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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. Th e testosterone depletion eff ects aft er 7 days post-EDS were verifi ed 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 quantifi ed. Th e germ cell apoptosis was identifi ed 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. Th us, overexpression of GZMK may indirectly regulate germ cell apoptosis by premature release of round spermatids from seminiferous tubule lumen.

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Depletion of testosterone (T), vasectomy or chemical insults are known to induce spermatic granuloma (SG) in epididymis, however, the exact mechanisms are unknown. We treated adult male rats with a Leydig cell specific toxicant ethylene dimethane sulfonate (EDS) to deplete T, causing SG formation in epididymis. Histological evaluation revealed the presence of testicular germ cells (TGC) in the caput of epididymis which were apoptotic. An increased proportion of tubular epithelial cells in caput were also apoptotic. Immunofluorescent staining showed extensive infiltration of monocytes/macrophages around the periphery of the shrunken tubules and SG. CD4+ and CD8+ lymphocytes were also detected inside the tubules and SG. In confirmation, quantitative real time PCR for mRNA of macrophage marker CD68, and lymphocyte markers CD4 and CD8 were increased in EDS treated epididymis compared to controls (19 fold, 3.5 fold and 7.5 fold respectively). Aquaporin 9 mRNA increased 11 fold in EDS treated epididymis which may be indicative of restricted testicular fluid flow into epididymis. These pathological conditions were reversed after T replacement in EDS treated rats. Thus, our data indicates that influx and accumulation of apoptotic TGCs in the epididymis due to the disintegration of the blood‐testis barrier and disruption of testicular fluid flow into epididymis may play a role in SG formation.