Intrafollicular paracrine function of ovarian androgen

During early antral stages of follicular development, granulosa cells are constantly exposed to a high local concentration of androgen. C19 steroids biosynthesized in LH-stimulated theca cells traverse the lamina basalis, enter granulosa cells and accumulate in antral fluid. Within the granulosa cell layer, androgens are metabolized to other androgens, oestrogens and catechol oestrogens, all of which may fulfil intrafollicular regulatory functions. Observations in vitro show that direct androgen action in FSH-stimulated granulosa cells leads to increased generation of cyclic AMP with amplification of key cyclic AMP-dependent functions associated with granulosa cell differentiation. This could help explain why high concentrations of androgen in follicular fluid are associated with normal preovulatory follicular development and maintenance of oocyte viability in the mammalian ovary.     

Steroid production from 17alpha-hydroxypregnenolone and dehydroepiandrosterone by human granulosa cells in vitro.

Granulosa cells were aspirated 3--4 h before the expected time of ovulation from 10 follicles of 4 patients treated with gonadotrophins: 4 of the follicles were immediately preovulatory. The granulosa cells were cultured for 10 h with 17alpha-hydroxypregnenolone or dehydroepiandrosterone and samples of medium removed at 3 and 10 h were assayed for 6 steroids. Granulosa cells were unable to synthesize androgens from endogenous substrate or undertake conversions via the delta5 pathway, but cells from all follicles were capable of aromatizing exogenous androgens to oestrogens although this capability was reduced in cells from follicles beginning to luteinize. Granulosa cells from preovulatory follicles synthesized more progesterone from endogenous substrate than cells from follicles which had not begun to luteinize. The results provide further support for the two-cell theory of oestrogen biosynthesis whereby granulosa cells aromatize androgens which are synthesized by the thecal cells in vivo.     

Effects of passive immunization against oestradiol-17 beta on some endocrine values of the male lamb

Passive immunization of male lambs against oestradiol-17 beta from 2 to 16 weeks of age significantly elevated androgen concentrations in plasma and depressed the median eminence content of dopamine. Removal of endogenous oestrogens had no significant effects on plasma FSH, LH or prolactin concentrations or on testicular growth and hypothalamic content of GnRH. These results suggest that endogenous oestrogens may indirectly suppress testicular androgen secretion by exerting a stimulatory influence on hypothalamic dopaminergic neurones, which in turn may inhibit GnRH secretion by the median eminence.     

Steroid regulation of kidney histidine decarboxylase and ornithine decarboxylase levels in mouse kidney: effects of the mutation testicular feminization, Tfm

1. Testosterone represses kidney histidine decarboxylase levels in both normal male and female mice. Tfm/Y mutant mice lack an androgen receptor and are phenotypically female. It has been suggested that the testosterone induction of HDC levels in these animals is a result of aromatisation to oestrogens in the absence of the androgen receptor; the oestrogens then induce the enzyme. 2. It is shown that the induction of HDC in Tfm/Y mice is specific to testosterone and not other androgens and can be mimiced by low doses of beta-oestradiol in normal female mice. 3. Analysis of Tfm/+ mice indicates that the testosterone induction effect is a function of individual kidney cells.     

Induction of Phosvitin by Oestradiol in Rooster Liver needs DNA Synthesis

THE synthesis of an egg yolk protein phosvitin, which is normally synthesized by the laying hen, can be induced in immature pullets¹ or roosters² by treatment with oestrogens. Phosvitin appears in the plasma of roosters with a lag period of 24 h after a single injection of oestradiol, reaching maximum values at 72 h³. The lag period is reduced on sequential injections of the hormone. The peak values of the protein in plasma as well as total amounts synthesized are enhanced progressively with successive exposures to the hormone³. The livers of hormone-treated birds become larger^4,5. These observations suggested that the amplified response to sequential injections of the hormone could be due to more cells that synthesize the protein being mobilized. Our experiments suggest that DNA synthesis is necessary before the target cells can be stimulated to synthesize phosvitin by oestrogens.     

