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

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 ␥3, restricted to elongating spermatids, is a putative binding partner of ␣6␤1-integrin localized in Sertoli cells at the apical ES. However, the identity of the ␣ and ␤ chains, which constitute a functional laminin ligand with the ␥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 ␣2, ␣3, ␤1, ␤2, ␤3, and ␥3 chains were found in germ cells, whereas ␣1, ␣2, ␣4, ␣5, ␤1, ␤2, ␥1, ␥2, and ␥3 chains were found in Sertoli cells, implying that ␣3 and ␤3 are the plausible laminin chains restricted to germ cells that may be the bona fide partners of ␥3. To verify this postulate, recombinant proteins based on domain G of ␣3 and domain I of ␤3 and ␥3 chains were produced and used to obtain the corresponding specific polyclonal antibodies. Additional studies have demonstrated that the laminin ␣3, ␤3, and ␥3 chains indeed are restricted to germ cells at the apical ES, co-localizing with each other and with ␤1-integrin. Furthermore, co-immunoprecipitation studies have confirmed the interactions among laminin ␣3, ␤3, and ␥3, as well as ␤1-integrin. When the functional laminin ligand at the apical ES was disrupted via blocking antibodies, such as using anti-laminin ␣3 or ␥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 bloodtestis barrier was also detected.During spermatogenesis, preleptotene spermatocytes residing at the basal compartment of the seminiferous tubules must traverse the BTB 3 to the apical compartment at late stage VII through early stage VIII of the epithelial cycle in adult rat testes (1). Once in the adluminal compartment, spermatocytes differentiate into round spermatids, and the elongating/elongate spermatids orientate themselves with heads and tails pointing toward the basal lamina and the tubule lumen, respectively. The primary anchoring device that facilitates this process of orientation, while maintaining adhesion at the Sertoli cell/spermatid interface, is the apical ectoplasmic specialization (ES), a cell/cell actin-based adherens junction (AJ) type (2-4). In recent years, numerous reports have been published identifying the adhesion molecules at the apical ES. These include the cadherin⅐catenin, nectin⅐afadin, and integrin⅐laminin protein complexes (5-10), which are the primary adhesion protein complexes at the Sertoli cell/developing spermatid interface. Interestingly, in virtually all other epithelia, the integrin⅐laminin complex is usually restricted to the basement membrane at the cell/matrix interface (11,12). Yet studies have shown that integrin is a crucial adhe...

An autocrine axis in the testis that coordinates spermiation and blood–testis barrier restructuring during spermatogenesis

“…This study was performed by using primary cultures of Sertoli cells (3) with negligible Leydig and germ cell contamination (16) that were shown to form functional BTB (such as a tight junctionpermeability barrier) with the ultrastructural features under electron microscopy that mimicked the in vivo barrier (17,18).…”

“…␤1-Integrin and ␣6-integrin form a functional adhesion complex of ␣6␤1-integrin at the apical ES residing in Sertoli cells (9,10,15), and ␣6␤1-integrin is the receptor of laminin-333 residing in elongating/elongated spermatids (3,11). However, the precise localization of ␤1-integrin at the basal compartment is not well defined.…”

“…Sertoli cells transfected with vector alone failed to express any laminin chains (Fig. 2 A), since laminin-333 is restricted to elongating/elongated spermatids in adult rat testes (3). The effects of overexpression of domain IV of the laminin ␥3 chain on the Sertoli cell-TJ barrier were also monitored vs. cells transfected with vector alone.…”

“…Endocytosis assay, RT-PCR, immunoblot analysis, fluorescent microscopy, Sertoli cell TJ-barrier function monitored by TER measurement, and statistical analyses were performed as described (3,17) (Table S1, SI Methods).…”

Intercellular adhesion molecule-1 is a regulator of blood–testis barrier function

“…The rabbit received yet another booster injection 3 weeks thereafter. Ten days thereafter blood was collected to obtain antiserum (Cheng and Bardin, 1987;Cheng et al, 1988b;Yan and Cheng, 2006), and IgG was isolated by (NH 4 ) 2 SO 4 precipitation and DEAEchromatography by using Macro-PrepH DEAE support (Bio-Rad Laboratories, Hercules, CA) (Cheng et al, 1988a;Page and Thorpe, 2001a;Page and Thorpe, 2001b).…”