Christine Légaré

P34H sperm protein is preferentially expressed by the human corpus epididymidis.

During epididymal transit, mammalian spermatozoa acquire new surface proteins that are necessary for gamete interaction. We have previously described a 34-kDa human epididymal sperm protein, P34H, that has been shown to be involved in sperm-zona pellucida interaction. In the present study, we report the cloning and characterization of the full-length complementary DNA encoding human P34H. The predicted amino acid sequence revealed 65% identity with P26h, the hamster counterpart of the P34H. The deduced P34H amino acid sequence revealed a 71% similarity with a pig lung tetrameric carbonyl reductase, a member of the short chain dehydrogenase/reductase family proteins. Northern blot analysis revealed that P34H messenger RNA (mRNA) was highly expressed in the human epididymis, principally in the corpus region. A single 912-bp P34H transcript was detected. In situ hybridization experiments showed that the P34H mRNA was predominantly expressed in the proximal and distal sections of the corpus epididymidis. The ...

Hamster sperm antigen P26h is a phosphatidylinositol-anchored protein

We have previously identified a hamster sperm protein, P26h, proposed to be involved in the interaction between spermatozoa and the eggs zona pellucida. In this study we investigated the mechanism of P26h accumulation on hamster spermatozoa during epididymal maturation. Immunocytochemical studies showed an accumulation of P26h on the acrosomal cap of hamster spermatozoa during epididymal transit. To document the anchoring mechanism of P26h, cauda epididymal spermatozoa were exposed to different treatments. High‐salt buffered solutions were unable to remove P26h from the surface of intact spermatozoa. P26h was released in a dose‐dependent manner when live spermatozoa were treated with a solution of phospholipase C specific to phosphatidylinositol. In contrast, the P26h remained associated to the sperm surface following treatment with trypsin. To document the transfer mechanisms of P26h on the maturing spermatozoa, prostasomes were isolated from the epididymal fluid and subjected to immunodetection. Western blots and immunogold studies showed that P26h was associated to epididymal prostasomes. Phospholipase C treatment performed on epididymal prostasomes, indicated that P26h also is anchored to these vesicles via a phosphatidylinositol. These results suggest that epididymal sperm maturation involves a cell to cell transfer of a phosphaditylinositol‐anchored protein and that prostasomes may be implicated in this process. Mol. Reprod. Dev. 52:225–233, 1999.

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.

Effects of Vasectomy on Gene Expression Profiling along the Human Epididymis

Abstract 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.

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.

Effect of vasectomy on P34H messenger ribonucleic acid expression along the human excurrent duct : A reflection on the function of the human epididymis

Abstract Sperm surface proteins involved in fertilization can be added or modified during epididymal transit. P34H, a human epididymal-sperm protein, appears on the sperm acrosomal cap in the distal caput-proximal corpus epididymis. In previous studies, it was shown that P34H is present on spermatozoa in men of proven fertility, is absent in 50% of men presenting with idiopathic infertility, and that a high proportion of men with normospermic vasovasectomy produce spermatozoa deficient in this sperm surface protein. P34H mRNA was expressed in the principal cells of the epididymis of normal men, predominantly in the corpus region. Recently, results coming from the assisted reproductive technologies have questioned the importance of the human epididymis in sperm maturation. In order to understand the effect of obstruction on the physiological state of the human epididymis and its function in sperm maturation, we have analyzed the expression of P34H mRNA at the level of the vas deferens and along the epididymis of normal and vasectomized men. In situ hybridization experiments showed that obstruction of the vas deferens alters the pattern of P34H mRNA expression compared with the tract of normal tissues. The P34H transcript was detected in the proximal caput epididymis of vasectomized men at a much higher intensity than that observed in the same region of normal tissues, being restricted to the principal cells of the epididymal epithelium. Compared with the normal duct, the lumen of vasectomized men was distended throughout the duct and the height of the epithelium was maximal in the caput. P34H mRNA was detectable in vas deferens, was not affected by vasectomy, and a 912-base pair P34H transcript was restricted to the epithelial cells of the vas deferens. Thus, using P34H as a marker, these results show that vasectomy alters the pattern of gene expression along the human epididymis, and suggest that the vas deferens can be a major contributor to sperm maturation in certain situations.

