Estrogen-induced spermatogenic cell apoptosis occurs via the mitochondrial pathway: role of superoxide and nitric oxide

The detrimental effects of estrogen on testicular function provide a conceptual basis to examine the speculative link between increased exposure to estrogens and spermatogenic cell death. Using an in vitro model, we provide an understanding of the events leading to estrogen-induced apoptosis in cells of spermatogenic lineage. Early events associated with estrogen exposure were up-regulation of FasL and increased generation of H(2)O(2), superoxide, and nitric oxide. The ability of anti-FasL antibodies to prevent several downstream biochemical changes and cell death induced by 17beta-estradiol substantiates the involvement of the cell death receptor pathway. Evidence for the amplification of the death-inducing signals through mitochondria was obtained from the transient mitochondrial hyperpolarization observed after estradiol exposure resulting in cytochrome c release. A combination of nitric oxide and superoxide but not H(2)O(2) was responsible for the mitochondrial hyperpolarization. Mn(III) tetrakis(4-benzoic acid)porphyrin chloride, an intracellular peroxynitrite scavenger, was able to reduce mitochondrial hyperpolarization and cell death. Although nitric oxide augmentation occurred through an increase in the expression of inducible nitric-oxide synthase, superoxide up-regulation was a product of estradiol metabolism. All of the above changes were mediated through an estrogen receptor-based mechanism because tamoxifen, the estrogen receptor modulator, was able to rescue the cells from estrogen-induced alterations. This study establishes the importance of the independent capability of cells of the spermatogenic lineage to respond to estrogens and most importantly suggests that low dose estrogens can potentially cause severe spermatogenic cellular dysfunction leading to impaired fertility even without interference of the hypothalamo-hypophyseal axis.     

Clinical aspects of male germ cell apoptosis during testis development and spermatogenesis

Apoptosis appears to have an essential role in the control of germ cell number in testes. During spermatogenesis germ cell deletion has been estimated to result in the loss of up to 75% of the potential number of mature sperm cells. At least three factors seem to determine the onset of apoptosis in male germ cells: (1) lack of hormones, especially gonadotropins and androgens; (2) the specific stage in the spermatogenic cycle; (3) and the developmental stage of the animal. Although male germ cell apoptosis has been well characterized in various animal models, few studies are presently available regarding germ cell apoptosis in the human testis. The first part of this review is focused on germ cell apoptosis in testes of prepubertal boys, with special emphasis on apoptosis in normal and cryptorchid testes. A higher percentage of apoptotic spermatogonia was seen in the cryptorchid testes than in the scrotal testes. The hCG-treatment increased the number of apoptotic spermatogonia. The hCG-treatment-induced apoptosis in spermatogonia had severe long-term consequences in reproductive functions in adulthood. Increased apoptosis after hCG-treatment was associated with subnormal testis volumes, subnormal sperm density and pathologically elevated serum FSH. This finding indicates that increased apoptosis in spermatogonia in prepuberty leads to disruption of testis development. To evaluate the role of apoptosis in human adult testes, apoptosis was induced in seminiferous tubules that were incubated under serum-free conditions in the absence or presence of testosterone. Most frequently apoptosis was identified in spermatocytes. Occasionally some spermatids also showed signs of apoptosis. In short term incubations apoptosis was suppressed by testosterone. Our findings lead to the conclusion that apoptosis is a normal, hormonally controlled phenomenon in the human testis. The role of apoptosis in disorders of spermatogenesis remains to be established.     

