Cisplatin-induced pulse of germ cell apoptosis precedes long-term elevated apoptotic rates in C57/BL/6 mouse testis.

Chemotherapeutic doses of cisplatin impair spermatogenesis and ultimately cause azoospermia and infertility in some men. The mechanism by which cisplatin damages testicular germ cells is poorly understood. Cisplatin's impact is first detected hours after exposure in the formation of DNA cross-links followed by weeks of testicular damage. Here, we report in 11-week-old male mice an early and massive rise of germ cell apoptosis after a single intraperitoneal (i.p.) injection of either 5 or 10 mg/kg cisplatin. For the lower dose, a roughly 9-fold peak increase in the apoptotic index over the control level is observed at 36 h, and for the higher dose, a 24-fold rise is seen at 24 h. At these peak levels, the lower dose produced a higher ratio of apoptotic early spermatocytes to apoptotic spermatogonia than did the higher dose. In addition to this early wave of germ cell die-off, our data show that while the post-wave apoptotic rates for both dose regimes diminish, at 12 days the apoptotic rates appear significantly higher (5 mg/kg) than controls. In summary, our findings show two events set in motion by acute cisplatin exposure: (1) a previously unreported massive apoptotic die-off of germ cells followed by (2) an elevated apoptotic rate possibly reflecting long-term or permanent damage to the seminiferous tubule.

The relevance of spontaneous- and chemically-induced alterations in testicular germ cell apoptosis to toxicology.

Elimination of germ cells via apoptosis occurs spontaneously under normal physiologic conditions and is often heightened after chemical-induced testicular injury. Though many different apoptosis-related elements have been identified in the testis, the molecular and cellular mechanisms that regulate germ cell apoptosis are not thoroughly understood. In this review, the role of germ cell apoptosis in spermatogenesis and possible key regulators of apoptosis is described. The involvement of the Fas-signaling pathway between Sertoli cells and germ cells is highlighted as a crucial paracrine-signaling mechanism that responds to both physiologic- or toxicant-induced declines in the supportive capacity of the testis and reduces the germ cell population accordingly.

Sensitivity of testicular germ cells to toxicant-induced apoptosis in gld mice that express a nonfunctional form of Fas ligand.

Germ cell apoptosis in testis is essential for functional spermatogenesis. Recent evidence suggests that the Fas signaling system is critical for the regulation of testicular germ cell apoptosis. To further evaluate the Fas signaling system in testis, we examined the incidence of germ cell apoptosis in gld mice that lack a functional Fas-signaling pathway. gld mice have a small, but significant, increase in testis weight and numbers of spermatid heads per testis compared with wild-type mice. In addition, gld mice have a small increase in the spontaneous incidence of germ cell apoptosis, as indicated by characteristic DNA fragmentation via the terminal deoxynucleotidyl-transferase-mediated deoxy-UTP nick end labeling assay. To test the role of the Fas system in toxicant-induced germ cell apoptosis, mice were exposed to either a Sertoli cell- or germ cell-specific toxicant [mono-(2-ethylhexyl)phthalate (MEHP; 1 g/kg) or 5 Gy radiation, respectively]. These two exposure paradigms induced extensive increases in germ cell apoptosis in wild-type mice. However, exposure of gld mice to MEHP caused only a minimal increase in germ cell apoptosis, whereas they were as sensitive as wild-type mice to radiation exposure. These data indicate that the Fas signaling pathway is 1) involved in regulating the numbers of germ cells in the testis, 2) crucial for the initiation of germ cell apoptosis after MEHP-induced Sertoli cell injury, and 3) differentially active in the cell-specific regulation of germ cell apoptosis that occurs as a consequence of Sertoli cell vs. germ cell injury.

Participation of the Fas-signaling system in the initiation of germ cell apoptosis in young rat testes after exposure to mono-(2-ethylhexyl) phthalate.

The Fas-signaling system is composed of the interacting proteins Fas (CD95/APO-1) and Fas ligand (FasL, CD95L, APO-1L) and is proposed to act in the testis as a paracrine signaling mechanism by which FasL-expressing Sertoli cells initiate apoptosis of Fas-bearing germ cells. Here we describe alterations in the expression of Fas and FasL in the testis after the intimate physical association between Sertoli cells and germ cells is disrupted by exposure to the Sertoli cell toxicant mono-2-(ethylhexyl) phthalate (MEHP). Young, 28-day-old Fisher rats were treated with MEHP (2 g/kg po) and killed 0, 3, 6, and 12 h after exposure. Immunohistochemical analyses revealed a significant increase in the numbers of Fas-positive germ cells as well as increases in the expression of Sertoli cell FasL. Western blot analysis demonstrated a time-dependent increase in the production of the soluble form of FasL after MEHP exposure and suggests that it may participate in triggering apoptosis in germ cells that have lost their intimate association with the Sertoli cells. Measurement of Fas in cytosolic and membrane fractions of testis homogenates by Western blot analysis revealed a significant shift of Fas expression into the membrane fraction after MEHP exposure. Taken together, these observations indicate that the Fas-mediated paracrine signaling mechanism participates in triggering apoptosis of germ cells despite the loss of their close physical association with Sertoli cells. A working model is presented to explain the involvement of the Fas-system in stimulating germ cell apoptosis after MEHP exposure.

