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Testosterone (T) deficiency (TD) is a common clinical condition, which contributes to co-morbidities including loss of muscle mass, increased fat mass, increased inflammation, insulin resistance, risk of vascular disease, sexual dysfunction, fatigue, depressed mood and reduced quality of life. T therapy attenuates inflammation, increases insulin sensitivity, muscle mass and reduces fat mass and adiposity. T therapy improves lipid profiles and endothelial function and reduces systolic and diastolic blood pressure. In addition, T therapy may reduce risk of vascular disease and mortality. T therapy improves bone mineral density and increases energy and vitality and improves mood and sexual function and overall quality of life. T therapy appears to be safe if treatment and monitoring are appropriately executed. The evidence available to date does not support alleged concerns regarding risk of cardiovascular disease and prostate cancer. Indeed, T therapy remains controversial. The data in the contemporary literature suggest that T therapy reduces cardiovascular risk and fears promoted by some recent studies should be re-evaluated. The cardiovascular risk and mortality with T therapy must await large prospective controlled clinical trials, which depend on many complex factors. Such studies may be prohibitive in the current environment due to logistical challenges, such as recruiting large number of men to be treated for long-durations with appropriate follow-up, requiring astronomical cost., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Testosterone and its metabolite, 5α-dihydrotestosterone are critical metabolic and vascular hormones, which regulate a host of biochemical pathways including carbohydrate, lipid and protein metabolism and modulate vascular function. Testosterone deficiency (TD) is a well-recognized medical condition with important health implications. TD is associated with a number of co-morbidities including increased body weight, adiposity and increased waist circumference, insulin resistance (IR) and type 2 diabetes mellitus (T2DM), hypertension, inflammation, atherosclerosis and cardiovascular disease, erectile dysfunction (ED) and increased incidence of mortality. In this review, we summarize the data in the literature on the prevalence of TD and its association with the various co-morbidities and suggest that T therapy is necessary to improve health outcomes in men with TD., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In this study, we investigated the binding characteristics of [3H]Delta(5)-androstene-3beta,17beta-diol to rabbit vaginal cytosolic and nuclear extracts and in freshly excised intact tissue strips. [3H]delta(5)-Androstene-3beta,17beta-diol bound to a protein(s) in the vaginal nuclear extract with high affinity (K(d)=3-5 nM) and limited capacity (50-100 fmol/mg protein). No specific binding was detected in the cytoplasmic extracts. Competitive binding studies showed that binding of [3H]delta(5)-androstene-3beta,17beta-diol was effectively displaced with unlabeled delta(5)-androstene-3beta,17beta-diol but not with dehydroepiandrosterone, testosterone, dihydrotestosterone, triamcinolone acetonide, or progesterone. However, estradiol at high concentrations partially displaced bound [3H]delta(5)-androstene-3beta,17beta-diol. Incubation of freshly excised vaginal tissue strips with [3H]delta(5)-androstene-3beta,17beta-diol in the absence or presence of excess unlabeled delta(5)-androstene-3beta,17beta-diol for 1h at 37 degrees C resulted in specific binding to a soluble macromolecule in the nuclear KCl extracts. In addition, quantitative measurement of estrogen receptor, androgen receptor and delta(5)-androstene-3beta,17beta-diol binding protein was performed by equilibrium ligand binding assays using extracts of distal vaginal tissue from intact animals or ovariectomized animals treated for 2 weeks with vehicle, estradiol, testosterone, or estradiol plus testosterone. These changes in steroid hormone levels resulted in opposing trends between the estrogen receptor and delta(5)-androstene-3beta,17beta-diol binding protein, suggesting that delta(5)-androstene-3beta,17beta-diol binding protein is regulated differently by the hormonal milieu than the estrogen receptor. These data suggest that rabbit vaginal tissue expresses a novel binding protein which specifically binds delta(5)-androstene-3beta,17beta-diol and is distinct from the androgen and estrogen receptors.

