Journal of Andrology, Vol. 22, No. 6, November/December 2001 Copyright 䉷 American Society of Andrology
Paternal Exposure to Drugs and Environmental Chemicals: Effects on Progeny Outcome
Review
BARBARA F. HALES* AND BERNARD ROBAIRE*†
cancer chemotherapeutic drugs, and social drugs such as alcohol or cigarette smoke have been evaluated for malemediated adverse effects on progeny outcome. The possibility has also been assessed that offspring conceived using assisted reproduction technologies may be at increased risk for adverse outcomes. Ionizing Radiation—Analyses of pregnancy outcome data among survivors of the bombing in Hiroshima and Nagasaki have suggested that either there is not an increased risk of congenital malformations (Neel and Schull, 1956), or that a small but not significant trend exists toward an increased risk with increasing parental radiation exposure (Otake et al, 1990). In a case-control study of the liveborn infants of workers in Canada’s largest nuclear electrical generating company, the risk of congenital malformation was not increased among children whose parents were occupationally exposed to ionizing radiation prior to conception (Green et al, 1997). In contrast, increased risk of stillbirth with increasing preconceptional exposure to ionizing radiation was reported for fathers working at the Sellafield nuclear site in Cambria, United Kingdom (Parker et al, 1999). A subsequent study by Doyle and coworkers (2000), based on a large cohort of nuclear industry workers in the United Kingdom, found no evidence of a link between exposure to lowlevel ionizing radiation before conception and an increased risk of adverse reproductive outcome among men working in the nuclear industry. Thus, little support exists for a link between preconceptional exposure to low-level ionizing radiation and increased risk of male-mediated adverse reproductive outcomes. Nevertheless, quantitative ultramorphological analyses of organelles in the sperm head have revealed significant differences between individuals who were engaged in clean-up operations after the Chernobyl nuclear plant disaster in Ukraine and control individuals of similar ages (Fischbein et al, 1997). Currently, it is not possible to rule out possible adverse effects of high-level exposures. Heavy Metals—Occupational exposure to metals such as lead and mercury are most prevalent in metal foundries; battery plants; metal scrapping, car repair and service, and glass and pottery industries; chloralkali plants; pesticide production; and the manufacture of electrical
From the Departments of *Pharmacology and Therapeutics and †Obstetrics and Gynecology, McGill University, Montre´al, Que´bec, Canada.
There are growing societal concerns about the consequences on reproduction and development of exposure to toxicants. The demonstration in 1977 that dibromochloropropane (DBCP), a testicular toxicant, caused permanent sterility among a high proportion of exposed workers was a major trigger for investigation of the effects of drugs and environmental chemicals on male reproductive health (Whorton et al, 1977; Potashnik et al, 1979). Data from epidemiological studies have suggested that paternal occupations such as welding, painting, auto mechanics, or firefighting, which involve exposure to metals, combustion products, solvents, and pesticides, may be associated with an increase in spontaneous abortions, birth defects, and childhood cancer (Olshan and Mattison, 1994). However, it has proven difficult to confirm some of these findings, both because the data are often conflicting and because they usually refer to broad and heterogeneous exposure paradigms. Yet, epidemiological studies still provide some of the most powerful evidence for understanding the potential consequences on the progeny of a man’s exposure to drugs and environmental chemicals. Highlights from some of these studies are summarized in the first part of this review. To obtain an accurate understanding of which chemicals can have deleterious effects on a man’s progeny and the mechanism by which they act, it is essential for ethical and practical reasons to use animal models. In the second part of this review we assess the mounting evidence that has accumulated on this subject from animal models.
Evidence From Human Studies Exposure to ionizing radiation, welding fumes, heavy metals such as lead, organic solvents, some pesticides, Supported by the Canadian Institutes for Health Research. Correspondence to: B. Hales, Department of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir-William-Osler, Montre´al, Que´bec, Canada H3G 1Y6. (e-mail:
[email protected]). Received for publication January 30, 2001; accepted for publication March 19, 2001.
