Berta Denduchis

Loss of Occludin Expression and Impairment of Blood-Testis Barrier Permeability in Rats with Autoimmune Orchitis: Effect of Interleukin 6 on Sertoli Cell Tight Junctions

ABSTRACT Inflammation of the male reproductive tract is accepted as being an important etiological factor of infertility. Experimental autoimmune orchitis (EAO) is characterized by interstitial lymphomononuclear cell infiltration and severe damage of seminiferous tubules with germ cells that undergo apoptosis and sloughing. Because the blood-testis barrier (BTB) is relevant for the protection of haploid germ cells against immune attack, the aim of this study was to analyze BTB permeability and the expression of tight junction proteins (occludin, claudin 11, and tight junction protein 1 [TJP1]) in rats during development of autoimmune orchitis. The role of IL6 as modulator of tight junction dynamics was also evaluated because intratesticular content of this cytokine is increased in EAO rats. Orchitis was induced in Sprague-Dawley adult rats by active immunization with testicular homogenate and adjuvants. Control rats (C) were injected with saline solution and adjuvants. Untreated (N) rats were also studied. Concomitant with early signs of germ cell sloughing, a reduced expression of occludin and delocalization of claudin 11 and TJP1 were detected in the testes of rats with EAO compared to C and N groups. The use of tracers showed increased BTB permeability in EAO rats. Intratesticular injection of IL6 induced focal testicular inflammation, which is associated with damaged seminiferous tubules. Rat Sertoli cells cultured in the presence of IL6 exhibited a redistribution of tight junction proteins and reduced transepithelial electrical resistance. These data indicate the possibility that IL6 might be involved in the downregulation of occludin expression and in the modulation of BTB permeability that occur in rats undergoing autoimmune orchitis.

Effects of di(2-ethylhexyl) phthalate on gap and tight junction protein expression in the testis of prepubertal rats.

The aim of this study was to analyze whether di(2‐ethylhexyl) phthalate (DEHP), a Sertoli and Leydig cell toxicant, is able to induce alterations in the expression of testicular gap and tight junction proteins. DEHP was administered by gavage (1 g/5 mL corn oil/kg body weight/day) to 25‐day‐old male Sprague–Dawley rats for 2 days (DEHP‐27d) and control rats were treated with corn‐oil vehicle for 2 days (C‐27d); animals were killed 24 h after the last treatment. Testes of DEHP‐27d rats showed different degrees of germ cell sloughing of seminiferous tubules (ST). No alterations of the blood testis barrier (BTB) by lanthanum tracer study were observed. ST of DEHP‐27d rats showed a milder immunofluorescence and more restricted expression of connexin‐43 (Cx43) in the adluminal and basal compartment compared to C‐27d. In DEHP‐27d rats, we found a discontinuous immunofluorescent (IF) pattern for zonula occludens (ZO‐1), contrasting with the continuous IF profile observed in C‐27d, and a delocalization of claudin‐11. A decrease in Cx43 and ZO‐1 and no changes in occludin expression were detected by Western blot in the testes of DEHP‐27d rats. Results from 57‐day‐old rats treated with DEHP for 2 days and held for 30 days without treatment showed that the alterations in protein expression induced by DEHP are reversible. However, a delay of spermatogenesis compared to C‐57d rats, occurred. Data demonstrated that DEHP does not impair BTB permeability but induces germ cell sloughing that might respond to a down regulation of Cx43 and ZO‐1 that alters cell junction proteins. Microsc. Res. Tech. 2009.

Experimental orchitis induced in rats by passive transfer of an antiserum to seminiferous tubule basement membrane.

A multifocal damage of the testis was obtained when rats were injected intravenously or under the tunica albuginea of the testis with a rabbit antiseminiferous tubule basement membrane serum. The damage was characterized by foci of perivascular and peritubular infiltrates of mononuclear round cells, infolding, thickening, and rupture of the seminiferous tubular wall and different degrees of injury of the germinal epithelium such as, cell disorganization, cell sloughing, and atrophy. Delamination and thickening of seminiferous tubule basement membrane and vacuolization of the Sertoli cell cytoplasm was often observed by electron microscopy. A linear deposit of rabbit gamma-globulin was detected by immunohistochemical techniques along the basement membranes of the seminiferous tubules and vessels. Testicular damage was not detected in rats injected with normal rabbit serum, used as control. In the kidneys of rats injected intravenously with the immune serum, a deposit of rabbit gamma-globulin was detected along glomerular basement membrane. Focal areas of mononuclear cell infiltrates, hypercellularity of glomeruli and thickening of glomerular capillary walls and Bowmans capsule were also observed.

