Biomedical subjects

P Robel

Publications and source records attributed to P Robel.

At least 73 records · Page 4Linked to original sources

PubMed1989

Neurosteroids: biosynthesis of pregnenolone and progesterone in primary cultures of rat glial cells.

Cells dissociated from newborn rat forebrains were established in long term primary cultures. The cultures were made up almost exclusively of oligodendrocytes and astrocytes, as confirmed by indirect immunofluorescence staining with monoclonal antibodies to galactocerebroside and glial fibrillary acidic protein, respectively. After 3 weeks of culture, the oligodendrocytes were also highly immunoreactive to monospecific polyclonal antibodies against cytochrome P450scc, an enzyme involved in the conversion of cholesterol to pregnenolone (P). Biosynthesis of [3H]cholesterol, [3H]P, and [3H]Pregn-5-ene-3 beta, 20 alpha-diol was demonstrated in these primary cultures by incubating cells with [3H]mevalonolactone in the presence of mevinolin and trilostane. The activity of the 2',3'-cyclic nucleotide 3'-phosphodiesterase enzyme, a documented indicator of oligodendrocyte differentiation, increased rapidly after day 10 of culture, together with the onset of steroid biosynthetic activity. Both reached a maximum at 3 weeks of culture and remained stable up to 6.5 weeks. In the absence of trilostane, [3H]P was converted by glial cell cultures to [3H]progesterone, [3H]5 alpha-pregnane-3,20-dione, and [3H]3 alpha-hydroxy-5 alpha-pregnan-20-one. The demonstration of P, pregn-5-ene-3 beta,20 alpha-diol, and progesterone synthesis by normal rat glial cells, once oligodendrocytes have undergone their differentiation process, brings additional support to the concept of "neurosteroids."

Animals↗

PubMed1989

Cell-specific variations and hormonal regulation of immunoreactive cytochrome P-450scc in the rat ovary.

Specific rabbit antibodies to the bovine cholesterol side-chain cleavage cytochrome P-450 (P-450scc) were used to cross-react with the enzyme in the rat ovary. The luteal cells of cyclic, pregnant, and pseudopregnant rats were immunostained. P-450scc was also expressed in the interstitial cells of prepubertal and cyclic adult rats, and in the thecal cells lining the preovulatory follicles. In cyclic females, RU 486 and oestradiol increased the intensity of P-450scc immunostaining. The granulosa cells of ovarian follicles whatever their stage of development, including preovulatory follicles, were not labelled, except after ovulation. The intensity of immunostaining of thecal and interstitial cells decreased during early pregnancy or pseudopregnancy, and disappeared after Day 9, whereas these cells were intensely labelled 24 h after parturition. The immunostaining of thecal and interstitial cells was again detected in 18-day pregnant rats, treated with the antiprogesterone RU 486. It is therefore concluded that both oestradiol and progesterone are involved in P-450scc regulation.

Animals↗

PubMed1989

[Immunocytochemical localization of cytochrome P-450scc in cultured rat oligodendrocytes].

Primary cultures of glial cells from newborn rat forebrain were tested after 3 to 4 weeks. Oligodendrocytes and astrocytes were characterized by immunofluorescence with monoclonal antibodies to galactocerebroside and glial fibrillary acidic protein, respectively. The cytoplasm of oligodendrocytes was specifically and intensely immunostained with monospecific polyclonal antibodies to the cytochrome P-450scc involved in the synthesis of pregnenolone from cholesterol. This observation brings additional support to the concept of "neurosteroids".

Animals↗

PubMed1988

Estrogen receptors in cultured rabbit articular chondrocytes: influence of age.

Primary cultures of pubertal and prepubertal rabbit articular cartilage cells were performed. Total homogenates or cell extracts were used to determine the specific binding of 17 beta-estradiol. A comparative study was undertaken with tissue minces homogenized without enzymatic treatment. Scatchard analysis of cell or tissue extracts revealed the presence of a high-affinity receptor with Kd values of 0.55 +/- 0.16 nM and 0.12 +/- 0.03 nM in prepubertal and pubertal rabbit chondrocytes respectively. A significant difference in the affinity of estrogen receptor for its ligand as a function of age was observed. In contrast there was no significant difference in the number of binding sites expressed as fmol per mg DNA between the two age groups. The ligand binding specificity was as expected for an estrogen receptor and the sedimentation coefficient was 3.2 S when analyzed by ultracentrifugation on sucrose density gradient in presence of 0.4 M KCl and 8.1 S in low salt conditions. The binding sites, labeled with [125I]estradiol, were specifically immunoprecipitated by a monoclonal antibody to the estrogen receptor (JS34/32).

