23. Effect of Nitric Oxide on Sertoli Cell Tight Junction-Associated Proteins

Makoto Onoda, Takanori Katsube and Hiroshi Inano

Keywords: Sertoli cell, nitric oxide, tight junction, occludin, ZO-1, cortactin, NOC18

Nitric oxide (NO) is an important biological molecule with a wide variety of functions. NO appears to be present in all parts of the male reproductive system and to play diverse roles in testicular, epididymal and vas deferent functions. We, therefore, undertook clarification of the effects of NO on the Sertoli cells (SC) tight junction (TJ)-associated proteins by using a culture system.

SC were isolated from 16-day-old immature rat testes by a three-step sequential enzyme digestion procedure, and cultured on 15-mm round glass cover slides coated with reconstituted extracellular matrix (Matrigel) in the well of a 24-multiwell plate. The cultures were maintained in serum free defined medium throughout the experimental period. 2,2'-(Hydroxynitrosohydrazino) bis-ethanamine (NOC 18), a NO donor, was added to the cultures, and the distribution and expression of the TJ-associated proteins, such as occludin, ZO-1 and actin cytoskeleton, were determined by immunocytochemistry and Western blot analysis, respectively, with specific antibodies. Cortactin (p80/85), a prominent substrate for Src protein tyrosine kinase, was also examined.

The amount of NO produced by SC in culture was 3.4 ±1.4 nmol/ml and 7.3 ± 0.9 nmol/ml for 24 hr- and 48 hr-incubation periods, respectively. This suggests that SC produce NO spontaneously under the culture condition, although the amount is relatively minute. On the other hand, the exogenously released NO from an NO donor into the medium was 147.8 ± 2.7 nmol/ml and 333.9 ±10.1 nmol/ml in the cultures with 200 µM and 400 µM of NOC 18, respectively, in a 24 hr-culture period. These values are in agreement with the theoretical values based on the half-life time (21 hr) of NOC 18. Immunostaining for occludin, ZO-1 and cortactin in untreated-SC showed continuous labeling around the cell periphery in the region of the cell-cell junctional complex, and these colocalized with TJ-associated F-actin filaments. However, incubation with NOC 18 (200 µM and 400 µM) caused complete disorganization of occludin staining and partial disruption of ZO-1 staining within 48 hr (Fig. 17), while, alteration in the immunolabeling pattern for cortactin and F-actin was inconclusive in NOC 18 treated SC. Furthermore, Western blotting of occludin, ZO-1 and cortactin exhibited descending expressions of these TJ-associated proteins in a dose-dependent manner of NOC 18 in the NO donor treated SC (Fig. 18). These results suggest that excessive production of NO within testis during acute or chronic pathophysiological conditions disrupts TJ-associated proteins of SC and that NO may perturb blood-testis barrier formation and then, in turn, the regulation of normal spermatogenesis.

Fig.17. Immunocytochemistry of tight junction-associated proteins in cultured rat Sertoli cells. Sertoli cells (1.5x106 cells/well) were isolated and cultured on Matrigel coated-cover glass in serum free defined medium with (A' and B') or without (A and B) NOC18 (400 µM). Sertoli cells were subjected to immunocytochemistry with specific antibodies. A and A': Cultures stained with anti-occludin antiserum, B and B': Cultures stained with anti-ZO-1 antiserum.

Fig.18. Effect of NOC18 on the expression of occludin in rat Sertoli cells. Sertoli cell -lysates (15 µg/lane) were loaded onto SDS-PAGE mini-gel system, and immunoblotting with a specific antibody was carried out. Values presented at the bottom of lanes are percentage of non-treated control. Std: standard rainbow marker proteins, (1): serum free defined medium alone, (2): with 200 µM NOC 18, (3): with 400 µM NOC 18.