Cell-, age- and stage-dependent distribution of connexin43 gap junctions in testes

Abstract

Immunocytochemical data demonstrate that the distribution of gap junction connexin43 (Cx43) in rodent testes is dependent on cell type, testis maturation, and stage of the mature seminiferous epithelium. Western blotting and indirect immunofluorescence microscopy using anti-peptide antisera to Cx43 revealed abundant Cx43 in rat and mouse testes and mouse TM3 and TM4 cells. Cx43 mRNA was detected in rat testes and mouse TM4 cells by Northern blot analysis. Cx43 was localized by immunogold electron microscopy to gap junctions on Sertoli cells and Leydig cells. A punctate distribution of Cx43 was observed on peritubular cell surfaces following indirect immunofluorescence of detergent-permeabilized tubule segments. In cryosections from testes of immature (to 30 days) rats, and mature rats and mice, Leydig cells showed a punctate surface distribution of Cx43 following indirect immunofluorescence. A diffuse cytoplasmic fluorescence was also seen in spermatocytes and spermatogonia. Cx43 staining associated with Sertoli cells was age- and stage-dependent. Over 90% of the tubules in immature tests (22-30 days) contained Cx43 in the region of Sertoli-Sertoli occluding junctions and in the adluminal compartment. In mature rat testes, however, Cx43 immunostaining was detected in only 60% of 1195 tubule sections where it was abundant proximal to the Sertoli cell occluding junctions. All strongly stained tubules were from stages I-VIII, while negatively stained tubules were at stages IX-XIV. Cx43 immunostaining in mature mouse testes was also stage-dependent with all positive tubules at stages VI-VIII. In contrast to Cx43, Cx26 and Cx32 were detected by immunofluorescence only in the apical regions of the seminiferous epithelia in 90% of tubules from mature rats. Consistent with the observed Cx43 immunostaining, octanol-sensitive in situ dye-coupling was observed between Leydig cells, between peritubular cells and between Sertoli cells, suggesting the occurrence of functional gap junctions in these cell types. These observations provide evidence for extensive gap junction-mediated communication between a variety of testis cell types important to the support of spermatogenesis.