Aging↗

PubMed1988

[Blood levels of dehydroepiandrosterone sulfate (DHEA-S) and the risk of breast cancer].

In North American women at low or high risk of developing breast cancer, as assessed by an epidemiologic questionnaire, the plasma concentration of dehydroepiandrosterone sulfate shows a statistically significant circannual variation. In adolescents, in all seasons, circulating dehydroepiandrosterone sulfate is a classifier of the risk of developing breast cancer, a relatively low concentration of this hormone being associated with an increased risk.

Adolescent↗

PubMed1988

[The role of dehydroepiandrosterone and pregnenolone in the expression of stress behavior towards lactating females in mice].

Triads of castrated male mice were chronically administered with either oil vehicle or 280 nmol of dehydroepiandrosterone (D) or pregnenolone (P). They were tested for their attack on a lactating intruder female introduced in their home-cage 2, 24 or 47 hr after their last injection. D significantly reduced male aggressive behavior for at least 24 hr. Other groups of castrated males were daily treated with vehicle or 280 nmol of D, dehydroepiandrosterone sulfate (DS) or androstenediol (ADIOL), a D metabolite with clear-cut oestrogenic properties. D, but neither DS nor ADIOL, significantly reduced their aggressive responses to lactating intruders. Finally, neural levels of D, DS and testosterone (T) were measured in intact and castrated males injected with either vehicle or D. Measurable amounts of D and DS were detected, with DS being the predominant chemical form. D and DS concentrations were unchanged by castration but neural D was increased more than twenty fold in castrates treated with D, whereas DS was unchanged. The concentration of T in the brain of the intact (sham-operated) mouse approached 3 ng/g but fell close to the detection limit after gonadectomy. D treatment caused a slight but significant increase in brain T concentration in castrated mice, although T remained far below the level measured in intact males.

Aggression↗

PubMed1987

Neurosteroids: cytochrome P-450scc in rat brain.

The steroid hormones corticosterone and testosterone are supplied to the central nervous system by endocrine glands, the adrenals and gonads. In contrast, the 3 beta-hydroxy-delta 5-derivatives of cholesterol, pregnenolone and dehydroepiandrosterone, accumulate in the rat brain through mechanisms independent of peripheral sources. Immunohistochemical studies have been performed with specific antibodies to bovine adrenal cytochrome P-450scc, which is involved in cholesterol side-chain cleavage and pregnenolone formation. The enzyme was localized in the white matter throughout the brain. Scarce clusters of cell bodies were also stained in the entorhinal and cingulate cortex and in the olfactory bulb. These observations strongly support the existence of "neurosteroids," which have been posited on the basis of biochemical, physiological, and behavioral studies.

Adrenal Glands↗

PubMed1987

Neuro-steroids: 3 beta-hydroxy-delta 5-derivatives in rat and monkey brain.

The rat brain accumulates pregnenolone (P) as the unconjugated steroid, the sulfate ester (S) and fatty acid esters (L). P + PS do not disappear from rat brain after combined adrenalectomy (adx) and castration (orx). PL does not serve a source of P after adx + orx. P is metabolized by several rat brain regions to progesterone and to PL. Brain microsomes contain the acyl-transferase which converts P to PL using endogenous substrates. Brain P and dehydroepiandrosterone (D) undergo a prominent circadian variation with their acrophases at the beginning of the dark span. The circadian variation of brain D persists after adx + orx. The monkey brain (Macaca fascicularis) also accumulates P and D. Adrenal suppression with dexamethasone for 4 days does not decrease the concentrations of brain P and 3rd ventricle CSFP and D. The concentrations of brain D are decreased to a much smaller extent than plasma D. D inhibits the aggressive behavior of castrated male mice exposed to lactating female intruders. This is not the case for DS or androst-5-ene-3 beta, 17 beta-diol. The D analog 3 beta-methyl-androst-5-en-17-one, which is not estrogenic and cannot be metabolized to testosterone or estradiol, is as active as D in inhibiting the aggressive behavior of castrated mice.

Adrenal Glands↗

PubMed1987

Cellular distribution and hormonal regulation of h-SBP in human hepatoma cells.

