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Male germ cell development: turning on the apoptotic pathways
Journal of Reproductive Immunology 83 (2009) 31–35
Contents lists available at ScienceDirect
Journal of Reproductive Immunology journal homepage: www.elsevier.com/locate/jreprimm
Male germ cell development: turning on the apoptotic pathways Rakshamani Tripathi a , Durga Prasad Mishra b , Chandrima Shaha a,∗ a b
Cell Death and Differentiation Research Laboratory, National Institute of Immunology, Aruna Asaf Ali Road, New Delhi 110067, India Endocrinology Division, Central Drug Research Institute, Lucknow, India
a r t i c l e
i n f o
Article history: Received 14 March 2009 Accepted 19 May 2009 Keywords: Testis Apoptosis Bax Bcl-2 Estrogen
a b s t r a c t From the viewpoint of improving germ cell production and treatment of testicular cancers, understanding the control of testicular cell death is of great relevance. One of the prominent features of spermatogenesis is apoptosis of germ cells at different stages of differentiation, by which excess and unfit cells are discarded to maintain proper tissue homeostasis. A phase of heightened apoptosis known as the ‘first wave of spermatogenesis’ occurs when the gonocytes differentiate into spermatogonia. The germ cells use an extrinsic pathway of apoptosis involving the Fas/FasL molecules as well as the mitochondrial pathway of death using the Bcl-2 family of proteins. A comprehensive view of the involvement of the different pro- and anti-apoptotic molecules has been defined through the use of mutant and knockout mice and toxin-induced cell death models. In addition, hormones such as estrogens in the male are of great interest. The presence of estrogen receptors on germ cells makes these cells susceptible to environmental agents which can mimic estrogens and potentially cause functional impairment of the male gamete. Post-industrialization, an increase in testicular cancers has been recorded and carcinoma of germ cell origin is susceptible to platinum-based compounds that induce multiple apoptotic pathways. This review covers recent progress made on the above issues. The challenge is now to identify the precise signaling pathways and the mechanisms by which germ cells and germ cell tumors initiate cell death processes, and to utilize this information for improving reproductive health related issues. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction In a multicellular body, substantial cell death occurs each day through various processes to maintain tissue homeostasis. One of the most well studied events for cell death is apoptosis, a process that is induced by an inherent cellular program and is an appropriate process of death because it does not elicit an immune response. The term apoptosis was first described by Kerr et al. (1972) describing some typical features by which the process can be identified. Apoptosis occurs during early development to provide proper shape and size to the organs, during repro-
∗ Corresponding author. Tel.: +91 1126703627; fax: +91 1126742125. E-mail address: [email protected] (C. Shaha). 0165-0378/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jri.2009.05.009
ductive life to maintain tissue homeostasis, and at the time of aging to predominantly discard sick and aged cells. Apoptosis also occurs as a defense mechanism such as in immune reactions or when cells are damaged by disease or toxins (Norbury and Hickson, 2001). The testis is one tissue where a large incidence of apoptosis occurs to discard excessive germ cells, or whereby germ cells damaged by toxins are removed (Hikim et al., 2003). Building a knowledge base on the molecular components of the apoptotic program in spermatogenic cells is an essential step towards the development of novel therapeutic regimens targeted to male contraception and treatment of germ cell tumors and infertility. Therefore, both from the perspective of improving gamete production and preventing effects of environmental agents, the study of cell death in the testis is of great relevance for the betterment of reproductive health.
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R. Tripathi et al. / Journal of Reproductive Immunology 83 (2009) 31–35
Fig. 1. A schematic diagram shows the various molecular components involved in the extrinsic and the intrinsic pathways of apoptosis. Three pathways are represented. The intrinsic pathway involving the mitochondria, the extrinsic pathway with the Fas/FasL and the Granzyme pathway with their corresponding downstream regulators are shown. The substrates of caspase 3, including lamins, fodrin and gelsolin are indicated as these are the primary components that are acted upon by caspase 3 leading to fragmentation of the cell. The activation of p53 after DNA damage is shown.
1.1. The process of apoptosis Ultrastructurally, apoptosis is characterized by membrane blebbing, cell volume shrinkage, chromatin condensation, cytoplasmic vacuolization and disassembly of the cell into membrane-bound remnants termed apoptotic bodies (Majno and Joris, 1995). The biochemical features of apoptosis include phosphatidylserine exposure to the external leaflet of the plasma membrane, activation of caspase cascades (Hengartner, 2000), DNA cleavage and formation of an oligonucleosomal ladder (King and Cidlowski, 1995). Many different types of signals—growth factor withdrawal, genotoxic stress, oxidative stress, radiation damage, cell cycle perturbations and DNA damage can initiate apoptosis. Apparently, the cell uses various sensing mechanisms to interpret and integrate the different death-inducing signals and then funnels the initiation message to activation of extrinsic (involving the death receptor) or intrinsic (mitochondrial) apoptotic pathway (Fig. 1). In the intrinsic pathway, proteins of the Bcl-2 family, consisting of various anti-apoptotic (Bcl-2, Bcl-xl, Bcl-w, A1, Bcl-G, Bc-Rmbo and Mcl-1) and pro-apoptotic (Bax, Bak, Bim/Bod, Bid, Bok, Bad, Bcl-xs, Noxa and Puma) members, are involved in the decision to activate the caspase cascade to induce apoptosis (Youle and Strasser, 2008). On
the other hand the engagement of Fas ligand to the Fas death receptor triggers the apoptosis via extrinsic pathway. Other proteins important in induction of apoptosis include p53, a tumor suppressor protein; activation of p38 MAP kinase and MEK/ERK signal transduction system that directly activates the caspase cascade (Pentikainen et al., 1999).
