A specific and rapid determination of human skin dihydrotestosterone cytosol receptor.

The 5 alpha-Dihydrotestosterone (DHT) receptor in cytosol prepared from foreskin fragments was identified and measured by polyacrylamide gel electrophoresis (PAGE). 14 normal subjects, 4 boys with hypospadias and two patients with male pseudohermaphroditism (MPH) were studied. The amount of DHT receptor was comparable in the normal subjects and those with hypospadias. No receptor was found in the 2 cases of MPH.

Adenosine 3'5'-cyclic monophosphate dependent protein kinase in human adrenocortical tumors.

Protein kinase activity has been studied in four human adrenocortical tumors and compared to the one of the normal human adrenal. In two cases where the lack of action of ACTH was related to an anomaly of ACTH receptor, the protein kinase activity was normal. In the other two cases the ACTH receptor was normal, but the protein kinase activity was different from that of the normal adrenal. In one of these cases where the steroidogenesis response of isolated tumor cells to ACTH and DcAMP was higher than in normal adrenal, basal and cAMP stimulated protein kinase activities were significantly higher than those of the normal adrenal, but the activation constants of both nucleotides were similar to those of the normal gland. In the other case, the basal and the cAMP stimulated protein kinase activities were significantly lower, as well as the activation constant of cAMP. However, the binding affinity of 3H-cAMP was normal. Normal adrenal cytosol contains three protein kinases, as resolved by DEAE-cellulose, two of which designated I and II, are cAMP-dependent. The DEAE-cellulose chromatography of the last tumor showed a loss of isoenzyme II. In addition, the protein kinase eluted at the same molarity as that of isoenzyme I of the normal adrenal was not activated by cAMP. Therefore, the lack of response to ACTH of some adrenocortical human tumors may be attributed either to an anomaly of the ACTH receptor or to some defect of the cAMP-dependent protein kinase.

Adenosine 3',5'-monophosphate-dependent protein kinase from normal human adrenal.

The protein kinase of normal human adrenal cytosol has been resolved by DEAE-cellulose chromatography into two major components, the protein kinases I and II, which are both adenosine 3',5'-monophosphate (cAMP) dependent. Both enzymes have similar substrate specificities, cAMP-dependency, and sensitivity to the stimulation by this nucleotide, but differ in their states of activation after preincubation with histone. The DEAE--cellulose charomatography of dissociated cytosol protein kinase reveals only one peak of kinase activity and two peaks of cAMP binding activity (A and B). Both binding proteins are able to inhibit the kinase activity of the catalytic subunit. Recombination experiments suggest that the regulatory subunit A originated from protein kinase I and subunit B from protein kinase II. The phosphorylation of histone by adrenal protein kinases is inhibited by a heat-stable protein inhibitor isolated from human fetal brain and human adult adrenal.

[Glucocorticoids receptors in rat testis: their role in Leydig cells specific function and DNA synthesis (author's transl)]. / RECEPTEURS DE GLUCOCORTICOIDES DANS LE TESTICULE DE RAT

Rat testicular cytosol binds specificity dexamethasone (KD = 5 X 10(=9)M). After incubation of testicular slices with 3H-dexamethasone, at 30 degrees C for 2 hours most of the intracellular radioactivity is found inthe nuclei. Administration of h CG at pharmacological doses for three days to male rat during puberty increases plasma testosterone levels and 3H-thymidine incorporation into testicular DNA. Simultaneous administration of dexamethasone partially blocks both effects of hCG. These results suggest that interaction of glucocorticoïds with specific receptors controls both the specific function and the cells multiplication of rat Leydig cells.

Gonadotropin stimulation of cyclic AMP levels in Chinese hamster ovary cells in culture.

When Chinese Hamster Ovary (CHO) cells, incubated in serum-free medium, are exposed to gonadotropins a transient increase in the intracellular concentration of cyclic AMP is observed. Maximum accumulation of cyclic AMP is noted 30 minutes after addition of either human chorionic gonadotropin (hCG) or follicle stimulating hormone (FSH). Within one to two hours after hormone addition, the intracellular concentrations of cyclic AMP have returned to basal levels. The enhancement of intracellular cyclic AMP levels by hCG is hormone concentration dependent, with maximal stimulation observed at 10 micrograms/ml hCG. The exogenous addition of gonadotropins also slows the growth rate of CHO cells. This effect on growth seems to be mediated through cyclic AMP since the growth rate of a mutant of CHO cells defective in the catalytic subunit of cyclic AMP dependent protein kinase is only slightly decreased.

Role of cyclic AMP and protein kinase on the steroidogenic action of ACTH, prostaglandin E1 and dibutyryl cyclic AMP in normal adrenal cells and adrenal tumor cells from humans.

The role of the cyclic AMP-protein kinase system in mediating the steroidogenic effect of ACTH, prostaglandin E1 and dibutyryl cyclic AMP, induced similar stimulations of protein kinase activity, cyclic AMP was studied using human adrenal cells isolated from normal and adrenocortical secreting tumors. At high concentrations of ACTH, complete activation of protein kinase of normal adrenal cells was observed within 3 min, at the time when cyclic AMP production was slightly increased and there was still no stimulation of steroidogenesis. At supramaximal concentrations, ACTH, PGE1 and dibutyryl cyclic AMP and cortisol productions in adrenal cells isolated from normal and from one adrenocortical tumor. In one tumor in which the adenylate cyclase activity was insensitive to ACTH, the hormone was unable to stimulate protein kinase or steroidogenesis, but the cells responded to both PGE1 and dibutyryl cyclic AMP. In another tumor in which the adenylate cyclase was insensitive to PGE1, this compound also did not increase protein kinase activity or steroidogenesis, but both parameters were stimulated by ACTH and dibutyryl cyclic AMP. After incubation of normal adrenal cells with increasing concentrations of ACTH (0.01-100 nM) marked differences were found between cyclic AMP formation and cortisol production. However at the lowest concentrations of ACTH exerting an effect on steroid production a close linked correlation was found between protein kinase activation and cortisol production, but half-maximal and maximal cortisol production occurs at lower concentration of ACTH than was necessary to induce the same stimulation of protein kinase. Similar findings were found after incubating the adrenal cells with dibutyryl cyclic AMP (0.01-10 mM). The results implicate an important role of the cyclic AMP-protein kinase system during activation of adrenal cell steroidogenesis by low concentrations of steroidogenic compounds.