The cellular distribution of human Sex Steroid Binding Plasma Protein (h-SBP) was studied in human cells and tissues by indirect immunofluorescence. h-SBP was detected in the cytoplasm of hepatocytes, of prostate and epididymis epithelial cells and in endometrium. Sexual and non-sexual skin, intestine epithelium, striated muscle and some rodent organs were not labelled. The intracellular localization of h-SBP indicate that h-SBP could be taken up from the extracellular compartment or synthesized in situ in sex steroid target organs, where it may play a role in hormone uptake. The hormonal regulation of h-SBP secretion by a human hepatoma cell line, H5A, showed that tri-iodothyronine was more potent than estradiol or tamoxifen, which acted as estrogen agonist, in increasing secreted h-SBP and the combined effect of both thyroid and estrogen hormones resulted in an additive stimulation of h-SBP secretion. As shown by Northern blot analysis, oligonucleotides synthesized from the known sequence of h-SBP hybridized with a RNA of approximately 2 kb which was more represented in H5A cells than in normal human liver, and was increased 2-3 times after hormonal stimulation of the cells. The presence of a poly(A+)RNA coding for h-SBP in the human liver indicated the hepatic synthesis of this protein.

Animals↗

PubMed1987

Neurosteroids: oligodendrocyte mitochondria convert cholesterol to pregnenolone.

Oligodendrocyte mitochondria from 21-day-old Sprague-Dawley male rats were incubated with 100 nM [3H]cholesterol. It yielded [3H]pregnenolone at a rate of 2.5 +/- 0.7 and 5-[3H]pregnene-3 beta, 20 alpha-diol at a rate of 2.5 +/- 1.1 pmol per mg of protein per hr. Cultures of glial cells from 19- to 21-day-old fetuses (a mixed population of astrocytes and oligodendrocytes) were incubated for 24 hr with [3H]mevalonolactone. [3H]Cholesterol, [3H]pregnenolone, and 5-[3H]pregnene-3 beta, 20 alpha-diol were characterized in cellular extracts. The formation of the 3H-labeled steroids was increased by dibutyryl cAMP (0.2 mM) added to the culture medium. The active cholesterol side-chain cleavage mechanism, recently suggested immunohistochemically and already observed in cultures of C6 glioma cells, reinforces the concept of "neurosteroids" applied to delta 5-3 beta-hydroxysteroids previously isolated from brain.

Animals↗

PubMed1987

Chronobiologic dynamics of delta 5-3 beta-hydroxysteroids and glucocorticoids in rat brain and plasma and human plasma.

While a different timing of circadian rhythms does not necessarily demonstrate the operation of independent mechanisms, it is one step in the isolation of separate interacting rhythmic factors underlying the dynamics of all life. With this step in mind, circadian rhythms are quantified in rat plasma and brain for corticosterone (B), pregnenolone (P), and dehydroepiandrosterone (D) by the rejection of the zero-amplitude assumption with the single cosinor method, from data obtained every 3 hr for 24 hr on groups of three male Sprague-Dawley rats, 11-12 weeks of age. The rats were killed; steroids were extracted from brain and plasma and were radioimmunoassayed. In relation to the acrophase (phi) of B, the phi of P in brain (P = 0.035) and of D in plasma (P = 0.0012) preceded the phi of B. By contrast, in clinically healthy women, the phi of plasma dehydroepiandrosterone sulfate (DHEA-S) lags behind that of cortisol (F), on the average by 6 hr 28 min. Such a lag is also seen in men. A species difference in the time relations of delta 5-3 beta-hydroxysteroids vs. glucocorticoids in plasma is obvious (P less than 0.01). A difference in time relations of human circulating D, DHEA-S, and F between schizophrenic and clinically healthy men renders the time relations of glucocorticosteroids and delta 5-3 beta-hydroxysteroids in brain particularly interesting, as do relations of an egocentric and expansive personality to the rhythm characteristics of DHEA-S. The fact that the acrophases for some of the steroids investigated in brain and plasma, respectively, are differently timed is in keeping with the assumption that they involve partly different mechanisms, even if differences in the metabolic handling of different steroids also remain to be investigated.

Animals↗

PubMed1986

Effect of aging on the dissociation kinetics and estradiol receptor nuclear interactions in mouse uteri: correlation with biological effects.

