Search

The mature spermatozoon, a highly differentiated cell equipped for the sole purpose of fertilization, lacks the protein machinery required for conventional endocytotic mechanisms. Perhaps contrary to expectation, cell-penetrating peptides (CPPs) rapidly translocate across the unique sperm plasma membrane to accrete within distinct intracellular compartments. Confocal microscopy, employing red-fluorescent CPPs and bioportides, is a convenient platform to study this membrane translocation process. In the virtual absence of genetic expression, rapid physiological responses of human sperm are dependent upon protein-protein interactions that may be regulated by posttranslational modifications including phosphorylation. This chapter provides an outline of the design of bioactive CPPs, or bioportides, which include protein-mimetic sequences from the interaction domains of sperm proteins. Protocols are included which enable the biological assessment of the impact of bioportides upon the viability and motility of spermatozoa.

Most techniques to study protein-protein interactions, gene expression or signal transduction, among others, in Sertoli cells, depend on obtaining a protein extract of such cells. This is accomplished by lysing the Sertoli cells and solubilizing the intracellular proteins. Depending on the purpose of your study, the technique used to lyse and consequently obtain protein extracts from Sertoli cells must be considered. In this chapter, we will focus on how to obtain a protein Sertoli cell extract suitable for co-immunoprecipitation technique.

Sertoli cells are crucial for germ cell support, create a suitable environment for spermatogenesis, and integrate information from the nervous system and germ cell line. To fully understand the role of Sertoli cells, it is necessary to characterize the protein-protein interactions. Identifying the interactome of a given protein may provide leads about the role and molecular mechanism of such protein in Sertoli cells. One of the techniques to characterize protein interactomes consists of co-immunoprecipitation followed by mass spectrometry or Western blot. Co-immunoprecipitation enables the isolation of a protein target and interactome under physiological conditions. In this chapter, we described how to perform an interactomic study using the co-immunoprecipitation technique in Sertoli cells. Moreover, we will focus on how to analyze and interpret the results of a co-immunoprecipitation before mass spectrometry analysis.