Juxtacrine effects of IL-1 alpha precursor promote iNOS expression in vascular smooth muscle cells

After injury to the blood vessel wall, vascular smooth muscle cells (SMC) synthesize interleukin (IL)-1 and inducible nitric oxide (NO) synthase (iNOS). The present study tested whether endogenous production of IL-1 alpha stimulates iNOS expression in vascular SMC, and assessed whether IL-1 alpha exerts autocrine effects on the cells producing IL-1 alpha or juxtacrine effects on cells that contact the IL-1 alpha producing cells. Rat aortic SMC were transiently transfected with expression plasmids encoding either IL-1 alpha precursor, which localizes to the plasma membrane, or mature IL-1 alpha, which remains cytosolic. iNOS mRNA levels, determined by RT-PCR, and production of nitrite, a stable oxidation product of NO, were markedly elevated in SMC overexpressing IL-1 alpha precursor, and modestly elevated in SMC overexpressing mature IL-1 alpha, relative to SMC transfected with vector alone. Exposure to exogenous IL-1 beta or TNF-alpha further stimulated iNOS gene expression in SMC producing IL-1 alpha; low levels of IL-1 beta (20 pg/ml) were effective in SMC transfected with IL-1 alpha precursor plasmid, whereas SMC transfected with mature IL-1 alpha plasmid or vector alone required higher concentrations of IL-1 beta (200 and 2,000 pg/ml, respectively). The increases in iNOS mRNA levels and NO production in SMC overexpressing IL-1 alpha precursor were prevented by exogenous IL-1 receptor antagonist, suggesting that these effects were mediated by the type I IL-1 receptor. Immunostaining studies indicated that IL-1 alpha precursor stimulates iNOS gene expression via cell-cell contact. Expression of iNOS was enhanced in cells that were in contact with a cell overexpressing IL-1 alpha precursor (identified by coexpression of green fluorescent protein), and in cells that were overexpressing IL-1 alpha themselves, but only when the cell contacted another cell. Together these results indicate that IL-1 alpha precursor acts by cell-cell contact as an autocrine and juxtacrine enhancer of iNOS gene expression, inducing moderate iNOS expression on its own, and markedly augmenting the responsiveness of rat aortic SMC to exogenous cytokines.     

No evidence for aromatization of [3H]testosterone in oestrogen receptor containing cells of the epididymis

Oestrogen receptors are found in the principal cells of the caput and in apical and clear cells of the epididymis of the mouse. The distribution pattern of oestrogen receptors is different from that of androgen receptors and suggests a physiological role for oestrogens in the epididymis. We examined by competition experiments and thaw-mount autoradiography to see whether aromatization of [3H]testosterone is the source of oestrogens in the epididymis. After injection of [3H]testosterone we found the same labeling pattern as after non-aromatizable [3H]dihydrotestosterone. In particular, apical and clear cells showed a low or no nuclear concentration of radioactivity as with [3H]dihydrotestosterone. Competition with oestradiol had no effect on the binding pattern of [3H]testosterone in the epididymis in contrast to its effects in the brain of the same animals. Competition with dihydrotestosterone abolished labeling in contrast to the brain, where no effect was observed. Thus no aromatization of [3H]testosterone to oestrogens but conversion to dihydrotestosterone seems to occur in the epididymis.     

Isolation and culture of rat seminal vesicle epithelial cells : The use of the secretory protein SVS IV as a functional probe

A method for the isolation and culture of seminal vesicle epithelial cells obtained from control and androgen-primed sexually-immature, uncastrated rats is described. This method allows the establishment of monolayer cultures from aggregates of seminal vesicle epithelial cells isolated after trypsin and collagenase digestion. Phase contrast and transmission electron microscopic methods demonstrate that cell aggregates, after attaching to the substrate, establish within 48 h a colony-like, epithelial-like growth pattern. Immunofluorescent localization studies of SVS IV, an androgen-dependent secretory protein purified from rat seminal vesicle secretion, show that cultured seminal vesicle epithelial cells are immunoreactive. An electrophoretic analysis of [35S]methionine-labeled secretory proteins immunoprecipitated with rabbit anti-SVS IV serum demonstrate that, whereas SVS IV is newly-synthesized and accumulated in the medium of cultured seminal vesicle cells established from androgen primed rats, cultured cells from control rats appear to synthesize and accumulate SVS IV in a precursor form. Results of this work show that seminal vesicle epithelial cells in culture not only retain several structural features representative of the tissue but also serve as a potential system for the study of androgen action.     

