Tritiated Steroids Research Articles

Role of OATP transporters in steroid uptake by prostate cancer cells in vivo.

BackgroundEpidemiologic and in vitro studies suggest SLCO-encoded OATP transporters influence the response of prostate cancer (PCa) to androgen deprivation by altering intra-tumor androgens. We have previously shown that castration resistant metastases express multiple SLCO transporters at significantly higher levels than primary PCa, suggesting OATP-mediated steroid transport is biologically relevant in advanced disease. However, whether OATP-mediated steroid transport can actually modify prostate tumor androgen levels in vivo has never been demonstrated.MethodsWe sought to determine whether OATP-mediated steroid transport can measurably alter PCa androgen levels in vivo. We evaluated uptake of DHEAS, E1S and testosterone in LNCaP cells engineered to express OATP1B1, 1B3, 2B1 or 4A1. We measured uptake via administration of tritiated steroids to castrate mice bearing vector control or OATP1B1, 2B1 or 4A1-expressing xenografts. We treated tumor-bearing mice with DHEAS and testosterone at physiologically relevant levels and measured intra-tumor accumulation of administered steroids by mass spectrometry.ResultsOATP1B1 and 2B-expressing xenografts each showed a 3-fold increase in tritiated-DHEAS uptake vs vector controls (p=0.002 and p=0.036, respectively). At circulating DHEAS levels similar to those in abiraterone-treated men (~15ug/dL) OATP1B1 and 2B1-expressing xenografts showed a 3.9-fold (p=0.057) and 1.9-fold (p=0.048) increase in tumor accumulation of DHEAS, and a 1.6-fold (p=0.057) and 2.7-fold (p=0.095) increase in DHEA, respectively. At testosterone levels found in eugonadal men, a consistent effect of OATP1B1, 2B1 or 4A1 on testosterone uptake in vivo was not detected.ConclusionsOATP transporters measurably alter DHEAS uptake and intra-tumor androgen levels in prostate tumors in vivo, even at circulating androgen levels achieved in abiraterone-treated patients. These novel data emphasize the continued need to inhibit ligand-mediated AR signaling in PCa tumors, and support prospective evaluation of studies designed to test inhibition of OATP-mediated DHEAS uptake and utilization.

Immunohistochemical localization and biological activity of 3β-hydroxysteroid dehydrogenase and 5α-reductase in the brain of the frog, Rana esculenta, during development

The occurrence of several enzymes responsible for the biosynthesis of neurosteroids in the brain of adult frogs is now firmly established but the expression of these enzymes during ontogenesis has not yet been investigated. In the present report, we describe the immunohistochemical distribution and biological activity of 3β-hydroxysteroid dehydrogenase (3β-HSD) and 5α-reductase (5α-R) in the brain of the European green frog, Rana esculenta, during larval development. The spatio-temporal distribution of 3β-HSD and 5α-R immunoreactivities in the tadpole brain was generally different, although these two enzymes were occasionally detected in the same areas such as the olfactory bulbs and cerebellum. Identification of neurons based on their morphological aspect as well as labeling of astrocytes with an antiserum against glial fibrillary acidic protein (GFAP) revealed that, in the tadpole brain, 3β-HSD- and 5α-R-immunoreactive materials were contained in both neurons and glial cells. Incubation of tadpole brain explants with [ 3H]-pregnenolone resulted in the formation of several tritiated steroids including progesterone, 17-hydroxyprogesterone, androstenedione, 5α-dihydroprogesterone and 5α-dihydrotestosterone. The present study provides the first immunocytochemical mapping of two key steroidogenic enzymes in the developing frog brain. The data also indicate that neurosteroid biosynthesis occurs in the brain of tadpoles, as previously shown for adult amphibians, birds and mammals. The transient expression of steroidogenic enzymes in several regions of the tadpole brain suggests that, in amphibians, neurosteroids may be implicated in neurotrophic activities during larval development.

Impaired Leydig cell function in vitro in testicular tissue from human males with "Sertoli cell only" syndrome.

