Mechanisms of Toxic Damage to Spermatogenesis

Abstract

Azoospermia and long-lasting testicular atrophy are common adverse consequences of cancer treatment. Chemotherapeutic agents may disrupt spermatogenesis by targeting various testicular cell types (Leydig cells, Sertoli cells, and germ cells) and by activating numerous molecular pathways involved in germ cell life-and-death decision making. Genetically modified animal models with deficiencies in specific proapoptotic and prosurvival pathways have become powerful tools in understanding the molecular regulation of spermatogenesis and the response of the seminiferous epithelium to toxic injury. In this brief review, selected examples of results of toxic exposures in genetically deficient animal models are discussed to highlight the roles of p53 and the Fas system as modulators of proapoptotic activity in the testis. A final section focuses on cisplatin, a cancer chemotherapeutic agent that produces male reproductive toxicity by targeting multiple cell types in the testis.

The testis produces mature gametes through a complex process of stem germ cell commitment to proliferation and differentiation, modulated by a network of endocrine and paracrine regulatory inputs. This brief review of mechanisms of toxic damage to spermatogenesis begins with the general background information necessary to understand the response of the seminiferous epithelium to injury and then focuses on specific target cells and molecular pathways. A final section uses the example of cisplatin toxicity to illustrate the diverse mechanisms and manifestations of testicular injury produced by a single chemotherapeutic agent.

BACKGROUND

The human testis is a known target organ for injury resulting from exposure to both therapeutic and toxic environmental agents. Although there are many possible mechanisms and manifestations of toxic damage to spermatogenesis (e.g., defects in spermiogenesis, retained spermatids, and sloughing of the seminiferous epithelium), this brief review focuses on one early consequence of a toxic exposure: induction of germ cell apoptosis, or programmed cell death. A feared long-term outcome of a toxic exposure is the development of persistent testicular atrophy, which if severe, can result in long-lasting azoospermia and infertility.

The testis has very active prosurvival and proapoptotic systems that work together to regulate the extent of germ cell apoptosis. The proliferation of germ cells is normally excessive, and a tightly regulated system of physiological apoptosis optimizes the output of germ cells to a level sustainable by the seminiferous epithelium. Both deficient and excessive apoptosis can result in decreased sperm output: Deficient apoptosis of early spermatogonia can result in an overcrowding of the seminiferous epithelium, displacing maturing germ cells, whereas excessive apoptosis of early spermatogonia may result in insufficient maturing germ cells. After exposure to a testicular toxicant, apoptosis is often markedly increased, indicating that the seminiferous epithelium is dysfunctional and cannot provide optimal support, or that direct damage to germ cells is severe. These observations of normal and toxicant-induced germ cell apoptosis point to the testis as having an active, highly developed, and tightly controlled apoptotic machinery. If the toxic exposure is brief or not too severe, the seminiferous epithelium can often recover and rapidly reestablish normal spermatogenesis. However, if the injury is protracted or severe, the seminiferous epithelium may become depleted of germ cells or irreversibly damaged, resulting in spermatogenic failure. Our increasing understanding of these processes has depended on the use of genetically modified animals and cell-specific toxic exposures.

TARGET CELLS AND MOLECULAR PATHWAYS

Within the testis, the three main target cells for toxicants that disrupt spermatogenesis are the somatic cells, the Leydig and Sertoli cells, and the germ cells themselves. In animal models of exposure, each of these cell types can be selectively targeted by specific toxicants, resulting in germ cell apoptosis and spermatogenic failure (Table 1).

Ethane-1,2-dimethanesulfonate is a cytotoxic alkylating agent that is selective for adult rat Leydig cells. Exposure causes Leydig cell ablation, a rapid decrease in testosterone levels, and a characteristic pattern of germ cell loss (1). This characteristic pattern of germ cell loss has been investigated using a variety of hormonally disruptive techniques (2) and agents (3) and is manifested as apoptosis of specific developmental stages of germ cells; over time, this stage-specific germ cell apoptosis results in the depletion of maturing germ cells from the seminiferous epithelium (Table 1). Presumably, the underlying molecular pathways affected in this hormone insufficiency response are the androgen-dependent transcriptional programs of testicular somatic cells (4). Recent data would indicate that the primary action of testosterone is to repress gene expression (5).

