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

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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

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

Peroxisomal multifunctional protein 2 is essential for lipid homeostasis in Sertoli cells and male fertility in mice

International audienceInactivation of peroxisomal beta-oxidation in mice, by knocking out multifunctional protein-2 (MFP-2; also called D-bifunctional enzyme), causes male infertility. In the testis, extensive accumulations of neutral lipids were observed in Sertoli cells, beginning in prepubertal mice and evolving in complete testicular atrophy by the age of 4 months. Spermatogenesis was already severely affected at the age of 5 wk, and pre- and postmeiotic germ cells gradually disappeared from the tubuli seminiferi. Based on cytochemical stainings and biochemical analyses, the lipid droplets consisted of cholesteryl esters and neutral glycerolipids. Furthermore, peroxisomal beta-oxidation substrates, such as very-long-chain fatty acids and pristanic acid, accumulated in the testis, whereas the concentration of docosapentaenoic acid, a polyunsaturated fatty acid and peroxisomal beta-oxidation product, was reduced. The testicular defects were also present in double MFP-2/peroxisome proliferator-activated receptor-alpha knockout mice, ruling out the possibility that they were mediated through the activation of this nuclear receptor. Immunoreactivity for peroxisomal proteins, including MFP-2, was detected in Sertoli cells as well as in germ cells and Leydig cells. The pivotal role of peroxisomal metabolism in Sertoli cells was also demonstrated by generating mice with a Sertoli cell-selective elimination of peroxisomes through cell type-specific inactivation of the peroxin 5 gene. These mice also developed lipid inclusions and were infertile, and their testes fully degenerated by the age of 4 months. In conclusion, the present data demonstrate that peroxisomal beta-oxidation is essential for lipid homeostasis in the testis and for male fertility

Role of androgens in fetal testis development and dysgenesis.

This study sought to establish whether reduced androgen levels/action in the fetal rat testis induced by di(n-butyl) phthalate (DBP) contributes to dysgenetic features, namely reduced Sertoli cell number, occurrence of multinucleated gonocytes (MNG), and Leydig cell aggregation. Pregnant rats were administered treatments or cotreatments designed to manipulate testosterone levels [DBP, testosterone propionate (TP)] or action [flutamide, 7,12-dimethyl-benz[a]anthracene (DMBA)]. The aforementioned end points were analyzed and related to intratesticular testosterone (ITT) levels and peripheral androgen action (anogenital distance). Dysgenetic features were also evaluated in mice with inactivation of the androgen receptor (testicular feminized or ARKO mice). Exposure to DBP alone, or combined with flutamide, DMBA, or TP, resulted in reduced Sertoli cell number and ITT levels, as did exposure to TP alone; coadministration of DBP + TP caused the most severe reduction in both parameters. A positive correlation between ITT levels and Sertoli cell number was found (r = 0.791; P = 0.019). Similarly, exposure to DBP alone, or as a cotreatment, significantly increased occurrence of MNG and Leydig cell aggregation, and these were negatively correlated with ITT levels. Exposure to flutamide or DMBA alone had no significant effect on these dysgenetic end points. These findings suggest that reduced ITT decreases fetal Sertoli cell numbers and might be involved in Leydig cell aggregation and MNG. However, of these three end points, only Sertoli cell number was affected significantly in ARKO/testicular feminized mice with absent androgen action. Therefore, induction of MNG and Leydig cell aggregation might result from DBP-induced effects other than suppression of ITT levels