Laminin α 3 Forms a Complex with β3 and γ3 Chains That Serves as the Ligand for α 6β1-Integrin at the Apical Ectoplasmic Specialization in Adult Rat Testes

Helen H.N. Yan,C. Yan Cheng

doi:10.1074/jbc.m513218200

Abstract

Apical ectoplasmic specialization (ES) is a testis-specific hybrid cell/cell actin-based adherens junction and cell/matrix focal contact anchoring junction type restricted to the interface between Sertoli cells and developing spermatids. Recent studies have shown that laminin gamma3, restricted to elongating spermatids, is a putative binding partner of alpha 6beta 1-integrin localized in Sertoli cells at the apical ES. However, the identity of the alpha and beta chains, which constitute a functional laminin ligand with the gamma3 chain at the apical ES, is not known. Using reverse transcription-PCR and immunoblotting to survey all laminin chains in cells of the seminiferous epithelium, it was noted that alpha 2, alpha 3, beta1, beta2, beta3, and gamma3 chains were found in germ cells, whereas alpha 1, alpha 2, alpha 4, alpha 5, beta1, beta2, gamma1, gamma2, and gamma3 chains were found in Sertoli cells, implying that alpha 3 and beta3 are the plausible laminin chains restricted to germ cells that may be the bona fide partners of gamma3. To verify this postulate, recombinant proteins based on domain G of alpha 3 and domain I of beta3 and gamma3 chains were produced and used to obtain the corresponding specific polyclonal antibodies. Additional studies have demonstrated that the laminin alpha 3, beta3, and gamma3 chains indeed are restricted to germ cells at the apical ES, co-localizing with each other and with beta1-integrin. Furthermore, co-immunoprecipitation studies have confirmed the interactions among laminin alpha 3, beta3, and gamma3, as well as beta1-integrin. When the functional laminin ligand at the apical ES was disrupted via blocking antibodies, such as using anti-laminin alpha 3 or gamma3 IgG, this treatment perturbed adhesion between Sertoli and germ cells (mostly spermatids), leading to germ cell loss from the epithelium. More important, a transient disruption of the blood-testis barrier was also detected.

Highlights

Elongating/elongate spermatids orientate themselves with heads and tails pointing toward the basal lamina and the tubule lumen, respectively
Identification of the Bona Fide Partners of Laminin ␥3 in Adult Rat Testes—As an initial step to identify the likely laminin ␣ and ␤ chains that interact with laminin ␥3 at the apical ectoplasmic specialization (ES), RT-PCR was used to survey different laminin chains in Sertoli and germ cells
It is interesting to note that laminin ␥3 was expressed predominantly in germ cells isolated from adult rat testes (containing spermatogonia:spermatocytes:elongating/elongate spermatids at a ratio of 16.7:18:65% as determined by DNA flow cytometry as reported previously [37]), whereas laminins ␣3 and ␤3 were restricted to germ cells

Summary

Introduction

Elongating/elongate spermatids orientate themselves with heads and tails pointing toward the basal lamina and the tubule lumen, respectively. The specificity of this anti-laminin ␥3 antibody was illustrated by immunoblotting, as shown in Fig. 5E in which a prominent immunoreactive band of 146 kDa was detected in lysates of seminiferous tubules and germ cells but not Sertoli cells.

Results

Conclusion