Germ cell apoptosis control during spermatogenesis

Contraception 72 (2005) 298 – 302

Original research article

Germ cell apoptosis control during spermatogenesis Claudia Giampietria,T, Simonetta Petrungaroa, Pierpaolo Colucciab, Alessio D’Alessioa, Donatella Staracea, Anna Ricciolia, Fabrizio Padulaa, Fioretta Palombia, Elio Ziparoa, Antonio Filippinia, Paola De Cesarisc a

Department of Histology and Medical Embryology, Istituto Pasteur-Fondazione Cenci Bolognetti, University of Rome bLa Sapienza Q, 00161 Rome, Italy b Department of Surgery bP. Valdoni Q, University bLa SapienzaQ, 00161 Rome, Italy c Department of Experimental Medicine, University of L’Aquila, 67100 L’Aquila, Italy Received 10 January 2005; revised 8 April 2005; accepted 10 April 2005

Abstract The aim of the present study was to investigate the expression and role of c-Flip long isoform (c-FlipL), a known anti-apoptotic protein. No data are currently available on c-FlipL in male gonad before puberty; therefore, this study was carried out in prepuberal mouse testis. We investigated pachytene spermatocytes and spermatogonia by immunostaining of testis sections and found a strong and specific expression of c-FlipL in pachytene spermatocytes, while spermatogonia expressed very low levels of c-FlipL. This finding inversely correlated with the caspases activity, which was higher in spermatogonia as compared to pachytene spermatocytes. Other experiments carried out in an organculture model revealed that Fas-induced apoptosis was higher in spermatogonia as compared to pachytene spermatocytes. These data suggest that c-FlipL may play a role as an anti-apoptotic molecule in the prepuberal mouse testis and open new perspectives in the comprehension of the mechanisms controlling germ cells apoptosis. D 2005 Elsevier Inc. All rights reserved. Keywords: c-Flip; Testis; Spermatocytes; Spermatogonia; Caspases

1. Introduction Apoptosis plays a central role both in development and in homeostasis [1,2]. Under physiological conditions, cells die by apoptosis in the embryo during development and morphogenesis, as well as in the adult during tissue remodeling and immune response. bDeath receptorsQ transduce apoptosis signals initiated by specific bdeath ligandsQ and activate the caspases cascade within seconds of ligand binding, leading to apoptosis within hours [3]. Several death receptors have been described; the best characterized are Fas (also called CD95 or Apo1) and TNFR1 (also known as p55 or CD120a) [4,5]. Downstream effectors of death receptors are cysteine proteases (caspases) which exist as inactive zymogens known as procaspases [6]. They are activated by proteolysis through different mechanisms including autoactivation, transactivation and proteolysis by other proteinases [7]. Adapter molecules link apoptotic sensors, namely, death receptors and mitochondria, to procaspases. For instance, Fas-associated T Corresponding author. Tel.: +39 06 49766582; fax: +39 06 4462854. E-mail address: [email protected] (C. Giampietri). 0010-7824/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.contraception.2005.04.011

death domain protein (FADD) couples Fas to procaspase-8. FADD contains a death domain (DD) that interacts with a similar domain on Fas and also contains a death effector domain (DED) which binds to the DEDs of procaspase-8. Fas activation allows interaction of Fas DD to the corresponding DD in FADD, which in turn recruits procaspase-8 by a homophilic interaction involving DEDs, thus leading to procaspase-8 autoactivation [3]. FADD also activates procaspase-10 via a similar mechanism [8]. The formation of a death-inducing signaling complex, containing FADD and caspase-8 or -10, triggers then a cascade activation initiating the apoptosis process. c-Flip proteins belong to a protein family of crucial relevance in the apoptosis control. They have been found in different cell systems to specifically interact with caspase-8 or -10, thus interfering with their recruitment/activation and inhibiting apoptosis [9]. The balance between germ cell proliferation, differentiation and apoptosis is critical to control spermatogenesis. Altering the fine regulation of any of these processes may lead to the onset of testicular diseases. During establishment of spermatogenesis at the puberal age, early germ cells apoptotic wave occurs, aimed at removing abnormal germ

