Germ cell apoptosis and its molecular trigger in mouse testes

Germ cell apoptosis is very common during various stages of mammalian testicular development. However, our understanding of the mechanisms underlying male germ cell apoptosis is still limited. This review firstly covers the general features of germ cell death in normal testes of fetal, neonatal, and adult mice from electron microscopy (EM) and terminal dUTP nick-end labeling (TUNEL) staining. The issue of whether the Fas and Fas ligand (FasL) system and/or the Bax and Bcl-2 system is involved in the induction of germ cell apoptosis in normal and damaged testes will then be addressed, including a special consideration of the ischemia-reperfusion model, the endocrine disruptor-treated model, and others. Finally, this review will propose that the process of normal spermatogenesis seems skillfull in taking advantage of apoptotic processes of germ cells and that different molecular pathways may be triggered to induce male germ cell apoptosis, depending upon the physiological and pathological states of germ cells.     

Fas ligand in bull ejaculated spermatozoa:A quantitative immunocytochemical study

Apoptosis, or programmed cell death, provides a way to remove redundant cells at the end of their lifespan and thus acts as a homeostatic mechanism, maintaining the correct number of cells in the body by balancing their production and death. In the testis, this process seemed to play a pivotal role in spermatogenesis. It is generally accepted that Sertoli cells control the germ cell population through one of the best-known apoptotic pathways, the Fas/Fas L paracrine signal transduction system, in which a Fas ligand (Fas L) expressed by Sertoli cells induces apoptosis when it binds with its receptor, Fas, expressed by the germ cells. Recently, we demonstrated the presence of Fas antigen in normal ejaculated spermatozoa from fertile bulls and suggested that this molecule might have a non-apoptotic, defensive role against injuries, especially oxidative stress. We have now investigated whether bull mature, fertile spermatozoa express not only the Fas receptor but also its natural ligand Fas L. Our results indicate that the whole sperm population expresses Fas L. We suggest that Fas L in bull spermatozoa, like in murine spermatozoa, might be able to kill activated lymphocytes and protect the male gamete from damage by the self-immune system or the cytotoxic activity of leukocytes in the female genital tract.     

A possible role of Fas antigen in ejaculated spermatozoa of fertile bulls: an immunocytochemical quantitative approach

The Fas/Fas L system is a widely recognized apoptosis signal transduction pathway in which transmembrane receptor protein (Fas) triggers a programmed cell death when bound by the Fas ligand (Fas L). This system in the testis is believed to be a paracrine signaling system by which Sertoli cells expressing Fas L can initiate killing of Fas-expressing germ cells during spermatogenesis. So far, the presence of Fas antigen in ejaculated spermatozoa was related only to subfertility or infertility conditions. We demonstrated for the first time that normal ejaculated spermatozoa also express Fas antigen. Our data showed that a large percentage of normal ejaculated spermatozoa of fertile bulls are immunocytochemically positive for Fas. Our observations provide further evidence of the “regionalization” of sperm membrane antigens. Furthermore, we suggest that in mature fertile ejaculated spermatozoa the Fas antigen may also provide resistance to programmed cell death, like in some other cells expressing molecules that inhibit the signals induced by Fas or the death program itself. In addition, we suggest that Fas antigen can partly protect the spermatozoa against apoptosis induced by lipoperoxidative damage that can occur spontaneously in the male gamete at various stages in its lifetime.     

Fas expression correlates with human germ cell degeneration in meiotic and post-meiotic arrest of spermatogenesis

Degeneration of human male germ cells was analysed by means of light (LM) and transmission electron (TEM) microscopy. The frequency of degenerating cells was correlated with that of Fas-expressing germ cells in human testes with normal spermatogenesis (n = 10), complete early maturation arrest (EMA) (n = 10) or incomplete late maturation arrest (LMA; n = 10) of spermatogenesis. LM analysis of testis sections with normal spermatogenesis indicated that degenerating germ cells were localized in the adluminal compartment of the seminiferous epithelium. TEM showed that apoptotic cells were mostly primary spermatocytes and, to a lesser extent, round or early elongating spermatids. Apoptotic germ cells appeared to be eliminated either in the seminiferous lumen or by Sertoli cell phagocytosis. An increased number of degenerating cells was observed in testes with LMA as compared with normal testes and testes with EMA of spermatogenesis (P < 0.001, Wilcoxon's rank sum test). Comparison of these results with those obtained from immunohistochemistry experiments demonstrated a tight correlation between the number of apoptotic cells and the number of Fas-expressing germ cells (P = 0.001, Spearman's rank = 0.69). These findings suggest that altered meiotic and post-meiotic germ cell maturation might be associated with an up-regulation of Fas gene expression capable of triggering apoptotic elimination of defective germ cells.     