Glucocorticoid effects on growth, and androgen receptor concentrations in DDT1MF-2 cell lines

The DDT1MF-2 smooth muscle tumor cell line contains receptors for and is differentially sensitive to androgens and glucocorticoids. Androgens stimulate and glucocorticoids inhibit growth. We now confirm that the latter involves the induction of a block in the G1 phase of the cell cycle. We have developed and characterized in vitro and in vivo a glucocorticoid resistant variant of this cell line, the DDT1MF-2-GR. Glucocorticoids specifically inhibit androgen induced androgen receptor augmentation in DDT1MF-2 cells, but not in the GR variant suggesting that growth inhibition is related to inhibition of androgen receptor augmentation. However, under optimal conditions for cell proliferation, when glucocorticoid inhibited growth is relieved by the exogenous addition of platelet derived growth factor, androgen receptor augmentation is still suppressed. Thus, androgen induced elevation in androgen receptor concentrations is not a prerequisite for cell proliferation. These results imply that in androgen responsive cells, although androgen stimulation of growth can be blocked by antagonism of androgen receptor mediated events, the antagonism can be bypassed by supplying the cells with exogenous growth factors. These results provoke speculation on how cells, which are dependent upon androgens for growth, become autonomous.     

Product inhibition of steroid-17 alpha-monooxygenase by endogenous 17 alpha-hydroxyprogesterone in microsomes and isolated Leydig cells from rat testis

Use of integrated rate equations for analysis of progesterone metabolism by isolated Leydig cells and microsomes from rat testis in presence of several progesterone concentrations within several periods reveals competitive product inhibition by endogenously formed 17 alpha-hydroxyprogesterone (Kpm = 0.1 microM) of steroid-17 alpha-monooxygenase activity (Ksm = 0.8 microM). The discrepancy between this very low interaction constant of endogenous 17 alpha-hydroxyprogesterone with the steroid-17 alpha-monooxygenase, and the respective values (from the literature) for exogenous 17 alpha-hydroxyprogesterone which are about 50-fold higher, may be explained by accumulation of endogenous 17 alpha-hydroxyprogesterone at the catalytic site of the steroid-17 alpha-monooxygenase. This mechanism may be important for intratesticular regulation of androgen biosynthesis from precursor steroids.     

Androgens and oestrogens in normal and cryptorchid stallions.

Total androgens, testosterone and total oestrogens were measured in twenty-one intact, nine unilaterally cryptorchid, three bilaterally cryptorchid stallions and four geldings. Total oestrogens were significantly higher (P less than 0-005) and total androgens significantly lower (P less than 0-05) in the bilateral cryptorchid compared to other groups. There was a significant (P less than 0-025) day and night variation in total androgen levels. Thyroidectomized and intact animals showed a marked decrease in total androgen as well as testosterone levels during the winter period thus showing an effect of season on androgenic function of the testis. Disappearance rate of total and androgens following castration was extremely rapid and levels were undectable within 12 hr. Sexual stimulation appeared to increase total androgen levels. Testosterone, androstenedione, dihydrotestosterone, androstandiols, and androstenediol were identified in spermatic vein blood. Dihydrotestosterone was measured in fluid from the cauda epididymidis.     

Histone H3 lysine-trimethylation markers are decreased by recombinant methioninase and increased by methotrexate at concentrations which inhibit methionine-addicted osteosarcoma cell proliferation

Methionine addiction is a fundamental and general hallmark of cancer cells, which require exogenous methionine, despite their ability to synthesize normal amounts of methionine from homocysteine. In contrast, methionine-independent normal cells do not require exogenous methionine in the presence of a methionine precursor. The methionine addiction of cancer cells is due to excess transmethylation reactions. We have previously shown that histone H3 lysine marks are over-methylated in cancer cells and the over-methylation is unstable when the cancer cells are restricted of methionine. In the present study, we show that methionine-addicted osteosarcoma cells are sensitive to both methotrexate (MTX) and recombinant methioninase (rMETase), but they affect histone H3 lysine-methylation in the opposite direction. Concentrations of MTX and rMETase, which inhibit osteosarcoma cells viability to 20%, had opposing effects on the status of histone methylation of H3K9me3 and H3K27me3. rMETase significantly decreased the amount of H3K9me3 and H3K27me3. In contrast, MTX significantly increased the amount of H3K9me and H3K27me3. The results suggest that increase or decrease in these methylated histone lysine marks is associated with proliferation arrest of methionine-addicted osteosarcoma.     