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928 supplies. A tendency toward reduced semen quality has been seen in men exposed to heavy metals (Bonde 1993; Irgens et al, 1999). Epidemiological studies have indicated that paternal exposure to heavy metals such as lead or mercury may be associated with an increased risk of spontaneous abortion (reviewed in Anttila and Sallme´n, 1995). Other studies have provided limited support for the hypothesis that low-level paternal exposure to lead is associated with decreased fertility (Sallme´n et al, 2000a); in an accompanying study, it was suggested that among fertile couples, lead exposure was related to childlessness rather than to a delay of pregnancy (Sallme´n et al, 2000b). An increased risk of spontaneous abortion was found in pregnancies in which the father worked as a stainless steel welder; there was no increased risk for spontaneous abortion in pregnancies in which the father was a welder of other metals (Hjollund et al, 2000). Fumes from the welding of stainless steel, but not mild steel, contain hexavalent chromium; this ion is mutagenic and, interestingly, when administered to a male rodent, it can induce an increase in the incidence of lung tumors in its offspring (Anderson et al, 1994). Organic Solvents—Several organic solvents to which men are occupationally exposed have been studied for their potential adverse effects on progeny outcome. These include toluene, n-hexane, xylene, ethyl acetate, carbon disulfide, ethylene glycol ethers and their acetates, styrene, trichloroethylene, tetrachloroethylene, and 1,1,1-trichloroethane. Wives of men exposed to perchloroethylene in the dry cleaning industry (Eskenazi et al, 1991) and to ethylene glycol ethers in semiconductor manufacturing (Correa et al, 1996) had slightly prolonged times to pregnancy. A study in Finland that monitored male workers for their organic solvent exposure found an increased time to pregnancy, but only among primigravida (Sallme´n et al, 1998). A study in the Netherlands found that implantation rates were decreased among couples undergoing in vitro fertilization-embryo transfer (IVF-ET) treatment in which men worked in occupations with high levels of organic solvent exposure; occupational exposure among women was not associated with a decrease in implantation rate (Tielemans et al, 2000). Analysis of semen quality during styrene exposure revealed a decline in sperm density, total sperm count, and the proportion of sperm with normal morphology (Kolstad et al, 1999b). Nevertheless, a multicenter study of European workers exposed to styrene in the reinforced plastics industry showed no detrimental effect of styrene exposure on male fecundity (Kolstad et al, 1999a). Pesticides and Other Polychlorinated Hydrocarbons— There is wide concern about the effects of pesticides on reproduction and the health of children. The results from a multicenter study in Europe of paternal pesticide exposure during the spraying season found no effect on se-
Journal of Andrology · November/December 2001 men quality or fertility, as assessed by time to pregnancy (Thonneau et al, 1999). Nevertheless, paternal pesticide exposure has been reported to decrease sperm fertilizing ability in vitro among those seeking IVF treatment (Tielemans et al, 1999). Preconceptional paternal pesticide exposure has been associated with an increased risk for acute lymoblastic leukemia in children aged 0–9 years (Infante-Rivard and Sinnett, 1999). Phenoxy herbicides, including 2,4,5-trichlororophenoxyacetic acid (2,4,5-T) and 2,4-dichlorophenoxyacetic acid (2,4-D), are among the most widely studied. Agent Orange, the predominant herbicide used in the Vietnam War, contained these 2 chemicals with dioxin (2,3,7,8-tetrachlorodibenzo--dioxin, TCDD) contamination. A number of published studies suggested there may be an increased risk of fetal death among the progeny of Vietnam War veterans who had been exposed to these chemicals (reviewed by Arbuckle and Sever, 1998); however, the relative risks for fetal death ranged from 0.87 to 3.2, and no causal relationship has been established to date. Some studies have attempted to assess the level of exposure. For example, in semen samples of Ontario farmers, 50% showed measurable levels of 2,4-dichlorophenoxyacetic acid, with the mean level similar to that in urine (Arbuckle et al, 1999). Because many active ingredients exist in pesticides, including solvents such as benzene, xylene, and toluene, it is a challenge to assign relative risk, to identify which agent is responsible for toxic effects, and to draw any definitive conclusions about the consequences on progeny of paternal exposure to pesticides. TCDD is one of the most toxic manmade substances. In 1976 in Sevoso, Italy, a plant that manufactured the herbicide 2,4,5-trichlorophenol exploded, releasing more than 30 kg of dioxin into the environment and exposing many people to TCDD. A study (Mocarelli et al, 2000) of the exposed population found that paternal, but not maternal, exposure to TCDD has been linked to a lowered male:female sex ratio in offspring. The 92 men in the study who had serum dioxin concentrations greater than 15 parts per trillion and who were younger than age 19 in 1976 fathered significantly more girls than boys, even in 1991, more than 15 years after the 1976 exposure. The mechanism for the regulation of the normal sex ratio (0.514) is not known. Dioxin has been shown to have antiandrogenic properties, and in animal studies, to permanently alter sperm transit time through the epididymis (Gray et al, 1997). In addition to effects on sperm maturation in the epididymis, chlorinated hydrocarbons may affect primordial germ cells in the embryo. In utero exposure of hamsters to polychlorinated biphenyls and polychlorinated dibenzofurans via contaminated cooking oil has resulted in sperm with increased abnormal morphology, reduced motility, and reduced capacity to penetrate oocytes after pu-
