The estrogen signaling pathway reprograms prostate cancer cell metabolism and supports proliferation and disease progression.

Just like the androgen receptor (AR), the estrogen receptor α (ERα) is expressed in the prostate and is thought to influence prostate cancer (PCa) biology. Yet the incomplete understanding of ERα functions in PCa hinders our ability to fully comprehend its clinical relevance and restricts the repurposing of estrogen-targeted therapies for the treatment of this disease. Using 2 human PCa tissue microarray cohorts, we first demonstrate that nuclear ERα expression was heterogeneous among patients, being detected in only half of the tumors. Positive nuclear ERα levels were correlated with disease recurrence, progression to metastatic PCa, and patient survival. Using in vitro and in vivo models of the normal prostate and PCa, bulk and single-cell RNA-Seq analyses revealed that estrogens partially mimicked the androgen transcriptional response and activated specific biological pathways linked to proliferation and metabolism. Bioenergetic flux assays and metabolomics confirmed the regulation of cancer metabolism by estrogens, supporting proliferation. Using cancer cell lines and patient-derived organoids, selective estrogen receptor modulators, a pure anti-estrogen, and genetic approaches impaired cancer cell proliferation and growth in an ERα-dependent manner. Overall, our study revealed that, when expressed, ERα functionally reprogrammed PCa metabolism, was associated with disease progression, and could be targeted for therapeutic purposes.

ERK5 Cooperates With MEF2C to Regulate Nr4a1 Transcription in MA-10 and MLTC-1 Leydig Cells.

Leydig cells produce hormones required for the development and maintenance of sex characteristics and fertility in males. MEF2 transcription factors are important regulators of Leydig cell gene expression and steroidogenesis. ERK5 is an atypical member of the MAP kinase family that modulates transcription factor activity, either by direct phosphorylation or by acting as a transcriptional coactivator. While MEF2 and ERK5 are known to cooperate transcriptionally, the presence and role of ERK5 in Leydig cells remained unknown. Our goal was to determine whether ERK5 is present in Leydig cells and whether it cooperates with MEF2 to regulate gene expression. We found that ERK5 is present in Leydig cells in testicular tissue and immortalized cell lines. ERK5 knockdown in human chorionic gonadotrophin-treated MA-10 Leydig cells reduced steroidogenesis and decreased Star and Nr4a1 expression. Luciferase assays using a synthetic reporter plasmid containing 3 MEF2 elements revealed that ERK5 enhances MEF2-dependent promoter activation. Although ERK5 did not cooperate with MEF2 on the Star promoter in Leydig cell lines, we found that ERK5 and MEF2C do cooperate on the Nr4a1 promoter, which contains 2 adjacent MEF2 elements. Mutation of each MEF2 element in a short version of the Nr4a1 promoter significantly decreased the ERK5/MEF2C cooperation, indicating that both MEF2 elements need to be intact. The ERK5/MEF2C cooperation did not require phosphorylation of MEF2C on Ser387. Taken together, our data identify ERK5 as a new regulator of MEF2 activity in Leydig cells and provide potential new insights into mechanisms that regulate Leydig cell gene expression and function.

Hedgehog signaling regulates Wolffian duct development through the primary cilium†.

Primary cilia play pivotal roles in embryonic patterning and organogenesis through transduction of the Hedgehog signaling pathway (Hh). Although mutations in Hh morphogens impair the development of the gonads and trigger male infertility, the contribution of Hh and primary cilia in the development of male reproductive ductules, including the epididymis, remains unknown. From a Pax2Cre; IFT88fl/fl knock-out mouse model, we found that primary cilia deletion is associated with imbalanced Hh signaling and morphometric changes in the Wolffian duct (WD), the embryonic precursor of the epididymis. Similar effects were observed following pharmacological blockade of primary cilia formation and Hh modulation on WD organotypic cultures. The expression of genes involved in extracellular matrix, mesenchymal-epithelial transition, canonical Hh and WD development was significantly altered after treatments. Altogether, we identified the primary cilia-dependent Hh signaling as a master regulator of genes involved in WD development. This provides new insights regarding the etiology of sexual differentiation and male infertility issues.

Arl13b controls basal cell stemness properties and Hedgehog signaling in the mouse epididymis.