The Fas system, a regulator of testicular germ cell apoptosis, is differentially up-regulated in Sertoli cell versus germ cell injury of the testis.

Sertoli cells, the supportive cells in the seminiferous epithelium, orchestrate spermatogenesis by providing structural and nutritional support to germ cells. In the rat, physiological apoptosis occurs continuously to limit the size of the germ cell population to numbers that can be adequately supported. This form of germ cell death is exaggerated after testicular insults such as toxicant treatment, radiation, and heat exposure. The Fas system has been proposed as a key regulator of the activation of germ cell apoptosis. According to this model, Fas ligand (FasL) and Fas, expressed by Sertoli cells and germ cells, respectively, respond to environmental conditions and initiate germ cell death. To assess the role of the Fas system in various testicular injury models, a semiquantitative RT-PCR technique was used to evaluate the expression kinetics of both FasL and Fas after induction of massive germ cell death. Radiation exposure, which targets actively dividing germ cells, produced an up-regulation of Fas gene expression, but not FasL gene expression. However, administration of mono-(2-ethylhexyl)phthalate and 2,5-hexanedione, two widely studied Sertoli cell toxicants, resulted in up-regulated expression of both FasL and Fas. These data support the following hypotheses: 1) up-regulation of Fas is a common and critical step for initiating germ cell death in vivo; and 2) if Sertoli cells are injured, Sertoli cells up-regulate FasL to eliminate Fas-positive germ cells, which cannot be supported adequately.

Expression of Fas system-related genes in the testis during development and after toxicant exposure.

The Fas system has been identified as a key regulator of testicular germ cell apoptosis. The goal of these experiments was to explore the expression of Fas system-related genes in the testis during development and after toxicant exposure. Both Fas ligand (FasL) and Fas receptor (Fas) were expressed postnatally in rat testis with peak expression associated with the high levels of germ cell apoptosis found during the first wave of spermatogenesis. The testicular expression of RIP and FAP-1, components of the Fas activating complex, increased after exposure to mono-(2-ethylhexyl)phthalate (MEHP), a Sertoli cell toxicant which induces massive germ cell death. Finally, the expression of additional apoptosis-inducing genes, including tumor necrosis factor receptor (TNFR), FADD, TRAIL, and DR5, was detected in mammalian testis. These results provide additional support for the following concepts: (1) Sertoli-germ cell interactions are important in the control of germ cell apoptosis; and (2) the Fas system and similar paracrine systems are important modulators of testicular homeostasis.

Perturbation of the mitosis/apoptosis balance: a fundamental mechanism in toxicology.

Perturbations of the balance between cell gain via mitosis and cell loss by apoptosis play a pivotal role in mediating and modifying the action of carcinogens and other toxicants in tissues such as liver, brain, the immune system, the gastrointestinal tract, and the reproductive organs. Apoptosis describes a highly conserved morphology associated with the death of many different cell types from diverse tissues. This symposium focused on induced changes in this critical balance as a key mechanism of action of a variety of diverse toxicants. In the colon, the "toxicology" of 5 fluorouracil (5FU) is entirely dependent on p53, since p53 knockouts lose the pathology of 5FU damage. Presumably, this is because DNA damage is not detected and there is no cell cycle arrest. In the testes, testicular germ cell survival is mediated by adjacent Sertoli cells via the Fas ligand (FasL)-Fas receptor (Fas) system. This system appears to mediate germ cell apoptosis after exposure to testicular toxicants such as the phthalate, mono(2-ethylhexyl) phthalate (MEHP). Interestingly, MEHP is a member of the peroxisome proliferator (PP) class of nongenotoxic carcinogens. PPs perturb both hepatocyte apoptosis and mitosis. This suppression of apoptosis occurs via activation of the peroxisome proliferator-activated receptor alpha (PPARalpha), providing a paradigm for the regulation of liver growth via activation of nuclear receptors. Similarly, the toxicological effects of dioxins are mediated via the Ah receptor (AHR), another ligand-activated nuclear receptor. This receptor upregulates a variety of genes (the Ah gene battery) associated with the toxicology of dioxins. Taken together, the data presented in this symposium illustrate to the toxicologist the need to quantitate and interpret modulations in apoptosis alongside more conventional assessments of S-phase. Although the toxicant may initiate cell damage, genes like Bcl-2, p53, Fas, PPARalpha, and AHR are final arbiters of the choice between death, survival, and proliferation.

