Micromethod for the determination of 3-β-HSD activity in cultured cells

doi:10.1016/0022-4731(89)90054-X

Journal of Steroid Biochemistry

Volume 33, Issue 4, Part 1, October 1989, Pages 643-646

https://doi.org/10.1016/0022-4731(89)90054-X Get rights and content

Abstract

A modified radioassay for the determination of the 3β-hydroxy-Δ5-steroid dehydrogenase (3-β-HSD) is described. The assay is based on the conversion of [³H]pregnenolone to [³H]progesterone followed by a digitonin precipitation step. The method was applied to neurons, glial cells, C6 glioma cells and adrenal tumor cells in culture. Adrenal tumor cells and C6 glioma cells showed higher enzyme activity than primary cultures of astrocytes and neurons. Dependence of enzyme activity on pH, protein concentration and reaction time was demonstrated for C6 cells. A pH optimum was shown between 7.5 and 8.1, and the reaction was linear up to 2 h. β-oestradiol inhibited 3-β-HSD activity completely. The assay presented is fast, highly reproducible, and offers the possibility of studying 3-β-HSD activity in differentiating cells in culture without preparation of microsomes or extraction of reaction products.

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The 3βHSD enzyme is responsible for the catalysis of progestagens and androgens, including the conversion of pregnenolone to progesterone. The activity of 3βHSD was determined by measuring the conversion of pregnenolone to progesterone as previously described (Bauer and Bauer, 1989; Raunig et al., 2011). ELISA and values were converted to ng/min/mg of protein using a standard curve of progesterone (manufacturer supplied).
Coral reefs are an indispensible worldwide resource, accounting for billions of dollars in cultural, economic, and ecological services. An understanding of coral reproduction is essential to determining the effects of environmental stressors on coral reef ecosystems and their persistence into the future. Here, we describe the presence of and changes in steroidal hormones along with associated steroidogenic and steroid removal enzymes during the reproductive cycle of the brooding, pan-Pacific, hermaphroditic coral, Pocillopora damicornis. Detectable levels of 17β-estradiol, estrone, progesterone and testosterone were consistently detected over two consecutive lunar reproductive cycles in coral tissue. Intra-colony variation in steroid hormone levels ranged between 1.5- and 2.2-fold and were not statistically different. Activities of the steroidogenic enzymes 3β-hydroxysteroid dehydrogenase and cytochrome P450 (CYP) 17 dehydrogenase were detectable and did not fluctuate over the reproductive cycle. Aromatase-like activity was detected during the lunar reproductive cycle with no significant fluctuations. Activities of regeneration enzymes did not fluctuate over the lunar cycle; however, activity of the clearance enzyme UDP-glucuronosyl transferases increased significantly (ANOVA, post hoc p < 0.01) during the two weeks before and after peak larval release (planulation), suggesting that the activity of this enzyme family may be linked to the reproductive state of the coral. Sulfotransferase enzymes could not be detected. Our findings provide the first data defining normal physiological and lunar/reproductive variability in steroidal enzymes in a coral species with respect to their potential role in coral reproduction.
Assisted reproduction technologies alter steroid delivery to the mouse fetus during pregnancy
2011, Journal of Steroid Biochemistry and Molecular Biology
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Percent areas were calculated from whole compound pictures and percentages presented are rounded to the nearest whole number. Assays for 3-β-hydroxysteroid dehydrogenase (3β-HSD) and cytochrome P450 17α-hydroxylase (CYP17) were performed using maternal livers (n = 3 each for IVF, ICSI and normal reproduction), ovaries (IVF, n = 2, ICSI, n = 4, normal reproduction n = 1), and fetal livers (IVF, n = 35, ICSI, n = 25, normal reproduction n = 26) using published protocols [13–15]. The assay for 3βHSD proceeded by addition of 10 μL of protein (0.05 mg/mL liver or 0.01 mg/mL ovaries), 79 μL of assay buffer (0.1 M Tris–HCl buffer with 50 mM MgCl2, pH 7.4) and 1 μL of pregnenolone (500 μM stock) to a 1.7 mL microtube.
Assisted reproduction technologies (ART) include in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), and are common treatments for infertility. Although generally successful, ART warrant further investigations due to emerging perinatal issues, especially low birth weight. Herein we extend our previous work demonstrating higher steroid clearance in murine ART placentas by examining steroid biosynthesis and the directional flow of steroids in the maternal–placental–fetal units. The activities of the major steroidogenic enzymes 3β-hydroxysteroid dehydrogenase (3β-HSD) and cytochrome P450 17-αhydroxylase (CYP17) were assessed in maternal liver and ovaries and fetal livers as were levels of cholesterol, progesterone, estrone (E1), and estradiol (E2) in the maternal, placental and fetal units. No structural abnormalities were found in placentas from ART. Although ART increased 3β-HSD activity in maternal livers, there were no other changes in 3β-HSD- or CYP17-mediated steroidogenesis. Cholesterol levels were significantly lower in maternal livers of ICSI pregnancies and in placentas from both IVF and ICSI pregnancies but not altered in the fetal livers. Progesterone levels were higher in maternal and fetal livers in IVF and ICSI, respectively, but were significantly lowered in ICSI placentas, compared to normal fertilization. For estrogenic hormones, no differences in E1 or E2 levels were observed in maternal livers but ICSI significantly increased both E1 and E2 levels in placentas while both IVF and ICSI significantly lowered E1 but raised E2 levels in fetal livers. In summary, while steroid production was normal, steroid diffusion/flow from mother to fetus was altered in murine pregnancies conceived by ART. This appears to occur, at least in part; through placental mechanisms. Impaired cholesterol and steroid transfer may affect correct regulation of fetal growth and development.
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The first data on the presence of 3β-HSD in the brain have been provided by Weidenfeld et al., (1980), who showed that homogenates of rat amygdala and septum could convert pregnenolone into progesterone. Subsequent studies have confirmed the existence of bioactive 3β-HSD in brain tissues and primary cultures of oligodendrocytes and neurons (Bauer and Bauer, 1989; Robel et al., 1986; Ukena et al., 1999). Type 1 3β-HSD mRNA has been detected in several regions of the rat brain including the olfactory bulb, the olfactory tubercle, the caudate putamen, the nucleus accumbens, the cerebral cortex, the thalamus, the hypothalamus, the hippocampus, the septum, the medial habenular nucleus, the nucleus vestibularis lateralis, the nucleus vestibularis medialis, the nucleus vestibularis spinalis and the cerebellum (Dupont et al., 1994; Furukawa et al., 1998; Guennoun et al., 1995; Kohchi et al., 1998; Meffre et al., 2007; Sanne and Krueger, 1995; Ukena et al., 1999).