2. Spermatogenesis Spermatogenesis is the process of sperm formation and maturation. It occurs in the seminiferous epithelium of the testis and is characterized by continuous germ cell maturation towards the center of the seminiferous tubules: mitotic proliferation of spermatogonia, meiotic division of spermatocytes, differentiation of spermatids and finally release of spermatozoa into the tubule lumen. As in many tissues throughout the body, the number of cells in the seminiferous tubules of the testis is determined by a dynamic balance between cell proliferation and apoptotic cell death (Shaha, 2008). Both spontaneous and increased cell death due to triggering stimuli (such as deprivation of intra-testicular testosterone and gonadotrophins, Sertoli cell toxicants or chemotherapeutic drugs) occur via apoptosis.
R. Tripathi et al. / Journal of Reproductive Immunology 83 (2009) 31–35
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Fig. 2. The components of the apoptotic pathways that are induced by various toxins to bring about cell death. Pathway components activated during the first wave of spermatogenesis to induce cell death are also shown.
3. Germ cell death during sperm development After formation of the testis, when the gonocytes differentiate into spermatogonia, a phase of heightened apoptosis has been observed which is known as the ‘first wave of spermatogenesis’ for adjustment of numbers in a growing testis. In mice this involves caspase 2 while in rats it involves caspases 3, 8 and 9, indicating involvement of both extrinsic and intrinsic pathway (Moreno et al., 2006). Bax is a pro-apoptotic member while Bcl-2 or Bcl-w is antiapoptotic member of the Bcl-2 family of proteins. Their involvement in the early spermatogenesis is well documented in Bax knockout transgenic mice or transgenic mice over-expressing Bcl-2 or Bcl-x, where the early wave of apoptosis is eliminated resulting in infertility (Furuchi et al., 1996). Data suggest that regulated spatial and temporal expression of Bcl-w is required for normal testicular development and spermatogenesis, and over-expression of Bcl-w inhibits germ cell cycle entry and/or cell cycle progression leading to disrupted spermatogenesis (Yan et al., 2003). Mice over-expressing Bcl-2 or Bcl-XL exhibit male sterility characterized by defects in spermatogenesis, apparently due to suppression of the early apoptotic wave. Therefore, a precise expression of both Bcl-2 and Bcl-xL is required since an alteration either way in the
balance of these proteins could lead to infertility. During the early wave, a high level of pro-apoptotic Bax expression is normally seen in germ cells of the testes, suggesting Bax-induced cell death (Jahnukainen et al., 2004). Thus, it appears that the early apoptotic wave in mice required for the formation of mature sperm is dependent on the proper balance of anti-apoptotic genes such as Bcl-XL , and proapoptotic genes such as Bax. It has been shown that not only the mitochondrial pathway, but the extrinsic pathway is also involved during the first wave of spermatogenesis, because upregulation of CD95 (Apo-1/Fas) is associated with spermatocyte apoptosis in the rat (Lizama et al., 2007). 4. Germ cell death induced by toxins and hormones Various toxin-induced germ cell death models have been used to study the involvement of various pro- and anti-apoptotic molecules. Amongst the toxins, ethane-1,2dimethanesulfonate (EDS) is a cytotoxic alkylating agent known as a selective toxin for adult rat Leydig cells, which causes an increase in testicular Fas with an accompanying elevation of germ cell apoptosis (Woolveridge et al., 2001). Phthalates, such as di-(2-ethylhexyl) phthalate that are ubiquitous environmental toxicants produce testicular atrophy in laboratory animals and impair germ cell
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development in the human fetal testis in vitro (Yao et al., 2007). Nitrobenzene (NB) is a testicular toxicant in vivo which is active even in the absence of Fas because adult gld mice (mice with FasL mutation) treated with a single oral dose of NB (800 mg/kg) show a higher apoptotic index compared with wild-type C57BL/6 mice (Richburg and Nanez, 2003). A high dose of bisphenol A, considered an endocrine disruptor, induces apoptosis of Leydig and germ cells in the mouse testis through the Fas signaling pathway (Li et al., 2009). Lindane, an organochlorine pesticide, is known to impair testicular functions and fertility though modulation of NFB and FasL (Saradha et al., 2009). Epidemiological surveys and animal experimental studies suggest that exposure to chemical 2-bromopropane (2BP), an intermediate in