Karel De Gendt

Conditional Inactivation of Androgen Receptor Gene in the Nervous System: Effects on Male Behavioral and Neuroendocrine Responses

Testosterone (T) profoundly influences central sexual differentiation and functions. In the brain, T signals either directly through androgen receptor (AR) or indirectly through estrogen receptor (ER) following aromatization into E2 (17-β-estradiol). As T, through AR, also controls peripheral male sexual differentiation, the relative contribution of central AR in T-mediated regulation of behavioral and neuroendocrine responses still remains unclear. To address this question, we generated, by using Cre-loxP technology, mice selectively lacking AR expression in the nervous system. The mutant male urogenital tract was normally developed, and mice were able to produce offspring. Nonetheless, sexual motivation and performance as well as aggressive behaviors were affected. Only a low percentage of males displayed a complete sexual behavior and offensive attacks. The latency to show masculine behaviors was increased and copulation length prolonged. Erectile activity during mating was also altered. These alterations occurred despite increased levels of T and its metabolites, and an unaffected number of ERα-immunoreactive cells. Olfactory preference and neuronal activation, mapped by Fos immunoreactivity, following exposure to estrus female-soiled bedding were also normal. At comparable T levels, greater differences in masculine behaviors were observed between gonadectomized control and mutant males. AR invalidation in the nervous system also disrupted the somatotropic axis since mutant males exhibited growth retardation and decreased serum levels of insulin-like growth factor I. Our findings show that central AR is required in T-induced regulation of male-typical behaviors and gonadotrope and somatotropic axes. This genetic model offers a unique opportunity in the understanding of ARs role in cerebral functions of T.

Relative impact of androgen and estrogen receptor activation in the effects of androgens on trabecular and cortical bone in growing male mice: a study in the androgen receptor knockout mouse model

The relative importance of AR and ER activation has been studied in pubertal male AR knockout and WT mice after orchidectomy and androgen replacement therapy, either with or without an aromatase inhibitor. AR activation dominates normal trabecular bone development and cortical bone modeling in male mice. Moreover, optimal periosteal bone expansion is only observed in the presence of both AR and ER activation.

Differential regulation of bone and body composition in male mice with combined inactivation of androgen and estrogen receptor-α

Osteoporosis and muscle frailty are important health problems in elderly men and may be partly related to biological androgen activity. This androgen action can be mediated directly through stimulation of the androgen receptor (AR) or indirectly through stimulation of estrogen receptor‐alpha (ERα) following aromatization of androgens into estrogens. To assess the differential action of AR and ERα pathways on bone and body composition, AR‐ERα double‐knockout mice were gener‐ated and characterized. AR disruption decreased trabec‐ular bone mass, whereas ERα disruption had no additional effect on the AR‐dependent trabecular bone loss. In contrast, combined AR and ERα inactivation additionally reduced cortical bone and muscle mass compared with either AR or ERα disruption alone. ERα inactivation—in the presence or absence of AR—increased fat mass. We demonstrate that AR activation is solely responsible for the development and maintenance of male trabecular bone mass. Both AR and ERα activation, however, are needed to optimize the acquisition of cortical bone and muscle mass. ERα activation alone is sufficient for the regulation of fat mass. Our findings clearly define the relative importance of AR and ERα signaling on trabecu‐lar and cortical bone mass as well as body composition in male mice.—Callewaert, F., Venken, K., Ophoff, J., De Gendt, K., Torcasio, A., van Lenthe, G. H., Van Ooster‐wyck, H., Boonen, S., Bouillon, R., Verhoeven, G., Vanderschueren, D. Differential regulation of bone and body composition in male mice with combined inactivation of androgen and estrogen receptor‐α. FASEB J. 23, 232‐240 (2009)

Androgens and spermatogenesis: lessons from transgenic mouse models.

Transgenic mouse models have contributed considerably to our understanding of the cellular and molecular mechanisms by which androgens control spermatogenesis. Cell-selective ablation of the androgen receptor (AR) in Sertoli cells (SC) results in a complete block in meiosis and unambiguously identifies the SC as the main cellular mediator of the effects of androgens on spermatogenesis. This conclusion is corroborated by similar knockouts in other potential testicular target cells. Mutations resulting in diminished expression of the AR or in alleles with increased length of the CAG repeat mimick specific human forms of disturbed fertility that are not accompanied by defects in male sexual development. Transcriptional profiling studies in mice with cell-selective and general knockouts of the AR, searching for androgen-regulated genes relevant to the control of spermatogenesis, have identified many candidate target genes. However, with the exception of Rhox5, the identified subsets of genes show little overlap. Genes related to tubular restructuring, cell junction dynamics, the cytoskeleton, solute transportation and vitamin A metabolism are prominently present. Further research will be needed to decide which of these genes are physiologically relevant and to identify genes that can be used as diagnostic tools or targets to modulate the effects of androgens in spermatogenesis.

