Testicular development in mice lacking receptors for follicle stimulating hormone and androgen

Post-natal testicular development is dependent on gonadotrophin and androgen stimulation. Follicle stimulating hormone (FSH) acts through receptors (FSHR) on the Sertoli cell to stimulate spermatogenesis while androgens promote testis growth through receptors (AR) on the Sertoli cells, Leydig cells and peritubular myoid cells. In this study we have examined the effects on testis development of ablating FSHRs (FSHRKO mice) and/or ARs ubiquitously (ARKO mice) or specifically on the Sertoli cells (SCARKO mice). Cell numbers were measured using stereological methods. In ARKO mice Sertoli cell numbers were reduced at all ages from birth until adulthood. FSHR ablation also caused small reductions in Sertoli cell numbers up to day 20 with more marked effects seen in the adult. Germ cell numbers were unaffected by FSHR and/or AR ablation at birth. By day 20 ubiquitous AR or FSHR ablation caused a marked reduction in germ cell numbers with a synergistic effect of losing both receptors (germ cell numbers in FSHRKO.ARKO mice were 3% of control). Germ cell numbers in SCARKO mice were less affected. By adulthood, in contrast, clear synergistic control of germ cell numbers had become established between the actions of FSH and androgen through the Sertoli cells. Leydig cell numbers were normal on day 1 and day 5 in all groups. By day 20 and in adult animals total AR or FSHR ablation significantly reduced Leydig cell numbers but Sertoli cell specific AR ablation had no effect. Results show that, prior to puberty, development of most testicular parameters is more dependent on FSH action than androgen action mediated through the Sertoli cells although androgen action through other cells types is crucial. Post-pubertally, germ cell numbers and spermatogenesis are dependent on FSH and androgen action through the Sertoli cells.     

Gene expression alterations by conditional knockout of androgen receptor in adult Sertoli cells of Utp14b jsd/jsd (jsd) mice

Spermatogenesis is dependent primarily on testosterone action on the Sertoli cells, but the molecular mechanisms have not been identified. Attempts to identify testosterone-regulated target genes in Sertoli cells have used microarray analysis of gene expression in mice lacking the androgen receptor (AR) in Sertoli cells (SCARKO) and wild-type mice, but the analyses have been complicated both by alteration of germ cell composition of the testis when pubertal or adult mice were used and by differences in Sertoli-cell gene expression from the expression in adults when prepubertal mice were used. To overcome these limitations and identify AR-regulated genes in adult Sertoli cells, we compared gene expression in adult jsd (Utp14b jsd/jsd, juvenile spermatogonial depletion) mouse testes and with that in SCARKO-jsd mouse testes, since their cellular compositions are essentially identical, consisting of only type A spermatogonia and somatic cells. Microarray analysis identified 157 genes as downregulated and 197 genes as upregulated in the SCARKO-jsd mice compared to jsd mice. Some of the AR-regulated genes identified in the previous studies, including Rhox5, Drd4, and Fhod3, were also AR regulated in the jsd testes, but others, such as proteases and components of junctional complexes, were not AR regulated in our model. Surprisingly, a set of germ cell–specific genes preferentially expressed in differentiated spermatogonia and meiotic cells, including Meig1, Sycp3, and Ddx4, were all upregulated about 2-fold in SCARKO-jsd testes. AR-regulated genes in Sertoli cells must therefore be involved in the regulation of spermatogonial differentiation, although there was no significant differentiation to spermatocytes in SCARKO-jsd mice. Further gene ontogeny analysis revealed sets of genes whose changes in expression may be involved in the dislocation of Sertoli cell nuclei in SCARKO-jsd testes.     

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 androgen-regulated gene expressed in Sertoli cells, were selected for confirmation by quantitative RT-PCR 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.     

Restoration of Spermatogenesis and Male Fertility Using an Androgen Receptor Transgene

Androgens signal through the androgen receptor (AR) to regulate male secondary sexual characteristics, reproductive tract development, prostate function, sperm production, bone and muscle mass as well as body hair growth among other functions. We developed a transgenic mouse model in which endogenous AR expression was replaced by a functionally modified AR transgene. A bacterial artificial chromosome (BAC) was constructed containing all AR exons and introns plus 40 kb each of 5'' and 3'' regulatory sequence. Insertion of an internal ribosome entry site and the EGFP gene 3’ to AR allowed co-expression of AR and EGFP. Pronuclear injection of the BAC resulted in six founder mice that displayed EGFP production in appropriate AR expressing tissues. The six founder mice were mated into a Sertoli cell specific AR knockout (SCARKO) background in which spermatogenesis is blocked at the meiosis stage of germ cell development. The AR-EGFP transgene was expressed in a cyclical manner similar to that of endogenous AR in Sertoli cells and fertility was restored as offspring were produced in the absence of Sertoli cell AR. Thus, the AR-EGFP transgene under the control of AR regulatory elements is capable of rescuing AR function in a cell selective, AR-null background. These initial studies provide proof of principle that a strategy employing the AR-EGFP transgene can be used to understand AR functions. Transgenic mice expressing selective modifications of the AR-EGFP transgene may provide crucial information needed to elicit the molecular mechanisms by which AR acts in the testis and other androgen responsive tissues.     

