Clémence Belleannée

Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals

Offspring affected by sperm small RNAs Paternal dietary conditions in mammals influence the metabolic phenotypes of offspring. Although prior work suggests the involvement of epigenetic pathways, the mechanisms remains unclear. Two studies now show that altered paternal diet affects the level of small RNAs in mouse sperm. Chen et al. injected sperm transfer RNA (tRNA) fragments from males that had been kept on a high-fat diet into normal oocytes. The progeny displayed metabolic disorders and concomitant alteration of genes in metabolic pathways. Sharma et al. observed the biogenesis and function of small tRNA-derived fragments during sperm maturation. Further understanding of the mechanisms by which progeny are affected by parental exposure may affect human diseases such as diet-induced metabolic disorders. Science, this issue p. 397, p. 391 Abundant transfer RNA fragments in maturing mammalian sperm regulate expression of endogenous retroelements in the embryo. Several recent studies link parental environments to phenotypes in subsequent generations. In this work, we investigate the mechanism by which paternal diet affects offspring metabolism. Protein restriction in mice affects small RNA (sRNA) levels in mature sperm, with decreased let-7 levels and increased amounts of 5′ fragments of glycine transfer RNAs (tRNAs). In testicular sperm, tRNA fragments are scarce but increase in abundance as sperm mature in the epididymis. Epididymosomes (vesicles that fuse with sperm during epididymal transit) carry RNA payloads matching those of mature sperm and can deliver RNAs to immature sperm in vitro. Functionally, tRNA-glycine-GCC fragments repress genes associated with the endogenous retroelement MERVL, in both embryonic stem cells and embryos. Our results shed light on sRNA biogenesis and its dietary regulation during posttesticular sperm maturation, and they also link tRNA fragments to regulation of endogenous retroelements active in the preimplantation embryo.

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

Significance On activation, blood platelets package components from their cytoplasm into microparticles (MPs), tiny vesicles released by cytoplasmic membrane budding and shedding. Given that MPs can impact other cellular lineages on internalization, we aimed to decipher the mechanisms promoting MP internalization by cellular recipients. We modeled MP internalization by neutrophils and identified a predominant lipid, 12(S)-hydroxyeicosatetranoic acid, as a mediator critical for the promotion of MP internalization. MPs were found inside neutrophils from individuals with rheumatoid arthritis, and their presence in neutrophils in the joints of mice treated with arthritogenic serum is dependent on the expression of enzymes implicated in the generation of 12(S)-hydroxyeicosatetranoic acid. These findings reveal a unique molecular mechanism implicated in MP internalization relevant to inflammatory processes. 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.

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.

Epididymosomes Convey Different Repertoires of MicroRNAs Throughout the Bovine Epididymis

ABSTRACT 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.

The contribution of proteomics to understanding epididymal maturation of mammalian spermatozoa

The acquisition of the ability of the male gamete to fertilize an ovum is the result of numerous and sequential steps of differentiation of spermatozoa that occur as they transit from the testis to the end of the epididymal tubule. The post gonadal sperm modifications are mostly related to motility, egg binding, and penetration processes. As the activity of the epididymis and its luminal fluid composition are believed to be directly related to ‘sperm maturation’, a review on epididymal proteins is presented. Comparative studies have shown that the epididymal activities are species specific. Nevertheless, for all mammalian species studied, similarities exist in the sequential proteomic changes of the luminal composition of the epididymal tubule and proteins on the sperm surface. The potential roles of these modifications are discussed.

Role of MicroRNAs in Controlling Gene Expression in Different Segments of the Human Epididymis

