Showing papers on "Pregnenolone published in 1987"
TL;DR: Observations strongly support the existence of "neurosteroids," which have been posited on the basis of biochemical, physiological, and behavioral studies.
Abstract: 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.
365 citations
TL;DR: The interaction of the 'neurosteroid', pregnenolone-sulfate (PS), with the GABA/benzodiazepine/chloride ionophore receptor complex was investigated in rat brain subcellular preparations, suggesting the regulation of GABAergic neurotransmission by various neurosteroids may be an important mechanism for controlling neuronal excitability.
320 citations
TL;DR: Results show that Leydig cells from animals deprived of LH had diminished capacity to convert pregnenolone to testosterone and reduced P450 C17-hydroxylase/C17,20-lyase content.
Abstract: Depriving rats of luteinizing hormone (LH) causes Leydig cells to lose smooth endoplasmic reticulum and diminishes their P450 C17-hydroxylase/C17,20-lyase activity (Wing et al., 1984). LH administration to hypophysectomized rats prevents these changes in Leydig cell structure and function (Ewing and Zirkin, 1983). We adopted a multistep procedure of rat Leydig cell isolation to study the trophic effects of LH on steroidogenesis in the Leydig cell. Our method employs vascular perfusion, enzymatic dissociation, centrifugal elutriation, and Percoll gradient centrifugation. The purified Leydig cell fraction obtained after Percoll density-gradient centrifugation contains 95% well-preserved 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD)-staining cells with ultrastructural characteristics of Leydig cells. These Leydig cells produced 248 and 29 ng of testosterone/10(6) Leydig cells when incubated for 3 h with and without a maximally stimulating concentration of ovine LH. Purified Leydig cells obtained from control rats and rats treated with testosterone-estradiol (T-E) implants for 4 days to inhibit LH production were incubated with a saturating concentration (2 microns) of pregnenolone. Leydig cells from control and T-E-implanted rats produced 537 and 200 ng of testosterone/10(6) Leydig cells X 3 h, respectively, suggesting a defect in the steroidogenic reactions converting pregnenolone to testosterone in Leydig cells from T-E-implanted rats. By using rabbit antibodies to the P450 C17-hydroxylase/C17,20-lyase pig microsomal enzyme, immunoblots of one-dimensional sodium dodecyl sulfate polyacrylamide gels of Leydig cell microsomal protein from control and 4- and 12-day T-E implanted rats revealed a continued loss of enzyme as the period of LH withdrawal continues. These results show that Leydig cells from animals deprived of LH had diminished capacity to convert pregnenolone to testosterone and reduced P450 C17-hydroxylase/C17,20-lyase content.
294 citations
TL;DR: 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.
Abstract: 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.
290 citations
TL;DR: The D analog 3β-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.
169 citations
TL;DR: It is suggested that thyroid hormones synergize with FSH to exert direct stimulatory effects on granulosa cell functions, including morphological differentiation, LH/human CG receptor formation and steroidogenic enzyme induction.
