Ariane Willems

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.

Early effects of Sertoli cell‐selective androgen receptor ablation on testicular gene expression

Evidence from several models of hormone depletion and/or replacement and from knockout animals points to a key role of androgens in the control of spermatogenesis. In testes of mice with a Sertoli cell-selective ablation of the androgen receptor (SCARKO), transcriptional profiling, using microarray technology, revealed that, already on postnatal day 10,692 genes are differentially expressed compared with testes of control mice. Further evaluation of a subset of these genes by quantitative RT-PCR suggested that differences in expression may already be evident on day 8 or earlier. As the androgen receptor in mouse Sertoli cells becomes immunologically detectable around day 5, we tried to identify the earliest responses to androgens by a new transcriptional profiling study on testes from 6-day-old SCARKO and control mice. No obvious and novel early androgen response genes, potentially acting as mediators of subsequent indirect androgen actions, could be identified. However, several genes differentially expressed on day 10 already displayed a response to androgen receptor ablation on day 6. Quantitative RT-PCR studies for 12 of these genes on 10 paired SCARKO and control testes from 4-, 6-, 8-, 10-, 20- and 50-day-old mice revealed significant differences in expression level from day 4 onwards for three genes (Eppin, PCI, Cldn11) and from day 6 onwards for one more gene (Rhox5). For at least two of these genes (Rhox5 and Eppin), there is evidence for direct regulation via the androgen receptor. For three additional genes (Gpd1, Tubb3 and Tpd52l1) significantly lower expression in the SCARKO was noted from day 8 onwards. For all the studied genes, an impressive increase in transcript levels was observed between day 4-50 and differential expression was maintained in adulthood. It is concluded that the SCARKO model indicates incipient androgen action in mouse Sertoli cells from day 4 onwards.

Expression of Tubb3, a Beta-Tubulin Isotype, Is Regulated by Androgens in Mouse and Rat Sertoli Cells

Our previous analysis of Sertoli cell androgen receptor (AR) knockout (SCARKO) mice revealed that several cytoskeletal components are a potential target of androgen action. Here, we found that one of these components, the beta-tubulin isotype Tubb3, is differentially regulated in testes from SCARKO mice (relative to littermate controls) from Postnatal Day 10 to adulthood. The Tubb3 gene is unique in this respect, as at Day 10, no other beta-tubulin genes are significantly regulated by AR. We further characterized androgen regulation of Tubb3 in vivo and in vitro and demonstrated that it is a conserved feature in both mice and rats. To investigate whether androgens directly regulate Tubb3 expression, we screened for androgen response elements (AREs) in the Tubb3 gene. In silico analysis revealed the presence of four ARE motifs in Tubb3 intron 1, two of which bind to AR in vitro. Mutation of one of these (ARE1) strongly reduced androgen-dependent reporter gene expression. These results, coupled with the finding that the AR binds to the Tubb3 ARE region in vivo, suggest that Tubb3 is a direct target of AR. Our data strengthen the contention that androgens exert their effects on spermatogenesis, in part, through modulation of the Sertoli cell cytoskeleton. Androgen regulation of beta-tubulin has also been described in neurons, fortifying the already known similarity in microtubule organization in Sertoli cell processes and neurons, the only other cell type in which Tubb3 is known to be expressed.

Characterization of the testicular, epididymal and endocrine phenotypes in the Leuven Vdr-deficient mouse model: targeting estrogen signalling.

Vitamin D is a key factor for calcium and bone homeostasis, but signalling through the vitamin D receptor (VDR) seems also to be important for testicular function. To test the functional role of vitamin D signalling we examined the male reproductive system of the Leuven Vdr-ablated (Vdr(-/-)) mice, previously established as a model for hereditary vitamin D-resistant rickets. We investigated reproductive hormones, changes in gene expression and histological phenotype of eleven Vdr(-/-), eight Vdr(+/-) and nine Vdr(+/+) mice. Testicular and epididymal histology were grossly normal in Vdr(-/-) mice. Accordingly, no differences were found in serum concentrations of testosterone, estradiol, LH, and FSH or testicular expression of Cyp19a1, Ersα, Cyp17a1, Star, Insl3, Inhbb, and Amh. However, a significantly lower ERβ expression was found in testis of Vdr(+/-) and Vdr(-/-) mice, conversely epididymal expressions of ERα and the estrogen-target gene Aqp9 were higher. In conclusion, vitamin D seems dispensable for murine spermatogenesis and sex hormone production, but aberrant estrogen-signalling may elicit some of the VDR-mediated effects on male reproduction.