Chinese hamster ovary cell population density affects intracellular concentrations of calcium-dependent regulator and ability of regulator to inhibit adenylate cyclase activity.

The adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] of crude Chinese hamster ovary cell membranes was inhibited 30-40% by low concentrations (6-600 ng/ml) of calcium-dependent regulator (CDR). This inhibitory effect was lost at concentrations of CDR above 600 ng/ml. The adenylate cyclase activity of membranes prepared from low population density Chinese hamster ovary cells was not appreciably altered by CDR. However, with increasing cell population density there was a significant increase in the ability of CDR to inhibit cyclic AMP formation. Further, the intracellular levels of CDR determined in the 12,000 x g supernatant and particulate fractions varied inversely with increasing cell population density. As cell number increased from 2 x 10(6) to 10 x 10(6) cells per dish the CDR concentration present in the supernatant fraction increased from 0.4 to 0.8 mug of CDR per mg of protein, while the amount of endogenous CDR associated with the particulate fraction decreased from 0.6 to 0.4 mug of CDR per mg of protein. This suggests that possible changes in the distribution of CDR between the supernatant and membrane fractions might serve as a regulatory mechanism for activities under CDR control.

[Ovarian insufficiency and galactosemia ]. / Insuffisance ovarienne et galactosémie congénitale.

A primitive gonadal failure was observed in 2, 20 and 18 years old galactosemic female patients. The conditions was clinically suspected because of primary amenorrhea in one case and delayed puberty, followed by spaniomenorrhea in the other case, and confirmed by very low plasma estradiol and very high plasma gonadotropin levels. Galactose-free diet was started at 9 months of age in the first case and soon after birth in the second. Several similar cases have been recently reported. Primitive gonadal failure associated with galactosemia suggests a possible toxic' destruction of the ovaries during the intrauterine life by a galactose metabolite.

Gonadotropin stimulation of pregnenolone metabolism in Chinese hamster ovary cells in culture.

To determine if Chinese Hamster Ovary (CHO) cells in culture are able to metabolize steroids, CHO cells were incubated in defined medium with [14C]pregnenolone. As shown, [14C]pregnenolone is metabolized to progesterone and other delta 53 beta steroids; this steroidogenic response is appreciably enhanced upon exposure of the cells to 50 nM gonadotropins (human chorionic gonadotropin and follicle-stimulation hormone). The primary metabolites that accumulate in the medium upon treatment with gonadotropins are 16 alpha-hydroxy-pregnenolone and 16 alpha-17 beta-dihydroxydehydroepiandrosterone. Exposure of the CHO cells to gonadotropins induces significant increases in the activities of 16 alpha-hydroxylase, 17 alpha-hydroxylase, and 17-20 lyase. Similar results are obtained when the CHO cells are treated with 0.1 mM 8-bromocyclic AMP, indicating that the gonadotropin enhancement of steroid metabolism is a cyclic AMP-mediated process. CHO cells apparently lack the cholesterol desmolase complex since 14C-cholesterol is not utilized by these cells to produce other steroid metabolites. These results indicate that CHO cells offer an in vitro system for the study of certain aspects of gonadotropin stimulation of steroidogenesis.

[Somatomedin activity and growth hormone secretion in children with idiopathic growth retardation (author's transl)]. / Activité somatomédine et sécrétion de l'hormone de croissance au cours des retards de croissance essentiels. Relation avec la croissance.

Somatomedin activity, as estimated by the porcine bioassay and GH response to stimulation were compared in prepubertal children with severe growth retardation and lack of evidence of pituitary dysfunction. Somatomedin activity was strongly correlated with growth rate. A group of patients had a low somatomedin activity in contrast with normal GH responsiveness. This appears to be a rather frequent situation and deserves further evaluation of the somatomedin system and a more critical approach of GH secretion.

Endocrine studies in male pseudohermaphroditism in childhood and adolescence.

The clinical and endocrine features of fifty cases of male pseudohermaphroditism and XY karyotype aged from 6 months to 20 years have been studied. Thirty-two subjects were pre-pubertal and eighteen, of whom ten developed gynaecomastia, were pubertal. A definite aetiology was established in 12%. 4% had deficient testosterone biosynthesis and 8% mixed gonadal dysgenesis. In the remaining 88% an aetiology of androgen unresponsiveness at the target areas is suggested. Hormonal investigations in these subjects showed that before puberty basal plasma testosterone and oestradiol were slightly but significantly elevated, whereas testosterone response to stimulation with human chorionic gonadotrophin was significantly diminished. In the pubertal subjects basal plasma testosterone, oestradiol, serum binding capacity of testosterone-oestradiol binding globulin and basal plasma LH were significantly elevated. These hormonal features in pubertal or post-pubertal male pseudohermaphrodites appear to be characteristic of androgen unresponsiveness. The presence in addition of elevated basal plasma testosterone and oestradiol in the pre-pubertal subjects suggest that some of these findings may be presented from early childhood.