyclophosphamide exposure has germ cell specific effects on the expression of stress response genes during rat spermatogenesis.

Exposure of male rats to cyclophosphamide, a commonly used anticancer and immunosuppressive drug, has been shown to alter fertility and progeny outcome in a male germ cell phase-specific manner. The effect of toxicant exposure on male germ cells depends in part on the stress response mechanisms present during the different stages of spermatogenesis. To assess how acute cyclophosphamide exposure affects the expression of stress response genes, we examined the expression of 216 genes, using gene expression arrays, in isolated rat spermatogenic cell types (pachytene spermatocytes, round spermatids, and elongating spermatids). Cyclophosphamide exposure affected gene expression in all cell types but most dramatically in round spermatids. Increased transcript levels were observed for 30 genes in round spermatids compared to seven genes in pachytene spermatocytes and two in elongating spermatids. The expression of genes involved in apoptosis, DNA-damage recognition and repair, transcriptional activation, and in the heat shock protein-chaperone response was most affected by cyclophosphamide in round spermatids. Our results demonstrate that cyclophosphamide alters the expression of stress response genes during spermatogenesis in a germ cell-specific manner. The greater response of round spermatids to cyclophosphamide suggests that this cell type may be more susceptible to the damaging effects induced by this drug, possibly due to the chromatin remodeling that is taking place at this stage of spermatogenesis. This observation is consistent with the reported higher level of abnormal progeny outcome seen when the germ cells were first exposed to cyclophosphamide as round spermatids.

Nucleotide excision repair gene expression in the rat conceptus during organogenesis.

DNA repair may be a determinant of the susceptibility of the conceptus to DNA damaging teratogens. The nucleotide excision repair (NER) pathway repairs a substantial amount of chemically induced DNA damage. The goals of this study were to assess the coordinate expression of NER genes in the midorganogenesis-stage rat conceptus and determine the consequences of exposure to the genotoxic teratogen, 4-hydroperoxycyclophosphamide (4-OOHCPA), on NER gene expression. Most NER genes were expressed at low levels in both yolk sac and embryo on gestational day (GD) 10, with the exception of XPD, XPE and PCNA. No significant alterations in gene expression occurred between GDs 10 and 11; in the yolk sac XPB expression increased on GD12 compared to either GD10 or 11. In the embryo, XPE expression increased between GDs 10 and 12, while hHR23B, XPB, ERCC1, and DNA polymerase epsilon expression increased on GD12 relative to both GDs 10 and 11. Contrary to gene expression data, XPB protein was found at high levels and XPD at low levels in GDs 10-12 embryos and yolk sacs. Mirroring gene expression, high levels of PCNA protein were found in both tissues; XPA protein levels were minimal in yolk sac from GDs 10-12 but increased in the embryo from moderate on GD10 to high on GD12. Therefore, NER gene expression during organogenesis was regulated in a developmental stage- and tissue-specific manner. Exposure of the conceptus to a teratogen, 4-OOHCPA, induced malformations without affecting NER transcript levels. Thus, NER gene expression in the conceptus was unresponsive to regulation by DNA alkylation.

Expression of stress response genes in germ cells during spermatogenesis.

During germ cell development different spermatogenic cell types show remarkable variation in their susceptibility to stressful stimuli. Various cellular mechanisms are triggered in germ cells after exposure to stress, but the expression of only a few of the genes involved in such pathways has been studied during spermatogenesis. In the present study we determined the expression profiles of 216 stress response genes in isolated rat germ cells (pachytene spermatocytes, and round and elongating spermatids) using cDNA atlas arrays. Of the 216 genes studied, 86 were detected in pachytene spermatocytes, 82 in round spermatids, and 52 in elongating spermatids. Fifty percent (48) of the total number of genes detected during spermatogenesis were detected in all three cell types while nearly 25% (25) were expressed exclusively in pachytene spermatocytes and round spermatids; some cell specific transcripts were observed also. The use of the K means clustering method allowed us to group genes by their pattern of expression during spermatogenesis; five specific expression profiles were obtained and analyzed. To determine how stress response genes are regulated throughout spermatogenesis, we examined the expression of genes involved in stress response mechanisms such as heat shock proteins-chaperones, DNA repair, and oxidative stress. Genes belonging to these families were differentially expressed during germ cell development. We suggest that the differential expression of stress response genes during spermatogenesis contributes to the selectivity of the susceptibility of germ cells to stress.

