Suzanne Samperez

Etude du récepteur cytoplasmique de la (1,2-3H) testosterone dans l'hypophyse antérieure et l'hypothalamus du rat

Abstract The anterior pituitary and the hypothalamus of the normal and castrated rat contain soluble receptors for testosterone, present in the 105.000 g supernatants. The capacity of macro-molecular fixation of testosterone is greater in anterior hypophysis than in hypothalamus. Raising the incubation temperature of slices and of homogenates results in a decrease of the binding capacity of testosterone. These receptors are macromolecules whose molecular weight most likely exceeds 100.000 and whose nature is, at least in part, protein. The bound radioactive material consists partly of testosterone and partly of an unidentified steroid (Compound X). Dihydrotestosterone and 4-androstene-3,17-dione are minor constituents.

Testosterone 5α-reductase of microsomes from rat anterior hypophysis: properties, increase by castration, and hormonal control

In this work, some properties of the microsomal 5α-reductase of the hypophysis from normal and castrated rat were studied. The apparent Km for this enzyme is 1.8 M in normal rat and 2.8 M in castrated rat. Castration increases several-fold the conversion of testosterone to dihydrotestosterone and 3α-androstanediol. A low increase in 5α-reductase activity has been observed 2 days after castration. A significant increase was noted after 4 days and the enzyme activity reached a plateau at a 20-day time. No variation of this enzyme activity was observed in the rat hypothalamus and brain cortex. Testosterone, dihydrotestosterone and 3α-androstanediol injected for 7 days immediately after castration prevented the rise in DHT, testosterone being more effective than its metabolites. Estrogens were without effects. Cycloheximide suppressed the 5α-reduction rise in castrated rats, while Actinomycin D seemed to have no effect. Results obtained are in favour of a progressive increase of enzyme level following castration and of a post transcriptional regulation of its synthesis.

Rapid and intensive conversion of 5α-androstane-3α,17β-diol into 5α-dihyorotestosterone in the male rat anterior pituitary: in vivo and in vitro studies

The in vivo and in vitro metabolism of (3H)-5α-androstane-α, 17β-diol by the male rat anterior pituitary was studied. A rapid and intensive conversion of 5α-androstane-3α,17β-diol into 5α-dihydrotestosterone was demonstrated, since following a 30 min. incubation time, 73 % of the recovered radioactivity were constituted by 5α-dihydrotestosterone. Studies on the subcellular distribution of steroids showed that 5α-dihydrotestosterone was the main steroid recovered except from the 105,000 × g pellet. From in vivo and in vitro experiments it was concluded that the transformation of 5α-dihydrotestosterone into 5α-androstane-3α,17β-diol was a reversible process, and that this last steroid could exert its biological action mainly via 5α-dihydrotestosterone.

Dihydrotestosterone-protein binding in the cytosol of rat anterior hypophysis in vitro: Evidence for a specific receptor

Abstract A study was designed to measure the specificity of the dihydrotestosterone binding in the cytosol of the anterior pituitary of immature male rats (40-day-old rats). The binding at equilibrium was studied after incubation of cytosol at 0°C with a constant dose of [3H]-dihydrotestosterone (DHT) for various times. The binding was measured after gel filtration on Sephadex G 25 medium. Equilibrium was reached at 6 h and remained constant for 24 h. No significant dihydrotestosterone metabolism was detected. The binding specificity was determined using different concentrations of [3H]-dihydrotestosterone. Cytosols were incubated for 6 h at 0°C with concentrations of [3H]-dihydrotestosterone ranging from 10−10 M to 10−7M. The protein-dihydrotestosterone complex was isolated by gel filtration on Sephadex G 25 medium. A Scatchard plot of the binding data produced a curved line showing that more than one binding component was involved. This curve was resolved into two linear components by the Rosenthal method. The dissociation constant (Kd) for the specific binding was 7.8 × 10−10 M and the estimated amount of specific receptor was 1.4 × 10−11 M, which represents 4.7 × 10−15 mol per mg of proteins. Unlabelled dihydrotestosterone and testosterone were potent and specific inhibitors of the [3H]-DHT binding. Other androgens, estrogens and progestagens were less potent competitors. 4-Androstenedione, corticosterone and estradiol-17α were without effect on the [3H]-DHT-binding.

Pituitary metabolism of 5α-androstane-3β-17β-diol: Intense and rapid conversion into 5α-androstane-3β,6α,17β-triol and 5α-androstane-3β,7α, 17β-triol

: In the male rat pituitary, 5alpha-androstane-3beta, 17beta-diol (3beta-diol) is extensively metabolized into polar steroids. They were identified as 5alpha-androstane-3beta, 6alpha-17beta-triol (6alpha-triol) and 5alpha-androstane-3beta, 7alpha, 17beta-triol (7alpha-triol). 6-alpha-Triol represents 53% and 7alpha-Triol 28% of the total 3beta-diol metabolites. The remaining percentage is related to 6beta and 7beta isomers. The biological role of triols is still unknown.

Androgen and estrogen receptors in the cytosol from male rat anterior hypophysis: further characteristics and differentiation between androgen and estrogen receptors.