Androgen regulation of stage-dependent cyclin D2 expression in Sertoli cells suggests a role in modulating androgen action on spermatogenesis

Regulation of spermatogenesis involves stage-dependent androgen action on Sertoli cells, but the pathways involved are unclear. We assessed if cyclin D2 could play a role. In rats, Sertoli cell nuclear, stage-dependent immunoexpression of cyclin D2 switched on after Day 10 and persisted through Day 35, but disappeared by adulthood. However, ethane dimethane sulfonate (EDS)-induced testosterone withdrawal in adult rats for 6 days induced stage-dependent cyclin D2 immunoexpression in Sertoli cells, with highest expression at stages IX-XII and nondetectable at stages VI-VIII (opposite that for androgen receptor [AR] immunoexpression). In EDS-treated rats, a single injection of testosterone but not of estrogen reversed this change in 4 h, and testosterone administration from the time of EDS treatment prevented expression of cyclin D2 in Sertoli cells. The EDS-induced changes in cyclin D2 immunoexpression were matched by changes in expression of Ccnd2 (cyclin D2) mRNA in isolated stage-dissected tubules. Treatment of adult rats with flutamide induced stage-dependent cyclin D2 immunoexpression in Sertoli cells within 18 h, and confocal microscopy revealed that immunoexpression of AR and cyclin D2 were mutually exclusive within individual seminiferous tubules in these animals. Sertoli cell-selective ablation of the AR in mice using Cre/loxP technology also resulted in stage-dependent Sertoli cell cyclin D2 immunoexpression. Downstream from cyclin D2 action is retinoblastoma 1 (RB1), a tumor suppressor protein, immunoexpression of which paralleled stage-dependent AR expression in Sertoli cells; RB1 stage specificity disappeared after EDS treatment. These results point to a non-cell cycle role for cyclin D2 and RB1 in mature Sertoli cells in the stage-dependent mechanisms regulated by AR expression and androgen action.     

Proteomic changes in rat spermatogenesis in response to in vivo androgen manipulation; impact on meiotic cells

The production of mature sperm is reliant on androgen action within the testis, and it is well established that androgens act on receptors within the somatic Sertoli cells to stimulate male germ cell development. Mice lacking Sertoli cell androgen receptors (AR) show late meiotic germ cell arrest, suggesting Sertoli cells transduce the androgenic stimulus co-ordinating this essential step in spermatogenesis. This study aimed to identify germ cell proteins responsive to changes in testicular androgen levels and thereby elucidate mechanisms by which androgens regulate meiosis. Testicular androgen levels were suppressed for 9 weeks using testosterone and estradiol-filled silastic implants, followed by a short period of either further androgen suppression (via an AR antagonist) or the restoration of intratesticular testosterone levels. Comparative proteomics were performed on protein extracts from enriched meiotic cell preparations from adult rats undergoing androgen deprivation and replacement in vivo. Loss of androgenic stimulus caused changes in proteins with known roles in meiosis (including Nasp and Hsp70-2), apoptosis (including Diablo), cell signalling (including 14-3-3 isoforms), oxidative stress, DNA repair, and RNA processing. Immunostaining for oxidised DNA adducts confirmed spermatocytes undergo oxidative stress-induced DNA damage during androgen suppression. An increase in PCNA and an associated ubiquitin-conjugating enzyme (Ubc13) suggested a role for PCNA-mediated regulation of DNA repair pathways in spermatocytes. Changes in cytoplasmic SUMO1 localisation in spermatocytes were paralleled by changes in the levels of free SUMO1 and of a subunit of its activating complex, suggesting sumoylation in spermatocytes is modified by androgen action on Sertoli cells. We conclude that Sertoli cells, in response to androgens, modulate protein translation and post-translational events in spermatocytes that impact on their metabolism, survival, and completion of meiosis.     

Intratesticular androgen levels, androgen receptor localization, and androgen receptor expression in adult rat Sertoli cells

In the rat, quantitatively normal spermatogenesis is maintained only when intratesticular testosterone (ITT) levels greatly exceed the peripheral T concentration. When ITT concentrations fall below a threshold, germ cells are lost at specific stages of the seminiferous cycle. Germ cells can be restored by high doses of T that binds to androgen receptors (AR) in Sertoli cells. However, the relationships between germ cell dynamics, AR-mediated molecular events, and ITT concentrations are not established. ITT levels may regulate germ cell life and death through an effect on AR localization and AR mRNA or protein levels within Sertoli cells at specific stages of the cycle. We determined AR localization and mRNA and protein expression in adult rat Sertoli cells in relation to reduced and then restored ITT concentrations in vivo. ITT levels were reduced by implanting rats with T- and estradiol (E)-filled capsules for 7-28 days and subsequently restored with large T-filled capsules. AR is normally localized within Sertoli cell nuclei at stages VII-VIII of the seminiferous epithelium. After T/E treatment, AR immunostaining in Sertoli cell nuclei became nondetectable by 14-28 days but was restored 6 h following T restoration. The loss of Sertoli cell nuclear AR localization correlated with increasing numbers of apoptotic germ cells. AR mRNA levels in isolated Sertoli cells did not change through 14 days of T/E treatment, increased significantly by Day 28, and remained elevated 24 h after T restoration. AR mRNA levels in microdissected tubules at stages II-IV, VI-VIII, and IX-XII did not decrease through 14 days of T/E treatment. In contrast, AR protein levels were reduced in seminiferous tubules by Day 14 and in testes at Day 28 post-T/E treatment but were restored within 24 h by T repletion. Therefore, the reduction of ITT concentration results in a time-dependent redistribution of AR and reduced AR protein but not AR mRNA levels in Sertoli cells. Repletion of T restored AR protein and it relocated to Sertoli cell nuclei. By an unknown mechanism, T regulates AR localization within Sertoli cells to determine germ cell life or death.     

