ArKO Males Research Articles

OR07-6 Disrupting DNA-binding Domain Dimerization In The Androgen Receptor In Mice Results In a Partial Androgen Insensitivity Model

Abstract The androgen receptor (AR) plays a crucial role in the development and maintenance of the male phenotype, as shown in patients with androgen insensitivity syndrome (AIS). The AR is a nuclear receptor that needs to homodimerize to execute its role as transcription factor. Dimerization can occur through three different modes: via the DNA-binding domain (DBD), via the ligand-binding domain and via an interaction between the LBD and the aminoterminal domain. Dimerization via the DBD is very well known and occurs through the D-box located in the second zinc finger. DBD dimerization is generally accepted for all steroid receptors, but some of them show that D-box mutations do not inactivate them completely. However, for the AR, no monomeric transactivation capacity was described yet. To study the role of D-box independent activity of the AR, we introduced a double point mutation in the D-box that is predicted to disrupt DBD dimerization (ARDmon). These two point mutations were found in separate patients suffering from partial AIS. In vitro studies with this mutant suggest that the remaining activity depends on the androgen response element under investigation. We show that ligand binding was not affected by the D-box mutation. Furthermore, BioID assays indicated no changes in coregulatory interactions. When introducing the corresponding mutation in mice, this led to a phenotype that is intermediate between global ARKO and wild type (WT) males. Based on their anogenital distance (AGD) and the absence of nipples at early age, the ARDmon/Y mice were identified as males. Follow-up of their AGD and body weight, however, revealed clear differences with both WT males and females. Furthermore, hypospadias was observed in the ARDmon/Y mice. Testes were smaller in size compared to the WT males and first analysis showed almost no expression of well-known AR regulated genes. Reminiscent structures of the epididymis were found. In summary, the ARDmon mouse model shows an intermediate AIS phenotype. Via orchidectomy in combination with androgen replacement therapy, we will now investigate remaining androgen responses in the ARDmon/Y mice and elucidate response elements on which the AR can act in a D-box independent way. Additionally, we generated the first rodent PAIS model with a genetic cause of hypospadias. Presentation: Saturday, June 11, 2022 12:45 p.m. - 1:00 p.m.

Oestradiol Exposure Early in Life Programs Daily and Circadian Activity Rhythms in Adult Mice.

Hormone signalling during critical periods organises the adult circadian timekeeping system by altering adult hormone sensitivity and shaping fundamental properties of circadian rhythmicity. However, the timing of when developmental oestrogens modify the timekeeping system is poorly understood. To test the hypothesis that alterations in postnatal oestrogenic signalling organise adult daily activity rhythms, we utilised aromatase knockout mice (ArKO), which lack the enzyme required for oestradiol synthesis. ArKO and wild-type (WT) males and females were administered either oestradiol (E) or oil (OIL) daily for the first 5 postnatal days (p1-5E and p1-5OIL , respectively) because this time encompasses the emergence of clock gene rhythmicity and light responsiveness in the suprachiasmatic nucleus, a bilateral hypothalamic structure regarded as the 'master oscillator'. After sexual maturation, gonadectomy and exogenous oestradiol supplementation, locomotor parameters were assessed. We determined that altered oestrogenic signalling in early life exerts organisational control over the expression of daily and circadian activity rhythms in adult mice. Specifically, p1-5E reduced total wheel running activity in male and female ArKO and female WT mice but had no effect on WT male activity levels. In females, wheel running was consolidated by p1-5E to the early versus late evening, a phenomenon characteristic of male mice. The time of peak activity was advanced by p1-5E in WT and ArKO females but not males. P1-5E shortened the length of the active phase (alpha) in WT males but had no effect on ArKO males or females of either genotypes. Finally, p1-5E altered the magnitude of photic-induced shifts, suggesting that developmental oestrogenic signalling impacts adult circadian functions. In the present study, we further define both a critical period of development of the adult timekeeping system and the role that oestrogenic signalling plays in the expression of daily and circadian activity rhythms throughout life.

