Neurosteroid levels are increased in vivo after LPS treatment and negatively regulate LPS-induced TNF production

Neuroactive steroids such as dehydroepiandrosterone sulfate and pregnenolone inhibit lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) production. Corticosteroids not only inhibit TNF production but their levels are increased in vivo after endotoxin injection, thus representing a feedback system that limits TNF production. We wondered whether the same could be true for neuroactive steroids. Thus, the possibility that neuroactive steroids might be increased concomitantly to TNF induction in vivo in mice treated with LPS was investigated. Increased plasma and hippocampal levels of allopregnanolone (but not of dehydroepiandrosterone or pregnenolone) were found 90 min after LPS injection. Allopregnanolone and progesterone (IC50 10- 7 and 10- 9 M, respectively) also inhibited TNF production by mouse peritoneal macrophages in vitro at concentrations in the range of those detected in vivo. These findings suggest that neuroactive steroids may act as endogenous inhibitors of cerebral and systemic TNF production.     

Pregnenolone sulfate modulates angiotensin II-induced inositol 1,4,5-trisphosphate changes in the anterior pituitary of female rat

The present study was designed to test whether pregnenolone sulfate can influence the angiotensin II (AngII) action in the anterior pituitary. Female rats were ovariectomized and 10 days after operation were treated with either 50 or 250 microg per animal in one of the following substances: oil (control), pregnenolone sulfate (PREG-S), estradiol benzoate (E(2),) progesterone (P), or dehydroepiandrosterone sulfate (DHEA-S), given in single intraperitoneal injection. Because AngII is known to act in the anterior pituitary through the phosphatidiloinositol breakdown, thus increasing the level of inositol-1,4,5-trisphosphate (IP(3)), the IP(3) concentration was determined 24 h after the injection in the anterior pituitary homogenate after in vitro exposure to AngII. Among the tested steroids only PREG-S enhanced the basal (without AngII) and AngII-induced level of IP(3) in both doses used. There was no difference between the effect of a high and low dose of PREG-S. These results show that PREG-S may modulate AngII action in the anterior pituitary, regardless of the presence of ovary.     

The Immediate Effect of HCG Upon Plasma Testosterone Levels in the Bull

No effect of HCG upon the plasma testosterone level in 16 bulls was seen the first 5 or 15 min after injection. In 8 bulls receiving 750 or 1500 i.u. HCG by intravenous injection a lag time of 30 min occurred before plasma testosterone response could be measured. The high plateau level of plasma testosterone concentration was reached approximately 1 h after injection. Following intramuscular injection of 750 i.u. or 1500 i.u. HCG a lag time of 45–60 min was observed for the testicular response measured as elevated plasma testosterone level. The high plateau levels of plasma testosterone in these bulls were reached approximately 1½ h post injection.     

Changes in oestrogen biosynthesis in preovulatory rat follicles after blockage of ovulation with pentobarbitone sodium

Follicles isolated 1 and 2 days after pentobarbitone sodium injection at pro-oestrus were incubated with C-21 steroids or aromatizable C-19 steroids. Addition of testosterone or androstenedione (50 ng/ml) increased oestradiol production by ovulation-blocked follicles, while addition of progesterone or 17 alpha-hydroxyprogesterone was ineffective. LH-stimulated oestradiol production was lower in follicles isolated 1 and 2 days after pentobarbitone sodium injection, but progesterone production was elevated compared to pro-oestrous follicles. Total steroidogenesis, measured by pregnenolone production in the presence of inhibitors of pregnenolone conversion, did not differ on the 3 days. The activity of C17-20 lyase, measured in follicular homogenates, decreased between pro-oestrus and the next day. Aromatase and 17 alpha-hydroxylase activities also decreased, but the activity of these enzymes was always considerably higher than that of C17-20 lyase. It is concluded that the decrease in follicular oestradiol production after injection of pentobarbitone sodium was due primarily to a decrease in the activity of the enzyme system responsible for the conversion of 17 alpha-hydroxyprogesterone to androstenedione, thereby limiting the amount of substrate available for aromatization to oestrogen.     