Abstract: To characterize thyroid hormone action on the ovary, the direct effects of T4 or T3 were investigated in vitro using a monolayer culture system of porcine granulosa cells. Monolayer cultures were maintained for 6 days in 4% serum-supplemented medium in the absence or presence of porcine FSH (20 ng/ml), with or without graded doses of T4 or T3. Combined treatment with FSH and T4 (10(-7) M) induced morphological alternation resembling epithelioid cells, while FSH alone or T4 alone failed to bring about the epithelioid morphology. Concomitant treatment with FSH and T4 (10(-7) M) markedly increased FSH-stimulated induction of [125I]iodo-human CG binding to cultured granulosa cells obtained from small follicles. The combined treatment with FSH and T4 (10(-7) M) also resulted in a significant increase in progesterone and estrogen secretion by the cultured cells relative to treatment with FSH alone. Increases in progesterone, 17 beta-estradiol, and estrone secretion caused by the combined treatment with FSH and T4 (10(-7) M) were further augmented in response to the addition of exogenously provided substrate pregnenolone, testosterone, and androstenedione, respectively. Furthermore, aromatase activity assessed by the release of [3H]water from [1 beta-3H, 4-14C]androstenedione was significantly higher in cells treated concomitantly with FSH and T4 (10(-7) M) than that in cells treated with FSH alone. All the stimulatory effects of T4 (10(-7) M) on the morphological and functional differentiation of cultured granulosa cells were also found in combined treatment with FSH and T3 (10(-9) M). Either treatment with higher or lower concentrations of T4 or T3 gave attenuated effects, and T4 or T3 alone without FSH was incapable of exhibiting these stimulatory effects. These findings suggest that thyroid hormones synergize with FSH to exert direct stimulatory effects on granulosa cell functions, including morphological differentiation, LH/human CG receptor formation and steroidogenic enzyme (3 beta-hydroxysteroid dehydrogenase and aromatase) induction. Hence, decreases in ovarian functions during the states of hypo- or hyperthyroidism may account for diminished responsiveness of the granulosa cells to FSH.
136 citations
TL;DR: Very high concentrations of progesterone, androst-4-enedione, pregnenolone, dehydroepiandrosterone and 17-hydroxyprogesterone pose the question of the existence of local biosynthesis pathways independent of the peripheral endocrine gland system as well as that of progressive accumulation of steroids over a lifetime.
133 citations
TL;DR: The results suggest that the enhanced loss of mitochondrial and microsomal cytochrome P-450 activities in cAMP-treated cultures is caused by the increased production of pregnenolone and testosterone, respectively, which generate reactive damaging species derived from reduced dioxygen.
Abstract: Treatment of primary cultures of rat Leydig cells with 1 mM 8-bromo-cAMP for 2 days at ambient oxygen tension (19%) caused a 59% decrease in mitochondrial cholesterol sidechain cleavage (P-450scc) activity. This decrease was completely prevented when the oxygen tension was reduced to 1% O2 or when steroid synthesis was inhibited by aminoglutethimide. When the endogenous concentration of pregnenolone was increased by inhibiting its further metabolism, P-450scc activity was reduced by 80% in unstimulated cultures and was completely eliminated in cAMP-treated cultures. These losses were prevented when cells were maintained at 1% O2. The amount of immunoreactive P-450scc was also decreased by treatments that reduced P-450scc activity. Stimulation with cAMP also lowered microsomal C17_20 lyase activity by an oxygen-mediated, steroid synthesis-dependent mechanism. Treatment of cultures with testosterone caused a similar oxygen tension-sensitive decrease in C17_20 lyase activity. These results suggest that the e...
114 citations
TL;DR: The results suggest that in the testis, as in other steroidogenic tissues, TGF-beta may play a role in the development and maintenance of differentiated function.
107 citations
TL;DR: The pattern of changes in steroid hormone biosynthesis during the bovine estrous cycle and in the ovarian content of steroidogenic enzymes is related to and probably dependent upon the pattern of change in levels of mRNAs for steroidsogenic enzymes and related proteins.
Abstract: To investigate the molecular basis for the pattern of ovarian steroid production during the bovine estrous cycle, the relative levels of mRNA specific for cholesterol side-chain cleavage cytochrome P-450,17αhydroxylase cytochrome P-450, adrenodoxin, and low density lipoprotein receptor were determined in ovarian antral follicles of differing size (<3–18 mm) and corpora lutea from the early, early-mid, late-mid, and regressionary stages. Total and poly(A)+ RNA was size-fractionated on agarose-formaldehyde gels, transferred to nylon filters and hybridized to specific 32P-labeled probes. The levels of mRNAs for the rate-limiting enzymes in the conversion of cholesterol into progesterone, namely cholesterol sidechain cleavage cytochrome P-450 and its electron donor, adrenodoxin, were higher in corpora lutea than in follicles. Conversely the levels of mRNA specific for the key regulatory enzyme in the conversion of pregnenolone or progesterone to androgen, namely 17α-hydroxylase cytochrome P-450, were high in ...