Epithelial cells orchestrate a series of intercellular signaling events in response to tissue damage. While the epididymis is composed of a pseudostratified epithelium that controls the acquisition of male fertility, the maintenance of its integrity in the context of tissue damage or inflammation remains largely unknown. Basal cells of the epididymis contain a primary cilium, an organelle that controls cellular differentiation in response to Hedgehog signaling cues. Hypothesizing its contribution to epithelial homeostasis, we knocked out the ciliary component ARL13B in keratin 5-positive basal cells. In this model, the reduced size of basal cell primary cilia was associated with impaired Hedgehog signaling and the loss of KRT5, KRT14, and P63 basal cell markers. When subjected to tissue injury, the epididymal epithelium from knock-out mice displayed imbalanced rates of cell proliferation/apoptosis and failed to properly regenerate in vivo. This response was associated with changes in the transcriptomic landscape related to immune response, cell differentiation, cell adhesion, and triggered severe hypoplasia of the epithelium. Together our results indicate that the ciliary GTPase, ARL13B, participates in the transduction of the Hedgehog signaling pathway to maintain basal cell stemness needed for tissue regeneration. These findings provide new insights into the role of basal cell primary cilia as safeguards of pseudostratified epithelia.

EV duty vehicles: Features and functions of ciliary extracellular vesicles.

The primary cilium is a microtubule-based organelle that extends from a basal body at the surface of most cells. This antenna is an efficient sensor of the cell micro-environment and is instrumental to the proper development and homeostatic control of organs. Recent compelling studies indicate that, in addition to its role as a sensor, the primary cilium also emits signals through the release of bioactive extracellular vesicles (EVs). While some primary-cilium derived EVs are released through an actin-dependent ectocytosis and are called ectosomes (or large EVs, 350-500 nm), others originate from the exocytosis of multivesicular bodies and are smaller (small EVs, 50-100 nm). Ciliary EVs carry unique signaling factors, including protein markers and microRNAs (miRNAs), and participate in intercellular communication in different organism models. This review discusses the mechanism of release, the molecular features, and functions of EVs deriving from cilia, based on the existing literature.

Expression of the pro-inflammatory P2Y14 receptor in the non-vasectomized and vasectomized human epididymis.

BACKGROUND: Vasectomy causes spermatozoa accumulation in the epididymis, which may cause epididymitis. Inflammation is triggered by alert molecules released following tissue stress or injury. These include uracil-diphosphate glucose (UDP)-glucose, which activates the pro-inflammatory P2Y14 receptor (P2Y14), and induces immune cell recruitment. However, little is known about P2Y14 in the epididymis and its potential activation following vasectomy. OBJECTIVES: (i) To localize P2Y14 in the human excurrent duct; and (ii) to examine the effect of vasectomy on P2Y14 protein and P2RY14 mRNA content, the production of selected cytokines and chemokines, and immune cell recruitment in the epididymis. MATERIAL AND METHODS: In situ hybridization, qRT-PCR, western blotting, immunohistochemistry, and immunofluorescence were performed in banked human epididymis samples. RESULTS: P2RY14 mRNA and P2Y14 protein were detected in epithelial cells in the efferent duct, epididymis and vas deferens in non-vasectomized men. Keratin 5 (KRT5)-positive basal cells were strongly labeled for P2Y14 in all epididymal segments. A progressive apical localization was detected in principal cells (negative for the proton pump V-ATPase) from the corpus to the cauda. A subset of V-ATPase-positive clear cells also showed strong P2Y14 labeling. Vasectomy induced an increase in P2RY14 mRNA in the corpus and cauda, and stronger apical labeling in principal cells in the corpus. CXCL10 mRNA increased in the cauda and CCL2 mRNA decreased in the corpus of vasectomized versus non-vasectomized men. No change in IL-8 and IL-1ß mRNA was detected. Numerous CD45+ leukocytes were detected in the interstitium of the corpus and cauda following vasectomy, while only a few were seen in non-vasectomized men. Several CD45+ leukocytes, some of which containing spermatozoa, were detected in the corpus lumen following vasectomy. DISCUSSION AND CONCLUSION: Our study indicates that vasectomy-induced spermatozoa congestion may lead to an inflamed-prone local environment characterized by potential activation of P2Y14 and recruitment of immune cells in the epididymis.

Platelet activation by SARS-CoV-2 implicates the release of active tissue factor by infected cells.