Synthesis of free and sulphoconjugated 16-androstene steroids by the Leydig cells of the mature domestic boar

This study examined the involvement of sulphoconjugation in the biosynthesis of the 16-androstene steroids in Leydig cells of the mature boar, since the formation of steroid sulphoconjugates can reduce the levels of these steroids that accumulate in fatty tissue. Leydig cells were purified from testes of mature male pigs and incubated with pregnenolone, or various individual 16-androstene steroids for 10 min, 1, 4 and 8h. Sulphoconjugated steroids were recovered by solid-phase extraction followed by solvolysis. Profiles of unconjugated and sulphoconjugated steroids were analysed by HPLC. Steroids present in the sulphoconjugated fractions were purified, derivatised as O-methoxime/trimethylsilyl ethers (MO-TMS), and subsequently identified using gas chromatography-mass spectrometry (GC-MS). The principal metabolite produced from incubations with pregnenolone, androstadienol, androstadienone and 5alpha-androstenone was 3beta-androstenol. 16-Androstene steroids that were sulphoconjugated included 5alpha-androstenone, 3beta-androstenol and 3alpha-androstenol. Approximately 70% of the total amount of each 16-androstene steroid was in its sulphoconjugated form after incubations for 4h or more. The finding that sulphoconjugated 5alpha-androstenone was present in large amounts suggests that this steroid may be converted from a 3-keto to a 3-enol form which is subsequently sulphoconjugated. These findings emphasise the need to consider the impact of sulphoconjugation of the 16-androstene steroids and their role in contributing to boar taint.     

A paradox: elevated 21-hydroxypregnenolone production in newborns with 21-hydroxylase deficiency

21-Hydroxypregnenolone and its metabolite 5-pregnene-3 beta, 20 alpha 21-triol have been measured in the sulfate fraction of neonatal urine. These two steroids are the major two 21-hydroxylated 5-pregnenes produced by neonates and are almost exclusively excreted as disulfates. The excretions of these steroids by normal infants and infants with 21-hydroxylase deficiency were compared. In addition to measurement of the absolute excretion, the excretion relative to the total 3 beta-hydroxy-5-ene output was also determined. The results show that 21-hydroxypregnenolone excretion is highly elevated in 21-hydroxylase deficiency (affected, mean 887 micrograms/24 h, range 453-1431 micrograms/24 h; normal, mean 117 micrograms/24 h, range 17-263 micrograms/24 h), but when compared to excretion of other delta 5 steroids the excretion is slightly low [(21-hydroxypregnenolone + 5-pregnene-3 beta, 20 alpha, 21-triol)/total 3-beta-hydroxy-5-ene steroids, 2.9% affected; 3.6% normal]. This difference was not statistically significant. There is thus no evidence that the 21-hydroxylase acting on pregnenolone is deficient in congenital adrenal hyperplasia. The explanation of the normal activity of "pregnenolone 21-hydroxylase," although not clearly defined, is probably associated with two recent findings by other workers: (a) that the human fetus has an active 21-hydroxylase distinct from the adrenal enzyme and (b) that a 21-hydroxylase structurally very different from the adrenal enzyme, with high activity towards pregnenolone (but no activity towards 17-hydroxyprogesterone), has been isolated from rabbit hepatic microsomes.     