71 citations
TL;DR: It is concluded that FSH and IGF-I can augment absolute rates of progestin biosynthesis by granulosa cells by activating dual mechanisms: stimulation of functional cholesterol side chain cleavage activity and enhancement of effective cellular uptake and utilization of low-density lipoprotein-borne sterol substrate.
TL;DR: It was shown that R2C cells constitutively synthesize an extramitochondrial cycloheximide-sensitive factor that is functionally identical to the factor produced in response to hCG in MA-10 cells, which enhanced mitochondrial pregnenolone biosynthesis.
Abstract: These studies were designed to characterize constitutive steroidogenesis in Leydig tumor cells. Constitutive steroidogenesis was investigated by comparing constitutively active R2C Leydig tumor cells to trophic hormone-responsive MA-10 Leydig tumor cells. Unlike the MA-10 cells, R2C cells appeared to synthesize steroid hormones independently of the cAMPprotein kinase pathway. Although the adenylate cyclase of R2C cells could be stimulated in the expected manner by cholera toxin, cAMP concentrations in these cells were low, and R2C cell steroidogenesis could be dissociated from other cAMPdependent processes. Two cAMP-dependent processes in steroidogenic cells, protein kinase activation and lactate formation, showed low basal activities in R2C cells and could be stimulated by (Bu)2cAMP with a dose dependence similar to that detected in MA-10 cells. Steroid hormone biosynthesis parallelled these other cAMP-dependent processes in MA-10 cells, but not in R2C cells. Cycloheximide, however, caused similar dose-d...
01 Jan 1987
TL;DR: Pharmacologically, DHA decreased a particular type of male mice agressive behavior linked to lactating female signal, and the “neurosteroid” concept is based on changes observed in a variety of physiological situations, including diurnal rhythm, development, and heterosexual exposure of male to female rats.
Abstract: Brain testosterone and corticosteroids arise from peripheral sources, whereas pregnenolone (Δ5-P) and dehydroepiandrosterone (DHA), the 3β-hydroxy-Δ5 derivatives of cholesterol which serve as precursors of steroid hormones in steroidogenic glands, accumulate in the brain by proper mechanism(s), even after surgical or pharmacological suppression of endocrine glands (adrenals, gonads). They are found in definite proportions as free steroids, or sulfate and fatty acid esters. Two enzymes involved in side-chain cleavage, cytochrome P-450 and adrenodoxin, have been immunohistochemi-cally localized in white matter, suggesting a possible modulatory/ trophic general function; they were also present in a few neurons of the olfactory bulb, entorhinal cortex and cingulum, evoking an olfactory pathway. The “neurosteroid” concept is based on changes observed in a variety of physiological situations, including diurnal rhythm, development, and heterosexual exposure of male to female rats. Pharmacologically, DHA decreased a particular type of male mice agressive behavior linked to lactating female signal. The mode of action of Δ5-P and DHA is yet unknown. It may include their transformation to classical steroid hormones acting on a paracrine mode, or directly their binding to unknown membrane or intra-cellular receptors, or even the control of neuronal functions by their insertion into membranes.
TL;DR: The cytochrome P-450-dependent cholesterol side chain cleavage system of the brain has been studied using nonsynaptic mitochondria as the source of enzymatic activity, and the formation of labeled pregnenolone after incubation of brain mitochondria with [4-14C]cholesterol has been obtained.
TL;DR: Tissue concentrations of progesterone are approximately 4 ng/mg protein, and it appears that local production rates could completely account for this, compatible with the hypothesis that local regulatory mechanisms may determine progestersone concentrations in fetal membrane tissues.
