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La question de savoir si les cellules de la lignee germinale possedent ou non des recepteurs aux androgenes reste debattue. Dans ce travail nous avons determine la distribution des recepteurs aux androgenes dans le testicule de rat et de souris a l'aide d'un anticorps polyclonal de lapin purifie par affinite, dirige contre un peptide de 21 acides amines de la region terminale du recepteur des androgenes (RA), marque par le systeme biotine-streptavidine immunoperoxidase. La specificite de l'anticorps est attestee par les points suivants: 1) Presence d'une bande specifique approximativement a 110 kD en Western immunoblot; 2) Elimination specifique du marquage par pre-adsorption de l'anticorps avec le peptide de 21 acides amines; 3) Mise en evidence d'une relation entre l'intensite du marquage dans toutes les cellules RA positives en fonction de la dilution des antiserums; 4) Mise en evidence dans tous les tissus connus pour exprimer de facon importante des RA (par exemple l'epididyme, la prostate, les vesicules seminales) d'un marquage extremement intense au niveau du noyau des cellules epitheliales; 5) Obtention d'un reaction RA positive avec les anticorps dans les lignees de cellules prostatiques connues pour exprimer les recepteurs aux androgenes; 6) Positivation de cellules COS-7 apres leur transfection par un vecteur exprimant le recepteur aux androgenes de rat. De plus la distribution intracellulaire des RA dans les cellules COS-7 est fonction de la presence d'androgenes dans le milieu. L'immunolocalisation des recepteurs aux androgenes montre les points suivants: a l'interieur du compartiment interstitiel chez le rat adulte des RA sont detectes dans quelques cellules de Leydig et dans toutes les cellules musculaires lisses formant la paroi des vaisseaux sanguins, alors que les cellules endotheliales restent negatives. Dans les tubes seminiferes les RA sont observes dans tous les noyaux des cellules myoides peritubulaires, mais pas dans les couches plus peripheriques correspondant aux cellules endotheliales des vaisseaux lymphatiques. Dans les cellules de Sertoli le marquage nucleaire des RA est specifique du stade de l'epithelium seminifere: la positivite du marquage commence a devenir evidente vers le stade IV tardif du cycle, atteint un pic tres fort aux stades VII et VIII puis disparait completement. Un immunomarquage specifique a egalement ete mis en evidence dans les noyaux des spermatides allongees stade XI, stade auquel l'elongation nucleaire est deja apparente mais sans que la condensation de la chromatine soit deja commencee. Des le debut de la condensation de la chromatine des spermatides allongees, l'immunomarquage nucleaire disparait en meme temps que le cytoplasme devient positif. Globalement les memes observations ont ete faites sur le testicule de Souris adulte, excepte le fait que, dans cette espece, toutes les cellules de Leydig sont tres fortement RA positives. Afin de confirmer l'immunomarquage des RA nous avons realise des etudes d'hybridation in situ dans le testicule de Rat adulte a l'aide d'une sonde antisens ARNc de 236 paires de bases (I'ADNc de RA nous a ete donne par le Docteur Chang, Universite du Wisconsin, Madison, WI). Un important signal d'hybridation a ete observe a la base de l'epithelium seminifere, dans la region occupee par les cellules de Sertoli et les spermatogonies. Ceci nous a conduit a reexaminer les resultats de l'immunomarquage afin de determiner si les spermatogonies etaient egalement positives pour les anticorps anti RA. Nos resultats preliminaires sont en faveur de la presence de RA dans certaines populations spermatogoniales. Au total, ces resultats concordent avec des observations anterieures suggerant que les RA sont presents dans les cellules somatiques du testicule; de ce fait, ce sont ces types cellulaires qui vraisemblablement repondent aux androgenes circulants pour controler la spermatogenese. Cependant nos resultats montrent egalement un immunomarquage de certains elements de la lignee germinale ce qui reactualise la controverse concernant le fait de savoir si la lignee germinale peut repondre ou non directement aux androgenes.

Despite the important role of calcium in the growth and differentiation of a variety of cell types, its exact location and function in the somatic and germ cells of the testis remain to be determined. In the present study, we examined the subcellular distribution of calcium in the immature and adult rat testis. Calcium was localized at the electron microscopic level by ion-capture cytochemistry using combined oxalate and pyroantimonate procedures. Calcium-containing precipitates localized primarily within the nuclei, mitochondria, and cytosol of somatic and germ cells. Differences in the size and quantity of the calcium precipitates were observed among the various cellular compartments. In the somatic cells (Sertoli, Leydig, and myoid), the nuclei exhibited large round-shaped calcium-containing precipitates, whereas the mitochondria in these cell types contained numerous smaller precipitates. The cytoplasmic vesicles possessed single precipitates. These vesicles could be calciosomes, which have been described in other non-muscle cell types. Among germ cells, round spermatids exhibited a large number of vesicular, calsiosome-like structures in the cytoplasm containing single precipitates. The elongating spermatids from adult testis showed calcium localization within the nuclear matrix unassociated with the nuclear envelope, or in a peripheral alignment of precipitates along the nuclear envelope. Calciosome-like structures were also seen in round spermatids. Spermatogonia and spermatocytes exhibited calcium in nuclei, mitochondria, and cytoplasmic vesicles. These results demonstrate a differential distribution of calcium within the various cell types of the testis. The presence of calcium in the nucleus may suggest a role in cell growth and differentiation; calsiosome-like structures may represent the active exchangeable pool of calcium, and the differential type of distribution of calcium in elongating spermatids suggests a role for calcium in spermatid differentiation.

Germ cells contain androgen receptor (AR). Germ cells do not contain androgen receptor. These diametrically opposed views are at the center of a long-running dispute regarding the cell site of androgen action within the testis (reviewed in 1, 2). In this chapter we examine AR distribution in the rat and mouse testis using an affinity-purified rabbit polyclonal antibody made to a 21 amino acid peptide of the amino terminus of the rat AR (3–5). Results reveal the presence of AR in testicular somatic cells, as well as in elongated spermatids.

Despite the important role of calcium in the growth and differentiation of a variety of cell types, its exact location and function in the somatic and germ cells of the testis remain to be determined. In the present study, we examined the subcellular distribution of calcium in the immature and adult rat testis. Calcium was localized at the electron microscopic level by ion-capture cytochemistry using combined oxalate and pyroantimonate procedures. Calcium-containing precipitates localized primarily within the nuclei, mitochondria, and cytosol of somatic and germ cells. Differences in the size and quantity of the calcium precipitates were observed among the various cellular compartments. In the somatic cells (Sertoli, Leydig, and myoid), the nuclei exhibited large round-shaped calcium-containing precipitates, whereas the mitochondria in these cell types contained numerous smaller precipitates. The cytoplasmic vesicles possessed single precipitates. These vesicles could be calciosomes, which have been described in other non-muscle cell types. Among germ cells, round spermatids exhibited a large number of vesicular, calsiosome-like structures in the cytoplasm containing single precipitates. The elongating spermatids from adult testis showed calcium localization within the nuclear matrix unassociated with the nuclear envelope, or in a peripheral alignment of precipitates along the nuclear envelope. Calciosome-like structures were also seen in round spermatids. Spermatogonia and spermatocytes exhibited calcium in nuclei, mitochondria, and cytoplasmic vesicles. These results demonstrate a differential distribution of calcium within the various cell types of the testis. The presence of calcium in the nucleus may suggest a role in cell growth and differentiation; calsiosome-like structures may represent the active exchangeable pool of calcium, and the differential type of distribution of calcium in elongating spermatids suggests a role for calcium in spermatid differentiation.