Infertility affects one out of every eight couples, with male dysfunction contributing to half of this incidence. The causes of male reproductive failure are usually unknown, though recent attention has focused on environmental toxicants and their effects. Microtubule disrupters - including model compounds (colchicine, taxol) and environmental contaminants (the benomyl metabolite, carbendazim, and the n-hexane metabolite, 2,5-hexanedione) - are male reproductive toxicants which target Sertoli cell microtubules and produce irreversible testicular injury and infertility. Past work using predominantly biochemical and cell biological in vitro approaches has suggested that these toxicants initiate a cascade of events: inhibition of Sertoli cell microtubule-dependent transport leads to decreased seminiferous tubule fluid formation and germ cell death. This working hypothesis is tested in this project with new methods developed to assess Sertoli cell functions in vivo. In preliminary studies, selective infection of rat Sertoli cells was accomplished by in vivo injection of replication-deficient adenovirus into the rete testis. Initial experiments will characterize this new technique, evaluating onset and persistence of adenovirus-directed gene expression. Next, two properties of this adenovirus injection system will be exploited to study the in vivo consequences of Sertoli cell microtubule disruption. First, since adenovirus is known to use microtubule tracks for translocation from the cell periphery to the nucleus, the time required for adenovirus to appear in the Sertoli cell nucleus after rete injection will be used to measure microtubule-dependent transport rates in this target cell. Second, the gene transfer capability of adenovirus will be used to over-express gamma-tubulin or to knock-out kinesin in the Sertoli cell, permitting germ cell viability to be evaluated in the setting of a selective failure of Sertoli cell microtubule-dependent transport. Together, these complimentary approaches will critically examine key steps in the pathogenetic sequence by which microtubule disrupters alter the supportive capacity of Sertoli cells resulting in loss of germ cells and infertility.