TL;DR: The data indicate that adrenal C‐19 steroids might be involved in the process of B PH, and whereas the estrogen glucuronide formation is diminished in men with BPH, the prostatic androgen metabolism as reflected by plasma dihydrotestosterone and androsterone glucuronides concentrations seems to be increased.
Abstract: Plasma steroids were analyzed in 16 normal men and in 10 men with prostatic benign hyperplasia (BPH). The steroids measured by radioimmunoassay include pregnenolone, 17-OH-pregnenolone, dehydroepiandrosterone, androst-5-ene-3 beta, 17 beta-diol, testosterone, dihydrotestosterone, androstane-3 alpha, 17 beta-diol, androstane-3 beta, 17 beta-diol, estrone, and estradiol as well as their glucuronide derivatives. In addition, cortisol and the sulphoconjugated form of dehydroepiandrosterone were determined. Whereas the levels of pregnenolone, pregnenolone glucuronide and 17-OH-pregnenolone glucuronide are not different in the two groups, the levels of 17-OH-pregnenolone in the BPH group (0.87 +/- 0.07 ng/ml) exceed by two-fold (p less than 0.01) those observed in normal men. Plasma dehydroepiandrosterone and androst-5-ene-3 beta, 17 beta-diol concentrations are markedly elevated in the BPH group (1.49 +/- 0.23 and 0.55 +/- 0.08 ng/ml vs the control groups 0.43 +/- 0.11 and 0.31 +/- 0.05 ng/ml, respectively). Since the plasma cortisol and pregnenolone levels are comparable in these two groups, our data suggest that the elevation of plasma 17-OH-pregnenolone, dehydroepiandrosterone, and androst-5-ene-3 beta, 17 beta-diol reflects an increase of adrenal 17-hydroxylase activity in patients with BPH. A slight increase of the plasma dihydrotestosterone and androsterone glucuronide concentration is also observed in men with BPH, indicating an increase of 5 alpha-reduced androgen formation. We have also observed, in the BPH group, a 50% decrease (p less than 0.01) of plasma glucuronidated androst-5-ene-3 beta, 17 beta-diol, estrone, and estradiol levels, suggesting that the transformation of unconjugated estrogenic steroids into glucuronide derivative is inhibited in BPH patients. In summary, our data indicate that adrenal C-19 steroids might be involved in the process of BPH. Furthermore, whereas the estrogen glucuronide formation is diminished in men with BPH, the prostatic androgen metabolism as reflected by plasma dihydrotestosterone and androsterone glucuronide concentrations seems to be increased.
TL;DR: The data indicate that the chronic administration of GnRH-A to elderly men results in inhibition in both the delta 4 and delta 5 pathways, with a subsequent decrease in the intratesticular T concentration.
Abstract: In an attempt to determine whether the chronic administration of GnRH agonist (GnRH-A) has a direct inhibitory effect on testicular steroidogenesis in the human, the testes of four men with disseminated prostatic cancer who were treated with GnRH-A daily for at least 1 yr were assayed for intrates-ticular pregnenolone (5-pregnen-3β-ol-20-one), progesterone, dehydroepiandrosterone, 17α-hydroxypregnenolone (5-pregnen-3β17α-diol-20-one), 17α-hydroxyprogesterone, androstenedione, and testosterone (T). In addition, testicular 17α-hydroxylase, 17,20-desmolase, and 17β-hydroxysteroid dehydrogenase enzyme activites of the Δ4 pathway were measured. These intra-testicular steroids and enzyme activities from four GnRH-A-treated patients were compared to those in five men (controls) who were orchiectomized as the primary treatment for their disseminated prostatic cancer and in three other men who were treated for 3–12 months with GnRH-A daily but received, in addition to the daily GnRH-A, 1000 IUhCG, im, every other ...
TL;DR: The present study confirms earlier reported results which showed that in the rat metabolism of pregnenolone to testosterone proceeds via the delta 4 pathway, but discloses for the first time that the conversion of pregnanolone proceeds very fast: progesterone, 17alpha-hydroxyprogesterone, and 17 alpha-hydroxypregnenolones as the only important delta 5 intermediate, peak and decline again to almost undetectable levels within the first 15 min of incubation.
