Dicer1 Ablation in the Mouse Epididymis Causes Dedifferentiation of the Epithelium and Imbalance in Sex Steroid Signaling

<div><h3>Background</h3><p>The postnatal development of the epididymis is a complex process that results in a highly differentiated epithelium, divided into several segments. Recent studies indicate a role for RNA interference (RNAi) in the development of the epididymis, however, the actual requirement for RNAi has remained elusive. Here, we present the first evidence of a direct need for RNAi in the differentiation of the epididymal epithelium.</p> <h3>Methodology/Principal Findings</h3><p>By utilizing the Cre-LoxP system we have generated a conditional knock-out of Dicer1 in the two most proximal segments of the mouse epididymis. Recombination of <em>Dicer1</em>, catalyzed by <em>Defb41^iCre/wt</em>, took place before puberty, starting from 12 days postpartum. Shortly thereafter, downregulation of the expression of two genes specific for the most proximal epididymis (lipocalin 8 and cystatin 8) was observed. Following this, segment development continued until week 5 at which age the epithelium started to regress back to an undifferentiated state. The dedifferentiated epithelium also showed an increase in estrogen receptor 1 expression while the expression of androgen receptor and its target genes; glutathione peroxidase 5, lipocalin 5 and cysteine-rich secretory protein 1 was downregulated, indicating imbalanced sex steroid signaling.</p> <h3>Conclusions/Significance</h3><p>At the time of the final epididymal development, Dicer1 acts as a regulator of signaling pathways essential for maintaining epithelial cell differentiation.</p> </div

Top genes differentially regulated in gremlin-1 transgenic lung.

<p>Top genes differentially regulated in gremlin-1 transgenic lung.</p

Reduced inflammatory gene response to silica.

<p>A. Gene expression microarray was performed using lung tissue mRNA isolated from 6 months old mice (n = 4 in each group). The number of upregulated or downregulated genes are indicated. B. Bubble plots for all immune-related annotations. It compares the most significant Gene Ontology (GO) terms from the “Immune-related Biological Process” ontology found across the different experimental conditions. The same selection strategy was applied for all conditions, which was a significance threshold of 0.05 for the adjusted enrichment p-value, at least five genes from the input list in the enriched category and the whole genome as reference background. C. and D. Quantitative RT-PCR analyses of selected genes identified as differentially expressed in the microarray. The results are presented as box blots. The p values were calculated using the Mann-Whitney U-test (at 2 weeks n = 8; at 2 months n = 4). WT = wild type mice; TG = gremlin-1 transgenic mice.</p

Reduced amount of inflammatory cell aggregates in gremlin-1 transgenic lung.

<p>A. Histological sections of gremlin-1 transgenic and wild type lung showing pleural thickening (original magnification 400x) and alveolar space enlargement (original magnification 100x) at 6 months of age. Results of histological scoring are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159010#pone.0159010.t001" target="_blank">Table 1</a>. B. Mice were treated with silica for 2 months and sacrificed at 6 months of age. Collagen 1 (<i>Col1A1</i>), collagen 3 (<i>Col3A1</i>) and tenascin-C (<i>Tnc</i>) mRNA expression levels analyzed from lung tissue RNA using quantitative RT-PCR (n = 4). The results are presented as box blots. The p values were calculated using the Kruskal-Wallis test or the Mann-Whitney U-test when comparing two groups. WT = wild type mice; TG = gremlin-1 transgenic mice. C. Representative histological pictures showing decreased amount of inflammatory cell aggregates in silica-treated gremlin-1 transgenic mice. Original magnification 200x, inset original magnification 400x. D. Immunofluorescence staining of wild type frozen lung tissue sections using CD4 and CD8 T-cell markers. DAPI staining was used to visualize the nuclei. Immunohistochemical staining of CD45R/B220 (brown color) is shown on the right.</p

Generation of gremlin-1 transgenic mice.

