Generation and characterization of androgen receptor knockout (ARKO) mice: an in vivo model for the study of androgen functions in selective tissues

By using a cre-lox conditional knockout strategy, we report here the generation of androgen receptor knockout (ARKO) mice. Phenotype analysis shows that ARKO male mice have a female-like appearance and body weight. Their testes are 80% smaller and serum testosterone concentrations are lower than in wild-type (wt) mice. Spermatogenesis is arrested at pachytene spermatocytes. The number and size of adipocytes are also different between the wt and ARKO mice. Cancellous bone volumes of ARKO male mice are reduced compared with wt littermates. In addition, we found the average number of pups per litter in homologous and heterozygous ARKO female mice is lower than in wt female mice, suggesting potential defects in female fertility and/or ovulation. The cre-lox ARKO mouse provides a much-needed in vivo animal model to study androgen functions in the selective androgen target tissues in female or male mice.     

A Sertoli cell-selective knockout of the androgen receptor causes spermatogenic arrest in meiosis

Androgens control spermatogenesis, but germ cells themselves do not express a functional androgen receptor (AR). Androgen regulation is thought to be mediated by Sertoli and peritubular myoid cells, but their relative roles and the mechanisms involved remain largely unknown. Using Cre/loxP technology, we have generated mice with a ubiquitous knockout of the AR as well as mice with a selective AR knockout in Sertoli cells (SC) only. Mice with a floxed exon 2 of the AR gene were crossed with mice expressing Cre recombinase ubiquitously or selectively in SC (under control of the anti-Müllerian hormone gene promoter). AR knockout males displayed a complete androgen insensitivity phenotype. Testes were located abdominally, and germ cell development was severely disrupted. In contrast, SC AR knockout males showed normal testis descent and development of the male urogenital tract. Expression of the homeobox gene Pem, which is androgen-regulated in SC, was severely decreased. Testis weight was reduced to 28% of that in WT littermates. Stereological analysis indicated that the number of SC was unchanged, whereas numbers of spermatocytes, round spermatids, and elongated spermatids were reduced to 64%, 3%, and 0% respectively of WT. These changes were associated with increased germ cell apoptosis and grossly reduced expression of genes specific for late spermatocyte or spermatid development. It is concluded that cell-autonomous action of the AR in SC is an absolute requirement for androgen maintenance of complete spermatogenesis, and that spermatocyte/spermatid development/survival critically depends on androgens.     

Suppressive function of androgen receptor in bone resorption

As locally converted estrogen from testicular testosterone contributes to apparent androgen activity, the physiological significance of androgen receptor (AR) function in the beneficial effects of androgens on skeletal tissues has remained unclear. We show here that inactivation of AR in mice using a Cre-loxP system-mediated gene-targeting technique caused bone loss in males but not in females. Histomorphometric analyses of 8-week-old male AR knockout (ARKO) mice showed high bone turnover with increased bone resorption that resulted in reduced trabecular and cortical bone mass without affecting bone shape. Bone loss in orchidectomized male ARKO mice was only partially prevented by treatment with aromatizable testosterone. Analysis of primary osteoblasts and osteoclasts from ARKO mice revealed that AR function was required for the suppressive effects of androgens on osteoclastogenesis supporting activity of osteoblasts but not on osteoclasts. Furthermore, expression of the receptor activator of NF-kappaB ligand (RANKL) gene, which encodes a major osteoclastogenesis inducer, was found to be up-regulated in osteoblasts from AR-deficient mice. Our results indicate that AR function is indispensable for male-type bone formation and remodeling.     

