17β-estradiol modulates NGF and BDNF expression through ERβ mediated ERK signaling in cortical astrocytes

17β-estradiol is known to exert neurotrophic and neuroprotective effects through classical estrogen receptors [ERs], ERα and ERβ, on a variety of cell types either by genomic or non-genomic actions. The actions of estradiol on glial cells are important to maintain metabolic functions of the nervous system. Astrocytes are considered to be active participants in brain activity because of their ability to release growth factors, including neurotrophins. Present in vitro studies show that 17β-estradiol modulates NGF and BDNF expression in time-dependent manner and ERK acts as secondary messenger for estradiol’s action. 17β-estradiol is involved in survival of cortical astrocytes. In conclusion, this study indicates vital role of ERβ mediated ERK signalling for regulation of NGF and BDNF expression along with cell viability of cortical astrocytes which further confirms the role of ERs, particularly ERβ in glial cells’ functions and viability.     

Binding of steroids to P.testosteroniΔ5-3-ketosteroid isomerase: Measurement of the number of binding sites by equilibrium dialysis

The binding of progesterone, 17β-estradiol and 19-nortestosterone acetate to the Δ⁵-3-ketosteroid isomerase from $\text{Pseudomonas}$$\text{testosteroni}$ has been investigated by the technique of equilibrium dialysis. Under the conditions used, all three steroids formed 2:1 complexes with each molecule of enzyme dimer (M.W. = 26,788). No evidence of any cooperative binding phenomena was obtained. The dissociation constants of the enzyme steroid complexes at 25°C were: progesterone, 2.2 μ$\text{M}$; estradiol, 2.5 μ$\text{M}$; 19-nortestosterone acetate, 9.2 μ$\text{M}$.     

Estrogen metabolism in nonhuman primates I. In vitro biosynthesis of estrogen glucosiduronates in rhesus monkey liver

Estrone glucosiduronate, 17β-estradiol-3-glucoslduronate, 17β-estradiol-17-glucosiduronate and estriol-16α-glucoslduronate have been biosynthesized in substantial yield by incubation of radioactive estrone, 17β-estradiol, estriol and uridlne diphosphoglucosiduronic acid with rhesus monkey liver homogenates. The metabolites were characterized by chromatography on Celite and DEAE-Sephadex, enzyme hydrolysis, derivative formation and crystallization to constant specific activity. The percent conversion to 17β-estradiol-17-glucosiduronate from 17β-estradlol ranged between 56–71%; from estrone, the conversion was 49–54%. Other metabolites formed from estradiol were estrone glucosiduronate(12–21%) and 17β-estradiol-3-glucosiduronate(5–12%). The same metabolites derived from estrone accounted for 18–28% and 10–14% respectively. After estriol incubation, more than 90% of the steroid was converted to estriol-16α-glucosiduronate with no detectable estriol-3-glucosiduronate. This report represents the first time that 17β-estradiol-17-glucosiduronate has been reported as a metabolite in the rhesus monkey and this is the only known species which forms 17β-estradiol-17-glucosiduronate as the predominant metabolite of either estrone or estradiol $\text{in vitro}$.Rhesus monkey liver is similar to the human and baboon in that it metabolizes estriol exclusively to estriol-16-glucosiduronate.     

Phosphorylation of calf uterus 17 beta-estradiol receptor by endogenous Ca2+-stimulated kinase activating the hormone binding of the receptor

Direct evidence is presented that uterus 17_β-estradiol receptor is phosphorylated $\text{in}\text{,}\text{vitro}$ by an endogenous kinase. Nuclear phosphatase, cytosol Ca²⁺-stimulated kinase (the former inactivating and the latter reactivating the hormone binding of the 17_β-estradiol receptor) and receptor were purified from calf uterus. 17_β-estradiol binding was inactivated by phosphatase, then reactivated by kinase in the presence of [_γ-³²P] ATP, Ca²⁺ and calmodulin, and the receptor was examined by various methods. The results of gel electrophoresis in non denaturating and denaturating conditions, and of centrifugation through sucrose gradients of receptor preincubated with monoclonal antibodies showed that the receptor is phosphorylated.     