Inhibition of sperm production in mice by annexin V microinjected into seminiferous tubules: possible etiology of phagocytic clearance of apoptotic spermatogenic cells and male infertility

Many differentiating spermatogenic cells die by apoptosis during the process of mammalian spermatogenesis. However, very few apoptotic spermatogenic cells are detected by histological examination of the testis, probably due to the rapid elimination of dying cells by phagocytosis. Previous in vitro studies showed that Sertoli cells selectively phagocytose dying spermatogenic cells by recognizing the membrane phospholipid phosphatidylserine (PS), which is exposed to the surface of spermatogenic cells during apoptosis. We examined here whether PS-mediated phagocytosis of apoptotic spermatogenic cells occurs in vivo. For this purpose, the PS-binding protein annexin V was microinjected into the seminiferous tubules of normal live mice, and their testes were examined. The injection of annexin V caused no histological changes in the testis, but significantly increased the number of apoptotic spermatogenic cells as assessed by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay. The number of Sertoli cells did not change in the annexin V-injected testes, and annexin V itself did not induce apoptosis in primary cultured spermatogenic cells. These results indicate that annexin V inhibited the phagocytic clearance of apoptotic spermatogenic cells and suggest that PS-mediated phagocytosis of those cells occurs in vivo. Furthermore, the injection of annexin V into the seminiferous tubules brought about a significant reduction in the number of spermatogenic cells and epididymal sperm in anticancer drug-treated mice. This suggests that the elimination of apoptotic spermatogenic cells is required for the production of sperm.     

Primate spermatogenesis: new insights into comparative testicular organisation, spermatogenic efficiency and endocrine control

Owing to the close phylogenetic relationship of Platyrrhini (New World monkeys) and Catarrhini (Old World monkeys) to man, nonhuman primates are often used as models for the study of male reproductive physiology and endocrinology. This review aims at providing new data and insights into comparative primate spermatogenesis, dealing specifically with quantitative aspects of germinal epithelial organisation and germ cell production, and with the roles of gonadotrophic hormones in this process. Typically, the seminiferous epithelium is composed of specific germ cell associations (spermatogenic stages). In rodents, prosimians and most Catarrhini, tubular cross sections contain a single spermatogenic stage whereas in Platyrrhini, great apes and man multi-stage tubules are present. Since Platyrrhini represent a more basal type of primate, this spermatogenic feature must have developed convergently. The primate multi-stage tubular arrangement was previously believed to be associated with low spermatogenic efficiency. However, recent studies using new methodological approaches and comparing primate species from all taxa have revealed that multistage organisation is compatible with highly efficient spermatogenesis. In fact, meta-analysis demonstrated that the efficiency of spermatogenesis in several nonhuman primate species is comparable to that of rodents which are considered as species with highly efficient germ cell production. The duration of the spermatogenic process was not related to organisation or efficiency of spermatogenesis. Sertoli cell work load was species-specific but had no impact on germ cell numbers and on the efficiency of spermatogenesis. The gonadotrophic hormones, luteinizing hormone (LH) and follicle stimulating hormone (FSH) are the primary regulators of primate testicular function. Recent studies revealed that in New World monkeys chorionic gonadotrophin (CG)--the primate pregnancy hormone--regulates testosterone production instead of LH. Receptor studies demonstrated a dual action of the closely related hormones LH and CG in primates. It is hypothesised that following the divergence of the Platyrrhini lineage from Catarrhini, the LH/CG system evolved independently with ancestral functions of the LH/CG system retained in the neotropical taxa. In summary, key spermatogenic features are preserved across all primate taxa whereas male reproductive endocrinology features appear substantially different in the neotropical primates compared to other primate lineages.     

Curcumin induces small cell lung cancer NCI-H446 cell apoptosis via the reactive oxygen species-mediated mitochondrial pathway and not the cell death receptor pathway

Curcumin (diferuloylmethane), an active component of the spice turmeric, induces apoptosis in several types of malignancies. However, little is known about its anticancer activity in small cell lung cancer (SCLC). SCLC represents a highly malignant and particularly aggressive form of cancer, with early and widespread metastases and a poor prognosis. In this study, we found that curcumin does not activate caspase-8 cleavage or alter the expression of apoptotic receptors FAS and TRAIL in NCI-H446 cells, suggesting that curcumin-induced apoptosis is not associated with death receptor-mediated pathways in these cells. Instead, curcumin caused apoptosis by increasing Bax expression while decreasing the expression of Bcl-2 and Bcl-xL. Curcumin induced a rapid decrease in mitochondrial membrane potential and the release of cytochrome c into the cytosol, followed by activation of caspase-9 and caspase-3. In addition, curcumin-induced apoptosis was accompanied by an increase of intracellular reactive oxygen species (ROS) level. These results indicated that a ROS-mediated mitochondrial pathway played an important role in the process of curcumin-induced apoptosis of human SCLC NCI-H446 cells.     

