Identification and partial purification of a germ cell factor that stimulates transferrin secretion by Sertoli cells.
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Animals↗
Biomedical subjects
C Y Cheng
Publications and source records attributed to C Y Cheng.
The identification of gonadotrophin surge inhibiting factor and its role in the regulation of pituitary gonadotrophin secretion.
Evidence from several laboratories suggests that the ovaries of rats, pigs, monkeys and women produce a non-steroidal factor which inhibits or attenuates the mid-cycle gonadotrophin surge. This substance is present in follicular fluid and we have called it gonadotrophin surge inhibiting factor (GnSIF). Utilizing a rat pituitary cell bioassay, we have monitored GnSIF activity during purification from porcine follicular fluid (PFF). Rat pituitary cells in short-term culture were incubated with GnSIF for 48 h, washed and then exposed to gonadotrophin releasing hormone (GnRH) plus GnSIF for 4 h. GnSIF activity was defined as suppression of GnRH-stimulated LH secretion over 4 h, and inhibin activity was expressed as suppression of basal follicle stimulating hormone (FSH) secretion over 48 h. Approximately 5 l of PFF was fractionated through heparin-Sepharose, Q-Sepharose, Mono-S, hydroxylapatite, and gel permeation chromatography steps. This fractionation removes all detectable inhibin and follistatin immunoactivity and bioactivity. The purified GnSIF inhibits GnRH-stimulated LH secretion with little or no effect on basal FSH release. In summary, we have obtained a purified preparation of GnSIF which contains undetectable inhibin and follistatin. As such, GnSIF appears to be distinct from other known gonadal proteins regulating reproductive function, and may participate along with the inhibins, activins and follistatins in the gonadal regulation of pituitary gonadotrophin secretion.
Animals↗
Regulation of Sertoli cell alpha 2-macroglobulin and clusterin (SGP-2) secretion by peritubular myoid cells.
alpha 2-Macroglobulin and clusterin are two putative Sertoli cell secretory products; however, the regulator(s) modulating their secretion by Sertoli cells is not known. Recent studies from this laboratory have shown that the testicular alpha 2-macroglobulin, unlike its liver homologue, is not an acute-phase reactant and its concentration is not affected by acute inflammation. We sought to determine whether FSH, testosterone, and other biomolecules would affect the secretion of alpha 2-macroglobulin and clusterin by Sertoli cells as well as whether peritubular myoid cells would affect the secretion of these proteins by Sertoli cells. It was noted that Sertoli cells cultured in vitro secreted increasing amounts of alpha 2-macroglobulin and clusterin as a function of time. FSH (50-1000 ng/ml) and testosterone (10(-11)-10(-5) M) had no apparent effect on the secretion of alpha 2-macroglobulin and clusterin by Sertoli cells. Addition of interleukin-6 to Sertoli cell-enriched cultures, in doses known to stimulate alpha 2-macroglobulin secretion by hepatocytes, did not affect the alpha 2-macroglobulin secretion. However, dexamethasone at 10(-7)-10(-5) M stimulated alpha 2-macroglobulin secretion by Sertoli cells dose-dependently while the addition of interleukin-6 had no synergistic effect on dexamethasone-stimulated alpha 2-macroglobulin secretion. These findings suggest that the synthesis and/or secretion of alpha 2-macroglobulin by Sertoli cells is regulated by a mechanism distinct from that of the liver.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Localization and molecular heterogeneity of sulfated glycoprotein-2 (clusterin) among ventral prostate, seminal vesicle, testis, and epididymis of rats.