Paternal exposure to cyclophosphamide induces DNA damage and alters the expression of DNA repair genes in the rat preimplantation embryo.

Chronic low dose treatment of male rats with cyclophosphamide, an anticancer alkylating agent, damages male germ cells, resulting in greater than 80% peri-implantation progeny loss. Little transcription or repair takes place in the DNA of post-meiotic male germ cells. The spermatozoal genome regains its transcriptional capacity in the fertilized oocyte. We hypothesized that as a consequence of exposure of male rats to cyclophosphamide DNA damage to the male genome is transmitted to the conceptus; furthermore, this damage leads to alterations in the expression profiles of DNA repair genes during preimplantation development. Male rats were treated with either saline or cyclophosphamide (6mg/kg/day, 4-6 weeks) and mated to control females; 1-8 cell stage embryos were collected. The alkaline comet assay was used to assess DNA damage in 1-cell embryos. A significantly higher percentage (68%) of the embryos fertilized by cyclophosphamide-exposed spermatozoa displayed a comet indicative of DNA damage, compared to those sired by control males (18%). The in situ transcription/antisense RNA approach was used to determine if DNA damage alters the expression of DNA repair genes in early embryos. Dramatic increases in the transcripts for selected members of the nucleotide excision repair family (XPC, XPE and PCNA), mismatch repair family (PMS1), and recombination repair family (RAD50) were found in 1-cell stage embryos sired by cyclophosphamide-treated males compared to controls, while decreases in the expression of base excision repair family members (UNG1, UNG2, and XRCC1) and in recombination repair transcripts (RAD54) were observed. By the 8-cell stage, transcripts for specific members of the nucleotide excision repair family (XPC) and mismatch repair family (MSH2, PMS2) were elevated greatly in control embryos compared to embryos sired by drug-treated males; in contrast, transcripts for other members of the nucleotide excision repair family (XPE, PCNA), as well as some of the base excision repair family (UNG1), were elevated in embryos sired by drug-treated males. Therefore, DNA damage incurred in spermatozoa, following cyclophosphamide exposure is associated with alterations in the expression profiles of DNA repair genes in preimplantation embryos as early as the 1-cell stage. Genotoxic stress may disturb the nuclear remodeling and reprogramming events that follow fertilization and precede zygotic genome activation.

Paternal exposure to cyclophosphamide dysregulates the gene activation program in rat preimplantation embryos.

Although there has been progress in determining the mechanisms by which maternal toxicant exposure affects progeny, there is little information on the actions of drugs administered to the father. We investigated the effects of pre-conceptional paternal exposure to cyclophosphamide, an anti-cancer agent, on embryonic gene activation in the rat. The male pronucleus was formed earlier in embryos sired by cyclophosphamide-treated male rats than in those sired by controls; early male pronucleus formation was followed by alterations in the gene activation program. BrUTP incorporation into RNA and Sp1 transcription factor immunostaining were increased and spread over both cytoplasmic and nuclear compartments in 2-cell embryos sired by cyclophosphamide-treated males compared to controls. Total RNA synthesis was constant in 1-8 cell embryos sired by drug-treated fathers, while in control embryos RNA synthesis increased four-fold to peak at the 4-cell stage. In 2-cell embryos sired by drug-treated males, the relative abundance of candidate imprinted genes was elevated significantly above control; a peak in the expression of these genes was not observed until the 8-cell stage in control embryos. Thus, paternal drug exposure temporally and spatially dysregulated rat zygotic gene activation, altering the developmental clock.