Testicular histopathology in patients with "Sertoli Cell Only" Syndrome is characterized by absence of germinal cells. Some of these patients have high levels of LH in their peripheral blood as well as subnormal serum testosterone levels. This indicates a possible correlation between the lack of germinal cells and an impairment of Leydig cell steroidogenic activity. It has previously been shown that in vitro conversion of tritiated steroid precursors within testicular tissue indicates the Leydig cell steroidogenic activity. In this study we have investigated whether or not testicular tubular cell disorder with lack of germinal cells is related to impairment of the intratesticular steroid metabolism in vitro. Ten patients with testicular histopathology characteristic of "Sertoli cell only" syndrome were investigated and their steroid metabolism patterns were compared with those of 22 control patients. Leydig cell dysfunction was found in the group of patients with SOS; 17 alpha-hydroxylation was significantly lower and the production of 20 alpha-dihydroprogesterone was significantly higher, as compared to the control patients. The Leydig cell impairment may be due to a disturbed influence from the damaged tubules.

The key steroidogenic enzyme 3β-hydroxysteroid dehydrogenase in Taenia solium and Taenia crassiceps (WFU)

Larval and adult stages of Taenia solium and Taenia crassiceps WFU strain were analyzed by histochemical and biochemical methods to determine the existence of steroid pathways. The presence of the key enzyme 3beta-hydroxisteroid-dehydrogenase (3beta-HSD) was examined in frozen sections of cysticerci obtained from mice and segments of tapeworms obtained from the intestine of hamsters. 3beta-HSD activity was detected by nitroblue-tetrazolium products after incubation with dehydroepiandrosterone, androstendiol, or pregnenolone. Tapeworm tissues exhibited 3beta-HSD activity in the subtegumentary areas of the neck and immature proglottids following incubation with androstendiol, as well as surrounding the testes in mature proglottids. T. solium cysticerci exhibited 3beta-HSD activity in the subtegumentary tissues. The synthesis of steroid hormones involving the activity of 3beta-HSD was studied in cysticerci or tapeworms incubated in the presence of tritiated steroid precursors. The culture media were analyzed by thin layer chromatography and showed synthesis of androstendiol, testosterone, and 17beta-estradiol by cysticerci, androstendiol, and 17beta-estradiol by tapeworms. The results strongly suggest the activity of 3beta-HSD in taeniid parasites that have at least a part of the enzymatic chain required for androgen and estrogen synthesis and that the enzymes are present in the larval stage and from the early strobilar stages to the mature proglottids.

Steroidogenic capacity of Trypanosoma cruzi trypomastigotes

American Trypanosomiasis is caused by the hemoflagellate Trypanosoma cruzi ( T. cruzi) and affects millions of persons causing variable degrees of digestive and heart disturbances. As far as we concerned, T. cruzi capacity to synthesize steroid hormones has not been investigated. Therefore, the aim of this work was to investigate the capacity of T. cruzi trypomastigotes to transform tritiated steroid precursors into androgens and estrogens. The T. cruzi Tulahuén strain was obtained from mice blood. The trypomastigotes were cultured for 6 and 24 h in Dulbbeco’s modified Eagle's medium plus FCS and antibiotics. Tritiated dehydroepiandrosterone or androstendione were added to the culture media and parasites were incubated for 6 or 24 h. The cultures were centrifuged and ether extracted. The steroids were analyzed by thin layer chromatography (TLC) in two solvent systems. After incubation with 3H-androstenedione, T. cruzi trypomastigotes synthesized 3H-testosterone (T), 3H-17β-estradiol (E 2) and 3H-estrone (E 1). Metabolism of 3H-DHEA by the parasites yielded 3H-androstendione and 3H-androstendiol at 6 h of incubation. The recrystallization procedure further demonstrated the 3H-androstendiol and 3H-17β-estradiol syntheses. Results indicate for the first time that T. cruzi trypomastigotes produce androgens and estrogens when incubated in the presence of steroid precursors and suggest the presence of active parasite steroidogenic enzymes.