In the rat reproductive tract, sulfated glycoprotein 2 (SGP-2) is present in the ventral prostate, seminal vesicle, testis, and epididymis. In the ventral prostate, SGP-2 is associated with the process of programmed cell death, while in the testis and epididymis a role for SGP-2 in sperm maturation has been proposed. Available information suggests that there are both inter- and intra-organ variations in SGP-2 localization, molecular forms, and response to androgen ablation. In the present study, localization of SGP-2 within the ventral prostate, seminal vesicle, and epididymis was compared by immunohistochemistry. In the ventral prostate of intact rats, immunoreactive SGP-2 was confined to a discrete population of epithelial cells lining the proximal ducts. Epithelial cells in other regions of the ventral prostate did not stain for SGP-2. A similar staining pattern was observed for the seminal vesicle; a small population of SGP-2-expressing epithelial cells was found in epithelium that did not stain for SGP-2. The epididymis also demonstrated a non-uniform staining pattern. The caput displayed strong immunoperoxidase reaction over the apical membrane and stereocilia of all principal cells. Principal cells also showed variable degrees of cytoplasmic staining ranging from weak to strongly positive. The corpus and cauda showed a similar staining pattern. After castration, all epithelial cells in the ventral prostate and seminal vesicle became intensely positive for SGP-2 staining. In the caput and cauda epididymis there was an increase in the number of principal cells demonstrating strong intracellular staining for SGP-2. These results suggest that as observed previously in the regressing ventral prostate, increased intracellular SGP-2 staining may also be associated with the regressing epididymis and seminal vesicle. Differences in molecular forms of SGP-2 were investigated by two-dimensional Western and lectin blots. Molecular forms of SGP-2 differed between testis and epididymis but were similar between ventral prostate and seminal vesicle. Prostate and seminal vesicle forms of SGP-2 differed from those of both testis and epididymis. Analysis of terminal carbohydrate present on the various SGP-2 molecular forms also confirmed the existence of heterogeneity. These results demonstrate the presence of multiple molecular forms of SGP-2 in various organs of the male reproductive tract in rats and suggest a possible variation in functional activity and/or half-life of SGP-2 in these organs.
Animals↗
Preparation and analysis of isogenic mutants in the transferrin receptor protein genes, tbpA and tbpB, from Neisseria meningitidis.
Isogenic mutants were constructed in the tbpA and tbpB genes from Neisseria meningitidis strain B16B6, which code for the transferrin receptor proteins, Tbp1 and Tbp2. Insertion mutants of the tbpA and tbpB genes were obtained by shuttle mutagenesis and by in vitro cassette mutagenesis, respectively. The isogenic mutants were verified by Southern blot and Western blot analysis. Isogenic mutants deficient in Tbp1 or Tbp2 demonstrated a reduced transferrin binding activity in intact cells and total membranes but were incapable of utilizing transferrin iron for growth. Tbp1 could be isolated by affinity methods from the mutant lacking Tbp2 but isolation of Tbp2 from the mutant lacking Tbp1 required the presence of exogenous Tbp1.
Bacterial Outer Membrane Proteins↗
Micropurification of two human cerebrospinal fluid proteins by high performance electrophoresis chromatography.
Using C8 reversed-phase HPLC in conjunction with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we have fractionated proteins contained in human CSFs obtained from patients with schizophrenic disorders. When these proteins were electrophoretically blotted onto polyvinylidene difluoride membrane for direct N-terminal amino acid sequencing, several CSF proteins were identified; these included albumin, transferrin, apolipoprotein A-I, beta 2-microglobulin, and prealbumin. We have also identified two structurally related human CSF proteins designated cerebrin 28 (M(r) 28,000) and cerebrin 30 (M(r) 30,000) that have an N-terminal amino acid sequence of NH2-APPAQVSVQPNF and NH2-APEAQVSVQPLFXQ, respectively. Comparison of these sequences with existing database at Protein Identification Resource (R 32.0), GenBank (R 72.0), SWISS-PROT (R 22.0), and EMBL (R 31.0) indicated that they are unique proteins. These proteins were subsequently purified by high performance electrophoresis chromatography (HPEC) using an Applied Biosystems 230A HPEC system. A specific polyclonal antibody was prepared and an ELISA was established for cerebrin 30. It was noted that HPEC is a powerful tool to purify microgram quantities of proteins from human, rabbit, and rat CSFs. Using such a system, we have been able to micropurify as many as 10 proteins simultaneously in a single experiment because the elution of proteins occurred strictly according to their molecular weights. More importantly, we routinely obtained a recovery of > 90%. The potential use of this technology for micropurification of proteins was discussed.