Hales and Robaire · Paternal Drug Exposure Affects Progeny berty (Guo et al, 2000). Whether these effects will result in reduced fecundity remains to be investigated. Anticancer Drugs—Long-term survivors of childhood cancer have an increased incidence of azoopermia and severe oligoasthenozoospermia (Lopez Andreu et al, 2000). A fertility deficit of about 60% has been associated with men who have undergone chemotherapy with alkylating agents (Byrne et al, 1987). It has been suggested that the infertility observed in humans after radiation and chemotherapy may be due to a failure in the differentiation of spermatogonia, rather than to the death of stem cells; fertility may be restored by hormone treatments that relieve the block (Meistrich, 1998). Significant increases in the frequency of disomy and diploidy were observed during chemotherapy, but these effects were transient; the increased risk of aneuploid sperm declined to pretreatment levels within 100 days of the end of chemotherapy (Robbins et al, 1997; Martin, 1998). Epidemiological studies have failed to demonstrate any increase in the risk of malformations or childhood cancer in the children of men who survive cancer treatment (Byrne et al, 1998; Byrne, 1999). In such studies, termination of exposure to chemotherapeutic agents usually occurs well before conception is attempted; hence, these studies test only the long-term consequences of such exposures on the quality of male germ stem cells. Animal experiments have provided clear evidence that paternal exposure to cancer therapeutic agents can induce adverse effects on offspring (Robaire and Hales, 1999; Trasler and Doerksen, 1999; Brinkworth, 2000); however, the most dramatic effects are found during treatment and shortly thereafter. Based on observations of increased aneuploidy in spermatozoa during chemotherapy and on animal studies, it is clear that men should be advised not to attempt to conceive children during exposure to such drugs or in the first cycle of spermatogenesis after termination of therapy. There appears to be little risk of fathering abnormal progeny after a prolonged discontinuation period. Social Drugs—Cigarette smoke, caffeine, and alcohol consumption do not appear to significantly affect sperm nuclear size, shape, or chromatin texture, as assessed by computer-aided semen analysis (CASA) in a study of 86 healthy male volunteers in North Carolina (Vine et al, 1997). Yet, in another study among 18-year-old smokers recruited from the Czech Republic, there was an elevated frequency of sperm aneuploidy (Y disomy), a reduced linearity of sperm motion, and more round-headed sperm (Rubes et al, 1998). Among these teenagers, smoking was highly correlated with alcohol, but not with caffeine, consumption. These authors concluded that active cigarette smoking, particularly in teenage men who consume alcohol, should be considered a potential genetic hazard capable of producing trisomy in future unexposed progeny. Other studies have shown higher levels of oxidative
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DNA adducts in the nuclei of spermatozoa in men who smoke (Fraga et al, 1996). Assisted Reproductive Technology—There are concerns that offspring conceived with the aid of assisted reproduction technologies may be at increased risk for adverse outcomes. Studies on the outcome of children conceived by conventional IVF-ET have shown no difference in the incidence of major malformations or developmental outcome compared with non-IVF controls (Olivennes et al, 1997). Intracytoplasmic sperm injection (ICSI) was introduced in 1993 for couples in whom fertilization and pregnancy rates were poor with IVF-ET. Martin (1998) found that infertile men who were candidates for ICSI had an increased frequency of disomy for chromosomes 1, 13, 21, and XY. It was suggested that infertile men have decreased recombination and pairing, leading to meiotic arrest (oligospermia) and nondisjunction. The incidence of major malformations detected at birth or in the perinatal period among offspring conceived by ICSI has been reported to range from 0.95% to 3.6% (Palermo et al, 1996; Bonduelle et al, 1999; Sutcliffe et al, 1999). Although most investigators concluded that there was no evidence of excess major birth defects among offspring conceived by ICSI, others arrived at an opposing conclusion. Sample sizes may be insufficiently large to ensure that the results are interpreted with confidence. Concerns were raised about the developmental potential of these children by a study in Australia that found that the mean Bayley mental development index at 1 year of age was significantly lower in children conceived by ICSI than in children conceived by IVF or naturally (Bowen et al, 1998). It was suggested that possible reasons for poorer performance among the children conceived by ICSI may include genetic abnormalities in the sperm as well as the ICSI procedure itself. In contrast, Bonduelle and coworkers reported no indication that children conceived by ICSI had slower mental development, as assessed by the Bayley test at the age of 2 years, than children conceived by IVF or than those in the general population (Bonduelle et al, 1998). Recently, Neuber and coworkers (1999) have shown that human sperm undergo nuclear envelope breakdown, chromatin reorganization, pronuclei formation, and DNA replication in an extract from Xenopus eggs. Two of the 8 samples in subfertile men displayed rates of DNA replication that were, interestingly, faster than any of those in proven fertile donors. Because DNA replication is stringently regulated, it will be to interesting to explore the mechanisms underlying this observation.