Paternal exposure to cyclophosphamide alters cell-cell contacts and activation of embryonic transcription in the preimplantation rat embryo.

Paternal exposure to chronic low doses of cyclophosphamide, an anticancer agent, results in aberrant embryonic development of the progeny. We hypothesized that paternal exposure to cyclophosphamide disturbs zygotic gene activity regulating proper progression through preimplantation development and that this disturbance results in improper cell-cell interactions. To test this hypothesis, we analyzed cell-cell interactions and the expression of cytoskeletal elements in preimplantation embryos sired by male rats gavaged with saline or 6 mg kg(-1) day(-1) cyclophosphamide for 5 wk. Embryos from control litters had 4-12 cells on Day 2 of gestation; cell-cell contacts were observed consistently. Embryos from litters sired by cyclophosphamide-treated males were frequently abnormal and had lower cell numbers and decreased cell-cell contacts. Steady state concentrations of the mRNAs for cell adhesion molecules (cadherins and connexin 43) and structural proteins (beta-actin, collagen, and vimentin) were low in two- and four-cell control embryos; expression increased dramatically by the eight-cell stage. In contrast, embryos sired by cyclophosphamide-treated males displayed the highest expression of most trancripts at the two-cell stage. In parallel with the mRNA profiles, E-cadherin immmunoreactivity was nearly absent in two-cell control embryos and was strong by the eight-cell stage; immunoreactivity in embryos sired by drug-treated fathers was strong at the two-cell stage but absent at later stages. Thus, drug exposure of the paternal genome led to dysregulated expression of structural elements and decreased cell interactions during preimplantation embryonic development.

Critical windows of exposure for children's health: the reproductive system in animals and humans.

Drugs and environmental chemicals can adversely affect the reproductive system. Currently, available data indicate that the consequences of exposure depend on the nature of the chemical, its target, and the timing of exposure relative to critical windows in development of the reproductive system. The reproductive system is designed to produce gametes in far greater excess than would seem to be necessary for the survival of species. Ten to hundreds of millions of spermatozoa are generated daily by most adult male mammals, yet very few of these germ cells succeed in transmitting their genetic material to the next generation. Although the number of oocytes produced in mammalian females is more limited, and their production occurs only during fetal life, most ovaries contain several orders of magnitude more oocytes than ever will be fertilized. Toxicant exposures may affect critical events in the development of the reproductive system, ranging from early primordial germ cell determination to gonadal differentiation, gametogenesis, external genitalia, or signaling events regulating sexual behavior. Although there are differences between the human reproductive system and that of the usual animal models, such models have been extremely useful in assessing risks for key human reproductive and developmental processes. The objectives for future studies should include the elucidation of the specific cellular and molecular targets of known toxicants; the design of a systematic approach to the identification of reproductive toxicants; and the development of sensitive, specific, and predictive animal models, minimally invasive surrogate markers, or in vitro tests to assess reproductive system function during embryonic, postnatal, and adult life.

Workshop to identify critical windows of exposure for children's health: reproductive health in children and adolescents work group summary.

This work group report addresses the central question: What are the critical windows during development (preconception through puberty) when exposure to xenobiotics may have the greatest adverse impact on subsequent reproductive health? The reproductive system develops in stages, with sex-specific organogenesis occurring prenatally and further maturational events occurring in the perinatal period and at puberty. Complex endocrine signals as well as other regulatory factors (genetics, growth factors) are involved at all stages. Evidence from animal models and human studies indicates that many specific events can be perturbed by a variety of toxicants, with endocrine-mediated mechanisms being the more widely studied. Prioritized research needs include basic studies on the cellular-molecular and endocrine regulation of sexual differentiation and development; increased efforts regarding potential adverse effects on development in females, including breast development; expanded animal studies on different classes of chemicals, comparing responses during development (prenatal and postnatal) with responses in adults; and, more extensive explorations regarding the reproductive biology and toxicology of puberty in humans.