Response of primary rabbit kidney proximal tubule cells to estrogens

The effects of estrogens on the growth and function of primary rabbit kidney proximal tubule (RPT) cells have been examined in hormonally defined phenol red–free medium. 17β-estradiol was observed to stimulate growth at dosages as low as 10⁻¹⁰ M. The growth stimulatory effects of 17β-estradiol were mitigated in the presence of hydrocortisone, suggesting that these two steroid hormones acted at least in part by common mechanisms. The effects of other steroids known to interact with the estrogen receptor were examined. Alpha estradiol was found to be growth stimulatory over a concentration range of 10⁻⁹ to 10⁻⁸ M, albeit to a lower extent than beta estradiol. In addition, the anti-estrogen tamoxifen was also growth stimulatory (unlike the case with the human mammary tumor cell line MCF-7). The effects of several metabolic precursors of 17β-estradiol were examined, including testosterone, which was growth stimulatory, and progesterone, which was growth inhibitory. The growth stimulatory effects of 17β-estradiol, alpha estradiol, and tamoxifen could possibly be explained by their interaction with an estrogen receptor. Indeed, metabolic labelling and immunoprecipitation studies indicated the presence of such an estrogen receptor in the primary cultures. The rate of biosynthesis of the estrogen receptor was found to be affected by the presence of exogenously added 17β-estradiol. 17β-estradiol was also observed to increase the activity of two brush border enzymes, alkaline phosphatase and gamma glutamyl transpeptidase, during the growth phase of the primary cultures. J Cell Physiol 178:35–43, 1999. © 1999 Wiley-Liss, Inc.     

Effect of conjugation to glucose on the uptake of 17 beta-(6,7-3-H)estradiol by rabbit liver subcellular fractions.

Homogenates and nuclear fractions from rabbit liver were incubated with 17β-[6,7-³H] estradiol and with the 3-glucoside and 3-glucuronide of this steroid. Localization of 17β-estradiol in the cell nucleus was enhanced when the glucoside rather than the free steroid was incubated with homogenates. It is suggested that conjugation to glucose may facilitate the transport of the steroid into the liver cell, or may protect it from metabolism. The effect was not observed in uterine tissue fractions.     

Sex hormone levels in the brain of d-aspartate-treated rats

d-Aspartate (d-Asp) is an endogenous amino acid present in the central nervous system and endocrine glands of various animal taxa. d-Asp is implicated in neurotransmission, physiology of learning, and memory processes. In gonads, it plays a crucial role in sex hormone synthesis. We have investigated the effects of chronic (30 days d-Asp drinking solution) and acute (i.p. injection of 2 μmol/g bw d-Asp) treatments on sex steroid synthesis in rat brain. Furthermore, to verify the direct effect of d-Asp on neurosteroidogenic enzyme activities, brain homogenates were incubated with different substrates (cholesterol, progesterone, or testosterone) with or without the addition of d-Asp. Enzyme activities were measured by evaluating the in vitro conversion rate of (i) cholesterol to progesterone, testosterone, and 17β-estradiol, (ii) progesterone to testosterone and 17β-estradiol, (iii) testosterone to 17β-estradiol. We found that d-Asp oral administration produced an increase of approximately 40% in progesterone, 110% in testosterone, and 35% in 17β-estradiol. Similarly, the results of the acute experiment showed that at 30 min after d-Asp treatment, the progesterone, testosterone, and 17β-estradiol levels increased by 29–35%, and at 8 h they further increased by a 100% increment. In vitro experiments demonstrate that the addition of d-Asp to brain homogenate + substrate induces a significant increase in progesterone, testosterone and 17β-estradiol suggesting that the amino acid upregulates the local activity of steroidogenic enzymes.     

Adenosylhomocysteine hydrolase inhibitors: synthesis of 5'-deoxy-5'-(isobutylthio)-3-deazaadenosine and its effect on Rous sarcoma virus and Gross murine leukemia virus.

One hour following administration of physiological concentrations of the steroid hormone antheridiol to a male strain of the water mold, $\text{Achlya}$$\text{ambisexualis}$, the rate of total cellular protein synthesis is increased. Further analysis revealed a sequential increase in the rate of syntheses for three classes of proteins following hormone stimulation. The rate of ribosomal protein synthesis increased as early as 20–30 minutes, followed by ribosomal salt wash proteins (40–60 minutes) and total soluble proteins after 60 minutes. Patterns of total cellular proteins, resolved by two-dimensional gel electrophoresis, during the first four hours after hormone treatment demonstrated the appearance of two newly synthesized peptides beginning at approximately 40 minutes followed by an increased rate of synthesis of three peptides after one hour. The synthesis of two peptides totally decreased after three hours of hormone induction.     

