Regulation Of Germ Cell Apoptosis Research Articles

The role of nitric oxide in the development of diseases of the male reproductive system and its potential applications in clinical practice

Nitric oxide (NO), a reactive nitrogen species, is a molecule of high physiological and pathological importance. Physiological mechanisms mediated by NO mainly include angiogenesis, growth, puberty, and senescence. NO has vital roles in normal reproduction, including steroidogenesis, gametogenesis, and the regulation of germ-cell apoptosis. In males, NO is a key player in steroidogenesis, erectile functions, sperm capacitation, and acrosome reaction. Moreover, NO is also a regulator of Sertoli cell-germ cell interaction and maintenance of the blood-testis barrier. In pathological conditions such as infections, increased nitric oxide synthase activities stimulate the excessive synthesis of NO which acts as a proinflammatory mediator inducing oxidative stress, detrimental to reproductive functions in males. Excessive NO synthesis disrupts gonadal functions and induces germ cell apoptosis and oxidative damage to the germ cells. This review elucidates how the differences in NO expression levels account for its beneficial and adverse impacts on male fertility.

The Role of Nitric Oxide on Male and Female Reproduction.

Nitric oxide (NO), a reactive nitrogen species, is a molecule of high physiological as well as pathological importance. Physiological mechanisms mediated by NO mainly include angiogenesis, growth, puberty and senescence. NO has vital roles in normal reproduction, including steroidogenesis, gametogenesis and the regulation of germ-cell apoptosis. In females, NO stimulates an inflammatory cascade to induce ovulation, decreases steroidogenesis in luteal and granulosa cells, and acts as a paracrine factor to mediate reproductive cycles and implantation. In males, NO is a key player for steroidogenesis, erectile functions, sperm capacitation and acrosome reaction. Moreover, NO is also a regulator of Sertoli cell-germ cell interaction and maintenance of the blood-testis barrier. In pathological conditions such as infections, increased nitric oxide synthase (NOS) activities stimulate the excessive synthesis of NO which acts as a proinflammatory mediator inducing oxidative stress (OS), which is detrimental to reproductive functions in both males and females. During impregnation, the overproduction of NO results in uterine epithelial cell inflammation and immune rejection of implantation. Excessive NO synthesis disrupts gonadal functions, and induces germ cell apoptosis and oxidative damage to the germ cells. This review elucidates how the differences in NO expression levels account for its beneficial and adverse impacts upon male and female fertility.

Germ cell apoptosis and expression of Bcl-2 and Bax in porcine testis under normal and heat stress conditions

The aim of this study was to examine whether an elevated ambient temperature (37–40°C) had an effect on the apoptosis of germ cells and the expression of Bcl-2 and Bax in porcine testis. Six boars were used. Three boars were subjected to an elevated ambient temperature (37–40°C, 7days, 3h per day) as a heat stress (HS) group. The other 3 boars were kept in a room temperature house (20–27°C) as a control group. All boars were castrated and the testes were harvested. TUNEL assay was used for the detection of apoptotic cells. Immunohistochemistry, Western blotting and quantitative real-time PCR were used to analyze protein and mRNA levels of Bcl-2 and Bax in response to heat treatment. The results showed that apoptotic signals increased under heat stress conditions compared with the control (P<0.01), and the cell types most affected by heat treatment were spermatocytes and spermatids. In both the control and experimental groups, Bcl-2 was expressed in the cytoplasm and nucleus of spermatogonia, spermatocytes and differentiating spermatids and Bcl-2 preferentially localized close to the seminiferous tubule's luminal surface in late spermatocytes and spermatids. Compared with the control group, the expression levels of Bcl-2 protein and mRNA significantly increased in heat treatment group, while the expression levels of Bax protein and mRNA did not show significant changes between the control and experimental group. Low to moderate Bax immunoreactivity staining was observed in all kinds of germ cells in the control group. Strong staining was observed in spermatogonia, and low to moderate Bax staining was observed in spermatocytes and spermatids. A redistribution of Bax from a cytoplasmic to perinuclear or nuclear localization could be observed in the spermatogonia, spermatocytes and spermatids obtained in the heat treated group. These results showed that elevated ambient temperatures induced germ cell apoptosis. In response to heat stress, the expression of Bcl-2 increased and a redistribution of Bax from a cytoplasmic to a perinuclear or nuclear localization. This indicates that Bcl-2 and Bax may be involved in regulation of germ cell apoptosis induced by heat stress in boars.