生精细胞凋亡相关基因

细胞凋亡(apoptosis)是一种基因控制的细胞生理性自杀行为,用以维持细胞数量的相对恒定,可由某种刺激或抑制剂的移除而激活。在哺乳动物精子发生过程中,各级生精细胞都会发生相应的凋亡,通过严格调控以确保成熟精子生成的数量和质量。生精细胞的凋亡是一个许多基因参与的复杂的不可逆过程,其中Bcl-2/Bax基因族、p53基因、Fas-Fasl基因、C-myc基因、CREM基因、HSP基因族、c-Kit/SCF基因、Insl3基因、iNOS基因、BMP8B基因、TR基因和存活蛋白(survivin)基因等发挥了重要作用。研究哺乳动物睾丸生精细胞凋亡相关基因,有利于了解生精细胞凋亡机制,为进一步阐明精子发生的调控机制,预防和治疗精子发生相关疾病提供重要的理论依据。     

Insights into male germ cell apoptosis due to depletion of gonadotropins caused by GnRH antagonists

The role of pituitary gonadotropins in the regulation of spermatogenesis has been unequivocally demonstrated, although, the precise mechanism of this regulation is not clearly understood. Previous studies have shown that specific immunoneutralization of LH/testosterone caused apoptotic cell death of meiotic and post-meiotic germ cells while that of FSH resulted in similar death of meiotic cells. In the present study, the death process of germ cells has been characterized by depleting both FSH and testosterone by administering two different potent GnRH antagonists, Cetrorelix and Acyline to both rats and mice. Pro-survival factors like Bcl-2 and Bcl-x/l were unaltered in germ cells due to GnRH antagonist treatment, although a significant increase in several pro-apoptotic markers including Fas and Bax were evident at both protein and RNA levels. This culminated in cytochrome C release from mitochondria and eventually increase in the activity of caspase-8 and caspase-3. These data suggest that both extrinsic and intrinsic apoptotic death pathways are operative in the germ cells death following decrease in FSH and testosterone levels. Multiple injections of GnRH antagonist resulted in complete disappearance of germ cells except the spermatogonial cells and discontinuation of the treatment resulted in full recovery of spermatogenesis. In conclusion our present data suggest that the principal role of FSH and testosterone is to maintain spermatogenic homeostasis by inhibiting death signals for the germ cells.     

Global gene expression in mouse vaginae exposed to diethylstilbestrol at different ages

Estrogens regulate proliferation and differentiation of cells in target organs such as the female reproductive tract. In mature mice, estrogens stimulate cell proliferation, whereas ovariectomy results in atrophy of the female reproductive tract. In contrast, perinatal exposure to estrogens, including diethylstilbestrol (DES), induces persistent, ovary-independent vaginal stratification and cervico-vaginal tumors later in life. These effects are due to altered cell fate following DES exposure during a critical developmental period. The detailed mechanisms underlying the reversible and irreversible cell proliferation in vaginae induced by DES at different ages has not been clarified. Therefore, we examined differences in gene expression pattern using DNA microarray analysis in mouse vaginae 6 hrs after a single injection of 2 mug DES per gram of body weight, and proliferation of vaginal epithelial and stromal cells 24 hrs after the injection at postnatal days (PNDs) 0, 5, 20, and 70. After DES stimulation, vaginal epithelial and stromal cells showed cell proliferation at PNDs 20 and 70, and at PNDs 0 and 5, respectively. DNA microarray analysis exhibited 54 DES-induced genes and 9 DES-repressed genes in vaginae at PND 0, whereas more than 200 DES-induced genes were found in vaginae at PNDs 5 and 20, and 350 genes at PND 70. Clustering analysis of DES-induced genes in the vaginae at different ages revealed that genes induced by DES at PND 5 were closer to the adult type than that of PND 0. Genes related to keratinocyte differentiation, such as Gadd45alpha, p21, 14-3-3 sigma, small proline-rich protein 2f (Sprr2f), and Krupple-like factor 4 (Klf4), were induced by DES. The number of DES-induced genes during the critical period, PND 0, was smaller than those found after the critical period. These results give insight toward understanding the molecular mechanisms underlying the critical period in mouse vaginae.     

