Laminin α3 forms a complex with β3 and γ3 chains that serves as the ligand for α6β1-integrin at the apical ectoplasmic specialization in adult rat testes

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 gamma 3, restricted to elongating spermatids, is a putative binding partner of alpha 6 beta 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 gamma 3 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, beta 1, beta 2, beta 3, and gamma 3 chains were found in germ cells, whereas alpha 1, alpha 2, alpha 4, alpha 5, beta 1, beta 2, gamma 1, gamma 2, and gamma 3 chains were found in Sertoli cells, implying that alpha 3 and beta 3 are the plausible laminin chains restricted to germ cells that may be the bona fide partners of gamma 3. To verify this postulate, recombinant proteins based on domain G of alpha 3 and domain I of beta 3 and gamma 3 chains were produced and used to obtain the corresponding specific polyclonal antibodies. Additional studies have demonstrated that the laminin alpha 3, beta 3, and gamma 3 chains indeed are restricted to germ cells at the apical ES, co-localizing with each other and with beta 1-integrin. Furthermore, co-immunoprecipitation studies have confirmed the interactions among laminin alpha 3, beta 3, and gamma 3, as well as beta 1-integrin. When the functional laminin ligand at the apical ES was disrupted via blocking antibodies, such as using anti-laminin alpha 3 or gamma 3 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.