In vitro studies were designed to determine whether Sertoli cell-delivered ABP could act on spermatogenetic events, whether such an action could occur via a paracrine or a juxtacrine pathway and whether sex steroids could be involved in this action. ABP delivery to germ cells was achieved using an in vitro model based on recombinant rat ABP-producing mouse Sertoli cells cocultivated with rat spermatids. Using semi-quantitative RT-PCR, the expression of the Tnp 1 gene encoding the Transition Protein 1, involved in the histone to protamine replacement during spermatid nuclear transformation, was analyzed. Our results provide clear evidence that Sertoli cell-derived ABP acts on spermatids by modifying the TP1 mRNA level. This outcome, strictly requiring juxtacrine conditions, is obtained in the absence of sex steroid hormones. To our knowledge this is the first evidence of an effect of ABP itself on male germ cells.

Dysregulation of androgen-binding protein (ABP) is associated with a number of endocrine and andrology diseases. However, the ABP metabolism in Sertoli cells is largely unknown. We report that autophagy degrades ABP in rat Sertoli cells, and the autophagic clearance of ABP is regulated by testosterone, which prolongs the ABP biological half-life by inhibiting autophagy. Further studies identified that the autophagic clearance of ABP might be selectively regulated by testosterone, independent of stress (hypoxia)-induced autophagic degradation. These data demonstrate that testosterone up-regulates ABP expression at least partially by suppressing the autophagic degradation. We report a novel finding with respect to the mechanisms by which ABP is cleared, and by which the process is regulated in Sertoli cells.

The draft mouse (Mus musculus) genome sequence revealed an unexpected proliferation of gene duplicates encoding a family of secretoglobin proteins including the androgen-binding protein (ABP) alpha, beta and gamma subunits. Further investigation of 14 alpha-like (Abpa) and 13 beta- or gamma-like (Abpbg) undisrupted gene sequences revealed a rich diversity of developmental stage-, sex- and tissue-specific expression. Despite these studies, our understanding of the evolution of this gene family remains incomplete. Questions arise from imperfections in the initial mouse genome assembly and a dearth of information about the gene family structure in other rodents and mammals.

The genes for salivary androgen-binding protein (ABP) subunits have been evolving rapidly in ancestors of the house mouse Mus musculus, as evidenced both by recent and extensive gene duplication and by high ratios of nonsynonymous to synonymous nucleotide substitution rates. This makes ABP an appropriate model system with which to investigate how recent adaptive evolution of paralogous genes results in functional innovation (neofunctionalization).

To better understand androgen function in the mammalian nose, we have determined the levels of testosterone (T) in the olfactory mucosa (OM, which harbors the olfactory receptor neurons) and the lateral nasal gland (LNG, which is the largest anterior nasal gland) of C57BL/6 mice. The results indicated that, in adult male mice, T levels in the LNG were substantially higher than those in the OM and other non-reproductive or non-endocrine tissues examined, including liver, kidney, and brain. Furthermore, in the LNG, the high T levels were accompanied by high levels of salivary androgen-binding protein (sABP) and low microsomal T-hydroxylase activities. The high abundance of T and sABP in the LNG suggests not only that the LNG is a storage site for androgen, but also the possibility that unusually high T levels may occur in other organs that have abundant expression of sABP but low expression of steroid-metabolizing enzymes. Our findings suggest a critical need to determine androgen levels in various organs, as well as to establish the functional significance of an unusually high T level in the LNG, a gland known for its secretion of biologically active molecules, such as odorant binding proteins and immunoglobulin A, to the nasal cavity.

This study aims to determine the ameliorative effect of vitamin E (vit E) on histological features and androgen binding protein (ABP) levels in rats induced by allethrin.

The androgen receptor (AR) plays a crucial role in normal physiology, development and metabolism as well as in the aetiology and treatment of diverse pathologies such as androgen insensitivity syndromes (AIS), male infertility and prostate cancer (PCa). Here we show that dimerization of AR ligand-binding domain (LBD) is induced by receptor agonists but not by antagonists. The 2.15-Å crystal structure of homodimeric, agonist- and coactivator peptide-bound AR-LBD unveils a 1,000-Å2 large dimerization surface, which harbours over 40 previously unexplained AIS- and PCa-associated point mutations. An AIS mutation in the self-association interface (P767A) disrupts dimer formation in vivo, and has a detrimental effect on the transactivating properties of full-length AR, despite retained hormone-binding capacity. The conservation of essential residues suggests that the unveiled dimerization mechanism might be shared by other nuclear receptors. Our work defines AR-LBD homodimerization as an essential step in the proper functioning of this important transcription factor.