Abstract: The time sequence of the metabolism of [4-14C] pregnenolone to testosterone in homogenates of human and rat testis was studied with special emphasis on the chain of events in the early 15 min of incubation. The incubations were performed at 32 C in the presence of NAD and a NADPH-generating system. The various intermediate steroids were separated by means of HPLC using a silica aliphatic diol column. Correction for procedural losses was performed by dual labeling. The present study confirms earlier reported results which showed that in the rat metabolism of pregnenolone to testosterone proceeds via the Δ4 pathway. However, this discloses for the first time that the conversion of pregnenolone proceeds very fast: progesterone, 17α-hydroxyprogesterone, and 17α-hydroxypregnenolone as the only important Δ5 intermediate, peak and decline again to almost undetectable levels within the first 15 min of incubation. Androstenedione and testosterone start to accumulate from 1 min on under the conditions used. In cont...
TL;DR: The results suggest that, like FSH, the stimulatory effect of the neuropeptide VIP on ovarian progesterone secretion involves regulation of P-450scc gene expression during functional maturation of the prepubertal ovary.
Abstract: Vasoactive intestinal peptide (VIP), a neuropeptide present in ovarian nerves, has been previously shown to induce synthesis of the side-chain cleavage cytochrome P-450 enzyme which catalyzes the conversion of cholesterol to pregnenolone (the rate-limiting step in progesterone synthesis). In the present study we demonstrate, by means of a bovine 3′-specific P-450scc cDNA probe, that this VIP effect is exerted at least partially at the level of gene expression in cultured granulosa cells that were isolated from estrogen-primed, immature rats. The size and level of the 2.0 kilobase P-450scc mRNA species was assessed by Northern blot analysis, while the translatability of this mRNA was assayed by immunoisolation of the 35S-labeled P-450scc precursor protein translated from total RNA of control and stimulated granulosa cells. FSH was much more effective than VIP at increasing P-450scc mRNA concentrations in cultured granulosa cells, whereas secretin treatment was ineffective. The results suggest that, like FS...
TL;DR: It is concluded that both the mixture of en- and zu-clomiphene and the isomers alone have direct inhibitory effects on oestradiol synthesis in human granulosa cells in vitro.
Abstract: The effect of en-clomiphene and zu-clomiphene (10(-9)-10(-5) M) on progestin synthesis in cultured human granulosa cells was studied under basal conditions and in the presence of LH (100 ng/ml). Granulosa cells were obtained from either pre-ovulatory follicles of clomiphene-HMG-stimulated cycles or from large follicles of mid-to-late follicular phase of spontaneous cycles. The basal and LH-stimulated progesterone accumulation was dose-dependently reduced by en- and zu-clomiphene (10(-6)-10(-5) M) in cells from both groups studied, an effect similar to that of oestradiol. In contrast to oestradiol, both en- and zu-clomiphene (10(-6)-10(-5) M) reduced the basal and LH-stimulated pregnenolone accumulation in cells of stimulated cycles. The effect of clomiphene on progesterone, and pregnenolone accumulation was more pronounced in LH-stimulated cells than under basal conditions. There were no qualitative differences between the two isomers and the results were principally the same in cells from both groups of follicles studied. It is concluded that en-clomiphene and zu-clomiphene have similar inhibitory effects on progestin synthesis in human granulosa cells in vitro.
TL;DR: A possible new role for certain steroids in neuroregulatory mechanisms related to behaviour is discussed in relation with a possible new roles for these steroid levels in the brain and in the retina of young adult male rats exposed to the scent or to the aroma of young cycling females.
TL;DR: Significant amounts of water-soluble steroid conjugates were found in the incubations with androstenedione and testosterone, indicating the presence of the UDP-glucuronosyl transferase in the catfish skin.
