Information AboutAndrogen |
| CATEGORIES ABOUT ANDROGEN | |
| sex steroids | |
| androgens | |
| SHOPPER'S DELIGHT | |
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TYPES OF ANDROGENS A subset of androgens, adrenal androgens, includes any of the 19-carbon steroids synthesized by the Adrenal Cortex , the outer portion of the Adrenal Gland , that function as weak steroids or steroid precursors, including Dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA-S), and androstenedione. Besides testosterone, other androgens include:
ANDROGEN FUNCTIONS Development of the male During mammalian development, the gonads are at first capable of becoming either Ovaries or testes. In humans, starting at about week 4 the gonadal rudiments are present within Intermediate Mesoderm adjacent to the developing kidneys. At about week 6, epithelial Sex Cords develop within the forming testes and incorporate the Germ Cell s as they migrate into the gonads. In males, certain Y Chromosome genes, particularly SRY , control development of the male phenotype, including conversion of the early bipotential gonad into testes. In males, the sex cords fully invade the developing gonads. By week 8 of human fetal development, Leydig cells appear in the differentiating gonads of males. The mesoderm-derived Epithelial cells of the sex cords in developing testes become the Sertoli cells which will function to support sperm cell formation. A minor population of non-epithelial cells exists between the tubules, these are the androgen-producing Leydig cells. The Leydig cells can be viewed as producers of androgens that function as Paracrine Hormone s required by the Sertoli cells in order to support sperm production. Soon after they differentiate, Leydig cells begin to produce androgens which are required for masculinization of the developing male fetus (including penis and scrotum formation). Under the influence of androgens, remnants of the Mesonephron , the Wolffian Ducts , develop into the Epididymis , Vas Deferens and Seminal Vesicles . This action of androgens is supported by a hormone from Sertoli cells, AMH , which prevents the embryonic Müllerian ducts from developing into fallopian tubes and other female reproductive tract tissues in male embryos. AMH and androgens cooperate to allow for the normal movement of testes into the scrotum. Before the production of the pituitary hormone LH by the embryo starting at about weeks 11-12, human chorionic gonadotrophin ( HCG ) promotes the differentiation of Leydig cells and their production of androgens. Androgen action in target tissues often involves conversion of testosterone to 5α-dihydrotestosterone ( DHT ). Spermatogenesis During puberty, androgen, LH and FSH production increase and the sex cords hollow out, forming the seminiferous tubules, and the germ cells start to differentiate into sperm. Throughout adulthood, androgens and FSH cooperatively act on Sertoli cells in the testes to support sperm production. Exogenous androgen supplements can be used as a Male Contraceptive . Elevated androgen levels caused by use of androgen supplements can inhibit production of LH and block production of endogenous androgens by Leydig cells. Without the locally high levels of androgens in testes due to androgen production by Leydig cells, the seminiferous tubules can degenerate resulting in infertility. Inhibition of fat deposition Males typically have less Adipose Tissue than females. Recent results indicate that androgens inhibit the ability of some fat cells to store lipids by blocking a signal transduction pathway that normally supports adipocyte function. Muscle mass Males typically have more Skeletal Muscle mass than females. Androgens promote the enlargement of skeletal muscle cells and probably act in a coordinated manner to enhance muscle function by acting on several cell types in skeletal muscle tissue. Brain Circulating levels of androgens can influence human behavior because some Neuron s are sensitive to steroid hormones. Androgen levels have been implicated in the regulation of human Aggression and libido. SCREENING METHODS FOR ANDROGENS
Whole Animal Methods (In Vivo) Intact animals used to measure androgenic activity (Allen and Doisy, 1924; Hershberger et al., 1953). In vivo methods - Take advantage of animal’s innate hormone-directed tissue development and maintenance responses and - Use these responses to identify whether administered substances alter the normal response. Animal’s inherent capacity to produce these hormones must be removed. # Castration. # Immature or juvenile animals used (M. Chaturvedia, P.C. Mali, A.S. Ansarib; Pharmacology: 2003; 68:38-48) For androgenicity evaluation of test subs. rats were divided into four groups: Group I- animals were castrated 30 days before the experiment to serve as controls Group II, III and IV subjected to castration 30 days before the experiments, followed by administration of test subs., testosterone propionate (0.01 mg/rat/alternate day s.c.), and test subs. with testosterone propionate, respectively, for 30 days. Studied-- cauda epididymis sperm motility and density, Number of pups, Fertility, The weights of testes, epididymis, seminal vesicle, and prostate Histoarchitecture of the testes Endocrine challenge test (ECT). (Anderson et al., 1992). Bioassay - to evaluate gonadal response to a substance by measuring the steroid hormone production and release following administration of test subs. The intact mature animal is challenged with GnRH or an LH- or FSH-like substance that stimulates a hormonal response. Serial blood samples collected and measured to evaluate whether the substance being tested causes increased testosterone Techniques used include tail vein sampling, tail vein treatments and/or jugular catheterization (Fail et al., 1995; Fail and Anderson, 2002). A number of different endpoints have been used.