Autocrine differentiation-inhibiting factor (ADIF) from chicken erythroleukemia cells acts on human and mouse early BFU-E erythroid progenitors

tsAEV-LSCC HD3 chicken erythroid cells transformed by the avian erythroblastosis virus (AEV) secrete an autocrine differentiation-inhibiting factor, ADIF, which blocks differentiation without affecting proliferation of the chicken erythroid cells that synthesize and secrete it into the culture medium. The chicken erythroleukemia cell ADIF activity is not restricted to avians. It prevents dimethylsulfoxide (DMSO) from stimulating murine Friend erythroleukemia cells to synthesize hemoglobin. ADIF also blocks erythroid differentiation in normal human and murine bone marrow where it selectively targets the early BFU-E (burst-forming) erythroid precursor cells without affecting the more advanced CFU-E erythroid precursor cells or cells of the different granulocyte-macrophage lineage.     

The control and biological importance of intratumoural aromatase in breast cancer

The existence of aromatase activity in human breast carcinomas has been established for about 20 years but the clinical and biological importance of this remains unclear. A number of studies in clinical material suggest that aromatase activity may be a prerequisite of response to aromatase inhibitors and that aromatase activity may be enhanced in those tumours relapsing during treatment with one such inhibitor, aminoglutethimide. These results would carry more significance, however, if it was demonstrable that the growth of breast carcinomas is affected by the conversion of androgens to oestrogens by intratumoural aromatase. We have tried to address this by establishing model systems with aromatase-transfected MCF7 breast cancer cells. We have demonstrated that these cells can be stimulated mitogenically with androgen and that this proliferation is suppressible with aromatase inhibitors. Similarly the growth of aromatase transfected cells but not wild type cells as xenografts is supported by androstenedione and inhibitable by both the steroidal aromatase inhibitor, 4-hydroxyandrostenedione and the non-steroidal inhibitor, CGS 20267. Work with the former of these, which is a suicide inhibitor allowed us to demonstrate that growth can proceed with aromatase activity approximating to the highest level seen in breast carcinomas indicating that at least at this extreme level the intratumoural conversion of androgens to oestrogens may indeed be able to support tumour growth. Further work with this model system should allow us to define the minimal amount of intratumoural activity which can support tumour growth.     

Characterization of light chain and light chain constant region fragment mRNAs in MPC 11 mouse myeloma cells and variants

Cultured MPC 11 mouse myeloma cells synthesize not only γ_2b heavy and κ light chains but also a carboxyl terminal (constant region) fragment of κ light chain. In vitro translational analysis of total cytoplasmic and microsomal RNA indicates that these cells contain RNA which directs synthesis of both a light chain precursor and a light chain fragment precursor. Variant clones which do not synthesize either heavy or light chains continue to synthesize the light chain fragment. One such “nonproducing” variant was studied in detail. It does not contain translatable mRNA for the intact light chain but does contain RNA which is translated into the light chain fragment precursor. Nucleic acid hybridization analysis with a cDNA probe specific for the constant region of κ light chains revealed that microsomal RNA from the wild-type cell contains both a 14S and a 10S species of κ specific RNA, whereas the variant contains only the 10S species. Translational analysis of these same RNAs indicates that the 14S species codes for the light chain precursor, while the 10S RNA codes for the light chain fragment precursor.     

Up-regulation of androgen receptor binding in male rat fat pad adipose precursor cells exposed to testosterone: study in a whole cell assay system

Binding of androgens to adipocytes has previously been evaluated using cytosol fractions without taking into account nuclear binding, although the latter is suggested to be close to the physiological site of action. In the present study, performed in differentiated fat pad adipose precursor cells, we describe a simple, reliable and reproducible androgen binding assay in a system with intact cells. Tritiated and unlabeled methyltrienolone (R1881) were used to define specific and unspecific androgen binding. Triamcinolone acetonide was added to prevent the binding of R1881 to other types of receptors. Differentiated adipose precursor cells contain a homogeneous class of high affinity androgen binding sites, and binding is saturable and reversible. Binding apparently occurs at one site, with a Kd in the range of physiological androgen concentration (about 4 nM). Competition studies indicate that the receptor is specific for R1881, testosterone and dihydrotestosterone, which have approximately the same affinity, while progesterone, estradiol and dexamethasone show much lower affinity. Androgen binding was markedly enhanced after cellular exposure to R1881 and testosterone but not dihydrotestosterone, and this increase was dependent on protein synthesis, suggesting the formation of new receptors by these androgens. In conclusion, fully differentiated adipocytes contain a specific, high affinity receptor, the density of which is dependent on androgens.     