Prostaglandin levels in preovulatory follicles from rabbit ovaries perfused invitro

Prostaglandin (PG) levels in follicular fluid from preovulatory follicles of rabbit ovaries perfused $\text{in}$$\text{vitro}$ were measured in order to compare PG changes in this model system with those that occur $\text{in}$$\text{vivo}$ and in isolated, LH-treated follicles $\text{in}$$\text{barvitro}$. One ovary from each rabbit was perfused without further treatment (control). The other ovary was exposed to LH (0.1 or 1 ug/ml) beginning 1 hour (h) after initiation of perfusion. Samples of perfusion medium were taken at frequent intervals for measurement of PGE, PGF, progesterone and estradiol 17β. The perfusions were terminated when the first ovulation occurred or appeared imminent as judged by changes in the size and shape of the follicles. Follicular fluid was then rapidly aspirated from all large follicles on both ovaries for PGE and PGF measurement.Ovulations occurred only in the LH-treated ovaries. Progesterone and estradiol levels were significantly elevated in the perfusion medium within 1 h of LH treatment in comparison to controls. PG levels in perfusion medium from the control and LH-treated ovaries were not different throughout perfusion and increased in both groups. In contrast, PG levels measured in follicular fluid from LH-treated ovaries were 4- to 5-fold greater than in fluid from control ovaries. It is concluded that ovulation induced by LH in this experimental model is accompanied by an increase in follicular PG levels similar to that seen in other $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$ models. This difference in follicular PG levels between the LH-treated and control ovaries is, however, not reflected in the perfusion medium.     

Differential effects of some novel synthetic oestrogen analogs on oxidative PC12 cell death caused by serum deprivation

Oestrogens with no or reduced oestrogen receptor (ER) binding properties are reported to have neuroprotective functions. However, we have previously shown that the hormonally inactive isomer of 17β-estradiol (17β-E), 17α-estradiol (17α-E), down-regulates glutathione (GSH) synthesis, and fails to rescue serum deprivation-induced cell death in the rat pheochromocytoma cell line PC12 in micromolar concentration. The present study examined cellular protective effects of new 17β-E analogs and 2-methoxyestradiol (2-ME) analogs with no or little oestrogen activity. 17β-E, 17α-E, 2-ME, and an antagonist of the G protein-coupled oestrogen receptor (GPER), G36, were also included. Both 17α-E and 2-ME protected against deprivation-induced cell death in PC12 cells at 1?nM, but they enhanced the deprivation-induced cell death accompanied by caspase 3 activity and decreased intracellular GSH levels during deprivation at 10?µM. In addition, 10?μM 17α-E activated the p38 mitogen activated protein kinase pathway, which was linked to the enhanced death and reduced GSH levels. Analogs of 2-ME modified with a 6-isoquinoline moiety (6iq) protected against deprivation-induced cell death at 1?nM and did not interfere with the GSH levels nor increase p38 protein levels at 10?µM. The promoter activity of the catalytic subunit of the rate-limiting enzyme, glutamate cysteine ligase (GCLC) in GSH synthesis as well as protein levels of GCLC and Nrf2, increased with the 2-ME analogs at 10?µM. In conclusion, the steroids have differential protective effects, and modifying 2-ME may give the steroid more favourable properties than 17α-E, 2-ME, and G36 in regard to GSH regulation.     

Pregnancy hormone concentrations in marijuana users

The maternal serum concentrations of human chorionic gonadotropin, pregnancy-specific beta-l-glycoprotein, placental lactogen, progesterone, 17-hydroxyprogesterone, estradiol and estriol were measured in 13 women who smoked marijuana regularly throughout pregnancy. Cannabinoid use in these women was confirmed by RIA measurements of their sum Δ ⁹- tetrahydrocannabinol (THC) concentrations. These THC using women were matched within $\text{2}\text{1}\text{2}$ weeks of gestational age with 13 pregnant non-THC using controls drawn from the same population. Placental protein and steroid hormone concentrations were within established normal ranges for gestational age and there were no significant differences between the groups in the concentrations of any of the protein and steroids measured. In addition, no significant differences between THC users were found following linear regression analysis of placental hormone concentrations as a function of gestational age. Thus, this study suggests that marijuana use during pregnancy does not significantly alter the circulating maternal concentrations of trophoblastic protein hormones or major fetoplacental steroid hormones.     

