John H. Richburg

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 deoxxynucleotidyltransferase-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 ...

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.

Sertoli Cell Toxicants

This chapter discusses toxicants that specifically target the Sertoli cell. The term toxicant encompasses any toxic agent of natural, biological, or synthetic origin. The accurate designation of the Sertoli cell as the target of a testicular toxicant depends on the quantity and quality of available information. Theoretically, any testicular toxicant may produce subtle alterations in germ cell function that trigger secondary manifestations of Sertoli cell dysfunction as the earliest observed sign of testicular injury. Such an unrecognized germ cell toxicant is considered a Sertoli cell toxicant until the knowledge base is improved by further experimentation. Alternatively, any testicular toxicant producing subtle, undetected alterations in Sertoli cell function followed by germ cell abnormalities as the earliest sign of testicular injury is incorrectly classified as a germ cell toxicant. The frequency of incorrect assignment of toxicant to target cell is unknown; however, given the central role of the Sertoli cell in germ cell maintenance, one would predict that cryptic Sertoli cell toxicants are more often misidentified as germ cell toxicants than vice versa.

Mono-(2-Ethylhexyl) Phthalate-Induced Disruption of Junctional Complexes in the Seminiferous Epithelium of the Rodent Testis Is Mediated by MMP2

Abstract Tight junctions between Sertoli cells of the testicular seminiferous epithelium establishes the blood-testis barrier (BTB) and creates a specialized adluminal microenvironment above the BTB that is required for the development of the germ cells that reside there. Actin filament-based anchoring junctions between Sertoli cells and germ cells are important for maintaining close physical contact between these cells as well as regulating the release of mature spermatids into the lumen. Previously, we reported that Sertoli cell injury in rodents after mono-(2-ethylhexyl) phthalate (MEHP) exposure results in the activation of matrix metalloproteinase 2 (MMP2) and increases the sensitivity of germ cells to undergo apoptosis. A disruption in the physical association between Sertoli cells and germ cells and premature loss of germ cells from the seminiferous epithelium has been widely described after phthalate treatment. In this study, we investigate the functional participation of MMP2 in the mechanism underlying MEHP-induced disruption of junction complexes and the resultant loss of germ cells. Exposure of C57BL/6J mice to MEHP (1 g/kg, oral gavage) decreased the expression of occludin in the tight junctions between Sertoli cells and caused gaps between adjacent Sertoli cells as observed by transmission electron microscopy. A reduced expression of laminin-gamma3 and beta1-integrin in apical ectoplasmic specializations between Sertoli cells and germ cells in a time-dependent manner was also observed. Treatment with specific MMP2 inhibitors (TIMP2 and SB-3CT) both in vitro and in vivo significantly suppressed MEHP-induced germ cell sloughing and changes in the expression of these junctional proteins, indicating that MMP-2 plays a primary role in this process. Furthermore, the detachment of germ cells from Sertoli cells appears to be independent of the apoptotic signaling process since MEHP-induced germ cell detachment from Sertoli cells could not be prevented by the addition of a pan-caspase inhibitor (z-VAD-FMK).

Transcriptional Regulation of FasL Expression and Participation of sTNF-α in Response to Sertoli Cell Injury

The Fas/FasL signaling pathway has previously been demonstrated to be critical for triggering germ cell apoptosis in response to mono-(2-ethylhexyl)phthalate (MEHP)-induced Sertoli cell injury. Although Sertoli cells ubiquitously express the FasL protein, MEHP-induced germ cell apoptosis appears to tightly correlate with increased levels of Sertoli cell FasL. Here we characterize the transcriptional regulation of the murine FasL gene in Sertoli cells after MEHP exposure. A serial deletion strategy for 1.5 kb of the 5′-upstream activating sequence of the FasL promoter was used to determine transcriptional activity in response to MEHP. Luciferase activity of the FasL promoter in the rat Sertoli cell line ASC-17D revealed that two regions, -500 to -324 and -1250 to -1000, were necessary to drive the inducible transcription of FasL. Sequence analysis of these two regions revealed two cis-regulatory elements, NF-κB and Sp-1. By site-directed mutagenesis, electrophoretic mobility shift and chromatin immunoprecipitation assays, it was confirmed that MEHP-induced FasL expression is enhanced through the transcriptional regulation of both NF-κB and Sp-1. Experiments performed both in vitro and in vivo revealed that MEHP exposure results in an increased production of sTNF-α and that sTNF-α-mediated NF-κB activation causes robust increases in FasL levels in both the ASC-17D Sertoli cell line and in primary rat Sertoli cell/germ cell co-cultures. In the seminiferous epithelium, Sertoli cells express TNFR1, whereas germ cells produce TNF-α. Therefore, sTNF-α released by germ cells after MEHP-induced Sertoli cell injury acts upon Sertoli cell TNFR1 and activates NF-κB and Sp-1 that consequently causes a robust induction of FasL expression. These novel findings point to a potential “feed-forward” signaling mechanism by which germ cells prompt Sertoli cells to trigger their apoptotic elimination.

