Contribution of recent transgenic models and transcriptional profiling studies to our understanding of the mechanisms by which androgens control spermatogenesis

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The effect of a sertoli cell-selective knockout of the androgen receptor on testicular gene expression in prepubertal mice

To unravel the molecular mechanisms mediating the effects of androgens on spermatogenesis, testicular gene expression was compared in mice with Sertoli cell-selective androgen receptor knockout (SCARKO) and littermate controls on postnatal d 10. Microarray analysis identified 692 genes with significant differences in expression. Of these, 28 appeared to be down-regulated and 12 up-regulated at least 2-fold in SCARKOs compared with controls. For nine of the more than 2-fold down-regulated genes, androgen regulation was confirmed by treatment of wild-type mice with an antiandrogen ( flutamide). Some of them were previously described to be androgen regulated or essential for spermatogenesis. Serine-type protease inhibitors were markedly overrepresented in this down-regulated subgroup. A time study (d 8-20), followed by cluster analysis, allowed identification of distinct expression patterns of differentially expressed genes. Three genes with a pattern closely resembling that of Pem, a prototypical an-drogen-regulated gene expressed in Sertoli cells, were selected for confirmation by quantitative RTPCR and additional analysis. The data confirm that the SCARKO model allows identification of novel androgen-regulated genes in the testis. Moreover, they suggest that protease inhibitors and other proteins related to tubular restructuring and cell junction dynamics may be controlled in part by androgens