(Fail et al., 1995; 1996). Combination of Whole Animal and Isolated Organs Method (ex vivo)
specimens collected, and the testis isolated.
Isolated Organ Methods (in vitro)
steroidogenic pathway.
# way in which the organ is processed.
The perfused testis # Provides a method to simulate biosynthesis and secretion of testicular steroid hormones Developed for the rabbit by Vandemark and Ewing (1963), modified for the rat by Chubb and Ewing (1979). # Apparatus is sterilized and then assembled. # The system must be air-free to prevent bubbles from entering the perfusate and blocking flow. # Testis is removed, spermatic artery cannulated, organ flushed of blood with perfusate solution. # The testis is placed in the organ chamber and perfusion initiated. # Test substance given after a preliminary perfusion for approximately 1 hour, the temperature (370 C), rate of flow (20 mL/hr), and pH (7.4) are monitored and maintained. # The perfusion can be maintained for 6 to 10 hours. # Samples are collected from the perfusate for analysis. Simple Whole Organ Incubation technique
Screening for AR activity using Gene expression assays
· KB2 assay (Vickie S. Wilson; 2002: Toxicological Sciences 66, 69-81) · Describe the effects of androgens in a stably transformed cell line that they developed (MDA-KB2), which expresses the human AR (hAR) and an AR-responsive promoter linked to a luciferase reporter gene (MMTV-luc). · Main advantage of KB2 assay -- it employs a genetically modified cell line, which eliminates the effort and inherent variability associated with repeated transient transfections. · The breast cancer cell line, MDA-MB-453, was stably transformed with the MMTV.luciferase.neo reporter gene construct. · Since both GR and AR are present in the MDA-MB-453 cells, and both receptors can act through the MMTV promoter, compounds that act through either AR or GR activate the MMTV luciferase reporter. · AR agonists such as dihydrotestosterone (DHT), and GR agonists such as dexamethasone (DEX), corticosterone, and aldosterone induce luciferase expression at appropriate concentrations. · DHT consistently produced 3–9-fold induction at concentrations from 0.1 to 10 nM. · To distinguish AR- from GR-mediated ligands, chemicals were assayed concurrently with the antiandrogen, hydroxyflutamide (OHF), which blocks AR- but not GR-mediated responses. · These cells are relatively easy to culture and maintain. · Responsiveness was monitored over time and was stable for more than 80 passages. · Some advantages -- relatively rapid (2 days), eliminates the need for transfection, can be conducted in a 96-well plate format, produces consistent reproducible results
MDA-MB-453 cells (ATCC No. HTB 131) used for transformation. Cells were seeded at 2 x 105 cells per 60-mm culture dish in maintenance medium, then transformed using Fugene 6 (Roche) per manufacturer's protocol, with 5 µg pMMTV.luc.neo per dish. To screen clones for responsiveness to an androgen agonist, cells grown from individual colonies were plated at 1 x 104 cells per well in 96-well luminometer plates, allowed to attach. When cells were attached, the medium was replaced with freshly prepared dosing medium containing ethanol only (negative control), an agonist (dihydrotestosterone, DHT; 1 nM), or the agonist plus a known competitor (hydroxyflutamide, OHF; 1 M). Cells were dosed with 100 µl of medium/well and incubated for 20–24 h, --harvested with 25 µl/well of lysis buffer and assayed for luciferase reporter activity. Relative light units per well determined using a 96-well MLX Luminometer. Clones with a minimum 2-fold response to 1 nM DHT were retained in culture. The final clone (MDA-kb2) was chosen based on appropriate ligand responsiveness and genetic stability over time.