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.     

Hormonal regulation of apoptosis and the Fas and Fas ligand system in human endometrial cells

The process of apoptosis is responsible for normal cellular turnover in numerous tissues throughout the body. The endometrial layer of the uterus shows steroid-dependent cyclic changes in structure and function. After a proliferative and secretory phase, steroid support is withdrawn and the uterine epithelium is shed. We hypothesize that the apoptosis observed in endometrial cells following hormonal withdrawal is mediated by the Fas/Fas ligand (FasL) system. Normal endometrial cells and endometrial cancer cells were cultured in the presence of estrogen and progesterone. In order to mimic physiological hormonal changes, estrogen and progesterone were removed from the media. Apoptosis was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-dephenyl tetrazolium bromide (MTT) assay and propidium iodide staining, while Fas and FasL expression were evaluated by Western blot analysis. The endometrial cells expressed Fas and low levels of FasL. Withdrawal of estrogen and/or progesterone from the culture induced apoptosis causing an approximately 50% decrease in cell viability. This coincided with increased Fas and FasL expression. Treatment of the cells with anti-FasL antibody prevented cell death following hormonal withdrawal. Estrogen and progesterone therefore represent survival factors which hamper cell death by impeding the expression of apoptotic factors. Our results indicate that Fas-mediated apoptosis is important for endometrial cycling and suggest that dysregulation of the Fas/FasL interactions may have an important role in the development of endometrial cancer.     

Involvement of the Fas/FasL pathway in the pathogenesis of germ cell tumours of the adult testis

Induction of apoptosis by Fas ligand (FasL) of Fas-containing cells is a known mechanism involved in the eradication of inappropriate cells during normal development. Alterations of the Fas/FasL pathway have been found in various types of cancer, leading to circumvention of attack of the tumour by the immune system. An alternative way to circumvent eradication by induction of apoptosis is through changes in the downstream inhibitors. For example, Fas-associating phosphatase-1 (Fap-1) binds directly to the Fas receptor and results in a block of the downstream signalling. To shed more light on the role of the Fas/FasL pathway in the development of human testicular germ cell tumours of the adult testis, this study investigated the presence of Fas, FasL, Fap-1, HLA class I and II molecules, CD45 (lymphocyte marker), and CD57 [natural killer (NK) cell marker] by immunohistochemistry on frozen sections of 41 cases of seminomas, non-seminomas, and spermatocytic seminomas. Every germ cell tumour was positive for Fap-1 and negative for HLA classes I and II, like their non-malignant cells of origin. The infiltrating lymphocytes, predominantly present in seminomas, showed consistently positive staining for Fas and CD45, but not for Fap-1. No Fas was found on NK cells. All seminomas and non-seminomas (except teratomas), including their precursor stages, carcinoma in situ, intratubular seminoma and intratubular non-seminoma, showed positive staining for FasL, but not for Fas. Teratoma showed no staining for FasL and was positive for Fas. In contrast, both Fas and FasL were detectable on spermatocytic seminoma. These data indicate a different regulation of the Fas/FasL system in seminoma and spermatocytic seminoma, supporting a separate pathogenesis for these germ cell-derived tumours. The presence of Fap-1 in all histological variants of germ cell tumours might be related to the consistently positive staining in cells of the germ lineage. This study indicates that production of FasL by the germ cell tumour cells might be involved in the early development of these types of adult testicular cancer by inducting apoptosis of Fas-positive, Fap-1-negative tumour-infiltrating lymphocytes.     

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.     

Fas expression and apoptosis in human B cells

Mechanisms of B cell apoptosis are critical in reducing aberrant B cell proliferations such as those that arise in autoimmune disease and in B cell malignancies. The physiologic interaction of CD4+ helper T cells and B lymphocytes has been extensively studied over the past two decades. Although CD4+ T cells are considered primarily to offer positive costimulatory signals for B cell differentiation into active immunoglobulin-secreting cells, recent studies have shown that CD4+ T cells are crucial in downregulating the humoral immune response. In the course of cognate interaction between CD40 ligand (CD40L)-bearing CD4+ T cells and CD40-expressing germinal center B cells, CD40 ligation results in augmented Fas expression at the B cell surface. Like CD40L, Fas ligand is expressed on activated CD4+ Th1 cells and when bound to Fas receptor on the B cell surface, initiates an apoptotic signal in that cell. Thus, CD4+ T cells limit the growth of autologous germinal center B cells by first inducing Fas expression and then instigating a death signal via Fas ligand. In this work, we will consider these observations about CD4+ T-cell-induced, Fas-mediated B cell death in the context of other factors that affect apoptosis in B cells, normal and malignant.     