The dissociation kinetics of uterine estradiol receptor (UER) from young, middle-aged, and old mice have been studied in vitro and correlated with interactions of the steroid receptor with nuclear suspensions in a cell-free system. Furthermore, we have determined in these various age groups of mice the concentration of uterine cytosolic progesterone receptors and the activity of glucose-6-phosphate dehydrogenase. Compared to UER from young mice, the receptor from middle-age and old mice displayed similar first order dissociation kinetics, but a significantly reduced fraction of the slow component resulting from transformation of the receptor into a state of higher affinity for estradiol. In all age groups, sodium molybdate markedly inhibited this heat activation of UER. Recombination studies using heat-treated cytosolic UER and enriched nuclear fractions of various age groups suggested a markedly reduced ability for nuclear binding with advancing age, despite unchanged affinity of activated UER for nuclear acceptor sites that ranged from 3-5 X 10(9) M-1. Cross-incubation studies of heat-activated cytosolic UER with nuclei from young, middle-aged, and old mice suggested that the activation defect of cytosolic UER was already present at middle age and that a reduced nuclear ability to support receptor binding followed the onset of reproductive acyclicity. In parallel with these endocrine defects, we observed, with aging, decreases in mean baseline progesterone receptor concentrations and glucose-6-phosphate activity in uteri of both intact and castrated mice. The diminution of these two parameters was present at middle age, further increased at old age, and persisted after administration of low (0.2 mg) or high (2.0 mg) doses of estradiol for 3 days before study. Our observations suggest that decreased receptor activation is primarily responsible for the decreased effects of estrogen in aging mice.

Aging↗

PubMed1986

Androgen regulation of the androgen receptor of the quail uropygial gland: application of a [3H]mibolerone exchange assay.

An exchange assay for androgen receptors in the quail uropygial gland using [3H]mibolerone was established. The most efficient exchange conditions were 3 days of incubation at 15 degrees C. Under these conditions, androgen receptors were stable in the presence of sodium molybdate, and the exchange of [3H]mibolerone with endogenous testosterone bound to cytosolic or nuclear androgen receptors was maximal. Less than 5% of [3H]mibolerone-binding sites occurred in the extracted nuclear pellets. Using this exchange technique, it was shown that androgen receptors in the uropygial gland of photostimulated male quail or castrated quail treated with testosterone were activated and that their concentrations in both cytosolic and nuclear fractions were increased. These results confirm the androgen dependency of the quail uropygial gland, and show that it is an organ which can be used as a model for the study of androgen action in sebaceous glands.

Animals↗

PubMed1986

[Hormone-dependency of the human prostate].

The development and normal and pathologic growth of the human prostate depends on testicular secretions. The human prostate contains receptors for the three sex steroids: testosterone, estradiol, and progesterone. The assumption that benign prostatic hyperplasia would result of a dihydrotestosterone excess is contradicted by the measurement of androgen receptors, which are not different in hyperplastic compared to normal prostate. Contrawise, hyperplastic prostate contains subnormal amounts of estradiol and progesterone receptors. The assay of hormone receptors does not seem to have any predictive value for the endocrine therapy of prostate cancer. However, tumors rich in nuclear androgen receptors or in dihydrotestosterone have longer remission interval and survival in response to hormone treatment.

Dihydrotestosterone↗

PubMed1986

[Persistence of the circadian rhythm of dehydroepiandrosterone in the brain, but not in the plasma, of castrated and adrenalectomized rats].

In adrenalectomized and orchidectomized rats, the removal of the steroidogenic endocrine glands is associated with a near-disappearance of corticosterone (B) from plasma and brain; circadian variations of B and of plasma dehydroepiandrosterone (D), characteristic of the intact rat, are no more detected. By contrast, analyses of brain D measurements by the cosinor method demonstrate a persisting circadian rhythm of large amplitude.

Adrenalectomy↗

PubMed1985

Chick oviduct glucocorticosteroid receptor. Specific binding of the synthetic steroid RU 486 and immunological studies with antibodies to chick oviduct progesterone receptor.