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cells and maintaining a proper ratio between maturing germ cells and Sertoli cells [10,11]. As result of this, up to 75% of the spermatogenic cells are eliminated during the maturation process [12,13]. It has been shown that the intracellular balance of BclxL and Bax proteins is crucial for this early apoptotic wave; in fact, Bax knockout mice and transgenic mice overexpressing BclxL are sterile, likely due to spermatogenic cell overproduction [14]. These data demonstrate that apoptosis control is crucial to achieve normal mature spermatogenesis and Fas/FasL system has been shown to be the major inducer of germ cells apoptosis under particular pathological conditions (viz., cryptorchid-, heated-, drug treated-, irradiated- or hormone-deprived testes) [15 – 22]. However, the fine molecular events controlling testicular germ cell apoptosis are still largely unknown. Fas expression in the testis has been reported by various authors in both germ cells [20,23] and Sertoli cell [24,25], while FasL expression is more controversial. In fact, some authors report its expression only in germ cells [24,26,27] while others claimed it is present only in Sertoli cells [16,23,28,29]. The tumor suppressor p53 protein is another widely described regulator of both cell proliferation and apoptosis. As for Bcl-2 family members, p53 modulates the intracellular death-signaling pathway. Many different reports suggest a role for p53 in testicular germ cell apoptosis, indicating that p53 plays a role in mediating both spontaneous and injury-induced spermatogonial apoptosis. Though the majority of these reports implicate p53 in the regulation of apoptosis in mitotically active spermatogonia, a recent report demonstrates that p53 may also regulate apoptosis in meiotic or postmeiotic germ cells [30,31]. While apoptosis is a massive event in the first puberal spermatogenenic wave, in the adult testis it is a rare event and affects mainly spermatogonia [14,32,33]. The reason of such change still remains to be clarified; according to some authors only spermatogonia exceeding the supportive capacity of Sertoli cells are eliminated to prevent seminiferous tubule overcrowding. Others suggest that spermatogonia elimination may represent an early selection of abnormal cells before the onset of meiosis [14]. The aim of the present work was to investigate the expression and function of c-Flip long isoform (c-FlipL) in prepuberal mouse testis, thus elucidating molecular mechanisms underlying the apoptosis control before puberty. 2. Materials and methods 2.1. Cell preparation Enriched populations of primary mouse spermatogonia and pachytene spermatocytes were obtained as previously described [34]. Purity of the preparation was N90%, according to morphological analysis. 2.2. Immunohistochemistry Testes of 18-day-old CD1 mice were fixed in Bouin solution and processed as already described [34]. Slides

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were incubated overnight at 48C with rabbit polyclonal anti-c-FlipL IgG (Upstate Biotechnologies, Lake Placid, NY, USA). Peroxidase reaction was performed using 0.06% diaminobenzidine (Sigma, St. Louis, MO). 2.3. Caspase activity assay Caspase activity was assayed on cytosolic extracts using the fluorogenic substrate DEVD-AFC (Calbiochem, San Diego, CA) as previously described [34]. 2.4. FACS analysis FACS analysis was carried out has previously described [26]. The expression of c-FlipL was determined by the rabbit anti-c-FlipL IgG polyclonal antibody #864 from Upstate Biotechnologies and by the secondary FITC-conjugated goat anti-rabbit antibody from Sigma. Analyses were performed on a Coulter Epics XL flow cytometer (Beckman Coulter, Fullerton, CA). Data were analyzed with the WinMDI free software. 2.5. Organ cultures Testes from prepuberal mice were microdissected on Petri dishes containing culture medium (MEM from Gibco, Paisley, Scotland). Segments of seminiferous tubules (about 1 mm length) were isolated and transferred to culture plates containing the same medium either in the presence or in the absence of anti-Fas antibody (Jo2 from BD-Pharmingen, San Diego, CA; 5 Ag/mL). Samples were then incubated overnight at 328C in serum-free conditions in a humidified atmosphere containing 5% CO2. Such experimental conditions were carefully selected to achieve maximal cell targeting. Apoptosis was revealed by TUNEL staining according to standard procedures. 3. Results Within the general aim of clarifying molecular mechanisms regulating germ cell apoptosis, we focused on differentiating germ cells, namely, spermatogonia and spermatocytes. Therefore, we investigated prepuberal (18 days post birth) mouse testis which lacks differentiated germ cells. c-Flip protein is known to be expressed in testis. In Fig. 1 (panel B) we show that c-FlipL is expressed in pachytene spermatocytes, while it is undetectable in Sertoli cells and is expressed at low levels in spermatogonia. These data indicated that c-FlipL expression pattern in prepuberal mouse testis was similar to the one observed in adult testis, according to previous data [34]. Fig. 2 presents flow cytometric (FACS) analysis performed on pachytene spermatocytes homogeneous population. The gray peak indicates isotype control while white peak indicates the staining with specific antibody. This quantitative assay confirms the specific high expression of c-FlipL in pachytene spermatocytes. Further experiments were carried out to measure caspases activity in both pachytene spermatocytes and spermatogonia.