Post-translational regulation of AP-1 transcription factor DNA-binding activity in the rat conceptus.

Activator protein-1 (AP-1) transcription factor DNA binding is induced during transient oxidative stress in the midorganogenesis rat conceptus in culture. L-2-Oxothiazolidine-4-carboxylate (OTC), a cysteine prodrug, prevented oxidative stress and the induction of AP-1 binding activity in the embryo but not in the yolk sac. Because AP-1 activity may be a significant determinant of developmental outcome after insult, we investigated the regulation of AP-1 activity in the conceptus. Supershift assays indicated that basal AP-1 binding in the embryo was due primarily to JunD, whereas in the yolk sac c-Jun and JunD were important. Under oxidative stress, c-Fos and c-Jun contributed to the AP-1 binding in the embryo; in the yolk sac, a c-Fos-shifted complex emerged. OTC protection from oxidative stress did not change the AP-1 composition, suggesting that increased AP-1 activity was due to post-translational modifications. Changes in AP-1 activity in embryos under oxidative stress or with OTC protection were not the result of alterations in the net phosphorylation state of Fos or Jun proteins or of changes in activities of the extracellular signal-regulated kinases 1 and 2 or stress-activated protein kinases. However, immunodepletion of redox factor 1 (Ref-1), a nuclear factor that promotes AP-1 binding, eliminated AP-1 activity from embryonic nuclear extracts under both basal and oxidative stress conditions. Therefore, Ref-1 plays a critical role in regulating AP-1 activity in the conceptus; it is plausible that Ref-1-mediated modulation of the AP-1 stress response is a determinant of embryonic fate.

Tissue-specific regulation of glutathione homeostasis and the activator protein-1 (AP-1) response in the rat conceptus.

Oxidative stress in the conceptus is characterized by an increased oxidized to reduced glutathione (GSSG:GSH) ratio and the induction of fos and jun mRNAs, transcripts for components of the activator protein-1 (AP-1) transcription factor. We investigated the role of glutathione homeostasis in the rat conceptus in the regulation of: (1) AP-1 expression and activity, and (2) the activities of glutathione-dependent cytoprotective enzymes. Glutathione content was enhanced with the addition of l-2-oxothiazolidine-4-carboxylate (OTC), a precursor of cysteine, a rate-limiting substrate in glutathione biosynthesis. Day 10 rat conceptuses were cultured for 44 hr with 0, 5, 10, or 20 mM OTC. High concentrations (10 and 20 mM) of OTC were embryotoxic. Incubation of the conceptus in 5 mM OTC caused mild (not statistically significant) embryotoxicity, increased significantly the embryonic glutathione content, prevented culture-induced oxidative stress, and inhibited the induction of AP-1 transcripts and DNA binding activity in the embryo. In contrast, in the yolk sac, 5 mM OTC failed to increase glutathione content or to prevent oxidative stress or AP-1 induction. Thus, regulation of glutathione status in the conceptus is tissue-specific. Glutathione S-transferase and glutathione peroxidase activities were increased approximately 50% in cultured embryos and yolk sacs. OTC treatment (5 mM) prevented this induction in the embryo, but not in the yolk sac, suggesting a role for glutathione homeostasis in the regulation of these enzymes. Tissue-specific regulation of glutathione status and of cytoprotective enzymes in the conceptus during organogenesis may impact on the consequences of insult with oxidative stress.

The role of p53 and cell death by apoptosis and necrosis in 4-hydroperoxycyclophosphamide-induced limb malformations.

