Spermatogenesis by Sisyphus: proliferating stem germ cells fail to repopulate the testis after 'irreversible' injury

2,5-Hexanedione is the toxic metabolite resulting from oxidation of the commonly used solvents n-hexane and methyl n-butyl ketone. Exposure to 2,5-hexanedione or its precursors results in a slowly progressive peripheral polyneuropathy and testicular injury. The chemical basis of the injury involves reaction of 2,5-hexanedione with protein amines, such as the epsilon-amine of lysine, to form pyrroles which further react to form protein-protein crosslinks. The target cell of injury in the testis is the supportive cell in the seminiferous epithelium, the Sertoli cell. A major function of the Sertoli cell is to nurture the dependent germ cell population by secreting seminiferous tubule fluid. 2,5-Hexanedione-induced crosslinking of the microtubule subunit protein, tubulin, leads to altered Sertoli cell microtubule-dependent transport and deficient formation of seminiferous tubule fluid, compromising germ cell viability. In an established model of testicular injury, rats are exposed to 1% 2,5-hexanedione in the drinking water for a period of 3-5 weeks. Three weeks after initiating exposure, decreased seminiferous tubule fluid secretion initiates a wave of germ apoptosis which peaks during the 5th week. The germ cell content of the injured testis continues to decline after cessation of the exposure, reaching a nadir during the 12th week. From this time onward, the testis is severely atrophic with less than 1% of seminiferous tubules in a testicular cross section containing germ cells more advanced than spermatogonia. Interestingly, this persistent state of post-injury 'irreversible' atrophy in the rat is characterized by the presence of a proliferating stem germ cell population which produces differentiating spermatogonia which then die by apoptosis. Serial cross sections of bromodeoxyuridine-labeled testis were analyzed to determine the kinetics of stem germ cell proliferation. Approximately 40% of stem cells (identified as single cells in the seminiferous epithelium) were actively proliferating with a cell cycle time of 8-14 days. Analysis of the total germ cell population present and modeling using the known cell cycle times of differentiating spermatogonia indicated a block in differentiation at the level of type A3/A4 spermatogonia. Quantitation of the frequency of apoptosis indicated that all of the germ cells died prematurely by this mechanism. Leuprolide is a gonadotropin-releasing hormone agonist which produces a profound suppression of testosterone levels with chronic administration. When delivered as a series of 3 depot injections 24 days apart, leuprolide resulted in a partial reversal of the 2,5-hexanedione-induced persistent atrophy. The reinitiation of spermatogenesis follows a lowering of the intratesticular testosterone concentration, indicating that intratesticular testosterone is at least partially responsible for the persistent atrophy. The efficacy of leuprolide-induced reversal of the persistent atrophy decreases with time after injury, suggesting that atrophic seminiferous tubules are initially capable of recovery and then enter a state of irreversible injury. Injection of ethane dimethane sulfonate at the beginning of leuprolide treatment eliminated Leydig cells during therapy and ablated the recovery of spermatogenesis, indicating that a Leydig cell-associated paracrine factor is required to restart spermatogenesis. The rat, therefore, has multiple states of testicular germ cell proliferation: normal spermatogenesis and at least two forms of persistent atrophy (leuprolide reversible and leuprolide non-reversible). Partial reversal of the persistent atrophy can be achieved by lowering intratesticular testosterone. Ongoing experiments are designed to address the role of the Leydig cell in post-injury recovery, and to further characterize the molecular events contributing to the different states of persistent atrophy.