Objective. Testosterone depletion induces increased germ cell apoptosis in testes. However, limited studies exist on genes that regulate the germ cell apoptosis. Granzymes (GZM) are serine proteases that induce apoptosis in various tissues. Multiple granzymes, including GZMA, GZMB and GZMN, are present in testes. Th us, we investigated which granzyme may be testosterone responsive and possibly may have a role in germ cell apoptosis aft er testosterone depletion. Methods. Ethylene dimethane sulfonate (EDS), a toxicant that selectively ablates the Leydig cells, was injected into rats to withdraw the testosterone. The testosterone depletion effects after 7 days post-EDS were verified by replacing the testosterone exogenously into EDS-treated rats. Serum or testicular testosterone was measured by radioimmunoassay. Using qPCR, mRNAs of granzyme variants in testes were quantified. The germ cell apoptosis was identified by TUNEL assay and the localization of GZMK was by immunohistochemistry. Results. EDS treatment eliminated the Leydig cells and depleted serum and testicular testosterone. At 7 days post-EDS, testis weights were reduced 18% with increased germ cell apoptosis plus elevation GZMK expression. GZMK was not associated with TUNEL-positive cells, but was localized to stripped cytoplasm of spermatids. In addition, apoptotic round spermatids were observed in the caput epididymis. Conclusions. GZMK expression in testes is testosterone dependent. GZMK is located adjacent to germ cells in seminiferous tubules and the presence of apoptotic round spermatids in the epididymis suggest its role in the degradation of microtubules in ectoplasmic specializations. Thus, overexpression of GZMK may indirectly regulate germ cell apoptosis by premature release of round spermatids from seminiferous tubule lumen.
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Amsterdam A Sasson R Keren-Tal I Aharoni D Dantes A Rimon E Land A Cohen T Dor Y Hirsh L. Alternative pathways of ovarian apoptosis: death for life. Biochem Pharmacol 66 1355-1362 2003.
Andersen MH Schrama D Th or Straten P Becker JC. Cytotoxic T cells. J Invest Dermatol 126 32-41 2006.
Anthony DA Andrews DM Watt SV Trapani JA Smyth MJ. Functional dissection of the granzyme family: cell death and infl ammation. Immunol Rev 235 73-92 2010.
Bhat GK Sea TL Olatinwo MO Simorangkir D Ford GD Ford BD Mann D R. Infl uence of a leptin defi ciency on testicular morphology germ cell apoptosis and expression levels of apoptosis-related genes in the mouse. J Androl 27 302-310 2006.
Blanco-Rodriguez J Martinez-Garcia C. Apoptosis is physiologically restricted to a specialized cytoplasmic compartment in rat spermatids. Biol Reprod 61 1541-1547 1999.
Bovenschen N Quadir R van den Berg AL Brenkman AB Vandenberghe I Devreese B Joore J Kummer JA. Granzyme K displays highly restricted substrate specifi city that only partially overlaps with granzyme A. J Biol Chem 284 3504-3512 2009.
Buzza MS Bird PI. Extracellular granzymes: current perspectives. Biol Chem 387 827-837 2006.
Chavez-Galan L Arenas-Del Angel MC Zenteno E Chavez R Lascurain R. Cell death mechanisms induced by cytotoxic lymphocytes. Cell Mol Immunol 6) 15-25 2009.
Chowdhury D Lieberman J. Death by a thousand cuts: granzyme pathways of programmed cell death. Annu Rev Immunol 26 389-420 2008.
Dutta D Guililat H Park I Sang S Mills N. Testosterone depletion following Leydig cell destruction by EDS induces testicular germ cell apoptosis and may involve granzyme variants. Biol Reprod 83(Suppl 1) 533-533 2010.
Dutta D Park I Guililat H Hanson L Sang S Mills N. Spermatic granuloma and infl ammatory reactions in epididymis of adult rats following testosterone depletion. Th e FASEB Journal 25(1 Suppl) 1002.9-1002.9 2011.
Dutta D Park I Mills NC. Fixation temperature aff ects DNA integrity in the testis as measured by the TUNEL assay. Toxicol Pathol 40 667-674 2012.
Eddy EM. Male germ cell gene expression. Recent Prog Horm Res 57 103-128 2002.