The exposure of embryonic murine limbs in vitro to an activated analog of cyclophosphamide, 4-hydroperoxycyclophosphamide (4OOH-CPA), induced limb malformations and apoptosis. The purpose of this study was to investigate the role of the tumor suppressor/cell cycle checkpoint gene, p53, and of cell cycle arrest in the response of the limbs to cyclophosphamide. Limbs, excised on day 12 of gestation from wild-type, heterozygous or homozygous p53-knockout transgenic murine embryos, were treated with vehicle (water) or 4OOH-CPA (0.3, 1.0 or 3.0 microgram/ml) and cultured for 6 days. Exposure of wild-type (+/+) limbs to 4OOH-CPA resulted in limb malformations, and reduced limb areas and developmental scores. The homozygous (-/-) limbs were dramatically more sensitive to the effects of 4OOH-CPA, as assessed by limb morphology, area and score. Heterozygous limbs exposed to the drug were intermediate for each parameter. Apoptosis, as assessed by the formation of a DNA ladder, was increased in drug-exposed wild-type limbs, but not in the drug-exposed homozygous limbs. Light and electron microscopy examination of the limbs revealed that drug treatment of wild-type limbs induced the morphological changes typical of apoptosis, particularly in the interdigital regions. In contrast, there was no evidence of apoptosis in homozygous limbs exposed to 4-OOH-CPA; morphological characteristics of necrosis such as cell membrane breakdown, mitochondrial swelling and cellular disintegration were evident throughout these limbs. Heterozygous limbs had cells dying with the characteristics of both apoptosis and necrosis. Fragments of poly(ADP-ribose) polymerase characteristic of necrosis predominated in the drug-treated heterozygous and homozygous limbs. 4-OOH-CPA-treatment of limbs from wild-type embryos led to arrest of the cell cycle at the G1/S phase. No cell cycle arrest was observed after drug treatment of homozygous limbs, in which populations of cells in S and G2/M phases, as well as a population of sub G1 cells, were found. Thus, the presence of p53 and of p53-dependent apoptosis protect organogenesis-stage limbs from insult with a teratogen. The absence of p53 may decrease DNA repair capacity and contribute to the accumulation of DNA damage in limb cells and their daughter cells; the failure of apoptosis to eliminate cells with DNA damage may result in increased cell death by necrosis and major limb malformations.

Induction of apoptosis in the germ cells of adult male rats after exposure to cyclophosphamide.

Treatment with cyclophosphamide, a commonly used anticancer drug, may result in oligozoospermia or azoospermia. The objective of this study was to determine whether exposure of male rats to cyclophosphamide induces apoptosis in male germ cells, and if so, when the peak of apoptosis occurs and at what specific stages of spermatogenesis. The presence of apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) detection in situ and by an increase in DNA fragmentation (DNA ladder). To determine the time course of drug-induced apoptosis, male Sprague-Dawley rats were treated with a single dose (70 mg/kg BW) of cyclophosphamide, and the testes were fixed 0, 4, 8, 12, 18, 24, and 48 h after treatment. To determine the dose response, rats were treated with doses of cyclophosphamide (0, 2, 7, 20, and 70 mg/kg), and the testes were fixed 12 h after treatment. A low spontaneous incidence of apoptosis was observed in controls, in particular in premeiotic germ cells of stages I-IV and XI-XIV of the seminiferous tubules. In cyclophosphamide-exposed rats, the incidence of apoptosis increased progressively at 4 h and 8 h, reached a peak at 12 h (about 3.5-fold above control), and then decreased rapidly to control levels by 48 h. A 70-mg/kg dose of cyclophosphamide induced a significant increase in apoptosis; lower doses did not. Although drug-induced apoptosis occurred in all stages of germ cells, it was most pronounced in spermatogonia and spermatocytes in stages I-IV and XI-XIV. Thus, apoptosis may be involved in the occurrence of oligozoospermia or azoospermia after cyclophosphamide treatment. Apoptosis of damaged premeiotic germ cells may serve a critical role in protecting subsequent generations from the diverse effects of toxicants.