It's all in your mind: determining germ cell fate by neuronal IRE-1 in C. elegans.

The C. elegans germline is pluripotent and mitotic, similar to self-renewing mammalian tissues. Apoptosis is triggered as part of the normal oogenesis program, and is increased in response to various stresses. Here, we examined the effect of endoplasmic reticulum (ER) stress on apoptosis in the C. elegans germline. We demonstrate that pharmacological or genetic induction of ER stress enhances germline apoptosis. This process is mediated by the ER stress response sensor IRE-1, but is independent of its canonical downstream target XBP-1. We further demonstrate that ire-1-dependent apoptosis in the germline requires both CEP-1/p53 and the same canonical apoptotic genes as DNA damage-induced germline apoptosis. Strikingly, we find that activation of ire-1, specifically in the ASI neurons, but not in germ cells, is sufficient to induce apoptosis in the germline. This implies that ER stress related germline apoptosis can be determined at the organism level, and is a result of active IRE-1 signaling in neurons. Altogether, our findings uncover ire-1 as a novel cell non-autonomous regulator of germ cell apoptosis, linking ER homeostasis in sensory neurons and germ cell fate.

Cell-nonautonomous inhibition of radiation-induced apoptosis by dynein light chain 1 in Caenorhabditis elegans

The evolutionarily conserved process of programmed cell death, apoptosis, is essential for development of multicellular organisms and is also a protective mechanism against cellular damage. We have identified dynein light chain 1 (DLC-1) as a new regulator of germ cell apoptosis in Caenorhabditis elegans. The DLC-1 protein is highly conserved across species and is a part of the dynein motor complex. There is, however, increasing evidence for dynein-independent functions of DLC-1, and our data describe a novel dynein-independent role. In mammalian cells, DLC-1 is important for cellular transport, cell division and regulation of protein activity, and it has been implicated in cancer. In C. elegans, we find that knockdown of dlc-1 by RNA interference (RNAi) induces excessive apoptosis in the germline but not in somatic cells during development. We show that DLC-1 mediates apoptosis through the genes lin-35, egl-1 and ced-13, which are all involved in the response to ionising radiation (IR)-induced apoptosis. In accordance with this, we show that IR cannot further induce apoptosis in dlc-1(RNAi) animals. Furthermore, we find that DLC-1 is functioning cell nonautonomously through the same pathway as kri-1 in response to IR-induced apoptosis and that DLC-1 regulates the levels of KRI-1. Our results strengthen the notion of a highly dynamic communication between somatic cells and germ cells in regulating the apoptotic process.

MicroRNA-34c Enhances Murine Male Germ Cell Apoptosis through Targeting ATF1

BackgroundMicroRNAs (miRNAs) play vital regulatory roles in many cellular processes. The expression of miRNA (miR)-34c is highly enriched in adult mouse testis, but its roles and underlying mechanisms of action are not well understood.Methodology/Principal FindingsIn the present study, we show that miR-34c is detected in mouse pachytene spermatocytes and continues to be highly expressed in spermatids. To explore the specific functions of miR-34c, we have established an in vivo model by transfecting miR-34c inhibitors into primary spermatocytes to study the loss-of-function of miR-34c. The results show that silencing of miR-34c significantly increases the Bcl-2/Bax ratio and prevents germ cell from apoptosis induced by deprivation of testosterone. Moreover, ectopic expression of the miR-34c in GC-2 cell trigger the cell apoptosis with a decreased Bcl-2/Bax ratio and miR-34c inhibition lead to a low spontaneous apoptotic ratio and an increased Bcl-2/Bax ratio. Furthermore, ectopic expression of miR-34c reduces ATF1 protein expression without affecting ATF1 mRNA level via directly binding to ATF1's 3′UTR, indicating that ATF1 is one of miR-34c's target genes. Meanwhile, the knockdown of ATF1 significantly decreases the Bcl-2/Bax ratio and triggers GC-2 cell apoptosis. Inhibition of miR-34c does not decrease the GC-2 cell apoptosis ratio in ATF1 knockdown cells.Conclusions/SignificanceOur study shows for the first time that miR-34c functions, at least partially, by targeting the ATF1 gene in germ cell apoptosis, providing a novel mechanism with involvement of miRNA in the regulation of germ cell apoptosis.