Platelets are hyperactivated in coronavirus disease 2019 (COVID-19). However, the mechanisms promoting platelet activation by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are not well understood. This may be due to inherent challenges in discriminating the contribution of viral vs host components produced by infected cells. This is particularly true for enveloped viruses and extracellular vesicles (EVs), as they are concomitantly released during infection and share biophysical properties. To study this, we evaluated whether SARS-CoV-2 itself or components derived from SARS-CoV-2-infected human lung epithelial cells could activate isolated platelets from healthy donors. Activation was measured by the surface expression of P-selectin and the activated conformation of integrin αIIbß3, degranulation, aggregation under flow conditions, and the release of EVs. We find that neither SARS-CoV-2 nor purified spike activates platelets. In contrast, tissue factor (TF) produced by infected cells was highly potent at activating platelets. This required trace amounts of plasma containing the coagulation factors FX, FII, and FVII. Robust platelet activation involved thrombin and the activation of protease-activated receptor (PAR)-1 and -4 expressed by platelets. Virions and EVs were identified by electron microscopy. Through size-exclusion chromatography, TF activity was found to be associated with a virus or EVs, which were indistinguishable. Increased TF messenger RNA (mRNA) expression and activity were also found in lungs in a murine model of COVID-19 and plasma of severe COVID-19 patients, respectively. In summary, TF activity from SARS-CoV-2-infected cells activates thrombin, which signals to PARs on platelets. Blockade of molecules in this pathway may interfere with platelet activation and the coagulation characteristic of COVID-19.

Sperm Heterogeneity Accounts for Sperm DNA Methylation Variations Observed in the Caput Epididymis, Independently From DNMT/TET Activities.

Following their production in the testis, spermatozoa enter the epididymis where they gain their motility and fertilizing abilities. This post-testicular maturation coincides with sperm epigenetic profile changes that influence progeny outcome. While recent studies highlighted the dynamics of small non-coding RNAs in maturing spermatozoa, little is known regarding sperm methylation changes and their impact at the post-fertilization level. Fluorescence-activated cell sorting (FACS) was used to purify spermatozoa from the testis and different epididymal segments (i.e., caput, corpus and cauda) of CAG/su9-DsRed2; Acr3-EGFP transgenic mice in order to map out sperm methylome dynamics. Reduced representation bisulfite sequencing (RRBS-Seq) performed on DNA from these respective sperm populations indicated that high methylation changes were observed between spermatozoa from the caput vs. testis with 5,546 entries meeting our threshold values (q value <0.01, methylation difference above 25%). Most of these changes were transitory during epididymal sperm maturation according to the low number of entries identified between spermatozoa from cauda vs. testis. According to enzymatic and sperm/epididymal fluid co-incubation assays, (de)methylases were not found responsible for these sperm methylation changes. Instead, we identified that a subpopulation of caput spermatozoa displayed distinct methylation marks that were susceptible to sperm DNAse treatment and accounted for the DNA methylation profile changes observed in the proximal epididymis. Our results support the paradigm that a fraction of caput spermatozoa has a higher propensity to bind extracellular DNA, a phenomenon responsible for the sperm methylome variations observed at the post-testicular level. Further investigating the degree of conservation of this sperm heterogeneity in human will eventually provide new considerations regarding sperm selection procedures used in fertility clinics.

Intra and intercellular signals governing sperm maturation.

After their production in the testis, spermatozoa do not have the capacity to move progressively and are unable to fertilise an oocyte. They sequentially acquire these abilities following their maturation in the epididymis and their capacitation/hyperactivation in the female reproductive system. As gene transcription is silenced in spermatozoa, extracellular factors released from the epididymal epithelium and from secretory glands allow spermatozoa to acquire bioactive molecules and to undergo intrinsic modifications. These modifications include epigenetic changes and post-translational modifications of endogenous proteins, which are important processes in sperm maturation. This article emphasises the roles played by extracellular factors secreted by the epididymis and accessory glands in the control of sperm intercellular signallings and fertilising abilities.

Contribution of epididymal epithelial cell functions to sperm epigenetic changes and the health of progeny.