The Fas system is a key regulator of germ cell apoptosis in the testis.

Apoptosis occurs in the testis as an important physiological mechanism to limit the number of germ cells in the seminiferous epithelium. Sertoli cells, which tightly regulate germ cell proliferation and differentiation, are implicated in the control of germ cell apoptosis. Fas (APO-1, CD95), a transmembrane receptor protein, transmits an apoptotic signal within cells when bound by Fas ligand (FasL). The Fas system has been implicated in immune regulation, including cytotoxic T cell-mediated cytotoxicity, activation-induced suicide of T cells, and control of immune-privileged sites. Here we propose the Fas system as a key regulator of spermatogenesis. In this model, FasL expressed by Sertoli cells initiates the apoptotic death of germ cells expressing Fas. Using immunohistochemistry, we localized Fas to germ cells and FasL to Sertoli cells. The expression of these genes was dramatically up-regulated after exposure to mono-(2-ethylhexyl) phthalate and 2,5-hexanedione, two widely studied Sertoli cell toxicants known to induce germ cell apoptosis. Mouse germ cells in vitro were susceptible to anti-Fas antibody-induced death, and the survival of rat germ cells was increased after disruption of FasL by antisense oligonucleotide treatment. Unlike its expression in other tissues, testicular expression of Fas in the lpr mouse, a spontaneous mutant of the Fas gene, is similar to that in the normal mouse, arguing for the importance of the Fas system in maintaining testicular homeostasis. These data implicate the Sertoli cell in the paracrine control of germ cell output during spermatogenesis by a Fas-mediated pathway.

Mono-(2-ethylhexyl) phthalate rapidly alters both Sertoli cell vimentin filaments and germ cell apoptosis in young rat testes.

Mono-(2-ethylhexyl) phthalate (MEHP) is a widely studied Sertoli cell toxicant. Here we describe alterations in Sertoli cell vimentin filament distribution and the incidence of testicular germ cell apoptosis in young (28-day-old) Fischer rats that were treated with MEHP (2 microgram/kg, po) and killed 0, 3, 6, or 12 hr after exposure. A collapse in vimentin filaments was observed 3 hr after MEHP exposure without accompanying changes in the pattern of Sertoli cell tubulin or actin. A progressive increase in the perinuclear condensation of the vimentin filaments was observed from 6 to 12 hr after exposure. To evaluate the consequences of these Sertoli cell changes on germ cells, the role of apoptosis in MEHP-induced testicular toxicity was examined. DNA isolated from testis of rats 6 and 12 hr after MEHP exposure showed a marked increase in low-molecular-weight DNA resulting from internucleosomal cleavage. In addition, DNA fragmentation visualized in frozen testis cross sections by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick end label (TUNEL) staining demonstrated a progressive increase in germ cell apoptosis from 6 to 12 hr after MEHP exposure. However, 3 hr after MEHP exposure, the incidence of TUNEL-positive germ cells was significantly decreased compared to that seen in controls. Taken together, the early collapse in Sertoli cell vimentin filaments and the concurrent decrease in germ cell apoptosis suggests that MEHP engenders Sertoli cell dysfunction resulting in the disruption of the physiological mechanism of germ cell apoptosis.

Seminiferous tubule fluid secretion is a Sertoli cell microtubule-dependent process inhibited by 2,5-hexanedione exposure.

Mammalian Sertoli cells are responsible for the formation and secretion of seminiferous tubule fluid (STF) which provides the nutritional and hormonal microenvironment necessary for spermatogenesis. Exposure of rats to 2,5-hexanedione (2,5-HD) results in testicular injury characterized by a decrease of STF secretion which immediately precedes bulk germ cell necrosis. The earliest biochemical change in 2,5-HD-exposed rats is an alteration in testicular microtubule assembly kinetics. In this study, we investigate the relationship between microtubule-dependent processes and STF secretion in adult Sprague-Dawley CD rats by exposing seminiferous tubules to two types of toxicants: (1) those which alter microtubules (colchicine and 2,5-HD) and (2) an inhibitor of protein secretion (brefeldin A, [BFA]). Secretion of STF is quantitated by monitoring the rate of transport of a microinjected oil droplet in the lumen of isolated seminiferous tubules using time-lapse stereoscopic microscopy. The rate of oil droplet transport in seminiferous tubules isolated from testis pretreated in vivo for 2 hr with colchicine (40 micrograms/testis) was significantly decreased. Exposure of isolated seminiferous tubules to BFA (10 micrograms/ml) for 40 min also significantly decreased the transport of lumenal oil droplets. Exposure of rats to 1%, 2,5-HD in drinking water decreased transport of injected oil droplets in seminiferous tubules beginning at 3 weeks of exposure in the absence of significant alterations in testicular morphology. These data demonstrate that normal STF secretion requires an intact, microtubule-dependent intracellular membrane transport pathway and strengthen the association between 2,5-HD-induced disruption of Sertoli cell STF secretion and 2,5-HD-induced alterations in Sertoli cell microtubules.