Ferlin A Garolla A Rigon F Rasi Caldogno L Lenzi A Foresta C. Changes in serum insulin-like factor 3 during normal male puberty. J Clin Endocrinol Metab 91 3426-3431 2006.
Grossman WJ Revell PA Lu ZH Johnson H Bredemeyer AJ Ley TJ. Th e orphan granzymes of humans and mice. Curr Opin Immunol 15 544-552 2003.
Guttman JA Takai Y Vogl AW. Evidence that tubulobulbar complexes in the seminiferous epithelium are involved with internalization of adhesion junctions. Biol Reprod 71 548-559 2004.
Hedger MP Wang J Lan HY Atkins RC Wreford NG. Immunoregulatory activity in adult rat testicular interstitial fl uid: relationship with intratesticular CD8+lymphocytes following treatment with ethane dimethane sulfonate and testosterone implants. Biol Reprod 58 935-942 1998.
Henriksen K Hakovirta H Parvinen M. Testosterone inhibits and induces apoptosis in rat seminiferous tubules in a stage-specifi c manner: in situ quantifi cation in squash preparations aft er administration of ethane dimethane sulfonate. Endocrinology 136 3285-3291 1995.
Hirst CE Buzza MS Sutton VR Trapani JA Loveland KL Bird PI. Perforin-independent expression of granzyme B and proteinase inhibitor 9 in human testis and placenta suggests a role for granzyme B-mediated proteolysis in reproduction. Mol Hum Reprod 7 1133-1142 2001.
Jackson CM Jackson H. Comparative protective actions of gonadotrophins and testosterone against the antispermatogenic action of ethane dimethanesulphonate. J Reprod Fertil 71 393-401 1984.
Katuru R Fernandes NV Elfakhani M Dutta D Mills N Hynds DL King C Mo H. Mevalonate depletion mediates the suppressive impact of geranylgeraniol on murine B16 melanoma cells. Exp Biol Med (Maywood) 236 604-613 2011.
Kerr JB Loveland KL O’Bryan MK de Kretser DM. Cytology of the Testis and Intrinsic Control Mechanisms. In: Knobil and Neill’s Physiology of Reproduction (Ed. JD Neill) 3rd ed. Vol. 1 pp. 837-947 Academic Press New York 2006.
Koeva YA Bakalska MV Atanassova NN Davidoff MS. INSLF3-LGR8 ligand-receptor system in testes of mature rats aft er exposure to ethane dimethanesulphonate (short communication). Folia Med (Plovdiv) 50 37-42 2008.
Livak KJ Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25 402-408 2001.
Mali P Kaipia A Kangasniemi M Toppari J Sandberg M Hecht NB Parvinen M. Stage-specifi c expression of nucleoprotein mRNAs during rat and mouse spermiogenesis. Reprod Fertil Dev 1 369-382 1989.
McCabe MJ Tarulli GA Meachem SJ Robertson DM Smooker PM Stanton PG. Gonadotropins regulate rat testicular tight junctions in vivo. Endocrinology 151 2911-2922 2010.
Meistrich ML Mohapatra B Shirley CR Zhao M. Roles of transition nuclear proteins in spermiogenesis. Chromosoma 111 483-488 2003.
Mendis-Handagama SM Ariyaratne HB Mrkonjich L Ivell R. Expression of insulin-like peptide 3 in the postnatal rat Leydig cell lineage: timing and eff ects of triiodothyronine-treatment. Reproduction 133 479-485 2007.
Mills NC. Androgen eff ects on Sertoli cells. Int J Androl 13 123-134 1990.
Mori H Christensen AK. Morphometric analysis of Leydig cells in the normal rat testis. J Cell Biol 84 340-354 1980.
Morris AJ Taylor MF Morris ID. Leydig cell apoptosis in response to ethane dimethanesulphonate aft er both in vivo and in vitro treatment. J Androl 18 274-280 1997.
Mruk DD Cheng CY. Sertoli-Sertoli and Sertoli-germ cell interactions and their signifi cance in germ cell movement in the seminiferous epithelium during spermatogenesis. Endocr Rev 25 747-806 2004.
Nandi S Banerjee PP Zirkin BR. Germ cell apoptosis in the testes of Sprague Dawley rats following testosterone withdrawal by ethane 12-dimethanesulfonate administration: relationship to Fas? Biol Reprod 61 70-75 1999.
