Ultrastructural Analysis of Kinesin-Related Motor Proteins During Spermatogenesis

Kinesins are a superfamily of microtubule-based motors that power intracellular traffic and play important roles in many fundamental cellular and developmental processes. Kinesins move on microtubules from their minus to plus end (conventional kinesin) or from plus to minus end (C-terminal kinesins), carrying cargoes to different destinations. A variety of cargoes such as vesicles, proteins, lipid drops, pigments, and the nucleus are moved by kinesins along cytoplasmic microtubules. Multiple mitotic kinesins and microtubule-associated proteins (MAPs) also have direct functions in spindle formation, chromosome segregation, and cytokinesis. Spermatogenesis provides an excellent model system to study the role of kinesin motor proteins during the dramatic cytoskeletal rearrangements that take place during male germ cell development. This chapter describes how to identify the multiple functions of kinesin motors during spermatogenesis by using ultrastructural analysis. Testis perfusion is described in detail, including how to anesthetize animals and how to select seminiferous tubules under transilluminated microscopy. Practical immunocytochemical staining is also described in detail in this chapter, especially methods to enhance staining and avoid contamination.