The development of an inducible androgen receptor knockout model in mouse to study the postmeiotic effects of androgens on germ cell development.

A mouse model with a Sertoli cell (SC)-selective ablation of the androgen receptor (AR) -the SCARKO mouse- demonstrated that the effects of androgens on spermatogenesis depend on the presence of an active AR in SC. This model has been extremely valuable in the study of the effects of androgens on the initiation of spermatogenesis. However, due to the early (prenatal) inactivation of the AR SCARKO mice develop a complete block in meiosis, making it impossible to study the effects of androgens on postmeiotic steps of germ cell development. It would therefore be of interest to develop a model in which the AR can be ablated at any chosen time point. Here we used a mouse line ubiquitously expressing a tamoxifen (TM)-inducible Cre recombinase to develop an inducible AR knockout model (iARKO). It is shown that treatment with TM (3 mg/day) for five consecutive days efficiently inactivates the AR in testicular tissue and decreases the expression of known AR-target genes in SC (Rhox5, Spinlw1) without markedly affecting testicular cell composition one day after the final injection. TM treatment did, however, decrease serum gonadotropin levels and the expression of several Leydig cell genes (StAR, Cyp17a1, Insl3), resulting in decreased testosterone levels. Nevertheless, the intratesticular testosterone concentration still far exceeds the estimated concentrations required to saturate the AR. It may be concluded that the study of androgen-responsive postmeiotic genes in SC may be feasible using a TM-inducible AR knockout model provided that appropriate controls are included correcting for off-target effects of TM.

Organotypic Cultures of Prepubertal Mouse Testes: A Method to Study Androgen Action in Sertoli Cells while Preserving their Natural Environment

Cluster analysis at Postnatal Day 8–20 of putative androgen-regulated genes in mice with Sertoli cell-selective knockout of the androgen receptor (SCARKO) has pinpointed three genes (Spinlw1, Gpd1, Drd4) with an expression pattern strongly resembling that of Rhox5, the definitive Sertoli cell (SC) androgen-regulated gene. We used organotypic testis cultures from Day 8 mice to study control of these genes by (anti)androgens and follicle-stimulating hormone (FSH). Testis morphology and androgen induction of the studied genes were preserved for 48 h. Preincubation with ketoconazole for 24 h to block endogenous androgen production, followed by 24-h incubation with the synthetic androgen R1881, resulted in 45-, 5-, 19-, and 6-fold induction of mRNA levels of Rhox5, Spinlw1, Gpd1, and Drd4, respectively. However, noticeable differences in control of the studied genes were observed. Rhox5 and Spinlw1 were fully induced by R1881 in the continuous (48 h) presence of ketoconazole, whereas only marginal effects were observed on expression of Gpd1 and Drd4. Similarly, FSH only marginally affected expression of Rhox5 and Spinlw1, whereas it markedly increased Gpd1 and Drd4 expression. Explant cultures of SCARKO testes confirmed the differential effects of FSH on the studied genes and, for Gpd1, showed that the effect did not depend on a functional androgen receptor in SC, whereas this was essential for the effects of FSH on Drd4. In conclusion, organotypic cultures represent the first in vitro approach to preserving androgen responsiveness of putative SC-expressed genes. This approach facilitates detailed analysis of their regulation in ways not possible in vivo.

Contribution of Recent Transgenic Models and Transcriptional Profiling Studies to Our Understanding of the Mechanisms by which Androgens Control Spermatogenesis

Androgens play a key role in the control of spermatogenesis and interference with their intratesticular secretion and action is a critical element in many contraceptive strategies. Nonetheless, the cellular and molecular mechanisms by which androgens control germ cell development remain poorly understood. Recent transgenic models in which the androgen receptor (AR) is selectively ablated in Ser- toli cells show unambiguously that the Sertoli cell is the main target for androgen action in the control of spermatogenesis. A number of additional mouse models have been developed mimicking human diseases in which mutations of the AR cause disturbed fertility without affecting male development. Transcriptional profiling studies in mice with Sertoli cell-selective AR ablation and in some other experi- mental paradigms have tried to identify androgen-regulated genes relevant to the control of spermatogenesis. The overlap in genes identi- fied in different studies is poor but this may be due mainly to dissimilarities in experimental setup. In all studies, relatively large numbers of genes rather than a few key genes seem to be affected by androgen action. Genes related to tubular restructuring, cell junction dynam- ics, cytoskeleton, solute transportation and vitamin A metabolism are prominently present. Although further work is obviously needed, it may be anticipated that these studies will result in the identification of subsets of genes that can be used as diagnostic tools as well as in the identification of targets for the development of novel contraceptives.