The Androgen-binding protein (Abp) region of the mouse genome contains 30 Abpa genes encoding alpha subunits and 34 Abpbg genes encoding betagamma subunits, their products forming dimers composed of an alpha and a betagamma subunit. We endeavored to determine how many Abp genes are expressed as proteins in tears and saliva, and as transcripts in the exocrine glands producing them. Using standard PCR, we amplified Abp transcripts from cDNA libraries of C57BL/6 mice and found fifteen Abp gene transcripts in the lacrimal gland and five in the submandibular gland. Proteomic analyses identified proteins corresponding to eleven of the lacrimal gland transcripts, all of them different from the three salivary ABPs reported previously. Our qPCR results showed that five of the six transcripts that lacked corresponding proteins are expressed at very low levels compared to those transcripts with proteins. We found 1) no overlap in the repertoires of expressed Abp paralogs in lacrimal gland/tears and salivary glands/saliva; 2) substantial sex-limited expression of lacrimal gland/tear expressed-paralogs in males but no sex-limited expression in females; and 3) that the lacrimal gland/tear expressed-paralogs are found exclusively in ancestral clades 1, 2 and 3 of the five clades described previously while the salivary glands/saliva expressed-paralogs are found only in clade 5. The number of instances of extremely low levels of transcription without corresponding protein production in paralogs specific to tears and saliva suggested the role of subfunctionalization, a derived condition wherein genes that may have been expressed highly in both glands ancestrally were down-regulated subsequent to duplication. Thus, evidence for subfunctionalization can be seen in our data and we argue that the partitioning of paralog expression between lacrimal and salivary glands that we report here occurred as the result of adaptive evolution.

Comparison of the androgen-binding protein (Abp) gene regions of six Mus genomes provides insights into the evolutionary history of this large murid rodent gene family. We identified 206 unique Abp sequences and mapped their physical relationships. At least 48 are duplicated and thus present in more than two identical copies. All six taxa have substantially elevated LINE1 densities in Abp regions compared with flanking regions, similar to levels in mouse and rat genomes, although nonallelic homologous recombination seems to have only occurred in Mus musculus domesticus. Phylogenetic and structural relationships support the hypothesis that the extensive Abp expansion began in an ancestor of the genus Mus. We also found duplicated Abpa27's in two taxa, suggesting that previously reported selection on a27 alleles may have actually detected selection on haplotypes wherein different paralogs were lost in each. Other studies reported that a27 gene and species trees were incongruent, likely because of homoplasy. However, L1MC3 phylogenies, supposed to be homoplasy-free compared with coding regions, support our paralog hypothesis because the L1MC3 phylogeny was congruent with the a27 topology. This paralog hypothesis provides an alternative explanation for the origin of the a27 gene that is suggested to be fixed in the three different subspecies of Mus musculus and to mediate sexual selection and incipient reinforcement between at least two of them. Finally, we ask why there are so many Abp genes, especially given the high frequency of pseudogenes and suggest that relaxed selection operates over a large part of the gene clusters.

ZIP9 is a Zn2+ transporter, testosterone receptor, and mediator of signaling events through G-proteins. Despite these pivotal properties, however, its physiological and pathophysiological significance has not yet been comprehensively addressed. Using a cell line that lacks the classical androgen receptor we show that ZIP9-mediated phosphorylation of Erk1/2, CREB, or ATF-1 and expression of claudin-5 and zonula occludens-1 by testosterone can be completely antagonized by bicalutamide (Casodex), an anti-androgen of significant clinical impact. Computational modeling and docking experiments with ZIP9 reveal typical characteristics of ZIP transporters and an extracellular binding site for testosterone capable of accommodating bicalutamide. The presence of this site is verified by our demonstration that the membrane-impermeable testosterone analogue T-BSA-FITC labels the membrane only when ZIP9 is expressed and that this labeling is completely prevented by bicalutamide. The study connects structural features of ZIP9 to its functions and indicates a possible relevance of ZIP9 as a pharmacological target.

The secretoglobins (SCGBs) comprise a family of small, secreted proteins found in animals exclusively of mammalian lineage. There are 11 human SCGB genes and five pseudogenes. Interestingly, mice have 68 Scgb genes, four of which are highly orthologous to human SCGB genes; the remainder represent an 'evolutionary bloom' and make up a large gene family represented by only six counterparts in humans. SCGBs are found in high concentrations in many mammalian secretions, including fluids of the lung, lacrimal gland, salivary gland, prostate and uterus. Whereas the biological activities of most individual SCGBs have not been fully characterised, what already has been discovered suggests that this family has an important role in the modulation of inflammation, tissue repair and tumorigenesis. In mice, the large Scgb1b and Scgb2b gene families encode the androgen-binding proteins, which have been shown to play a role in mate selection. Although much has been learned about SCGBs in recent years, clearly more research remains to be done to allow a better understanding of the roles of these proteins in human health and disease. Such information is predicted to reveal valuable novel drug targets for the treatment of inflammation, as well as designing biomarkers that might identify tissue damage or cancer.

