Sertoli Cell Microtubules and Testicular Injury

Kim Boekelheide, M.D., Ph.D.

Summary New data provide further support for the hypothesis that toxicants which inhibit Sertoli cell microtubule-dependent transport lead to decreased seminiferous tubule fluid formation and germ cell death. This hypothesis explains the testicular injury produced by an entire class of toxicants, microtubule disrupters. A total of four microtubule disrupters which are known testicular toxicants have now been studied by the lumenal oil droplet technique. These microtubule disrupters-colchicine, taxol, carbendazim, and 2,5-hexanedione-are heterogeneous in terms of their effects on microtubules (inhibitors versus promoters of microtubule assembly) and toxicant properties (model compound versus environmental contaminant), and yet they are tied together by their shared ability to alter microtubule assembly, inhibit seminiferous tubule fluid formation, and cause germ cell death.

At first it seems surprising that both inhibitors and promoters of microtubule assembly disrupt microtubule-dependent transport. After all, it is easy to understand why colchicine, which destroys microtubules, would inhibit the transport of motor-bearing vesicles along microtubule tracks (Parczyk et al., 1989); but, taxol, which causes an excess of microtubules to be formed, also inhibits this process (Rindler et al., 1987). The explanation for this apparent contradiction lies in the disorganization and superstability of taxol-generated microtubules, and the effect of this altered cytoskeleton on organelle structure and function (van Zeijl & Matlin, 1990). Taxol causes superstable microtubules to form throughout the cytoplasm, obviating the usual controls on microtubule assembly and disassembly which allow the cell to adapt to a changing environment.

The environmental toxicants carbendazim and 2,5-hexanedione also differ in their mode of microtubule disruption, since carbendazim is a colchicine-like inhibitor of microtubule assembly while 2,5-hexanedione is a taxol-like promoter of microtubule assembly. Although acting by different mechanism to alter microtubule assembly, both of these toxicants inhibit seminiferous tubule fluid formation and produce testicular injury. The lumenal oil droplet method (Cheung et al., 1977; Richburg et al., 1994) is an in vitro technique which offers several advantages over the commonly employed in vivo technique of efferent duct ligation (Setchell, 1970) for the determination of seminiferous tubule fluid secretion, including 1) rapidity of analysis, 2) greater sensitivity, 3) minimal animal use and discomfort, and 4) potential use as an in vitro screening assay. In the efferent duct ligation method, both general anesthesia and survival surgery are required; these stressful interventions have the potential to complicate the evaluation. In this technique, the testes are exposed through an abdominal or scrotal incision, the efferent ducts of one testis are ligated, and the wound is sutured closed. After waiting 16 - 24 hours for seminiferous tubule fluid to accumulate, the animal is killed, and the testes are removed and weighed; the increased weight of the ligated testis compared to the unligated testis reflects the accumulation of seminiferous tubule fluid. Each animal provides only a single data point. With the lumenal oil droplet method, however, multiple seminiferous tubules may be analyzed simultaneously in a couple of hours. Therefore, this approach may be particularly appropriate for the in vitro screening of compounds that inhibit seminiferous tubule fluid secretion. Seminiferous tubule fluid secretion has been examined by both the lumenal oil droplet method (Richburg et al., 1994) and efferent duct ligation (Johnson et al., 1991) following 2,5-hexanedione exposure. Using the lumenal oil droplet method, a statistically significant decrease in seminiferous tubule fluid secretion was observed after 3 weeks of 2,5-hexanedione exposure while 4 weeks of exposure was required before a decrease was observed by efferent duct ligation. Therefore, the lumenal oil droplet method is more sensitive than efferent duct ligation in the ability to detect inhibited seminiferous tubule fluid secretion. In summary, the lumenal oil droplet method is a rapid and sensitive in vitro technique for the assessment of seminiferous tubule fluid secretion. Exposure to microtubule disrupters which are known testicular toxicants consistently results in decreased seminiferous tubule fluid secretion and germ cell loss, indicating that Sertoli cell microtubules are critical to normal testicular homeostasis.

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