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.

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.

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.

(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.

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.

Platelet microparticles are internalized in neutrophils via the concerted activity of 12-lipoxygenase and secreted phospholipase A2-IIA.

Platelets are anucleated blood elements highly potent at generating extracellular vesicles (EVs) called microparticles (MPs). Whereas EVs are accepted as an important means of intercellular communication, the mechanisms underlying platelet MP internalization in recipient cells are poorly understood. Our lipidomic analyses identified 12(S)-hydroxyeicosatetranoic acid [12(S)-HETE] as the predominant eicosanoid generated by MPs. Mechanistically, 12(S)-HETE is produced through the concerted activity of secreted phospholipase A2 IIA (sPLA2-IIA), present in inflammatory fluids, and platelet-type 12-lipoxygenase (12-LO), expressed by platelet MPs. Platelet MPs convey an elaborate set of transcription factors and nucleic acids, and contain mitochondria. We observed that MPs and their cargo are internalized by activated neutrophils in the endomembrane system via 12(S)-HETE. Platelet MPs are found inside neutrophils isolated from the joints of arthritic patients, and are found in neutrophils only in the presence of sPLA2-IIA and 12-LO in an in vivo model of autoimmune inflammatory arthritis. Using a combination of genetically modified mice, we show that the coordinated action of sPLA2-IIA and 12-LO promotes inflammatory arthritis. These findings identify 12(S)-HETE as a trigger of platelet MP internalization by neutrophils, a mechanism highly relevant to inflammatory processes. Because sPLA2-IIA is induced during inflammation, and 12-LO expression is restricted mainly to platelets, these observations demonstrate that platelet MPs promote their internalization in recipient cells through highly regulated mechanisms.

Evidences of Biological Functions of Biliverdin Reductase A in the Bovine Epididymis.

Epididymal sperm binding protein 1 (ELSPBP1) is secreted by the epididymal epithelium via epididymosomes and is specifically transferred to dead spermatozoa during epididymal transit. We identified biliverdin reductase A (BLVRA) as a partner of ELSPBP1 by immunoprecipitation followed by tandem mass spectrometry. Pull down assays showed that these two proteins interact in the presence of zinc ions. The BLVRA enzyme is known to convert biliverdin to bilirubin, both of which possess antioxidant activity. Assessment by real-time RT-PCR showed that BLVRA is highly expressed in the caput and the corpus epididymis, but is expressed at lower levels in the testis and the cauda epididymis. It is primarily found in the soluble fraction of the caput epididymal fluid, is barely detectable in the cauda fluid, and is detectable to a lesser extent in the epididymosome fraction of both caput and cauda fluids. Immunocytometry on epididymal sperm showed that BLVRA is found on all sperm recovered from the caput region, whereas it is undetectable on cauda sperm. Biliverdin and bilirubin are found in higher concentrations in the caput epididymal fluid, as measured by mass spectrometry. Lipid peroxidation was limited by 1 µM of biliverdin, but not bilirubin when caput spermatozoa were challenged with 500 µM H2O2. Since immature spermatozoa are a source of reactive oxygen species, BLVRA may be involved in the protection of maturing spermatozoa. It is also plausible that BLVRA is implicated in haemic protein catabolism in the epididymal luminal environment.

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.

Epididymosomes convey different repertoires of microRNAs throughout the bovine epididymis.

Epididymosomes are small membrane vesicles that are secreted by epididymal epithelial cells and are involved in posttesticular sperm maturation. Although their role in protein transfer to the sperm membrane is well documented, we report their capacity to transport microRNAs (miRNAs), which are potent regulators of posttranscriptional gene expression. Using a microperfusion technique combined with a global microarray approach, we demonstrated that epididymosomes from two discrete bovine epididymal regions (caput and cauda) possess distinct miRNA signatures. In addition, we also established that miRNA repertoires contained within epididymosomes differ from those of their parent epithelial cells, suggesting that miRNA populations released from the cells may be selectively sorted. Binding of DilC12-labeled epididymosomes to primary cultured epididymal cells was measured by flow cytometry, and the results indicated that epididymosomes from the median caput and their miRNA content may be incorporated into distal caput epithelial cells. Overall, these findings reveal that distinct miRNA repertoires are released into the intraluminal fluid in a region-specific manner and could be involved in a novel mechanism of intercellular communication throughout the epididymis via epididymosomes.

microRNA signature is altered in both human epididymis and seminal microvesicles following vasectomy.

