The Blood-Testes Barrier (abbreviated as BTB) acts as a physical barrier between the blood vessels and the seminiferous tubules of the testes. This barrier is formed by tight and adherens connections between the Sertoli cells, which are sustentacular cells (supporting cells) of the seminiferous tubules, and nourish the spermatogonia (Ref.1). In the testis, tight and adherens junctions are dynamically remodeled to allow the movement of post-meiotic germ cells across the seminiferous epithelium and the timely release of spermatids into the tubular lumen. Three main functions are ascribed to the blood-testis barrier; (i) creates a specialized environment; (ii) regulates the passage of molecules; and (iii) serves as an immunological barrier. When the blood-testes barrier is breached, and sperm enters the bloodstream, the immune system mounts an autoimmune response against the sperm. The anti-sperm antibodies generated by the immune system bind to various antigenic sites on the surface of the sperm. If they bind to the head, the sperm may be less able to fertilize an egg, and if they bind to the tail, the motility of the sperm can be reduced. These junctions that form the BTB maintain tissue integrity and their turnover plays a crucial role in cell development and morphogenesis (Ref.1 & 2).
Morphologically, tight junctions form a continuous circumferential seal above the basal lamina of the seminiferous tubules in the testis. The major proteins of BTB tight junctions include Occludins, Claudins and JAMs (Junctional Adhesion Molecules). Occludins directly interacts with ZO1 (Zona Occludens-1), ZO2, ZO3, ZONAB, PALS2 (Protein Associated with Lin7), ZAK (Sterile Alpha Motif and Leucine Zipper Containing Kinase-AZK), Spectrin/Fodrin, Protein 4.1, PILT, Cgn (Cingulin), 7H6 Antigen, Sympk (Symplekin) and Actin. Claudin clusters interact with ZO1, ZO2, Spectrin/Fodrin and PILT, whereas the main adhesion molecules near the JAM clusters includes ZO1, PILT, Spectrin/Fodrin and Cgn. Such binding association activates cytoskeletal proteins like Alpha-Actn (Alpha-Actinin), Fimbrin, Epsin , Tubulin , KEAP1 (Kelch-like ECH-Associated Protein-1) and Myosin7A that in turn activate F-Actin polymerization to enhance cell adhesion between two neighbouring sertoli cells (Ref.2). Similarly the adherens junction between sertoli cells consists of Nectin and CdhE (E-Cadherin) family cell adhesion molecules, which are linked to the Actin cytoskeleton through their binding proteins Afadin and Catenins, respectively. Cadherins comprise two domains; the C-terminal distal Ctnn-Beta (Catenin-Beta)-binding domain (D-Beta-D); and the Juxtamembrane domain (JMD), which is the p120Ctn (p120-Catenin)-binding site. p120Ctn binds to c-Src and tyrosine phosphatase, MTMR2 (Myotubularin-Related Protein-2) complex to dephosphorylate inositol lipids and modulate cytoskeletal reorganization. Likewise formation of Cdh/Ctnn-Beta/Ctnn-Alpha/Ctnn-Gamma plaques recruits and activates cytoskeletal modulators like ZO1, Vcl (Vinculin), Axin, WASP (Wiskott-Aldrich Syndrome Protein) and Alpha-Actn . Nectin/Afadin junctional complexes recruit ZO1, Ctnn-Alpha and Ponsin. These proteins at adherens junction plaques activate Epsin, Fimbrin and KEAP1 to increase F-Actin /Myosin7A/Tubulin cross bridging and thereby control regulation of adherens junction dynamics in testes. Further Rab8B activation by CdhE leads to Cfl (Cofilin) disintegration and binding of Ctnn-Alpha to Occludin plaques results in Actin polymerization and strong adhesion (Ref.2 & 3).
Junction dynamics is affected by cytokines like TNF-Alpha (Tumor Necrosis Factor-Alpha) and TGF-Beta3 (Transforming Growth Factor-Beta-3), however their signals are ought to be finely-tuned to allow regulation and remodeling of various junction types in the testes. Remodeling of junctions in the testes is necessary for germ cell migration. The BTB dynamics are chiefly regulated by TGF-Beta3/TGF-BetaR3 (Transforming Growth Factor-Beta Receptor-Type III) and TNF-Alpha/TNF-AlphaR (Tumor Necrosis Factor-Alpha-Receptor), via various defined signaling pathways. The TGF-Beta3/TGF-BetaR3 routes include CDC42/Rac >MEKK >JNKK >JNK; CDC42/Rac >MEKK >MKK3 >p38; and Ras >Raf >MEK1/2>ERK1/2 . The TNF-Alpha/TNF-AlphaR routes include Itg (Integrin)>ILK>GSK3 >p130CAS>JNK ; whereas the CDC42/Rac >MKK >JNK route activates Alpha2M (Alpha-2-Macroglobulin) that counterbalances the effect of cytokines by inhibiting their action through binding (Ref.3 & 4). Again interaction of TNF-AlphaR and TGF-BetaR3 with Occludin plaques activates NOS (Nitric Oxide Synthase) which catalyzes L-Arginine into nitric oxide (NO). First, nitric oxide stimulates sAC (Soluble Adenylate Cyclase) and sGC (Soluble Guanylate Cyclase) to synthesize cAMP (Cyclic Adenosine 3,5-Monophosphate) and cGMP (Cyclic Guanosine 3,5-Monophosphate), respectively. cAMP and cGMP in turn activate their respective kinases PKA (cAMP-dependent Protein Kinase-A) and PKG (cGMP-Dependent Protein Kinase). Activation of PKA, PKG , JNK and p38 leads to inhibition of Occludin function and disruption of tight junctions, whereas ERK1/2, p38 and JNK activation stimulates TFs (Transcription Factors) to control changes in testicular junction dynamics, cell division and differentiation, apoptosis and cell migration (Ref.5 & 6). Similarly, E-Cadherin/Ctnn-Alpha/Ctnn-Beta/Vcl/ F-Actin induced activation of MAGI2/3 (Membrane Associated Guanylate Kinases-2/3) prevents PTEN degradation in sertoli cells, which significantly decreases the cell proliferation activity of the Akt (v-Akt Murine Thymoma Viral Oncogene Homolog) through conversion of PIP3 (Phosphatidylinositol-3,4,5-Trisphosphate) to PIP2 (Phosphatidylinositol-4,5-Bisphosphate). Simultaneous effect of PIP2 generation and Akt inhibition decrease GSK3 activation by Akt. Such signaling creates a unique microenvironment for germ cell development around BTB and avoids passage of cytotoxic agents into the seminiferous tubules through BTB. Needless to say, understanding the junction dynamics of the seminiferous epithelium may unfold new targets for non-hormonal male contraceptive development (Ref.7).