Is the cadherin/catenin complex a functional unit of cell-cell actin-based adherens junctions in the rat testis?

Abstract

Much controversy exists regarding the presence of the cadherin/catenin complex and its intracellular attachment site in the testis, which is the functional unit for actin-based cell-cell adherens junctions (AJs) in multiple epithelia. Furthermore, whether germ and Sertoli cells are equipped with the necessary AJ-associated signaling molecules to regulate this cadherin/catenin complex during spermatogenesis is not known. In the present study, it was shown that both Sertoli and germ cells indeed express N-cadherin, E-cadherin, α-catenin, β-catenin, and p120ctn by semiquantitative reverse transcription-polymerase chain reaction and immunoblotting. Furthermore, the assembly of AJs between Sertoli and germ cells was associated with a transient induction in the steady-state mRNA and protein levels of cadherins and catenins. These analyses reveal, to our knowledge for the first time, that the testis may indeed be using the cadherin/catenin complex as one of the functional units to regulate AJ dynamics between Sertoli and germ cells in addition to α6β1, integrin and the nectin/afadin complex. To further confirm the existence of such a complex between Sertoli and germ cells, immunoprecipitation experiments were performed using Sertoligerm cell lysates during AJ assembly. An anti-N-cadherin antibody can pull out β-catenin, whereas N-cadherin can also be pulled out using an anti-β-catenin antibody. To further expand and validate these in vitro biochemical studies, immunofluorescent histochemistry was performed, which colocalized N-cadherin and β-catenin to the same site of Sertoli-Sertoli and Sertoli-germ cell AJs, possibly ectoplasmic specializations near the basal compartment, at the lower third of the seminiferous epithelium in vivo as well as between Sertoli cells cultured in vitro. Furthermore, studies by cross-linking using dithiobis(succinimidylpropionate) confirmed that the cadherin/catenin complex between Sertoli cells as well as between Sertoli and germ cells indeed structurally linked to actin but not to vimentin (an intermediate filament protein) or to tubulin (a microtubule protein). These results thus unequivocally demonstrate that the cadherin/catenin complex, which can be up-regulated by testosterone, is indeed present between Sertoli and germ cells and is used for the assembly of functional AJs.