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

The “Reproductive system disease” network is the most significantly modified in the cauda epididymis of Dicer1 CKO compared to control mice according to Ingenuity Pathway Analysis (IPA) analysis.

<p>Only probe-sets displaying a fold change >1.5 and a p-value <0.01 were considered.</p

Protein Expression level of Zinc-alpha-2-glycoprotein (AZGP1) in the epididymis of Dicer1 cKO and control mice.

<p><b>(A)</b> Relative quantification of AZGP1 protein in the cauda epididymis of Dicer1 cKO and control mice by western blot. Statistical significance was assessed by Student t-test from n = 3 replicates per group. <b>(B)</b> Longitudinal section of a mouse epididymis from a Dicer1 cKO mouse stained by immunohistochemistry for AZGP1. Image taken at 2.5 X. <i>Fp</i>: Fat pad. <b>(C)</b> Immuno-detection of AZGP1 on the proximal caput (a, b), distal caput (c, d), corpus (e, f) and cauda (g, h) epididymidis of control (a, c, e, g) and Dicer1 cKO (b, d, f, h) mice. Bar = 200 μm. Negative controls in absence of primary antibody are shown in insets (a’, c’, e’, g’). Bar = 250 μm.</p

miRNA signature change in the proximal epididymis region (<i>i</i>.<i>e</i>. initial segment/caput) of Dicer1 cKO mice.

<p><b>(A)</b> The different miRNA profiles of Dicer1 cKO and control mice are plotted on the volcano plot according to two-way ANOVA parameters, <i>i</i>.<i>e</i>. p-value and fold-change. Only miRNAs with a fold change above 2 and a p-value below 0.01 (red lines) are annotated. <b>(B)</b> Changes in mature miRNA production in Dicer1 cKO were assessed by qRT-PCR. Data represent experimental duplicates on n = 3 biological samples per group and are normalized to Let-7b expression. ns: non significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.</p

Secretion of Dicer1-dependent miRNAs from DC2 cells via extracellular vesicles (EVs).

<p><b>(A)</b> Schematic view of DC2 derived EVs analysis. DC2 cells were cultured, labelled with CMFDA dye and stimulated with calcium ionophore to induce EVs production. EVs were characterized according to i) their surface antigens (<i>e</i>.<i>g</i>. CD9 marker, phosphatidylserine (PS) by flow cytometry, and to ii) their miRNA content by RT-PCR. <b>(B)</b> Detection of <i>Cftr</i> (principal cell marker) and <i>Atp6v1b1</i> (B1 V-ATPase subunit, clear cell marker) in cauda epididymis and DC2 cell line extracts by end-point PCR. Immunostaining for cytokeratin (green) on DC2 cells. Nuclei were counterstained with DAPI. <b>(C)</b> Size distribution of EVs released from DC2 cells in culture measured in a Zetasizer. This plot is representative of acquisitions performed twice on three distinct biological replicates. <b>(D)</b> Detection of CMFDA-positive EVs released from CMFDA-labeled DC2 cells by High-Sensitivity Flow Cytometry (HS-FCM). Supernatants from DC2 cells stimulated or not with 1 μM calcium ionophore for 30 min or 24 h were analyzed by HS-FCM for EV detection after annexin V (phosphatidylserine detection) and CD9 labeling. Controls include Triton X-100 0.05% that solubilizes most membranous particles, and EDTA 50 μM that inhibits annexin V labelling. <b>(E)</b> Detection of Dicer1-dependent miRNAs in DC2-derived EVs by end-point PCR in cells, whole tissue, and extracellular medium extracts. CM: culture medium; EVs: purified extracellular vesicles; DC2: culture medium-free DC2 cells; WE: whole epididymis; NTC: no-template control.</p

List of murine mature miRNAs displaying a reduced or increased intensity of detection in Dicer1 cKO <i>vs</i>. control (Ctrl) mice.

<p>List of murine mature miRNAs displaying a reduced or increased intensity of detection in Dicer1 cKO <i>vs</i>. control (Ctrl) mice.</p

Impact of Dicer1-dependent factors on epididymal gene expression.

<p><b>(A)</b> Transcripts with the most significantly altered expression in the corpus (left) or the cauda (right) epididymidis of the Dicer1 cKO mouse model are displayed on heat maps. Significance threshold: fold change > 2 or < −2, p-value < 0.002. n = 3 mice per group. <b>(B)</b> Relative Log 2 intensity levels of Prostate and testis expressed 4 (<i>Pate4</i>), Zinc-alpha-2-glycoprotein (<i>Azgp1</i>), Succinyl-CoA:3-ketoacid coenzyme A transferase 2B (<i>Oxct2b</i>), and Phosducin-like protein 2 (<i>Pdcl2</i>) in the corpus and cauda epididymidis of Dicer1 cKO and control mice after microarray analyses on n = 3 biological samples per group. <b>(C)</b> Expression changes of <i>Pate4</i>, <i>Azgp1</i>, <i>Oxct2b and Pdcl2</i> were validated by qRT-PCR in the caput, corpus and cauda regions from control and Dicer1 cKO mice. qRT-PCR data shown as means and standard deviations of results obtained from n = 3 biological samples per group after normalization to <i>Gapdh</i> expression. Unpaired t-test corrected for multiple comparison using the Holm-Sidak method. ns: non significant, *p<0.05, ***p < 0.001, ****p < 0.0001.</p