Morphology and gene expression profile of the submandibular gland of androgen-receptor-deficient mice

ObjectivesIn the submandibular gland (SMG) of mice, the granular convoluted tubule (GCT) develops preferentially in males dependent on androgens. To clarify the molecular mechanism of androgen action in SMG, we examined the SMG of mice deficient for the androgen receptor (ARKO). DesignThe morphological features and gene expression in the SMG of control and ARKO mice with or without hormone treatments were analysed by immunohistochemistry, DNA microarray, and RT-PCR. ResultsThe development of GCT and expression of GCT-specific products such as NGF were even lower in ARKO male SMG than in control female SMG. The administration of androgens to ARKO males had no effect on SMG, whereas the administration of thyroid hormone (T4) caused the extensive conversion of striated duct cells to GCT cells with the increase of NGF mRNA. Gene expression profiles in control and ARKO male SMG were analysed by DNA microarrays, and genes with higher or lower expression in ARKO male SMG were determined. They were then classified into groups according to their responsiveness to the administration of dihydotestosterone (DHT) or T4 to ARKO males. RT-PCR revealed that, while no gene was responsive to DHT, expression of many genes was up- or down-regulated by T4. ConclusionsThese results confirmed that GCT cell differentiation induced by androgens is dependent on the classical androgen receptor (AR), whereas that by T4 is independent of AR. Differential reactivity of genes to androgens and thyroid hormone in ARKO mice may shed light on the mechanism of androgen action in the SMG.

Androgen resistance in female mice increases susceptibility to DMBA-induced mammary tumors.

Hormones, notably estrogens, are pivotal in the origins of breast cancer but androgenic effects, while supported by persistence of AR expression in breast cancers, remain controversial. This study determined the role of the androgen actions via androgen receptor (AR) in experimental mammary cancer. Androgen-resistant female and male mice (ARKO) were generated using Cre/loxP technique and featured a global AR inactivation. The effect of AR inactivation and influence of genetic background on 7,12-dimethylbenz[a]anthracene (DMBA)-induced tumorigenesis was confirmed using two separate ARKO models with different genetic backgrounds. The onset of palpable mammary tumors was significantly faster in ARKO females (median time 22 vs 34weeks, respectively; (p = 0.0024; multivariate Cox regression) compared to WT and independent of the mouse genetic background. The cumulative incidence at 9months was 81 ± 10% [mean ± SE] for ARKO compared to 50 ± 13% in WT females. The increased DMBA susceptibility of ARKO females was associated with a higher epithelial proliferation index but not with major structural or receptor (estrogen or progesterone) expression differences between the virgin WT or ARKO female mammary glands. AR inactivation allowed substantial ductal extension in ARKO males while WT males displayed only rudimentary epithelial branches or complete regression of epithelial structures. Yet, DMBA did not induce epithelial mammary tumors in WT or ARKO males, demonstrating that AR inactivation alone is insufficient to promote mammary tumors. These results demonstrate that AR inactivation accelerates mammary carcinogenesis in female mice exposed to the chemical carcinogen DMBA regardless of mouse genetic background but require prior exposure to endogenous ovarian hormones.

Estradiol deficiency during development modulates the expression of circadian and daily rhythms in male and female aromatase knockout mice

Gonadal steroids modify the phase, amplitude and period of circadian rhythms. To further resolve the role of estradiol, we examined daily patterns of activity, circadian free running period and behavioral responses to light pulses using aromatase deficient (ArKO) mice. These animals lack the enzyme necessary to produce estradiol. We hypothesized that circulating estrogens during development and adulthood modulate the amount of activity, the temporal relationship of activity patterns relative to a light:dark cycle, and the free running period. Intact and gonadectomized male and female ArKO and wildtype (WT) littermates were used. WT males, but not ArKO males, retained the ability to respond to steroid hormones; the time of activity onset, free running period in constant darkness, and total daily activity were significantly different in gonadectomized compared to intact males. In contrast, gonadectomy did not alter the expression of these variables in ArKO males. ArKO females had a longer free running period in constant darkness compared to WT females regardless of gonadal state. Ovariectomized ArKO females had a significantly delayed activity onset when compared to intact ArKO females and ovariectomized WT females, despite all 3 groups being estrogen deficient. Phase shifts in response to light pulses given at different times of the day revealed an interaction between genotype, sex, and circulating steroids. These results from ArKO animals strongly suggest an organizational effect of estradiol during a critical period of development on the expression of biological rhythms.