Immunohistochemical localization and biological activity of the steroidogenic enzyme cytochrome P450 17α‐hydroxylase/C17, 20‐lyase (P450C17) in the frog brain and pituitary

It is now clearly established that the brain has the capability of synthesizing various biologically active steroids including 17-hydroxypregnenolone (17OH-Delta(5)P), 17-hydroxyprogesterone (17OH-P), dehydroepiandrosterone (DHEA) and androstenedione (Delta(4)). However, the presence, distribution and activity of cytochrome P450 17alpha-hydroxylase/C17, 20-lyase (P450(C17)), a key enzyme required for the conversion of pregnenolone (Delta(5)P) and progesterone (P) into these steroids, are poorly documented. Here, we show that P450(C17)-like immunoreactivity is widely distributed in the frog brain and pituitary. Prominent populations of P450(C17)-containing cells were observed in a number nuclei of the telencephalon, diencephalon, mesencephalon and metencephalon, as well as in the pars distalis and pars intermedia of the pituitary. In the brain, P450(C17)-like immunoreactivity was almost exclusively located in neurons. In several hypothalamic nuclei, P450(C17)-positive cell bodies also contained 3beta-hydroxysteroid dehydrogenase-like immunoreactivity. Incubation of telencephalon, diencephalon, mesencephalon, metencephalon or pituitary explants with [(3)H]Delta(5)P resulted in the formation of several tritiated steroids including 17OH-Delta(5)P, 17OH-P, DHEA and Delta(4). De novo synthesis of C(21) 17-hydroxysteroids and C(19) ketosteroids was reduced in a concentration-dependent manner by ketoconazole, a P450(C17) inhibitor. This is the first detailed immunohistochemical mapping of P450(C17) in the brain and pituitary of any vertebrate. Altogether, the present data provide evidence that CNS neurons and pituitary cells can synthesize androgens.

Taenia solium cysticerci synthesize androgens and estrogens in vitro

Cysticerci from Taenia solium develop in the pig muscle and cause severe diseases in humans. Here we report on the capacity of T. solium cysticerci to synthesize sex steroid hormones. T. solium cysticerci were dissected from infected pork meat. Parasites were incubated for different periods in culture media plus antibiotics and tritiated steroid precursors. Blanks and parasite culture media were extracted and analyzed by thin-layer chromatography (TLC) in two different solvent systems. In some experiments, the scoleces were incubated separately. Results showed that T. solium cysticerci transform [(3)H]androstenedione to [(3)H]testosterone in a time-dependent manner. The production was confirmed in two different solvent systems. The incubation with [(3)H]testosterone yielded only small amounts of [(3)H]androstenedione. The recrystallization procedure further demonstrated that the metabolite identified by TLC was testosterone. The isolated scoleces incubated in the presence of [(3)H]androstenedione yielded [(3)H]testosterone and small quantities of [(3)H]17beta-estradiol. The results reported here demonstrate that T. solium cysticerci have the capacity to synthesize steroid hormones.

The rate of uptake of sex steroids from water by Tinca tinca is influenced by their affinity for sex steroid binding protein in plasma