Changes in ovarian follicles and in vitro sex hormone release in the lizard Podarcis sicula sicula

An in vitro superfusion method was used to test sex hormone release from different kinds of ovarian follicle (growing follicles, postovulatory follicles, and atretic follicles) in the lizard Podarcis sicula sicula. Sex hormone output changes with the stage of follicle evolution and sexual cycle. Previtellogenetic follicles prevail in early-spring quiescent ovaries and secrete mainly progesterone, which is probably utilized at that phase to delay ovarian resumption. In the active ovary, progesterone output from previtellogenetic follicles decreases, whereas vitellogenetic follicles produce a significant amount of 17β-estradiol, which is necessary for sustaining vitellogenin synthesis by the liver and oviduct growth. As follicles become ripe, progesterone production is resumed, and it increases in young postovulatory follicles. This is in line with the functions assigned to the hormone at that phase of the sexual cycle, i.e., the induction of oocyte maturation and the regulation of egg retention in the oviduct. Postovulatory follicles can also synthetize 17β-estradiol. After oviposition, this hormone, which is secreted by the old postovulatory follicles, can reinitiate vitellogenin synthesis, allowing the development of a new oocyte set. Our data confirm that active, although ephemeral, corpora lutea are also formed in oviparous species. A limited contribution to ovarian sex steroid production derives also from atretic follicles, at least at the early stages of the breeding cycle. © 1993 Wiley-Liss, Inc.     

17beta-Estradiol inhibits osteoprotegerin production by the estrogen receptor-alpha-positive human breast cancer cell line MCF-7

Estrogen regulates various cytokines and growth factors in estrogen receptor (ER)-positive human breast cancer. Receptor activator of NF-κB ligand (RANKL) is an essential cytokine for osteoclasts, whereas osteoprotegerin (OPG) is a soluble inhibitor for RANKL. We analyzed the regulation of the RANKL/OPG system by estrogens and androgens in the ER-positive breast cancer cell line MCF-7 and the ER-negative breast cancer cell line MDA-MB-231. In MCF-7 cells, which predominantly express ER-α, 17β-estradiol and testosterone dose-dependently decreased OPG mRNA levels and protein secretion by 70 and 65%, respectively (p < 0.0001 by ANOVA). The inhibition of OPG production by 17β-estradiol and testosterone was specifically prevented by the pure anti-estrogen ICI 182,780, and the testosterone effect was prevented by an aromatase inhibitor. In conclusion, 17β-estradiol suppressed OPG production by human breast cancer cell lines in a dose-dependent and specific manner, indicating that the RANKL/OPG cytokine system is an estrogen-responsive target in breast cancer.     

A radioimmunoassay for 11-oxotestosterone: Its application in the measurement of levels in blood sebum of rainbow trout (S. Gairdneri).

17β-Hyd.roxyandrost-4-ene-3,11-dione was linked via its 3-(O-carboxymethyl) oxime to bovine serum albumin to give a conjugate which was used to generate antiserum in rabbits. The antiserum, at an overall dilution of 1 in 16,000, together with [1,2-³H] 17β-hydroxyandrost-4-ene-3,11-dione synthesized from [1,2-³H] cortisone have been used to develop a radioimmunoassay for the parent steroid. The assay incorporates a purification step in which serum or plasma extracts are chromatographed on silica gel layers bound to plastic or aluminium sheets and the steroid, containing zones cut out and eluted directly with assay buffer. The cross-reactivities of several steroids with the antiserum and the specificity, sensitivity, accuracy and precision of the assay are described. Blood sera from Immature male rainbow trout contain $\text{ca}$ 0.2–0.4 μg/100 ml of 17β-hydroxyandrost-4-ene-3,11-dione. As male fish mature, serum levels rise sharply to reach values of 2 to >9 μg/100 ml. Levels in immature females rarely exceeded the assay sensitivity but serum from three ripe females showed low but detectable levels ($\text{ca}$ 0.2 μg/100 ml) of steroid. The assay has found application in sexing live fish for experimental purposes.     

