Regulation of blood-testis barrier dynamics: an in vivo study

Abstract

IntroductionA single type A1 spermatogonium (diploid, 2n) in rodents, such as rats and mice, gives rise to 256 mature spermatids (haploid, n) in the seminiferous epithelium during spermatogenesis (for reviews, see de Kretser and Kerr, 1988;Cheng and Mruk, 2002;Lui et al., 2003b). For this event to happen, preleptotene and leptotene spermatocytes must traverse the blood-testis barrier (BTB, also known as the seminiferous epithelium barrier) at late stage VIII and early IX of the epithelial cycle (Russell, 1977). The BTB is constituted largely by inter-Sertoli cell tight junctions (TJ). However, cellcell actin-based adherens junctions (AJ, e.g. ectoplasmic specialization, a testis-specific AJ type) and possibly cell-cell intermediate filament-based desmosome-like junctions (for reviews, see de Kretser and Kerr, 1988;Pelletier, 2001;Cheng and Mruk, 2002), are also found at the BTB site. As such, these junctions must undergo extensive restructuring during this process of germ cell migration. Yet the mechanism(s) that regulates BTB dynamics remains largely unknown (for a review, see Cheng and Mruk, 2002).Recently completed in vitro studies have shown that Sertoli cell TJ dynamics are regulated, at least in part, by transforming growth factor-β3 (TGF-β3) (Lui et al., 2001;Lui et al., 2003a) and tumor necrosis factor-α (TNF-α) (Siu et al., 2003a) via the TGF-β3/MEKKs/p38 mitogen activated protein (MAP) kinase and the TNF-α/integrin-linked kinase (ILK)/p130 Crkassociated substrate (Cas)/MAP kinase signaling pathways, respectively. More importantly, a preliminary in vivo study has shown that the event of BTB disruption induced by cadmium chloride (CdCl2) indeed was mediated via the TGF-β3/MEKKs/p38 MAP kinase pathway (Lui et al., 2003d). Yet it remains to be determined whether proteases, protease inhibitors, AJ integral membrane proteins and their associated peripheral adaptors, and signaling molecules are also involved in TJ restructuring and, in particular, the subsequent germ cell loss from the epithelium as a result of BTB damage. In brief, in this study we sought to investigate whether a primary loss of the BTB function can lead to a secondary disruption of the AJs. We also investigated the roles of proteases and protease inhibitors in this event, since recent in vitro studies have shown that AJ dynamics in the testis are regulated by the intricate interactions between proteases and protease inhibitors under An induction of α α 2-macroglobulin (a non-specific protease inhibitor) was also observed during BTB damage and when the seminiferous epithelium was being depleted of germ cells. These data illustrate that a primary disruption of the BTB can lead to a secondary loss of cell adhesion function at the site of AJs, concomitant with an induction in protease inhibitor, which apparently is used to protect the epithelium from unwanted proteolysis. α α 2-Macroglobulin was also shown to associate physically with TGF-β β3, afadin and nectin 3, but not occludin, E-cadherin or N-cadherin, indicating its possible role in jun...

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

“…6Ha,b), illustrating BTB disruption. This is in line with previously published reports, which showed CdCl 2 to disrupt BTB integrity (Durieu-Trautmann et al, 1994;Elkin et al, 2010;Prozialeck et al, 2006;Siu et al, 2009;Wong et al, 2004). For the negative control (pCI-neo/MOCK), inulin-FITC was restricted to the basal compartment (Fig.…”

“…2A). It is worth noting that previous studies have shown all of these proteins to localize to the Sertoli cell barrier/BTB (Cheng and Mruk, 2012;Cyr et al, 1999;Hellani et al, 2000;Mruk and Cheng, 2004;Su et al, 2010a;Wang et al, 2007;Wong et al, 2004). To validate that ICAM-1 was indeed a constituent protein of the BTB, Sertoli cells (previously cultured for 4 days and having a functional barrier that mimicked the BTB in vivo) were fluorescently immunostained and analyzed by confocal microscopy (Fig.…”

“…Three-dimensional images were processed with ImageJ software (version 1.46, NIH), and the Richardson-Lucy deconvolution algorithm was used to reduce blur and/or noise associating with acquired images (Lucy, 1974;Richardson, 1972). Electron microscopy was performed as previously described Wong et al, 2004). Confocal and electron microscopy were performed at the Bio-Imaging and Electron Microscopy Resource Centers, respectively, The Rockefeller University.…”

“…and using these animals for BTB integrity assays 3 days after the administration of CdCl 2 . CdCl 2 was used as a positive control because its disruptive effects on BTB function have been described in previously published reports (Elkin et al, 2010;Wong et al, 2004).…”

Blood–testis barrier dynamics are regulated by an engagement/disengagement mechanism between tight and adherens junctions via peripheral adaptors

“…Immunofluorescent microscopy was performed as described in ref. 19 by using different primary antibodies at different dilutions, which were obtained from different vendors as described in refs. 14, 19, and 20.…”