Two experiments were carried out in which male and female tench Tinca tinca were placed in individual containers and tritiated steroids then added to the water. Water samples were collected over the next 6 or 7 h and the fish then sacrificed, bled and the gall bladder removed. Radioactivity was counted in all the samples. Over the course of the exposure period in the first experiment (7 h), radioactivity of 11‐ketotestosterone (11‐KT) in the water was depleted by 11%, 17,20β‐dihydroxypregn‐4‐en‐3‐one (17,20ß‐P) and 17,20α‐dihydroxypregn‐4‐en‐3‐one (17,20α‐P) by 28%, testosterone (T) by 56% and androstenedione (AD) by 68%. HPLC analysis of water samples at 3 h indicated that none of the steroids was extensively metabolized during the experiment. Females had a faster rate of uptake of AD than males. In the second experiment (6 h), radioactivity of cortisol in the water was depleted by 5%, 11‐KT by 7%, 17‐hydroxypregnen‐4‐ene (17‐P) by 17%, 17β‐oestradiol (E2) by 35%, T by 37% and AD by 44%. In both experiments, the amounts of radioactivity that were recovered from the gall bladder and plasma were positively correlated with the rate of disappearance of radioactivity from the water. The ability of the steroids to bind to sex steroid binding protein (SBP) of tench plasma was tested by incubating plasma with radioactive steroids and then separating bound and free with ice cold dextran‐coated charcoal. When plasma at a final dilution of 1 : 60 (v/v) was incubated with 5 nM of each steroid, the percentage of radiolabel bound to SBP was: T 48% AD 44%, E2 30%, 17‐P 17%, 11‐KT 13·2%, 17,20α‐P 10·3%, 17,20β‐P 4·5% and cortisol 0%. Saturation analysis established dissociation constants (Kd; mean ± s.e.) of 3·4 ± 0·4, 2·2 ± 0·2, 4·0 ± 0·3. 9·0 ± 2·8 and 51·8 nM and binding capacities (Bmax) of 201 ± 29, 201 ± 33, 165 ± 3, 187 ± 15 and 13·4 nM for T, AD, E2,17‐P and 17,20β‐P respectively. The ability of steroids to displace tritiated T and AD from SBP was in the rank order AD > T > E2 > 17,20αP = 17,20β‐P = 11‐KT = 17‐P > cortisol. Thus, the ability of tench plasma to bind certain steroids showed a relatively strong correlation with the ability of the fish to take up these steroids from water. Modelling of data for AD and 17,20β‐P helped to show why and how plasma binding had a strong influence on the rate of uptake (and hence release) of the steroids.

Metabolism of estrogens and androgens by scleractinian corals

Estrogens and androgens are steroids that act as reproductive hormones in vertebrates. These compounds have also been detected in reef-building corals and other invertebrates, where they are hypothesized to act as bioregulatory molecules. Experiments were conducted using labeled steroid substrates to evaluate metabolism of estrogens and androgens by coral homogenates. GC-MS analysis of 13C-labeled steroids showed that Montipora capitata coral homogenates or fragments could convert estradiol to estrone and testosterone to androstenedione and androstanedione, evidence that M. capitata contains 17β-hydroxysteroid dehydrogenase and 5α-reductase. When homogenates from three coral species and symbiotic dinoflagellates (zooxanthellae) were incubated with tritiated steroid substrates, metabolites separated by thin-layer chromatography confirmed that 17β-hydroxysteroid dehydrogenase activity occurred in all species tested. NADP + was the preferred cofactor in dehydrogenation reactions with coral homogenates. Reduction of estrone and androstenedione occurred at lower rates and aromatization of androgens was not observed. It is unclear whether estrogens detected previously in coral tissues are produced endogenously or sequestered in coral tissue from dietary or environmental sources. Previous studies have demonstrated that corals can take up estrogens from the water column overlying coral reefs. Considered in total, these observations suggest corals could alter the concentration or form of steroids available to reef organisms.

Steroid synthesis in follicular cells and induction of germinal vesicle breakdown in spotted wolffish (Anarhichas minor) oocytes

Incubation of follicular cells from postvitellogenic spotted wolffish ovaries with tritiated steroid precursors revealed that granulosa cells were able to convert 17-hydroxyprogesterone (17-P) to 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and androstenedione. Theca cells had limited ability to synthesise additional steroids from 17-P but converted 17,20β-P to 17,20β-P sulphate. Neither of the two cell layers was able to synthesise 5β-pregnane-3α,17,20β-triol-20-sulphate (3α,17,20β-P-5β-S) which is found in high concentrations in plasma. 17,20β,21-trihydroxy-4-pregnen-3-one (17,20β,21-P), 17,20β-dihydroxy-5β-pregnan-3-one (17,20β-P-5β) and 17,20β-P were most potent in inducing germinal vesicle breakdown (GVBD). Sulphation of 17,20β-P resulted in loss of GVBD inducing activity.