Evidence From Animal Models A major difficulty in human studies is that with few exceptions, there are no data on the specific chemical or exposure, dose, duration of exposure, and potential chem-
930 ical interactions. In addition, infertility and pregnancy loss are frequent events in humans; to demonstrate a significant increase requires both a large study population and a marked augmentation in risk. In animal studies it is possible to define the exposure chemical, dose, and duration. Extensive evidence exists in animal models to show that exposure to specific drugs, radiation, or environmental chemicals alters male reproductive health, resulting in adverse progeny outcome. Furthermore, these models have proven useful in elucidating the mechanisms that underlie the adverse effects of paternal chemical exposures on progeny outcome. Exposure of males to drugs, radiation, or environmental chemicals may adversely affect progeny via two major mechanisms. The first is by direct exposure of the embryo or developing fetus during mating to a chemical in the seminal fluid. The second is by a direct genetic or epigenetic effect of the toxicant on the male germ cell either during spermatogenesis in the testis or sperm maturation in the epididymis. Direct Exposure of the Conceptus—Most drugs or chemicals to which males are exposed have access to the seminal fluid (Pichini et al, 1994) and may enter a female’s reproductive tract during mating. The literature contains evidence suggesting that methadone and morphine administered to a man have adverse effects on progeny outcome as a consequence of the presence of the chemical in seminal fluid (Soyka et al, 1978). In contrast, no adverse developmental effects were observed when cisplatin-treated males were mated to untreated females 6–24 hours after drug administration, at a time when cisplatin DNA adducts were present in spermatozoa (Hooser et al, 2000). However, another anticancer alkylating agent, cyclophosphamide (and its metabolites), administered to male rats, was found in seminal fluid, transmitted to the female partner during mating, and caused a dose-dependent increase in preimplantation loss (Hales et al, 1986). It is likely that most drugs will be present in seminal fluid only at low levels; the potency of the toxicant, level of exposure, and window of exposure during development are likely to be determinants of the adverse effects on development resulting from direct exposure via this route. Unlike humans, animals used as research subjects do not mate after the female has become pregnant; therefore, such models do not assess the consequences that may arise from multiple exposures of pregnant women during intercourse to drugs in semen. The Male Germ Cell as a Target—Toxicants may directly affect male germ cells either during sperm maturation in the epididymis or spermatogenesis in the testis, and radiation and drugs both affect spermatozoa during transit through the epididymis and vas deferens. A twofold increase in mutation frequency was found in bone marrow cells in the F1 generation of male mice irradiated 1–7 days prior to mating (Luke et al, 1997). Treatment of
Journal of Andrology · November/December 2001 male rats with cyclophosphamide resulted in an increase in postimplantation loss (dominant lethality) when the exposed spermatozoa originated in the head or body, but not the tail, of the epididymis (Qiu et al, 1992). Methyl chloride induced an increase in dominant lethal mutations that was believed to be a consequence of the selective inflammatory action of this drug on the epididymis; the increase in embryo loss was reversed by an anti-inflammatory agent (Chellman et al, 1986a,b). Most studies of the mechanisms by which drugs administered to the male have adverse effects on progeny outcome have emphasized the germ cell in the testis as the target. Spermatogenesis is a highly ordered and regulated process. Establishment of the primordial germ cell lineage occurs early during development; in the mouse on GD 8, 10–100 primordial germ cells can be identified; primordial germ cells replicate by mitosis so that their numbers depend on mitotic activity. In mice, in utero exposure of male fetuses to a toxicant such as N-ethyl-Nnitrosourea induced a high rate of recessive mutations in the primordial germ cells of male progeny (Shibuya et al, 1996). Spermatogonia undergo several mitotic cell divisions (5 in the rat) in the adult testis to become spermatocytes, which then undergo 2 meiotic cell divisions to form spermatids (spermacytogenesis). Spermatids differentiate into spermatozoa primarily through the condensation of nuclear elements, development of a propulsion mechanism, and by shedding most of their cytoplasm (spermiogenesis; Clermont, 1972). One can deduce the stage specificity of the susceptibility of the germ cells during spermatogenesis by timing the effect of toxicant exposure (Clermont, 1972; Russell, 1994). An effect on progeny outcome during the first week after exposure of an adult male rat to a drug or x-rays may be a consequence of an effect on spermatozoa in the epididymis (Qiu et al, 1992; Russell, 1994). Spermatozoa are also maximally sensitive to the specific locus mutations induced by acrylamide monomer and the dominant lethality induced by ethylnitrosourea (Russell, 1994). In rodent models, exposure to a drug 2–4 weeks prior to conception represents an effect on germ cells that were first exposed to the toxicant when they were spermatids; for example, following exposure of mice to anticancer drugs such as chlorambucil, a peak in mutation yield was observed when offspring were conceived from germ cells that were exposed as spermatids (Russell, 1994). Toxicant exposure 5–6 weeks prior to mating results in fertilization by germ cells that were first exposed as spermatocytes; exposures of 7–9 weeks or longer prior to conception represent an effect on germ cells that were first exposed as spermatogonia (Trasler et al, 1985; 1986). Germ cells at the spermatogonial stage are very susceptible to drugs such as procarbazine or to x-ray exposure, resulting in reduced sperm numbers and an increased percentage of
Hales and Robaire · Paternal Drug Exposure Affects Progeny morphologically abnormal surviving sperm (Russell, 1994; Kangasniemi et al, 1995). Whereas the nature of a toxicant will effect its germ cell stage specificity, chromatin remodeling during sperm maturation and spermatogenesis will determine accessibility of the paternal genome. The capacity of the germ cell to repair incurred damage is a second important determinant of progeny outcome. Male-mediated developmental toxicants such as cyclophosphamide, acrylamide, and ethylnitrosourea induce dominant lethality, heritable translocations, and specific locus mutations. Other manifestations of exposure to a male-mediated developmental toxicant include behavioral alterations and an increased incidence of tumors in F1 progeny. Distinct manifestations are observed with different compounds. For example, in F1 progeny, Edwards et al (1999) observed an increase in incidence and a decrease in time of onset of liver tumors, without increasing dominant lethality, when males were treated for 10 weeks with the drug ethylcarbamate (urethane). The anticancer alkylating agent cyclophosphamide has served in several laboratories as a model male-mediated developmental toxicant. Cyclophosphamide has marked dose-dependent and time-specific effects on progeny outcome (Trasler et al, 1985, 1986, 1987; Qiu et al, 1992; Anderson et al, 1995). An increase in postimplantation loss was found when male rats were treated for 2 weeks with chronic, low-dose cyclophosphamide; by the 4th week of treatment this postimplantation loss rose dramatically to plateau at a level that was dependent on drug dosage, and was reversed within 4 weeks of drug treatment termination (Hales and Robaire, 1990). Thus, cyclophosphamide-induced postimplantation loss was associated primarily with germ cell exposure during spermiogenesis. Postmeiotic germ cells were susceptible to the induction of learning abnormalities in progeny after paternal exposure to cyclophosphamide (Fabricant et al, 1983). Heritable translocations were found in mice after exposure of spermatids and spermatozoa to a single high dose of cyclophosphamide (Generoso et al, 1981). Exposure of rat spermatocytes to cyclophosphamide resulted in synaptic failure, fragmentation of the synaptonemal complex, and altered centromeric DNA sequences (Backer et al, 1988). An increase in malformed and growth-retarded fetuses was observed after 7–9 weeks of drug treatment; the malformations observed were principally hydrocephaly, edema, and micrognathia (Trasler et al, 1986). Therefore, external malformations and growth retardation were produced in progeny sired by germ cells first exposed to cyclophosphamide as spermatogonia. Spermatogonia were, interestingly, reported to be at low risk for the induction of heritable translocations by cyclophosphamide (Sotomayor and Cumming, 1975; Generoso et al, 1981).