CD147 regulates apoptosis in mouse spermatocytes but not spermatogonia

Spermatogenesis is maintained by a dynamic balance between germ cell proliferation and apoptosis. Previous study has demonstrated that CD147 knockout mice are infertile with arrested germ cells. However, the question of whether and how CD147 may be involved in the apoptotic process during spermatogenesis remains elusive. The aim of this study was to evaluate the role of CD147 in the regulation of germ cell apoptosis in mice. CD147 function was blocked by anti-CD147 antibody in GC-1 (immortalized spermatogonia) and GC-2 (immortalized spermatocytes) cell lines and in testicular germ cells in vivo. Testes size and weight were examined after injection of anti-CD147 antibody into the seminiferous tubules of severe combined immunodeficiency mice. Germ cell apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and levels of p53 and two effectors, caspase 3 and poly ADP-ribose polymerase (PARP), using western blots. The size and weight of the CD147-immunodepleted testes were decreased compared with that in control testes (P < 0.001). The TUNEL assay showed an increase in the number of apoptotic spermatocytes (P < 0.001 versus control) but not spermatogonia in Stages XI-XII of CD147-immunodepleted testes. In addition, in vitro experiments demonstrated that CD147 immunodepletion induced an increase in apoptosis in GC-2 cells (P < 0.001 versus control) but had no effect on GC-1 cells. Moreover, deprivation of CD147 induced apoptosis in spermatocytes through a p53-independent mechanism, which led to caspase 3 and PARP activation. We have demonstrated that immunodepletion of CD147 induces p53-independent apoptosis in mouse spermatocytes but not spermatogonia.

P2-221 The effect of environmental exposure to phthalates on testicular carcinogenesis

Testicular germ cell tumour is the most common malignancy in young males and its incidence has been rising in recent years. Environmental and occupational exposures are believed to increase the...

FasL Gene–Deficient Mice Display a Limited Disruption in Spermatogenesis and Inhibition of Mono-(2-ethylhexyl) Phthalate–Induced Germ Cell Apoptosis

FasL (TNFSF6, CD95L) is hypothesized to trigger testicular germ cell apoptosis that normally occurs during a distinct peripubertal period as well as in response to toxicant-induced Sertoli cell injury. To test this hypothesis, we evaluated the testis of FasL gene–deficient mice (FasL−/−) at two distinct developmental ages (postnatal day [PND] 28 and 44) and after toxicant-induced Sertoli cell injury. Testicular cross sections from peripubertal (PND 28) FasL−/− mice showed significant increases in the basal germ cell apoptotic index (AI; 20.58 ± 4.59) as compared to the testis of C57BL/6J wild-type mice (5.16 ± 0.08) and closely correlated with increased expression of TRAIL protein in the testis of FasL−/− mice. A limited, but significant, number of seminiferous tubules in the testis of PND 28 FasL−/− mice showed a severe loss of germ cells with only Sertoli cells present. In contrast, no apparent gross histological changes were observed in the testis of adult (PND 44) FasL−/− mice. However, PND 44 FasL−/− mice did show a 51% reduction in homogenization-resistant elongate spermatids as compared to age-matched C57BL/6J mice. Exposure of PND 28 FasL−/− mice to mono-(2-ethylhexyl) phthalate (MEHP), a well-described Sertoli cell toxicant, unexpectedly caused a rapid decrease in the germ cell AI that paralleled increased levels of the CFLAR (c-FLIP) protein, a known inhibitor of death receptor signaling. In contrast, MEHP treatment did not decrease c-FLIP levels in PND 28 C57BL/6J mice. Taken together, these findings indicate that FasL protein expression is required during the peripubertal period for the proper regulation of germ cell apoptosis that occurs normally during this period. The influence of FasL on the cellular regulation of c-FLIP protein levels appears to be a unique mechanism for modulating germ cell apoptosis after toxicant-induced Sertoli cell injury.