Follicle-Stimulating Hormone Regulates Steroidogenic Enzymes in Cultured Cells of the Chick Embryo Ovary

This investigation addresses the potential regulation of enzymes involved in the biosynthesis of steroid hormones during early stages of gonadal development by follicle-stimulating hormone (FSH). Gonadal cells of 10-day-old chick embryo and cells of the left ovary of 18-day-old chick embryo were cultured for 60 h in a defined medium with or without the addition of FSH (2.0 IU/ml). At the end of the culture, cells were recovered and evaluated by biotransformation of tritiated steroid precursors and mRNA levels were evaluated by RT-PCR. The production of estrone from androstenedione was increased in the FSH-treated cells, both human FSH (hFSH) and recombinant human FSH (rhFSH), indicating a stimulatory effect on aromatase (P450arom). Similarly, the intensity of the band corresponding to P450arom mRNA was higher in hFSH and rhFSH than in control and chorionic gonadotropin (hCG) groups. The P450arom stimulation was observed in the ovary of 10- and 18-day-old chick embryo. The transformation of dehydroepiandrosterone to androstenedione was taken as evidence of 3β-hydroxysteroid dehydrogenase function. This enzyme was stimulated in the cultured ovarian cells of 18-day-old chick embryos treated with hFSH and rhFSH compared with controls. The production of pregnenolone in the mitocondrial fraction of 18-day-old chick embryo ovary was increased when cultured with hFSH and rhFSH. This observation together with the increase in the band intensity corresponding to mRNA of P450 cholesterol side-chain cleavage indicates stimulation by FSH treatment; hCG produced a similar effect. Somatic cells of the medullary cords are proposed to be FSH target cells in the ovary of the chick embryo.

Human granulosa cells are a site of sulphatase activity and are able to utilize dehydroepiandrosterone sulphate as a precursor for oestradiol production.

Dehydroepiandrosterone sulphate (DHEAS) is the most abundant androgen in the circulation and in ovarian follicular fluid. A steroid sulphatase accepting DHEAS as a substrate has been identified in the follicle, but the cellular location has not been determined. As DHEAS is also a potential source of oestrogen for endocrine-dependent tumours, a potent steroid sulphatase inhibitor oestrone-3-O-sulphamate (EMATE) has been developed which inhibits this activity in rat liver and mammary tumour. The aim of this study was to investigate human granulosa cells as a site of steroid sulphatase activity, to determine whether DHEAS can be utilized as a precursor for oestrogen synthesis and to investigate the inhibitory capacity of EMATE in these cells. Conversion of DHEAS to DHEA was assessed in luteinized granulosa cells by tritiated steroid assay following incubation with or without LH or insulin and steroid accumulation in the medium measured by RIA. The effects of EMATE were assessed by addition of a range of doses during the measurement of conversion of DHEAS to DHEA. Cells from three sizes of small follicles from an unstimulated ovary were also assessed for their ability to produce oestradiol from DHEAS. Sulphatase enzyme activity was present in all cells; the mean conversion of tritiated DHEAS to DHEA was 50% (range 4-65%). LH and EMATE inhibited and insulin stimulated this activity. Addition of DHEAS to granulosa cells caused a dose-dependent increase in oestradiol and androstenedione production with no change in progesterone concentration. LH increased the accumulation of oestradiol in the medium. DHEAS also stimulated oestradiol production by granulosa cells from small follicles. This is the first demonstration that granulosa cells are a site of sulphatase activity and that DHEAS can be utilized as a substrate for androstenedione and oestrogen production. This may be of physiological importance for both normal folliculogenesis and oestrogen-dependent tumour growth.

Androgen-induced changes in Leydig cell ultrastructure and steroidogenesis in juvenile African catfish, Clarias gariepinus.