-MDA-kb2 cells stably transformed with the pMMTV.neo.luc reporter gene construct were maintained in L-15 media supplemented with, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B at 37°C, without CO2. -For experiments, cells were plated at 1 x 104 cells per well in 100 µl of medium in 96-well luminometer plates. -When cells were attached (4–6 h), medium was removed and replaced with dosing medium. Stock solutions for each chemical were prepared at 1000x in ethanol. - Dosing medium was prepared at the time of treatment by aliquoting 1 µl of stock solution into 1 ml of maintenance medium -Vehicle control wells contained 100 µl medium/ well with1 µl of ethanol/ml of medium. Each plate also contained either 0.1 or 1.0 nM DHT, or DHT/plus 1 µM OHF as agonist control. -Cells were incubated overnight at 37°C without CO2. Luciferase activity was assayed --using an MLX microtiter plate luminometer and quantified as relative light units (RLU). -Relative light units were converted to fold induction above the vehicle control value for each replicate for statistical analysis. -Stability of Androgenic Response and Baseline Activity Androgen-induced luciferase activity measured over approximately 9 months of continuous culture to assess the stability and responsiveness of the MDA-kb2 clone. -cells were assayed over more than 80 passages for their response to 1 nM DHT. -At early passages, luciferase induction relative to solvent controls was about 10 fold. -In continuous culture, luciferase induction dropped over time, but responsiveness stabilized at 30 to 40 passages. -In later passages, a minimum of 5–6-fold induction in response to 1 nM DHT was consistently maintained over the remaining test period for more than 80 passages, demonstrating stable expression of the luciferase gene. P. C. Hartig,1, Toxicological Sciences 66, 82-90 (2002)
· Twenty-four h prior to transduction, 5 x 104 cells were plated per well. · Medium was removed and replaced with 20 µl of control medium or medium with diluted virus. · MDA cells (which contain endogenous AR) were transduced with Ad/mLuc7 reporter virus at a multiplicity of infection (MOI) of 50 (i.e., 50 virions per cell). · CV-1 cells (which lack native AR) were transduced with Ad/mLuc7 at a MOI of 50 and Ad5hAR at a MOI of one. · Dishes were rocked every 15 min for 1 h, incubated 3 additional h, · 200 µl medium/medium + test chemicals added to each well · followed by a 48-h incubation. · Plates were either frozen at –80°C, or assayed immediately for luciferase activity. · Luciferase activity was quantitated in an MLX microtiter plate luminometer, data expressed in relative light units (RLU). · For androgen agonists, which stimulate luciferase expression, treatments compared to the media/ethanol control group · Relative light units were converted to fold induction above the media value for each replicate (L. G. Parks; Toxicological Sciences 62, 257-267 (2001) COS whole cell human androgen receptor (hAR) binding assay
(0.13 M ethylhexadecyldimethylammonium bromide with 3% glacial acetic acid).
· 1 µM hydroxyflutamide was added to half of the samples to see if this potent antiandrogen would block the test substance-induced luciferase activity. · Cells also were exposed to 1 nM DHT as a positive control. CV-1 AR and GR 40-dependent transcriptional activation assays
· Twenty-four h after transfection, medium was aspirated and replaced with 2 ml of medium containing test substance. · Cells then incubated at 37°C under 5% carbon dioxide. · After 5 h of exposure, medium was removed, and cells were washed once with phosphate-buffered saline and harvested with 500 µl lysis buffer. · Relative light units of 0.05-ml aliquots of lysate determined using a Monolight 2010 luminometer. COS cell immunocytochemistry Experiment conducted to visualize, by immunofluorescence, the ligand-induced nuclear translocation of hAR in COS cells. · Two chamber slides were seeded with 100,000 cells/chamber in 2 ml DMEM supplemented with 10% FBS · Cells transfected with 0.5 µg pCMVhAR · Following transfection, 2 ml medium containing test substance, added to slides, · incubated for 24 h at 37°C under 5% CO2. · The next day, medium removed, cells washed once with DPBS (Dulbecco's phosphate buffered saline), allowed to dry for 45 min at room temperature, fixed for 10 min with 95% ethanol (-20°C), blocked with 5% BSA, and incubated overnight with primary AR antibody (1:1000) at 4°C. · The following day, cells were washed once with DPBS and incubated with fluorescently labeled secondary antibody for 30 min at room temperature. · To visualize the nuclei, cells were counter-stained with DAPI, a DNA stain, mounted with fluoromount · slides examined using Microscope at 200 x magnification. · The localization of the AR was classified as either perinuclear or nuclear in a blinded fashion from 10 randomly selected fields from a slide for each site. · In another experiment, cells were exposed to 1 nM DHT, as a positive control. Novel yeast bioassay system for detection of androgenic compounds. Lee HJ, Lee YS, Kwon HB, Lee K.;Toxicol In Vitro. :2003 Apr;17(2):237-44. Report the development of a rapid, simple, effective yeast detection system for androgenic compounds, based on the yeast two-hybrid protein interaction.
INSENSITIVITY TO ANDROGEN IN HUMANS Reduced ability of a XY Karyotype fetus to respond to androgens can result in one of several problems, including infertility and several forms of Intersex conditions. See Androgen Insensitivity Syndrome (AIS). REFERENCES |