TNF Alpha-Mediated Disruption of Spermatogenesis in Response to Sertoli Cell Injury in Rodents Is Partially Regulated by MMP2

Abstract Mono-(2-ethylhexyl) phthalate (MEHP)-induced Sertoli cell injury in peripubertal rodents results in the stimulation of germ cell apoptosis through an interaction of FAS/FASL between these two cell types. During this peripubertal period, an early spike in the incidence of germ cell apoptosis occurs during the first wave of spermatogenesis and is essential for the development of functional spermatogenesis in adults. Our previous observations revealed that soluble tumor necrosis factor alpha (sTNFA) released by germ cells after MEHP exposure consequently resulted in a robust induction of FASL by Sertoli cells. Metalloproteinases (MPs) are essential for processing the TNFA precursor to its soluble form and its ability to bind to TNFRSF1A. The activity of MPs is regulated by the tissue inhibitors of MPs (TIMPs) family. Herein we report that TIMP2 is predominately expressed in Sertoli cells and that protein levels decrease in a time-dependent manner after MEHP exposure. The secretion of matrix MP 2 (MMP2) in primary rat Sertoli cell-germ cell cocultures is induced after MEHP exposure, and its activity increases in a time-dependent manner. The addition of SB-3CT, a specific gelatinase inhibitor, decreases the activity of MMP2 and significantly reduces MEHP-enhanced sTNFA production in primary cocultures. In vivo challenges with SB-3CT decrease sTNFA and reduce MEHP-induced testicular germ cell apoptosis. In primary cocultures, MEHP exposure causes a 9.46-fold increase in sTNFA, while the addition of recombinant MMP2 protein results in a 5.4-fold increase in sTNFA, suggesting that MEHP-induced MMP2 is in part responsible for the activation of TNFA in the testis. Taken together, these observations indicate the distinct role of specific MPs in response to toxicant-induced Sertoli cell injury, providing further insights into the mechanism by which Sertoli cells control the sensitivity of germ cells to undergo apoptosis.

Defining the cellular and molecular mechanisms of toxicant action in the testis.

A symposium was held at the 41st annual meeting of the Society of Toxicology with presentations that emphasized novel molecular and cellular pathways that modulate the response to testicular toxicants. The first two presentations described cellular alterations after exposure to the Sertoli cell toxicant, mono-(2-ethylhexyl) phthalate (MEHP). The expression of flamingo1, a G protei coupled receptor family member that may couple cell-cell adhesion to G protein-dependent signaling in Sertoli cells, was rapidly altered after MEHP exposure. Sertoli cell associated flamingo1 immunostaining was redistributed early (within 2 h) after MEHP exposure and disappeared by 12 h, suggesting that flamingo1 is a proximal phthalate target. MEHP was also described to alter the expression and activity of the newly identified death receptors DR4, 5 and 6 in the testis. The differential cellular changes in the levels of DR4, 5 and 6 after phthalate exposure suggested that they may act as surrogates or in concert with the widely described Fas-signaling pathway in the initiation of germ cell apoptosis after MEHP exposure. The next two presentations focused on revealing mechanisms that may explain the persistent post-exposure testicular atrophy that is observed in rodents after a variety of chemical or physical insults (radiation, chemotherapeutics, toxicants) and possible strategies to re-initiate spermatogenesis in the atrophic testis. Hormonal manipulations that lower testosterone and serum FSH levels allow for re-initiation of spermatogonial development. Recent investigation of additional models of persistent atrophy such as mutant mice, the aged Brown Norway rat, EDS-induced Leydig cell deficient rat, and primates, have broadened insight into the mechanisms responsible for persistent atrophy. The last presentation described the use of cDNA arrays in the investigation of cellular elements and mechanisms responsible for disruption of spermatogenesis by the drinking water disinfectant bromochloroacetic acid (BCA). A custom mouse testis cDNA array interrogating 950 genes was used for analysis of testis mRNA. BCA altered the expression of 53 of the 950 genes, including two encoding sperm proteins known to be significant for male fertility, and other genes involved in spermatogenesis, stress response, and cell communication/adhesion. These observations strengthen the hypothesis that BCA disrupts spermatogenesis by altering the process of spermiogenesis.