Investigation of male infertility using quantitative comparative proteomics.

Male factors account for 40% of infertility cases. The identification of differentially expressed proteins on spermatozoa from fertile and infertile men can help in the elucidation of the molecular basis of male infertility. The aim of this study was to compare sperm proteomes from 3 different groups: fertile men, normozoospermic men consulting for infertility, and normozoospermic men with an impaired capacity for fertilization (IVF-failure). We used differential proteomics with isobaric tags for relative and absolute quantitation (iTRAQ) labeling, and LC-MS analysis to identify proteins that are differentially expressed. A total of 348 unique proteins were identified and quantified. The analysis identified 33 proteins that were differentially expressed in the IVF-failure group vs the fertile group. Comparison of the infertile and fertile groups revealed that 18 proteins appeared to be differentially expressed. Four proteins were similarly altered in the IVF-failure and infertile groups: semenogelin 1 (SEMG1), prolactin-induced protein (PIP), glyceraldehyde-3-phosphate dehydrogenase (GAPDHS), and phosphoglycerate kinase 2 (PGK2). These protein markers were selected for validation using multiple reactions monitoring mass spectrometry (MRM-MS) and further confirmed by Western blot analysis. Overall, these results suggest that a panel of proteins may be used as biomarkers for future studies of infertility.

Expression of the Hamster Sperm Protein P26h During Spermatogenesis

Abstract P26h is a hamster sperm protein of 26 kDa that has been previously characterized as a surface protein covering the acrosome acquired during epididymal transit. P26h is involved in sperm-egg interactions. Recently, it has been shown that the P26h transcript is highly expressed in the testis, and the P26h cDNA has been cloned from a hamster testicular cDNA library. Herein we report the production of a fusion protein (maltose binding protein-P26h) with the whole P26h cDNA encoding sequence and the production of a polyclonal antiserum against it. In Western blots, this antiserum recognized both the P26h extracted from cauda epididymal spermatozoa and the MBP-P26h. We also determined the age of appearance of P26h and which germ cell types express P26h mRNA and its translational product. Northern blots and in situ hybridization analysis showed that P26h transcripts appear at 3 wk of age, within the first round of spermatogenesis in the golden hamster. In situ hybridization showed that P26h transcripts are expressed in spermatocytes and round spermatids, whereas immunostaining revealed the presence of P26h in the cytoplasm of round spermatids and elongated spermatids. P26h was undetectable in testicular spermatozoa. Both in situ hybridization and immunostaining showed P26h expression to be dependent of the testicular cell type and the epithelium cycle. The implications for P26h in sperm-egg interaction and the testicular origin of P26h are discussed.

Effect of Vasectomy on Gene Expression in the Epididymis of Cynomolgus Monkey

Abstract Vasectomy has been shown to affect the pattern of mRNA expression of P34H, a human sperm protein added to the acrosomal cap during epididymal transit. It has been reported that vasectomy alters the histology of the reproductive tract in various species as a result of the increased pressure in the epididymis. The aim of this study was to evaluate if other epididymis-specific mRNAs, which are expressed in different patterns along the duct, are altered by vasectomy as well. We analyzed the expression of P31m (a monkey homologue of human P34H) and three different HE-like (HE-l) mRNAs along the epididymis in the cynomolgus monkey (Macaca fascicularis). Sexually mature cynomolgus monkeys were vasectomized unilaterally; then the epididymides were surgically removed at different time points. The ipsilateral normal epididymis was used as a control. Histomorphometric measurements showed that the height of the epididymal epithelial cells started to be affected only at 14 wk postsurgery. However, Northern blot and in situ hybridization analysis showed that the expression pattern of P31m, HE1, and HE5-like mRNA along the epididymis was not affected by vasectomy. Only the HE2-like mRNA predominantly expressed in the normal corpus epididymidis was significantly lowered 14 wk after vasectomy. Thus, ductal obstruction differentially alters mRNA expression along the epididymis of the cynomolgus monkey.

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.