Testis

Interactions of Proteases, Protease Inhibitors, and the 1 Integrin/Laminin 3 Protein Complex in the Regulation of Ectoplasmic Specialization Dynamics in the Rat Testis

Abstract
During spermatogenesis, developing germ cells migrate progressively across the seminiferous epithelium. This event requires extensive restructuring of cell-cell actin- based adherens junctions (AJs), such as ectoplasmic specialization (ES, a testis-specific AJ type), between Sertoli cells and elongating/elongate spermatids. It was postulated that proteases and protease inhibitors worked in a "yin-yang" relationship to regulate these events. If this is true, it is anticipated both proteases and protease inhibitors are found at the ES. Indeed, matrix metalloprotease (MMP)-2, membrane-type 1 (MT1)-MMP and their inhibitor, TIMP-2, were shown to localize at apical ES. In order to identify the putative MMP substrate as well as the unknown binding ligand for the 61 integrin in the ES structure, immunofluorescent microcopy coupled with immunoprecipitation technique were used to demonstrate that laminin 3, largely a germ cell product, was present at the apical ES and could form a bona fide complex with 1-integrin. Furthermore, the structural interactions of MMP-2 and MT1-MMP with laminin 3 and 1-integrin, but not with N-cadherin or nectin-3, have implicated the crucial role of MMP- 2/MT1-MMP in the regulation of integrin/laminin-based ES dynamics. Using an in vivo model to study AJ dynamics where adult rats were treated with 1-(2,4-dichlorobenzyl)- indazole-3-carbohydrazide (AF-2364) to disrupt Sertoli- germ cell adhesive function, an induction of active-MMP- 2, active-MT1-MMP and TIMP-2 but not active-MMP-9 was detected between 0.5-8 h after AF-2364 treatment. This time frame coincided with the depletion of elongating/elongate spermatids from the epithelium, illustrating the synergistic relationships between MMP-2, MT1-MMP and TIMP-2 in AJ disassembly. Perhaps, the most important of all, the use of a specific MMP-2 and MMP-9 inhibitor, (2R)-2-[(4-biphenylylsulfonyl)amino]-3- phenylpropionic acid, could effectively delay the AF-2364- induced elongating/elongate spermatid loss from the epithelium, demonstrating unequivocally the pivotal role of MMP-2 activation in ES disassembly. Collectively, these studies illustrate the 1-integrin/laminin 3 complex is a putative ES-structural protein complex, which is regulated, at least in part, by the activation of MMP-2 involving MT1-MMP and TIMP-2 at the apical ES. The net result of this interaction likely regulates germ cell movement in the seminiferous epithelium.

Key words: Testis • Sertoli cells • Signal transduction

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