the synthesis of pharmaceuticals, dyes, and other organic chemicals could result in reproductive and hematopoietic disorders in animals as well as humans (Kim et al., 1999). The effects of 2-BP treatment are mediated through both the extrinsic and the intrinsic pathway of apoptosis (Yu et al., 2001). The effects of chronic exposure of 4-tert-octylphenol on the testicular development of prepubertal male rats show that expression of bcl-xL mRNA was significantly decreased in the treated groups, whereas the expressions of bcl-2 and bax mRNA were not significantly changed (Kim et al., 2004). Using an in vitro spermatogenic cell apoptosis model, we showed that Ca2+ regulates the Bcl-xS and Bcl-xL expression when exposed to 2,5-hexanedione, a metabolite of the common industrial solvent n-hexane (Mishra et al., 2006). Fig. 2 summarizes the pro- and anti-apoptotic molecules involved during toxin-induced death. In the mammalian testes, hormones such as follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), and testosterone have all been shown to regulate germ cell survival (Griswold, 1998). Excess testosterone can cause death, for example, after injection of testosterone undecanoate, both the apoptotic signal in germ cells and the expression of Fas/FasL in monkey testis increase correlatively in a time-dependent manner, reaching a maximum level on day 30 (Zhou et al., 2001). In contrast, testosterone withdrawal stimulates caspase activity and produces DNA fragmentation in Sertoli cells, with a weak effect on DNA fragmentation and caspase activity in germ cells suggesting a caspase-independent way of death in germ cells which is controlled by Sertoli cells via an as yet undetermined mechanism (Tesarik et al., 2002). Like testosterone, estrogens have a profound effect on cell death in the testis and evidence has accumulated over several decades that estrogen is essential for spermatogenesis and that intra-testicular concentrations of estrogen are very high (Hess, 2003). Mouse germ cells express both estrogen receptor-␣ and - and mice lacking estrogen receptor-␣ are infertile (Chen et al., 2009). Therefore, agents able to mimic estrogens can potentially alter the action of the hormone on spermatogenic cells leading to functional impairment of the male gamete. Reports of lowered fertility rates as a consequence of exposure to agents with estrogenic activity termed as endocrine disruptors are well documented in wild life populations (Hutchinson et al., 2000), although its effect on humans remain controver-
sial (Storgaard et al., 2006). Our studies show that exposure to 17-estradiol induces Fas ligand expression and subsequent ligation of this ligand with the Fas receptor increases nitric oxide formation through the activation of inducible nitric oxide synthase in the rat both in vitro and in vivo (Nair and Shaha, 2003; Mishra and Shaha, 2005). 5. Cell death in testicular carcinoma Over the past several decades, the incidence of testicular cancers has increased. The chemotherapy regimens most commonly used for these cancers are platinum-based compounds like cisplatin (Bokemeyer et al., 1996). The human testicular germ cell tumor cell lines show an extraordinary sensitivity to treatment with cisplatin (Johnstone et al., 2002) which causes DNA intra-stand and inter-stand crosslinks that when not repaired, lead to apoptosis. DNA damage by cisplatin results in transcription inhibition, activation of multiple signaling pathways including upregulation of p53 levels, phosphorylation of p38 MAP kinase and Erk1/2 and the other signaling pathways both in vitro and in vivo (Chauhan et al., 2009; Wu et al., 2005), leading to apoptosis in testicular germ cell tumors (TGCT). In addition, cisplatin generates reactive oxygen species (ROS), which are known as one of the intermediates following chemotherapy (Wu et al., 2005). The high toxicity of cisplatin poses a major challenge to develop alternate therapies for the treatment of testicular cancers. 