Amino Acid Sequence↗
Germ cell control of testin production is inverse to that of other Sertoli cell products.
Recent studies have shown that germ cells can regulate testins, two newly identified Sertoli cell proteins that are associated with junctional complexes. To investigate this possibility, several parameters of Sertoli cell function were investigated over 2-120 days post exposure of the rat testes to x-rays (3 Grays). The irradiation-induced loss of spermatogonia resulted in a maturation-depletion process progressively affecting all germ cell classes. Testis weight began to decrease when the most numerous germ cell type (spermatids) began to decline. A complete or near complete recovery of spermatogenesis and of the testis weight had occurred by day 120 post irradiation. There was no significant change in FSH, epididymal androgen-binding protein, and tubule fluid levels during the first weeks after irradiation, when the seminiferious epithelium was depleted of spermatogonia and germ cells up to early spermatids. In contrast, when the number of the more mature forms of spermatids declined (between day 21 and 54), FSH rose and androgen-binding protein as well as fluid production declined. The subsequent recovery of these parameters was also highly correlated with the number of late spermatids. By contrast, testicular testin contents reacted to the depletion of germ cells with a biphasic increase; a doubling occurred when spermatogonia, spermatocytes, and early spermatids were absent (days 4-28), and a 7-fold rise occurred by day 37 when the number of late spermatids had decreased by 50%. By day 54, when the sperm counts had reached a nadir, testin contents had returned to levels corresponding to about four times the control levels; they progressively recovered thereafter. These observations support the postulate that germ cells negatively regulate testins. This possibility was investigated with in vitro experiments showing that addition of germ cell-conditioned medium to Sertoli cell monolayers inhibited testin secretion in a dose-dependent manner. In conclusion this study; 1) highlights the complex interplay between the various germ cell classes in the control of the Sertoli cell function in the adult testis; 2) establishes that germ cell effects may be opposite on different Sertoli cell products; 3) demonstrates that several classes of germ cells negatively control testicular testin contents; and 4) emphasizes the particular role of late spermatids in Sertoli cell regulation.
Androgen-Binding Protein↗
Multi-adjustable post-operative orthosis for congenital muscular torticollis.
A multi-adjustable torticollis orthosis is described for the post-operative bracing of patients after surgical correction of congenital muscular torticollis. The orthosis can be put on in the early post-operative period and the head and neck position can be maintained in the corrected, and later over-corrected position by the built-in multi-adjustable joint-mechanism. The details of the manufacturing are described. Twenty-five patients (13 girls and 12 boys) from age 1 to 22 with congenital muscular torticollis were fitted with the orthosis post-operatively for an average duration of 10 weeks. Satisfactory compliance with the orthosis was found in 23 cases. Complications were minimal (3 cases) and were related to scalp irritation which improved after minor adjustments of the halo.
Adolescent↗
Urinary clusterin in chronic nephrotoxicity in the rat.
The excretion of clusterin was compared with that of N-acetyl-beta-glucosaminidase (NAG) in rats given gentamicin daily for 2 months to determine whether clusterin excretion stays elevated after NAG excretion falls during chronic gentamicin administration. Clusterin was measured by radioimmunoassay and NAG by the hydrolysis of 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide. Gentamicin at 110 mg/kg was given daily for 44 days and thereafter, at 90 mg/kg daily. The excretion rate of both proteins rose rapidly, peaked, and then declined; however, the clusterin values stayed significantly above control values for the entire study, whereas NAG values were close to normal during the last 10 days, even though tubulointerstitial disease was active at that time. For this reason, the further evaluation of clusterin as a marker of renal tubular cell injury or death is warranted.