Progesterone-synthesizing ability of preovulatory follicles of cows relative to the peak of LH

Preovulatory cow follicles (n = 34) were collected at different times after the onset of oestrus until shortly before ovulation. In-vitro conversion of tritiated pregnenolone in the presence of NAD+ by homogenates of the follicular wall was compared in phases relative to the LH peak. During phase 0 (before the LH surge) a moderate conversion into progesterone occurred, but it was subsidiary to that into 17 alpha-hydroxypregnenolone and other unidentified steroids. During phases 1 (0-6 h after the LH peak), 2A (6-14 h) and 2B (14-20 h) the production of progesterone and 17 alpha-hydroxypregnenolone remained constant; at phase 2B the percentage of remaining pregnenolone was higher than in the preceding phases. In phase 3 (20 h after the LH peak until ovulation) conversion into progesterone had increased about 4-fold to the highest levels observed (97% after 2 h incubation), and production of 17 alpha-hydroxypregnenolone and unidentified steroids was low. In an additional experiment, homogenates of the wall of 3 follicles at phase 3 were also incubated with tritated progesterone in the presence of NADPH. The percentage of remaining progesterone was high, and a moderate conversion into 17 alpha-hydroxyprogesterone occurred. In the main experiments, however, production of this steroid was not observed. The results indicate that steroid synthesis in the preovulatory follicle of the cow changes to the production of progesterone shortly before ovulation.     

Extraction of corticosterone from cell homogenates and subcellular fractions of the rat adrenal cortex. III. ACTH-induced temporal subcellular redistributions of steroid precursors to corticosterone

Cholesterol, pregnenolone, progesterone, 11-deoxycorticosterone (11-DOC) and corticosterone were quantitated in subcellular fractions isolated from in vivo adrenocorticotropin (ACTH)-stimulated rat adrenal zona fasciculata/reticularis. Six adrenal subcellular fractions separated by discontinuous sucrose gradient centrifugation (lipid, 0.125 M sucrose, cytosolic, microsomal, mitochondrial and nuclear) were extracted with alkaline ether/ethanol and assayed by high pressure liquid chromatography (HPLC). Lipid fractions contained the major cholesterol stores, while most pregnenolone and progesterone was found in lipid, microsomal and mitochondrial fractions. The 0.125 M sucrose and cytosol fractions together contained approximately 75% of the total 11-DOC and corticosterone. The five steroids were only present in small amounts in organelle fractions containing steroidogenic enzymes. Homogenate and lipid fraction cholesterol decreased between 10 and 15 min and again 30 min after ACTH injection. In the homogenate, lipid, microsomal and mitochondrial fractions, pregnenolone and progesterone were increased after ACTH injection; peak pregnenolone and progesterone concentrations were often measured in adrenal gland sucrose, cytosolic, microsomal and mitochondrial fractions 15 to 20 min after rats were injected with ACTH. Although ACTH increased 11-DOC and corticosterone in all but the mitochondrial and nuclear fractions, the sucrose, cytosolic and microsomal 11-DOC, and cytosolic corticosterone increased most dramatically. In many fractions, peak 11-DOC and corticosterone concentrations were most often observed between the 10 and 15 min periods and again at 30 min.     

Isolated Sertoli cells from immature rats produce 20α-hydroxy-pregn-4-en-3-one from progesterone and 3β,20α-dihydroxy-5α-pregnane from pregnenolone

Sertoli cells from 17 day old rats convert progesterone to 20α-hydroxy-pregn-4-en-3-one and pregnenolone to 3β,20α-dihydroxy-5α-pregnane after 72 hours $\text{in vitro}$. The metabolites were identified by several systems of thin layer and gas chromatography, derivative formation and crystallization with authentic steroids. The production of 20α-hydroxy-pregn-4-en-3-one and 3β,20α-dihydroxy-5α-pregnane amounted to 1380 and 740 pmoles/h/mg protein which can account for the total amounts of these steroids reported in the testis. It is the first direct evidence that Sertoli cells can metabolize progesterone and pregnenolone and suggests that Sertoli cells contain the major, if not the only, amounts of 20α-hydroxysteroid dehydrogenase in the immature rat testis.     

