The blood-testis barrier (BTB) remains semi-permeable to tracers of increasing molecular weight in an adult rat model of spermatogenic re-initiation1. Complete closure of the BTB occurred when steps 2-7 round spermatids re-appeared in the epithelium, and coincided with the localisation of a new tight junction (TJ) protein, claudin-12 (Cldn12), at the BTB. We hypothesised that meiotic and/or post-meiotic germ cells can up-regulate Sertoli cell TJs, and aimed to demonstrate that isolated germ cells stimulate TJs in rat Sertoli cells in vitro.
Material and Methods:
Sertoli cells from 20d Sprague-Dawley rats were cultured for 5 days, and then purified pachytene spermatocytes (PSC) or round spermatids (rST) were added in co-culture for 24hrs. TJ function was monitored by trans-epithelial electrical resistance (TER). Clnd11 and Cldn12 were examined by qPCR and immunofluorescence microscopy.
Results and Discussion:
TER increased 2-2.7-fold over 24hrs when PSC or rST were added to Sertoli cells, but only when cells were in direct contact. Tight junction protein Cldn11 mRNA expression was not altered by germ cells, while Cldn12 expression was up-regulated 1.5-2.4-fold. Cldn11 protein localisation was largely unaffected by germ cells, however Cldn12 localisation was intensely present at cell junctions when germ cells were added.
This data provides strong evidence that an extra group of TJ proteins, in addition to the androgen-dependent claudins (Cldn11, Cldn3) exist in rat Sertoli cells and are up-regulated by meiotic and post-meiotic germ cells. This is the first demonstration that TJs at the BTB are stimulated by germ cells, and suggests a new mechanism by which BTB function may be regulated in normal spermatogenesis, of likely importance in diseases or conditions which impact these germ cell types.