Acetylglucosaminidase↗
Chronic inflammatory response in the rat can be blocked by bindarit.
In the rat, injection of Freund's complete adjuvant was accompanied by a significant increase in concanavalin A (Con A)-reactivity of selected plasma proteins along with an increase in concentrations of selected proteins known as acute phase proteins. We have evaluated the effect of bindarit, (2-[(1-benzyl-indazol-3-yl)methoxy]-2-methyl propionic acid), on the expression of alpha 2-macroglobulin, a known acute-phase protein in the rat. This compound has previously been shown to inhibit heat-induced denaturation of rat serum albumin and to strongly reduce the secondary phase response of adjuvant induced arthritis. Adult rats were induced with chronic inflammation by injection with Freund's complete adjuvant. Bindarit was administered to the chronic inflamed rats as a 0.5% medicated diet. Indomethacin, given by gavage daily at a dose of 1 mg/kg body weight, was used as a reference drug. Qualitative and quantitative changes of Con A-reactive proteins and alpha 2-macroglobulin were examined by lectin- and immuno-blots, and by radioimmunoassay. It was noted that the concentration of alpha 2-macroglobulin increased in rats with adjuvant induced arthritis. The addition of bindarit and indomethacin were able to reduce the concentration of alpha 2-macroglobulin as well as the Con A-reactivity of various proteins to normal level 37 days following treatment. We have also examined the effects of chronic inflammation on the levels of rat clusterin, a testicular and serum glycoprotein related to programmed cell death, tissue regression, and complement cascade reaction; and testibumin, a testicular FSH and testosterone-responsive protein with unknown function. It was noted that chronic inflammation did not induce significant changes in both the clusterin and testibumin concentrations in these experimental groups. The involvement of protein glycosylation and denaturation in the production of new antigenic determinants, their role in the development of chronic inflammatory disease and the potential use of bindarit to investigate the relationship between abnormal glycosylation and autoimmune disease were discussed.
Acute-Phase Proteins↗
Selective reversible and irreversible ligands for the kappa opioid receptor.
(+-)-(5 beta,7 alpha,8 beta)-3,4-Dichloro-N-methyl-N-[3-methylene-2- oxo-8-(1-pyrrolidinyl)-1-oxaspiro[4,5]dec-7-yl]benzeneacetamide (14) and its (5 alpha,7 alpha,8 beta) diastereomer 15 have been synthesized from 1,4-cyclohexanedione monoethylene ketal (1) in 10 steps. Compound 14, which we have designated SMBU-1, was found to bind with moderate affinity (Ki = 109 nM) and good selectivity (mu/kappa = 29) to the kappa opioid receptor, while 15 was only 1/10 as potent as a kappa ligand. Preincubation of brain membranes with 14 resulted in wash-resistant inhibition of kappa-receptor binding (69 +/- 6% of control at 10(-6) M). The ketone precursor trans-N-methyl-N-[5-oxo-2-(1- pyrrolidinyl)cyclohexyl]benzeneacetamide (12) showed a higher kappa-affinity (Ki = 78 nM) and a much higher kappa-selectivity (mu/kappa = 166) than 14. Compound 10, the ethylene ketal precursor of 12, exhibited a similar receptor binding profile to 14, with increased kappa-selectivity (mu/kappa = 55), while ketal 11, being a regioisomer of 10 and an oxygen isostere of the kappa-selective analgesic spiradoline (U-62,066), demonstrated the highest kappa-affinity (Ki = 1.5 nM) and kappa-selectivity (mu/kappa = 468) observed in this series.
Benzeneacetamides↗
The effect of 3,3-di-pyridyl-methyl-1-phenyl-2-indolinone on the nerve terminal currents of mouse skeletal muscles.