STUDY QUESTION: Does vasectomy impact microRNA (miRNA) expression in the epididymis and seminal microvesicles (SMVs) in a non-reversible manner? SUMMARY ANSWER: The miRNA signature in the epididymis and SMVs is altered by vasectomy and only partially restored after vasovasostomy surgery. WHAT IS KNOWN ALREADY: Vasectomy modifies the epididymal transcriptome and triggers non-reversible changes that affect sperm function. Some vasovasostomized men experience a reduced fertility outcome. STUDY DESIGN, SIZE, DURATION: Human epididymides provided by three control donors and three vasectomized donors were collected under artificial circulation through Transplant Quebec (Quebec, QC, Canada). Semen from three normal, three vasectomized and five vasovasostomized donors was provided by the andrology clinic. PARTICIPANTS/MATERIALS, SETTING, METHODS: Epididymides and semen were collected from donors between 26 and 50 years of age with no known pathologies that could potentially affect reproductive function. After RNA extraction, epididymal miRNA profiles were determined by microarray (Affimetrix), compared by ANOVA and confirmed by real-time PCR. The correlation between miRNA and gene expression profiles was investigated by an integrated genomic approach. miRNA signature from purified SMVs was established by microarray. MAIN RESULTS AND THE ROLE OF CHANCE: Vasectomy significantly modified the expression of epididymal miRNAs, which were mainly correlated with mRNAs for transcription factors. Vasectomy also impacted the detection of 118 of the miRNAs found in SMVs from normal donors, including miRNAs of epididymal origin contained in epididymosomes. Among seminal miRNAs changes, 52 were reversible according to the expression levels of miRNA in the semen samples from vasovasostomized donors, while 66 were non-reversible. LIMITATIONS, REASONS FOR CAUTION: Identification of miRNAs responsive to vasectomy was determined with a limited number of samples due to the low number of human specimen samples available. WIDER IMPLICATIONS OF THE FINDINGS: According to the critical role played by miRNAs in all biological systems, we believe that miRNA changes occurring upstream and downstream of the vasectomy site may be related to the reduced fertility outcome reported following surgically successful vasectomy reversal. This study may provide new tools for predicting vasovasostomy success and open avenues for the identification of the molecular players involved in male infertility.

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.

ATP secretion in the male reproductive tract: essential role of CFTR.

Extracellular ATP is essential for the function of the epididymis and spermatozoa, but ATP release in the epididymis remains uncharacterized. We investigated here whether epithelial cells release ATP into the lumen of the epididymis, and we examined the role of the cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-) and HCO(3)(-) conducting ion channel known to be associated with male fertility, in this process. Immunofluorescence labelling of mouse cauda epididymidis showed expression of CFTR in principal cells but not in other epithelial cells. CFTR mRNA was not detectable in clear cells isolated by fluorescence-activated cell sorting (FACS) from B1-EGFP mice, which express enhanced green fluorescent protein (EGFP) exclusively in these cells in the epididymis. ATP release was detected from the mouse epididymal principal cell line (DC2) and increased by adrenaline and forskolin. Inhibition of CFTR with CFTR(inh172) and transfection with CFTR-specific siRNAs in DC2 cells reduced basal and forskolin-activated ATP release. CFTR-dependent ATP release was also observed in primary cultures of mouse epididymal epithelial cells. In addition, steady-state ATP release was detected in vivo in mice, by measuring ATP concentration in a solution perfused through the lumen of the cauda epididymidis tubule and collected by cannulation of the vas deferens. Luminal CFTR(inh172) reduced the ATP concentration detected in the perfusate. This study shows that CFTR is involved in the regulation of ATP release from principal cells in the cauda epididymidis. Given that mutations in CFTR are a leading cause of male infertility, we propose that defective ATP signalling in the epididymis might contribute to dysfunction of the male reproductive tract associated with these mutations.