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Significantly, an increase in postimplantation loss and malformations persisted to the F2 generation (Hales et al, 1992). Observed malformations included open eyes, omphalocele, generalized edema, syndactyly, gigantism and dwarfism; these F2 generation malformations were similar to those found in the F1 generation (Trasler et al, 1986). Some of the behavioral abnormalities caused by paternal cyclophosphamide treatment also persisted in subsequent generations (Auroux et al, 1988, 1990). Nagao and Fujikawa (1996) suggested that the types of malformations induced by male-mediated developmental toxicants such as ethylnitrosourea are the same as those that occur spontaneously. Future research is needed to determine whether specific loci encode these specific malformations, and if so, whether these loci serve as ‘‘hot spots’’ in the male genome with increased susceptibility to damage. Genetic Effects—The male genome is a central focus for studies on the mechanism of the effects of paternal exposure to toxicants on progeny outcome. Studies with mice doubly heterozygous for Robertsonian translocations provided evidence that there is no selection against aneuploid sperm during spermiogenesis, fertilization, or the first cell cycle of zygotic development (Marchetti et al, 1999). The results of specific-locus mutation studies have been used to suggest that exposure of spermatogonia to chemicals or radiation yields few large lesions, whereas large lesions are common after exposure of postspermatogonial germ cells. At the chromosome level using cytogenetic approaches, it has proven difficult to detect even bulky deletions, aneuploidy, or chromosomal duplications in spermatozoa (Allen et al, 1994). Mature spermatozoa do not undergo mitosis. Hence it was only by allowing denuded hamster eggs to be fertilized by spermatozoa that chromosomal structures in the male pronucleus could be analyzed. This approach was used to demonstrate that age, x-irradiation, and drugs affect the chromosomal banding pattern of human sperm (Martin et al, 1989). More recently, fluorescent in situ hybridization (FISH) has been developed for analysis of aneuploidy in the male genome (Wyrobek et al, 1994; Martin et al, 1995; Lowe et al, 1998). Using this approach, Marchetti et al (1997) showed that high levels of chromosomally defective zygotes were detected among the progeny of male mice after treatment with acrylamide for 5 days. Moreover, the proportion of zygotes with chromosomal aberrations was highly correlated to the proportions of dominant lethality and heritable translocations among the progeny. Using the Comet assay, we have shown that cyclophosphamide-exposed spermatozoa imparted significantly greater DNA damage to the newly fertilized egg at the one-cell stage than did control spermatozoa (Harrouk et al, 2000a). It is clear that sperm with DNA damage have the ability to fertilize an oocyte, and it is interesting that the extent of
932 DNA damage by gamma radiation was not a factor in determination of fertilization rate (Ahmadi and Ng, 1999). The overall consequence of germ cell exposure to a toxicant depends on both the extent and specificity of DNA damage and on the DNA repair processes that are active at various stages of spermatogenesis. Treatment with cyclophosphamide for 1 week caused DNA singlestrand breaks; no DNA cross-links were observed (Qiu et al, 1995a). By contrast, 6 weeks of treatment with cyclophosphamide induced a significant increase in DNA single-strand breaks and cross-links, primarily DNA-DNA, in spermatozoal nuclei (Qiu et al, 1995a). When the appropriate DNA repair systems are either absent or overwhelmed, a male germ cell with damaged DNA has two options: 1) it may survive and fertilize an oocyte; DNA repair in the oocyte may rescue the zygote or abnormal progeny may result; or, alternatively, 2) the damaged male germ cell may undergo apoptosis. A low incidence of spontaneous apoptosis is observed in the seminiferous tubules of testes from control rats (Cai et al, 1997; Sinha Hikim and Swerdloff, 1999; Brinkworth and Nieschlag, 2000); in cyclophosphamide-exposed rats, the incidence of apoptosis increased to a level 3.5-fold above control by 12 hours after treatment (Cai et al, 1997). Drug-induced apoptosis was most pronounced in premeiotic germ cells (spermatogonia and spermatocytes; Cai et al, 1997; Sinha Hikim and Swerdloff, 1999; Brinkworth and Nieschlag, 2000). We hypothesize that apoptosis of damaged premeiotic germ cells serves a critical role in protecting subsequent generations from the diverse effects of toxicants. Postmeiotic germ cells may have lost the components that are essential to undergo apoptosis. Thus, cyclophosphamide may exert its maximal effects on elongating spermatids and