The androgen receptor mRNA is up-regulated by testosterone in both the harderian gland and thumb pad of the frog, Rana esculenta

³²P-labelled cDNA probe from plasmid containing rat androgen receptor (rAR) has been tested in hybridization experiments using RNAs from the Harderian gland and thumb pad of the edible frog, Rana esculenta. Northern blot analysis has shown a high degree of homology between the rAR cDNA and the frog androgen receptor mRNA (fAR mRNA); this has been supported by both the hybridization conditions (high stringency) and the molecular size of fAR mRNA which is quite similar to those described in mammals (9.4 kb). The role of androgens has been further investigated with respect to the kinetics of expression of fAR mRNA in in vivo experiments. In both the Harderian gland and thumb pad, testosterone has increased the levels of fAR mRNA as compared with the untreated groups. The use of cyproterone acetate (CPA) in combination with testosterone has resulted in a loss of the increase in fAR mRNA as compared to testosterone-treated groups, while CPA alone has resembled the control group. In primary cultures of frog Harderian gland and thumb pad cells, the steady-state levels of fAR mRNA have been increased in the cells exposed to testosterone as compared to those not exposed. These findings confirm that, in these androgen target tissues, testosterone exerts an up-regulation on its own receptors, increasing the accumulation of fAR mRNA in the same way as oestrogens up-regulate the expression of their own receptors in Xenopus liver and oviduct cells.     

Biosynthesis of human alpha-N-acetylgalactosaminidase: defective phosphorylation and maturation in infantile alpha-NAGA deficiency

The biosynthesis of human alpha-N-acetylgalactosaminidase (alpha-NAGA) was studied in normal fibroblasts and in cells from patients with infantile alpha-NAGA deficiency. Normal alpha-NAGA is synthesized as a 52 kDa precursor which matures to a 49 kDa species through phosphorylation and carbohydrate triming. Fibroblasts from the patients synthesize normal amounts of a 52 kDa precursor, however phosphorylation does not occur and this precursor is subsequently degraded intracellularly.     

Steroidogenesis inhibitors alter but do not eliminate androgen synthesis mechanisms during progression to castration-resistance in LNCaP prostate xenografts

In castration-resistant prostate cancer (CRPC) many androgen-regulated genes become re-expressed and tissue androgen levels increase despite low serum levels. We and others have recently reported that CRPC tumor cells can de novo synthesize androgens from adrenal steroid precursors or cholesterol and that high levels of progesterone exist in LNCaP tumors after castration serving perhaps as an intermediate in androgen synthesis.Herein, we compare androgen synthesis from [³H-progesterone] in the presence of specific steroidogenesis inhibitors and anti-androgens in steroid starved LNCaP cells and CRPC tumors. Similarly, we compare steroid profiles in LNCaP tumors at different stages of CRPC progression.Steroidogenesis inhibitors targeting CYP17A1 and SRD5A2 significantly altered but did not eliminate androgen synthesis from progesterone in steroid starved LNCaP cells and CRPC tumors. Upon exposure to inhibitors of steroidogenesis prostate cancer cells adapt gradually during CRPC progression to synthesize DHT in a compensatory manner through alternative feed-forward mechanisms. Furthermore, tumors obtained immediately after castration are significantly less efficient at metabolizing progesterone (36%) and produce a different steroid profile to CRPC tumors. Optimal targeting of the androgen axis may be most effective when tumors are least efficient at synthesizing androgens. Confirmatory studies in humans are required to validate these findings.     

Choline stimulates the synthesis and accumulation of acetate in a cholinergic neuroblastoma clone

The NS 20 mouse neuroblastoma clone was shown to synthesize acetylcholine from labelled glucose or acetate as precursor of the acetyl moiety of acetylcholine; in both cases, the synthesis was stimulated by the presence of exogenous choline. In addition, we report that acetate is accumulated in the NS 20 clone by a mechanism that is highly temperature-dependent and is stimulated by the presence of externally added choline.