Expression of perforin and granzyme variants in adult rat testes after ethylene dimethane sulfonate (EDS)-induced testosterone depletion
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Testosterone is essential for regulation of spermatogenesis and testosterone withdrawal results in germ cell apoptosis. However, the mechanism for germ cell apoptosis is unknown. In testes, mature Leydig cells produce testosterone. Using ethane dimethane sulfonate (EDS), a Leydig cell-specific toxicant, to deplete testosterone, we investigated the relationship between granzyme and perforin, and germ cell apoptosis. To confirm effects of testosterone depletion, EDS-treated rats were compared with testosterone-replaced and testosterone-supplemented rats. At 5 and 7 days post-EDS treatment, rats were euthanized for tissue collection. Leydig cell loss was confirmed by the significant reduction in mRNA for Lhr (90%) and Insl3 (99%), and testosterone depletion was confirmed by the undetectable level of testosterone in serum and testes of EDS-treated rats. Due to ablation of testosterone, there was testicular weight loss (18%) along with a significant increase in germ cell apoptosis (135 cells/mm2 of testicular cross-section). However, no difference in testicular weight or numbers of apoptotic germ cells was observed in testosterone-replaced and testosterone-supplemented rats. Hence, although EDS eliminated Leydig cells, testosterone replacement maintained testicular weight and germ cell viability. In addition to increased germ cell apoptosis, the mRNA levels for grankyme K (GzmK) and perforin (Prf) were also significantly higher (2- and 2.5-fold, respectively) in EDS-treated rats compared to controls. After testosterone replacement, the levels of GzmK and Prf mRNAs were restored. The mRNA levels for Cd4 and Cd8 were also significantly elevated (2- and 5-fold, respectively) following EDS treatment, whereas testosterone replacement partially reversed it. Therefore, it was hypothesized that testosterone depletion resulted in disruption of the blood-testis barrier allowing CD4+ and CD8+ T cells to migrate inside the seminiferous tubule. With recruitment of CD8+ T cells inside the seminiferous tubule, GzmK and Prf were released to induce germ cell apoptosis. However, CD8+ T cells were detected in the interstitium, whereas GZMK and PRF were detected inside the seminiferous tubule. This indicated that GZMK and PRF expression in testes may not be associated with CD8 + T cells. Moreover, detection of GZMK and PRF proximal to residual bodies indicated their possible role in germ cell release during spermiation.