The present report focuses on the mechanism(s) involved in the steroid-induced decrease of androgen production in immature African catfish testes that was observed in previous studies. Juvenile animals were implanted with Silastic pellets containing different 11-oxygenated androgens (11-ketotestosterone, KT; 11 beta-hydroxyandrostenedione, OHA; 11-ketoandrostenedione, KA), testosterone (T) or estradiol-17 beta (E2). Control groups received steroid-free pellets. Two weeks later, testis tissue fragments were either incubated with increasing concentrations of catfish luteinizing hormone (LH), or incubated with [3H]-pregnenolone ([3H]-P5) or [3H]-androstenedione ([3H]-A). Tissue fragments were also prepared for the quantitative assessment of Leydig cell morphology. Most of the parameters studied were not affected significantly by implantation of E2. Implantation of all androgens inhibited both the basal and the LH-stimulated androgen secretory capacity in vitro. This was associated with a reduced size of the Leydig cells and loss of half of their mitochondria. The studies on the metabolism of tritiated steroid hormones indicated that steroidogenic steps prior to 11 beta-hydroxylation, probably C17-20 lyase activity, were affected by all androgens. Although the effects of 11-oxygenated androgens and T on Leydig cells were mostly similar, previous work showed that only the 11-oxygenated androgens stimulated spermatogenesis, suggesting that distinct mechanisms of action are used by 11-oxygenated androgens and T. These mechanisms, however, seem to merge on the same target(s) to impair Leydig cell androgen production. Such a negative feedback mechanism may be of relevance in the context of the decline in androgen secretion per milligram testis tissue that accompanies the first wave of spermatogenesis in pubertal African catfish.

Isolation and Characterization of a Novel cDNA Encoding a Human UDP-Glucuronosyltransferase Active on C19 Steroids

To isolate cDNA clones encoding novel UGT2B enzymes, human prostate and LNCaP cell cDNA libraries were screened using a pool of steroid-specific UGT2B cDNA probes. In approximately 10(6) recombinants, we isolated 3 cDNA clones of 2.1 kilobases that encode a novel UGT2B enzyme. UGT2B17 is 95% identical with UGT2B15 and 91% identical with UGT2B8. Primary structure analysis of UGT2B17 based on the nucleotide sequence revealed a putative amino-terminal membrane insertion signal peptide, a carboxyl-terminal membrane-spanning region, and three potential asparagine-linked glycosylation sites. UGT2B17 cloned in the pBK-CMV expression vector was transfected into HK293 cells to obtain a stable clonal cell line expressing a high level of the active 53-kDa UGT2B17 enzyme. Of the over 60 endogenous and exogenous substances tested, 25 compounds revealed reactivity. The major substrates are eugenol > 4-methylumbelliferone > dihydrotestosterone > androstane-3alpha, 17beta-diol (3alpha-diol) > testosterone > androsterone (ADT). The apparent Km values obtained with tritiated steroids in intact cells were 0.4 microM for ADT, 0.7 microM for dihydrotestosterone, 1.0 microM for 3alpha-diol, and 3.4 microM for testosterone. Southern blot analysis of reverse transcription-polymerase chain reaction products revealed expression of UGT2B17 mRNA in various tissues including the liver, kidney, testis, uterus, placenta, mammary gland, adrenal gland, skin, and prostate. UGT2B17 is the first human uridine diphosphoglucuronosyltransferase enzyme expressed in extrahepatic tissues to have a specificity for ADT as well as testosterone, dihydrotestosterone, and 3alpha-diol.

Myometrial steroid concentration and oxytocin receptor density in parturient women at term

The aim of this study was to measure oxytocin receptor concentration in myometrial tissue from term pregnant women with normal and dysfunctional labor and to relate this concentration to the progress of labor and to the levels of estradiol and progesterone in the same myometrium. Myometrial biopsies were obtained from 50 term pregnant women undergoing cesarean section. The patients were categorized as follows: not in labor, normal labor, successful oxytocin-augmented labor, and oxytocin-resistant labor. Specific binding of [ 3H]oxytocin to high-affinity sites in membrane preparations from myometrial tissues was determined. Estradiol and progesterone were assayed using tritiated steroids with a sensitive radioimmunoassay technique. Oxytocin receptor density was significantly lower in oxytocin-resistant labor compared to successful oxytocin-augmentated labor ( P < 0.04) and to spontaneously active normal labor ( P < 0.02). Oxytocin receptor concentration was also significantly lower in non-labor patients compared to normal spontaneous labor ( P < 0.01), and successful oxytocinaugmented labor ( P < 0.02). There was a positive relationship between the progress of cervical dilatation (cm/h) and oxytocin receptor density in the myometrium ( r = 0.408, P < 0.025). The concentration of progesterone and estradiol in the pregnant myometrium did not differ in patients with different types of labor or with the state of uterine contractile activity. Our results suggest that individual myometrial sensitivity is an important determinant of the response to administered oxytocin in humans. Furthermore, myometrial oxytocin receptor expression in vivo seems not be related to ovarian steroid concentration in the myometrium. The low oxytocin receptor density in oxytocin-resistant dystocia needs further investigation.