spermatozoa (postmeiotic germ cells) because these cells have lost both the ability to repair DNA and to undergo apoptosis. Exposure to cyclophosphamide altered in vitro spermatozoal decondensation (Qiu et al, 1995b); spermatozoa from drug-treated males remained quite compacted, whereas chromatin from control spermatozoa dispersed completely. Decreased decondensation and DNA synthesis were observed when sperm isolated from rats treated with cyclophosphamide for 6 weeks were incubated with cytoplasmic extracts of Xenopus laevis eggs (Sawyer and Brown, 2000). However, in denuded hamster oocytes, the decondensation of spermatozoa from cyclophosphamidetreated males was more rapid than that of control spermatozoa (Harrouk et al, 2000b). It is not clear whether effects on spermatozoal decondensation are primarily due to DNA damage or protamine cross-linking. Exposure to lead, another male-mediated developmental toxicant, caused a conformational change in protamine, binding at two different sites; this interaction between lead and protamine resulted in a dose-dependent decrease in the extent
Journal of Andrology · November/December 2001 of protamine-DNA binding (Quintanilla-Vega et al, 2000). These authors suggested that alterations in protamine-DNA interaction may disturb sperm chromatin condensation or decondensation and reduce fertility. A disturbance in male germ cell chromatin condensation, remodeling, and pronucleus formation may result in adverse effects on embryo development. Fertilization with damaged or poor quality sperm may lead to aberrant pronuclear development. A significantly higher incidence of mosaicism was found in human embryos that exhibited two pronuclei differing in size by at least 4 m (Sadowy et al, 1998). Indeed, male pronucleus formation was early in rat oocytes sired by cyclophosphamide-treated males (Harrouk et al, 2000b). It is likely that chromatin remodeling is required to make the male genome accessible to DNA polymerases for DNA replication. The first round of DNA replication is important for reprogramming gene expression in the zygote (Davis and Schultz, 1997). Restriction of sperm template function is released in vitro in sperm that are decondensed by treatment with disulfidereducing agents (Qiu et al, 1995b). The in vitro template function of spermatozoal DNA was markedly affected after 6 weeks of treatment with cyclophosphamide (Qiu et al, 1995b). One of the mechanisms underlying male-mediated adverse effects on progeny outcome may be dysregulation of zygotic gene activation. Lead was found to affect the proteins synthesized in 2-cell embryos fathered by male rats with blood lead levels in the range of 15–23 g/dL (Gandley et al, 1999). Total RNA synthesis was constant in 1- to 8-cell embryos sired by cyclophosphamide-treated fathers, whereas in control embryos, RNA synthesis increased fourfold to peak at the 4-cell stage. Moreover, both BrUTP incorporation into RNA and Sp1 transcription factor immunostaining were increased and spread over the cytoplasmic and nuclear compartments in 2-cell embryos sired by cyclophosphamide-treated males (Harrouk et al, 2000b). The profile of expression of specific genes, even as early as the 1-cell stage, was altered in embryos sired by drug-treated males (Harrouk et al, 2000a,b,c). By the 2-cell stage, the relative abundance of transcripts for candidate genes was elevated significantly above control in embryos sired by cyclophosphamidetreated males; a peak in the expression of many of these genes was not observed until the 8-cell stage in control embryos (Harrouk et al, 2000b). One consequence of a disturbance in zygotic gene activity as a result of paternal exposure to cyclophosphamide may be improper cell-cell interactions. Steady state concentrations of the messenger RNAs for cell adhesion molecules were low in 2- and 4cell control embryos, but increased dramatically by the 8-cell stage. In contrast, embryos sired by cyclophosphamide-treated males displayed the highest expression of most of these transcripts at the 2-cell stage (Harrouk et
Hales and Robaire · Paternal Drug Exposure Affects Progeny al, 2000c). Embryos from litters sired by cyclophosphamide-treated males had lower cell numbers and decreased cell-cell contacts (Austin et al, 1994; Harrouk et al, 2000c). Cell-cell interactions are essential for normal blastocyst development. Epigenetic Mechanisms—The effects of a male-mediated developmental toxicant may be mediated via an epigenetic mechanism if the toxicant affects gene expression, genomic imprinting, or DNA methylation. Paternal and maternal genomes have complementary roles during mammalian development; both are required for development to term. The expression of genomically imprinted genes is dependent on whether the allele is inherited from the spermatozoa or from the