Abstract Further characteristics of androgen and estrogen receptors from male rat pituitary cytosol were studied. Following ultra-centrifugation in a linear sucrose gradient (5–20%) two radioactive peaks for testosterone were observed, one at the top of the gradient, and one in the 7–8 S region. For dihydrotestosterone there were also two peaks one in the 3–4 S region, the other in the 7–8 S region. Only the 7–8 S peaks were specific for androgens. Under the same conditions, estradiol-17β exhibited only one peak in the 8–10 S region. When rats were previously castrated, an increase of the 7–8 S peak for testosterone and for dihydrotestosterone was observed. Thus, orchidectomy did not abolish androgen receptors in pituitary cytosol. It was likely that androgen and estrogen receptors were proteins. Indeed, the hormone-receptor binding was abolished to a great extent by proteolytic enzymes action. Taking into account some characteristics of these receptors: dissociation constants, specific binding capacity, sedimentation constants, sensitivity to heat or to enzyme action, it seemed easy to differentiate androgen receptors from estrogen receptors. Thus, it was concluded that pituitary cytosol from male rats contained two kinds of receptors, one for androgens, and one for estrogens. Contrarily, it is not certain at the moment that there are two androgen receptors, one for testosterone and one for dihydrotestosterone, or that there is only one receptor for both hormones.

Stimulation by estradiol-17β of thymidine kinase activity in the rat uterus

Abstract The action of estradiol-17β(E 2 ) on thymidine kinase (TK) activity was studied in uteri from immature female rats. It was demonstrated that a single injection of E 2 highly stimulated the enzyme activity which reached its maximum level 24 h after hormone administration. Physiological amounts of E 2 were efficient and changes in TK activity were observed exclusively in uterus and liver. A single injection of Tamoxifen produced the same effect as E 2 but repeated administration resulted in the complete inhibition of enzyme activity. Using antibiotics it was demonstrated that E 2 induced the synthesis of new enzyme molecules rather than an increase in enzyme activity. This statement was corroborated by the fact that after hormone administration the increase in TK activity was preceded by an increase in RNA-polymerase activity and followed by that in DNA-polymerase α activity. Moreover, the separation of TK isoenzymes on DEAE-Sephadex and the use of d -CTP as inhibitor of the adult isozyme suggested that E 2 induced the “fetal” form of the enzyme. In addition, it was demonstrated that TK activity in uteri from ovariectomized adult female rats was enhanced by E 2 administration, and that the increase was due to the stimulation of the fetal isoenzyme. It was suggested that TK could be used as a marker of the action of estrogens and antiestrogens in target organs.

Properties of 3α-hydroxysteroid dehydrogenase of the male rat pituitary gland

The 3α-hydroxysteroid dehydrogenase activity was mainly concentrated in the cytosol fraction of the male rat anterior hypophysis. The product of 5α-dihydrotestosterone reduction at 3 position was identified as 5α-androstane-3α, 17β-diol. No formation of 3β,17β-isomer was detected in these experiments. Some properties of the 3α-hydroxysteroid dehydrogenase in the soluble fraction from the male rat pituitary gland were examined. Optimal pH of the reaction was around 6.5–7.0. Its apparent Km was 2.0 × 10−6 M. The activity of 3α-hydroxysteroid dehydrogenase required NADPH as cofactor. Whereas the 5α-reductase activity in the rat anterior hypophysis was increased by castration, the rate of 3α-reduction of 5α-dihydrotestosterone in this gland was not influenced by orchidectomy.

A specific testosterone receptor in the cytosol of rat anterior hypophysis

To search for and to measure the specificity of the cytosol testosterone protein association in the rat anterior hypophysis 40-43 day old male Wistar rats were used. Anterior hypophyses were removed and homogeneized in a phosphate buffer and centrifuge. The 105000 g supernatant (cytosol) was used for binding analysis. Cytosol fractions were incubated with (tritiated)-testosterone at 0 degrees C from 5 minues to 24 hours. At times samples were removed and bound and unbound testosterone were measured after gel filtration on Sephadex G25 columns. After gel filtration was fraction containing the radioactivity bound to the molecules was extracted. Androgens were chromatographed. The radioactivity of the chromatograms was measured. The amount of testosterone bound was increased up to 4-6 hours and then remained constant up to 24 hours. During the incubation at 0 degrees C testosterone metabolism did not occur. Results show 2 binding components. Thus the cytosol of the immature rat anterior hypophysis contains a specific receptor for testosterone and some specific associations. Results are similar to those reported by others showing a specific estradiol receptor in the female rat anterior hypophysis. Thus the presence of a soluble and specific receptor for testosterone in the male rat anterior hypophysis seems to be well established. Discussion by others follows this presentation.

Studies on the 5α-androstane-3β, 17β-diol-6α-hydroxylase and on the 5α-androstane-3β, 17β-diol-7α-hydroxylase in anterior hypophysis of male rats.

In anterior pituitaries from male rats, it appeared that 5α-androstane-3β, 17β-diol was quickly metabolized into 5α-androstane-3β,6α-17β-triol and 5α-androstane-3β,7α, 17β-triol by action of 6α- and 7α-hydroxylases. Hydroxysteroid hydroxylases were located in endoplasmic reticulum and were dependent on NADPH+. Their optimum pH was 8.0, optima temperature, 37°C, and their apparent Km was 2.7 μM. Hydroxylative reactions were not reversible and not modified by gonadectomy. Hydroxylation seemed an efficient control of the pituitary level of 5α-andros-tane-3β, 17β-diol.