<p>A. Schematic representation of the breeding strategy. B. SPC-lox-gremlin-1 mice were crossbread with Rosa26CreERT mice. Part of the mice were treated with tamoxifen for five days before they were sacrificed. Tissue DNA was isolated followed by genotyping for SPC-loxP-gremlin1 (floxed) and R26CreERT2. The recombination event was monitored using primers surrounding the NEO cassette (deleted). Results of SPC-lox-gremlin-1/ R26CreERT2 positive (+/+) and SPC-lox-gremlin-1/- positive (+/-) mice are shown. Expression of gremlin-1 protein was analyzed using Western blotting of lung and kidney tissue lysates. C. Gremlin-1 protein expression was analyzed by immunofluorescence staining of frozen lung tissue sections. Original magnification 200x. WT = wild type mice; TG = gremlin-1 transgenic mice.</p

Role of Dicer1-Dependent Factors in the Paracrine Regulation of Epididymal Gene Expression

<div><p>Dicer1 is an endoribonuclease involved in the biogenesis of functional molecules such as microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs). These small non-coding RNAs are important regulators of post-transcriptional gene expression and participate in the control of male fertility. With the knowledge that 1) Dicer1-dependent factors are required for proper sperm maturation in the epididymis, and that 2) miRNAs are potent mediators of intercellular communication in most biological systems, we investigated the role of Dicer1-dependent factors produced by the proximal epididymis (initial segment/caput)- including miRNAs- on the regulation of epididymal gene expression in the distal epididymis regions (<i>i</i>.<i>e</i>. corpus and cauda). To this end, we performed comparative microarray and ANOVA analyses on control <i>vs</i>. <i>Defb41</i>^{<i>iCre/wt</i>}<i>;Dicer1</i>^<i>fl/fl</i> mice in which functional Dicer1 is absent from the principal cells of the proximal epididymis. We identified 35 and 33 transcripts that displayed significant expression level changes in the corpus and cauda regions (Fold change > 2 or < −2; p < 0.002), respectively. Among these transcripts, Zn-alpha 2-glycoprotein (<i>Azgp1</i>) encodes for a sperm equatorial protein whose expression in the epididymis of Dicer1 cKO mice is significantly increased compared to controls. In addition, 154 miRNAs, including <i>miR-210</i>, <i>miR-672</i>, <i>miR-191</i> and <i>miR-204</i>, showed significantly impaired biogenesis in the absence of Dicer1 from the principal cells of the proximal epididymis (Fold change > 2 or < −2; p < 0.01). These miRNAs are secreted via extracellular vesicles (EVs) derived from the DC2 epididymal principal cell line, and their expression correlates with target transcripts involved in distinct biological pathways, as evidenced by <i>in silico</i> analysis. Albeit correlative and based on <i>in silico</i> approach, our study proposes that Dicer1-dependent factors trigger- directly or not—significant genes expression changes in distinct regions of this organ. The paracrine control of functions important to post-testicular sperm maturation by Dicer1-dependent factors may open new avenues for the identification of molecular targets important to male fertility control.</p></div

Differential cell counts.

<p>Differential cell counts.</p

Gremlin-1 does not alter the overall innate immune response to silica.

<p>A. Immunohistochemical staining of lung tissue sections using CD11b antibody after two-week silica-exposure. Original magnification 200x. Staining scores are indicated below the photomicrographs (mean ± SEM, n = 8). B. Quantitative RT-PCR analyses of macrophage migration inhibitory factor (<i>Mif</i>) and tumor necrosis factor-α (<i>Tnf</i>) after two-week (n = 8) or two-month (n = 4) silica-exposure. The results are presented as box blots. The p values were calculated using the Mann-Whitney U-test. WT = wild type mice; TG = gremlin-1 transgenic mice.</p

Histological scoring.

<p>Histological scoring.</p

CXCL10 chemokine levels correlate negatively with gremlin-1 levels in mouse and human lung.

<p>A. Inflammatory cytokines in BAL fluid of wild type and gremlin-1 transgenic mice exposed to silica for two weeks were analyzed using a mouse cytokine array. B. Quantification of positive array signals. Mean pixel density of the signal in transgenic BAL fluid is divided by the signal in wild type BAL fluid. The error bars represent standard deviation (n = 2). C. Quantitative RT-PCR analyses of <i>Cxcl10</i> after two-week or two-month silica-exposure. The results are presented as box blots. The p value was calculated using the Mann-Whitney U-test (n = 8). WT = wild type mice; TG = gremlin-1 transgenic mice. D. Quantitative RT-PCR analyses of human gremlin-1 (<i>GREM1</i>) and <i>CXCL10</i> in control (ctrl) and idiopathic pulmonary fibrosis patient (IPF) lung tissue. E. Cultured human fibroblasts isolated from control (ctrl) and IPF patient lung tissue (IPF) were analyzed for <i>GREM1</i> and <i>CXCL10</i> expression by quantitative RT-PCR. The error bars represent standard deviation (n = 3).</p