oocyte (Barton et al, 1984; Surani et al, 1984). Parthenogenetic and gynogenetic mouse embryos (with 2 maternal genomes) have poor extraembryonic tissue development and die at or before day 10 of gestation (Barton et al, 1984; Surani et al, 1984). Exposure of the male gamete to a toxicant may damage the paternal genome by selectively targeting imprinted genes expressed from the paternal allele, such as Peg1/ Mest, Igf 2, or Snrpn. This ‘‘insult’’ could be manifested as expression from neither allele, failure to mark the paternal allele, or as expression from both alleles due to relaxation of the paternal imprint. Differential DNA methylation at cytosine residues in CpG dinucleotides in critical domains plays a role in the monoallelic expression of imprinted genes (Bartolomei et al, 1993; Li et al, 1993). Methylation is erased and reset during male and female gametogenesis, when the differential marking of imprinted genes is believed to occur (Obata et al, 1998; Kerjean et al, 2000). Methylation is the only known epigenetic signal that is retained by the paternal genome throughout male gamete maturation, when chromatin is reorganized and histones are replaced by protamines. The acquisition of the typical paternal methylation profile of imprinted genes may represent a critical component of spermatogenesis. In addition, mutations arising through deamination of methylated cytosine account for approximately a third of all point mutations, making CpG dinucleotides ‘‘hot spots’’ in the genome. DNA methylation may have an essential role in transcriptional silencing in the embryo. Transient depletion of Xenopus DNA methyltransferase 1 led to premature gene activation two cell cycles earlier then normal (Stancheva and Meehan, 2000). Chronic treatment of male rats with 5-azacytidine, a drug that alters DNA methylation, affected male germ cells, resulting in abnormal early embryo development among their progeny (Doerksen and Trasler, 1996). The extent to which exposure to other toxicants targets genomic imprinting in animal models, or in humans, deserves further investigation.
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Future Directions In human studies, significant progress is being made in exposure assessment and the use of markers of male fecundity, such as seminal characteristics or reproductive hormone profiles, time to pregnancy, and fertility rates. An explosion in the development and availability of new scientifically based biomarkers for risk assessment in male reproductive health will permit important advances in this field and have clinical implications. Recently developed markers of semen quality include CASA, sperm chromatin structure assays with acridine orange, and FISH to detect aneuploidy. Analysis of human sperm activation, pronucleus formation, and DNA replication is now feasible using model systems such as Xenopus egg extracts. Studies of the mechanisms by which paternal exposures adversely affect progeny outcome remain a high priority. There is no quick, easy screen for male-mediated developmental toxicity. It is an expensive and time consuming proposition to test extensive lists of chemicals. Molecular characterization of the male reproductive system and its regulation will help us understand how toxicant-induced alterations affect fertility and progeny outcome. Numerous questions need to be addressed, such as the following: ● To what extent are toxicant insults to primordial germ cells in the embryo or fetus responsible for adverse effects on male reproductive health and progeny outcome? ● What is the role of perinatal exposure in impaired testicular function in adulthood? ● What is the nature of toxicant-induced damage in male germ cells? ● Are specific targets in the male genome responsible for male-mediated developmental toxicity? ● What factors determine the germ cell stage specificity of this insult? ● What is the role of the paternal genome in embryonic development and hereditary damage? Answers to some of these questions are essential in order to develop approaches to predict and prevent male-mediated adverse progeny outcomes. It is the integration of the results of basic science and mechanistic studies with epidemiology and clinical investigations that will permit us to define the role of paternal exposures in male reproductive health and the fate of offspring. More effective collaborations between clinicians, epidemiologists, and basic scientists are essential in order to identify specific chemicals or groups of chemicals that affect progeny outcome and to determine the cellular and molecular mechanisms underlying such actions. This is increasingly important in the era of genome sequencing, cloning, epigenetics, proteomics, and assisted reproductive technologies.
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