6. Conclusions and perspectives This review has attempted to highlight the recent developments in the field of apoptosis in general and the role of this mode of cell death in spermatogenesis. Studies using genetically altered mice and toxin-induced apoptosis models indicate that germ cell apoptosis, like that of other cell systems, is regulated by multiple genes that either inhibit or promote cell death. The challenge is now to identify the full complement of pro- and anti-apoptotic molecules involved in germ cell or germ cell tumors and to determine the precise signaling pathways under different situations. Future studies of genetically modified animals like knockouts or knock-ins for particular genes, especially those carrying conditional or germ cell type-specific genetic abnormalities will reveal more about the involvement of the various apoptosis-associated molecules. This type of work will without doubt, lay the foundation for future rational treatment of male infertility and testicular neoplasia, as well as providing a basis for the rational design of male contraceptive agents. Recent developments showing the involvement of micro-RNAs which are a group of highly conserved, non-coding endogenous small RNAs (ValenciaSanchez et al., 2006) in the control of gene transcription during spermatogenesis opens up a new era of investigations. Dicer is an essential component for micro-RNA processing and it has been shown that conditional knockout mouse lacking proper Dicer1 function have abnormal germ cell differentiation, elongation and sperm motility resulting in infertility (Maatouk et al., 2008). Therefore, there are many aspects of regulation of spermatogenic cell apoptosis that need to be addressed in order to formu-
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late areas of interference and augmentation of pro- and anti-apoptotic events. The potential use of anti-apoptotic compounds in humans in vivo requires careful appraisal of the capability of the seminiferous epithelium for functional spermatogenesis after the treatments. Accordingly, studies employing experimental animals should be carefully evaluated for production of healthy offspring after apoptosis-inhibiting treatments. It is obvious from the data available on germ cell apoptosis that we have progressed substantially from what was known a decade ago on germ cell death. These observations has opened up major challenges to take the field further and emergence of newer technologies have opened avenues to investigate and resolve several steps involved in germ cell apoptosis. Acknowledgement Part of the work described from authors laboratory was supported by grants to the National Institute of Immunology from the Department of Biotechnology and a grant from Indo-US Collaboration on ‘Contraceptive and Reproductive Health Research (CRHR)’, USA. References Bokemeyer, C., Kohrmann, O., Tischler, J., Weissbach, L., Rath, U., Haupt, A., Schoffski, P., Harstrick, A., Schmoll, H.J., 1996. A randomized trial of cisplatin, etoposide and bleomycin (PEB) versus carboplatin, etoposide and bleomycin (CEB) for patients with ‘good-risk’ metastatic non-seminomatous germ cell tumors. Ann. Oncol. 7, 1015– 1021. Chauhan, P., Sodhi, A., Shrivastava, A., 2009. Cisplatin primes murine peritoneal macrophages for enhanced expression of nitric oxide, proinflammatory cytokines, TLRs, transcription factors and activation of MAP kinases upon co-incubation with L929 cells. Immunobiology 214, 197–209. Chen, M., Hsu, I., Wolfe, A., Radovick, S., Huang, K., Yu, S., Chang, C., Messing, E.M., Yeh, S., 2009. Defects of prostate development and reproductive system in the estrogen receptor-alpha null male mice. Endocrinology 150, 251–259. Furuchi, T., Masuko, K., Nishimune, Y., Obinata, M., Matsui, Y., 1996. Inhibition of testicular germ cell apoptosis and differentiation in mice misexpressing Bcl-2 in spermatogonia. Development 122, 1703–1709. Griswold, M.D., 1998. The central role of Sertoli cells in spermatogenesis. Semin. Cell Dev. Biol. 9, 411–416. Hengartner, M.O., 2000. The biochemistry of apoptosis. Nature 407, 770–776. Hess, R.A., 2003. Estrogen in the adult male reproductive tract: a review. Reprod. Biol. Endocrinol. 1, 1–14. Hikim, A.P., Lue, Y., Yamamoto, C.M., Vera, Y., Rodriguez, S., Yen, P.H., Soeng, K., Wang, C., Swerdloff, R.S., 2003. Key apoptotic pathways for heat-induced programmed germ cell death in the testis. Endocrinology 144, 3167–3175. Hutchinson, T.H., Brown, R., Brugger, K.E., Campbell, P.M., Holt, M., Lange, R., McCahon, P., Tattersfield, L.J., van Egmond, R., 2000. Ecological risk assessment of endocrine disruptors. Environ. Health Perspect. 108, 1007–1014. Jahnukainen, K., Chrysis, D., Hou, M., Parvinen, M., Eksborg, S., Soder, O., 2004. Increased apoptosis occurring during the first wave of spermatogenesis is stage-specific and primarily affects midpachytene spermatocytes in the rat testis. Biol. Reprod. 70, 290– 296. Johnstone, R.W., Ruefli, A.A., Lowe, S.W., 2002. Apoptosis: a link between cancer genetics and chemotherapy. Cell 108, 153–164. Kerr, J.F., Wyllie, A.H., Currie, A.R., 1972. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239–257. Kim, S.K., Lee, H.J., Yang, H., Kim, H.S., Yoon, Y.D., 2004. Prepubertal exposure to 4-tert-octylphenol induces apoptosis of testicular germ cells in adult rat. Arch. Androl. 50, 427–441.