Social isolation-induced increase in the sensitivity of rats to the steroidogenic effect of ethanol

Social isolation of rats for 30 days immediately after weaning results in marked decreases in the cerebrocortical and plasma concentrations of pregnenolone, progesterone, 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-TH PROG), and 3alpha,5alpha-tetrahydrodeoxycorticosterone (3alpha,5alpha-TH DOC), as well as a moderate increase in the plasma concentration of corticosterone. This mildly stressful condition has now been shown to increase the sensitivity of rats to the effect of acute ethanol administration on the cerebrocortical and plasma concentrations of neuroactive steroids. The percentage increases in the brain and plasma concentrations of pregnenolone, progesterone, 3alpha,5alpha-TH PROG, and 3alpha,5alpha-TH DOC, apparent 20 min after a single intraperitoneal injection of ethanol (1 g/kg), were thus markedly greater in isolated rats than in group-housed animals. A subcutaneous injection of isoniazid (300 mg/kg) also induced greater percentage increases in the concentrations of these steroids in isolated rats than in group-housed animals. These results suggest that mild chronic stress, such as that induced by social isolation, enhances the steroidogenic effect of ethanol, a drug abused by humans under stress or affected by neuropsychiatric disorders. Social isolation also induced hyper-responsiveness of the hypothalamic-pituitary-adrenal (HPA) axis, as was apparent after reduction of GABA-mediated inhibitory tone by isoniazid administration.     

Effects of indomethacin and prostaglandins on ovulation of goldfish.

A technique is described whereby elevated temperature and HCG injection yield a high percentage of ovulation in gravid goldfish. Indomethacin (10 mug/g; i.p. injection) completely inhibits ovulation if given within 6 hours following HCG (4 IU/g); the unovulated oocytes develop rapidly into corpora atretica. PGE1, PGE2, and PGF2alpha (5 mug/g; i.p. injection) induce ovulation in fish treated with indomethacin and HCG; PGE2 was most effective when given 11 hours after HCG. The results suggest that the ovulatory action of prostaglandins following HCG stimulation is at the level of the ovary and that it is restricted to a period between 7 and 12 hours after the gonadotropin injection.     

Studies on neurosteroids: VII. Determination of pregnenolone and its 3-stearate in rat brains using high-performance liquid chromatography–atmospheric pressure chemical ionization mass spectrometry

An assay method for pregnenolone and its 3-stearate in rat brains has been developed using LC–atmospheric pressure chemical ionization (APCI) isotope dilution MS. The extraction of the rat brain homogenate containing deuterated internal standards (ISs) with organic solvent followed by silica gel mini-column chromatography gave two fractions containing pregnenolone and its 3-stearate together with the respective IS. Each fraction was derivatized into 3-acetate-20-methyloxime and 20-methyloxime, respectively, followed by silica gel mini-column chromatography to remove the excess reagents, and then each derivatized fraction was applied onto reversed-phase LC–APCI-MS. The method was applied to the determination of these steroids in the gray matter and olfactory bulbs of rat brains, which were divided into control and acute stressed specimens. Although pregnenolone in both regions of the rat brains increased more than five times after stress, its 3-stearate decreased after stress.     

Neuropsychopharmacological properties of neuroactive steroids.

In addition to the well-known genomic effects of steroid molecules via intracellular steroid receptors, certain steroids rapidly alter neuronal excitability through interaction with neurotransmitter-gated ion channels. Several of these steroids accumulate in the brain after local synthesis or after metabolism of adrenal steroids. The 3alpha-hydroxy ring A-reduced pregnane steroids allopregnanolone and tetrahydrodeoxycorticosterone have been thought not to interact with intracellular receptors, but enhance gamma-aminobutyric acid (GABA)-mediated chloride currents, whereas pregnenolone sulfate and dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABA(A) receptors. We demonstrated that these neuroactive steroids can regulate also gene expression via the progesterone receptor after intracellular oxidation. Thus, in physiological concentrations these neuroactive steroids regulate neuronal function through their concurrent influence on transmitter-gated ion channels and gene expression. When administered in animal studies, memory-enhancing effects have been shown for pregnenolone sulfate and DHEA. The 3alpha-hydroxy ring A-reduced neuroactive steroids predominantly display anxiolytic, anticonvulsant, and hypnotic activities. Sleep studies evaluating the effects of progesterone as a precursor molecule for these neuroactive steroids revealed a sleep electroencephalogram pattern similar to that obtained by the administration of benzodiazepines. These findings extend the concept of a "cross-talk" between membrane and nuclear hormone effects and provide a new role for the therapeutic application of these steroids in neurology and psychiatry.     