Effects of Organisational Oestradiol on Adult Immunoreactive Oestrogen Receptors (α and β) in the Male Mouse Brain

Steroid hormones act on developing neural circuits that regulate the hypothalamic-pituitary-gonadal axis and are involved in hormone-sensitive behaviours. To test the hypothesis that developmental exposure to oestradiol (E(2)) organises the quantity of adult oestrogen receptors (ERalpha and ERbeta), we used male mice with a targeted mutation of the aromatase enzyme gene (ArKO) and their wild-type (WT) littermates. These mice are unable to aromatise testosterone to E(2), but still express both ERalpha and beta. To evaluate adult responsiveness to E(2), gonadectomised males were implanted with Silastic capsules containing E(2), or an empty implant, 5 days prior to sacrifice. Immunoreactivity for ERalpha and ERbeta was quantified in the caudal ventromedial nucleus (VMN) and the medial preoptic area (POA). Regardless of genotype, adult treatment with E(2) reduced ERalpha-immunoreactive (ir) and ERbeta-ir cell numbers in the POA, as well as ERbeta-ir, but not ERalpha-ir, cell numbers in the VMN. Genotype, and thus endogenous exposure to E(2), produced opposite effects on ER expression in the two brain areas. In the VMN, ArKO males had more ERalpha-ir and ERbeta-ir cells than did WT males. In the POA, ArKO males had fewer ERalpha-ir and ERbeta-ir cells than did WT males. Thus, numbers of immunoreactive neurones containing both ERs in the adult ArKO male were enhanced in the POA, but decreased in the VMN, and most likely these patterns were established during the developmental critical period. Furthermore, although both ERalpha and beta-ir cell numbers are altered by the disruption of the aromatase gene, ERbeta is altered in a more robust and region-specific manner.

The Pituitary Function of Androgen Receptor Constitutes a Glucocorticoid Production Circuit

Androgen receptor (AR) mediates diverse androgen actions, particularly reproductive processes in males and females. AR-mediated androgen signaling is considered to also control metabolic processes; however, the molecular basis remains elusive. In the present study, we explored the molecular mechanism of late-onset obesity in male AR null mutant (ARKO) mice. We determined that the obesity was caused by a hypercorticoid state. The negative feedback system regulating glucocorticoid production was impaired in ARKO mice. Male and female ARKO mice exhibited hypertrophic adrenal glands and glucocorticoid overproduction, presumably due to high levels of adrenal corticotropic hormone. The pituitary glands of the ARKO males had increased expression of proopiomelanocortin and decreased expression of the glucocorticoid receptor (GR). There were no overt structural abnormalities and no alteration in the distribution of cell types in the pituitaries of male ARKO mice. Additionally, there was normal production of the other hormones within the glucocorticoid feedback system in both the pituitary and hypothalamus. In a cell line derived from pituitary glands, GR expression was under the positive control of the activated AR. Thus, this study suggests that the activated AR supports the negative feedback regulation of glucocorticoid production via up-regulation of GR expression in the pituitary gland.

Androgen receptor functions in male and female reproduction.

The androgen receptor (AR) is a ligand-dependent transcription factor involved in the regulation of many different physiological processes. Dysfunction of AR causes diverse clinical conditions, such as testicular feminization mutation (Tfm) syndrome and prostate cancer. However, the molecular basis of the AR in these disorders largely remains unknown, as a result of a lack of genetic models. Using conditional targeting technique with Cre-loxP system, we successfully generated null AR mutant (ARKO) mice. The ARKO males grew healthily, but they showed typical Tfm abnormalities. The ARKO males exhibited late onset of obesity with impaired bone metabolism and sexual behaviors. No overt abnormality was found in female ARKO mice, but a premature ovarian failure-like phenotype was found with impaired folliculogenesis. Thus, andorogen/AR system supports normal reproduction as well as normal female reproduction. (Reprod Med Biol 2007; 6: 11-17).

Male aromatase knockout mice acquire a conditioned place preference for cocaine but not for contact with an estrous female

We have previously shown that male mice carrying a targeted mutation in the Cyp19 gene which encodes the aromatase enzyme (aromatase knockout or ArKO), showed a reduced interest to investigate volatile odors from conspecifics in a Y-maze. We asked here whether the incentive value of reproductively relevant odors is reduced in ArKO males by comparing the ability of male wild-type (WT) and ArKO mice to learn a conditioned place preference using exposure to reproductively relevant odors as incentive stimuli. When the presence of an anesthetized estrous female or soiled bedding from estrous females was used as incentive stimuli, only WT and not male ArKO mice showed conditioned place preference suggesting that the reward value of these stimuli is reduced in ArKO males. However, ArKO males showed conditioned place preference when cocaine was used as incentive stimulus, indicating that ArKO males are able to learn the conditioned place preference procedure. These results thus further confirm the important role of estradiol in sexually related behavioral responses in male mice.