A physiological role for androgen actions in the absence of androgen receptor DNA binding activity

We tested the hypothesis that androgens have physiological actions via non-DNA binding-dependent androgen receptor (AR) signaling pathways in males, using our genetically modified mice that express a mutant AR with deletion of the 2nd zinc finger of the DNA binding domain (AR^ΔZF2) that cannot bind DNA. In cultured genital skin fibroblasts, the mutant AR^ΔZF2 has normal ligand binding ability, phosphorylates ERK-1/2 in response to 1 min DHT treatment (blocked by the AR antagonist bicalutamide), but has reduced androgen-dependent nuclear localization compared to wildtype (WT). AR^ΔZF2 males have normal baseline ERK-1/2 phosphorylation, with a 1.5-fold increase in Akt phosphorylation in AR^ΔZF2 muscle vs WT. To identify physiological actions of non-DNA binding-dependent AR signaling, AR^ΔZF2 males were treated for 6 weeks with dihydrotestosterone (DHT). Cortical bone growth was suppressed by DHT in AR^ΔZF2 mice (6% decrease in periosteal and 7% decrease in medullary circumference vs untreated AR^ΔZF2 males). In conclusion, these data suggest that non-DNA binding dependent AR actions suppress cortical bone growth, which may provide a mechanism to fine-tune the response to androgens in bone.     

Targeted overexpression of androgen receptor with a liver-specific promoter in transgenic mice.

The rodent liver displays marked age- and sex-dependent changes in androgen sensitivity due to the sexually dimorphic and temporally programmed expression of the androgen receptor (AR) gene. We have altered this normal phenotype by constitutive overexpression of the rat AR transgene in the mouse liver by targeting it via the human phenylalanine hydroxylase (hPAH) gene promoter. These transgenic animals in their heterozygous state produce an approximately 30-fold higher level of the AR in the liver as compared with the nontransgenic control. Androgen inactivation via sulfonation of the hormone by dehydroepiandrosterone sulfotransferase (DST), an androgen-repressible enzyme, also contributes to the age- and sex-dependent regulation of hepatic androgen sensitivity. DST has a broad range of substrate specificity and is responsible for the age- and sex-specific activation of certain polycyclic aromatic hepatocarcinogens as well, by converting them to electrophilic sulfonated derivatives. In the transgenic female, the hepatic expression of DST was approximately 4-fold lower than in normal females, a level comparable to that in normal males. The hPAH-AR mice will serve as a valuable model for studying the sex- and age-invariant expression of liver-specific genes, particularly those involved in the activation of environmental hepatocarcinogens such as the aromatic hydrocarbons.     

Targeted overexpression of androgen receptor in osteoblasts: unexpected complex bone phenotype in growing animals

The androgen receptor (AR), as a classic steroid receptor, generally mediates biologic responses to androgens. In bone tissue, both AR and the estrogen receptor (ER) are expressed in a variety of cell types. Because androgens can be converted into estrogen via aromatase activity, the specific role of the AR in maintenance of skeletal homoeostasis remains controversial. The goal of this study was to use skeletally targeted overexpression of AR as a means of elucidating the specific role(s) for AR transactivation in bone homeostasis. Rat AR cDNA was cloned downstream of a 3.6-kb alpha1(I)-collagen promoter fragment and used to create AR-transgenic mice. AR-transgenic males gain less weight and body and femur length is shorter than wild-type controls, whereas females are not different. AR-transgenic males also demonstrate thickened calvaria and increased periosteal but reduced endosteal labeling by fluorescent labeling and reduced osteocalcin levels. High-resolution micro-computed tomography shows normal mineral content in both male and female AR-transgenic mice, but male AR-transgenics reveal a reduction in cortical area and moment of inertia. Male AR-transgenics also demonstrate an altered trabecular morphology with bulging at the metaphysis. Histomorphometric analysis of trabecular bone parameters confirmed the increased bone volume comprised of more trabeculae that are closer together but not thicker. Biomechanical analysis of the skeletal phenotype demonstrate reduced stiffness, maximum load, post-yield deflection, and work-to-failure in male AR-transgenic mice. Steady-state levels of selected osteoblastic and osteoclastic genes are reduced in tibia from both male and female transgenics, with the exception of increased osteoprotegerin expression in male AR-transgenic mice. These results indicate that AR action is important in the development of a sexually dimorphic skeleton and argue for a direct role for androgen transactivation of AR in osteoblasts in modulating skeletal development and homeostasis.     

