Infertility is a problem faced by 15% of married couples. Of these couples, 50% are infertile due to inadequate production of competent sperm. Environmental and work-related causes of male infertility are well documented and include exposures to heat, metals, and organic chemicals. Despite the magnitude of this problem and the recognition of an ongoing environmental risk, little is known about the underlying mechanisms by which these environmental agents cause infertility. One intensely studied model of chemical-induced testicular injury is 2,5- hexanedione exposure in the rat. The earliest biochemical change in 2,5- hexanedione-exposed rats is an alteration in testicular microtubule assembly. Three to four weeks after beginning exposure, seminiferous tubule fluid production is impaired followed closely by an abrupt loss of germ cells which appears to be """"""""irreversible"""""""" because it persists for more than a year after termination of exposure. These fundamental observations have led to the first working hypothesis which guides this project: 2,5- hexanedione alters microtubule dynamics compromising Sertoli cell microtubule-dependent organelle transport resulting in a failure of seminiferous tubule fluid formation and germ cell necrosis. This hypothesis will be tested by the following Specific Aims: 1) use video microscopy to compare the movement of 2,5-hexanedione-treated and control microtubules by rat testicular microtubule motors, 2) characterize 2,5- hexanedione-induced alterations in Sertoli cell microtubule motors and microtubule-associated proteins involved in stability, 3) measure the rate of microtubule-dependent transport directly in isolated seminiferous tubules, 4) investigate the role of microtubule-dependent transport in seminiferous tubule fluid formation, and 5) correlate these functional changes with ultrastructural alterations induced by 2,5-hexanedione exposure. Long after 2,5-hexanedione exposure has ended, there is persistence of abnormal Sertoli cells which fail to maintain differentiating germ cells, leading to a second working hypothesis: the long-term """"""""irreversible"""""""" testicular atrophy induced by 2,5-hexanedione is caused by abnormal Sertoli cells which fail to create an appropriate growth factor environment to support spermatogenesis. This hypothesis will be tested by the following Specific Aims: 1) investigate the roles of cell-cell contact and second messengers in persistence of the Sertoli cell abnormalities, 2) delineate the kinetics of stem and committed germ cell turnover, 3) compare Sertoli cell growth factor mRNA and protein levels and their distributions in models of reversible and """"""""irreversible"""""""" testicular atrophy, and 4) determine the germ cell response to growth factor repletion in Sertoli-germ cell co-culture and in the 2,5-hexanedione- treated atrophic testis. This investigation of 2,5-hexanedione-induced testicular injury in the rat will examine fundamental spermatogenetic processes which are susceptible to chemical perturbation and provide clues to environmentally-sensitive testicular targets in humans.
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