Diisopropyl fluorophosphate inhibits receptor-activated Ca2+ influx in isolated rat hepatocytes.

The influence of diisopropyl fluorophosphate (DFP) on receptor-activated increases in cytosolic free Ca2+ concentration ([Ca2+]i) in isolated rat hepatocytes was monitored by measuring phosphorylase a activity and the fluorescence ratio of the Ca2+ sensitive dye Indo-1. Pretreatment (2 min) of hepatocytes with DFP (1 mM) inhibited maximal increases in phosphorylase a activity stimulated by phenylephrine (1 microM), angiotensin II (5 nM), or vasopressin (10 nM) by 36, 35, and 17%, respectively, when the cells were incubated in Ca2+ (1 mM)-containing medium. In contrast, agonist-stimulated increases in phosphorylase a activity were similar in control and DFP-pretreated cells when cells were incubated in medium containing very low (10 nM) Ca2+. Addition of Ca2+ (1 mM) to hepatocytes maintained in the low Ca2+ buffer and exposed to agonists rapidly increased phosphorylase a activity in control cells; however, increases in DFP-pretreated cells were markedly attenuated. Changes in [Ca2+]i similar to those noted with phosphorylase a were observed using Indo-1. Addition of calcium ionophore A23187 to control or DFP-pretreated hepatocytes increased phosphorylase a activity to a similar extent in control and DFP-pretreated cells, demonstrating that DFP pretreatment did not alter the ability of the enzyme to respond to elevation in [Ca2+]i. Collectively, these data indicate that DFP pretreatment of hepatocytes irreversibly inhibits one or more components of the Ca2+ influx pathway.

Microtubules with altered assembly kinetics have a decreased rate of kinesin-based transport.

Microtubules treated with the gamma-diketone 2,5-hexanedione (2,5-HD) have altered assembly behavior characterized by precocious nucleation and rapid elongation. By measuring the rate of microtubule transport, we have examined the potential functional significance of this 2,5-HD-induced microtubule modification. 2,5-HD-treated microtubules were transported at only 70% of the rate of control microtubules in a simple kinesin-based motility assay on glass coverslips using video and computer enhanced differential interference contrast microscopy. Since 2,5-HD is capable of forming both pyrrole adducts and crosslinks with tubulin, the contributions of pyrrole formation and crosslinking to slowed microtubule transport were determined. 3-Acetyl-2,5-hexanedione (AcHD), a pyrrole forming, non-crosslinking congener of 2,5-HD which does not alter microtubule assembly, did not produce slowed microtubule transport as occurs with 2,5-HD. However, glutaraldehyde, a pyrrole-independent crosslinking agent which alters microtubule assembly in the same way as 2,5-HD, slowed microtubule transport. These results indicate that a 2,5-HD-induced microtubule modification, possibly a crosslink-related conformational change, produces both an alteration in the kinetics of assembly and an alteration in the microtubule-motor interaction.

Reversible and irreversible oxidant injury to PC12 cells by hydrogen peroxide.

A simple and reproducible model to identify biochemical changes associated with the transition from reversible to irreversible oxidant injury and cell death was established using rat pheochromocytoma PC12 cells. Cells were subjected to a transient oxidative stress induced by exposure to hydrogen peroxide (H2O2). Reversible loss of high-energy phosphates, induced by exposing cells to 0.2 mM H2O2, was preceded by transient increases in cytosolic calcium with no loss of plasma membrane integrity, as indexed by release of cytosolic enzymes. In contrast, permanent loss of high-energy phosphates, induced by treating cells with 0.5 mH H2O2, was associated with sustained rises in cytosolic-free calcium and increased oxidation of pyruvate and palmitate, two mitochondrial substrates. Initial production of pyruvate and lactate was inhibited by exposure to 0.5 mM H2O2 but returned to values comparable to control values at one hour after treatment with H2O2. Compromise of the plasma membrane was a late event, occurring between 1 and 2 hours after exposure to 0.5 mM H2O2. Collectively, these data indicate that irreversible loss of high-energy phosphates and cell death caused by oxidative stress is more closely associated with altered mitochondrial function than with impaired glycolysis.