The role of estrogens in regulating cellular metabolism in many tissues is well documented. Estrogens regulate cellular activity by interacting with specific intracellular receptor proteins. Two estrogen receptor (ER) isoforms have been isolated, cloned and characterized. Estrogen receptor alpha (ERalpha) and beta (ERbeta) are ligand dependent transcriptional activators, which regulate gene expression via complex mechanisms requiring ligand binding, transformation, dimerization, and interaction with specific unique cis DNA hormone response elements (EREs) and co-activators and co-repressors. Studies of ER structure and function have been tremendously facilitated by the development of molecular and biologic probes. Cloning and functional studies of the ERalpha and ERbeta have delineated some of the structural requirements involved in receptor function. Immunochemical analyses together with biochemical and molecular approaches have contributed to our understanding of ER structure and function. Although antibodies to ER have been developed and utilized for the past two decades, there has yet to be a comprehensive review that discusses the utility and usefulness of these antibodies in receptor detection and analysis. In this review, we summarize a plethora of information concerning the development and characterization of site-directed monoclonal and polyclonal antibodies to the ERalpha and ERbeta. We provide critical discussion on the characteristics and utility of ER antibodies in analyses, characterization and localization of ER isoforms in various tissues. We also provide a comparison of the potential utility of the available antibodies in various immunochemical assays. An epitope map detailing the specific sites of antibody-receptor interactions is constructed based on the available information. The advent of antibodies with high specificity and titer had facilitated detection of ER isoforms in normal and neoplastic tissues. The advent of new antibodies remains a powerful tool for assessment of ER expression and post-translational modification and receptor function in many experimental systems.

The objective of this study was to assess the potential utility of a new site-directed, monoclonal anti-estrogen receptor antibody (EVG F9) in detection and analyses of human breast tumor estrogen receptor (ERalpha), using immunoblotting and immunohistochemical assays. Using Western Blot analyses, we demonstrated that EVG F9 monoclonal antibody binds specifically to ERalpha and does not cross-react with ERbeta. Furthermore, binding of EVG F9 to ERalpha was effectively displaced with the immunogenic peptide in Western Blots and in immunohistochemical analyses. In Western Blot analyses, EVG F9 detected ERalpha at low concentrations approaching 5 to 10 fmol/sample. Determination of ERalpha status of a series of human breast tumor samples by Western Blot analyses or immunohistochemistry using EVG F9 correlated well with ERalpha values measured by ligand binding assays. These observations suggest that EVG F9 monoclonal anti-ERalpha antibody is a valuable immunochemical tool for detection and analyses of ERalpha in human breast tumors.

The interactions of estrogen receptor (ER) with monoclonal antibody (Mab) F9, developed against a synthetic 30-mer hybrid oligopeptide, were determined in the presence or absence of Mab NMT-1, raised against 15-mer peptide from the N-terminal A/B region (amino acids 140- 154) or Mab 213, raised to a peptide AT3 in the DNA-binding domain (amino acids 247-263). Mab F9 bound ER and formed a complex sedimenting at the approximately 11S region of the gradients. Mabs 213 and NMT-1 bound ER and formed complexes sedimenting at approximately 7S and 9S, respectively. Preincubation of ER with Mab 213, followed by reincubation with Mab F9, resulted in a complex sedimenting at the approximately 11S region of the gradients. Similarly, preincubation of ER with Mab NMT-1 followed by reincubation with Mab F9 also produced an approximately 11S complex on the gradients. These observations suggest that binding of Mab F9 to ER induced conformational changes causing the release of Mab 213 and Mab NMT-1 from ER. Furthermore, binding of Mab NMT-1 to the A/B region of ER also produced conformational changes causing the release of Mab 213 from its epitope in the DNA-binding region. These results indicate that binding of Mab F9 and Mab NMT-1, with epitopes located within amino acids 140-154 of the A/B region of ER, induced conformational changes in the DNA-binding domain, as determined by the inability of Mab 213 to remain bound to its epitope. These data further suggest that the DNA-binding region is sensitive to conformational changes induced in the native protein.