Excretion of Free and Conjugated Steroids in Rainbow Trout (Oncorhynchus mykiss): Evidence for Branchial Excretion of the Maturation-Inducing Steroid, 17,20β-Dihydroxy-4-pregnen-3-one

The goal of this study was to identify excretory routes of three main steroids produced by sexually mature male and female rainbow trout: 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P), sulfated 17,20β-P (17,20β-P-S), and testosterone glucuronide (TG). Spermiating males or maturing trout were cannulated via the dorsal aorta and urinary bladder and injected with tritiated steroids. Blood, water, and urine were sampled over the next 12 hr when the fish were killed and bile was collected. The identities of the excreted products were determined by anion-exchange chromatography, reverse-phase high-performance liquid chromatography, enzyme hydrolysis, acid solvolysis, microchemical modification, and thin-layer chromatography. Following the injection of tritiated 17,20β-P, 25% of the radioactivity rapidly appeared unmodified in the water; 15% appeared slowly in the urine, mainly as 17,20β-P-S; and 40% was recovered in the bile, mainly as 17,20β-P-glucuronide. 17,20β-P was shown to be released into the water via the gills. Over the 12-hr sampling period, 20% of the 17,20β-P released into the water was taken up again by the fish (also branchially). A mathematical analysis showed that 40% of the 17,20β-P would have been released into the water in the absence of uptake. Following the injection of tritiated 17,20β-P-S, 63% appeared very rapidly, in an unmodified form, in the urine, and 15% was recovered in the bile. Following the injection of tritiated TG, 9% appeared slowly, mainly untransformed, in the urine, and 59% was recovered in the bile. These results show that the three types of steroids are released into the water by three different routes: free steroids, gills; sulfated steroids, urine; and glucuronidated steroids, bile.

Steroid Metabolism in Theca Externa Cells from Preovulatory Follicles of Domestic Hen (Gallus domesticus)

The purpose of this study was to evaluate the ability of theca externa cells from preovulatory follicles of the domestic hen to metabolize tritiated steroid precursors. Theca externa cells were isolated from ovarian preovulatory follicles at three different developmental stages; F1 (35 mm), F3 (26 mm), and F5 (13 mm). Tritiated pregnenolone (P5), progesterone (P4), dehydroepiandrosterone (DHEA), androstenedione (A4), and testosterone (T) were employed as precursors and their metabolic products were evaluated after separation by thin-layer chromatography. The major metabolite of P5 by theca externa cells was 5-pregnen-3β,20β-diol (F5, 47%; F3, 39%; and F1, 24%), but minimal quantities of P4 were detected. Progesterone metabolism yielded mainly 4-pregnen-20β-ol-3-one (F5, 52%; F3, 34%; and F1, 49%). When DHEA was used as precursor, A4 was produced in higher amounts (F5, 29%; F3, 23%; and F1, 11%) than estrone (E1) (F5, 1.5%; F3, 0.9%; and F1, 0.4%). Androstenedione was mainly transformed into E1 (F5, 11.9%; F3, 12.2%; and F1, 0.2%), but lower quantities of T and 17β-estradiol (E2) were found. Testosterone was actively transformed into A4 (F5, 50%; F3, 50%; and F1, 30%), but a low transformation to E2 (F5, 1.9%; F3, 1.7%; and F1, 1.4%) and E1 (F5, 2%; F3, 1%; and F1, 0.5%) was found. These results show that theca externa cells from preovulatory follicles of hen have enzymatic activities of 20β-reductase (from P5 and P4), 3β-hydroxysteroid dehydrogenase/5-4 isomerase (from P5 and DHEA), 17β-hydroxysteroid dehydrogenase (from A4 and T), and aromatase (from A4 and T). Furthermore, the enzyme activities decrease with follicular maturation, except for 20β-reductase which is constant. These data support the concept that theca externa cells have the ability to synthesize different steroids than reported in theca externa cells. In addition, since theca externa cells did not show the capacity to produce androgens but these steroids were aromatized to estrogens by these cells, it was suggested that the interaction between theca interna cells and theca externa cells occursin vivo,thus supporting the multicellular theory for estrogen production.

