The unspecific effect of PP on thymocyte maturation is not reversed by exogenous AA and PGE₂.

<p><b>A.</b> Representative thymocyte subpopulation distribution in WT cPLA₂α FTOC after 5 days of culture in absence or presence of 1μM of PP and exogenous (1μM) AA and PGE₂. Thymocytes were identified cytofluorometrically using fluorochrome-conjugated antibodies directed against CD3, CD4 and CD8. <b>B.</b> WT cPLA₂α fetal thymuses were cultured during 5 days as FTOCs in absence or presence of 1μM of PP, and exogenous (1μM) AA and PGE₂. After mechanical dissociation of fetal thymuses, the thymocytes were identified with fluorochrome-conjugated antibodies directed against CD3, CD4, and CD8 and analyzed by flow cytometry. Data are mean ± SEM of 3 independent experiments and the number of fetal thymuses for each condition is: Diluent (n = 5); 1μM PP (n = 6); 1μM PP and 1μM AA (n = 5); 1μM PP and 1μM PGE₂ (n = 5). * P< .05; ** P< .01; *** P< .001.</p

Eicosanoid profiles of cPLA₂α WT and KO FTOC supernatants and adult mouse thymuses.

<p><b>A.</b> Expression distribution of the eicosanoids present in cPLA₂α WT FTOCs. The supernatants of FTOCs were collected at the indicated time of culture and the eicosanoid profiles were determined by combined liquid chromatography/tandem mass spectrometry. Data are mean of 3 different supernatants. <b>B.</b> Eicosanoid profiles of cPLA₂α WT and KO FTOC supernatants. The supernatants of FTOCs were collected at the indicated time of culture and eicosanoid profiles were determined by combined liquid chromatography/tandem mass spectrometry. Data are mean ± SEM of 3 different supernatants. <b>C.</b> Eicosanoid profiles of cPLA₂α WT and KO thymuses from adult mice. Adult thymuses were mechanically disrupted and eicosanoid profiles were determined by combined liquid chromatography/tandem mass spectrometry. ** P< .01, data are means ± SEM of 3 cPLA₂α WT thymuses and 2 cPLA₂α KO thymuses.</p

The disruption of the cPLA₂α gene does not impact thymocyte maturation in FTOC.

<p><b>A.</b> Representative thymocyte subpopulation distribution in WT and KO cPLA₂α FTOC. After 5 days of culture, the identification of thymocytes with fluorochrome-conjugated antibodies directed against CD3, CD4 and CD8 was determined by flow cytometry. <b>B.</b> WT and KO cPLA₂α fetal thymuses were cultured during 5 days as FTOCs. After mechanical dissociation of fetal thymuses, the thymocytes were labeled with fluorochrome-conjugated antibodies directed against CD3, CD4, and CD8, and analyzed by flow cytometry. Data are mean ± SEM of 6 independent experiments and the number of fetal thymuses for each genotype is: cPLA₂^+/+ (n = 17); cPLA₂^-/- (n = 9). NS (non significant).</p

cPLA₂α inhibition does not impact human thymocyte maturation.

<p><b>A.</b> Representative thymocyte subpopulation distribution in human FTOC after 5 days of culture in absence or presence of indicated concentrations of PP. Thymocytes were identified cytofluorometrically using fluorochrome-conjugated antibodies directed against CD3, CD4 and CD8. <b>B.</b> Human FTOCs were cultured during 5 days in absence or presence of indicated concentrations of PP. After mechanical dissociation of human FTOCs, the thymocytes were labeled with fluorochrome-conjugated antibodies directed against CD3, CD4, and CD8 and analyzed by flow cytometry. Data are mean ± SEM of 3 independent experiments and the number of thymuses for each condition is: Diluent (n = 6); 10nM PP (n = 6); 100nM PP (n = 6); 1000nM PP (n = 6). NS (non significant).</p

Study of the Role of Cytosolic Phospholipase A₂ Alpha in Eicosanoid Generation and Thymocyte Maturation in the Thymus

<div><p>The thymus is a primary lymphoid organ, home of maturation and selection of thymocytes for generation of functional T-cells. Multiple factors are involved throughout the different stages of the maturation process to tightly regulate T-cell production. The metabolism of arachidonic acid by cyclooxygenases, lipoxygenases and specific isomerases generates eicosanoids, lipid mediators capable of triggering cellular responses. In this study, we determined the profile of expression of the eicosanoids present in the mouse thymus at different stages of thymocyte development. As the group IVA cytosolic phospholipase A₂ (cPLA₂α) catalyzes the hydrolysis of phospholipids, thereby generating arachidonic acid, we further verified its contribution by including cPLA₂α deficient mice to our investigations. We found that a vast array of eicosanoids is expressed in the thymus, which expression is substantially modulated through thymocyte development. The cPLA₂α was dispensable in the generation of most eicosanoids in the thymus and consistently, the ablation of the cPLA₂α gene in mouse thymus and the culture of thymuses from human newborns in presence of the cPLA₂α inhibitor pyrrophenone did not impact thymocyte maturation. This study provides information on the eicosanoid repertoire present during thymocyte development and suggests that thymocyte maturation can occur independently of cPLA₂α.</p></div

Pharmacological blockade of cPLA₂α does not affect thymocyte maturation in FTOC.

<p><b>A.</b> Representative thymocyte subpopulation distribution in WT cPLA₂α FTOC after 5 days of culture in absence or presence of indicated concentrations of PP. Thymocytes were identified with fluorochrome-conjugated antibodies directed against CD3, CD4 and CD8 by flow cytometry. <b>B.</b> WT cPLA₂α fetal thymuses were cultured during 5 days as FTOCs in absence or presence of indicated concentrations of PP. After mechanical dissociation of fetal thymuses, the thymocytes were labeled with fluorochrome-conjugated antibodies directed against CD3, CD4, and CD8 and analyzed by flow cytometry. Data are mean ± SEM of 5 independent experiments and the number of fetal thymuses for each condition is: Diluent (n = 10); 10nM PP (n = 8); 100nM PP (n = 12). NS (non significant).</p

cPLA₂α inhibition by high concentration of PP impacts thymocyte maturation in FTOC.

<p><b>A.</b> Representative thymocyte subpopulation distribution in WT cPLA₂α FTOC after 5 days of culture in absence or presence of 1μM of PP. Thymocytes were identified cytofluorometrically using fluorochrome-conjugated antibodies directed against CD3, CD4 and CD8. <b>B.</b> WT cPLA₂α fetal thymuses were cultured during 5 days as FTOCs in absence or presence of 1μM of PP. After mechanical dissociation of fetal thymuses, the thymocytes were labeled with fluorochrome-conjugated antibodies directed against CD3, CD4, and CD8 and analyzed by flow cytometry. Data are mean ± SEM of 9 independent experiments and the number of fetal thymuses for each condition is: Diluent (n = 22); 1μM PP (n = 13). *** P< .001.</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

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