Sertoli cell-specific ablation of <i>miR-17-92</i> cluster significantly alters whole testis transcriptome without apparent phenotypic effects

<div><p>MicroRNAs are frequently organized into polycistronic clusters whose transcription is controlled by a single promoter. The <i>miR-17-92</i> cluster is expressed in most embryonic and postnatal organs. It is a potent oncogene associated to several types of cancer and it is involved in several important developmental processes. In the testis, expression of the <i>miR-17-92</i> cluster in the germ cells is necessary to maintain normal spermatogenesis. This cluster is also expressed in Sertoli cells (the somatic cells of the seminiferous tubules), which require miRNAs for correct cell development and survival. To study the possible role of <i>miR-17-92</i> in Sertoli cell development and function and, in order to overcome the postnatal lethality of <i>miR-17-92</i>^<i>-/</i> mice, we conditionally deleted it in embryonic Sertoli cells shortly after the sex determination stage using an <i>Amh-Cre</i> allele. Mutant mice developed apparently normal testes and were fertile, but their testis transcriptomes contained hundreds of moderately deregulated genes, indicating that testis homeostasis is tightly controlled in mammals and that <i>miR-17-92</i> expression in Sertoli cells contribute to maintain normal gene expression levels, but is unnecessary for testis development and function. Our results show that significant deregulation of hundreds of genes might have no functional consequences.</p></div

Unaltered expression of both somatic and germ cell markers in <i>SC-miR-17-92</i> KO testes.

<p>No difference between mutant and control testes was observed in the expression pattern of protein markers specific for Sertoli cells (SOX9 and WT1; A <b>a-f</b>), germ cells (PCNA and DMC1; <b>B a-f</b>), peritubular mioid cells (ACTA2; <b>C a</b> and <b>d</b>) and seminiferous tubule basement membrane (LAMININ; <b>C b</b> and <b>e; C c</b> and <b>f</b> show merged images), and Leydig cells (P450scc; <b>D a</b> and <b>b</b>). Scale bar shown in Db represent 100μm for all pictures in Fig. 3.</p

Transcriptome analysis of <i>SC-miR-17-92</i> KO testes.

<p><b>A)</b> Hierarchical clustering analysis showed that the replicate testis samples of both <i>SC-miR-17-92</i> KO and controls grouped together. <b>B)</b> Smear plot of the differential expression test showed that the vast majority of the deregulated genes changed their expression levels by less than one log2 fold change (log2FC). <b>C)</b> GO analysis of <i>SC-miR-17-92</i> KO deregulated genes revealed a significant enrichment (P adj. < 0.05, blue dashed line) in terms associated to normal testicular functions.</p

Analysis of the blood-testis barrier and apoptosis in <i>SC-miR-17-92</i> KO testes.

<p>(<b>A</b>) Both the expression pattern of CLAUDIN 11 (<b>a</b> and <b>d</b>) and the BTB function (<b>b</b> and <b>e</b>) are normal in mutant testes when compared to controls, (<b>c</b>) merged images a+b, (<b>f</b>) merged images d+e. (<b>B</b>) The incidence of apoptosis was similar in both mutant (<b>a</b>) and control (<b>b</b>) testes, as cell counts evidenced no significant differences between them (<b>c</b>). Scale bars shown in Af and Bb represent 100μm in A and B, respectively.</p

Confirmation that Sertoli cell-specific conditional <i>miR-17-92</i> mutant mice were generated.

<p>β-galactosidase staining of testes shows Cre-mediated recombination of the <i>R26R-LacZ</i> allele in Sertoli cells at both embryonic E15.5 (<b>A</b>) and P60 (<b>B</b>). (<b>C</b>) PCR genotyping shows that the 441 bp band specific for the deleted <i>miR-17-92</i>^<i>flox</i> allele (arrowheads) appears in testes of <i>SC-KO</i> mice, but not in those of control males or in other tissues (kid, kidney; liv, liver). (<b>D</b>) Quantitative RT-PCR for the detection of <i>miR-17-92hg</i> transcript levels in E15.5 control and <i>SC-miR-17-92</i> KO testes. Scale bars represent 20μm in A and 100μm in B.</p

Testicular phenotype of <i>SC-miR-17-92</i> KO males at P60 and P365.

<p>Testis mass (<b>A</b>), histological features (<b>B</b>), and sperm counts (<b>C</b>) were similar in both mutant and control males. Scale bar shown in B represent 100μm for all pictures in B.</p

Assignation of differentially expressed genes to the five major cell types in the testis of <i>SC-miR-17-92</i> KO males.

