Jean-Louis Dacheux

A PORCINE HOMOLOG OF THE MAJOR SECRETORY PROTEIN OF HUMAN EPIDIDYMIS, HE1,SPECIFICALLY BINDS CHOLESTEROL

A porcine homolog of the major secretory protein of human epididymis, HE1, was for the first time purified from the porcine cauda epididymal fluid. The HE1 homolog was secreted into the epididymal fluid as a 19-kDa glycoprotein, whose sugar moiety was gradually processed to form a 16-kDa protein during transit through the epididymis. The HE1 homolog mRNA was detected only in the caput and corpus epididymis among the porcine tissues examined. The purified HE1 homolog specifically bound cholesterol with high affinity (Kd=2. 3 microM). The binding stoichiometry was determined to be 0.94 mol/mol, suggesting that 1 mol of cholesterol binds to 1 mol of the protein. It was also found that the HE1 homolog is a major cholesterol-binding protein in the porcine epididymal fluid. The possibility that the HE1 homolog is involved in the regulation of the lipid composition of the sperm membranes during the maturation in epididymis is discussed.

Stallion Epididymal Fluid Proteome: Qualitative and Quantitative Characterization; Secretion and Dynamic Changes of Major Proteins

Abstract Proteins present in and secreted into the lumen of various regions of the stallion epididymis were characterized qualitatively and quantitatively by two-dimensional electrophoresis. Using this proteomic approach, 201 proteins were found in the lumen and 117 were found that were secreted by the epithelium in various parts of the organ. Eighteen proteins made up 92.6% of the total epididymal secretory activity, lactoferrin (41.2%) and clusterin (24.8%) being the most abundant. Procathepsin D, HE1/CTP (cholesterol transfer protein), GPX (glutathione peroxidase), beta-N-acetyl-hexosaminidase, and PGDS (prostaglandin D2 synthase) were the other major compounds secreted. The most abundant proteins found in the luminal fluid were albumin and the secreted proteins: lactoferrin, PGDS, GPX, HE1/CTP, and hexosaminidase. Three main secretory epididymal regions were identified from the protein pattern, i.e., regions E0–E2, E3–E5, and E6–E9. Region E0–E2 was characterized by the secretion of clusterin (53%), PGDS (44%), and GPX (6%). Region E3–E5 had the highest number of secreted proteins, the highest protein concentrations (60–80 mg/ml), and the highest spermatocrit value (85%). Lactoferrin (60% in E4), clusterin (29% in E3), hexosaminidase (10% in E3), and procathepsin D (6.9% in E4) were the most abundant proteins in this region. Region E6–E9, in which few region-specific secreted compounds were found, was characterized by a high quantity of lactoferrin in the luminal fluid (2–14 mg/ml). Comparison between the secretion of the major proteins and their concentrations in the lumen throughout the organ showed that the behavior of each protein is specific, in particular for the three isoforms of clusterin.

IDENTIFICATION, PROTEOMIC PROFILING AND ORIGIN OF RAM EPIDIDYMAL FLUID EXOSOME-LIKE VESICLES.

Abstract Small membranous vesicles, between 25- and 75-nm diameter, were collected by high-speed centrifugation from the ram cauda epididymal fluid and were found to be normal constituents of this fluid and of the seminal plasma. The SDS-PAGE protein pattern of these vesicles was specific and very different from that of the caudal fluid, seminal plasma, sperm extract, and cytoplasmic droplets. After two-dimensional electrophoresis separation and mass spectrometry analysis, several proteins were identified and grouped into i) membrane-linked enzymes, such as dipeptidyl peptidase IV (DPP-IV), neprilysin (NEP), phosphodiesterase-I (E-NPP3), and protein G-beta; ii) vesicle-associated proteins, such as lactadherin (MFEG8-PAS6/7) and vacuolar ATPase; iii) several cytoskeleton-associated proteins, such as actin, ezrin and annexin; and iv) metabolic enzymes. The presence of some of these proteins as well as several different hydrophobic proteins secreted by the epididymis was further confirmed by immunoblotting. These markers showed that the majority of the vesicles originated from the cauda epididymal region. The physical and biochemical characteristics of these vesicles suggest they are the equivalent of the exosomes secreted by several cell types and epithelium. The main membrane-linked proteins of the vesicles were not retrieved in the extract from cauda or ejaculated sperm, suggesting that these vesicles did not fuse with sperm in vivo.

New insights into epididymal function in relation to sperm maturation

Testicular spermatozoa acquire fertility only after 1 or 2 weeks of transit through the epididymis. At the end of this several meters long epididymal tubule, the male gamete is able to move, capacitate, migrate through the female tract, bind to the egg membrane and fuse to the oocyte to result in a viable embryo. All these sperm properties are acquired after sequential modifications occurring either at the level of the spermatozoon or in the epididymal surroundings. Over the last few decades, significant increases in the understanding of the composition of the male gamete and its surroundings have resulted from the use of new techniques such as genome sequencing, proteomics combined with high-sensitivity mass spectrometry, and gene-knockout approaches. This review reports and discusses the most relevant new results obtained in different species regarding the various cellular processes occurring at the sperm level, in particular, those related to the development of motility and egg binding during epididymal transit.