The glucocorticosteroid receptor (GR) has been studied in oviduct cytosol prepared from estrogen-primed, 4-week-withdrawn chicken. The equilibrium dissociation constant was 6 nM for dexamethasone, and 18 300 receptor sites/cell were measured assuming that all cells contain identical concentrations of GR. Dexamethasone, used in most studies investigating glucocorticosteroid action, was found not to be the best GR ligand. The affinities of several natural and synthetic glucocorticosteroids for GR increased in the following order: cortisol less than deoxycorticosterone less than dexamethasone less than corticosterone less than triamcinolone acetonide. The synthetic steroid RU 486 was the most specific ligand of GR (its affinity was approximately equal to 10-fold higher than that of triamcinolone acetonide), while it did not bind either to plasma transcortin (which binds dexamethasone nor, surprisingly, to progesterone receptor (PR), contrary to what occurs in mammalian species. The molybdate-stabilized, 8-S form of GR was prepared from withdrawn chick oviduct, whole chick embryo or cultured chick embryo fibroblasts (which do not contain PR), and was labeled with either [3H]dexamethasone or [3H]RU 486. The sedimentation coefficient of radioactive ligand--8-S GR complexes was shifted towards heavier forms after incubation with polyclonal (IgG-G3) or monoclonal (BF4) antibodies generated against the molybdate-stabilized, 8-S form of the chick oviduct PR. Since neither IgG-G3 nor BF4 interacted with the steroid binding 4-S form of GR, it is suggested that these antibodies recognized a non-steroid binding protein common to molybdate-stabilized, 8-S forms of GR and PR.

Animals↗

PubMed1985

Sex steroid receptors in normal and hyperplastic human prostate.

The concentrations of sex steroid receptors (per unit DNA) were measured in normal periurethral and peripheral prostatic tissue samples from seven men (mean age 64 years; range 54-71 years) undergoing cystectomy for bladder cancer, and in hyperplastic nodules from 15 men with BPH (mean age 69 years; range 60-89). Occupied androgen (AR) and estrogen (ER) receptors were measured with an improved exchange procedure, where receptor-binding sites were stabilized by a combinatorial procedure involving careful washout of extracellular secretory products (including proteases) prior to homogenization, inclusion of 0.5 mM phenylmethyl sulfonylfluoride (PMSF) and 20 mM molybdate in the exchange medium, and long-term incubation at 0-4 degrees C. Bound radioligands were separated by a hydroxylapatite (HAP) batch adsorption procedure. Maximal specific exchange binding of 3H-R 1881 or 3H-estradiol in total homogenates of human prostate samples was achieved after incubation periods of about 72 h at 0-4 degrees C. In contrast, progestin receptors (PR) were readily available for binding 3H-R 5020; thus overnight binding at 0-4 degrees C was routinely used to measure PR. Binding specificities and equilibrium binding constants (calculated from 8-point Scatchard plots, correcting for nonsaturable binding) were found to be characteristic for AR, PR, and ER, respectively. The receptor results obtained in this study demonstrate that no significant differences existed in total AR per unit DNA between hyperplastic and either central or peripheral prostatic tissue samples; PR was present in both zones of normal prostatic tissue as often as in BPH samples, with PR concentrations significantly lower in hyperplastic samples; and ER was irregularly detected in both normal and hyperplastic tissue in low concentration relative to AR and PR; the frequency of ER detection was much lower in BPH than in normal prostate tissue. Studies of steroid receptor content relative to enzyme markers specific for epithelial and stromal cells in BPH samples showed a positive correlation between acid phosphatase activity (a specific marker for epithelial cells) and both AR and PR. No correlation was observed between AR or PR with either prolyl hydroxylase or myosin ATPase (specific markers for stromal cells). These observations suggest that PR, as well as AR, is primarily associated with the epithelial elements of prostate. Because of the relative infrequency of ER, similar correlation of ER with enzyme markers was not possible.

Acid Phosphatase↗

PubMed1985

Studies on the effects of dehydroepiandrosterone and its metabolites on attack by castrated mice on lactating intruders.

Triads of castrated male mice (residents) were injected each day for 2 weeks with oil vehicle or 280 nmol of dehydroepiandrosterone (D). They were tested for their attack on a lactating female (intruder) introduced in their home-cage 2,24 or 48 hr after their last injection. D significantly reduced male aggressive behavior for at least 24 hr. Other groups of castrated males were injected each day with vehicle or 280 nmol of D, dehydroepiandrosterone sulfate (DS) or androstenediol (ADIOL). D, but neither DS nor ADIOL, significantly reduced their aggressive responses to intruders. Neural levels of D, DS and testosterone (T) were measured in intact males injected with vehicle or D. The concentration of D in brain was unchanged by castration and was increased about 20 fold in D injected males. The concentration of neural T was 10 nmol/g of tissue in intact males, was almost undetectable in castrated males, and was slightly increased in D injected males. Further work will be needed to identify the steroid molecule(s) responsible for this behavioral effect of D.

Aggression↗