Essential roles of androgen signaling in Wolffian duct stabilization and epididymal cell differentiation

The epididymis is a male accessory organ and functions for sperm maturation and storage under the control of androgen. The development of the epididymis is also androgen dependent. The Wolffian duct (WD), anlagen of the epididymis, is formed in both male and female embryos; however, it is stabilized only in male embryos by testicular androgen. Androgen drives subsequent differentiation of the WD into the epididymis. Although the essential roles of androgen in WD masculinization and epididymal function have been established, little is known about cellular events regulated precisely by androgen signaling during these processes. It is also unclear whether androgen signaling, especially in the epithelia, has further function for epididymal epithelial cell differentiation. In this study we examined the cellular death and proliferation controlled by androgen signaling via the androgen receptor (AR) in WD stabilization. Analyses using AR knockout mice revealed that androgen signaling inhibits epithelial cell death in this process. Analysis of AP2α-Cre;AR(flox/Y) mice, in which AR function is deleted in the WD epithelium, revealed that epithelial AR is not required for the WD stabilization but is required for epithelial cell differentiation in the epididymis. Specifically, loss of epithelial AR significantly reduced expression of p63 that is essential for differentiation of basal cells in the epididymal epithelium. We also interrogated the possibility of regulation of the p63 gene (Trp63) by AR in vitro and found that p63 is a likely direct target of AR regulation.     

Targeting the stromal androgen receptor in primary prostate tumors at earlier stages

To differentiate roles of androgen receptor (AR) in prostate stromal and epithelial cells, we have generated inducible-(ind)ARKO-TRAMP and prostate epithelial-specific ARKO TRAMP (pes-ARKO-TRAMP) mouse models, in which the AR was knocked down in both prostate epithelium and stroma or was knocked out in the prostate epithelium, respectively. We found that loss of AR in both mouse models resulted in poorly differentiated primary tumors with expanded intermediate cell populations. Interestingly, knockdown of both epithelial and stromal AR in ind-ARKO-TRAMP mice at earlier stages resulted in smaller primary prostate tumors with lower proliferation rates, and knockout of AR in pes-ARKO-TRAMP mice resulted in larger primary prostate tumors with higher proliferation rates. The differential proliferation rates, yet with similarly expanded intermediate cell populations, indicated that the prostate stromal AR might play a more dominant role than the epithelial AR to promote primary tumor proliferation at an early stage of tumor. Tissue recombination of human prostate stromal cell lines (WPMY1-v or WPMY1-ARsi) with human prostate cancer epithelial cell lines (PC3-v or PC3-AR9) further demonstrated that the AR might function as a suppressor in epithelial cells and a proliferator in stromal cells in the primary prostate tumors. The dual roles of the AR in prostate epithelium and stroma may require us to reevaluate the target and timing of androgen-deprivation therapy for prostate cancer patients and may suggest a need to develop new drugs to selectively target stromal AR in the primary prostate tumors at earlier stages.     

Complete androgen insensitivity syndrome caused by a novel mutation in the ligand-binding domain of the androgen receptor: functional characterization

Mutations in the X-linked androgen receptor (AR) gene cause the androgen insensitivity syndrome by impairing androgen-dependent male sexual differentiation to varying degrees. Complete androgen insensitivity (CAIS) yields an external female phenotype, whereas affected cases of partial androgen insensitivity have various ambiguities of the genitalia. Here we describe a 46,XY phenotypically female patient with all of the characteristics of CAIS, i.e. primary amenorrhea, no axillary or pubic hair, female external genitalia, no uterus, and undescended testes. Defects in testosterone and dihydrotestosterone synthesis were excluded. The molecular basis of the disease was clarified by means of direct sequencing of PCR-amplified exonic fragments of the AR gene. An A to C transition in exon 4 of the AR gene led to a novel missense His(689)Pro mutation in the ligand-binding domain of the AR protein. Functional studies demonstrated that the mutated AR is unable to efficiently bind its natural ligand dihydrotestosterone and to trans-activate known androgen response elements. Analysis of the structural consequences of the His(689)Pro substitution suggests that this mutation is likely to perturb the conformation of the second helix of the AR ligand-binding domain, which contains residues critical for androgen binding.     