BACKGROUND: Spermatozoa acquire their motility and fertilizing abilities during their maturation through the epididymis. This process is controlled by epididymal epithelial cells that possess features adapted to sense and respond to their surrounding environment and to communicate with spermatozoa. During the past decade, new intercellular communication processes have been discovered, including the secretion and transport of molecules from the epithelium to spermatozoa via extracellular vesicles (EVs), as well as sensing of the intraluminal milieu by cellular extensions. OBJECTIVE AND RATIONALE: This review addresses recent findings regarding epididymal epithelial cell features and interactions between spermatozoa and the epididymal epithelium as well as epigenetic modifications undergone by spermatozoa during transit through the epididymal microenvironment. SEARCH METHODS: A systematic search was conducted in Pubmed with the keyword 'epididymis'. Results were filtered on original research articles published from 2009 to 2021 and written in the English language. One hundred fifteen original articles presenting recent advancements on the epididymis contribution to sperm maturation were selected. Some additional papers cited in the primary reference were also included. A special focus was given to higher mammalian species, particularly rodents, bovines and humans, that are the most studied in this field. OUTCOMES: This review provides novel insights into the contribution of epididymal epithelium and EVs to post-testicular sperm maturation. First, new immune cell populations have been described in the epididymis, where they are proposed to play a role in protecting the environment surrounding sperm against infections or autoimmune responses. Second, novel epididymal cell extensions, including dendrites, axopodia and primary cilia, have been identified as sensors of the environment surrounding sperm. Third, new functions have been outlined for epididymal EVs, which modify the sperm epigenetic profile and participate in transgenerational epigenetic inheritance of paternal traits. WIDER IMPLICATIONS: Although the majority of these findings result from studies in rodents, this fundamental research will ultimately improve our knowledge of human reproductive physiopathologies. Recent discoveries linking sperm epigenetic modifications with paternal environmental exposure and progeny outcome further stress the importance of advancing fundamental research on the epididymis. From this, new therapeutic options for infertile couples and better counseling strategies may arise to increase positive health outcomes in children conceived either naturally or with ART.

Altered expression of the vitamin D metabolizing enzymes CYP27B1 and CYP24A1 under the context of prostate aging and pathologies.

Low circulating levels of vitamin D are common at older ages and have been linked to an increased risk of prostate disease, including cancer. However, it has not yet been determined whether aging affects the ability of prostate cells to locally metabolize vitamin D into its active metabolite calcitriol and thus mediate the vitamin D signaling in autocrine and paracrine ways. By using a suitable rat model to interrogate spontaneous prostatic modifications over the course of aging, here we showed that both CYP27B1 and CYP24A1 enzymes, which are key players respectively involved with calcitriol synthesis and deactivation, were highly expressed in the prostate epithelium. Furthermore, as the animals aged, a drastic reduction of CYP27B1 levels was detected in total protein extracts and especially in epithelial areas of lesions, including tumors. On the other hand, CYP24A1 expression significantly increased with aging and remained elevated even in altered epithelia. Such intricate unbalance in regard to vitamin D metabolizing enzymes was strongly associated with reduced bioavailability of calcitriol in the senile prostate, which in addition to decreased expression of the vitamin D receptor, further limits the protective actions mediated by vitamin D signaling. This evidence was corroborated by the increased proliferative activity exactly at sites of lesions where the factors implicated with calcitriol synthesis and responsiveness had its expression inhibited. Taken together, our results emphasize a set of modifications over the course of aging with a high potential to hamper vitamin D signaling on the prostate. These findings highlight a crosstalk between vitamin D, aging, and prostate carcinogenesis, offering new potential targets in the prevention of malignancies and other aging-related disorders arising in the gland.

Platelets release mitochondrial antigens in systemic lupus erythematosus.

The accumulation of DNA and nuclear components in blood and their recognition by autoantibodies play a central role in the pathophysiology of systemic lupus erythematosus (SLE). Despite the efforts, the sources of circulating autoantigens in SLE are still unclear. Here, we show that in SLE, platelets release mitochondrial DNA, the majority of which is associated with the extracellular mitochondrial organelle. Mitochondrial release in patients with SLE correlates with platelet degranulation. This process requires the stimulation of platelet FcγRIIA, a receptor for immune complexes. Because mice lack FcγRIIA and murine platelets are completely devoid of receptor capable of binding IgG-containing immune complexes, we used transgenic mice expressing FcγRIIA for our in vivo investigations. FcγRIIA expression in lupus-prone mice led to the recruitment of platelets in kidneys and to the release of mitochondria in vivo. Using a reporter mouse with red fluorescent protein targeted to the mitochondrion, we confirmed platelets as a source of extracellular mitochondria driven by FcγRIIA and its cosignaling by the fibrinogen receptor α2bß3 in vivo. These findings suggest that platelets might be a key source of mitochondrial antigens in SLE and might be a therapeutic target for treating SLE.

