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.

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 software's. 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.

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.

Gene expression in the epididymis of normal and vasectomized men: what can we learn about human sperm maturation?

Anatomically, the human epididymis is unusual when compared with the excurrent duct of other eutherian mammals. Furthermore, clinical observations suggest that it may not be as important for sperm maturation as is the case for laboratory animals. In contrast, hierarchical clustering of microarray data of epididymides from normal men revealed 2274 modulated qualifiers between the epididymal segments, 1184, 713, and 269 of them being highly expressed in the caput, corpus, and cauda, respectively. The organization of qualifiers according to their similarities by gene ontology indicated that caput transcripts are dedicated to cell-cell adhesion, whereas the corpus is characterized by genes involved in response to other organisms (ie, defense mechanisms) and the cauda transcriptome is specialized in muscle contraction and establishment of localization. A region-specific gene expression pattern thus characterizes the human epididymis as in animal models. In humans, vasectomies have consequences on the epididymal transcriptome. Cluster analysis revealed that 1363 genes are expressed in both normal and vasectomized epididymides, whereas 911 and 660 of them are specifically expressed in normal and vasectomized epididymides, respectively. Three of the affected genes are particularly interesting because of their involvement in sperm biochemical remodeling during epididymal transit: dicarbonyl/l-xylulose reductase, Niemann-Pick disease, type C2, and cysteine-rich secretory protein 1. In some vasovasostomized men, these modifications in gene expression induced by vasectomy are irreversible, thus affecting the biochemical parameters, and potentially, the function of their ejaculated sperm. This may explain the discrepancies between a surgically successful vasovasostomy and fertility recovery.

Vasectomy affects cysteine-rich secretory protein expression along the human epididymis and its association with ejaculated spermatozoa following vasectomy surgical reversal.

The epididymis is essential for the acquisition of sperm fertilizing ability and forward motility. After vasectomy, the flux and composition of the epididymal fluid are modified, causing possible sequelae to the occluded excurrent duct. Some of these sequelae may not be reversible following vasovasostomy, affecting sperm physiology and their fertilizing ability. We previously demonstrated that the epididymal expression in men of a major glycoprotein secreted by the epididymis, cysteine-rich secretory protein 1 (CRISP1), and its encoding mRNA are affected by vasectomy. In this study we showed that following vasectomy, the increased level of CRISP1 is not due to a secretory defect but to its accumulation in the intraluminal compartment of the cauda epididymidis. Western blot analyses were performed to determine the amount of CRISP1 associated with spermatozoa of men who had undergone surgical vasectomy reversal. Spermatozoa of vasovasostomized men are characterized by a significant increase (P < .05) in CRISP1 levels when compared with normal donors. There was no linear correlation between CRISP1 levels and the period of time elapsed between vasectomy and vasovasostomy. CRISP1 was also present in seminal plasma of normal and vasovasostomized men, but not in vasectomized individuals. The soluble concentration of CRISP1 was significantly higher (P < .05) in seminal plasma of vasovasostomized men when compared with normal men. Knowing that one of the proposed functions of CRISP1 is to modulate sperm capacitation, we evaluated the level of tyrosine protein phosphorylation of 2 AXAP proteins of the fibrous sheath, p81 and p105. Spermatozoa of vasovasostomized men were characterized by a 50% increase of protein tyrosine phosphorylation when compared to spermatozoa of normal men (P < .05). Our results are discussed with regard to the fertilizing ability of ejaculated spermatozoa of some vasovasostomized men.

Localization of Hsp60 and Grp78 in the human testis, epididymis and mature spermatozoa.