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Fluorescence units (FU)

400

300

200

100

A 0

Spermatogonia

Pachytene Spermatocytes

Fig. 3. Caspases activity evaluated by DEVD-AFC cleavage, in cytosolic extracts of spermatogonia and pachytene spermatocytes. Data represent FU absolute values and are representative of three independent experiments.

B Fig. 1. Immunohistochemical localization of c-FlipL in mouse testis at 18 days after birth. Panel A represents a section stained with an isotype antibody, as a negative control. Panel B shows a section stained with anticFLIPL antibody (magnification 400). Results are representative of three independent experiments.

full agreement with data reported in Figs. 1 and 2, therefore indicating that c-FlipL levels inversely correlate with caspases activity level in germ cells from prepuberal testis. Finally, organ cultures were performed to study the apoptotic response in conditions closer to the physiological setup. Namely, Fas-induced apoptosis of germ cells was investigated in the seminiferous tubules organ culture model. Such experiments showed an inverse relation between Fas-induced apoptosis and c-Flip expression levels;

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Caspases activity was measured by DEVD-AFC cleavage assay (Fig. 3). Caspases activity was found to be markedly higher in spermatogonia than in pachytene spermatocytes, in

Iso Flip

0

E v e n t s

100

101

102

Flip-FITC Fig. 2. Flow cytometric (FACS) analysis of c-FlipL expression in pachytene spermatocytes. The gray peak indicates isotype control and the white peak indicates the staining with specific antibody. Results are representative of three independent experiments.

Fig. 4. TUNEL staining of anti-Fas antibody-treated seminiferous tubules. The same microscopic field was pictured by fluorescence and phasecontrast microscopy (panels A and B, respectively; magnification 300).

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more specifically, high Fas-dependent apoptosis was found at the tubule periphery, where spermatogonia are located, as compared to low Fas-dependent apoptosis found at the center of the tubule, where pachytene spermatocytes are located (Fig. 4). In conclusion, our studies (1) show a cell-specific expression pattern of c-FlipL, in prepuberal mouse testis, (2) suggest its role in controlling germ cells caspases activity in vitro and Fas sensitivity in seminiferous tubules explants and (3) suggest a role of c-FlipL in protecting specific germ cell populations from apoptosis.

4. Discussion We have previously shown that c-FlipL controls Fasmediated germ cell apoptosis and that it protects specific germ cells populations from apoptosis in adult mouse testis. Experiments carried out in the present study aimed to address c-FlipL expression in prepuberal mouse testis in order to clarify events proceeding the first spermatogenic wave. Data collected confirmed the anti-apoptotic role of c-FlipL in this still poorly investigated model. In fact, immunohistochemical analyses on 18-day-old mouse testis demonstrated that c-FlipL is expressed at high levels in pachytene spermatocytes while it is expressed at low levels in spermatogonia. It is well accepted that c-FlipL is an inhibitor of caspases activation. We, therefore, hypothesized that mouse spermatogonia and pachytene spermatocytes might show difference in caspases activity, since they express different levels of c-FlipL. Interestingly, we found caspases activity higher in spermatogonia as compared to pachytene spermatocytes. Consistent with these findings, organ-culture experiments confirmed that spermatogonia showed higher Fas-induced apoptosis as compared to pachytene spermatocytes. These and other data [34] indicate c-FlipL as a key molecule protecting germ cells from Fas-mediated apoptosis and might explain, at least in part, why spermatogonia are one of the testis populations most sensitive to apoptosis in vivo. Testicular germ cells apoptosis is a critical prerequisite for normal spermatogenesis under physiological conditions and alterations of its regulation are associated with various diseases. In fact, increased rate of germ cell apoptosis has been observed in testis biopsies of infertile men [35 –44]. In addition, massive Fas-mediated germ cell apoptosis may be associated with seminiferous tubules atrophy in autoimmune orchitis. Interestingly, different ethnic groups show different rates of spontaneous apoptosis (found to be higher in Chinese than in Caucasian men) [45]. This suggests that different populations may differ in their sensitivity to chemicalinduced apoptosis of their germ cells. We believe this might likely be based on differences in the signaling machinery involved, for example, Fas, caspases, c-FlipL. In conclusion, this study suggests a key role of c-FlipL in modulating testis apoptotic response, although further studies are needed to clarify c-Flip role in male infertility and other testis diseases.

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Acknowledgment This study was supported in part by funds from the Ministry of Instruction, University and Research and funds from the University La Sapienza. We also thank Stefania Fera for technical assistance and Dr. Antonio Facchiano for useful discussions.