雄激素通过雄激素受体途径调控垂体肿瘤转化基因(PTTG1)的表达

雄激素能上调大鼠前列腺中垂体肿瘤转化基因1(PTTG1)的表达,在前列腺癌标本中,发现PTTG1的表达明显高于正常组织.因此,用雄激素依赖的前列腺癌细胞LNCaP来研究雄激素调控PTTG1表达的分子机制.在LNCaP细胞中,通过雄激素刺激后,PTTG1的表达明显升高.根据序列分析以及5-缺失体实验(deletion)和突变实验(mutation),发现PTTG1的启动子上游-950到-933的序列上有1个雄激素受体反应元件(ARE),染色体免疫共沉淀实验(CHIP)也证实了雄激素能促进雄激素受体与PTTG1启动子上ARE结合,从而在转录水平调控PTTG1的表达.     

Transfection with steroid-responsive reporter constructs shows glucocorticoid rather than androgen responsiveness in cultured Sertoli cells

It remains unclear why it has proven so difficult to identify androgen target genes in cultured Sertoli cells. Given the lack of useful endogenous reporter genes, we studied the androgen and glucocorticoid responsiveness of these cells by transfection with three different steroid-responsive reporter constructs. The constructs were driven by the tyrosine aminotransferase steroid-responsive region (TAT-GRE4x-Luc), the mouse mammary tumor virus promoter (MMTV-Luc) and the Pem homeobox gene proximal promoter respectively (Pem-Luc). These constructs can be activated either by both the glucocorticoid receptor (GR) and the androgen receptor (AR) (TAT-GRE4x-Luc and MMTV-Luc) or selectively by the AR (Pem-Luc). Despite high transfection efficiency (30-40%) none of the constructs could be activated by treatment of the Sertoli cells with testosterone, 5alpha-dihydrotestosterone or synthetic androgens. Even pretreatment with follicle-stimulating hormone to raise AR levels (from 31 up to 82fmol/mg protein) did not result in androgen responsiveness. In contrast, treatment with dexamethasone markedly stimulated TAT-GRE4x-Luc and MMTV-Luc activity. GR levels reached a value of 172fmol/mg protein in the cultured cells and both AR and GR displayed homogeneous distribution by immunocytochemical evaluation. Androgen responsiveness was restored and glucocorticoid responsiveness was increased by cotransfection with AR or GR expression constructs. Under cotransfection conditions, 1nM of testosterone (a concentration that is some 100 times lower than that estimated to be present in the testis) was sufficient to stimulate the TAT-GRE4x-Luc maximally. Our data indicate that cultured Sertoli cells respond better to glucocorticoids than to androgens and that one of the factors limiting androgen responsiveness is the availability of AR. Other factors limiting the transactivation capacity of the (endogenous) AR, however, cannot be excluded.     

Sertoli cell-specific deletion of the androgen receptor compromises testicular immune privilege in mice

In the mammalian testis, meiotic and postmeiotic germ cell antigens are granted immune privilege. Both local immune suppression and specialized intercellular junctions between somatic Sertoli cells have been proposed to contribute to a highly restricted and effective blood-testis barrier (BTB) that helps maintain tolerance to germ cell antigens. Several studies have suggested that androgens play a role in immune suppression, although direct evidence for this is lacking. We previously reported that Sertoli cell-specific ablation of the androgen receptor (Ar) decreases expression of Cldn3, an androgen-regulated gene and component of Sertoli cell tight junctions, and increases the permeability of the BTB to biotin, a small-molecular-weight tracer. The physiological consequences of Sertoli cell-specific Ar (S-Ar) ablation on immune privilege are unknown. Here we show that in the testes of S-Ar mutant mice, the ultrastructure of Sertoli cell tight junctions is defective and testicular IgG levels are elevated. The interstitium of S-Ar mutant testes becomes populated with macrophages, neutrophils, plasma cells, and eosinophils, and serum samples of mutant mice contain antibodies against germ cell antigens. Together, these results suggest that Sertoli cell-specific deletion of the androgen receptor results in loss of testicular immune privilege. Suppressed levels of androgen signaling may be a contributing factor in idiopathic male infertility.