Antigens of the basement membranes of the seminiferous tubules induce autoimmunity in Wistar rats

A preparation enriched in basement membranes from seminiferous tubules was isolated from rat testes (STBM) and injected with complete Freunds adjuvant into Wistar rats. In 60% of animals a mild multifocal orchitis was observed. In damaged areas, perivascular and peritubular mononuclear cell infiltrates and different degrees of cell sloughing of some seminiferous tubules were observed. Electron microscopy revealed focal thickenings and delamination of the basement membrane of the seminiferous tubules as well as vacuolization of Sertoli cell cytoplasm. Using immunofluorescence discontinuous linear deposits of IgG were detected along the seminiferous tubular wall. Moreover, the same pattern of immunofluorescence was observed when the IgG eluted from the testes of the immunized rats was layered on sections of normal rat testis. Circulating antibodies to STBM were detected using passive haemagglutination in approximately 45% of the immunized rats, with titers ranging from 1:20 to 1:80. Leukocyte migration was inhibited when the spleen cells of the immunized rats were incubated with antigens from the basement membrane of seminiferous tubules, whilst a negative reaction was obtained when the soluble fraction of testis homogenate was used.

Passive Immunization with Anti-Laminin Immunoglobulin G Modifies the Integrity of the Seminiferous Epithelium and Induces Arrest of Spermatogenesis in the Guinea Pig

Abstract In the testis, the base of the Sertoli cells is in contact with the basement membrane matrix, in which the laminins constitute the major noncollagenous components. We have previously demonstrated that antibodies against a preparation enriched in basement membranes of seminiferous tubules (STBM) or a noncollagenous fraction of STBM passively transferred induced modifications to the basement membranes and focal sloughing of the seminiferous epithelium in the rat. In the present report, we tested the effect of passive immunization with anti-laminin IgG on the limiting membrane of the seminiferous tubules, spermatogenesis, and maintenance of the blood-testis barrier in the adult guinea pig. Rabbit antibodies to laminin 1 (IgG fraction) were injected in adult male guinea pigs (GP). Nonimmunized GP and GP immunized with normal rabbit serum IgG were used as controls. Measurements of variations in the diameter and lumen of the tubules and in the size of individual components of the tubular limiting membrane showed that the highest percentage of tubules with reduced lumen occurred 30 days after passive immunization with anti-laminin, when the limiting membrane was thickest and lesions to the seminiferous epithelium were most severe. The lesions included thickening of the limiting membrane, infolding in the basal lamina, deposits of immune complexes coincident with sloughing of pachytene spermatocytes and spermatids, and vacuolization of the Sertoli cells. Mononuclear cell infiltration of the tubules was rare. Permeability tracer studies revealed that Sertoli cell tight junctions remained impermeable. Fifty and 80 days after treatment, the basement membrane of the tubules and the progression of the spermatogenesis were normal. Passive immunization with anti-laminin IgG provided a valuable experimental model for the in vivo study of the influence of the basement membrane on the issue of spermatogenesis and the integrity of the seminiferous epithelium.

Multifocal damage of the testis induced in rats by passive transfer of antibodies prepared against non-collagenous fraction of basement membrane

Multifocal damage of the testis was induced in 70% of the rats injected with an antiserum against a non-collagenous fraction (D-STBM) obtained from a preparation enriched in basement membranes of seminiferous tubules. The damaged areas were characterized by perivascular and peritubular cell infiltrates, changes in the walls of small vessels and seminiferous tubules, and sloughing of the germinal epithelium. By electron microscopy, the most frequent changes observed in basement membrane of the seminiferous tubules were folding, focal thickening, and delamination. By immunofluorescence, discontinuous linear deposits of rabbit IgG were observed along the walls of the seminiferous tubules. In the same localization, faint immunofluorescence showing the presence of rat IgG was also detected. The same pattern was obtained when rabbit and rat IgG eluted from the testes of these rats were layered on sections of normal rat testis. Moreover, by immunoelectron microscopy, discontinuous deposits of rabbit IgG were detected along the basement membranes of the seminiferous tubules. Neither C3 deposits nor changes in the serum CH 50 were observed. By leucocyte migration inhibition reaction (LMIR) a cellular immune response to basement membrane antigens was detected. In the control group, 12% of the rats injected with normal rabbit serum presented mild interstitial cell infiltrates and occasional sloughing of the germinal epithelium. Neither deposits of rat IgG or rabbit IgG nor a cellular immune response were observed.