TL;DR: Six murine monoclonal antibodies against human hepatic cytochrome P-450 have been raised, using human liver microsomes (microsomal fractions) or semi-purified human cy tochromeP-450 as immunogen, showing four different banding patterns.
Abstract: Six murine monoclonal antibodies against human hepatic cytochrome P-450 have been raised, using human liver microsomes (microsomal fractions) or semi-purified human cytochrome P-450 as immunogen. All six antibodies recognized the same highly purified of human liver cytochrome P-450 of molecular mass 53 kDa and gave rise to a single band at 53 kDa on immunoblots of human liver microsomes from 11 individuals. The antibodies also recognized proteins at 52 kDa and 54 kDa on immunoblots of control and induced male-rat liver microsomes, showing four different banding patterns. Antibodies HL4 and HP16 recognized a 52 kDa protein that was only weakly expressed in untreated rats and which was strongly induced by pregnenolone 16 alpha-carbonitrile (PCN) but not by phenobarbitone (PB), 3-methylcholanthrene (3MC), isosafrole (ISF), Aroclor 1254 (ARO), clofibrate or imidazole. HP10 and HL5 recognized a constitutive 52 kDa protein that was weakly induced by PCN but not by the other agents and was suppressed by 3MC and ARO. HP3 recognized a 54 kDa protein that was undetectable in control rats but was strongly induced by PB, PCN, ISF and ARO. HL3 appeared to recognize a combination of the proteins recognized by the other antibodies plus a 54 kDa protein that was weakly expressed in control rats. The constitutive proteins recognized were male-specific.
TL;DR: The locus of gonadotropin-induced acute stimulation of pregnenolone production by cholesterol side-chain cleavage (CSCC) enzyme containing cytochrome P450 (cytP450scc) was examined in rat corpus luteum and suggests that the transport of cholesterol to inner mitochondrial membranes from outer membranes is regulated by hCG.
Abstract: The locus of gonadotropin-induced acute stimulation of pregnenolone production by cholesterol side-chain cleavage (CSCC) enzyme containing cytochrome P450 (cyt P450scc) was examined in rat corpus luteum. Mitochondria were isolated from pseudopregnant rat ovaries after treatment with different doses of human CG (hCG) (25–200 IU) for 30 min; Electron Paramagnetic Resonance (EPR) spectra of high spin cholesterol complex of cyt P450scc (type I high spin EPR signal) and the cyt P450scc activity were determined. hCG treatment increased the formation of type I EPR spectra compared to that obtained with saline-treated controls, and pretreatment with cycloheximide (30 mg/kg BW) before hCG abolished this increase. The magnitude of type I EPR signal diminished with increasing pH over the range of 6.2–7.3. The type I EPR signal increased with doses of hCG and correlated well with the pregnenolone production. Aminoglutethimide treatment (competitive inhibitor of CSCC) before hCG injection led to an increased accumulat...
TL;DR: Pregnenolone and its sulphate evidently have modulatory actions at ileal GABAA-receptor complexes in keeping with those seen using neurochemical studies in the central nervous system.
TL;DR: Experiments using calcium antagonists suggest that Ca2+, acting through calmodulin at some specific step(s) distal to cAMP elevation and prior to pregnenolone formation, is involved in FPH-induced progesterone accumulation, apparently with the participation of both extracellular and intracellular pools of Ca2+.