1,25-Dihydroxyvitamin D3 binding sites in the eye and associated tissues of the green lizard Anolis carolinensis

Receptor autoradiography was used for the demonstration of specific binding of the tritiated steroid hormone 1,25-dihydroxyvitamin D3 in the eyes and associated tissues of Anolis carolinensis. A 100-fold excess of non-labelled 1,25-dihydroxyvitamin D3 abolished specific nuclear binding of tracer. Nuclear [3H]-1,25-dihydroxyvitamin D3 binding was present in all animals in the retina stratum ganglionare and stratum nuclear externum as well as in the cornea; however, binding was absent in the optic nerve, except in cells of the surrounding arachnoidea. Additional cranial tissues such as chondrocytes in the sclera, parasphenoid, skeletal muscle cells, and epithelial cells of the lacrimal and Harderian glands exhibited nuclear labelling. The results suggest that 1,25-dihydroxyvitamin D3 has genomic regulatory actions that involve cell proliferation, differentiation, and functions of certain cells of the eye and associated cranial tissues. The presence of vitamin D receptors in tissues of the eye and skeletal muscle in the reptile is in part different from that observed in mammals. In general, receptors for vitamin D and related target tissues appear to be even more extensive in lizards than has been observed in rodents, which may reflect a more extensive dependency of these tissues on solar environment and active seasonal and circadian regulation.

Changes in oestrogen, progesterone and epidermal growth factor receptor concentrations and affinities during the oestrous cycle in the normal mammary gland and uterus of dogs.

Changes in the concentrations and affinities of receptors for oestrogen (ER), progesterone (PR) and epidermal growth factor (EGF-R) were studied in mammary glands of healthy bitches with regard to age, the location in the mammary chain and the stage of the oestrous cycle. Uterus was used as the reference tissue for the evaluation of steroid receptors. Mammary and uterine samples from 7 healthy bitches were taken at five stages of the oestrous cycle in such a way that all the locations in the mammary chain were represented at each stage of the cycle (10 samples/dog). ER, PR and EGF-R were detected by biochemical assays using increasing concentrations of tritiated (steroids) or iodinated (EGF) ligands. A significant direct correlation was found between the ER and PR concentrations for mammary and uterine samples. No significant correlation was found between the steroid receptors and EGF-R concentrations. Mammary ER concentrations were significantly higher in bitches of 5 years of age or older than in younger ones; in posterior glands (4th and 5th pairs) than in anterior glands; and in the mid-luteal phase. Mammary PR did not vary significantly with age or location but was significantly lower in the early luteal phase than in other phases. A similar decrease in PR concentrations was observed in the uterus during the early luteal phase and uterine ER and PR concentrations were very low in the mid-luteal phase. Mammary EGF-R were not significantly higher in the early or mid-luteal phase than in pro-oestrus or anoestrus. The differences observed between the uterine and mammary steroid receptor concentrations during the oestrous cycle could be due to different mechanisms for regulating steroid receptor expression in the two tissues. Mammary EGF-R concentrations may be linked, as in other species, to cellular proliferation and/or to the serum progesterone concentrations.

Exchange labelling strategies for tritiated steroids

AbstractA number of commonly used tritium exchange procedures for steroids are described. Reference is also made to other available methods.