It is now well documented that brain tissue is capable of synthesizing de novo bioactive steroids, named neurosteroids, which are involved in the regulation of various functions in the brain, including behavioral, neuroendocrine and metabolic processes. In this chapter, we have summarized the current knowledge about the expression of enzymes involved in the biosynthesis and metabolism of steroids in the brain with special emphasis on the morphological localization of those enzymes. The results obtained following use of immunocytochemistry and/or in situ hybridization indicate that the enzymes are expressed in both neurons and glial cells distributed throughout the brain with some species-related variations. As observed at the electron microscopic level, the enzymes localized in the neurons or glial cells are not associated with any specific organelles, being distributed throughout the cytoplasm. Since usually one nerve cell expresses only one enzyme, it might be suggested that the neuroactive steroid synthesis that requires the action of several enzymes involves the intervention of several cells (neurons and/or glial cells). More work is required to fully establish the migration of precursors and active steroids in the brain.
Neurosteroid biosynthesis: Enzymatic pathways and neuroendocrine regulation by neurotransmitters and neuropeptides
2009, Frontiers in Neuroendocrinology
Citation Excerpt :
i) The occurrence of steroidogenic enzymes or their mRNAs has been evidenced, respectively, by immunohistochemistry or in situ hybridization studies, either in neurons or in glial cells, depending on the species and the enzyme considered [486,487,492,493,494,495,524,623,625,626,720,745,782,792]. ( ii) The corresponding enzymatic activities were demonstrated through the ability of brain tissue to convert tritiated precursors such as cholesterol or 5P into radioactive metabolites including 7α-hydroxypregnenolone (7αΟΗ-Δ5P), 17-hydroxypregnenolone (17OH-5P), progesterone (P), 17-hydroxyprogesterone (17OH-P), DHEA, 7α-hydroxydehydroepiandrosterone (7αOH-DHEA), dihydroprogesterone (DHP), tetrahydroprogesterone (THP, allopregnanolone), Δ5PS and DHEAS [54,61,141,177,304,325,454–456,486,492–495,775,779–781,785,792,793]. Concurrently, it became clear that neurosteroids exert a large array of biological activities in the brain [65,382,431,568,739] either through a conventional genomic action or through interaction with membrane receptors.
Neuroactive steroids synthesized in neuronal tissue, referred to as neurosteroids, are implicated in proliferation, differentiation, activity and survival of nerve cells. Neurosteroids are also involved in the control of a number of behavioral, neuroendocrine and metabolic processes such as regulation of food intake, locomotor activity, sexual activity, aggressiveness, anxiety, depression, body temperature and blood pressure. In this article, we summarize the current knowledge regarding the existence, neuroanatomical distribution and biological activity of the enzymes responsible for the biosynthesis of neurosteroids in the brain of vertebrates, and we review the neuronal mechanisms that control the activity of these enzymes. The observation that the activity of key steroidogenic enzymes is finely tuned by various neurotransmitters and neuropeptides strongly suggests that some of the central effects of these neuromodulators may be mediated via the regulation of neurosteroid production.
Anatomical and biochemical evidence for the synthesis of unconjugated and sulfated neurosteroids in amphibians
2001, Brain Research Reviews
Various studies have shown that, in mammals, neurons and glial cells are capable of synthesizing bioactive steroids, or neurosteroids, which regulate the activity of the central nervous system (CNS). However, although steroid hormones are involved in the regulation of behavioral and neuroendocrine processes in amphibians, neurosteroid biosynthesis has never been studied in the CNS of non-mammalian vertebrates. Reviewed here are several data sets concerning the production of unconjugated and sulfated neurosteroids in amphibians. These data were obtained by investigating the immunohistochemical localization and activity of 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD) and hydroxysteroid sulfotransferase (HST), in the frog brain. Numerous 3β-HSD-immunoreactive neurons were detected in the anterior preoptic area, nucleus of the periventricular organ, posterior tuberculum, ventral and dorsal hypothalamic nuclei. 17β-HSD-like immunoreactivity was found in ependymal gliocytes bordering the lateral ventricles of the telencephalon. Two populations of HST-immunoreactive neurons were localized in the anterior preoptic area and the dorsal magnocellular nucleus of the hypothalamus. High amounts of progesterone (PROG), 17-hydroxyprogesterone (17OH-PROG), testosterone (T) and dehydroepiandrosterone sulfate (DHEAS) were measured in the frog brain by combining HPLC analysis of tissue extracts with radioimmunoassay detection. Incubation of telencephalic or hypothalamic explants with tritiated pregnenolone ([³H]PREG) yielded the synthesis of various metabolites including PROG, 17OH-PROG, DHEA and T. Incorporation of [³⁵S]3′-phosphoadenosine 5′-phosphosulfate ([³⁵S]PAPS) and [³H]PREG or [³H]DHEA into frog brain homogenates led to the formation of [³H,³⁵S]pregnenolone sulfate ([³H,³⁵S]PREGS) or [³H,³⁵S]DHEAS, respectively. Altogether, these results demonstrate that the process of neurosteroid biosynthesis occurs in amphibians as previously seen in mammals.
Biosynthesis of Neurosteroids and regulation of their synthesis
2001, International Review of Neurobiology
The brain, like the gonads, adrenal glands, and placenta, is a steroidogenic organ. The steroids synthesized by the brain and by the nervous system, given the name neurosteroids, have a wide variety of diverse functions. In general, they mediate their actions not through classic steroid hormone nuclear receptors but through ion-gated neurotransmitter receptors. This chapter summarizes the biochemistry of the enzymes involved in the biosynthesis of neurosteroids, their localization during development and in adulthood, and the regulation of their expression, highlighting both similarities and differences between expression in the brain and in classic steroldogenlc tissues.