LPS-induced transient testicular dysfunction accompanied by apoptosis of testicular germ cells in mice

The aim of this study was to clarify effects of inflammation on spermatogenesis in LPS-administered mice. ICR mice were treated by intraperitoneal injection for 7 days with either physiological saline (control) or 0.1 mg lipopolysaccharide (LPS)/kg body weight/day. Control mice were killed at 24 h after the last injection and the LPS-treated group after 24 h or 1, 3, or 5 weeks. Sperm concentration and motility in the cauda epididymis were examined as well as immunohistochemical localization of Fas and FasL and germ cell apoptosis. Sperm concentration and motility markedly fluctuated in LPS-treated mice. Increase of apoptotic cells was common in all post-LPS treatment groups, with a peak at 24 h after LPS injection. In contrast to the lack of Fas immunoreactivity in control testes, LPS-treated groups demonstrated Fas in many germ cells, especially in spermatocytes and spermatids. Immunoreactivity for FasL, on the other hand, was positive for some Sertoli cells, Leydig cells, and germ cells in both control and LPS-treated groups at all time points. The results suggest that the Fas/FasL system mediates apoptosis of germ cells in LPS-treated mice testes. LPS-administered mice thus provide a good experimental model for the study of transient disruption of spermatogenesis.     

Fas and FasL in the homeostatic regulation of immune responses

Studies of the biological effects of Fas signaling, using transformed cell lines as targets, indicate that ligation of the Fas receptor induces an apoptotic death signal. Chronically activated normal human T cells are also susceptible to Fas-mediated apoptosis. However, interactions between Fas and Fas ligand can also yield a costimulatory signal. Here, David Lynch, Fred Ramsdell and Mark Alderson present a model for the role of Fas and FasL in the homeostatic regulation of normal immune responses. They discuss how dysregulation of the Fas apoptotic pathway may contribute to certain disease states, including autoimmune disease and human immunodeficiency virus (HIV)-induced depletion of CD4⁺ T cells.     

Expression of Fas and Fas Ligand on Mouse Renal Tubular Epithelial Cells in the Generalized Shwartzman Reaction and Its Relationship to Apoptosis

Previously we reported that the consecutive injection of lipopolysaccharide (LPS) into LPS-sensitized mice for the generalized Shwartzman reaction (GSR) appeared to induce the injury of renal tubular epithelial cells via apoptosis. The aim of this study was to characterize the mechanism of renal tubular epithelial cell injury in GSR. The expression of Fas and Fas ligand was immunohistochemically detected on renal tubular epithelial cells from GSR-induced mice, although neither Fas nor Fas ligand was found in cells from untreated control mice or in cells from mice receiving a single injection of LPS. GSR-induced renal tubular epithelial cell injury was produced in neither Fas-negative MRL-lpr/lpr mice nor Fas ligand-negative MRL-gld/gld mice. The administration of anti-gamma interferon antibody together with a preparative injection of LPS prevented the expression of Fas and Fas ligand and the apoptosis of renal tubular epithelial cells. A provocative injection of tumor necrosis factor alpha into LPS-sensitized mice augmented Fas and Fas ligand expression and the apoptosis of renal tubular epithelial cells. The administration of tumor necrosis factor alpha to interleukin-12-sensitized mice resulted in Fas and Fas ligand expression and the apoptosis. Sensitization with interleukin-12 together with anti-gamma interferon antibody did not cause the apoptosis of renal tubular epithelial cells. It was suggested that the Fas/Fas ligand system probably plays a critical role in the development of renal tubular epithelial cell injury through apoptotic cell death.     

Germ cell apoptosis in autoimmune orchitis: involvement of the Fas-FasL system

PROBLEM: The aim of this study was to determine the mechanism of germ cell death in experimental autoimmune orchitis (EAO) and the involvement of the Fas-FasL system in this process. METHOD OF STUDY: The EAO was induced in rats by immunization with testis homogenate and adjuvants. Apoptosis was studied by light microscopy, in situ end labeling of apoptotic DNA and DNA fragmentation techniques. Fas, FasL and caspase 3 expression was detected by immunohistochemistry. RESULTS: In rats with orchitis the number of Fas+ and FasL+ apoptotic germ cells increased from day 50, when the lesion develops, to 150 days, and correlates with the degree of testicular damage. Most spermatocytes expressing Fas were apoptotic. Many Fas+ germ cells were also immunoreactive for FasL. Moreover, these cells also expressed caspase 3. CONCLUSIONS: In rats with EAO germ cell death occurs through an apoptotic mechanism preceding germ cell sloughing. Immunohistochemical data suggest that the Fas-FasL system mediates germ cell apoptosis in an autocrine and/or paracrine way.     