Use of combined in silico expression data and phylogenetic analysis to identify new oocyte genes encoding RNA binding proteins in the mouse

During folliculogenesis, oocytes accumulate maternal mRNAs in preparation for the first steps of early embryogenesis. The processing of oocyte mRNAs is ensured by heterogeneous nuclear ribonucleoproteins (hnRNPs) genes that encode RNA binding proteins implied in mRNA biogenesis, translation, alternative splicing, nuclear exportation, and degradation. In the present work, by combining phylogenetic analyses and, when available, in silico expression data, we have identified three new oocyte-expressed genes encoding RNA binding proteins by using two strategies. Firstly, we have identified mouse orthologs of the Car1 gene, known to be involved in regulation of germ cell apoptosis in C. elegans, and of the Squid gene, required for the establishment of anteroposterior polarity in the Drosophila oocyte. Secondly, we have identified, among genes whose ESTs are highly represented in oocyte libraries, a paralog of Poly(A) binding protein--Interacting Protein 2 (Paip2) gene, known to inhibit the interaction of the Poly(A)-Binding Protein with Poly(A) tails of mRNAs. For all of these genes, the expression in oocyte was verified by in situ hybridization. Overall, this work underlines the efficiency of in silico methodologies to identify new genes involved in biological processes such as oogenesis.

Follicle-Stimulating Hormone Affects Spermatogonial Survival by Regulating the Intrinsic Apoptotic Pathway in Adult Rats1

Follicle-stimulating hormone plays a key role in spermatogonial development in adult rats via poorly understood mechanisms. We aimed to identify the role of this hormone in the regulation of germ cell apoptosis and proliferation in adult rats by suppression of FSH action following passive immunoneutralization with a rat FSH antibody for 4 and 7 days. Apoptosis and proliferation were identified by TUNEL and proliferating cell nuclear antigen labeling methods, respectively. Intrinsic and extrinsic apoptotic pathways were identified by immunohistochemistry, stereological techniques, and RT-PCR by assessing pathway-specific proteins and genes. Following FSH suppression for 4 and 7 days, we have previously reported a 30% decrease in spermatogonial number, with increased apoptosis in a stage-specific manner. The present study also shows stage-specific increases in apoptosis with no changes in proliferation. This increase in apoptosis was attributable to an increase in spermatogonial apoptosis via the intrinsic rather than extrinsic pathway, as shown by increased activated caspase 9-positive spermatogonia. The concomitant suppression of FSH and LH/testosterone showed that testosterone alone or together with FSH was more important in spermatocyte and spermatid survival by regulating both apoptotic pathways. A reduction in the level of the intrinsic pathway transcript Bcl2l2 (apoptosis suppressor gene) following FSH suppression for 4 days shows that FSH regulates some components of the intrinsic pathway. This study reveals that FSH predominantly acts as a survival factor for spermatogonia by regulating the intrinsic pathway while having no affect on germ cell proliferation in rats in vivo.

