Is the Cadherin/Catenin Complex a Functional Unit of Cell-Cell Actin-Based Adherens Junctions in the Rat Testis?1

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, alpha-catenin, beta-catenin, and p120(ctn) 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 alpha(6)beta(1) 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 Sertoli-germ cell lysates during AJ assembly. An anti-N-cadherin antibody can pull out beta-catenin, whereas N-cadherin can also be pulled out using an anti-beta-catenin antibody. To further expand and validate these in vitro biochemical studies, immunofluorescent histochemistry was performed, which colocalized N-cadherin and beta-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.

Anchoring Junctions As Drug Targets: Role in Contraceptive Development

“…The effects of adjudin on Sertoli-germ cell adhesion have been well documented in both in vitro and in vivo studies Grima et al, 2001;Chen et al, 2003b;Lau and Mruk, 2003;Lee et al, 2003;Lui et al, 2003;Siu et al, 2003;Mruk et al, 2006;Wolski et al, 2006) (Table 1), and these reports have shown that adjudin exerts its effects on Sertoli-germ cell adhesion by activating two separate but related signaling cascades. It was found that integrin and RhoB were activated within hours of adjudin treatment (Lui et al, 2003;Siu et al, 2003).…”

“…In the testis, Sertoli and germ cells have been shown to express both E-and N-cadherin (Wu et al, 1993;Lee et al, 2003), but the expression of E-cadherin by germ cells was considerably higher than that of N-cadherin . In addition, E-cadherin expression in undifferentiated type A spermatogonia was recently demonstrated in the mouse testis (Tokuda et al, 2007), which clearly illustrates that spermatogonia use this cell adhesion protein predominantly to adhere to Sertoli cells.…”

“…ANCHORING JUNCTIONS AND MALE CONTRACEPTION ticular E-and N-cadherin were also shown to interact indirectly with intermediate filaments (Johnson and Boekelheide, 2002;Lee et al, 2003 (Fig. 1).…”

“…Classic catenins. Catenins comprise a family of well studied proteins that support adherens junction function in different epithelia Scott and Yap, 2006) including the testis (Chen et al, 2003b;Lee et al, 2003;. Catenins have long been regarded as proteins that link cadherins to the actin cytoskeleton either directly or indirectly ( Fig.…”

“…3). After decades of morphological and biochemical studies, we know that Sertoli cells isolated from rat testes become polarized and establish functional junctions when they are plated on a reconstituted basement membrane such as Matrigel at high cell density (ϳ0.5-1.0 ϫ 10 6 cells/cm 2 ) in a two-dimensional environment (Chung et al, 1999;Grima and Cheng, 2000;Wong et al, 2000;Chung and Cheng, 2001;Lee et al, 2003). We also know that Sertoli cells cultured under these conditions are functionally similar to those found in the testis in vivo, because they secrete transferrin, androgen binding protein (ABP), testin, and ␣ 2 -macroglobulin in a polarized fashion (Grima et al, 1992).…”