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Kim, Y., Park, J., Moon, Y., 1999. Hematopoietic and reproductive toxicity of 2-bromopropane, a recently introduced substitute for chlorofluorocarbons. Toxicol. Lett. 108, 309–313. King, K.L., Cidlowski, J.A., 1995. Cell cycle and apoptosis: common pathways to life and death. J. Cell Biochem. 58, 175–180. Li, Y.J., Song, T.B., Cai, Y.Y., Zhou, J.S., Song, X., Zhao, X., Wu, X.L., 2009. Bisphenol A exposure induces apoptosis and up-regulation of Fas/FasL and caspase-3 expression in the testes of mice. Toxicol. Sci. 108, 427–436. Lizama, C., Alfaro, I., Reyes, J.G., Moreno, R.D., 2007. Up-regulation of CD95 (Apo-1/Fas) is associated with spermatocyte apoptosis during the first round of spermatogenesis in the rat. Apoptosis 12, 499–512. Maatouk, D.M., Loveland, K.L., McManus, M.T., Moore, K., Harfe, B.D., 2008. Dicer1 is required for differentiation of the mouse male germline. Biol. Reprod. 79, 696–703. Majno, G., Joris, I., 1995. Apoptosis, oncosis, and necrosis. An overview of cell death. Am. J. Pathol. 146, 3–15. Mishra, D.P., Shaha, C., 2005. Estrogen-induced spermatogenic cell apoptosis occurs via the mitochondrial pathway: role of superoxide and nitric oxide. J. Biol. Chem. 280, 6181–6196. Mishra, D.P., Pal, R., Shaha, C., 2006. Changes in cytosolic Ca2+ levels regulate Bcl-xS and Bcl-xL expression in spermatogenic cells during apoptotic death. J. Biol. Chem. 281, 2133–2143. Moreno, R.D., Lizama, C., Urzua, N., Vergara, S.P., Reyes, J.G., 2006. Caspase activation throughout the first wave of spermatogenesis in the rat. Cell Tissue Res. 325, 533–540. Nair, R., Shaha, C., 2003. Diethylstilbestrol induces rat spermatogenic cell apoptosis in vivo through increased expression of spermatogenic cell Fas/FasL system. J. Biol. Chem. 278, 6470–6481. Norbury, C.J., Hickson, I.D., 2001. Cellular responses to DNA damage. Annu. Rev. Pharmacol. Toxicol. 41, 367–401. Pentikainen, V., Erkkila, K., Dunkel, L., 1999. Fas regulates germ cell apoptosis in the human testis in vitro. Am. J. Physiol. 276, E310–E316. Richburg, J.H., Nanez, A., 2003. Fas- or FasL-deficient mice display an increased sensitivity to nitrobenzene-induced testicular germ cell apoptosis. Toxicol. Lett. 139, 1–10. Saradha, B., Vaithinathan, S., Mathur, P.P., 2009. Lindane induces testicular apoptosis in adult Wistar rats through the involvement of Fas–FasL and mitochondria-dependent pathways. Toxicology 255, 131–139. Shaha, C., 2008. Estrogens and spermatogenesis. In: Cheng, C.Y. (Ed.), Molecular Mechanisms of Spermatogenesis. Landes Biosciences. Austin, TX, pp. 42–64. Storgaard, L., Bonde, J.P., Olsen, J., 2006. Male reproductive disorders in humans and prenatal indicators of estrogen exposure. A review of published epidemiological studies. Reprod. Toxicol. 21, 4–15. Tesarik, J., Martinez, F., Rienzi, L., Iacobelli, M., Ubaldi, F., Mendoza, C., Greco, E., 2002. In-vitro effects of FSH and testosterone withdrawal on caspase activation and DNA fragmentation in different cell types of human seminiferous epithelium. Hum. Reprod. 17, 1811–1819. Valencia-Sanchez, M.A., Liu, J., Hannon, G.J., Parker, R., 2006. Control of translation and mRNA degradation by miRNAs and siRNAs. Genes Dev. 20, 515–524. Woolveridge, I., Taylor, M.F., Rommerts, F.F., Morris, I.D., 2001. Apoptosis related gene products in differentiated and tumorigenic rat Leydig cells and following regression induced by the cytotoxin ethane dimethanesulphonate. Int. J. Androl. 24, 56–64. Wu, Y.J., Muldoon, L.L., Neuwelt, E.A., 2005. The chemoprotective agent N-acetylcysteine blocks cisplatin-induced apoptosis through caspase signaling pathway. J. Pharmacol. Exp. Ther. 312, 424–431. Yan, W., Huang, J.X., Lax, A.S., Pelliniemi, L., Salminen, E., Poutanen, M., Toppari, J., 2003. Overexpression of Bcl-W in the testis disrupts spermatogenesis: revelation of a role of BCL-W in male germ cell cycle control. Mol. Endocrinol. 17, 1868–1879. Yao, P.L., Lin, Y.C., Sawhney, P., Richburg, J.H., 2007. Transcriptional regulation of FasL expression and participation of sTNF-alpha in response to sertoli cell injury. J. Biol. Chem. 282, 5420–5431. Youle, R.J., Strasser, A., 2008. The BCL-2 protein family: opposing activities that mediate cell death. Nat. Rev. Mol. Cell Biol. 9, 47–59. Yu, X., Kubota, H., Wang, R., Saegusa, J., Ogawa, Y., Ichihara, G., Takeuchi, Y., Hisanaga, N., 2001. Involvement of Bcl-2 family genes and Fas signaling system in primary and secondary male germ cell apoptosis induced by 2-bromopropane in rat. Toxicol. Appl. Pharmacol. 174, 35–48. Zhou, X.C., Wei, P., Hu, Z.Y., Gao, F., Zhou, R.J., Liu, Y.X., 2001. Role of Fas/FasL genes in azoospermia or oligozoospermia induced by testosterone undecanoate in rhesus monkey. Acta Pharmacol. Sin. 22, 1028–1033.