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

Androgen action in Sertoli cells

Androgens have clearly been shown to be essential for spermatogenesis. The mechanisms by which they exert their effects on germ cell development, however, have remained elusive. Although at the initiation of this project the target cell(s) which would mediate the effects of androgens on spermatogenesis was (were) not unambiguously identified, Sertoli cells were generally considered prime candidates given their intimate morphological and functional interactions with the developing germ cells and the fact that they express the AR in a stage-dependent manner during spermatogenesis. The development – during the course of these studies – of a mouse model with a Sertoli cell-selective knockout of the AR, and the demonstration that these mice (SCARKO) display a spermatogenic arrest in meiosis, unequivocally demonstrates that the AR in Sertoli cells plays a pivotal role in the control of germ cell development. To explore the mechanisms of androgen action in Sertoli cells, we first performed transfection studies with androgen-responsive promoter-reporter constructs and used microarray analysis to search for endogenous androgen-regulated genes in isolated Sertoli cells. Unfortunately, the major conclusion of these studies was that Sertoli cells loose many aspects of their androgen responsiveness upon isolation and culture. Nevertheless, we could identify a couple of endogenous genes with a limited response to androgens and, after cotransfection with an exogenous AR plasmid, also the promoter-reporter constructs responded to androgen treatment with a moderate but reproducible increase in expression. Interestingly, the androgen responses observed under these conditions were maximal at concentrations of androgens (~10-9 M) that are markedly lower than those required to maintain spermatogenesis. After the successful development of SCARKO mice in our laboratory, this unique in vivo model was used to further study the molecular mechanisms by which androgens affect spermatogenesis through Sertoli cells. By comparing gene expression in testes of 10-day-old control and SCARKO mice using microarray technology, 692 genes were found to be differentially expressed. Twenty eight of these genes displayed a more than twofold lower expression in SCARKO vs. control testes (further indicated as strongly downregulated) suggesting that these genes are strongly upregulated by androgens. Three of these genes were previously shown to be essential for spermatogenesis and for five of them direct or indirect evidence for androgen responsiveness existed in the testis or in other target tissues. Twelve genes were strongly upregulated in SCARKO testes and accordingly supposedly downregulated by androgen action in Sertoli cells. For a selection of 9 of the 28 genes which were strongly downregulated in SCARKO testes, differential expression was also shown by Q-RT-PCR and androgen regulation was confirmed in an independent experimental model. Interestingly, the rat homologues of 6 of these selected genes, also proved to be androgen regulated in the rat testis and 4 of the latter even responded to androgens in cultured rat Sertoli cells. Altogether these results support the power of our approach to identify genes mediating the effects of androgens on spermatogenesis through Sertoli cells. In addition, functional analyses and follow-up studies on the microarray data have put forward serine protease inhibitors and enzymes involved in ganglioside biosynthesis as major targets of androgen action in Sertoli cells, suggesting that androgens might influence spermatogenesis through effects on tubular restructuring events, cell junction dynamics and cell-cell communication. In conclusion, our gene expression studies on day 10 support the contention that androgens influence spermatogenesis through limited effects on the expression of an array of different genes in Sertoli cells rather than switching on/off the expression of one or several key genes. These diverse effects on gene expression, however, appear to be directed towards a confined set of molecular processes including tubular restructuring, cell junction dynamics and cell-cell communication. Future studies are warranted to examine the physiological effect of androgens on each of these processes in more detail. Moreover, gene expression studies at younger ages will be required to investigate if the differences in gene expression observed at day 10 represent primary and direct effects of androgens or if they are secondary to earlier effects of androgens on the expression of a different and possibly more limited set of genes.TABLE OF CONTENTS I ABBREVIATIONS V CHAPTER 1: INTRODUCTION 1 1.1. ANDROGENS 1 1.1.1. Androgen production and distribution 1 1.1.2. Molecular mechanisms of androgen action 2 1.1.2.1. Genomic effects of androgens 3 1.1.2.2. Non-genomic effects of androgens 6 1.1.2.2.1. AR-dependent non-genomic effects of androgens 6 1.1.2.2.2. AR-independent non-genomic effects of androgens 7 1.2. SPERMATOGENESIS 8 1.2.1. The spermatogenic environment 8 1.2.2. Organisation of spermatogenesis 10 1.2.3. Hormonal control of spermatogenesis 12 1.2.3.1. Hormonal control of the initiation of spermatogenesis 13 1.2.3.2. Hormonal control of the maintenance or reinitiation of spermatogenesis 14 1.3. ANDROGEN ACTION ON SPERMATOGENESIS 16 1.3.1. What is the cellular target of androgen action on spermatogenesis? 