<p><b>A</b>) Circos plot showing the assignation to the different cell types of the differentially expressed genes in <i>SC-miR-17-92</i> KO testes. The color in the Heatmap track show relative expression level in <i>SC-miR-17-92</i> KO testes respect to control normal testes. Red indicate overregulated and green underregulated genes. <b>B-E</b>) Gene ontology enrichment analysis. Genes assigned to the different cell types appears to belong to GO terms normally related to the cell type to which they have been assigned according to the algorithm described. P adj. < 0.05, blue dashed line.</p

DataSheet1_Contribution of TEX15 genetic variants to the risk of developing severe non-obstructive oligozoospermia.PDF

Background: Severe spermatogenic failure (SPGF) represents one of the most relevant causes of male infertility. This pathological condition can lead to extreme abnormalities in the seminal sperm count, such as severe oligozoospermia (SO) or non-obstructive azoospermia (NOA). Most cases of SPGF have an unknown aetiology, and it is known that this idiopathic form of male infertility represents a complex condition. In this study, we aimed to evaluate whether common genetic variation in TEX15, which encodes a key player in spermatogenesis, is involved in the susceptibility to idiopathic SPGF.Materials and Methods: We designed a genetic association study comprising a total of 727 SPGF cases (including 527 NOA and 200 SO) and 1,058 unaffected men from the Iberian Peninsula. Following a tagging strategy, three tag single-nucleotide polymorphisms (SNPs) of TEX15 (rs1362912, rs323342, and rs323346) were selected for genotyping using TaqMan probes. Case-control association tests were then performed by logistic regression models. In silico analyses were also carried out to shed light into the putative functional implications of the studied variants.Results: A significant increase in TEX15-rs1362912 minor allele frequency (MAF) was observed in the group of SO patients (MAF = 0.0842) compared to either the control cohort (MAF = 0.0468, OR = 1.90, p = 7.47E-03) or the NOA group (MAF = 0.0472, OR = 1.83, p = 1.23E-02). The genotype distribution of the SO population was also different from those of both control (p = 1.14E-02) and NOA groups (p = 4.33–02). The analysis of functional annotations of the human genome suggested that the effect of the SO-associated TEX15 variants is likely exerted by alteration of the binding affinity of crucial transcription factors for spermatogenesis.Conclusion: Our results suggest that common variation in TEX15 is involved in the genetic predisposition to SO, thus supporting the notion of idiopathic SPGF as a complex trait.</p

Table1_Contribution of TEX15 genetic variants to the risk of developing severe non-obstructive oligozoospermia.XLSX

Background: Severe spermatogenic failure (SPGF) represents one of the most relevant causes of male infertility. This pathological condition can lead to extreme abnormalities in the seminal sperm count, such as severe oligozoospermia (SO) or non-obstructive azoospermia (NOA). Most cases of SPGF have an unknown aetiology, and it is known that this idiopathic form of male infertility represents a complex condition. In this study, we aimed to evaluate whether common genetic variation in TEX15, which encodes a key player in spermatogenesis, is involved in the susceptibility to idiopathic SPGF.Materials and Methods: We designed a genetic association study comprising a total of 727 SPGF cases (including 527 NOA and 200 SO) and 1,058 unaffected men from the Iberian Peninsula. Following a tagging strategy, three tag single-nucleotide polymorphisms (SNPs) of TEX15 (rs1362912, rs323342, and rs323346) were selected for genotyping using TaqMan probes. Case-control association tests were then performed by logistic regression models. In silico analyses were also carried out to shed light into the putative functional implications of the studied variants.Results: A significant increase in TEX15-rs1362912 minor allele frequency (MAF) was observed in the group of SO patients (MAF = 0.0842) compared to either the control cohort (MAF = 0.0468, OR = 1.90, p = 7.47E-03) or the NOA group (MAF = 0.0472, OR = 1.83, p = 1.23E-02). The genotype distribution of the SO population was also different from those of both control (p = 1.14E-02) and NOA groups (p = 4.33–02). The analysis of functional annotations of the human genome suggested that the effect of the SO-associated TEX15 variants is likely exerted by alteration of the binding affinity of crucial transcription factors for spermatogenesis.Conclusion: Our results suggest that common variation in TEX15 is involved in the genetic predisposition to SO, thus supporting the notion of idiopathic SPGF as a complex trait.</p