Staging Seminiferous Tubules in Unfixed Mouse Testis Sections Using Hoechst and SCP3

Abstract

Spermatogenesis within mammals occurs in the seminiferous epithelium of the testes. When viewing seminiferous tubule cross-sections, the developmental stages of germ cells within the epithelium are not easily distinguishable. The traditional method for staging is performed using Peanut Agglutinin (PNA) lectin staining, which stains acrosomes of developing spermatids. Since acrosomes are internalized organelles, they are not accessible at the surface. Therefore, this method of staging requires tissue permeabilization, which may not be desirable depending on experimental conditions. Permeabilization is typically a result of tissue fixation, which wipes away the cell membrane and the enzymes within it. Some experiments such as enzyme activity staining require unfixed, and unpermeabilized tissue. Therefore, we propose a new staging method that uses Synaptonemal Complex Protein 3 (SCP3), a protein complex used in meiosis that facilitates genetic recombination and stains the spermatocytes, along with Hoechst, a nuclear stain that penetrates DNA. These stains will enable visualization of cell organization within the epithelium and allow for identification of distinct stages within spermatids meiotic progression without tissue permeabilization.