Inability to respond to the circulating androgens is named as androgen insensitivity syndrome (AIS). Mutations in the androgen receptor (AR) gene are the most common cause of AIS. A cause and effect relationship between some of these mutations and the AIS phenotype has been proven by in vitro studies. Several other mutations have been identified, but need to be functionally validated for pathogenicity. Screening of the AR mutations upon presumptive diagnosis of AIS is recommended. We analyzed a case of complete androgen insensitivity syndrome (CAIS) for mutations in the AR gene. Sequencing of the entire coding region revealed C>G mutation (CTT-GTT) at codon 712 (position according to the NCBI database) in exon 4 of the gene, resulting in replacement of leucine with valine in the ligand-binding domain of the AR protein. No incidence of this mutation was observed in 230 normal male individuals analyzed for comparison. In vitro androgen binding and transactivation assays using mutant clone showed approximately 71% loss of ligand binding and about 76% loss of transactivation function. We conclude that CAIS in this individual was due to L712V substitution in the androgen receptor protein.

Androgen signaling via the androgen receptor (AR) is involved in normal prostate development and prostate cancer progression. In addition to androgen binding, a variety of protein kinases, including cyclic AMP-dependent protein kinase A (PKA), can activate the AR. Although hormone deprivation, especially that of androgen, continues to be an important strategy for treating prostate cancer patients, the disease ultimately progresses to castration-resistant prostate cancer (CRPC), despite a continuous hormone-deprived environment. To date, it remains unclear which pathways in this progression are active and targetable. Here, we performed a proteomic analysis of VCaP cells stimulated with androgen or forskolin to identify proteins specific for androgen-induced and androgen-bypassing signaling, respectively. Patterns of differentially expressed proteins were quantified, and eight proteins showing significant changes in expression were identified. Functional information, including a Gene Ontology analysis, revealed that most of these proteins are involved in metabolic processes and are associated with cancer. The mRNA and protein expression of selected proteins was validated, and functional correlations of identified proteins with signaling in VCaP cells were assessed by measuring metabolites related to each enzyme. These analyses offered new clues regarding effector molecules involved in prostate cancer development, insights that are supported by the demonstration of increased expression levels of the eight identified proteins in prostate cancer patients and assessments of the progression-free interval. Taken together, our findings show that aberrant levels of eight proteins reflect molecular changes that are significantly regulated by androgen and/or PKA signaling pathways, suggesting possible molecular mechanisms of CRPC.

In the domestic horse; failure of normal masculinization and virilization due to deficiency of androgenic action leads to a specific disorder of sexual development known as equine androgen insensitivity syndrome (AIS). Affected individuals appear to demonstrate an incoherency between their genetic sex and sexual phenotype; i.e., XY-sex chromosome constitution and female phenotypic appearance. AIS is well documented in humans. Here we report the finding of two novel genetic variants for the AR-gene identified in a Tennessee Walking Horse and a Thoroughbred horse mare; each in individual clinical cases of horse AIS syndrome.

Although more than 1,000 androgen receptor (AR) mutations have been identified and these mutants are pathologically important, few theoretical studies have investigated the role of AR protein folding stability in disease and its relationship with the phenotype of the patients. Here, we extracted AR variant data from four databases: ARDB, HGMD, Cosmic, and 1,000 genome. 905 androgen insensitivity syndrome (AIS)-associated loss-of-function mutants and 168 prostate cancer-associated gain-of-function mutants in AR were found. We analyzed the effect of single-residue variation on the folding stability of AR by FoldX and guanidine hydrochloride denaturation experiment, and found that genetic disease-associated mutations tend to have a significantly greater effect on protein stability than gene polymorphisms. Moreover, AR mutants in complete androgen insensitivity syndrome (CAIS) tend to have a greater effect on protein stability than in partial androgen insensitive syndrome (PAIS). This study, by linking disease phenotypes to changes in AR stability, demonstrates the importance of protein stability in the pathogenesis of hereditary disease.

