Germ cells contribute to the function of the Sertoli cell barrier

Abstract

One of the most important but still poorly understood cellular phenomena occurring during spermatogenesis is the movement of preleptotene/leptotene spermatocytes across the blood-testis barrier (BTB), an ultrastructure comprised of tight junctions (TJs), basal ectoplasmic specializations, gap junctions, and desmosomes. Previous studies have shown cytokines and androgens to mediate BTB restructuring, but it is not yet entirely known if germ cells can regulate barrier function, and if yes, how. To address this question, we utilized a previously characterized Sertoli–germ cell coculture model coupled with transepithelial electrical resistance (TER), immunoblotting, and immunolocalization experiments. When freshly isolated germ cells from adult rat testes were added to Sertoli cells at a Sertoli:germ cell ratio of 1:5 (Sertoli cells were previously cultured at high density on Matrigel™-coated culture inserts for 3 d to allow assembly of a functional permeability barrier that mimicked the Sertoli cell BTB in vivo), there was a significant increase in TER compared with time-matched controls (i.e., Sertoli cells only), illustrating that germ cells promote Sertoli cell barrier function. This increase in barrier function was not likely the result of TJ gene expression by germ cells. Instead, germ cells upregulated the steady-state levels of several TJ proteins, including occludin, tricellulin, claudin, junctional adhesion molecule-A, and coxsackievirus and adenovirus receptor (CAR) in Sertoli cells. These results were corroborated in part by immunofluorescence staining when an increase in occludin at Sertoli–Sertoli cell borders was observed in vitro. Taken collectively, our results illustrate that germ cells contribute to BTB integrity, which is essential for spermatogenesis and fertility.