Sertoli-Germ Cell Anchoring Junction Dynamics in the Testis Are Regulated by an Interplay of Lipid and Protein Kinases

Abstract

When Sertoli and germ cells were co-cultured in vitro in serum-free chemically defined medium, functional anchoring junctions such as cell-cell intermediate filament-based desmosome-like junctions and cell-cell actin-based adherens junctions (e.g. ectoplasmic specialization (ES)) were formed within 1-2 days. This event was marked by the induction of several protein kinases such as phosphatidylinositol 3-kinase (PI3K), phosphorylated protein kinase B (PKB; also known as Akt), p21-activated kinase-2 (PAK-2), and their downstream effector (ERK) as well as an increase in PKB intrinsic activity. PI3K, phospho (p)-PKB, and PAK were co-localized to the site of apical ES in the seminiferous epithelium of the rat testis in immunohistochemistry studies. Furthermore, PI3K also co-localized with p-PKB to the same site in the epithelium as determined by fluorescence microscopy, consistent with their localization at the ES. These kinases were shown to associate with ES-associated proteins such as ␤1-integrin, phosphorylated focal adhesion kinase, and c-Src by co-immunoprecipitation, suggesting that the integrin⅐laminin protein complex at the apical ES likely utilizes these protein kinases as regulatory proteins to modulate Sertoli-germ cell adherens junction dynamics via the ERK signaling pathway. To validate this hypothesis further, an in vivo model using AF-2364 (1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide) to perturb Sertoli-germ cell anchoring junction function, inducing germ cell loss from the epithelium in adult rats, was used in conjunction with specific inhibitors. Interestingly, the event of germ cell loss induced by AF-2364 in vivo was also associated with induction of PI3K, p-PKB, PAK-2, and p-ERK as well as a surge in intrinsic PKB activity. Perhaps the most important of all, pretreatment of rats with wortmannin (a PI3K inhibitor) or anti-␤1-integrin antibody via intratesticular injection indeed delayed AF-2364-induced spermatid loss from the epithelium. In summary, these results illustrate that Sertoli-germ cell anchoring junction dynamics in the testis are regulated, at least in part, via the ␤1-integrin/PI3K/PKB/ERK signaling pathway.In the seminiferous epithelium of the rat testis, Sertoli-germ cell adhesion is maintained by cell-cell actin-based adherens junctions (AJs) 1 and intermediate filament-based desmosomelike junctions (for reviews, see Refs. 1-3). The best studied testis-specific AJ type is ectoplasmic specialization (ES). The ES is confined between Sertoli cells (known as the basal ES) at the site of the blood-testis barrier (BTB) as well as between Sertoli cells and spermatids (known as the apical ES) in the adluminal compartment of the epithelium (for reviews, see Refs. 3 and 4). Most of the studies on cell adhesion function in the testis in the past 2 decades have focused on the apical ES because the biochemical composition of the desmosome-like junction remains largely unexplored in the testis (for reviews, see Refs. 1, 3, and 5). The apical ES is an important anchoring junction device t...

Coxsackie and adenovirus receptor (CAR) is a product of Sertoli and germ cells in rat testes which is localized at the Sertoli–Sertoli and Sertoli–germ cell interface

“…4 where cells were used for staining on day 3 following plating). By then, functional inter-Sertoli tight junction and adherens junctions were mostly established as shown by transepithelial electrical resistance measurement (TER) ( Fig 5) and electron microscopy [23]. CAR (red fluorescence) is observed to be concentrated at cell-cell contacts of Sertoli cells (Fig.…”

“…To obtain Sertoli cell cultures with purity greater than 98%, cells were hypotonically treated 36 hours after plating with 20 mM Tris (pH 7.4), for 2.5 min at 22°C to lyse contaminating germ cells [22]. The wells were then washed twice with F12/DMEM, media were replaced every 24 hours, and cells were incubated for an additional 5-7 days [23], however, it is noted that functional tight and anchoring junctions were established within 2-3 days after cell plating (see below) [23]. To terminate cultures at specified time points, cells were rinsed with cold PBS once and then scraped from the wells with lysis buffer for protein lysate preparation.…”

“…The net value of electrical resistance was then computed by subtracting the background, which was measured on Matrigel-coated cell-free chambers, from values of Sertoli cell-plated chambers [26]. Under these conditions, inter-Sertoli tight junctions were mostly formed at ~2-3 days [23]. Each time point had triplicate cultures, and each experiment was repeated 3 times using different batches of primary Sertoli cell cultures.…”

“…Sertoli cells were cultured alone for 4 days at 0.5 x 10 6 cells/cm 2 on Matrigel-coated dishes with established functional tight and anchoring junctions that mimicked the in vivo cellular physiology and morphology as earlier reported [23] prior to their used for lysate preparation. Testis or Sertoli cell lysates were first pretreated with 2 μg of rabbit or mouse IgG for approximately 1 hour, followed by incubation with 10 μL Protein A/G-PLUS agarose (Santa Cruz, CA) for 2 hours to eliminate nonspecific binding of protein with IgG or agarose.…”

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

“…On day 2, cells were transfected with empty pCI-neo vector (MOCK, control), ICAM-1, sICAM-1 or ICAM-2 plasmids using EffecteneH transfection reagent (Qiagen). It should be noted that by this time in vitro, Sertoli cells had formed a polarized epithelium with functional TJs, basal ESs, desmosomes and GJs Lie et al, 2009;Siu et al, 2005;Wong et al, 2000), thereby mimicking the BTB in vivo (Byers et al, 1986;Chung et al, 1999;Siu et al, 2005;Steinberger and Jakubowiak, 1993;Yan et al, 2008). Thus, this excellent in vitro model has been, and continues to be, used by many investigators to study BTB dynamics.…”