Contents lists available at ScienceDirect
Journal of Reproductive Immunology journal homepage: www.elsevier.com/locate/jreprimm
Male germ cell development: turning on the apoptotic pathways Rakshamani Tripathi a , Durga Prasad Mishra b , Chandrima Shaha a,∗ a b
Cell Death and Differentiation Research Laboratory, National Institute of Immunology, Aruna Asaf Ali Road, New Delhi 110067, India Endocrinology Division, Central Drug Research Institute, Lucknow, India
a r t i c l e
i n f o
Article history: Received 14 March 2009 Accepted 19 May 2009 Keywords: Testis Apoptosis Bax Bcl-2 Estrogen
a b s t r a c t From the viewpoint of improving germ cell production and treatment of testicular cancers, understanding the control of testicular cell death is of great relevance. One of the prominent features of spermatogenesis is apoptosis of germ cells at different stages of differentiation, by which excess and unfit cells are discarded to maintain proper tissue homeostasis. A phase of heightened apoptosis known as the ‘first wave of spermatogenesis’ occurs when the gonocytes differentiate into spermatogonia. The germ cells use an extrinsic pathway of apoptosis involving the Fas/FasL molecules as well as the mitochondrial pathway of death using the Bcl-2 family of proteins. A comprehensive view of the involvement of the different pro- and anti-apoptotic molecules has been defined through the use of mutant and knockout mice and toxin-induced cell death models. In addition, hormones such as estrogens in the male are of great interest. The presence of estrogen receptors on germ cells makes these cells susceptible to environmental agents which can mimic estrogens and potentially cause functional impairment of the male gamete. Post-industrialization, an increase in testicular cancers has been recorded and carcinoma of germ cell origin is susceptible to platinum-based compounds that induce multiple apoptotic pathways. This review covers recent progress made on the above issues. The challenge is now to identify the precise signaling pathways and the mechanisms by which germ cells and germ cell tumors initiate cell death processes, and to utilize this information for improving reproductive health related issues. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction In a multicellular body, substantial cell death occurs each day through various processes to maintain tissue homeostasis. One of the most well studied events for cell death is apoptosis, a process that is induced by an inherent cellular program and is an appropriate process of death because it does not elicit an immune response. The term apoptosis was first described by Kerr et al. (1972) describing some typical features by which the process can be identified. Apoptosis occurs during early development to provide proper shape and size to the organs, during repro-
∗ Corresponding author. Tel.: +91 1126703627; fax: +91 1126742125. E-mail address: [email protected] (C. Shaha). 0165-0378/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jri.2009.05.009
ductive life to maintain tissue homeostasis, and at the time of aging to predominantly discard sick and aged cells. Apoptosis also occurs as a defense mechanism such as in immune reactions or when cells are damaged by disease or toxins (Norbury and Hickson, 2001). The testis is one tissue where a large incidence of apoptosis occurs to discard excessive germ cells, or whereby germ cells damaged by toxins are removed (Hikim et al., 2003). Building a knowledge base on the molecular components of the apoptotic program in spermatogenic cells is an essential step towards the development of novel therapeutic regimens targeted to male contraception and treatment of germ cell tumors and infertility. Therefore, both from the perspective of improving gamete production and preventing effects of environmental agents, the study of cell death in the testis is of great relevance for the betterment of reproductive health.
32
R. Tripathi et al. / Journal of Reproductive Immunology 83 (2009) 31–35
Fig. 1. A schematic diagram shows the various molecular components involved in the extrinsic and the intrinsic pathways of apoptosis. Three pathways are represented. The intrinsic pathway involving the mitochondria, the extrinsic pathway with the Fas/FasL and the Granzyme pathway with their corresponding downstream regulators are shown. The substrates of caspase 3, including lamins, fodrin and gelsolin are indicated as these are the primary components that are acted upon by caspase 3 leading to fragmentation of the cell. The activation of p53 after DNA damage is shown.
1.1. The process of apoptosis Ultrastructurally, apoptosis is characterized by membrane blebbing, cell volume shrinkage, chromatin condensation, cytoplasmic vacuolization and disassembly of the cell into membrane-bound remnants termed apoptotic bodies (Majno and Joris, 1995). The biochemical features of apoptosis include phosphatidylserine exposure to the external leaflet of the plasma membrane, activation of caspase cascades (Hengartner, 2000), DNA cleavage and formation of an oligonucleosomal ladder (King and Cidlowski, 1995). Many different types of signals—growth factor withdrawal, genotoxic stress, oxidative stress, radiation damage, cell cycle perturbations and DNA damage can initiate apoptosis. Apparently, the cell uses various sensing mechanisms to interpret and integrate the different death-inducing signals and then funnels the initiation message to activation of extrinsic (involving the death receptor) or intrinsic (mitochondrial) apoptotic pathway (Fig. 1). In the intrinsic pathway, proteins of the Bcl-2 family, consisting of various anti-apoptotic (Bcl-2, Bcl-xl, Bcl-w, A1, Bcl-G, Bc-Rmbo and Mcl-1) and pro-apoptotic (Bax, Bak, Bim/Bod, Bid, Bok, Bad, Bcl-xs, Noxa and Puma) members, are involved in the decision to activate the caspase cascade to induce apoptosis (Youle and Strasser, 2008). On
the other hand the engagement of Fas ligand to the Fas death receptor triggers the apoptosis via extrinsic pathway. Other proteins important in induction of apoptosis include p53, a tumor suppressor protein; activation of p38 MAP kinase and MEK/ERK signal transduction system that directly activates the caspase cascade (Pentikainen et al., 1999).