Protein kinase C activation modulates pro- and anti-apoptotic signaling pathways

Activation of protein kinase C (PKC) by TPA in human U937 myeloid leukemia cells is associated with induction of adherence, differentiation, and G0/G1 cell cycle arrest. In this study, we demonstrate that in addition to these differentiating cells about 25% of U937 cells accumulated in the subG1 phase after TPA treatment. This effect proved to be phorbol ester-specific, since other compounds such as retinoic acid or vitamin D3 failed to induce apoptosis in conjunction with differentiation. Only a specific inhibitor of PKC, GF109203X, but not the broad-spectrum kinase inhibitor staurosporine or a tyrosine kinase inhibitor genistein could reverse the induction of apoptosis. Bryostatin-1, another specific PKC activator with distinct biochemical activity failed to induce apoptosis. Moreover, bryostatin-1 completely abolished the induction of apoptosis in U937 cells even if added 8 hours after TPA treatment. Apart from apoptosis induced by various chemotherapeutic drugs, TPA-related cell death is not mediated by an autocrine Fas-FasL loop and could not be prevented by a blocking antibody to the Fas receptor. However, a 75% reduction in the number of apoptotic cells after TPA stimulation was achieved by preincubation with a blocking antibody to the TNFalpha receptor. Tetrapeptide cleavage assays revealed a four-fold increase in the DEVD-cleavage activity in U937 cells compared to a three-fold increase in TUR cells. Immunoblotting demonstrated that TUR cells did not activate significant levels of caspase-3 or -7, whereas in U937 cells a 20-kDa cleavage product corresponding to activated caspase-3 was detectable after 3 d TPA exposure. Moreover, immunoblots revealed a strongly reduced expression of the adaptor molecule APAF-1, which is required for cytochrome c-dependent activation of caspase-9 and subsequently caspase-3. APAF-1 proved to be inducible after PKC activation with phorbol ester in U937, but not in TUR cells. Thus, APAF-1 expression may, at least in part, be regulated by PKC activity and reduced APAF-1 levels are associated with resistance to various inducers of apoptosis. Furthermore, TPA exposure of U937 cells is associated with increased levels of the pro-apoptotic proteins Bak and Bcl-xs, whereas simultaneously a decline in the Bcl-2 expression was noticable.     

HLJ1 is a novel caspase-3 substrate and its expression enhances UV-induced apoptosis in non-small cell lung carcinoma

Carcinogenesis is determined based on both cell proliferation and death rates. Recent studies demonstrate that heat shock proteins (HSPs) regulate apoptosis. HLJ1, a member of the DnaJ-like Hsp40 family, is a newly identified tumor suppressor protein closely related to relapse and survival in non-small cell lung cancer (NSCLC) patients. However, its role in apoptosis is currently unknown. In this study, NSCLC cell lines displaying varying HLJ1 expression levels were subjected to ultraviolet (UV) irradiation, followed by flow cytometry. Interestingly, the percentages of apoptotic cells in the seven cell lines examined were positively correlated with HLJ1 expression. Enforcing expression of HLJ1 in low-HLJ1 expressing highly invasive cells promoted UV-induced apoptosis through enhancing JNK and caspase-3 activation in NSCLC. Additionally, UV irradiation led to reduced levels of HLJ1 predominantly in apoptotic cells. The pan-caspase inhibitor, zVAD-fmk and caspase-3-specific inhibitor, DEVD-fmk, prevented UV-induced degradation of HLJ1 by the late stage of apoptosis. Further experiments revealed a non-typical caspase-3 cleavage site (MEID) at amino acid 125–128 of HLJ1. Our results collectively suggest that HLJ1 is a novel substrate of caspase-3 during the UV-induced apoptotic process.     