The effects of 3,3-dipyridyl-methyl-1-phenyl-2-indolinone (DPMPI), a new cognition enhancer, on perineural waveforms were assessed on triangularis sterni nerve-muscle preparations in the mouse. The perineural waveforms were recorded with extracellular electrodes placed in the perineural sheaths of motor nerves. At 64.5 microM, DPMPI decreased the fast potassium current of the nerve terminal. The sodium current, calcium currents and calcium-dependent potassium current of the nerve terminal were not affected. At a greater concentration (215 microM), DPMPI decreased all of the components of the waveforms associated with sodium, potassium and calcium currents. It is concluded that DPMPI affects potassium, as well as sodium currents in the nerve terminal. The effect may contribute to its pharmacological actions on synaptic transmission.
Animals↗
The effect of 3,3-dipyridylmethyl-1-phenyl-2-indolinone on the neuromuscular transmission in the rodent skeletal muscles.
The effects of a cognition enhancer, 3,3-dipyridylmethyl-1-phenyl-2-indolinone (DPMPI) (21.5-645 microM), on neuromuscular transmission were studied electrophysiologically on diaphragms of mouse and rat and the soleus muscle of rat. The drug DPMPI (21.5-645 microM) increased both direct and indirect twitch tension of mouse diaphragm. It also increased (a) the frequency of miniature endplate potentials and (b) the quantal content of endplate potential. However, DPMPI (64.5 microM) affected neither the amplitude of the directly elicited action potential of soleus muscle in the rat nor the magnitude of the resting membrane potential of mouse diaphragm, although DPMPI (215 microM) decreased the amplitude of the compound action potential of phrenic nerve. Based on these results, it is concluded that DPMPI had several effects on neuromuscular transmission, i.e. it (a) facilitated the transmitter releasing process of the motor nerve terminal, (b) decreased the conduction in the phrenic nerve and (c) increased the directly elicited twitch tension.
Action Potentials↗
Rat Sertoli cell clusterin, alpha 2-macroglobulin, and testins: biosynthesis and differential regulation by germ cells.
Clusterin, alpha 2-macroglobulin and testins are three novel Sertoli cell proteins whose physiological functions may be related to cell-cell interactions in the seminiferous epithelium of the testis. We have demonstrated the biosynthesis of clusterin, alpha 2-macroglobulin, and testins by Sertoli cells in vitro using pulse-chase labeling analysis. For clusterin, two precursors with an apparent molecular weight (M(r)) of 72,000 (PH) and 66,000 (PL) were detected in the Sertoli cell cytosol in addition to the alpha (M(r) 43,000) and beta (M(r) 35,000) subunits of the mature protein. However, the precursors were not secreted into the medium since only the alpha and beta subunits of clusterin were detected. For alpha 2-macroglobulin and testins, no precursor molecules were detected either in the Sertoli cell cytosol or culture medium. The polarized secretory pattern of these proteins and their regulation by follicle stimulating hormone (FSH) and testosterone (T) were examined using a bicameral culture chamber that mimics the in vivo physiological conditions. Clusterin was secreted almost exclusively into the apical chamber of the bicameral culture unit with an apical:basal ratio of 30:1. In contrast, alpha 2-macroglobulin and testins had an apical:basal ratio of 1:1 and 1.5:1, respectively. Thus, the polarized secretory pattern for clusterin is different from alpha 2-macroglobulin and testins. It was noted that FSH and T, the known Sertoli cell regulators, did not affect the secretion of either clusterin or alpha 2-macroglobulin. Due to the morphological intimacy between Sertoli cells and germ cells in the adluminal compartment of the testis, the effects of germ cell-conditioned medium were investigated. Addition of germ cell-conditioned medium (1-30 micrograms protein) to the apical chamber of the bicameral culture unit caused a dose-dependent inhibition of clusterin and testins apical secretion and a slight but statistically significant stimulation of their basal secretion. In contrast, the secretion of alpha 2-macroglobulin by Sertoli cells was stimulated both apically and basally. These observations suggest that germ cell-conditioned medium contains a biological factor(s) that differentially regulates the bidirectional secretion of Sertoli cell proteins. These studies therefore reveal the complicated regulatory processes involved in cell-cell interactions in the seminiferous epithelium.