2. Spermatogenesis Spermatogenesis is the process of sperm formation and maturation. It occurs in the seminiferous epithelium of the testis and is characterized by continuous germ cell maturation towards the center of the seminiferous tubules: mitotic proliferation of spermatogonia, meiotic division of spermatocytes, differentiation of spermatids and finally release of spermatozoa into the tubule lumen. As in many tissues throughout the body, the number of cells in the seminiferous tubules of the testis is determined by a dynamic balance between cell proliferation and apoptotic cell death (Shaha, 2008). Both spontaneous and increased cell death due to triggering stimuli (such as deprivation of intra-testicular testosterone and gonadotrophins, Sertoli cell toxicants or chemotherapeutic drugs) occur via apoptosis.
R. Tripathi et al. / Journal of Reproductive Immunology 83 (2009) 31–35
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Fig. 2. The components of the apoptotic pathways that are induced by various toxins to bring about cell death. Pathway components activated during the first wave of spermatogenesis to induce cell death are also shown.
3. Germ cell death during sperm development After formation of the testis, when the gonocytes differentiate into spermatogonia, a phase of heightened apoptosis has been observed which is known as the ‘first wave of spermatogenesis’ for adjustment of numbers in a growing testis. In mice this involves caspase 2 while in rats it involves caspases 3, 8 and 9, indicating involvement of both extrinsic and intrinsic pathway (Moreno et al., 2006). Bax is a pro-apoptotic member while Bcl-2 or Bcl-w is antiapoptotic member of the Bcl-2 family of proteins. Their involvement in the early spermatogenesis is well documented in Bax knockout transgenic mice or transgenic mice over-expressing Bcl-2 or Bcl-x, where the early wave of apoptosis is eliminated resulting in infertility (Furuchi et al., 1996). Data suggest that regulated spatial and temporal expression of Bcl-w is required for normal testicular development and spermatogenesis, and over-expression of Bcl-w inhibits germ cell cycle entry and/or cell cycle progression leading to disrupted spermatogenesis (Yan et al., 2003). Mice over-expressing Bcl-2 or Bcl-XL exhibit male sterility characterized by defects in spermatogenesis, apparently due to suppression of the early apoptotic wave. Therefore, a precise expression of both Bcl-2 and Bcl-xL is required since an alteration either way in the
balance of these proteins could lead to infertility. During the early wave, a high level of pro-apoptotic Bax expression is normally seen in germ cells of the testes, suggesting Bax-induced cell death (Jahnukainen et al., 2004). Thus, it appears that the early apoptotic wave in mice required for the formation of mature sperm is dependent on the proper balance of anti-apoptotic genes such as Bcl-XL , and proapoptotic genes such as Bax. It has been shown that not only the mitochondrial pathway, but the extrinsic pathway is also involved during the first wave of spermatogenesis, because upregulation of CD95 (Apo-1/Fas) is associated with spermatocyte apoptosis in the rat (Lizama et al., 2007). 4. Germ cell death induced by toxins and hormones Various toxin-induced germ cell death models have been used to study the involvement of various pro- and anti-apoptotic molecules. Amongst the toxins, ethane-1,2dimethanesulfonate (EDS) is a cytotoxic alkylating agent known as a selective toxin for adult rat Leydig cells, which causes an increase in testicular Fas with an accompanying elevation of germ cell apoptosis (Woolveridge et al., 2001). Phthalates, such as di-(2-ethylhexyl) phthalate that are ubiquitous environmental toxicants produce testicular atrophy in laboratory animals and impair germ cell
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development in the human fetal testis in vitro (Yao et al., 2007). Nitrobenzene (NB) is a testicular toxicant in vivo which is active even in the absence of Fas because adult gld mice (mice with FasL mutation) treated with a single oral dose of NB (800 mg/kg) show a higher apoptotic index compared with wild-type C57BL/6 mice (Richburg and Nanez, 2003). A high dose of bisphenol A, considered an endocrine disruptor, induces apoptosis of Leydig and germ cells in the mouse testis through the Fas signaling pathway (Li et al., 2009). Lindane, an organochlorine pesticide, is known to impair testicular functions and fertility though modulation of NFB and FasL (Saradha et al., 2009). Epidemiological surveys and animal experimental studies suggest that exposure to chemical 2-bromopropane (2BP), an intermediate in the synthesis of pharmaceuticals, dyes, and other organic chemicals could result in reproductive and hematopoietic disorders in animals as well as humans (Kim et al., 1999). The effects of 2-BP treatment are mediated through both the extrinsic and the intrinsic pathway of apoptosis (Yu et al., 2001). The effects of chronic exposure of 4-tert-octylphenol on the testicular development of prepubertal male rats show that expression of bcl-xL mRNA was significantly decreased in the treated groups, whereas the expressions of bcl-2 and bax mRNA were