Human epididymal secretome and proteome

The proteins that are neosynthesized and secreted in the different regions of the human epididymis were determined by in vitro biosynthesis of epididymal tubules, and the luminal proteins were collected by microperfusion of each tubule. The preparations were analyzed by two-dimensional gel electrophoresis and the proteins were identified by mass spectrometry. Some of the major proteins identified corresponded to serum compounds such as albumin, transferrin and alpha-1-antitrypsin. The other proteins identified included lactotransferrin, clusterin, PEBP, NCP2/CTP/HE1, HE3, Crisp, actin, calmodulin, E12, PGDS, l-lactate dehydrogenase, malate dehydrogenase, carbonic anhydrase, triose phosphate isomerase, glutamyltransferase, glutathione S-transferase P, thioredoxin peroxidase, superoxide dismutase, cathepsin D and cystatin. Epididymal activity is highly regionalized in most species. However, in this study in humans, there were only minor changes in the major proteins secreted. It is suggested that this specificity might be related to the difference between species in the location of the epididymis where sperm become fertile.

Identification of luminal and secreted proteins in bull epididymis.

The epididymis plays a major role in the acquisition of sperm fertility. In order to shed light on specific features of epididymal function in mammalian species, we characterized the luminal proteins (luminal proteome) and secreted proteins (secretome) in the bovine epididymis. We identified 172 different luminal proteins in 9 distinct epididymal regions. The concentration and secretory activity of luminal proteins were quantified throughout the epididymis. Among the most abundant secreted proteins, we found lipocalin 5, (LCN5), NADP(+)dependent prostaglandin dehydrogenase (PTGDS), Niemann-Pick disease type C2 protein (NPC2), glutathione peroxidase type 5 (GPX 5), clusterin (CLU), hexosaminidase B (HEXB) and galactosidase (GLB1), each of which is released in distinct epididymal regions. Gelsolin, (GSN) previously not described in mammalian epididymal fluid, appeared to be a major protein secreted exclusively in the distal region of the bovine epididymis, where fully mature spermatozoa are stored. Although the major epididymal proteins are conserved between mammalian species, this study highlights the specificity and mechanisms of protein processing of epididymal secretion in the bull. In addition, this study provides a major insight into the sequential changes occurring in the sperm environment while gaining fertilizing capacity and could provide new information for the future identification of potential fertility markers.

Comparison, Characterization, and Identification of Proteases and Protease Inhibitors in Epididymal Fluids of Domestic Mammals. Matrix Metalloproteinases Are Major Fluid Gelatinases

Abstract The testicular and epididymal fluids of ram, boar, and stallion were analyzed by means of one-dimensional and two-dimensional gelatin gel zymography. Five main gelatinolytic bands were revealed in the ram and at least seven were observed in the boar and stallion. These proteolytic bands showed regionalized distribution throughout the organs. The two main proteolytic activities at around 54–66 kDa retrieved in all three species were inhibited by EDTA and phenanthroline, indicating that they were metallo-dependent enzymes. The activity of some of the low-molecular-weight gelatinases was also decreased by EDTA, whereas others were inhibited by serine protease inhibitors. One of the main proteases at 60–62 kDa from the caput fluid of the stallion and the ram was N-terminal sequenced; in both cases, high sequence homology was found with the N-terminal of the matrix-metalloproteinase-2 pro-form (pro-MMP-2). Antibodies against MMP-2, MMP-3, and MMP-9 gelatinases confirmed the regional distribution in the fluids of pre -, pro-, active, or degraded forms of these metalloproteases in all three species. We also observed the presence of acrosin in epididymal fluids, which was probably released by dead spermatozoa, but this enzyme did not explain all the serine protease activity. Moreover, the majority of this enzyme is bound to the protease inhibitor α2-macroglobulin, which is present in the fluids of all three species. TIMP-2, a potent inhibitor of MMPs, was present in the fluid of the caput regions in the ram and boar, and in the caput and caudal fluids of the stallion. This study demonstrated that similar types of proteases and inhibitors are regionally distributed in the epididymal fluids of three domestic species, suggesting an identical role in the sperm maturation process, the plasticity of this organ, or both.

Mammalian epididymal proteome.

In all mammalian species, the final differentiation of the male germ cell occurs in the epididymal duct where the spermatozoa develop the ability to be motile and fertilize an ovum. Understanding of these biological processes is the key to understanding and controlling male fertility. Comparative studies between several mammals could be an informative approach to finding common sperm modifications which are not species-specific. The new global biological approaches such as transcriptomes and proteomes provide considerable information which can be used for such comparative approaches. This report summarizes our proteomic studies of the epididymis of several mammals, including humans.

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.

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.