Enrichment and disassembly of ectoplasmic specializations in the rat testis

Ectoplasmic specializations are testis specific intercellular adhesion junctions found in Sertoli cells. They are tripartite structures consisting of the plasma membrane of the Sertoli cell, a submembrane layer of actin filaments and an attached cistern of endoplasmic reticulum. Ectoplasmic specializations occur in areas of attachment to spermatids and as part of the basal junction complex between neighboring Sertoli cells. They are functionally related to a number of fundamental events that occur during spermatogenesis, including attachment and then release of developing sperm cells and the translocation of spermatocytes through the blood-testis barrier. The structures may contain viable molecular targets for the development of contraceptives. Here we describe techniques for obtaining, from rat testes, testicular fractions enriched for spermatids with attached ectoplasmic specializations and for disassembling the complexes with gelsolin to obtain supernatants enriched for plaque components. The techniques involve stripping the epithelium from tubule walls, mechanically fragmenting the epithelium, using step sucrose gradients to enrich for spermatids with attached junction plaques, and then incubating with exogenous gelsolin to release plaque components into solution.