Primary Sertoli Cell Cultures from 30‐Day Old Rats Form Cords with a Morphologically Differentiated Phenotype

Abstract

The seminiferous epithelium lines the seminiferous tubules of the mammalian testes and is comprised of Sertoli cells and germ cells. Sertoli cells are tall columnar cells that extend from the base of the seminiferous epithelium to the tubule lumen. They play a key role in spermatogenesis by providing support and nourishment for the developing spermatogenic cells. We have been working to establish a primary culture system for studying junction turnover in morphologically differentiated Sertoli cells. We have found that when the age of the rats used for isolating Sertoli cells are older than 20 days, the cells respond much differently to the same culture conditions than cells from younger animals. The purpose of this study is to characterize the differences in growth and morphology between these two populations of cells. Sertoli cells were enzymatically dissociated from the testes of 20‐day or older rats and were plated at a density of 1.3×106 cells /0.2 mL on Transwell culture inserts coated with undiluted Matrigel. Sertoli cells were cultured in serum‐free defined medium (SFDM) containing hormones (FSH and testosterone), vitamins and antibiotics and incubated at 34°C in 5% CO2 for either 9 days or 2 weeks. The cultured cells were treated with a brief hypotonic wash after two days to lyse any contaminating germ cells. Finally, the cells were fixed and processed for immunofluorescence microscopy or transmission electron microscopy. Sertoli cells isolated from 20‐day old rats formed a confluent layer within 24–48 hours with cells having a cuboidal to columnar shape. Cells formed a monolayer interrupted by areas where they formed focal clumps. The cells generally were polarized with nuclei and junction networks basally located. Sertoli cells isolated from 25–30 day old rats rapidly reorganized to form a condensed mass from which cylindrical cords of cells projected into the surrounding remodeled Matrigel. Sertoli cells in the cords were columnar and their irregularly shaped nuclei were located basally around the periphery of the cords. Intercellular junctions appeared better formed at the base of Sertoli cells than in cultures from 20‐day old animals. Claudin‐11 staining confirmed the presence of numerous tight junctions within ectoplasmic specializations. All the primary cultures were predominantly comprised of Sertoli cells with few contaminating germ and myoid cells. This study presents an improved primary Sertoli cell culture system that more closely resembles the in vivo phenotype and may be a more effective model for studying junction turnover.Support or Funding InformationSupported by a NSERC Discovery Grant (#155397‐2013) to AWV.