Molecular chaperones of the heat shock proteins (HSP) family are important in numerous cellular processes. In this study, the expression of Hsp60 and Grp78 proteins was investigated in the male reproductive tract. The cellular distribution of Hsp60 and Grp78 proteins was analysed in the human testis and epididymis by immunohistochemical approaches. DNA microarray technology was used to analyse HSP60 and GRP78 gene expression along human epididymis. The cellular localization of these chaperone proteins in ejaculated spermatozoa was investigated by indirect immunofluorescence and by Western blot following sperm sub-cellular fractionation. In the human testis, Hsp60 was detected in spermatogonia, whereas a strong Grp78 staining was observed in spermatocytes and round spermatids. Grp78 protein was also observed in the epididymal epithelium, whereas no Hsp60 staining was observed in this organ by immunohistochemistry. The presence of both Hsp60 and Grp78 RNA in human epididymis was confirmed by microarrays. In ejaculated spermatozoa, Hsp60 was localized in the mid-piece, whereas Grp78 was detected in the neck region. These results indicate that in addition to being expressed in human testis spermatogenic cells, both Hsp60 and Grp78 proteins persist in ejaculated spermatozoa. These findings are in agreement with the involvement of Hsp60 and Grp78 during spermatogenesis and in sperm functions such as fertilization.

Protein composition of human epididymosomes collected during surgical vasectomy reversal: a proteomic and genomic approach.

BACKGROUND: The epididymal epithelium secretes membranous vesicles, called epididymosomes, with which a complex mixture of proteins is associated. These vesicles transfer to spermatozoa selected proteins involved in sperm maturation. Epididymosomes in the human excurrent duct have been described, but their protein composition and possible functions are unknown. METHODS AND RESULTS: Epididymosomes were collected during vasovasostomy procedures, purified and submitted to liquid chromatography with hybrid quadrupole time-of-flight mass spectrometry. From all the mass spectra generated, 1022 peptides allowed the identification of 146 different proteins. Identification of some of these proteins was confirmed by western blots. Furthermore, western blot showed that the protein composition of epididymosomes differed from that characterizing prostasomes; membranous vesicles secreted by the prostate. Organization of the epididymosomes proteome according to common functional features suggests that epididymosomes have multiple functions. In order to understand the origin of epididymosomes collected distally, microarray databases of caput, corpus and cauda epididymidis were analysed to determine where along the excurrent duct the encoded proteins associated to epididymosomes are synthesised. Results suggest that some proteins synthesized in the caput and corpus epididymidis are associated with epididymosomes collected distally. CONCLUSIONS: Epididymosomes thus transit along the excurrent duct, and vesicles collected distally represent a mixed population.

Effects of vasectomy on gene expression profiling along the human epididymis.

Worldwide, almost 100 million men rely on vasectomy for male contraceptive purposes. Due to changes in their personal lives, an increasing number of these men request surgical vasectomy reversal. Unfortunately, a significant proportion of these men remain infertile, despite the reestablishment of patent ducts, possibly due to epididymal damage caused by vasectomy. In animal models, vasectomy affects different epididymal physiological and biochemical parameters. However, the consequences of vasectomy on epididymal function are poorly understood. Furthermore, results obtained with animal models cannot be extrapolated to humans to understand the consequences of vasectomy on epididymal function. Gene expression along the epididymis is highly regulated. We previously showed that the human epididymal expression pattern of two genes is altered after vasectomy. To complete the list of epididymal genes affected by vasectomy, we analyzed the epididymal gene expression pattern of three vasectomized donors using the Affymetrix human GeneChip U133 Plus 2. These results were compared with the gene expression pattern of three "normal" donors. The data generated allowed the identification of many human epididymal genes for which expression is modified after vasectomy. Quantitative (Qt)-PCR and Western blot analysis of six selected genes known to be expressed in specific epididymal segments were performed. The Qt-PCR results confirmed the selected transcripts expression pattern deduced from microarray data. However, Western blot analysis revealed some differences in protein distribution along the epididymis when compared with the encoding transcripts expression pattern. These results contribute to an understanding of the reasons why fertility is not recovered in vasovasostomized men, even though spermogram values suggest surgical success of vasectomy reversal.

One- and two-dimensional SDS-PAGE zymography with quenched fluorogenic substrates provides identification of biological fluid proteases by direct mass spectrometry.

We describe a simple and direct zymographic method for the detection of proteases using quenched fluorescent substrates. The proteases were separated using one- and two-dimensional electrophoresis, and the gel subsequently was incubated with the quenched fluorescent substrate. After a short incubation, the released fluorescence allowed the localization of the proteases directly using UV light. The protease spots could then be cut directly from the gel and processed for identification by mass spectrometry. This method could easily be used to develop or test whether a substrate is specific or not and also to detect the proteases that are able to cleave this substrate in a complex biological fluid. This also allowed direct identification of proteases without complex purification.

