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

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.e. corpus and cauda). To this end, we performed comparative microarray and ANOVA analyses on control vs. Defb41iCre/wt;Dicer1fl/fl 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 (Azgp1) 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 miR-210, miR-672, miR-191 and miR-204, 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 in silico analysis. Albeit correlative and based on in silico 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.

Dicer1是一种核糖核酸内切酶（endoribonuclease），参与微小RNA（microRNAs, miRNAs）和内源性小干扰RNA（endogenous small interfering RNAs, endo-siRNAs）等功能分子的生物发生过程。这类小型非编码RNA是转录后基因表达的重要调控因子，参与雄性生育力的调控。已知两点：1）Dicer1依赖因子在附睾（epididymis）的精子正常成熟过程中不可或缺；2）miRNAs在绝大多数生物系统中均为细胞间通讯的有效介质。基于此，我们探究了近端附睾（proximal epididymis，即初始段/帽区（initial segment/caput））的主细胞（principal cells）所产生的Dicer1依赖因子（包括miRNAs），对远端附睾区域（distal epididymis regions，即体部（corpus）与尾部（cauda））基因表达的调控作用。为此，我们对对照组与Defb41iCre/wt;Dicer1fl/fl小鼠开展了比较微阵列（microarray）分析与方差分析（ANOVA, analysis of variance）——该模型小鼠的近端附睾主细胞中功能性Dicer1已被敲除。我们分别在附睾体部与尾部区域鉴定出35和33个转录本（transcripts），其表达水平发生显著变化（倍数变化（Fold change）>2或<−2；p < 0.002）。在这些转录本中，Zn-α2-糖蛋白（Zn-alpha 2-glycoprotein, Azgp1）编码一种精子赤道蛋白，其在Dicer1条件性敲除小鼠（cKO, conditional knockout mice）附睾中的表达水平较对照组显著升高。此外，包括miR-210、miR-672、miR-191及miR-204在内的154个miRNAs，在近端附睾主细胞缺失Dicer1的情况下，其生物发生过程受到显著损害（倍数变化>2或<−2；p < 0.01）。此类miRNAs通过源自DC2附睾主细胞系的细胞外囊泡（extracellular vesicles, EVs）进行分泌，且经生物信息学分析（in silico）证实，其表达与参与不同生物学通路的靶转录本密切相关。尽管本研究仅为相关性分析且基于生物信息学方法，但我们提出：Dicer1依赖因子可直接或间接引发该器官不同区域的显著基因表达变化。由Dicer1依赖因子介导的、对睾丸后精子成熟（post-testicular sperm maturation）至关重要的功能的旁分泌调控（paracrine control），或可为鉴定雄性生育调控（male fertility control）相关的分子靶点开辟全新方向。