Conformational differences between type I antiestrogen-liganded estrogen receptor and estradiol (E2)-liganded estrogen receptor (ER) are thought to be responsible for differentiating agonist versus antagonist ER activity at individual genes. To examine the impact of ER ligand on estrogen-response element (ERE) binding kinetics and receptor conformation, we quantitated the effect of site-directed, ER-specific antibodies raised against synthetic peptides corresponding to the DNA-binding domain of human ER on ER-ERE binding in vitro. Although 4-hydroxytamoxifen-liganded-ER (4-OHT-ER) and E2-ER bind a consensus ERE with equal high affinity, the stoichiometry of 4-OHT-ER-ERE binding at saturation is approximately 50% lower than that of E2-ER binding to all ERE sequences tested. In contrast, the ERE binding stoichiometry of tamoxifen aziridine-liganded ER (TAz-ER) is identical to that of E2-ER: one receptor dimer bound per ERE. The difference in binding stoichiometry is caused by dissociation of one molecule of 4-OHT from the ER as the dimeric receptor binds DNA. Addition of low concentrations of ER-specific polyclonal antibodies AT3A or AT3B prevented 4-OHT ligand dissociation, yielding an increase in specific 4-OHT-ER-ERE binding to a level equal to that of E2-ER- or TAz-ER-ERE binding. However, higher amounts of AT3A or AT3B inhibited specific ERE binding of both 4-OHT- and E2-ER. We conclude that differences in ER conformation when liganded with 4-OHT versus E2 are revealed by these antibodies and that such differences in receptor conformation may influence subsequent interaction of the receptor with other proteins necessary for transactivation.

We have developed and characterized site-directed monoclonal (MAb) and polyclonal antibodies to a specific domain in the N-terminal A/B region in order to assess estrogen receptor (ER) structural integrity in human breast tumor samples. The antibodies (Abs) reacted specifically with the native (undenatured) ER from various species. The synthetic peptides competed effectively for ER binding to the Abs, suggesting site-specificity. The Abs recognized the activated (4S) and transformed (5S) but not the unactivated, untransformed, molybdate-stabilized (8S) ER, suggesting that the epitope is inaccessible in the 8S form. Some of these Abs reacted with ER bound to its responsive elements, as determined by gel mobility shift assay. To evaluate the structural integrity of ER in breast cancer, we have utilized a) ligand binding analysis for the hormone binding domain; b) site-directed MAb to the DNA-binding domain; and c) site-directed MAb to the N-terminal transactivation domain. Analysis of ER from 29 human breast tumors revealed that 10 out of 29 tumors (35%) contained ER with intact hormone-, DNA-, and N-terminal domains. Thirteen out of 29 tumors (approximately 45%) contained ER with intact hormone binding and N-terminal domains but were defective only in the DNA-binding domain. Three out of 29 tumors (approximately 10%) contained ER defective only in the N-terminal domain. Another subgroup of tumors (3/29; approximately 10%) had ER with normal hormone binding domain but were defective in both the DNA-binding and the N-terminal activation domains.(ABSTRACT TRUNCATED AT 250 WORDS)

Using synthetic peptides with sequences derived from specific regions of human estrogen (ER) and progesterone (PR) receptors, we have developed site-directed monoclonal and polyclonal antibodies to specific domains of these receptors. These antibodies interact specifically with the native (nondenatured) receptors and detect changes in the conformation of these proteins. Monoclonal antibody PR-AT 4.14 bound more tightly to PR-ORG 2058 complexes than to PR-7 alpha,17 alpha-dimethyl-19-nor-testosterone (7 alpha,17 alpha, DMNT; mibolerone) complexes, suggesting possible ligand-induced conformational changes in PR. In the absence of the antibody, PR-[3H]ORG 2058 complexes sedimented as 4S-5S entity on sucrose density gradients (SDG) containing 0.4 M KCl. In the presence of the antibody, PR-[3H]ORG 2058 complexes sedimented as 7-8S complexes. In contrast, at the same concentrations of antibody, PR-[3H]7 alpha,17 alpha, DMNT complexes sedimented at 4S-5S region in the absence of the antibody and as two populations in the presence of the antibody, suggesting that the antibody did not recognize all of the PR-7 alpha,17 alpha, DMNT complexes. To exclude the possibility that the inability of the antibody to recognize receptor-[3H]7 alpha,17 alpha, DMNT complexes was due to its binding to androgen receptors, unlabeled 5 alpha-dihydrotestosterone (5 alpha-DHT) (50 nM) was added to the incubation to inhibit 7 alpha,17 alpha, DMNT binding to androgen receptors. While PR-[3H]ORG 2058 complexes were immunoprecipitated in the presence of the antibody, PR-[3H]7 alpha,17 alpha, DMNT complexes were only partially immunoprecipitated, further confirming the results obtained with SDG.(ABSTRACT TRUNCATED AT 250 WORDS)