O’Donnell L McLachlan RI Wreford NG Robertson DM. Testosterone promotes the conversion of round spermatids between stages VII and VIII of the rat spermatogenic cycle. Endocrinology 135 2608-2614 1994.
O’Donnell L Meachem SJ Stanton PG McLachlan RI. Endocrine Regulation of Spermatogenesis. In: Knobil and Neill’s Physiology of Reproduction (Ed. JD Neill) 3rd ed. Vol. 1 pp. 1017-1070 Academic Press New York 2006.
Plasman K Demol H Bird PI Gevaert K Van Damme P. Substrate specifi cities of the granzyme tryptases A and K. J Proteome Res 13 6067-6077 2014.
Perey B Clermont Y Leblond CP. Th e wave of the seminiferous epithelium in the rat. Am J Anat 108 47-77 1961.
Print CG Loveland KL. Germ cell suicide: new insights into apoptosis during spermatogenesis. Bioessays 22 423- 430 2000.
Redenbach DM Boekelheide K Vogl AW. Binding between mammalian spermatid-ectoplasmic specialization complexes and microtubules. Eur J Cell Biol 59 433-448 1992.
Romero V Andrade F. Non-apoptotic functions of granzymes. Tissue Antigens 71 409-416 2008.
Sharpe RM. Regulation of Spermatogenesis. In: Knobil and Neill’s Th e Physiology of Reproduction (Eds. E. Knobil JD Neill) 2nd ed Vol 1 pp. 1363-1434 Raven Press New York 1994.
Suemoto T Taniguchi M Shiosaka S Yoshida S. cDNA cloning and expression of a novel serine protease in the mouse brain. Brain Res Mol Brain Res 70) 273-281 1999.
Sutton KA Maiti S Tribley WA Lindsey JS Meistrich ML Bucana CD Sanborn BM Joseph DR Griswold MD Cornwall GA Wilkinson MF. Androgen regulation of the Pem homeodomain gene in mice and rat Sertoli and epididymal cells. J Androl 19 21-30 1998.
Takano N Matusi H Takahashi T. Granzyme N a novel granzyme is expressed in spermatocytes and spermatids of the mouse testis. Biol Reprod 71 1785-1795 2004.
Taylor MF Woolveridge I Metcalfe AD Streuli CH Hickman JA Morris ID. Leydig cell apoptosis in the rat testes aft er administration of the cytotoxin ethane dimethanesulphonate: role of the Bcl-2 family members. J Endocrinol 157 317-326 1998.
Taylor MF de Boer-Brouwer M Woolveridge I Teerds KJ Morris ID. Leydig cell apoptosis aft er the administration of ethane dimethanesulfonate to the adult male rat is a Fas-mediated process. Endocrinology 140 3797-3804 1999.
Vogl AW Pfeiff er DC Mulholland D Kimel G Guttman J. Unique and multifunctional adhesion junctions in the testis: ectoplasmic specializations. Arch Histol Cytol 63 1-15 2000.
Wang RS Yeh S Tzeng CR Chang C. Androgen receptor roles in spermatogenesis and fertility: lessons from testicular cell-specifi c androgen receptor knockout mice. Endocr Rev 30 119-132 2009.
Wiegand U Corbach S Minn A Kang J Muller-Hill B. Cloning of the cDNA encoding human brain trypsinogen and characterization of its product. Gene 136 167-175 1993.
Woolveridge I de 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 60 461-470 1999.
Woolveridge I Taylor MF Rommerts FF Morris ID. Apoptosis related gene products in diff erentiated and tumorigenic rat Leydig cells and following regression induced by the cytotoxin ethane dimethanesulphonate. Int J Androl 24 56-64 2001.
Yin Y Hawkins KL DeWolf WC Morgentaler A. Heat stress causes testicular germ cell apoptosis in adult mice. J Androl 18 159-165 1997.
Zar JH. Biostatistical analysis 4th ed. Prentice-Hall New Jersey 1999.
Zhang YF Yuan KM Liang Y Chu YH Lian QQ Ge YF Zhen W Sottas CM Su ZJ Ge RS. Alterations of gene profi les in Leydig-cell-regenerating adult rat testis aft er ethane dimethane sulfonate-treatment. Asian J Androl 17 253-260 2015.