References [1] Steller H. Mechanisms and genes of cellular suicide. Science 1995; 267:1445 – 9. [2] Jacobson MD, Weil M, Raff MC. Programmed cell death in animal development. Cell 1997;88:347 – 54. [3] Ashkenazi A, Dixit VM. Death receptors: signaling and modulation. Science 1998;281:1305 – 8. [4] Smith CA, Farrah T, Goodwin RG. The TNF receptor superfamily of cellular and viral proteins: activation, costimulation, and death. Cell 1994;76:959 – 62. [5] Nagata S. Apoptosis by death factor. Cell 1997;88:355 – 65. [6] Thornberry NA, Lazebnik Y. Caspases: enemies within. Science 1998; 281:1312 – 6. [7] Wolf BB, Green DR. Suicidal tendencies: apoptotic cell death by caspase family proteinases. J Biol Chem 1999;274:20049 – 52. [8] Vincenz C, Dixit VM. Fas-associated death domain protein interleukin-1beta-converting enzyme 2 (FLICE2), an ICE/Ced-3 homologue, is proximally involved in CD95- and p55-mediated death signaling. J Biol Chem 1997;272:6578 – 83. [9] Irmler M, Thome M, Hahne M, et al. Inhibition of death receptor signals by cellular FLIP. Nature 1997;388:190 – 5. [10] de Rooij DG, Grootegoed JA. Spermatogonial stem cells. Curr Opin Cell Biol 1998;10:694 – 701. [11] Koji T. Male germ cell death in mouse testes: possible involvement of Fas and Fas ligand. Med Electron Microsc 2001;34:213 – 22. [12] Bartke A. Apoptosis of male germ cells, a generalized or a cell typespecific phenomenon? Endocrinology 1995;136:3 – 4. [13] Billig H, Furuta I, Rivier C, Tapanainen J, Parvinen M, Hsueh AJ. Apoptosis in testis germ cells: developmental changes in gonadotropin dependence and localization to selective tubule stages. Endocrinology 1995;136:5 – 12. [14] Rodriguez I, Ody C, Araki K, Garcia I, Vassalli P. An early and massive wave of germinal cell apoptosis is required for the development of functional spermatogenesis. EMBO J 1997;16:2262 – 70. [15] Pentikainen V, Erkkila K, Dunkel L. Fas regulates germ cell apoptosis in the human testis in vitro. Am J Physiol 1999;276(2 Pt 1):E310 – 6. [16] Lee J, Richburg JH, Younkin SC, Boekelheide K. The Fas system is a key regulator of germ cell apoptosis in the testis. Endocrinology 1997; 138:2081 – 8. [17] Ohta Y, Nishikawa A, Fukazawa Y, et al. Apoptosis in adult mouse testis induced by experimental cryptorchidism. Acta Anat (Basel) 1996; 157:195 – 204. [18] Lee J, Richburg JH, Shipp EB, Meistrich ML, Boekelheide K. The Fas system, a regulator of testicular germ cell apoptosis, is differentially up-regulated in Sertoli cell versus germ cell injury of the testis. Endocrinology 1999;140:852 – 8. [19] Blanco-Rodriguez J, Martinez-Garcia C. Apoptosis precedes detachment of germ cells from the seminiferous epithelium after hormone suppression by short-term oestradiol treatment of rats. Int J Androl 1998;21:109 – 15. [20] Francavilla S, D’Abrizio P, Rucci N, et al. Fas and Fas ligand expression in fetal and adult human testis with normal or deranged spermatogenesis. J Clin Endocrinol Metab 2000;85:2692 – 700. [21] Rockett JC, Mapp FL, Garges JB, Luft JC, Mori C, Dix DJ. Effects of hyperthermia on spermatogenesis, apoptosis, gene expression, and fertility in adult male mice. Biol Reprod 2001;65:229 – 39.