Regulation of Germ Cell Apoptosis via the Activity of the Itch E3 Ligase

Testicular germ cell numbers are continuously regulated by apoptosis. The balance of anti- and pro-apoptotic proteins determine whether the cell favors death or survival. We have shown that stress generated with the testicular toxicant MEHP sensitizes germ cells to death receptor-mediated apoptosis. Experiments with the spermatocyte-like germ cell line GC2-spd (which possesses a TS mutation that controls p53 activity at defined temperatures) suggests that cell death is achieved via ubiquitinylation of the anti-apoptotic protein c-FLIP. Furthermore, p53 expression appears to be an important contributor to this process as robust ubiquitinylation occurs only at the p53 permissive temperature. Initial experiments suggest that the modulatory protein Itch is a candidate for the post-transcriptional regulation of c-FLIP because it possesses a ubiquinylating E3 ligase. Itch is also present in testis and highly expressed in the germ cell subtypes most sensitive to apoptosis. Immunohistochemical detection of Itch in mouse testicular tissue reveals that Itch protein levels increase upon MEHP treatment and subsequently localize to the cytoplasm of meiotic spermatocyte germ cells. Western blotting of these tissues also show a correlation between c-FLIP reduction and the activated Itch protein, indicated by the phosphorylation of thr-222. These data provide the first evidence that the Itch protein may play an important role in the regulation of spermatocyte germ cell numbers and ensure apoptosis occurs following toxicant stress.

The Fas/FasL system is a possible regulator of germ cell apoptosis in the testis of Torpedo marmorata

The role played by the Fas/FasL system in the activation of apoptosis during the spermatogenesis of the spotted ray Torpedo marmorata was investigated. By immunohistochemical and western blot techniques it was demonstrated that the Fas/FasL system is amply represented during spermatogenesis of T. marmorata and it could act both via a paracrine and autocrine route: paracrine at the level of immature and mature cysts, autocrine only in mature cysts.

Expression of HSP105 and HSP60 during germ cell apoptosis in the heat-treated testes of adult cynomolgus monkeys (macaca fascicularis)

To confirm that transient increase in temperature of the testis (43C for 30 minutes once daily for 2 consecutive days) could induce apoptosis of germ cells in non-human primates and to investigate the possible roles of Hsp105 and Hsp60 in regulation of germ cell loss, we conducted the study on eight cynomolgus monkeys. The sperm concentration on day 28 after heat shock decreased to 8.4% of pretreatment levels and recovered to baseline on day 144. Using the TUNEL assay, increased numbers of apoptotic spermatocytes and round spermatids were detected on days 3, 8, and 30 post heat treatment. Hsp105 and Hsp60 mRNA and protein levels were analyzed using in situ hybridization, RT-PCR, immunohistochemical and Western blot methods. Hsp105 was confined to nuclei of spermatids before treatment, decreased dramatically with the loss of spermatids on days 3, 8, and 30, before returning to baseline levels on days 84 and 144. The expression of Hsp60 was high on days 3, 8, 30 and was only detected in Sertoli cells and spermatogonia. These results suggested that exposure of the testis to heat resulted in selective, but reversible damage to the seminiferous epithelium via increased germ cell apoptosis. Temporal changes in the expression pattern of Hsp105 and Hsp60 in relation to germ cell death suggests they may be involved in key processes in regulation of germ cell apoptosis.

Developmental expression of p63 in the mouse testis.

p63 is a member of the p53 gene family and have structural similarities with p53. p63 encodes for multiple isotypes either with N-terminal transactivation domain (TAp63) or without it (DeltaNp63). In the mammalian testis, it has been shown that p53 plays important roles in the regulation of germ cell apoptosis and meiosis. However, little is known for the physiological function of p63 in the mammalian spermatogenesis. To investigate the potential roles of p63 in the developing mouse testis, we examined the expression pattern of p63 in the mouse testis from birth to adulthood. In addition to the TAp63 mRNA which was continuously expressed in the developing testis, transcripts encoding DeltaNp63 was detected at specific stages of testicular development by RT-PCR, from postnatal day 1 to day 7 and from 3 weeks to 4 weeks after birth. Western blot analysis of whole testis lysates with anti-p63 antibody revealed an approximately 68 kD band throughout development and a less abundant protein at 60 kD in the earlier period of postnatal development. Immunopositive reactions for p63 were observed as early as 10 days after birth and p63 protein was localized to the nuclei of spermatocytes and round spermatids. These findings strongly suggest that p63 might be involved in the regulation of proliferation and differentiation of spermatogenic cells in the developing mouse testis.