Animals↗
Testins are localized to the junctional complexes of rat Sertoli and epididymal cells.
UNLABELLED: Testin I and Testin II were originally identified as Sertoli cell products with similar NH2-terminal amino acid sequences. Secretion of testins is stimulated by testosterone in Sertoli cell-enriched cultures. By contrast the secretion of testins from intact seminiferous tubules appears to be inversely related to germ cell number. In the present study testin antiserum that recognized both Testin I and Testin II ("testin") was used to localize these proteins in tissue secretions by immunofluorescence. Testin was localized at the base of the seminiferous epithelium at Sertoli-Sertoli junctions. Fluorescence also appeared to be located at the sites of interaction between spermatoids and Sertoli cells. A punctate pattern of fluorescence was also present in the cytoplasm of Leydig cells; without electron microscopic studies it was not possible to determine which structures the antibodies bound to in these cells. In the epididymis the reaction product was localized at the apices of the epithelial cells adjacent to the lumen at the sites of known junctional complexes. A variety of positive and negative controls indicated that staining was specific for testins. CONCLUSIONS: This is the first study to associate testins with junctional complexes. Relative to other junctional proteins, testins are unusual because of their small size and because they are secreted proteins.
Animals↗
Expression of 11 beta-hydroxylase and 21-hydroxylase in long-term cultures of bovine adrenocortical cells requires extracellular matrix factors.
In order to elucidate mechanisms for the loss of expression of 11 beta-hydroxylase and 21-hydroxylase, induction of these genes in long-term cultures of bovine adrenocortical cells was reassessed and compared with induction of 17 alpha-hydroxylase. We previously showed that both 11 beta- and 21-hydroxylases require insulin-like growth factor-I (IGF-I) as well as cAMP for induction; these are the only factors needed by primary cultures. Cells at population doubling level 10 grown on fibronectin-coated polystyrene dishes and incubated with cholera toxin and IGF-I did not express 11 beta-hydroxylase and 21-hydroxylase. They showed a truncated steroidogenic pathway, converting 25-hydroxycholesterol to some 11-deoxycortisol but little cortisol. However, when population doubling level 10 cells were grown for 5 days in extracellular matrix Matrigel, cholera toxin and IGF-I induced a complete steroidogenic pathway to cortisol. Northern blotting also showed that expression of 11 beta-hydroxylase messenger RNA (mRNA) after cholera toxin/IGF-I induction was observed only in cultures grown in Matrigel and was undetectable in cultures grown on plastic. 21-Hydroxylase mRNA was observed in cultures grown on plastic but was greatly enhanced by Matrigel; however, 17 alpha-hydroxylase mRNA was induced to a similar extent with and without Matrigel. In other middle passage cultures, whether grown as mass cultures, SV40 T antigen-transfected clones, or normal (nontransfected) clones, cells did not express 11 beta-hydroxylase except when grown in Matrigel; 21-hydroxylase was low and expression was enhanced by Matrigel, whereas 17 alpha-hydroxylase expression was unaffected. As previously determined, in late-passage cells and clones only side-chain cleavage enzyme and 3 beta-hydroxysteroid dehydrogenase activities were detected and 17 alpha-hydroxylase was not expressed. In such cells 11 beta-hydroxylase and 21-hydroxylase were also not expressed, even in the presence of Matrigel. Thus, prior to the previously described loss of expression of 17 alpha-hydroxylase, Matrigel permits the cholera toxin/IGF-I-induced expression of a complete steroidogenic pathway in bovine adrenocortical cells in long-term culture.