We have generated three polyclonal antisera to the DNA-binding domain of the human estrogen receptor (hER). Antiserum AT2A was generated against a peptide spanning amino acids 231-245 of hER, while antisera AT3A and AT3B were generated against a peptide spanning amino acids 247-261 of hER. The interaction of these three antisera with ER has been characterized by sucrose density gradient analysis. The antisera bound to the unactivated (8S), salt-activated (4S), and heat transformed (5S) ER complex. All the antisera appeared to be site-specific since binding of salt-activated ER to the antisera was blocked by the presence of excess free synthetic peptides. Antisera AT3A and AT3B inhibited the binding to DNA of the KCl-activated 4S ER and the heat-transformed 5S ER. Although antiserum AT2A bound to ER, it did not inhibit DNA binding of activated ER complexes. The ability of antisera AT3A and AT3B to inhibit ER binding to DNA was concentration dependent. Once bound to the DNA, ER complexes were not significantly affected by incubation with the antisera, suggesting that binding of DNA to ER inhibits antibody ER interaction and renders that domain inaccessible to the antibodies. These results demonstrate that site-directed antibodies to ER inhibit binding of activated ER complexes to DNA in vitro.

We have synthesized three peptides with amino acid sequences identical to those spanning amino acids 201-215, 231-245, and 247-261 of the human estrogen receptor (hER). These peptides were conjugated to keyhole limpet hemocyanin and used as immunogens to develop monoclonal antibodies (MoAbs) to hER. Antibody responses were only elicited by the peptide with amino acid sequence 247-261. Splenocytes from immunized mice were used for hybridoma production. Of the seven MoAbs that recognized the native (functional) form of the ER, four (MoAbs 16, 33, 114, and 213) recognized the ER with high affinity, as demonstrated by the increased sedimentation coefficient of the antibody-complexed ER in sucrose density gradients. Antibodies 318, 35, and 36 bound to ER with low affinity since they immunoprecipitated ER, but the ER-antibody complex appeared to dissociate on sucrose density gradients. The high-affinity MoAbs appear to be site-specific since the peptide competed effectively for binding of the receptor by the antibody. The fact that they reacted with ER from human breast cancer and calf, rat, and mouse uterine tissues suggests that this epitope of the receptor is conserved in these species. Although the DNA-binding region appears to be conserved among the various steroid receptors, these MoAbs did not recognize the native forms of progesterone, androgen, or glucocorticoid receptors. These MoAbs bound to the KCl-activated 4S ER and heat-transformed 5S ER, suggesting that the antibody-binding site is accessible in the monomeric and dimeric forms of ER. The antibodies did not recognize the untransformed 8S ER in the presence of molybdate and without KCl, suggesting that the antibody-binding site in the oligomeric form of ER is inaccessible. The fact that the antibodies did bind to the unoccupied 4S ER was demonstrated by the data obtained with sucrose density gradient analysis followed by postlabeling of ER with [3H]estradiol. The antibodies bound to ERs with high affinity (KD = 0.4 to 1.8 nM). At a fixed concentration of antibody, ERs ranging from 20 to 1,000 fmol were detectable. These MoAbs did not inhibit nuclear or DNA binding of ER in vitro. This can be attributed to the dissociation of the antibodies from ER when the latter interacts with its acceptor site. These results demonstrate the development of site-specific MoAbs to the native form of the hER using synthetic peptides as immunogens.