Haploinsufficiency of Bcl-x leads to male-specific defects in fetal germ cells: differential regulation of germ cell apoptosis between the sexes

In germ cells, the function of which is to form the next generation, apoptotic cell death occurs during development, as in the case of somatic cells. In this study, we show that Bcl-x knockout heterozygous ( Bcl-x +/−) mice exhibit severe defects in male germ cells during development. A substantial increase in apoptosis of male germ cells occurs at around embryonic day 13.5 (E13.5) in Bcl-x +/− embryos, leading to hypoplasia of postnatal testes and reduced fertility. On the other hand, female germ cells at the same stages do not show discernible differences between wild-type and Bcl-x +/− embryos. This phenotype of Bcl-x haploinsufficiency shows that regulation of apoptosis becomes different between the sexes at around the onset of sex differentiation. Through this study, we found that, in wild-type embryos, (1) apoptosis is much more frequent (approximately 10 times) in the male than in female germ cells, and (2) expression of Bcl-x L, but not that of Bax, is higher in female than in male germ cells, at around E13.5. Male fetal germ cells, cultured with gonadal somatic cells in vitro, showed higher frequencies of apoptosis than those cultured without gonadal somatic cells. On the other hand, in the absence of gonadal somatic cells, both male and female fetal germ cells in vitro showed similar frequencies of apoptosis to female fetal germ cells in vivo. Therefore, male germ cell apoptosis, of which the default pathway is similar to that of the female, is likely to be influenced by male gonadal environments.

The attractive Achilles heel of germ cell tumours: an inherent sensitivity to apoptosis-inducing stimuli.

Testicular germ cell tumours (TGCTs) are extremely sensitive to cisplatin-containing chemotherapy. The rapid time course of apoptosis induction after exposure to cisplatin suggests that TGCT cells are primed to undergo programmed cell death as an inherent property of the cell of origin. In fact, apoptosis induction of germ cells in the testis is an important physiological mechanism to control the quality and quantity of the gametes produced. Although p53 protein is highly expressed in the majority of TGCTs, almost no p53 mutations have been detected. Interestingly, p53 overexpression is associated with loss of p21 and gain of mdm2 expression, which might indicate a partial loss in functionality of the p53 regulatory pathway in TGCTs. Besides p21, TGCTs often show low expression of other proteins involved in the regulation of cell cycle progression, such as the retinoblastoma protein and members of the INK4 family. It can be postulated that the deregulated G(1)-S phase checkpoint results in premature entry into the S phase upon DNA damage. In addition to Bcl-2 family members that are involved in the regulation of germ cell apoptosis in the normal testis via the mitochondrial death pathway, the Fas death pathway is also known to regulate apoptosis of germ cells in the testis. Since chemotherapy has been shown to activate the Fas death pathway and TGCTs co-express both Fas and its ligand FasL, TGCT cells might undergo apoptosis upon cisplatin treatment via autocrine or paracrine activation of the Fas system by FasL. The hypothesis suggested here is that the lack of cell cycle arrest following a cisplatin-containing treatment, together with the activation of the Fas death pathway and the mitochondrial death pathway, explains the rapid and efficient apoptosis of TGCT cells. Defining the mechanisms involved in the cisplatin sensitivity of TGCTs will provide tools to increase cisplatin sensitivity in other human tumours with acquired or intrinsic resistance.

Expression of p63 in the testis of mouse embryos.

Apoptosis of testicular germ cells during fetal development is regulated by both p53-dependent and independent mechanisms. However, its precise mechanisms are largely unknown. A member of p53 gene family, p63, is required for p53-dependent apoptosis and can induce apoptosis in the absence of p53 through the activation of p53-target genes. In this study, we examined the expression pattern of p63 in the mouse testis from embryonic day (E) 13.5 to E18.5 to clarify their possible role in the regulation of germ cell apoptosis. Immunostaining for p63 was found in the nucleus of germ cells at E13.5, and continuously observed until E18.5. The RT-PCR using specific primers for two p63 isotypes, those containing the transactivation domain and others not, showed that both transcripts were expressed in the fetal gonads. Possible roles of p63 in the embryonic testes were discussed.