Role of class B scavenger receptor type I in phagocytosis of apoptotic rat spermatogenic cells by Sertoli cells

Rat Sertoli cells phagocytose apoptotic spermatogenic cells, which consist mostly of spermatocytes, in primary culture by recognizing phosphatidylserine (PS) exposed on the surface of degenerating spermatogenic cells. We compared the mode of phagocytosis using spermatogenic cells at different stages of spermatogenesis. Spermatogenic cells were separated into several groups based on their ploidy, with purities of 60-90%. When the fractionated spermatogenic cell populations were subjected to a phagocytosis assay, cells with ploidies of 1n, 2n, and 4n were almost equally phagocytosed by Sertoli cells. All the cell populations exposed PS on the cell surface, and phagocytosis of all cell populations was similarly inhibited by the addition of PS-containing liposomes. Class B scavenger receptor type I (SR-BI), a candidate for the PS receptor, was detected in Sertoli cells. Overexpression of the rat SR-BI cDNA increased the PS-mediated phagocytic activity of Sertoli cell-derived cell lines. Moreover, phagocytosis of spermatogenic cells by Sertoli cells was inhibited in the presence of an anti-SR-BI antibody. Finally, the addition of high density lipoprotein, a ligand specific for SR-BI, decreased both phagocytosis of spermatogenic cells and incorporation of PS-containing liposomes by Sertoli cells. In conclusion, SR-BI functions at least partly as a PS receptor, enabling Sertoli cells to recognize and phagocytose apoptotic spermatogenic cells at all stages of differentiation.     

Thiosulfinates from Allium tuberosum L. induce apoptosis via caspase-dependent and -independent pathways in PC-3 human prostate cancer cells

This study was aimed to evaluate the apoptotic effects of thiosulfinates purified from Allium tuberosum L. on PC-3 human prostate cancer cells, and to elucidate detailed apoptosis mechanisms. Thiosulfinates significantly decrease viable cell numbers in dose- and time-dependent manners by apoptotic cell death via DNA fragmentation, chromatin condensation, and an increased sub-G1 phase. Apoptosis induced by thiosulfinates is associated with the activation of initiator caspase-8 and -9, and the effector caspase-3. In this study, thiosulfinates stimulated Bid cleavage, indicating that the apoptotic action of caspase-8-mediated Bid cleavage leads to the activation of caspase-9. Thiosulfinates decreased the expression of the anti-apoptotic protein Bcl-2 and increased the expression of the pro-apoptotic protein Bax. Thiosulfinates also increased the expression of AIF, a caspase-independent mitochondrial apoptosis factor, in PC-3 cells. These results indicate that thiosulfinates from A. tuberosum L. inhibit cell proliferation and induce apoptosis in PC-3 cells, which may be mediated via both caspase-dependent and -independent pathways.     

Apoptosis induced by interferon-alpha and antagonized by EGF is regulated by caspase-3-mediated cleavage of gelsolin in human epidermoid cancer cells

We have previously reported that interferon-alpha (IFNalpha) induces apoptosis and EGF can antagonize this effect in human epidermoid cancer KB cells. Since apoptosis occurs together with cytoskeleton reorganization we have evaluated if IFNalpha and EGF could modulate cell remodeling in our experimental conditions. We have found that 48 h 1,000 IU/ml IFNalpha induced structural reorganization of stress fibers and membrane delocalization and partial capping of the actin severing protein gelsolin. The transfection of KB cells with both a wild type (WT) or a C-terminal truncated form of gelsolin caused overexpression of the protein and an increase of both the spontaneous and IFNalpha-induced apoptosis and cell cytoskeletal modifications. In fact, after 48 h of treatment IFNalpha induced 45% of apoptotic cell death in parental cells while an approximately 80% of cell population was apoptotic in transfected cells. These effects occurred together with an increase of the expression and consequent degradation of gelsolin. Again the addition of EGF to IFNalpha-treated transfected cells caused a recovery of the apoptosis. Notably, IFNalpha and EGF did not modify the expression of other molecules associated to cytoskeleton such as focal adhesion kinase and vinculin. In the same experimental conditions IFNalpha induced also gelsolin cleavage that occurred together with caspase-3 activation and release of cytochrome c. All these effects were antagonized by the exposure of IFNalpha-treated KB to 10 nM EGF for the last 12 h. Moreover, the specific inhibition of caspase-3 with 20 microM DEVD completely abrogated apoptosis and gelsolin cleavage induced by IFNalpha. In conclusion, our data are the first demonstration that IFNalpha can induce morphological cell changes that are peculiar of apoptosis onset through the caspase-3-mediated cleavage of gelsolin. Furthermore, we have demonstrated that EGF is able to antagonize these effects through the inhibition of caspase-3 activation.     