Anaplastic and atypical meningiomas express high levels of Fas and undergo apoptosis in response to Fas ligation

In this study we characterized the expression of Fas and Fas ligand in different types of meningiomas and examined the effect of Fas ligation on the death of meningioma cells in culture. Using Western blot analysis, we found that extracts derived from anaplastic and atypical meningiomas expressed high levels of Fas, whereas benign meningiomas did not express detectable levels of this protein. All of the meningiomas examined expressed low levels of Fas ligand. Cultures of anaplastic meningiomas also expressed Fas and treatment of these cells with anti-Fas antibody induced cell death. The results of this study indicate that Fas is preferentially expressed in atypical and anaplastic meningiomas and suggest that it is involved in the increased apoptosis observed in these tumors.     

Apoptosis induced in T cells by human neuroblastoma cells: role of Fas ligand

The CD95/CD95L (Fas/Fas ligand) receptor/ligand system plays an important role in regulation of cell survival and induction of a programmed cell death. It is also involved in regulation of effector phase of T and NK cell cytotoxicity, establishment of immune privilege sites, and tumor escape from immune recognition. In this study, we assessed expression of CD95L in tumors obtained from patients with neuroblastoma (NB) and in established NB cell lines. We measured the presence of intratumoral T cell infiltrates and T cell survival in tumor tissue samples. High levels of apoptosis were observed in tumor-associated lymphocytes as well as in Jurkat T cells cocultured with NB cells in vitro. T cell death was reduced after treatment of NB cells (in vitro) with antibody to FAS ligand (FasL). Overall, our data suggest that NB-induced apoptosis of Fas-sensitive Jurkat T cells is mediated by functional FasL expressed on NB and Fas/FasL interaction may be responsible for the elimination of T cells in the NB microenvironment.     

Serum Levels of Soluble Fas in Patients with Multinodular Goiter

Objective: Fas is a cell-surface receptor responsible for induction of apoptosis in human thyrocytes upon interaction with Fas Ligand. Fas protein expression on thyroid cells and Fas-mediated apoptosis is decreased in multinodular goiter (MNG) resulting in thyroid cell proliferation. The soluble form of Fas (sFas) produced by alternative mRNA splicing may inhibit Fas-Fas Ligand binding and apoptosis. The aim of this study was to examine whether sFas is differentially expressed in multinodular goiter (MNG), which is associated with decreased Fas-mediated apoptosis. Method: We determined serum sFas levels using enzyme-linked immunosorbent assay (ELISA) in 42 patients with MNG and 23 normal controls. Results: Serum sFas levels were increased in patients with MNG (7.47 ± 2.55 ng/ml) compared to normal controls (2.26 ± 0.9 ng/ml). Levels of sFas were not significantly correlated with age, sex or clinical parameters, such as serum levels of FT4 or TSH. Discussion: Increased sFas in MNG may indicate increased expression of alternatively spliced Fas mRNA variant and decreased expression of cell-surface Fas protein, and may enhance thyroid cell proliferation by protecting thyroid cells from Fas-mediated apoptosis.     

Oleandrin-Mediated Expression of Fas Potentiates Apoptosis in Tumor Cells

Chemotherapeutic agent is characterized by its concentration in tumor cells with minimum side effects. Oleandrin, a polyphenolic cardiac glycoside is known to induce apoptosis in tumor cells. However, no report is available on its efficacy in primary cells. In this report we are providing the evidence that oleandrin induces apoptosis, not necrosis in tumor cells but not in primary cells like peripheral blood mononuclear cells (PBMC) and neutrophils. Oleandrin inhibited NF-κB activation in tumor cells but not in primary cells. It induced cell death in NF-κB-overexpressed tumor cells. Oleandrin induced Fas expression thereby inducing apoptosis in tumor cells but not in primary cells. Dominant negative FADD inhibited oleandrin-induced cell death in tumor cells. Overall, these results suggest that oleandrin mediates apoptosis in tumor cells by inducing Fas but not in primary cells indicating its potential anti-cancer property with no or slight side effect.