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Journal of Steroid Biochemistry

Abstract

References (16)

Competitive inhibition of adrenal delta-5-3β-hydroxysteroid dehydrogenase and delta 5-4-ketosteroid isomerase activities by adenosine 3'5'mono-phosphate

J. biol. Chem.

Partial characterization of placental 3β-hydroxysteroid dehydrogenase (EC 1.1:1.145) delta-4-delta-5-isomerase (EC 5.3:3.1) in human term placental mitochondria

J. steroid Biochem.

Reversed-phase high performance liquid chromatography of steroid 3-sulfates and the corresponding unconjugated steroids

J. Chromat.

A modified digitonin-precipitation radioassay for 3β-hydroxy-delta-5-steroid dehydrogenase

Analyl. Biochem.

Neural cell markers and their application to Neurology

J. Neuroimmun.

Protein measurement with the Folin Phenol Reagent

J. biol. Chem.

In vitro glucocorticoid studies in human lymphoma: clinical and biological significance

J. steroid Biochem.

Steroids and brain activity

Biochem. Pharmac.

Cited by (26)

Molecular reproductive characteristics of the reef coral Pocillopora damicornis

Assisted reproduction technologies alter steroid delivery to the mouse fetus during pregnancy

Steroidogenic enzymes in the brain: Morphological aspects

Neurosteroid biosynthesis: Enzymatic pathways and neuroendocrine regulation by neurotransmitters and neuropeptides

Anatomical and biochemical evidence for the synthesis of unconjugated and sulfated neurosteroids in amphibians

Biosynthesis of Neurosteroids and regulation of their synthesis

General reviewMicromethod for the determination of 3-β-HSD activity in cultured cells

Abstract

J. biol. Chem.

J. steroid Biochem.

J. Chromat.

Analyl. Biochem.

J. Neuroimmun.

J. biol. Chem.

J. steroid Biochem.

Biochem. Pharmac.

General review
Micromethod for the determination of 3-β-HSD activity in cultured cells