Abstract: In the amphibian ovarian follicle, progesterone production is thought to induce maturation of the enclosed oocyte. Intracellular mechanisms regulating these events in the somatic and germ cells are incompletely understood. However, calcium appears to play a role in the production and action of progesterone. Experiments using calcium antagonists were carried out to delineate the role of extra- and intracellular calcium during in vitro stimulation of follicular steroidogenesis and oocyte maturation. Calcium-free medium, verapamil, and La3+ were used to block Ca2+ influx and inhibited follicular progesterone accumulation in response to frog pituitary homogenate (FPH) or exogenous cAMP + IBMX. Progesterone accumulation was not impaired under identical conditions when pregnenolone was added to cultured follicles. TMB-8, an inhibitor of intracellular Ca2+ mobilization, partially inhibited progesterone levels stimulated by FPH at low doses but not higher doses of the inhibitor. However, TMB-8 inhibited FPH-induced oocyte germinal vesicle breakdown (GVBD) in a dose-dependent manner, as well as maturation due to exogenous progesterone or La3+. Calmodulin antagonists, W-7, R24571, and trifluoperazine, were used to assess the involvement of calmodulin in the responses of these two cell types. All three antagonists inhibited progesterone accumulation induced by FPH with the apparent order of potency being R24571 greater than W-7 greater than TFP. W-7 inhibited cAMP-induced progesterone elevation, but had no effect on conversion of pregnenolone to progesterone. Of these three calmodulin antagonists, only R24571 exhibited a dramatic ability to inhibit GVBD induced by exogenous progesterone and was associated with morphologic alterations in the oocytes. These data suggest that Ca2+, acting through calmodulin at some specific step(s) distal to cAMP elevation and prior to pregnenolone formation, is involved in FPH-induced progesterone accumulation, apparently with the participation of both extracellular and intracellular pools of Ca2+. In the oocyte, mobilization of Ca2+ from intracellular stores appears to be of primary importance to maturation while extracellular Ca2+ is not. These data provide further evidence that Ca2+ mediates the hormonally provoked responses in both cell types in the intact follicle, but that the source of Ca2+ may differ. Using intact follicles it seems apparent that exploiting this difference with selective inhibitors provides a means for differential modulation and functional uncoupling of these cells with regard to steroidogenesis and steroid action.
TL;DR: Titers of pregnenolone progesterone, testosterone, 5α-dihydrotestosterone and estradiol did not substantially fluctuate among the developmental stages the authors examined in Locusta migratoria.
Abstract: Summary Titers of pregnenolone, progesterone, testosterone, 5α-dihydrotestosterone, estrone and estradiol were measured by radioimmunoassay in purified haemolymph extracts of larval and adult male and female Locusta migratoria They varied between following values (in pg/ml): pregnenolone: 467–757; progesterone: 37–119; testosterone: 11–54; 5α-dihydrotestosterone: 13–41; estrone: 25–1392; estradiol: 12–26 Titers of pregnenolone progesterone, testosterone, 5α-dihydrotestosterone and estradiol did not substantially fluctuate among the developmental stages we examined Peaks of estrone were found in males and females in the middle of the fifth larval instar and in 3 week old adult males The titers of most of the above six steroids are about 5 to 10 times lower than the concentrations found in purified extracts of several tissues of this insect
TL;DR: The acute and the long-term effects of protein kinase C activators, phorbol 12 myristate 13-acetate (PMA) and 1-oleoyl-2-acetyl-sn-glycerol, and the calcium ionophore A23187 on cultured pig Leydig cell functions were investigated to explain the hCG-induced down regulation.