Androgen insensitivity syndrome (AIS) is the most common cause of 46,XY disorders of sex development (46,XY DSD). This syndrome is an X-linked inheritance disease and it is caused by mutations in the human androgen receptor (AR) gene. Non-synonymous point AR mutations are frequently described in this disease, including in the complete phenotype. We present a novel synonymous mutation in the human AR gene (c.1530C > T) in four 46,XY patients from two unrelated families associated with complete androgen insensitivity syndrome (CAIS). The analysis of mRNA from testis showed that synonymous AR mutation changed the natural exon 1 donor splice site, with deletion of the last 92 nucleotides of the AR exon 1 leading to a premature stop codon 12 positions ahead resulting in a truncate AR protein. Linkage analyses suggested a probable founder effect for this mutation. In conclusion, we described the first synonymous AR mutation associated with CAIS phenotype, reinforcing the disease-causing role of synonymous mutations.

Prostate cancer is the second leading cause of cancer-related deaths among men in North America. Almost all prostate cancers begin in an androgen-dependent state, so androgen deprivation therapy is administered and results in improved clinical outcomes. However, over time, some cancerous cells are able to survive and grow during this treatment, resulting in androgen-independent prostate cancer. At this point, the disease is fatal, as there are no effective targeted therapies available. Most prostate cancer tumors require androgen receptor (AR) signalling for survival. During the progression to androgen-independence, this signalling cascade has been found to be altered at many levels within prostate cancers. Mechanisms that enhance AR signalling during androgen deprivation include: AR gene amplifications, AR gene mutations, changes in expression of AR co-regulatory proteins, changes in expression of steroid-generating enzymes, ligand-independent activation of AR via 'outlaw' pathways, and AR-independent pathways that become activated, termed 'bypass' pathways. One or more of these aforementioned changes can lead to prostate cancer cells to gain androgen-independent properties. Understanding the molecular alterations that occur during this process will allow for improved therapeutic strategies to target key molecules and pathways important for this progression.

The classical action of androgen receptor (AR) is to regulate gene transcriptional processes via AR nuclear translocation, response element binding and recruitment of, or crosstalk with, transcription factors. AR also utilises non-classical, non-genomic mechanisms of signal transduction. These precede gene transcription or protein synthesis, and involve steroid-induced modulation of cytoplasmic or cell membrane-bound regulatory proteins. Despite many decades of investigation, the role of AR in gene regulation of cells and tissues remains only partially characterised. AR exerts most of its effects in sex hormone-dependent tissues of the body, but the receptor is also expressed in many tissues not previously thought to be androgen sensitive. Thus it is likely that a complex, more over-arching, role for AR exists. Each AR domain co-ordinates a multitude of individual and vital roles via a diverse array of interacting partner molecules that are necessary for cellular and tissue development and maintenance. Aberrant AR activity, promoted by mutations or binding partner misregulation, can present as many clinical manifestations including androgen insensitivity syndrome and prostate cancer. In the case of malignant prostate cancer, treatment generally revolves around androgen deprivation therapies designed to interfere with AR action and the androgen signalling axis. Androgen therapies for prostate cancer often fail, highlighting a real need for increased research into AR function.

The androgen insensitivity syndrome may cause developmental failure of normal male external genitalia in individuals with 46,XY karyotype. It results from the diminished or absent biological action of androgens, which is mediated by the androgen receptor in both embryo and secondary sex development. Mutations in the androgen receptor gene, located on the X chromosome, are responsible for the disease. Almost 70% of 46,XY affected individuals inherited mutations from their carrier mothers.

Androgen binding to the androgen receptor (AR) in the cytoplasm induces the AR to translocate to the nucleus, where it regulates the expression of target genes. Here, we found that androgens rapidly activated a plasma membrane-associated signaling node that enhanced nuclear AR functions. In murine primary osteoblasts, dihydrotestosterone (DHT) binding to a membrane-associated form of AR stimulated plasma membrane-associated protein kinase G type 2 (PKG2), leading to the activation of multiple kinases, including ERK. Phosphorylation of AR at Ser515 by ERK increased the nuclear accumulation and binding of AR to the promoter of Ctnnb1, which encodes the transcription factor β-catenin. In male mouse osteoblasts and human prostate cancer cells, DHT induced the expression of Ctnnb1 and CTNN1B, respectively, as well as β-catenin target genes, stimulating the proliferation, survival, and differentiation of osteoblasts and the proliferation of prostate cancer cells in a PKG2-dependent fashion. Because β-catenin is a master regulator of skeletal homeostasis, these results explain the reported male-specific osteoporotic phenotype of mice lacking PKG2 in osteoblasts and imply that PKG2-dependent AR signaling is essential for maintaining bone mass in vivo. Our results suggest that widely used pharmacological PKG activators, such as sildenafil, could be beneficial for male and estrogen-deficient female patients with osteoporosis but detrimental in patients with prostate cancer.