Identification of gene pathways altered by deletion of the androgen receptor specifically in mineralizing osteoblasts and osteocytes in mice

Androgens play a key role in skeletal growth and maintenance in males and can mediate their actions, at least in part, via the androgen receptor (AR) in osteoblasts. To investigate the mechanisms by which androgens exert their effects via the AR in mineralizing osteoblasts and osteocytes, we identified gene targets/pathways regulated by the AR using targeted gene expression and microarray approaches on bone isolated from mice in which the AR is specifically deleted in mineralizing osteoblasts and osteocytes (mOBL-ARKOs). Gene ontology mining indicated a number of biological processes to be affected in the bones of mOBL-ARKOs including skeletal and muscular system development and carbohydrate metabolism. All genes identified to have altered expression in the bones of mOBL-ARKOs were confirmed by Q-PCR for their androgen responsiveness in an androgen deprivation and replacement mouse model. The osteoblast genes Col1a1 and Bglap and the osteoclast genes Ctsk and RANKL (Tnfs11) were upregulated in the bones of mOBL-ARKOs, consistent with the increased matrix synthesis, mineralization, and bone resorption observed previously in these mice. Of significant interest, we identified genes involved in carbohydrate metabolism (adiponectin and Dpp4) and in growth and development (GH, Tgfb (Tgfb2), Wnt4) as potential targets of androgen action via the AR in mineralizing osteoblasts.     

Developmental basis of sexually dimorphic digit ratios

Males and females generally have different finger proportions. In males, digit 2 is shorter than digit 4, but in females digit 2 is the same length or longer than digit 4. The second- to fourth-digit (2D:4D) ratio correlates with numerous sexually dimorphic behavioral and physiological conditions. Although correlational studies suggest that digit ratios reflect prenatal exposure to androgen, the developmental mechanism underlying sexually dimorphic digit development remains unknown. Here we report that the 2D:4D ratio in mice is controlled by the balance of androgen to estrogen signaling during a narrow window of digit development. Androgen receptor (AR) and estrogen receptor α (ER-α) activity is higher in digit 4 than in digit 2. Inactivation of AR decreases growth of digit 4, which causes a higher 2D:4D ratio, whereas inactivation of ER-α increases growth of digit 4, which leads to a lower 2D:4D ratio. We also show that addition of androgen has the same effect as inactivation of ER and that addition of estrogen mimics the reduction of AR. Androgen and estrogen differentially regulate the network of genes that controls chondrocyte proliferation, leading to differential growth of digit 4 in males and females. These studies identify previously undescribed molecular dimorphisms between male and female limb buds and provide experimental evidence that the digit ratio is a lifelong signature of prenatal hormonal exposure. Our results also suggest that the 2D:4D ratio can serve as an indicator of disrupted endocrine signaling during early development, which may aid in the identification of fetal origins of adult diseases.     

Tamoxifen-inducible gene deletion in the cardiac conduction system

Temporally controlled gene deletion provides a powerful technique for examination of gene function in vivo. To permit use of this technology in the study of cardiac pacemaking, we attempted to generate a mouse line expressing an inducible Cre recombinase selectively in cardiac pacemaker cells. The tamoxifen-inducible CreER^T2 construct was ‘knocked in’ into the pacemaker channel HCN4 locus. In the absence of inducing agent, recombination was undetectable in HCN4-KiT mice. After injection of tamoxifen, highly selective and efficient recombination was observed in the sinoatrial and atrioventricular node. Expression of Cre and tamoxifen per se did not affect cardiac rhythm, basal heart rate and heart rate modulation. By crossing these animals with floxed HCN4 mice, complete deletion of this gene in the sinoatrial node could be achieved. HCN4-KiT mice represent the first tool for the temporally controlled inactivation of floxed target genes selectively in the conduction system of the murine heart.     