Phthalates, such as di-(2-ethylhexyl) phthalate, are ubiquitous environmental toxicants capable of producing testicular atrophy in laboratory animals. Mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of di-(2-ethylhexyl) phthalate, targets Sertoli cells, making them dysfunctional, and also results in a rapid induction of testicular germ cell apoptosis, with spermatocytes as the most sensitive population (Table 1) (6). The Fas system is a ligand-receptor system that triggers the death of cells expressing Fas through an interaction with caspase 8, initiating the execution cascade of apoptosis. Fas ligand is expressed by Sertoli cells, and Fas receptor is expressed by germ cells (6), indicating this as a paracrine pathway of apoptotic control in the testis. Animal experiments have indicated that the Fas system is involved in MEHP-induced germ cell apoptosis (6,7). The attenuated germ cell apoptotic response to MEHP exposure in mice lacking a functional Fas ligand (called gld mice), when compared to wild-type mice, strongly supports a role for the Fas system in regulating the testicular response to injury (8). Mice lacking functional p53 are also protected from MEHP-induced germ cell apoptosis (unpublished data).

The effects on spermatogenesis of ionizing irradiation have been studied extensively [reviewed in (9)]. Germ cell death occurs by apoptosis (10) and results from free radical–induced alterations in DNA. As a consequence, the actively dividing spermatogonia are the most susceptible, followed by spermatocytes [Table 1; for review, see (11)]. p53, most commonly known as a tumor suppressor, also has roles as a cell cycle check, an inducer of apoptosis [for review see (12)], and a DNA repair factor. During spermatogenesis, p53 mRNA and protein are present in primary spermatocytes (13–16), indicating that p53 plays a role in the prophase of meiosis. p53 is closely associated with the nuclear membrane, and on induction, it is translocated to the nucleus, where it may serve as a transcription factor (17).

p53 has been identified as a major proapoptotic factor regulating the testicular response to x-irradiation (18). Experiments were designed to determine whether Fas receptor is involved in p53-dependent, radiation-induced germ cell death. After exposure to 5 Gy x-irradiation, Fas mRNA expression remained at pretreatment levels in p53 knock-out mice. However, Fas mRNA increased in a time-dependent manner in wild-type mice following exposure to 5 Gy radiation, indicating that radiation-induced Fas expression is p53 dependent (19). The functional significance of Fas involvement was demonstrated when lpr^cg mice, having a nonfunctional Fas receptor, were exposed to 5 Gy radiation and had a reduced number of apoptotic seminiferous tubules 12 hours postdose (19). In addition, lpr^cg mice exposed to 0.5 Gy radiation had increased spermatid head counts 29 days postdose, relative to wild-type mice. Interestingly, gld mice with a nonfunctional Fas ligand were just as sensitive to radiation as wild-type animals, and levels of Fas ligand mRNA were not affected by radiation treatment (19).

In summary, the apoptotic response to the germ cell toxicant, x-irradiation, is p53 dependent. Furthermore, although Fas receptor is necessary, in part, for radiation-induced p53-dependent apoptosis, Fas ligand is not. In contrast, the apoptosis that results from the Sertoli cell toxicant MEHP is at least partially dependent on both FasL and p53. Therefore, these mouse models provide useful insight into the mechanisms involved in the response to injuries such as ionizing radiation and phthalate exposure, and they help to discern the differences in germ cell apoptosis induced by germ cell toxicants versus Sertoli cell toxicants.

CISPLATIN-INDUCED TESTICULAR INJURY

Cisplatin is an important chemotherapeutic agent that is noted for its activity against testicular germ cell cancer. As a heavy metal coordination compound, cisplatin produces cross-links, including the DNA cross-links that are presumably responsible for its antineoplastic effect (20). Chemotherapy with cisplatin can have profound and long-lasting effects on spermatogenesis (21).

