Molecular characterization of epididymal proteins (original) (raw)
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Function of human epididymal proteins in sperm maturation
Andrologia, 2009
The epididymis is necessary for post-testicular sperm maturation as it provides the milieu required for spermatozoa to gain the ability for progressive movement and fertilization. In the epididymis the sperm protein, lipid and small RNA content are heavily modified due to interaction with luminal proteins secreted by the epididymal epithelium and extracellular vesicles, epididymosomes. This review focuses on epididymal proteins demonstrated to have an effect on sperm functions, such as motility, capacitation, acrosome reaction, sperm-zona pellucida binding and sperm-egg binding, as well as on embryonic development.
Identification of epididymal proteins associated with hamster sperm
Journal of Experimental Zoology, 1991
The electrophoretic analysis of the proteins that were extracted from immature caput and mature cauda sperm showed evidence of accumulation of several proteins during the epididymal transit of the sperm. An antiserum, raised against detergent-extracted proteins from mature spermatozoa, immunostained six epididymal proteins with apparent molecular masses of 16,22.5, 26'37, 60, and 80 kDa on Western blots of epididymal fluid. Of these proteins, only the 26 kDa protein was significantly immunodetected in proximal caput epididymal fluid. Its biosynthesis by caput epididymis was confirmed by immunoprecipitation of an in vitro translated product of caput poly (A) RNA. The homology of the 26 kDa epididymal protein with the 26 kDa sperm protein was verified by epitope mapping. The other epididymal proteins were found in the fluid of the more distal portions of the organ. Their presence in the epididymal fluid coincided with their detection on the sperm. These epididymal proteins were considered to be sperm-coating proteins.
Biology of Reproduction, 1992
A specific 135-Wa protein was purified from porcine cauda epididymal fluid. Analysis of its N.terminal amino acid sequence revealed it to be a new protein. Stable clones of hybridomas that produced monoclonal antibodies against the purified 135-Wa protein were established. A clone, B-ui, reacting both with epididymal fluid and with sperm plasma membranes was selected and used in this study. Immunoblotting analysis showed that B-il reacted only with a I 35-kDa protein among epididymal fluid proteins. In contrast, B-li did not recognize a similar 135-Wa sperm protein but did strongly react with a 27.kDa protein among sperm membrane proteins, extracted by f4P.40 in the presence of protease inhibitors. B-li also reacted only with a 27-kDa protein fragment among trypsin digests of the 135-Wa epididymai protein. The 135-Wa protein was first detected, by EUSA or immunoblotting analysis, at the beginning of the corpus epididymis. Maximal levels were reached in the distal corpus and levels were slightly decreased in the cauda epididymis. On the other hand, the surface of caput sperm were found to contain small amounts of antigen(s), the concentration of which gradually increased during epididymal transit. In immunocytochemical studies, the antigen was detectable in the epithelial cells from the initial segment to the corpus of the epididymis but not in the caudal cells. In the lumen, the presence of the 135 Wa protein was apparent in the corpus (at a maximum in the middle and distal corpus) and to a lesser degree in the caudal lumen. The 27-Wa protein was distributed all over the equatorial region of the acrosome of less than iO% of caput epididymal sperm. As sperm passed through the corpus epididymis, the percentage of immunoreactive cells increased and the protein was restricted to specific domains of the sperm head. Thus, on the mature sperm, antigen was localized in a crescent-shaped area of the equatorial segment just behind the anterior part of the acrosome and on the apical rim of the sperm head. This is the first observation of a sperm surface antigen derived from an epididymal protein as a proteolytic fragment that interacts with specific regions of the sperm membrane during the process of spermatozoa maturation.