not significantly changed (Kim et al., 2004). Using an in vitro spermatogenic cell apoptosis model, we showed that Ca2+ regulates the Bcl-xS and Bcl-xL expression when exposed to 2,5-hexanedione, a metabolite of the common industrial solvent n-hexane (Mishra et al., 2006). Fig. 2 summarizes the pro- and anti-apoptotic molecules involved during toxin-induced death. In the mammalian testes, hormones such as follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), and testosterone have all been shown to regulate germ cell survival (Griswold, 1998). Excess testosterone can cause death, for example, after injection of testosterone undecanoate, both the apoptotic signal in germ cells and the expression of Fas/FasL in monkey testis increase correlatively in a time-dependent manner, reaching a maximum level on day 30 (Zhou et al., 2001). In contrast, testosterone withdrawal stimulates caspase activity and produces DNA fragmentation in Sertoli cells, with a weak effect on DNA fragmentation and caspase activity in germ cells suggesting a caspase-independent way of death in germ cells which is controlled by Sertoli cells via an as yet undetermined mechanism (Tesarik et al., 2002). Like testosterone, estrogens have a profound effect on cell death in the testis and evidence has accumulated over several decades that estrogen is essential for spermatogenesis and that intra-testicular concentrations of estrogen are very high (Hess, 2003). Mouse germ cells express both estrogen receptor-␣ and - and mice lacking estrogen receptor-␣ are infertile (Chen et al., 2009). Therefore, agents able to mimic estrogens can potentially alter the action of the hormone on spermatogenic cells leading to functional impairment of the male gamete. Reports of lowered fertility rates as a consequence of exposure to agents with estrogenic activity termed as endocrine disruptors are well documented in wild life populations (Hutchinson et al., 2000), although its effect on humans remain controver-
sial (Storgaard et al., 2006). Our studies show that exposure to 17-estradiol induces Fas ligand expression and subsequent ligation of this ligand with the Fas receptor increases nitric oxide formation through the activation of inducible nitric oxide synthase in the rat both in vitro and in vivo (Nair and Shaha, 2003; Mishra and Shaha, 2005). 5. Cell death in testicular carcinoma Over the past several decades, the incidence of testicular cancers has increased. The chemotherapy regimens most commonly used for these cancers are platinum-based compounds like cisplatin (Bokemeyer et al., 1996). The human testicular germ cell tumor cell lines show an extraordinary sensitivity to treatment with cisplatin (Johnstone et al., 2002) which causes DNA intra-stand and inter-stand crosslinks that when not repaired, lead to apoptosis. DNA damage by cisplatin results in transcription inhibition, activation of multiple signaling pathways including upregulation of p53 levels, phosphorylation of p38 MAP kinase and Erk1/2 and the other signaling pathways both in vitro and in vivo (Chauhan et al., 2009; Wu et al., 2005), leading to apoptosis in testicular germ cell tumors (TGCT). In addition, cisplatin generates reactive oxygen species (ROS), which are known as one of the intermediates following chemotherapy (Wu et al., 2005). The high toxicity of cisplatin poses a major challenge to develop alternate therapies for the treatment of testicular cancers. 6. Conclusions and perspectives This review has attempted to highlight the recent developments in the field of apoptosis in general and the role of this mode of cell death in spermatogenesis. Studies using genetically altered mice and toxin-induced apoptosis models indicate that germ cell apoptosis, like that of other cell systems, is regulated by multiple genes that either inhibit or promote cell death. The challenge is now to identify the full complement of pro- and anti-apoptotic molecules involved in germ cell or germ cell tumors and to determine the precise signaling pathways under different situations. Future studies of genetically modified animals like knockouts or knock-ins for particular genes, especially those carrying conditional or germ cell type-specific genetic abnormalities will reveal more about the involvement of the various apoptosis-associated molecules. This type of work will without doubt, lay the foundation for future rational treatment of male infertility and testicular neoplasia, as well as providing a basis for the rational design of male contraceptive agents. Recent developments showing the involvement of micro-RNAs which are a group of highly conserved, non-coding endogenous small RNAs (ValenciaSanchez et al., 2006) in the control of gene transcription during spermatogenesis opens up a new era of investigations. Dicer is an essential component for micro-RNA processing and it has been shown that conditional knockout mouse lacking proper Dicer1 function have abnormal germ cell differentiation, elongation and sperm motility resulting in infertility (Maatouk et al., 2008). Therefore, there are many aspects of regulation of spermatogenic cell apoptosis that need to be addressed in order to formu-
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