Relationship of F9 antigens to spermatogenic cell antigens

Mice immunized with teratocarcinoma F9 cells or human blood group substances A, B or H exhibited significant inhibition of spermatogenesis comparable to the inhibition induced by immunization with testicular cells. All these immunization schemes resulted in the production of antibodies which recognize antigens common to F9 and spermatogenic cells. In addition to these antigens, both anti-F9 and anti-ABH sera also recognize antigens which are specific for F9 cells. One of them was identified as SSEA-1. These results support the hypothesis that oncofetal F9 antigens are carbohydrate structures, which may play an important role in spermatogenic cell differentiation.     

Fas-Fas ligand system as a possible mediator of spermatogenic cell apoptosis in human maturation-arrested testes

To elucidate the mechanism of maturation arrest, known as one of the male infertility, we addressed whether germ cell apoptosis occurs during maturation arrest, and if so, whether Fas and Fas ligand expressions are involved in the apoptosis. By electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), typical apoptotic features were frequently found around the spermatocytic stage in maturation arrest, compared to that in normal testes. When paraffin-embedded sections reacted with anti-Fas antiserum, staining for Fas was found in the plasma membranes of spermatocytes in the maturation-arrested testes, while no positive spermatogenic cells were seen in the normal testes. On the other hand, positive immunostaining for Fas ligand was restricted to Sertoli cells in the maturation-arrested testes as well as in the normal testes, although the intensity of staining for Fas ligand in normal testicular Sertoli cells was much weaker than that of maturation-arrested ones. Thus, these findings demonstrate that "maturation arrest" is characterized by frequent apoptosis of spermatocytes, and that Fas and Fas ligand staining are associated with a high frequency of apoptosis.     

Mono-(2-ethylhexyl) phthalate (MEHP) induces testicular alterations in male guinea pigs at prepubertal stage

We have recently shown that MEHP induces spermatogenic cell apoptosis in guinea pigs at prepubertal stage in vitro. To evaluate the effects of MEHP on the testicular tissues of guinea pigs in vivo, we conducted this research work. Five weeks old male guinea pigs were used in this experiment. They received a single oral dose of 2000 mg/ml of MEHP in corn oil by gavage at a volume equal to 4 ml/kg. Control group received a similar volume of corn oil vehicle. Vehicle- and MEHP-treated guinea pigs were sacrificed at the interval of 3, 6, and 9 h, and the testicular tissues were processed for histopathological studies. Distinct histopathological changes were recognized in testes. Detachment and displacement of spermatogenic cells, thin seminiferous epithelia, vacuolization of Sertoli cells were prominent at 6 h after MEHP treatment. The lumina of the efferent ductules were frequently occupied with sloughed seminiferous epithelia from 6 to 9 h after MEHP treatment. Apoptotic spermatogenic cells appeared at 3 h in the control group. The incidence of apoptotic spermatogenic cells significantly increased (*p<0.05) from 3 to 9 h, and the maximal increase of apoptotic spermatogenic cells were observed at 9 h after MEHP treatment. Time-dependent increases of apoptotic spermatogenic cells was recognized throughout the experimental period. It may be suggested here that MEHP also induces spermatogenic cell apoptosis in guinea pigs in vivo and guinea pigs may be considered as a useful animal model for sensitivity test of the reproductive toxicity to some phthalate esters at their earlier stage in vivo.