Estrogen response in the hFOB 1.19 human fetal osteoblastic cell line stably transfected with the human estrogen receptor gene

The gene coding for the human wild-type estrogen receptor (ER) was stably transfected into the human fetal osteoblastic cell line hFOB 1.19, a clonal cell line which is conditionally immortilized with a temperature sensitive mutant of SV40 large T antigen (tsA58). Five subclones were obtained which express various levels of ER mRNA and protein. The subclone with the highest level of functional (nuclear bound) ER, hFOB/ER9, contained 3,931 (±1,341) 17β-estradiol molecules bound/nucleus as determined by the nuclear binding (NB) assay. Using the dextran coated charcoal (DCC) method, the level of total cytosolic ER measured was 204 (±2) fmol/mg protein. This subclone was examined further for estradiol (E₂) responsiveness. The ER expressed in hFOB/ER9 cells was shown to be functional using a transiently transfected ERE-TK-luciferase construct. Expression of luciferase from this construct increased ～25-fold in hFOB/ER9 cells following 10^?9M E₂ treatment. This effect on ERE-TK-luciferase expression was both dose and steroid dependant. Further, treatment of hFOB/ER9 cells with 10^?9M E₂ resulted in a 2.5–4.0-fold increase in endogenous progesterone receptor (PR) levels detected by steroid binding assays, and a noticeable increase in both the A and B forms of PR by western blot assay. The establishment of this estrogen responsive human osteoblastic cell line should provide an excellent model system for the study of estrogen action on osteoblast function. © 1995 Wiley-Liss, Inc.     

On the mechanism of the glucagon-induced inhibition of hepatic protein synthesis.

The intraperitoneal administration of glucagon (200 μg) to rats produced a transient increase of the hepatic polypeptide chain completion time, the increase being maximum at 5 min returning to control values at 20 min. This inhibitory effect was sustained when glucagon was constantly supplied by continuous infusion. Postmitochondrial supernatants from livers of the control group or rats treated with glucagon for 5 min showed no difference in their protein synthetic activity. After 20 min of intraperitoneal administration of the hormone, that is, when the effect on protein synthesis had vanished, the levels of cAMP were still 40% above those of the control group, and the ribosomal proteins were 110% more phosphorylated. These results suggest that the observed effect of glucagon is not due to its direct action on the protein synthesis machinery. On the other hand, the variations in the hepatic amino acid content brought about by glucagon do not appear to be quantitatively significant to account for the observed inhibition of protein synthesis. The effect of glucagon was always paralleled by a decrease in the $\text{[ATP]}\text{[ADP]}$ ratio which may be responsible for the observed decrease in the rates of elongation and/or termination steps of protein synthesis. Glucagon also produced a rise in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ ratio in both cellular compartments, cytosol and mitochondria, as reflected by the rise in the lactate to pyruvate and the β-hydroxybutyrate to acetoacetate ratios. This shift of the NAD⁺ couple to a more reduced state seems to be the result of an increased mobilization and oxidation of fatty acids brought about by the hormone. It is postulated then that the primary effect of glucagon leading to a decrease in protein synthesis is probably to increase the state of reduction of the hepatic nicotinamide nucleotide system. This point of view is supported by the fact that the nicotinamide and adenine nucleotide systems in rat liver are in equilibrium through cytosolic equilibrium reactions, so that a decrease in the $\text{[ATP]}\text{[ADP]}$ ratio brought about by glucagon may be secondary to the increase in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ ratio. This hypothesis is supported by the fact that glucagon was not effective in inhibiting hepatic protein synthesis in rats pretreated with a drug, 2-benzene-sulfonamido-5-(β-methoxy-ethoxy)pyrimidine, that prevents fatty acid mobilization and the subsequent changes in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ and $\text{[ATP]}\text{[ADP]}$ ratios. Furthermore, the administration of exogenous fatty acid brings about an inhibition of the rate of hepatic protein synthesis accompanied by a decrease in the ATP levels and an increase in the state of reduction of the NAD⁺ system.     

