Limited Tryptic Proteolysis of the Benzodiazepine Binding Proteins in Different Species Reveals Structural Homologies

Peptide mapping can be used to elucidate further the structural similarities of the benzodiazepine binding proteins in different vertebrate species. Crude synaptic membrane preparations were photoaffinity-labeled with [3H]flunitrazepam and subsequently degraded with various concentrations of trypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fluorography allowed a comparison of the molecular weights of photolabeled peptides in different species. Tryptic degradation led to a common peptide of 40K in all species investigated, a finding indicating that the benzodiazepine binding proteins are structurally homologous in higher bony fishes and tetrapods.     

Differential distribution of3H dihydrotestosterone and3H estradiol nuclear binding sites in mouse male accessory sex organs

Summary The distribution of specific nuclear binding sites for androgens and estrogens in the male accessory sex organs of the mouse was assessed by autoradiography with³H dihydrotestosterone (³H DHT) and³H estradiol (³H E₂). With³H DHT nuclear labeling differed among the epithelia of the organs. It was high in seminal vesicle and ampullary gland, moderate in ventral prostate, urethral gland, prostatic excretory ducts and the ampulla ductus deferentis, low in dorsal prostate and low or absent in coagulation gland. With³H E₂, in contrast, epithelial nuclear labeling was high only in coagulation gland, moderate or low in seminal vesicle, low or absent in ventral and dorsal prostate and absent in ampullary gland and ampulla ductus deferentis. In the lamina propria of all organs nuclear labeling with³H DHT was generally moderate and existed only in some cells, with the highest number in the ampulla ductus deferentis. With³H E₂, nuclear labeling in the lamina propria showed a high intensity in all organs, except in ventral and dorsal prostate which remained unlabeled. Many labeled cells were found in the deferent duct and its ampulla, while in the other organs only a few cells showed nuclear labeling with³H E₂. In the smooth muscle sheath of all organs, some muscle cells were moderately labeled with³H DHT, but not with³H E₂. The results indicate the presence of nuclear receptors in male accessory sex organs for both dihydrotestosterone and estradiol. The differential patterns of³H DHT and³H E₂ nuclear uptake suggest differential sensitivities of the individual organs and their tissue compartments for androgens and estrogens. Supported by PHS grant NSO9914 to W.E.S. and Deutsche Forschungsgemeinschaft Dr94/4 to U.D. The work of Dr. Schleicher and his stay in Chapel Hill were additionally sponsored by Studienstiftung des Deutschen Volkes and Boehringer-Ingelheim Fonds     

Embryonic cholinesterase activity during morphogenesis of the mouse genital tract

Summary In the genital tract of male and female mouse embryos cholinesterase activity is described that is independent from innervation. The enzyme activity is localized in the mesenchyme at the junction of Wolffian and Müllerian ducts with the urogenital sinus. During male development prostate buds and vesicular glands grow out into the cholinesterase-active mesenchyme. During female development the active mesenchyme participates in the downgrowth of the vaginal anlage. Ultrastructurally the cholinesterase activity is localized in the perinuclear cisterna and in smooth endoplasmic reticulum of the mesenchymal cells. The enzyme activity disappears with definitive differentiation of the tissue. The embryonic cholinesterase is a component of a primitive muscarinic system. Its relation to the morphogenetic action of testosterone and its possible general functions are discussed.     

Nuclei in testicular feminization (Tfm) are not activated by intact testosterone receptor complexes: A morphometric study in striated urethral muscle of mosaic mice

Summary Sex reversed mice heterozygous for the X-linked Tfm mutation are mosaics with respect to the Tfm locus. In the androgen-dependent striated urethral muscle, nuclei coding for the intact testosterone receptor protein and nuclei coding for the defective Tfm receptor protein are incorporated in the same multinucleate muscle fibres. The intact testosterone receptor complex can thus be expected to enter the Tfm nuclei. Our measurements show that the fibre diameters of the mosaic muscle form a homogeneous population, intermediate in size between induced male and non-inducible Tfm phenotypes. By contrast, the nuclear size shows a bimodal distribution, the subpopulations corresponding to Tfm and wild type nuclei. The results indicate that the Tfm nuclei are not activated by the intact testosterone receptor complex.     

Intact testosterone receptor complex does not induce RNA synthesis of tfm-nuclei in multinucleated urethral muscle fibres of mosaic mice

Summary The X-linked testicular feminization mutation (Tfm) of the mouse leads to androgen insensitivity of target cells. Through the autosomal sex reversed (Sxr) factor we have converted female heterozygotes into males. Due to X-inactivation, mosaic animals arise which are composed of androgen sensitive wild-type and androgen insensitive Tfm cells. In the androgen dependent striated urethral muscle, Tfm and wild-type cells fuse and form multinucleated muscle fibres. In the muscle fibres the Tfm nuclei are exposed to the intact cytoplasmic testosterone receptor complex coded for by the wildtype nuclei. We ask the question whether under these conditions RNA synthesis can be stimulated in the Tfm nuclei. Castrated mosaic animals were injected with testosterone, and incorporation of ³H-uridine was studied by autoradiography. We found two classes of muscle cell nuclei, those with low grain counts corresponding to the Tfm controls and those with high grain counts corresponding to the stimulated male controls. The results indicate that the Tfm nuclei are not stimulated by the intact testosterone receptor complex.This study is dedicated to Prof. Dr. W. Graumann on occasion of his 65th birthday