Sertoli cell androgen receptor signalling in adulthood is essential for post-meiotic germ cell development.

Androgens are key drivers of spermatogenesis, and germ cells in mice lacking androgen receptor (AR), specifically from Sertoli cells, arrest in meiosis (reviewed in Smith and Walker, 2014). When Sertoli‐cell AR is ablated during fetal life (De Gendt and Verhoeven, 2012), however, it is impossible to determine whether the meiotic‐arrest phenotype observed in adults results from perturbed Sertoli cell development or perturbed function in adulthood. We used a lentiviral approach to determine if Sertoli‐cell AR is essential for supporting spermatogenesis specifically in adult testes—an organ where tamoxifen‐inducible knockout may present off‐target effects. Specifically, we introduced Cre recombinase into the Sertoli cells of adult male ARflox mice (De Gendt et al., 2004) to generate adult Sertoli‐Cell AR Knockout (aSCARKO) mice. Lentiviral particles contained both CMV‐Cre recombinase and tRFP635 (red fluorescent protein) transgenes separated by an IRES, or CMV‐tRFP635 alone. Shuttle vectors were packaged using a third‐generation lentiviral vector pseudotyped for VSV‐G, produced at a viral titer of >1 × 109. Virus was introduced into the seminiferous tubules of adult male ARflox/Y via injection into the efferent ducts, using 10 μl of Cre virus, tRFP635 control virus, or optiMEM (vehicle); an additional sham operated, but not injected, control was also evaluted. To control for systemic effects, combinations of Cre/control Cre/optiMEM, Cre/sham, control/optiMEM, control/sham, were generated in testes from individual mice (one treatment per testis; n = 10 per group). Tissues were collected 40 days after surgery (one complete cycle of spermatogenesis). Body and seminal vesicle weight (a biomarker of circulating androgen concentrations) did not differ between any treatment group (data not shown), but the weight of testes injected with Cre recombinase virus was significantly reduced (sham, 92.52 ± 4.29; OptiMEM, 100.12 ± 4.96; tRFP635 control, 70.22 ± 17.31; Cre, 35.48 ± 3.45 mg) to a final weight consistent with developmental‐SCARKO mice (De Gendt et al., 2004). tRFP635 was specifically detected in the cytoplasm of Sertoli cells (Fig. ​(Fig.1A,B),1A,B), but not in other testicular cell types. AR expression was observed in all somatic cells in both sham‐operated and control tRFP635 lentivirus‐injected testes. In contrast, injection of testes with Cre recombinase virus resulted in Sertoli cell‐specific localisation of tRFP with a loss of AR expression only in Sertoli cells; AR was retained in other testicular somatic cell types. Thus, AR had been selectively ablated in adult Sertoli cells whilst leaving the remainder of the testis untouched. Figure 1 Immunolocalisation of tRFP635 and AR, and testicular and epididymal morphology in virus‐injected adult testes. A–B: Sertoli cells retain AR expression (white arrows) following injection of control virus (A), whereas injection of Cre‐encoding ... Forty days post‐injection, seminiferous tubules from control testes retained normal spermatogenesis, with no obvious defects (Fig. ​(Fig.1A,C).1A,C). Testes injected with Cre recombinase virus, however, displayed evidence of germ‐cell arrest during meiosis (Fig. ​(Fig.1B,D),1B,D), similar to that observed in SCARKO mice (De Gendt et al., 2004). Furthermore, epididymides continuous with the Cre‐encoding virus‐injected testes contained no mature spermatozoa (Fig. ​(Fig.1F).1F). Therefore, loss of Sertoli‐cell AR in adulthood recapitulates the spermatogenic‐block phenotype observed in developmentally induced SCARKO models, unequivocally demonstrating that Sertoli cell AR is essential for continuous spermatogenesis in adults.