16 1.3.1.1. The role of Sertoli cells in spermatogenesis 17 1.3.1.2. Sertoli cells as a target for androgen action in the control of spermatogenesis 18 1.3.2. The need for high intratesticular testosterone levels in the maintenance of spermatogenesis? 21 1.3.3. Molecular mechanisms by which androgen action in Sertoli cells controls spermatogenesis 24 1.3.3.1. Androgen-regulated genes/proteins in Sertoli cells 24 1.3.3.2. Mechanism of androgen action in Sertoli cells 28 1.4. AIM AND SCOPE OF THE STUDY 30 CHAPTER 2: MATERIALS AND METHODS 33 2.1. STANDARD DNA MANIPULATIONS 33 2.1.1. Ligation of DNA fragments in vectors 33 2.1.2. Transformation of bacteria 33 2.1.3. Plasmid preparation 34 2.1.4. DNA sequencing 34 2.2. STANDARD RNA MANIPULATIONS 34 2.2.1. RNA isolation from cultured Sertoli cells 35 2.2.2. RNA isolation from mouse and rat testes 35 2.2.3. cDNA preparation 35 2.3 STUDIES ON ISOLATED AND CULTURED TESTICULAR CELLS 36 2.3.1. Preparation of Sertoli cells and peritubular myoid cells 36 2.3.2. Collection of peritubular myoid cell conditioned medium (PTCM) 37 2.3.3. Transfection studies 37 2.3.3.1. Origin and description of plasmids used for transfections 37 2.3.3.2. Transfection of isolated rat Sertoli cells 38 2.3.4. Monolayer androgen and glucocorticoid binding assay in isolated Sertoli cells 39 2.3.5. Western blot analysis 39 2.3.6. Immunocytochemical stainings on Sertoli cell monolayers 40 2.4. IN VIVO STUDIES INVOLVING TRANSGENIC AND HORMONALLY MANIPULATED ANIMALS 40 2.4.1. Breeding of SCARKO mice 41 2.4.2. In vivo treatment of prepubertal mice or rats with androgens and anti-androgens 41 2.4.3. In vivo treatment of adult rats with EDS 42 2.4.4. Immunohistochemical stainings on testicular sections 42 2.4.5. Enzymatic separation of interstitial and tubular fractions from mouse testes 43 2.5. GENE EXPRESSION ANALYSES 43 2.5.1. Northern blot analysis 43 2.5.2. Microarray analysis 44 2.5.2.1. Monitoring of RNA quality 44 2.5.2.2. cDNA array analysis 45 2.5.2.3. Oligonucleotide array analysis 45 2.5.2.3.1. Oligonucleotide array target synthesis and processing 45 2.5.2.3.2. Oligonucleotide array data analysis 46 2.5.3. Q-RT-PCR analysis 48 2.5.3.1. Preparation of standards for Q-RT-PCR analysis 48 2.5.3.2. Q-RT-PCR analysis 48 2.6. STATISTICAL ANALYSIS 49 CHAPTER 3: STUDY OF THE MECHANISM OF ANDROGEN REGULATION IN ISOLATED AND CULTURED SERTOLI CELLS 53 3.1. INTRODUCTION 53 3.2. TRANSFECTION STUDIES WITH ANDROGEN- AND GLUCOCORTICOID-RESPONSIVE REPORTER CONSTRUCTS 54 3.3. DOSE-RESPONSE STUDIES WITH AR-CONTAINING PLASMIDS 58 3.4. DISCUSSION AND CONCLUSION 60 CHAPTER 4: A PILOT STUDY EXPLORING THE POSSIBILITY TO IDENTIFY ENDOGENOUS ANDROGEN-REGULATED GENES IN ISOLATED AND CULTURED SERTOLI CELLS 65 4.1. INTRODUCTION 65 4.2. MICROARRAY SCREENING FOR ANDROGEN-REGULATED GENES IN ISOLATED AND CULTURED MOUSE SERTOLI CELLS 66 4.3. EFFECT OF ANDROGEN TREATMENT ON THE EXPRESSION OF IDENTIFIED CANDIDATE TARGET GENES IN ISOLATED AND CULTURED RAT SERTOLI CELLS 66 4.4. HORMONAL REGULATION OF GS EXPRESSION AND GS PROTEIN LEVELS 68 4.5. DISCUSSION AND CONCLUSION 70 CHAPTER 5: GENE EXPRESSION ANALYSIS IN TESTES OF MICE WITH A SERTOLI CELL-SELECTIVE AR KNOCKOUT 75 5.1. INTRODUCTION 75 5.2. IDENTIFICATION OF DIFFERENTIALLY EXPRESSED TRANSCRIPTS IN CONTROL VERSUS SCARKO TESTES AT D 10 76 5.3. EXPRESSION PATTERNS OF TRANSCRIPTS DIFFERENTIALLY EXPRESSED AT D 10 IN SCARKO AND CONTROL MICE DURING PREPUBERTAL DEVELOPMENT 78 5.3. GENES WITH AN EXPRESSION PATTERN RESEMBLING THAT OF PEM/RHOX5 82 5.4. EFFECTS OF SERTOLI CELL-SELECTIVE AR INACTIVATION ON GENES RELATED TO TUBULAR REMODELLING AND JUNCTION DYNAMICS 83 5.5. DISCUSSION AND CONCLUSION 84 CHAPTER 6: FURTHER EVALUATION AND VALIDATION OF GENES DIFFERENTIALLY EXPRESSED IN TESTES OF SCARKO AND CONTROL MICE AS ANDROGEN TARGET GENES 91 6.1. INTRODUCTION 91 6.2. VERIFICATION OF MICROARRAY RESULTS FOR A SELECTION OF 9 DIFFERENTIALLY EXPRESSED GENES USING Q-RT-PCR 92 6.3. SITE OF EXPRESSION AS STUDIED BY ENZYMATIC SEPARATION OF INTERSTITIAL AND TUBULAR COMPARTMENT 94 6.4. VALIDATION OF ANDROGEN RESPONSIVENESS IN AN ALTERNATIVE MODEL: PREPUBERTAL MICE AND RATS TREATED WITH ANTI-ANDROGENS 95 6.4.1. Effects of anti-androgens and androgens on the expression of selected genes in prepubertal mice 95 6.4.2. Effects of anti-androgens and androgens on the expression of the corresponding subset of genes in prepubertal rats 98 6.5. EFFECTS OF AR ABLATION OR ANDROGEN ABLATION ON THE EXPRESSION OF THE SELECTED GENES IN ADULT TESTES FROM MICE AND RATS 99 6.6. CONTROL OF THE EXPRESSION OF THE SELECTED GENES IN ISOLATED RAT SERTOLI CELLS 101 6.6.1. Effects of testosterone, dexamethasone, FSH and PTCM on the expression of the rat homologues of the selected mouse genes 101 6.6.2. Dose response and time studies for the effects of testosterone 102 6.6.3. Effects of anti-androgens on the observed androgen responses 103 6.7. DISCUSSION AND CONCLUSION 104 CHAPTER 7: GENERAL CONCLUSIONS AND PERSPECTIVES 109 7.1 THE ENDOGENOUS AR IN CULTURED SERTOLI CELLS IS UNABLE TO ACTIVATE TRANSFECTED REPORTER CONSTRUCTS BUT STIMULATES THE EXPRESSION OF SOME ENDOGENOUS TARGET GENES TO A LIMITED EXTENT 110 7.2. THE SCARKO MOUSE IS A VALUABLE MODEL TO STUDY ANDROGEN ACTION IN SERTOLI CELLS IN VIVO 113 SUMMARY 119 SAMENVATTING 121 REFERENCE LIST 125 CURRICULUM VITAE 147nrpages: 148status: publishe