Adrenal Cortex↗
Rat epididymal retinoic acid-binding protein: development of a radioimmunoassay, its tissue distribution, and its changes in selected androgen-dependent organs after orchiectomy.
Rat epididymal retinoic acid-binding protein (EP-RABP) has been purified to apparent homogeneity from extracts of the epididymis by HPLC. N-Terminal amino acid sequence analysis revealed that the HPLC-purified protein consisted of two molecular variants, in that one has three extra N-terminal amino acids of NH2-TEG. These two molecular variants were subsequently separated by high performance electrophoresis chromatography. A specific and sensitive RIA has been developed to quantify this protein in various organ extracts of both male and female rats. Rat EP-RABP is a male-specific protein, since it was detected in male organ extracts, including epididymis, testis, prostate, seminal vesicles, liver, spleen, and brain, with a negligible concentration in the female liver and spleen. It was noted that this protein was absent in the systemic circulation of both male and female rats. It was first detected in the epididymis and testis of rats at 22 and 27 days of age, respectively. Both the concentrations (micrograms per g tissue) and the organ content (micrograms per organ pairs) of this protein in the testis and epididymis increased progressively with an increase in age and reached the highest levels at 60 and 120 days of age, respectively. At 120 days of age, its concentrations in all organs examined, with the exception of the epididymis, showed a dramatic decrease compared to levels in rats at 60 days of age. Orchiectomy decreased its concentrations in the caput, corpus, and cauda epididymis and in the ventral prostate, but had no apparent effect on seminal vesicles. Administration of dihydrotestosterone to castrated rats restored only 30% of the level of this protein in the caput epididymis compared to that in intact animals, but had no apparent effect on the corpus, cauda epididymis, or ventral prostate. These observations suggest that this protein is under multihormonal control in the epididymis and selected androgen-dependent organs.
Amino Acid Sequence↗
Expression of 17 alpha-hydroxylase and 3 beta-hydroxysteroid dehydrogenase in fetal human adrenocortical cells transfected with SV40 T antigen.
Primary fetal human adrenocortical cells of definitive zone origin were transfected by electroporation with pSV3neo, a plasmid coding for SV40 T antigen and neo, which confers resistance to the antibiotic G418. The clones obtained proliferated for 30 to 40 population doublings after isolation when grown under standard medium conditions, and then entered 'crisis'. When early-passage clones were incubated with cyclic AMP (1:1 N6-monobutyryl and 8-bromo analogues), cell rounding was observed, as in primary cultures of human adrenocortical cells. As previously shown in bovine adrenocortical cells, rounding was inhibited with a monoclonal antibody against urokinase plasminogen activator but not with a monoclonal antibody against tissue plasminogen activator. The regulation of the steroidogenic pathway in clones was investigated. The effects of cyclic AMP and activation of protein kinase C were examined in cells maintained in defined medium or in the presence of serum. 17 alpha-Hydroxylase was strongly induced by cyclic AMP, as evidenced by Northern blotting and by the conversion of progesterone or 25-hydroxy-[1,2-3H]cholesterol, this induction being blocked by low concentrations of 12-O-tetradecanoylphorbol-13-acetate (TPA). Cholesterol side-chain cleavage enzyme was strongly induced by cyclic AMP, and clones also showed low activities of 21-hydroxylase and 11 beta-hydroxylase. Under all circumstances levels of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), as assessed by Northern blotting or by conversion of 25-hydroxycholesterol, were very low. 3 beta-HSD was not induced by cyclic AMP or TPA alone, but was induced by the combination of the two agents. The regulation of 17 alpha-hydroxylase and 3 beta-HSD resembles that previously described in primary cultures of human fetal adrenocortical cells. Thus, transfection with SV40 T antigen resulted in the production of clones which preserve the unique characteristics of the human adrenal cortex.
3-Hydroxysteroid Dehydrogenases↗