Studies in mice have demonstrated acute damage to spermatogenesis following intraperitoneal or intravenous injection of cisplatin. At low doses, cisplatin was selectively toxic for spermatogonia, with an LD₅₀ of 1.1 mg/kg (22). At higher doses, cisplatin had broad activity, killing some cells in all stages, including spermatocytes and spermatids in the adlumenal compartment (22). This activity against cells in the adlumenal compartment, which is normally protected by the blood–testis barrier, indicated that cisplatin might induce Sertoli cell toxicity (23). Stem cells appeared to be relatively resistant to cisplatin-induced toxicity (22).

Detailed investigations in animal models of potential cellular targets and mechanisms of toxicity within the testis indicate that cisplatin has broad activity, targeting Leydig cells, Sertoli cells, and germ cells. This is not surprising, given that cisplatin is chemically reactive as a crosslinking agent.

Adult rats exposed to cisplatin (7–9 mg/kg intravenously) had a dramatic reduction in serum and intratesticular testosterone 7 days after exposure—an effect that was largely reversed by treatment with human chorionic gonadotropin (hCG) (24,25). Cisplatin exposure did not decrease serum luteinizing hormone (LH) or follicle stimulating hormone levels. The cisplatin-induced changes in testosterone were associated with a decreased number of LH receptors on Leydig cells and with inhibited p450 side-chain cleavage activity (25). A gonadotropin-releasing hormone (GnRH) challenge test led to prompt multifold increases in serum LH as well as to increases in serum and intratesticular testosterone levels, indicating that high levels of LH could still stimulate steroidogenesis. These cisplatin-induced changes in the hormonal regulation of spermatogenesis were interpreted as a primary effect on Leydig cells, with relative sparing of the hypothalamus and pituitary (25).

Changes in Sertoli cell structure and function following cisplatin exposure have indicated that this cell is a target cell for toxicity. As early as 24 hours after intraperitoneal injection of cisplatin, 2 mg/kg per day, for 5 days, inter-Sertoli cell junctions (the blood–testis barrier) became leaky—an effect that persisted for at least 40 days (26,27). This alteration in Sertoli cell junctions could explain observed changes in seminiferous tubule fluid electrolytes (26). A decrease in serum and epididymal androgen binding protein levels further indicated an effect of cisplatin exposure on Sertoli cell function (28). Primary cultures of Sertoli cells exposed to cisplatin had reduced production of transferrin, androgen binding protein, lactate, and estradiol (28,29).

In addition to modifying DNA, cisplatin produces protein adducts and cross-links and a chemical reactivity similar to that of the Sertoli cell toxicant, 2,5-hexanedione. Because 2,5-hexanedione alters Sertoli cell microtubule function (30), this molecular target was examined following cisplatin exposure. Assembly of tubulin purified from the testes of rats exposed to cisplatin was abnormal, with a marked slowing of the rate of cold-induced disassembly (31). This cisplatin-induced increase in microtubule stability was a taxol-like effect that was similar to that observed with 2,5-hexanedione exposure (30). Additional in vitro experiments confirmed that cisplatin altered microtubule assembly, producing cold stable, and short microtubules. The cisplatin effect could be ameliorated by diethyldithiocarbamate, indicating that a sulfhydryl site on the microtubules was involved in the covalent interaction with cisplatin (31).

Recently, the dynamics of cisplatin-induced germ cell apoptosis have been evaluated. These studies confirmed that the germ cell death that results from cisplatin exposure is apoptotic, as the germ cells were positive by terminal deoxy-nucleotidyl transferase-mediated dUTP nick end labeling staining (32,33). The induction of germ cell apoptosis was rapid, reaching a peak within days of cisplatin exposure (33). Germ cell apoptosis remained above control levels 12 days after cisplatin exposure, indicating the presence of long-term damage to the seminiferous epithelium (33). Spermatocytes appeared to be more sensitive than spermatogonia to the apoptosis-inducing effects of cisplatin at all doses examined (33).

In summary, cisplatin exposure in men is capable of producing long-lasting azoospermia and testicular atrophy. Animal studies have provided evidence for cisplatin-induced dysfunction of Leydig cells, Sertoli cells, and germ cells. Therefore, it is likely that cisplatin targets multiple cell types and molecular pathways while producing testicular injury.

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