Death Receptor Response in Rodent Testis after Mono-(2-ethylhexyl) Phthalate Exposure

Apoptosis of testicular germ cells is critical for the maintenance of functional spermatogenesis. Previously, we have demonstrated that the Fas (Apo-1, CD95) receptor participates in the regulation of germ cell apoptosis, particularly after toxicant-induced Sertoli cell injury. In this study, we show that germ cells from B6.SMNC3H-Fas gld,gld ( gld) mice that express a dysfunctional form of FasL still undergo significant apoptosis, albeit at a lower incidence than seen in B6 mice, following mono-(2-ethylhexyl) phthalate (MEHP)-induced Sertoli cell injury. In addition, we show the presence of Fas, TRAIL-R1 (Death Receptor-4, DR4), and TRAIL-R2 (DR5) in the testis of C57BL/6 (B6) and gld mice (4 weeks old), and Sprague–Dawley rats (5 weeks old) and their responsiveness after MEHP treatment. More importantly, Western blot analysis of cellular fractions showed an increase in death receptor localization on the membrane fractions taken from Sprague–Dawley rats. Immunohistochemical analysis indicated localization of Fas and DR5 primarily to the spermatocyte subpopulation of germ cells. Examination of downstream receptor-mediated signals (i.e., cleavage of procaspase-8 and NFκB activation) revealed an early increase in NFκB–DNA binding and an increase in procaspase-8 processing in mutant gld mice. In summary, germ cell-associated death receptors, as well as downstream signaling elements, appear to be responsive to MEHP-induced Sertoli cell injury. Whether this is directly responsible for the increases in germ cell apoptosis after MEHP exposure is yet to be determined. The observed robust and early increase in Fas in wild-type testis and diminished rates of germ cell apoptosis in mutant testis ( gld and lpr cg ) reiterates the importance of the Fas signaling pathway.

Frequent Fas Gene Mutations in Testicular Germ Cell Tumors

The Fas (Apo-1/CD95)/Fas ligand (L) system is involved in cell death signaling, and has been suggested to be important for the regulation of germ cell apoptosis in the testis. Mutations of the Fas gene may result in accumulation of germ cells and thus might contribute to testicular carcinogenesis. The open reading frame of Fas cDNA was examined in 24 cases of testicular germ cell tumors (TGCTs), comprised of 19 pure histological type (15 seminomas, 3 embryonal carcinomas, 1 immature teratoma) and 5 mixed-type tumors. Mutations of the Fas gene were found in nine (37.5%) of these cases. Each lesion with a homogeneous histological picture was selectively microdissected using a laser capture microdissection method: samples consisted of 18 lesions from seminomas, 7 embryonal carcinomas, 4 immature teratomas, 2 choriocarcinomas, and 1 from a yolk sac tumor. Microdissected genomic DNA was examined to determine which mutations were derived from which kind of histological lesion. Eleven mutations were detected in 10 TGCT lesions from nine cases, but none were found in benign lesions. All were point mutations, and eight missense mutations occurred in exon 9 encoding the core protein of the death domain essential for apoptotic signal transduction. Three were silent mutations. Mutations were found in the seminoma (27.8%) and embryonal carcinoma lesions (62.5%), but none were found in the one yolk sac tumor, two choriocarcinomas, or four immature teratoma lesions. Each seminoma and embryonal carcinoma lesion found in the same case had a different type of Fas mutation from the others. Mouse T-cell lymphoma cells transfected with missense mutated genes were resistant to apoptosis induced by anti-Fas antibody, indicating these to be loss-of-function mutations. These findings suggested a role of Fas gene mutations in the pathogenesis of TGCTs.