Region-specific gene expression profiling along the human epididymis.

During their transit through the epididymis, spermatozoa undergo many biochemical modifications necessary to acquire flagellar motility and fertilizing ability. These modifications, collectively called sperm maturation, are well orchestrated along the epididymis and depend on highly regionalized gene expression patterns. Based on clinical observations, the role of the epididymis in human sperm maturation has been questioned. To further understand the function of the excurrent duct in humans, we analysed gene expression of three donors on 'Affymetrix human GeneChip U133 plus 2' representing 47,000 transcriptional variants. More than 50% of transcripts were detected in each epididymal region. The analysis of hierarchical clustering performed from 2274 modulated qualifers between the three regions revealed that 1184, 713 and 269 were highly expressed in the caput, corpus and cauda region, respectively, in a very specific manner. The expressed qualifers were grouped according their similarity by Gene Ontology to give an overview of the functional features of the encoded proteins and to elucidate their potential roles in the epididymis. Northern blot analysis of eight gene transcripts predicted by microarray data to be highly expressed in the human epididymis was performed. All the transcript expression patterns confirmed the microarrays results. The data generated in this study demonstrate a region-specific gene expression pattern along the human epididymis that seems to coincide with the morphologically distinctive features of the excurrent duct.

Analysis of furin ectodomain shedding in epididymal fluid of mammals: demonstration that shedding of furin occurs in vivo.

Sperm cell surface proteins and proteins of their surrounding fluids are reported to be proteolytically processed in relation to acquisition of sperm fertility during epididymal transit. Several of these proteins might be potential targets for subtilisin-like pro-protein convertase. Using immunochemistry and mass spectrometry analysis, we found that an 80 kDa form of furin (EC 3.4.21.75) is present in the fluid from the mid-caput to the distal corpus regions of the epididymis of various domestic mammals. This protein is absent from the fluid of the caudal region, suggesting that it is reabsorbed or degraded. The cDNA sequence of ovine furin was obtained and the mRNA was found throughout this organ, although in greater amounts in the mid and distal caput regions. Metabolic labeling with (35)S-amino acids indicated that the protein was synthesized and released from the epithelium only in a restricted area of the mid-caput, suggesting a specific regionalized mechanism of secretion. The fluid protein is not pelleted at 100 000 g and did not react with a C-terminal antibody indicating that it is not bound to membranous materials. These findings demonstrate that a furin ectodomain shedding occurs naturally in vivo in the epididymis where this enzyme could be involved in fluid and/or sperm membrane protein processing.

Shedding of the germinal angiotensin I-converting enzyme (gACE) involves a serine protease and is activated by epididymal fluid.

The present report describes how the soluble germinal angiotensin I-converting enzyme (gACE) appears in the epididymal fluid, where it has been identified in some laboratory rodents and domestic ungulates. We showed that this gACE results from an active proteolytic process that releases the enzyme's extracellular domain from sperm in a precise spatiotemporal location during epididymal transit and that this process involves serine protease activity. Using polyclonal antibodies against the C-terminal intracellular sequence of ACE, a fragment of approximately 10 kDa was detected on the sperm extract only in the epididymal region, where the gACE release occurs. The fluid enzyme was purified, and the cleavage site was determined by mass spectrometry to be between Arg622 and Leu623 of the mature sheep gACE sequence (equivalent to Arg627 and Arg1203 of the human mature gACE and somatic ACE sequences, respectively). Thereafter, the C-terminal Arg was removed, leaving Ala621 as a C-terminal. Using an in vitro assay, gACE cleavage from sperm was strongly increased by the presence of epididymal fluid from the release zone, and this increase was inhibited specifically by the serine protease-inhibitor AEBSF but not by para-aminobenzamidine. None of the other inhibitors tested, such as metallo- or cystein-protease inhibitors, had a similar effect on release. It was also found that this process did not involve changes in gACE phosphorylation.