Abstract: The acute and the long-term (24 h) effects of protein kinase C activators, phorbol 12 myristate 13-acetate (PMA) and 1-olcoyl-2-acetyl-sn-glycerol, and the calcium ionophore A23187 on cultured pig Leydig cell functions were investigated. None of these drugs modified basal cAMP production, but they induced a small (3–4-fold) increase in testosterone secretion. The stimulatory effects of human choriogonadotropin (hCG; 1 nM) on both cAMP and testosterone productions were inhibited by short-term incubation with these drugs. In addition, they suppressed the stimulation of testosterone output by forskolin and 8-bromo-adenosine 3′,5′-monophosphate, whereas the forskolin-dependent cAMP production was unaffected. The inhibitory effects of PMA on hCG stimulation of both cAMP and testosterone were due mainly to a decrease of the Vmax without modification of the ED50. Moreover, PMA did not modify the binding of 125I-hCG. Pretreatment of Leydig cells with the three drugs for 24 h induced more pronounced modifications, such as a reduction in the number of hCG binding sites and a decreased responsiveness to hCG and forskolin, the testosterone production being drastically reduced. The effects of PMA were dose- and time-dependent; however, the concentration of PMA required to induce half-maximal effects on hCG receptors (10 nM) was about one order of magnitude higher than those required to reduce cAMP and testosterone productions. Further, the inhibitory effects on cAMP and testosterone secretions appeared within the first 3 h, whereas the hCG receptor number remained constant for at least 8 h. It appears therefore, that the main alteration responsible for the steroidogenic refractoriness of PMA-treated Leydig cells is located beyond cAMP formation. Moreover, since conversion of exogenous pregnenolone to testosterone by control and PMA-treated cells was similar, the alteration was probably located before pregnenolone formation. Kinetic studies with 125I-hCG showed that the rate of internalization of the hormone-receptor complexes was similar in control cells and in PMA-treated cells, suggesting that the decline in receptor number observed in the latter group after an 8-h delay is not due to an increased rate of internalization nor to sequestration of the internalized receptors inside the cells. Since cycloheximide blocked the effects of PMA on hCG down-regulation, it is likely that the phorbol esters and 1-oleoyl-2-acetyl-sn-glycerol induce the synthesis of some proteins which blocked the recycling of internalized receptors. A similar hypothesis has been put forward recently to explain the hCG-induced down regulation. Taken together, these results indicate that activation of protein kinase C probably plays a minor role in the acute regulation of testosterone secretion by pig Leydig cells. However, protein kinase C, as well as cAMP-dependent protein kinase activation, might play a role in the regulation of Leydig cell responsiveness to luteinizing hormone/human choriogonadotrophin.
TL;DR: The data indicate that gonadotropin suppression by GnRH agonist most likely affects testicular steroidogenesis by inhibiting 3 beta-hydroxysteroid dehydrogenase and a step(s) prior to pregnenolone formation.
Abstract: Endogenous testosterone (T), LH and FSH receptors, and in vitro production of cyclic adenosine-3':5'-monophosphate (cAMP), T and some of its steroid precursors were measured in testicular tissue obtained at orchiectomy from seven prostatic cancer patients treated for 6 months with a potent gonadotropin-releasing hormone (GnRH) agonist analog (buserelin, Hoechst, 600 micrograms 3 times a day intranasally). In addition, histologic and morphometric studies were carried out on the testicular tissue. Testicular tissue from age-matched prostatic cancer patients (n = 14), whose first therapy was orchiectomy, served as controls. The peptide treatment decreased intratesticular T by 95% (P less than 0.01) and FSH receptors by 57% (P less than 0.01), but had no effect on LH receptors. The in vitro production of T decreased by 94% (P less than 0.01), but that of cAMP was unaffected. Besides T, the in vitro production of testicular 17-hydroxyprogesterone (17-OHP-4), androstenedione and 5 alpha-dihydrotestosterone (5 alpha-DHT) dropped by 71 to 90% (P less than 0.01 to 0.05) during buserelin treatment, but those of pregnenolone, progesterone and dehydroepiandrosterone (DHEA) were not affected. Histologic studies revealed considerable variation in the seminiferous epithelium of the control group, but spermatogenesis was highly suppressed in nearly all of the buserelin-treated group. The number of Sertoli cells was unaffected, but tubular diameters were reduced (P less than 0.05) by buserelin treatment. Leydig cells appeared dedifferentiated in this group, although their number per testis was not altered. These data indicate that gonadotropin suppression by GnRH agonist most likely affects testicular steroidogenesis by inhibiting 3 beta-hydroxysteroid dehydrogenase and a step(s) prior to pregnenolone formation. The treatment does not impair testicular LH binding or cAMP production, but clearly suppresses FSH receptors. Spermatogenesis in general is suppressed but with considerable variation.