Adenosine regulates Sertoli cell function by activating AMPK

This work evaluates adenosine effects on Sertoli cell functions, which are different to those resulting from occupancy of purinergic receptors. The effects of adenosine and N(6)-cyclohexyladenosine (CHA) - an A(1) receptor agonist resistant to cellular uptake - on Sertoli cell physiology were compared. Adenosine but not CHA increased lactate production, glucose uptake, GLUT1, LDHA and MCT4 mRNA levels, and stabilized ZO-1 protein at the cell membrane. These differential effects suggested a mechanism of action of adenosine that cannot be solely explained by occupancy of type A(1) purinergic receptors. Activation by adenosine but not by CHA of AMPK was observed. AMPK participation in lactate production and ZO-1 stabilization was confirmed by utilizing specific inhibitors. Altogether, these results suggest that activation of AMPK by adenosine promotes lactate offer to germ cells and cooperates in the maintenance of junctional complex integrity, thus contributing to the preservation of an optimum microenvironment for a successful spermatogenesis.

Immunodetection of Cell Adhesion Molecules in Rat Sertoli Cell Cultures

PROBLEM: The presence of cell adhesion molecules (CAMs) in Sertoli cells has not been explored extensively. The expression of CAMs involved in cell‐matrix and cell‐to‐cell interactions in Sertoli cell cultures was examined.

Expression of cell adhesion molecules, chemokines and chemokine receptors involved in leukocyte traffic in rats undergoing autoimmune orchitis

The testis is considered an immunologically privileged site where germ cell antigens are protected from autoimmune attack. Yet in response to infections, inflammatory diseases, or trauma, there is an influx of leukocytes to testicular interstitium. Interactions between endothelial cells (EC) and circulating leukocytes are implicated in the initiation and evolution of inflammatory processes. Chemokines are a family of chemoattractant cytokines characterized by their ability to both recruit and activate cells. Thus, we investigated the expression of CCL3, its receptors, and adhesion molecules CD31 and CD106 in an in vivo model of experimental autoimmune orchitis (EAO). In EAO, the highest content of CCL3 in testicular fluid coincides with onset of the disease. However, CCL3 released in vitro by testicular macrophages is higher during the immunization period. The specific chemokine receptors, CCR1 and CCR5, were expressed by testicular monocytes/macrophages and an increased number of CCR5+ cells was associated with the degree of testicular lesion. EC also play an essential role by facilitating leukocyte recruitment via their ability to express cell surface adhesion molecules that mediate interactions with leukocytes in the bloodstream. Rats with EAO showed a significant increase in the percentage of CD31+ EC that upregulate the expression of CD106. The percentage of leukocytes isolated from peripheral blood and lymph nodes expressing CD49d (CD106 ligand) also increases during orchitis. These data suggest that cell adhesion molecules, in conjunction with chemokines, contribute to the formation of a chemotactic gradient within the testis, causing the leukocyte infiltration characteristic of EAO histopathology.

Isolation and Immunological Reactivity of Soluble Fractions from Rat Seminiferous Tubule Basement Membrane

A preparation rich in basement membranes isolated from rat testes (STBM) was exposed to pepsin, collagenase, trypsin, and pronase to obtain soluble fractions. The immunological reactivity of these fractions was studied by gel immunodiffusion or by passive hemagglutination tests against an anti-STBM serum. All fractions reacted with the antiserum, but the highest titer was detected when the antiserum was reacted with a fraction that contained only traces of hydroxyproline (fraction 1), whereas low titers were obtained with collagen or collagen fragments isolated from STBM. Antibodies in the anti-STBM serum were mainly directed to the glycoproteins of STBM not related to collagen. Fraction 1, obtained by subsequent collagenase and trypsin digestion of STBM and purification by Sephadex G-200, was a high molecular weight glycoprotein that was free of half-cystine and methionine, had only traces of hydroxyproline, and contained 7.2% neutral sugars, 0.26% sialic acid, and 8.7 residues of glucosamine per 1000 residues of amino acids.