From Sperm Motility to Sperm-Borne microRNA Signatures: New Approaches to Predict Male Fertility Potential.

In addition to the paternal genome, spermatozoa carry several intrinsic factors, including organelles (e.g., centrioles and mitochondria) and molecules (e.g., proteins and RNAs), which are involved in important steps of reproductive biology such as spermatogenesis, sperm maturation, oocyte fertilization and embryo development. These factors constitute potential biomarkers of "viable sperm" and male fertility status and may become major assets for diagnosing instances of idiopathic male infertility in both humans and livestock animals. A better understanding of the mechanism of action of these sperm intrinsic factors in the regulation of reproductive and developmental processes still presents a major challenge that must be addressed. This review assembles the main data regarding morpho-functional and intrinsic sperm features that are associated with male infertility, with a particular focus on microRNA (miRNA) molecules.

Hedgehog signaling pathway regulates gene expression profile of epididymal principal cells through the primary cilium.

Primary cilia (PC) are organelles that sense and respond to dynamic changes of the extracellular milieu through the regulation of target genes. By using the epididymis as a model system, we determined the contribution of primary cilia in the regulation of epithelial cell functions through the transduction of the Hedgehog (Hh) signaling pathway. Both Sonic (SHH) and Indian Hedgehog (IHH) ligands were detected in epididymal epithelial cells by confocal microscopy and found secreted in the extracellular space. Gene expression profiling preformed on ciliated epithelial cells indicated that 153 and 1052 genes were differentially expressed following treatment with the Hh agonist SAG or the Hh antagonist cyclopamine (Cyclo), respectively. Strikingly, gene ontology analysis indicated that genes associated with immune response were the most affected following Hh modulation. The contribution of epididymal PC to canonical Hh pathway transduction was validated by ciliobrevin D treatment, which induced a significant decrease in PC length and a reduction in the expression Hh signaling targets. Such findings bring us closer to a molecular understanding of the subtle immune balance observed in some epithelia, including the epididymis and the intestine, which are organs featuring both tolerance toward autoimmune spermatozoa (or commensal bacteria) and defense against pathogens.

Sperm-borne miR-216b modulates cell proliferation during early embryo development via K-RAS.

Semen fertilizing potential is dependent upon the morphological, functional and molecular attributes of sperm. Sperm microRNAs (miRNAs) were recently shown to hold promise regarding their association with different fertility phenotypes. However, their role in fertility regulation remains to be determined. We postulated that sperm miRNAs might regulate early embryonic development. From this perspective, sperm quality and 380 sperm miRNAs were investigated in frozen-thawed semen from high (HF; 54.3 ± 1.0% pregnancy rate) and low (LF; 41.5 ± 2.3%) fertility bulls. Out of nine miRNAs that showed different levels in sperm cells, miR-216b was present at lower levels in HF sperm cells and zygotes. Among miR-216b target genes (K-RAS, BECN1 and JUN), K-RAS, related to cell proliferation, revealed a higher level in HF two-cell embryos. First cleavage rate, blastocyst cell number and division number were also higher in HF. In addition, by using a model based on polyspermy embryos, we demonstrated an increase in miR-216b levels in zygotes associated with sperm cell entry. Our results shed light on a possible mechanism of paternal contribution involving sperm-borne miR-216b that modulates levels of miR-216b in zygotes and K-RAS in two-cell embryos. This modulation might regulate early development by interfering with the first cleavage and blastocyst quality.

Primary cilia: biosensors of the male reproductive tract.

BACKGROUND: The primary cilium is a microtubule-based organelle that extends transiently from the apical cell surface to act as a sensory antenna. Initially viewed as a cellular appendage of obscure significance, the primary cilium is now acknowledged as a key coordinator of signaling pathways during development and in tissue homeostasis. OBJECTIVES: The aim of this review was to present the structure and function of this overlooked organelle,with an emphasis on its epididymal context and contribution to male infertility issues. MATERIALS AND METHODS: A systematic review has been performed in order to include main references relevant to the aforementioned topic. RESULTS: Increasing evidence demonstrates that primary cilia dysfunctions are associated with impaired male reproductive system development and male infertility issues. DISCUSSION: While a large amount of data exists regarding the role of primary cilia in most organs and tissues, few studies investigated the contribution of these organelles to male reproductive tract development and homeostasis. CONCLUSION: Functional studies of primary cilia constitute an emergent and exciting new area in reproductive biology research.