Selective ablation of the androgen receptor in mouse sertoli cells affects sertoli cell maturation, barrier formation and cytoskeletal development

The observation that mice with a selective ablation of the androgen receptor (AR) in Sertoli cells (SC) (SCARKO mice) display a complete block in meiosis supports the contention that SC play a pivotal role in the control of germ cell development by androgens. To delineate the physiological and molecular mechanism responsible for this control, we compared tubular development in pubertal SCARKO mice and littermate controls. Particular attention was paid to differences in SC maturation, SC barrier formation and cytoskeletal organization and to the molecular mediators potentially involved. Functional analysis of SC barrier development by hypertonic perfusion and lanthanum permeation techniques and immunohistochemical analysis of junction formation showed that SCARKO mice still attempt to produce a barrier separating basal and adluminal compartment but that barrier formation is delayed and defective. Defective barrier formation was accompanied by disturbances in SC nuclear maturation (immature shape, absence of prominent, tripartite nucleoli) and SC polarization (aberrant positioning of SC nuclei and cytoskeletal elements such as vimentin). Quantitative RT-PCR was used to study the transcript levels of genes potentially related to the described phenomena between day 8 and 35. Differences in the expression of SC genes known to play a role in junction formation could be shown from day 8 for Cldn11, from day 15 for Cldn3 and Espn, from day 20 for Cdh2 and Jam3 and from day 35 for ZO-1. Marked differences were also noted in the transcript levels of several genes that are also related to cell adhesion and cytoskeletal dynamics but that have not yet been studied in SC (Actn3, Ank3, Anxa9, Scin, Emb, Mpzl2). It is concluded that absence of a functional AR in SC impedes the remodeling of testicular tubules expected at the onset of spermatogenesis and interferes with the creation of the specific environment needed for germ cell development.

Androgen Signaling in Myocytes Contributes to the Maintenance of Muscle Mass and Fiber Type Regulation But Not to Muscle Strength or Fatigue

Muscle frailty is considered a major cause of disability in the elderly and chronically ill. However, the exact role of androgen receptor (AR) signaling in muscle remains unclear. Therefore, a postmitotic myocyte-specific AR knockout (mARKO) mouse model was created and investigated together with a mouse model with ubiquitous AR deletion. Muscles from mARKO mice displayed a marked reduction in AR protein (60-88%). Interestingly, body weights and lean body mass were lower in mARKO vs. control mice (-8%). The weight of the highly androgen-sensitive musculus levator ani was significantly reduced (-46%), whereas the weights of other peripheral skeletal muscles were not or only slightly reduced. mARKO mice had lower intra-abdominal fat but did not demonstrate a cortical or trabecular bone phenotype, indicating that selective ablation of the AR in myocytes affected male body composition but not skeletal homeostasis. Furthermore, muscle contractile performance in mARKO mice did not differ from their controls. Myocyte-specific AR ablation resulted in a conversion of fast toward slow fibers, without affecting muscle strength or fatigue. Similar results were obtained in ubiquitous AR deletion, showing lower body weight, whereas some but not all muscle weights were reduced. The percent slow fibers was increased, but no changes in muscle strength or fatigue could be detected. Together, our findings show that myocyte AR signaling contributes to the maintenance of muscle mass and fiber type regulation but not to muscle strength or fatigue. The levator ani weight remains the most sensitive and specific marker of AR-mediated anabolic action on muscle.

Loss of androgen receptor binding to selective androgen response elements causes a reproductive phenotype in a knockin mouse model

Androgens influence transcription of their target genes through the activation of the androgen receptor (AR) that subsequently interacts with specific DNA motifs in these genes. These DNA motifs, called androgen response elements (AREs), can be classified in two classes: the classical AREs, which are also recognized by the other steroid hormone receptors; and the AR-selective AREs, which display selectivity for the AR. For in vitro interaction with the selective AREs, the androgen receptor DNA-binding domain is dependent on specific residues in its second zinc-finger. To evaluate the physiological relevance of these selective elements, we generated a germ-line knockin mouse model, termed SPARKI (SPecificity-affecting AR KnockIn), in which the second zinc-finger of the AR was replaced with that of the glucocorticoid receptor, resulting in a chimeric protein that retains its ability to bind classical AREs but is unable to bind selective AREs. The reproductive organs of SPARKI males are smaller compared with wild-type animals, and they are also subfertile. Intriguingly, however, they do not display any anabolic phenotype. The expression of two testis-specific, androgen-responsive genes is differentially affected by the SPARKI mutation, which is correlated with the involvement of different types of response elements in their androgen responsiveness. In this report, we present the first in vivo evidence of the existence of two functionally different types of AREs and demonstrate that AR-regulated gene expression can be targeted based on this distinction.