We have investigated the binding of cyproterone acetate (CA) to cytosolic androgen receptors (RC) and translocation of the RCCA complex into the nucleus. In a cell-free system (3H)CA binds to cytosolic androgen receptors with high affinity (KD = 11.6 nM) and limited capacity (180-200 femtomoles/mg protein). (3H)CA, however, dissociates very rapidly from the cytosolic and nuclear androgen receptors (Rn) at 0 degree C. Incubation of RC (3H)CA at 20 degrees C increased its ability to bind to nuclei. Translocation of RC (3H)CA to nuclei of intact cells was demonstrated after incubation of prostatic tissue with (3H)CA in tissue culture medium at 37 degrees C. In vivo administration of CA to castrated rats promoted RCCA translocation but did not induce androgen receptor replenishment. These data demonstrate that CA binds to and translocates androgen receptors to nuclei without concomitant receptor replenishment.

The binding characteristics of (3H)7 alpha,17 alpha-dimethyl-19-nortestosterone [3H)DMNT) to progesterone receptors (PgR) of calf uterine tissue cytosol were determined and compared to those of the synthetic progestins (3H)ORG2058 and (3H)R5020. Scatchard plot analysis of the equilibrium binding data showed that (3H)DMNT binds to calf uterine PgR with a KD of 2.35 +/- 1.1 nM. This value is slightly higher than that of (3H)R5020 (KD = 1.16 +/- 0.4 nM) and (3H)ORG2058 (KD = 1.04 +/- 0.65 nM). Analysis of dissociation kinetics showed that (3H)DMNT dissociates much more rapidly from the receptor than the other two ligands. Competition experiments showed that ORG2058 has a lower inhibition constant (Ki) than DMNT. Sucrose density gradient (SDG) analysis of PgR showed that (3H)ORG2058-PgR complexes sediment as 8S, (3H)R5020-PgR complexes sediment as 8S and 4S, and (3H)DMNT-PgR complexes sediment as 8S entities along with dissociated (3H)steroid. These data suggest that (3H)DMNT binds to PgR with lower affinity than (3H)ORG2058 and (3H)R5020. The number of binding sites detected with (3H)DMNT are significantly lower than those measured with (3H)ORG2058.

In cell-free systems androgen receptor (AR) labeled with (3H)DHT at 0 degrees C in the presence of 50mM molybdate remains unactivated (less than 3% binding to nuclei) and untransformed (7-8S on sucrose density gradients containing 0.4M KCl and 50mM molybdate). In the absence of molybdate, however, these complexes undergo activation and transformation even at 0 degrees C, albeit, very slowly. Incubation of unactivated, untransformed AR complexes at 18 degrees C, or at 0 degrees C in the presence of 0.4M KCl, greatly accelerated both activation and transformation. Activation and transformation are also associated with formation of high affinity (3H)DHT-receptor complexes as indicated by decreased rates of (3H)DHT dissociation from the receptor. Cytosolic AR complexes labeled with (3H)DHT in tissue slices at 37 degrees C, or in vivo, undergo rapid activation, transformation and nuclear translocation. The data suggest that activation and transformation of cytosolic AR in cell-free systems is associated with changes in the physicochemical properties of AR similar to those occurring upon hormone binding in intact cells and in vivo.

The effects of sucrose on androgen binding to its receptor were investigated. Sucrose decreased the rate of thermal inactivation of unoccupied and occupied androgen receptor (AR) and the rates of [3H]5 alpha-dihydrotestosterone [( 3H]DHT) dissociation from both activated and nonactivated AR complexes. Binding of [3H]DHT to AR in vivo, or in intact cells at 37 degrees C, caused reduction of [3H]DHT dissociation from cytosolic and nuclear complexes, as compared to in vitro labeled receptor complexes. Further, exposure of these complexes to sucrose at 0 degrees C caused an additional reduction of dissociation rates. Thus, the decrease of [3H]DHT dissociation induced by sucrose is independent of the reaction that reduces DHT dissociation from activated and transformed AR. Sucrose also reduced the ability of mersalyl acid to inactivate AR complexes. This effect of sucrose was markedly diminished in the presence of 2M urea. Sucrose did not significantly affect the association rate, sedimentation properties, or nuclear binding ability of AR complexes, but it did decrease the equilibrium dissociation constant. Other monosaccharides and disaccharides also stabilized AR. These data suggest that sucrose induces conformational changes in the steroid binding domain of androgen receptor, thereby reducing the rates of inactivation, steroid dissociation, and the accessibility of sulfhydryl groups to mersalyl.