Male aromatase-knockout mice exhibit normal levels of activity, anxiety and “depressive-like” symptomatology

It is well known that estradiol derived from neural aromatization of testosterone plays a crucial role in the development of the male brain and the display of sexual behaviors in adulthood. It was recently found that male aromatase knockout mice (ArKO) deficient in estradiol due to a mutation in the aromatase gene have general deficits in coital behavior and are sexually less motivated. We wondered whether these behavioral deficits of ArKO males could be related to changes in activity, exploration, anxiety and “depressive-like” symptomatology. ArKO and wild type (WT) males were subjected to open field (OF), elevated plus maze (EPM), and forced swim tests (FST), after being exposed or not to chronic mild stress (CMS). CMS was used to evaluate the impact of chronic stressful procedures and to unveil possible differences between genotypes. There was no effect of genotype on OF, EPM and FST behavioral parameters. WT and ArKO mice exposed to CMS or not exhibited the same behavioral profile during these three types of tests. However, all CMS-exposed mice (ArKO and WT) spent less time in the center of the EPM. Additionally, floating duration measured in the FST increased between two tests in both WT and ArKO mice, though that increase was less prominent in mice previously subjected to CMS than in controls. Therefore, both ArKO and WT males displayed the same behavior and had the same response to CMS however CMS exposure slightly modified the behavior displayed by mice of both genotypes in the FST and EPM paradigms. These results show that ArKO males display normal levels of activity, exploration, anxiety and “depressive-like” symptomatology and thus their deficits in sexual behavior are specific in nature and do not result indirectly from other behavioral changes.

Androgen receptor functions from reverse genetic models

The androgen receptor (AR) is a ligand-dependent transcription factor involved in the regulation of many different physiological processes. AR dysfunction causes a diverse range of clinical conditions, including testicular feminization mutation (Tfm) syndrome, prostate cancer, and motor neuron disease (Kennedy’s disease). However, due to lack of genetic models, the molecular basis of the AR in these disorders remains largely unknown. Using a conditional targeting technique based on the Cre-loxP system, we successfully generated null AR mutant (ARKO) mice. ARKO males exhibited normal healthy growth, but showed typical Tfm abnormalities. Hormonal assay of ARKO males revealed that while serum androgen levels were very low, estrogen levels were normal. Another hallmark of ARKO males was late-onset obesity, with marked accumulation of white adipose tissue. To clarify the role of human AR (hAR) mutants with expanded polyQ stretches as observed in neurodegenerative disease, we also established a Dorsophila model in which either wild-type or polyQ-expanded hAR were ectopically expressed. Although no overt phenotype was detected in adult fly-eye neurons expressing mutant hAR, the ingestion of androgen caused marked neurodegeneraton.

Alternations in hepatic expression of fatty-acid metabolizing enzymes in ArKO mice and their reversal by the treatment with 17β-estradiol or a peroxisome proliferator

We generated aromatase gene knockout mice (ArKO mice) by targeting disruption of Cyp19, which encodes an enzyme responsible for conversion of androgens to estrogens. We found that ArKO males developed hepatic steatosis spontaneously with aging, indicating that the function of Cyp19 is required to maintain constitutive lipid metabolism in male mice. Plasma lipoprotein analysis using a gel permeation chromatography revealed that high density lipoprotein (HDL)-cholesterol levels were slightly higher in ArKO males than in wild-type males, whereas no other obvious alternations in the profiles were detected. Nevertheless, analysis of lipoprotein compositions by SDS-polyacrylamide gel electrophoresis demonstrated apparent reduction in the amounts of apolipoprotein E, functioning in receptor-mediated clearance of lipoproteins in the liver, in the IDL/LDL fraction of ArKO males as compared with that of wild-type males. Biochemical analysis on the ArKO livers revealed suppression of mRNA expression and activity of enzymes involved in fatty acid β-oxidation. The impairment was reversed to the wild-type levels by treatment with 17β-estradiol or bezafibrate, the latter is a synthetic peroxisome proliferator. These findings indicated a pivotal role of estrogen in supporting constitutive hepatic expression of genes involved in fatty acid β-oxidation and in maintaining lipid homeostasis.