Fetal programming of adult Leydig cell function by androgenic effects on stem/progenitor cells

Significance Men are defined by androgens (testosterone), which drive fetal masculinization (male development) and puberty and maintain masculinity in adulthood, including sex drive, erectile function, and fertility. Moreover, Western cardiometabolic diseases are all associated with lowered testosterone levels in men. Therefore, influences on testosterone levels in adulthood have pervasive importance for masculinity and health. Our study shows, for the first time, to our knowledge, that testosterone levels during fetal masculinization can (re)program adult testosterone levels through effects on stem cells, which develop into adult Leydig cells (the source of testosterone) after puberty. These stem cells are present in fetal testes of humans and animals, and using the latter, we show how these cells are reprogrammed to affect adult testosterone levels. Fetal growth plays a role in programming of adult cardiometabolic disorders, which in men, are associated with lowered testosterone levels. Fetal growth and fetal androgen exposure can also predetermine testosterone levels in men, although how is unknown, because the adult Leydig cells (ALCs) that produce testosterone do not differentiate until puberty. To explain this conundrum, we hypothesized that stem cells for ALCs must be present in the fetal testis and might be susceptible to programming by fetal androgen exposure during masculinization. To address this hypothesis, we used ALC ablation/regeneration to identify that, in rats, ALCs derive from stem/progenitor cells that express chicken ovalbumin upstream promoter transcription factor II. These stem cells are abundant in the fetal testis of humans and rodents, and lineage tracing in mice shows that they develop into ALCs. The stem cells also express androgen receptors (ARs). Reduction in fetal androgen action through AR KO in mice or dibutyl phthalate (DBP) -induced reduction in intratesticular testosterone in rats reduced ALC stem cell number by ∼40% at birth to adulthood and induced compensated ALC failure (low/normal testosterone and elevated luteinizing hormone). In DBP-exposed males, this failure was probably explained by reduced testicular steroidogenic acute regulatory protein expression, which is associated with increased histone methylation (H3K27me3) in the proximal promoter. Accordingly, ALCs and ALC stem cells immunoexpressed increased H3K27me3, a change that was also evident in ALC stem cells in fetal testes. These studies highlight how a key component of male reproductive development can fundamentally reprogram adult hormone production (through an epigenetic change), which might affect lifetime disease risk.

Tissue- and cell-specific functions of the androgen receptor revealed through conditional knockout models in mice.

This review aims to evaluate the contribution of individual cell-selective knockout models to our current understanding of androgen action. Cre/loxP technology has allowed the generation of cell-selective knockout models targeting the androgen receptor (AR) in distinct putative target cells in a wide variety of organs and tissues including: testis, ovary, accessory sex tissues, muscle, bone, fat, liver, skin and myeloid tissue. In some androgen-regulated processes such as spermatogenesis and folliculogenesis this approach has lead to the identification of a key cellular mediator of androgen action (Sertoli and granulosa cells, respectively). In many target tissues, however, the final response to androgens appears to be more complex. Here, cell-selective knockout technology offers a platform upon which we can begin to unravel the more complex interplay and signaling pathways of androgens. A prototypic example is the analysis of mesenchymal-epithelial interactions in many accessory sex glands. Furthermore, for some actions of testosterone, in which part of the effect is mediated by the active metabolite 17β-estradiol, conditional knockout technology offers a novel strategy to study the relative contribution of AR and estrogen receptor-mediated signaling. The latter approach has already resulted in a better understanding of androgen action in brain and bone. Finally, cell-selective knockout technology has generated valuable models to search for AR-controlled molecular mediators of androgen action, a strategy that has successfully been applied to the study of androgen action in the testis and in the epididymis. Although some conditional knockout models have provided clear answers to physiologic questions, it should be noted that others have pointed to unexpected complexities or technical limitations confounding interpretation of the results.

Androgen Receptor-Dependent and Independent Atheroprotection by Testosterone in Male Mice

The atheroprotective effect of testosterone is thought to require aromatization of testosterone to estradiol, but no study has adequately addressed the role of the androgen receptor (AR), the major pathway for the physiological effects of testosterone. We used AR knockout (ARKO) mice on apolipoprotein E-deficient background to study the role of the AR in testosterone atheroprotection in male mice. Because ARKO mice are testosterone deficient, we sham operated or orchiectomized (Orx) the mice before puberty, and Orx mice were supplemented with placebo or a physiological testosterone dose. From 8 to 16 wk of age, the mice consumed a high-fat diet. In the aortic root, ARKO mice showed increased atherosclerotic lesion area (+80%, P < 0.05). Compared with placebo, testosterone reduced lesion area both in Orx wild-type (WT) mice (by 50%, P < 0.001) and ARKO mice (by 24%, P < 0.05). However, lesion area was larger in testosterone-supplemented ARKO compared with testosterone-supplemented WT mice (+57%, P < 0.05). In WT mice, testosterone reduced the presence of a necrotic core in the plaque (80% among placebo-treated vs. 12% among testosterone-treated mice; P < 0.05), whereas there was no significant effect in ARKO mice (P = 0.20). In conclusion, ARKO mice on apolipoprotein E-deficient background display accelerated atherosclerosis. Testosterone treatment reduced atherosclerosis in both WT and ARKO mice. However, the effect on lesion area and complexity was more pronounced in WT than in ARKO mice, and lesion area was larger in ARKO mice even after testosterone supplementation. These results are consistent with an AR-dependent as well as an AR-independent component of testosterone atheroprotection in male mice.