Estrogen-Dependent and -Independent Estrogen Receptor-α Signaling Separately Regulate Male Fertility

Estrogen receptor-alpha (ERalpha) plays a critical role in male reproductive tract development and fertility. To determine whether estrogen-dependent and -independent ERalpha mechanisms are involved in male fertility, we examined male estrogen nonresponsive ERalpha knock-in mice. These animals have a point mutation (G525L) in the ligand-binding domain of ERalpha that significantly reduces interaction with, and response to, endogenous estrogens but does not affect growth factor activation of ligand-independent ERalpha pathways. Surprisingly, we found that ligand-independent ERalpha signaling is essential for concentrating epididymal sperm via regulation of efferent ductule fluid reabsorption. In contrast, estrogen-dependent ERalpha signaling is required for germ cell viability, most likely through support of Sertoli cell function. By treating estrogen nonresponsive ERalpha knock-in (ENERKI) mice with the ERalpha selective synthetic agonist propyl pyrazole triol, which is able to bind and activate G525L ERalpha in vivo, we discovered male fertility required neonatal estrogen-mediated ERalpha signaling. Thus, our work indicates both estrogen-dependent and -independent pathways play separable roles in male murine reproductive tract development and that the role of ERalpha in human infertility should be examined more closely.

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. Cisplatins 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.

Fas- or FasL-deficient mice display an increased sensitivity to nitrobenzene-induced testicular germ cell apoptosis

We have previously reported that the Fas/Apo-1/CD95-mediated apoptosis-inducing signaling system participates in the initiation of toxicant-induced testicular germ cell apoptosis. The contribution of Fas-mediated signaling is especially evident in the initiation of germ cell apoptosis after mono-(2-ethylhexyl)phthalate (MEHP)-induced Sertoli cell injury. In previous work, we demonstrated that the incidence of germ cell apoptosis after MEHP exposure is significantly reduced in B6.SMNC3H-Fas(gld,gld) (gld) mice that express a dysfunctional form of the FasL protein (the associated ligand that activates Fas). This has led to the hypothesis that activation of the Fas-mediated signaling pathway is a common mechanism for the initiation of germ cell apoptosis after toxicant-induced Sertoli cell injury. To test this hypothesis, we evaluated the sensitivity of testicular germ cells of wild-type, gld- and Fas-deficient CBA/KlJms-Tnfrsf6lpr-cg((lpr-cg)) (lpr(cg)) mice to undergo apoptosis after exposure to the Sertoli cell toxicant nitrobenzene (NB). Adult, 8-week-old gld mice treated with a single oral dose of NB (800 mg/kg) were observed to have a higher apoptotic index (AI; 66.1+/-1.3) 24 h after exposure as compared with the wild-type C57BL/6 (C57) mice (50.4+/-1.8). Similarly, 8-week-old lpr(cg) mice treated with NB displayed a higher AI 24 h after exposure (45.1+/-4.6) as compared with the wild-type CBA/KlJms (CBA) mice (32.1+/-3.8). Interestingly, exposure of both peri-pubertal 4-week-old C57 and gld mice showed a similar increase in the incidence of germ cell apoptosis after NB (600 mg/kg) exposure. Taken together, these findings indicate that Fas-mediated signaling is not required for NB-induced germ cell apoptosis and imply that a dysfunctional Fas signaling system sensitizes adult mice to NB-induced germ cell elimination.