Relationship between gut environment,feces-to-food ratio,and androgen deficiency-induced metabolic disorders

ABSTRACT

Androgen action generates sex-related differences that include changes in the gut microbiota composition. Hypoandrogenism and hyperandrogenism in males and females, respectively, are associated with the prevalence of metabolic disorders. Our recent work showed that male androgen receptor knockout (ARKO) mice developed high-fat diet (HFD)-dependent sarcopenic abdominal obesity, hyperglycemia, and hepatic steatosis, leading to early death. The ARKO mice also exhibited alterations in intestinal microbiota but did not experience metabolic abnormalities when administered with antibiotics. Here, we show that time-dependent changes in feed efficiency (ratio of body weight gain to food intake) and weight of dried feces-to-food ratio could be good markers for changes in gut microbiota. Turicibacter spp., Lactobacillus spp., and L. reuteri increased in the gut in both HFD-fed ARKO and castrated mice having metabolic abnormalities. HFD-fed ARKO mice showed increased plasma levels of aspartate, but not alanine, aminotransferase. Changes in the gut microbiome appear to provoke androgen deficiency-induced metabolic diseases, leading to early mortality.     

Androgens and bone

Loss of estrogens or androgens increases the rate of bone remodeling by removing restraining effects on osteoblastogenesis and osteoclastogenesis, and also causes a focal imbalance between resorption and formation by prolonging the lifespan of osteoclasts and shortening the lifespan of osteoblasts. Conversely, androgens, as well as estrogens, maintain cancellous bone mass and integrity, regardless of age or sex. Although androgens, via the androgen receptor (AR), and estrogens, via the estrogen receptors (ERs), can exert these effects, their relative contribution remains uncertain. Recent studies suggest that androgen action on cancellous bone depends on (local) aromatization of androgens into estrogens. However, at least in rodents, androgen action on cancellous bone can be directly mediated via AR activation, even in the absence of ERs.Androgens also increase cortical bone size via stimulation of both longitudinal and radial growth. First, androgens, like estrogens, have a biphasic effect on endochondral bone formation: at the start of puberty, sex steroids stimulate endochondral bone formation, whereas they induce epiphyseal closure at the end of puberty. Androgen action on the growth plate is, however, clearly mediated via aromatization in estrogens and interaction with ERalpha. Androgens increase radial growth, whereas estrogens decrease periosteal bone formation. This effect of androgens may be important because bone strength in males seems to be determined by relatively higher periosteal bone formation and, therefore, greater bone dimensions, relative to muscle mass at older age. Experiments in mice again suggest that both the AR and ERalpha pathways are involved in androgen action on radial bone growth. ERbeta may mediate growth-limiting effects of estrogens in the female but does not seem to be involved in the regulation of bone size in males.In conclusion, androgens may protect men against osteoporosis via maintenance of cancellous bone mass and expansion of cortical bone. Such androgen action on bone is mediated by the AR and ERalpha.     

Differential effects of spermatogenesis and fertility in mice lacking androgen receptor in individual testis cells

Using a Cre-Lox conditional knockout strategy, we generated a germ cell-specific androgen receptor (AR) knockout mouse (G-AR(-/y)) with normal spermatogenesis. Sperm count and motility in epididymis from AR(-/y) mice are similar to that of WT (G-AR(+/y)) mice. Furthermore, fertility tests show there was no difference in fertility, and almost 100% of female pups sired by G-AR(-/y) males younger than 15 weeks carried the deleted AR allele, suggesting the efficient AR knockout occurred in germ cells during meiosis. Together, these data provide in vivo evidence showing male mice without AR in germ cells can still have normal spermatogenesis and fertility, suggesting the essential roles of AR during spermatogenesis might come from indirect cell-cell communication in a paracrine fashion. We then compared the consequences of AR loss in the spermatogenesis and fertility of G-AR(-/y) mice with two other testicular cell-specific AR(-/y) mice and total AR knockout male mice. The results provide clear in vivo evidence that androgen/AR signaling in Sertoli cells plays a direct important role in spermatogenesis and in Leydig cells plays an autocrine regulatory role to modulate Leydig cell steroidogenic function. Total AR knockout male mice have the most severe defects among these mice. These contrasting data with G-AR(-/y) mice suggest AR might have different roles in the various cells within testis to contribute to normal spermatogenesis and male fertility in mice.