Testis

Regulation of Sertoli-Germ Cell Adherens Junction Dynamics in the Testis via the Nitric Oxide Synthase/Cyclic Guanosine 5'- Monophosphate/Protein Kinase G/-Catenin Signaling Pathway: An In Vitro and In Vivo Study

Abstract
During spermatogenesis, extensive restructuring of cell junctions takes place in the seminiferous epithelium to facilitate germ cell movement. However, the mechanism that regulates this event remains largely unknown. Recent studies have shown that nitric oxide (NO) likely regulates tight junction (TJ) dynamics in the testis via the cGMP/protein kinase G (cGMP-dependent protein kinase, PRKG) signaling pathway. Due to the proximity of TJ and adherens junctions (AJ) in the testis, in particular at the bloodtestis barrier, it is of interest to investigate if NO can affect AJ dynamics. Studies using Sertoli-germ cell cocultures in vitro have shown that the levels of NOS (nitric oxide synthase), cGMP and PRKG were induced when anchoring junctions were being established. Using an in vivo model in which adult rats were treated with Adjudin [a molecule that induces adherens junction disruption, formerly called AF- 2364, 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide], the event of AJ disruption was also associated with a transient iNOS (inducible nitric oxide synthase, NOS2) induction. Immunohistochemistry has illustrated that NOS2 was intensely accumulated in Sertoli and germ cells in the epithelium during Adjudin-induced germ cell loss, with a concomitant accumulation of intracellular cGMP and an induction of PRKG, but not cAMP or protein kinase A (cAMP-dependent protein kinase, PRKA). To identify the NOS-mediated downstream signaling partners, co-immunoprecipitation was used to demonstrate that NOS2 and eNOS (endothelial nitric oxide synthase, NOS3) were structurally associated with the Ncadherin (CDH2)/-catenin (CATNB)/actin complex, but not the nectin-3 (poliovirus receptor-related 3, PVRL 3)/afadin (myeloid/lymphoid or mixed lineage-leukemia translocation to 4 homolog, MLLT4) nor the ITGB1-mediated protein complexes, illustrating the spatial vicinity of NOS with selected AJ-protein complexes. Interestingly, CDH2 and CATNB were shown to dissociate from NOS during the Adjudinmediated AJ disruption, implicating the CDH2/CATNB protein complex is the likely downstream target of the NOS signaling. Furthermore, PRKG, the downstream signaling protein of NOS, was shown to interact with CATNB in the rat testis. In addition, pretreatment of testes with KT5823, a specific PRKG inhibitor, can indeed delay the Adjudin-induced germ cell loss, further validating NOS/NO regulates Sertoli-germ cell AJ dynamics via the cGMP/PRKG pathway. These results illustrate that the CDH2/CATNB-mediated adhesion function in the testis is regulated, at least in part, via the NOS/cGMP/PRKG/CATNB pathway.

Key words: Testis • Kinases • Sertoli cells • Signal transduction • Spermatogenesis

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