Steroid metabolism by purified adult rat leydig cells in primary culture

To characterize Leydig cell steroidogensis, we examined the metabolism of (³H)pregnenolone (3β-hydroxy-5-pregnen-20-one) to androgens in the presence and absence of human chorionic gonadotropin (hCG) as a function of culture duration. Approximately 20–30% of the (³H)pregnenolone was converted to testosterone (17_β-hydroxy-4-androsten-3-one) by purified Leydig cells at 0, 3 and 5 days (d) of culture. Androstenedione (4-androstene-3,17-dione) and dihydrotestosterone (17_β-hydroxy-5_α-androstan-3-one) were also produced while on day 5 of culture, significant amounts of progesterone (4-pregnene-3, 20-dione) were isolated. The Δ⁵ intermediates, 17-hydroxypregnenolone (3β, 17-dihydroxy-5-pregnen-20-one) and dehydroepiandrosterone (3β-hydroxy-5-androsten-17-one), accounted for less than 1% of substrate conversion, indicating a clear preference for Leydig cells to metabolize (³H)pregnenolone via the Δ⁴ pathway. On day 0 of culture, unidentified metabolites consisted of predominately polar steroids while on day 5 of culture, the unidentified metabolites consisted of predominately nonpolar steroids. In the presence of hCG, (³H)pregnenolone metabolism did not differ from basal on day 0 or 3 of culture. HCG increased the conversion of pregnenolone to progesterone and 17-hydroxyprogesterone (17-hydroxy-4-pregnene-3, 20-dione) on 5d. This suggests that Leydig cells cultured for 5d have decreased C_17–20 desmolase activity or that hCG acutely stimulates 3β-hydroxysteroid dehydrogenase and Δ⁴-Δ⁵ isomerase activities.     

Comparison of serum steroid responses to a single injection of hCG in man and rat

The responses of peripheral serum steroids to a single injection of hCG (80 IU/kg b wt) were compared in adult male rats and humans. Before hCG, the quantitatively dominating steroids were dehydroepiandrosterone, testosterone and 17-hydroxypregnenolone in the men, and testosterone and progesterone in the rats. One hour after hCG the concentrations of testosterone and all its precursors measured except for pregnenolone were significantly elevated in the rat serum, whereas a clear rapid response was not observed in the men. Transient blockade of C21 steroid side-chain cleavage was seen in both species at about 24-36 h after hCG, which occurred at the same time as the maximum concentration of estradiol in the men. No changes in rat serum estradiol concentrations were observed. Both species showed a secondary stimulation of testosterone and androstenedione formation at around 3 days. Our findings are compatible with the concept that the main difference in the gonadotropin-stimulated steroidogenesis in man and rat is the magnitude of the rapid steroidogenic response to hCG, which is very small in man and indicates smaller supply or lesser metabolism of mitochondrial cholesterol in human testis.     

Long-term effect of HCG on plasma testosterone in bulls.

The spontaneous diurnal variation of peripheral plasma testosterone concentrations in four bulls was established and then the long-term effect of a single intravenous or intramuscular injection of HCG on testosterone levels was studied. Intravenous and intramuscular HCG injections produced, within 1/2 hr and 3 hr, respectively, a rapid rise of testosterone to levels equivalent to the highest values seen in the diurnal pattern. A second increase of up to x2 to x3 the highest values of the diurnal cycle was observed 2 days after the injection of HCG, and the testosterone level remained high for at least 3 to 4 days after plasma levels of HCG were no longer detectable. The pattern of diurnal variation after HCG revealed an attenuation of the extensive spontaneous variation and high levels with only slight fluctuations were maintained.     