Bone phenotype of the aromatase deficient mouse

Estrogens are important for normal bone growth and metabolism. The mechanisms are incompletely understood. Thus, we have undertaken characterization of the skeletal phenotype of aromatase (ArKO) deficient mice. No abnormalities have been noted in skeletal patterning in newborns. Adult ArKO mice show decreased femur length and decreased peak Bone Mineral Density (BMD) with accelerated bone loss by 7 months of age in females. Magnetic resonance microscopy (MR) and microCT (μCT) imaging disclosed decreased cancellous connectivity and reduced cancellous bone volume in ArKO females. Bone formation rate (BFR) is increased in ArKO females and decreased in ArKO males. Estradiol therapy reverses these changes. This anabolic effect of estradiol in the male skeleton is supported by 18-F − Positron Emission Tomography (PET) imaging, which clearly demonstrates decreased spinal uptake, but marked increase after estradiol therapy. Serum IGF-1 levels are high in young female ArKO mice but low in young ArKO males. The reduced BMD in ArKO females, despite the presence of elevated serum IGF 1, suggests that other mechanism(s) are operative. There is increased B-cell lymphopoiesis in adult female ArKO bone marrow cells. These results show that ArKO mice show the effects of estrogen deficiency on bone growth, mass, metabolism, microarchitecture and the hematopoietic microenvironment.

Oestrogen at the neonatal stage is critical for the reproductive ability of male mice as revealed by supplementation with 17beta-oestradiol to aromatase gene (Cyp19) knockout mice.

Aromatase P450 (CYP19) is an enzyme responsible for the conversion of androgens to oestrogens. We generated CYP19 knockout (ArKO) mice by targeted disruption of Cyp19 and studied the role of oestrogens in male reproductive ability. Approximately 85% of ArKO males were unable to sire offspring. However, no obvious difference was found in testicular and epididymal weights, numbers of sperm in the epididymis or the ability of sperm to fertilize eggs in vitro between wild-type and ArKO males. An examination of mating behaviour demonstrated that ArKO males showed an impairment in mounting behaviour against sexually mature females. The inability of more than 90% of ArKO males to sire offspring was reversed by repeated subcutaneous injections of 17beta-oestradiol when initiated on the day of birth. The effects of 17beta-oestradiol on reproduction were concentration dependent and evident when supplementation was initiated on day 7, but not on day 15 after birth. These findings suggest that oestrogens acting during neonatal life are required for normal mating behaviour in adulthood.

A loss of aggressive behaviour and its reinstatement by oestrogen in mice lacking the aromatase gene (Cyp19).

Aromatase P450 (CYP19) is an enzyme responsible for conversion of androgens to oestrogens. We generated CYP19 knockout (ArKO) mice by targeting disruption of the CYP19 gene and observed that the ArKO males exhibited a complete loss of aggressive behaviour against intruder mice when examined using a resident-intruder paradigm. The defect in the behaviour of ArKO males was reinstated when the mice received supplements of 17beta-oestradiol soon after birth. Nevertheless, the cumulative duration of the behaviour displayed by the treated mice during the test period of 15 min was 19+/-10 s, which was much shorter than that displayed by wild-type males, 90+/-17 s. When the supplementation was started at 7 days after birth, the defect was not restored. These findings illustrate an absolute requirement for oestrogen during the neonatal stage of a male's life for the development of the potential for aggression observed in adulthood. Furthermore, the present study demonstrates that ArKO males are a useful model in which to investigate the neural mechanisms by which aggressive behaviour is controlled.

Sex- and Age-Related Response to Aromatase Deficiency in Bone

Deficiency of sex steroids causes osteoporosis, but the relationship between estrogen and androgen is not clear because androgen is converted into estrogen by aromatase. In this study, we characterized bone metabolism in the aromatase-deficient (ArKO) mouse. At 9 weeks old, a marked loss of cancellous bone due to increased bone resorption was observed not only in female ArKO mice but also in males. The degree of bone loss in ArKO males was similar to that in females, and treatment with 17β-estradiol completely restored the bone mass in both sexes. At 32 weeks old, female ArKO mice showed severe loss of cancellous and cortical bone. Male ArKO mice of this age also showed reduced bone mass, but the degree of bone loss in females was more marked than that in males. Here, we report sex- and age-related responses to aromatase deficiency in bone.