Bovine sperm raft membrane associated Glioma Pathogenesis-Related 1-like protein 1 (GliPr1L1) is modified during the epididymal transit and is potentially involved in sperm binding to the zona pellucida.

Glioma pathogenesis-related 1-like protein1 (GliPr1L1) was identified by liquid chromatography-tandem mass spectrometry analyses of proteins associated to bovine sperm lipid raft membrane domains. This protein belongs to the CAP superfamily including cysteine-rich secretory proteins, Antigen 5 and pathogenesis-related 1 protein. PCR analysis revealed that GliPr1L1 is expressed in testis and, at a much lower level, all along the epididymis. Western blotting showed a similar distribution of GliPr1L1 in testicular and epididymal tissue extracts. In the epididymal lumen, GliPr1L1 was associated with the maturing spermatozoa and epididymosomes all along the excurrent duct but was undetectable in the soluble fraction of epididymal fluid. The protein was detectable as multiple isoforms with a higher MW form in the testis and proximal caput. Treatments with PNGase F revealed that N-glycosylation was responsible of multiple bands detected on Western blots. These results suggest that the N-glycosylation moiety of GliPr1L1 is processed during the transit in the caput. Western blots demonstrated that GliPr1L1 was associated with the sperm plasma membrane preparation. GliPr1L1 is glycosyl phosphatidyl inositol (GPI) anchored to caput and cauda spermatozoa as demonstrated by the ability of phosphatidylinositol specific phospholipase C to release GliPr1L1 from intact sperm cells. Lipid raft membrane domains were separated from caput and cauda epididymal spermatozoa. GliPr1L1 was immunodetectable in the low buoyant density fractions where lipid rafts are distributed. GliPr1L1 was localized on sperm equatorial segment and neck. In vitro fertilization performed in presence of anti-GliPr1L1 showed that this protein is involved in sperm-zona pellucida interaction.

Region-specific gene expression in the epididymis.

The epididymis is responsible for post-testicular sperm maturation, which consists in the acquisition of forward motility and fertilizing ability. This organ is composed of three main anatomical regions - the caput, corpus and cauda epididymidis - which possess distinct gene expression profiles, ensuring different epididymal functions essential to the different steps of sperm maturation. Since many genes display spatially restricted expression in the epididymis, this organ constitutes a model of choice to study the mechanisms that govern region-specific gene expression. Factors such as steroid hormones, lumicrine factors and temperature affect the pattern of gene expression in the epididymis. Recently, the contribution of small RNAs in epididymal gene regulation has been investigated and constitutes a promising avenue for clinical application with regard to male fertility.

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.

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.

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.

Purification and identification of sperm surface proteins and changes during epididymal maturation.

Surface membrane proteins have a key role in the sequential interactions between spermatozoa and oocytes. The aim of this study was to characterize protein changes occurring during post-testicular differentiation using a new overall approach to study surface membrane proteins of spermatozoa. A dedicated protocol based on specific purification of surface membrane proteins labeled with sulfo-NHS-SS-biotin was developed for this purpose. Appropriate gel electrophoresis separation and purification methods combined with standard proteomic methods were then used to identify and quantify surface membrane proteins from immature and mature spermatozoa. Membrane-associated proteins were discriminated from integral membrane proteins by differential solubilization. Protein regionalization on the spermatozoon surface was achieved by comparative analysis of the surface protein extracts from the entire spermatozoa and from periacrosomal sperm plasma membranes. Identification of several known proteins and of new proteins related to the process of epididymal maturation showed the reliability of this protocol for specific purification of a subproteome and identification of new sperm membrane proteins. This approach opens up a new area in the search for male fertility markers.