Fertility, ovulation and maturation of eggs in mares injected with HCG

Pony mares were observed from January to August for incidence of oestrus, duration of oestrus, length of the oestrous cycle and for ovulation and fertility after injection of HCG. From January to 15 May most mares showed oestrus but the duration of oestrus was quite variable and few mares ovulated in response to HCG. From 15 May to 17 August oestrous cycles were more regular and ovulation was induced within 40-50 h by an intramuscular injection of 1500-5000 i.u. HCG. Pregnancy was established by one mating at a fixed time after HCG in 20 of 69 mares. Degenerate eggs were recovered from the oviducts of anoestrous recently ovulated, mated, unmated and pregnant mares. The first polar body was formed before ovulation in 2 eggs and had not formed in 2 recently ovulated eggs flushed from the oviduct. The second polar body formed after sperm penetration 10-12 h after ovulation. After formation of pronuclei, the first cleavage division occurred at 20 h and the second at 32 h after ovulation. Oestrus was inhibited by progesterone administered by vaginal devices but occurred within 1-3 days in 12 of the 20 mares after withdrawal of the devices.     

Simultaneous radioimmunoassay of progesterone, androst-4-enedione, pregnenolone, dehydroepiandrosterone and 17-hydroxyprogesterone in specific regions of human brain

Simultaneous determination of progesterone, androst-4-enedione, pregnenolone, dehydroepiandrosterone (DHEA) and 17-hydroxyprogesterone has been developed for human cerebral tissue. Before immunoassay, steroids were separated on a Celite column with propylene glycol as stationary phase with hexane containing increasing proportions of dichloromethane as mobile phase. This system allowed separation of steroids of similar polarity, especially of pregnenolone and progesterone. The brain regions studied cortex (prefrontal, parietal and temporal), cerebellum and corpus callosum, were obtained after autopsy from 9 women and 1 man between 76 and 93 years of age. Steroids were found in all regions. The overall concentrations expressed in nmol/kg of tissue were: 10.1, 7.6, 120.7, 19.6 and 10.4 respectively, for progesterone, androst-4-enedione, pregnenolone, dehydroepiandrosterone and 17-hydroxyprogesterone, corresponding to 7.3, 4.9, 74, 6.5 and 9.2 times the plasma levels. These very high concentrations, not previously described in human brain tissue, pose the question of the existence of local biosynthetic pathways independent of the peripheral endocrine gland system as well as that of progressive accumulation of steroids over a lifetime. Concentrations of each steroid in each subject varied little among the various brain regions studied, but there was much variation among the subjects with respect to the concentrations of a given steroid.     

Effects of pregnenolone,cyanoketone, and equilenin on the human malignant trophoblast in vitro

Summary Cultures of human malignant trophoblast cells were studied to determine the basis of inhibition of human chorionic gonadotropin (HCG) secretion and depletion of glycogen following incubation of the cells in the presence of pregnenolone (3β-hydroxypregn-5-en-20-one). Incubation of the cells for as long as 3 days with 5 or 10 μg of pregnenolone per ml resulted in decreased protein content, inhibition of DNA synthesis, diminished glucose utilization, and marked accumulation of acellular debris in the medium. These changes became more pronounced with time of incubation and were related to the dose of pregnenolone employed. The effect of pregnenolone on all of the parameters measured was mimicked and potentiated by either equilenin (3-hydroxy-1,3,5(10),6,8-estrapentaen-17-one) or cyanoketone (androst-5-en-2α-cyano-17β-hydroxy-4,4,17α-trimethyl-3-one), inhibitors of pregnenolone conversion to progesterone. These results suggested that the glycogenolysis and inhibition of HCG secretion that occur when the trophblast cells are incubated in the presence of pregnenolone result from toxicity rather than from cellular differentiation, and that prior conversion of pregnenolone to progesterone is not necessary for the manifestation of the toxicity. This work was supported in part by Contract PH 43-NCI-E-68-1010 from the Special Virus Cancer Program, National Cancer Institute, United States Public Health Service.