302

C. Giampietri et al. / Contraception 72 (2005) 298 – 302

[22] Lue YH, Hikim AP, Swerdloff RS, et al. Single exposure to heat induces stage-specific germ cell apoptosis in rats: role of intratesticular testosterone on stage specificity. Endocrinology 1999;140:1709 – 17. [23] Xu JP, Li X, Mori E, et al. Expression of Fas–Fas ligand in murine testis. Am J Reprod Immunol 1999;42:381 – 8. [24] Woolveridge I, Boer-Brouwer M, Taylor MF, Teerds KJ, Wu FC, Morris ID. Apoptosis in the rat spermatogenic epithelium following androgen withdrawal: changes in apoptosis-related genes. Biol Reprod 1999;60:461 – 70. [25] Riccioli A, Starace D, D’Alessio A, et al. TNF-alpha and IFN-gamma regulate expression and function of the Fas system in the seminiferous epithelium. J Immunol 2000;165:743 – 9. [26] D’Alessio A, Riccioli A, Lauretti P, et al. Testicular FasL is expressed by sperm cells. Proc Natl Acad Sci U S A 2001;98:3316 – 21. [27] Riccioli A, Salvati L, D’Alessio A, et al. The Fas system in the seminiferous epithelium and its possible extra-testicular role. Andrologia 2003;35:64 – 70. [28] Richburg JH, Nanez A, Williams LR, Embree ME, Boekelheide K. Sensitivity of testicular germ cells to toxicant-induced apoptosis in gld mice that express a nonfunctional form of Fas ligand. Endocrinology 2000;141:787 – 93. [29] Huang DC, Hahne M, Schroeter M, et al. Activation of Fas by FasL induces apoptosis by a mechanism that cannot be blocked by Bcl-2 or Bcl-x(L). Proc Natl Acad Sci U S A 1999;96:14871 – 6. [30] Richburg JH. The relevance of spontaneous- and chemically-induced alterations in testicular germ cell apoptosis to toxicology. Toxicol Lett 2000;112,113:79 – 86. [31] Beumer TL, Roepers-Gajadien HL, Gademan IS, et al. The role of the tumor suppressor p53 in spermatogenesis. Cell Death Differ 1998;5: 669 – 77. [32] Allan DJ, Harmon BV, Roberts SA. Spermatogonial apoptosis has three morphologically recognizable phases and shows no circadian rhythm during normal spermatogenesis in the rat. Cell Prolif 1992;25:241 – 50. [33] Xu X, Toselli PA, Russell LD, Seldin DC. Globozoospermia in mice lacking the casein kinase II alpha’ catalytic subunit. Nat Genet 1999;23: 118 – 21. [34] Giampietri C, Petrungaro S, Coluccia P, et al. FLIP is expressed in mouse testis and protects germ cells from apoptosis. Cell Death Differ 2003;10:175 – 84.

[35] Gandini L, Lombardo F, Paoli D, et al. Study of apoptotic DNA fragmentation in human spermatozoa. Hum Reprod 2000;15: 830 – 9. [36] Aravindan GR, Bjordahl J, Jost LK, Evenson DP. Susceptibility of human sperm to in situ DNA denaturation is strongly correlated with DNA strand breaks identified by single-cell electrophoresis. Exp Cell Res 1997;236:231 – 7. [37] Barroso G, Morshedi M, Oehninger S. Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa. Hum Reprod 2000;15: 1338 – 44. [38] Gorczyca W, Traganos F, Jesionowska H, Darzynkiewicz Z. Presence of DNA strand breaks and increased sensitivity of DNA in situ to denaturation in abnormal human sperm cells: analogy to apoptosis of somatic cells. Exp Cell Res 1993;207:202 – 5. [39] Host E, Lindenberg S, Smidt-Jensen S. The role of DNA strand breaks in human spermatozoa used for IVF and ICSI. Acta Obstet Gynecol Scand 2000;79:559 – 63. [40] Jurisicova A, Lopes S, Meriano J, Oppedisano L, Casper RF, Varmuza S. DNA damage in round spermatids of mice with a targeted disruption of the Pp1cgamma gene and in testicular biopsies of patients with non-obstructive azoospermia. Mol Hum Reprod 1999;5: 323 – 30. [41] Oosterhuis GJ, Mulder AB, Kalsbeek-Batenburg E, Lambalk CB, Schoemaker J, Vermes I. Measuring apoptosis in human spermatozoa: a biological assay for semen quality? Fertil Steril 2000;74:245 – 50. [42] Sakkas D, Mariethoz E, St John JC. Abnormal sperm parameters in humans are indicative of an abortive apoptotic mechanism linked to the Fas-mediated pathway. Exp Cell Res 1999;251:350 – 5. [43] Sun JG, Jurisicova A, Casper RF. Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol Reprod 1997;56:602 – 7. [44] Tesarik J, Greco E, Cohen-Bacrie P, Mendoza C. Germ cell apoptosis in men with complete and incomplete spermiogenesis failure. Mol Hum Reprod 1998;4:757 – 62. [45] Hikim AP, Wang C, Lue Y, Johnson L, Wang XH, Swerdloff RS. Spontaneous germ cell apoptosis in humans: evidence for ethnic differences in the susceptibility of germ cells to programmed cell death. J Clin Endocrinol Metab 1998;83:152 – 6.