Anti-mitochondrial autoantibodies in systemic lupus erythematosus and their association with disease manifestations.

Mitochondria are organelles that govern energy supply and control cell death. Mitochondria also express bacterial features, such as the presence of inner membrane cardiolipin and a circular genome rich in hypomethylated CpG motifs. While mitochondrial extrusion by damaged organs or activated cells is thought to trigger innate immunity, it is unclear whether extracellular mitochondria also stimulate an adaptive immune response. We describe the development of novel assays to detect autoantibodies specific to two distinct components of the mitochondrion: the mitochondrial outer membrane and mitochondrial DNA. Antibodies to these two mitochondrial constituents were increased in both human and murine systemic lupus erythematosus (SLE), compared to controls, and were present at higher levels than in patients with antiphospholipid syndrome or primary biliary cirrhosis. In both bi- and multi-variate regression models, antibodies to mitochondrial DNA, but not whole mitochondria, were associated with increased anti-dsDNA antibodies and lupus nephritis. This study describes new and optimized methods for the assessment of anti-mitochondrial antibodies, and demonstrates their presence in both human and murine SLE. These findings suggest that different mitochondrial components are immunogenic in SLE, and support the concept that extracellular mitochondria may provide an important source of circulating autoantigens in SLE.

Cell-lineage specificity of primary cilia during postnatal epididymal development.

STUDY QUESTION: Where are primary cilia (PC) organelles located during postnatal epididymal development? SUMMARY ANSWER: Our findings unveil the existence of PC sensory organelles in different epididymal cell types according to postnatal development stage. WHAT IS KNOWN ALREADY: Primary cilia are sensory organelles that orchestrate major signaling pathways during organ development and homeostasis. Epididymal PC have been detected in the horses, donkey and mules but their cell-lineage specificity has never been investigated in this organ. STUDY DESIGN, SIZE, DURATION: A longitudinal study was performed by examining tissue from n = 3 to n = 10 transgenic mice at different times of postnatal development. Tissues were fixed by intracardiac perfusion and the epididymides collected. PARTICIPANTS/MATERIALS, SETTING, METHODS: Transmission electron microscopy and confocal microscopy/3D reconstruction were used on a double transgenic mouse model expressing endogenous fluorescence in PC and centrioles (Arl13b-mCherry/Centrin2-GFP). Several PC parameters (i.e. length, orientation relative to the lumen) were quantified by using an image-processing pipeline. Epididymal tissues and serum-free cultures of DC2 immortalized epididymal principal murine cell lines were used to identify primary ciliary signaling components. MAIN RESULTS AND THE ROLE OF CHANCE: We report here a constitutive localization of PC in peritubular myoid cells and a dynamic profiling in epithelial cells throughout postnatal epididymal development. While PC are present at the apical pole of the undifferentiated epithelial cells from birth to puberty, they are absent from the apical pole of the epithelium in adults, where they appear exclusively associated with cytokeratin 5-positive basal cells. We determined that PC from epididymal cells are associated with polycystin 1 (PC1), polycystin 2 (PC2), and Gli-3 Hedgehog signaling transcription factor. No inter-individual variability was observed within each age group. LIMITATIONS, REASONS FOR CAUTION: As our present study is descriptive and performed exclusively in the mouse, future functional studies will be required to unravel the contribution of these organelles in the control of reproductive functions. WIDER IMPLICATIONS OF THE FINDINGS: Acknowledging the important roles played by PC sensory organelles in organ homeostasis and development in humans, our work opens new avenues of research concerning the cellular control of epididymal functions, which are essential to male fertility. STUDY FUNDING/COMPETING INTEREST(S): Study funded by an NSERC operating grant to CB (RGPIN-2015-109194). No competing interest to declare.

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.

(Dicer)phering roles of microRNA in platelets.

In this issue of Blood, Rowley et al report that noncoding RNAs precisely regulate the messenger RNA (mRNA) profile in platelets. Interfering in this process using genetically engineered mice affects hemostatic and thrombotic functions of platelets.