In vitro on an HCG responsive, testosterone secreting adrenal cortical adenoma

Clinical and biochemical investigation of a virilized woman has shown an adrenal cortical adenoma to be the source of elevated plasma testosterone levels and to be responsive to gonadotropin administration $\text{in vivo}$ (Givens et al.) (2). In the present study, the gonadotropin responsiveness and biosynthetic potential of the adenoma were evaluated $\text{in vitro}$. Incubation of minced adrenal tumor with hCG resulted in increased ¹⁴C-acetate incorporation into pregnenolone, progesterone, dehydroepiandrosterone (DHA), androstenedione, and testosterone. Androstenedione and testosterone were the major products, accounting for 27% and 20% respectively of the total radio-activity added, when ³H-pregnenolone was incubated with homogenized tissue. Estrogen synthesis was not observed in the tumor. The adenoma contained 9.0 μg/g testosterone and 1.9 μg/g androstenedione as determined by radio immunoassay. 17β-hydroxysteroid oxido-reductase was active in the adenoma. Androstenedione was reduced to testosterone at a rate of 0.6 μg/100mg/hr. Under the same conditions, reduction of estrone to estradiol was undetectable. The reductase activity was present in both the mitochondrial and microsomal fractions. NADPH was the required cofactor. When NADH was substituted, the rate was less than 10% of that with NADPH in both particulate fractions.The experimental results indicate the presence of steroid path-way(s) necessary to synthesize testosterone, and represent the first $\text{in vitro}$ demonstration of gonadotropin sensitive steroidogenesis in an adrenal cortical adenoma.     

Synthesis and pyrogenic effect of 3α,7α-dihydroxy-5β-androstan-17-one and 3α-hydroxy-5β-androstane-7,17-dione

The first chemical synthesis of 3α,7α-dihydroxy-5β-androstan-17-one and 3α-hydroxy-5β-androstane-7,17-dione is reported. In this method, the 17β-side chain of commercial chenodesoxycholic acid was degraded in 6 steps after selective protection of the hydroxyl groups : 3α-OH by a $\text{tert}$-butyldimetfaylsilyl group and 7α-OH by an acetoxy group. The capacity of 3α,7α-dihydroxy-5β-androstan-17-one and 3α-hydroxy-5β-androstane-7, 17-dione to release a pyrogen by human leukocytes was investigated by two independent methods : supernatants from leukocytes incubated with a steroid are injected to rabbits whose fever is measured, or tested by the Limulus Test (a pyrogen detection technique). The 7-keto substituted etiocholanolone still possessed pyrogenic activity, while the 7α-hydroxyl substituted one did not.     

Estradiol-mediated suppression of CYP1B1 expression in mouse MA-10 Leydig cells is independent of protein kinase A and estrogen receptor

Estrogens have multifaceted roles in mammalian testis. In the present study, we focused on estradiol as a potential regulator of testicular cytochrome P450 1B1 (CYP1B1) expression and investigated the possible mechanisms involved in the estradiol-mediated suppression. CYP1B1 protein levels were measured in the testes of rats that were treated with 17β-estradiol benzoate (1.5 mg/kg) at different stages of development. In addition, CYP1B1 mRNA levels were measured in mouse MA-10 Leydig tumor cells treated with (a) various concentrations of 17β-estradiol benzoate, (b) 17β-estradiol benzoate in the presence of exogenous luteinizing hormone (LH), or (c) 17β-estradiol benzoate in the presence of ICI 182,780, a competitive steroidal antagonist of estrogen receptors (ERs). Treatment of neonatal, pubertal, or adult rats with 17β-estradiol benzoate was associated with a reduction of approximately 90% in testicular CYP1B1 protein content compared to age-matched controls. Treatment of MA-10 cells with 17β-estradiol benzoate (10-500 nM) produced a concentration- and time-dependent decrease in CYP1B1 mRNA levels, but had no effect on LH receptor mRNA levels or on protein kinase A (PKA) activity. However, 17β-estradiol benzoate (10-500 nM), regardless of the concentration tested, failed to attenuate the LH-elicited increase in CYP1B1 mRNA or PKA activity in MA-10 cells that were co-treated with LH and estradiol. Similarly, ICI 182,780 (10-1000 μM) did not reverse the suppressive effect of estradiol on CYP1B1 mRNA expression in MA-10 cells co-treated with estradiol and ICI 182,780. The results indicate that downregulation of testicular CYP1B1 by estradiol was independent of PKA activity and was not mediated by ERs in MA-10 cells.