Background The molecular mechanisms implicated in regionalized gene expression in the human epididymis have not yet been fully elucidated. Interestingly, more than 200 microRNAs (miRNAs) have been identified in the human epididymis and could be involved in the regulation of mRNA stability and post-transcriptional expression in this organ. Methods Using a miRNA microarray approach, we investigated the correlation between miRNA signatures and gene expression profiles found in three distinct regions (caput, corpus and cauda) of human epididymides from 3 donors. In silico prediction of transcript miRNA targets was performed using TargetScan and Miranda softwares. FHCE1 immortalized epididymal cell lines were cotransfected with mimic microRNAs and plasmid constructs containing the 3′UTR of predicted target genes downstream of the luciferase gene. Results We identified 35 miRNAs differentially expressed in the distinct segments of the epididymis (fold change ≥2, P-value≤0.01). Among these miRNAs, miR-890, miR-892a, miR-892b, miR-891a, miR-891b belonging to the same epididymis-enriched cluster located on the X chromosome, are significantly more expressed in the corpus and cauda regions than in the caput. Interestingly, a strong negative correlation (r = −0,89, P-value≤0.001) was found between the pattern of expression of miR-892b and its potential mRNA target Esrrg (Estrogen Related Receptor Gamma) and with miR-145 and Cldn10 mRNA (r = −0,92, P-value≤0.001). We confirmed that miR-145 and miR-892b inhibit the expression of the luciferase reporter via Cldn10 and Esrrg 3′ UTRs, respectively. Conclusion Our study shows that the expression of miRNAs is segmented along the human epididymis and correlates with the pattern of target gene expression in different regions. Therefore, epididymal miRNAs may be in control of the maintenance of gene expression profile in the epididymis, which dictates segment-specific secretion of proteins and establishes physiological compartments that directly or indirectly affect sperm maturation and fertility.

Detection and Quantification of Microparticles from Different Cellular Lineages Using Flow Cytometry. Evaluation of the Impact of Secreted Phospholipase A2 on Microparticle Assessment

Microparticles, also called microvesicles, are submicron extracellular vesicles produced by plasma membrane budding and shedding recognized as key actors in numerous physio(patho)logical processes. Since they can be released by virtually any cell lineages and are retrieved in biological fluids, microparticles appear as potent biomarkers. However, the small dimensions of microparticles and soluble factors present in body fluids can considerably impede their quantification. Here, flow cytometry with improved methodology for microparticle resolution was used to detect microparticles of human and mouse species generated from platelets, red blood cells, endothelial cells, apoptotic thymocytes and cells from the male reproductive tract. A family of soluble proteins, the secreted phospholipases A2 (sPLA2), comprises enzymes concomitantly expressed with microparticles in biological fluids and that catalyze the hydrolysis of membrane phospholipids. As sPLA2 can hydrolyze phosphatidylserine, a phospholipid frequently used to assess microparticles, and might even clear microparticles, we further considered the impact of relevant sPLA2 enzymes, sPLA2 group IIA, V and X, on microparticle quantification. We observed that if enriched in fluids, certain sPLA2 enzymes impair the quantification of microparticles depending on the species studied, the source of microparticles and the means of detection employed (surface phosphatidylserine or protein antigen detection). This study provides analytical considerations for appropriate interpretation of microparticle cytofluorometric measurements in biological samples containing sPLA2 enzymes.

CD9-positive microvesicles mediate the transfer of molecules to Bovine Spermatozoa during epididymal maturation.

Acquisition of fertilization ability by spermatozoa during epididymal transit occurs in part by the transfer of molecules from membranous vesicles called epididymosomes. Epididymosomes are heterogeneous in terms of both size and molecular composition. Exosomes and other related small membranous vesicles (30–120 nm) containing tetraspanin proteins on their surface are found in many biological fluids. In this study, we demonstrate that these vesicles are present in bovine cauda epididymal fluid as a subpopulation of epididymosomes. They contain tetraspanin CD9 in addition to other proteins involved in sperm maturation such as P25b, GliPr1L1, and MIF. In order to study the mechanism of protein transfer to sperm, DilC12-labeled unfractionated epididymosomes or CD9-positive microvesicles were coincubated with epididymal spermatozoa, and their transfer was evaluated by flow cytometry. CD9-positive microvesicles from epididymal fluid specifically transferred molecules to spermatozoa, whereas those prepared from blood were unable to do so. The CD9-positive microvesicles transferred molecules to the same sperm regions (acrosome and midpiece) as epididymosomes, with the same kinetics; however, the molecules were preferentially transferred to live sperm and, in contrast to epididymosomes, Zn2+ did not demonstrate potentiated transfer. Tetraspanin CD9 was associated with other proteins on the membrane surface of CD9-positive microvesicles according to coimmunoprecipitation experiments. CD26 cooperated with CD9 in the molecular transfer to sperm since the amount of molecules transferred was significantly reduced in the presence of specific antibodies. In conclusion, CD9-positive microvesicles are present in bovine cauda epididymal fluid and transfer molecules to live maturing sperm in a tissue-specific manner that involves CD9 and CD26.

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.

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. J. Cell. Physiol. 227: 3876–3886, 2012.