Biology of Reproduction, 1990
The sequential interactions of epididymal secretory proteins with spermatozoa during epididymal transit were examined. Mice received injections of #{176}5S-methionine, and the radiolabeled luminal fluid and sperm-associated proteins were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis at various times after injection. The majority of the luminal fluid and spermassociated proteins were found in the caput epididymidis at 8 h; by 7 days, many of these proteins had been transported to the cauda epididymidis. Two classes of epididymal protein-sperm interactions were distinguished on the basis of regional synthesis and secretion. The major class consisted of proteins that were synthesized, secreted, and bound to spermatozoa in the caput epididymidis. In this class, however, the binding of proteins to the spermatozoa was variable. For example, a protein of 25 kDa remained associated with spermatozoa in substantial amounts during epididymal transit, while proteins of 40 and 35 kDa decreased in amount. Other proteins such as a protein of 18 kDa did not remain associated with spermatozoa. Another class of proteins (54, 44, 29 kDa) were synthesized and secreted from all epididymal regions but bound only to caput spermatozoa. Most of the epididymal proteins appeared to be tightly bound to the spermatozoa since spermatozoa already saturated with the unlabeled protein in the distal epididymis remained so even though the spermatozoa were surrounded by labeled proteins in the luminal fluid. These studies demonstrate that a variety of specific interactions occur between epididymal secretory proteins and spermatozoa as they migrate and mature in the epididymis.
Protein synthesis and secretion in the human epididymis and immunoreactivity with sperm antibodies
Molecular Reproduction and Development, 1990
The synthesis and secretion of proteins in the different regions of the human epididymis were studied in vitro. Epididymal tissues obtained from patients undergoing castration for prostatic carcinoma or from cadavers were incubated in the presence of [35S]methionine, and the resulting radiolabeled proteins were analysed on SDS-PAGE. The corpus region was found to be the most active segment in total protein synthesis. Significant qualitative and quantitative changes were observed in the pattern of proteins secreted from the different epididymal regions. To establish those epididymal proteins that interact with maturing sperm, the secreted products were immunoreacted with antibodies raised against a Triton X-100 extract of ejaculated human sperm heads. The antibodies react mainly with the head region of ejaculated spermatozoa as judged by indirect immunofluorescence. Protein A-gold labeling of freeze-fracture images showed gold particle distribution on the sperm plasma membrane. Western blot analysis of the secreted proteins revealed four bands (66, 37, 32, and 29 kDa) in the proximal regions and six additional bands (80, 76, 48, 27, 22, and 17 kDa) in the distal part of the epididymis. lmmunoprecipitation of the secreted proteins with these antibodies revealed six radioactive bonds of 170, 80, 76, 60, 48, and 37 kDa, which indicates that certain proteins of epididymal origin bind to the sperm plasma membrane.
Human epididymal proteins and sperm function during fertilization: un update
Biological Research, 2001
utilized as a new marker for semen abnormalities. In a recent report (Sutovsky et al, 2001b), a "sperm-ubiquitin tag immunoassay (SUTI)" was described as a valuable new tool for infertility diagnosis and prediction of IVF success in subfertile men diagnosed with idiopathic infertility. CONCLUSIONS This review has briefly shown some findings reported by several investigators on a set of human epididymal proteins identified and partially characterized in the recent years. The results suggest that the epididymis produces and secretes numerous proteins that would associate to the spermatozoa while they are transiting through the organ. These components would dramatically affect sperm functionality, allowing the male gamete to recognize the oocyte. However, much still is waiting to be done to comprehend this phenomenon. Implementation of genomics and proteomics will help, in the near future, to further characterize some already identified proteins, as well as to describe novel epididymal components, anticipating great advances in the elucidation of the sperm maturation process in humans.
Biochemical Characterization of Two Ram Cauda Epididymal Maturation-Dependent Sperm Glycoproteins1
Biology of Reproduction, 2000
Rabbit polyclonal antibodies were raised against ram cauda epididymal sperm proteins solubilized by N-octyl--D-glucopyranoside (anti-CESP) and against proteins of the fluid obtained from the cauda epididymidis (anti-CEF). The anti-CESP polyclonal antibody reacted with several bands from 17 to 111 kDa with different regionalization throughout the epididymis. The strongest epitopes at 17 kDa and 23 kDa were restricted to the cauda epididymidis. The anti-CEF polyclonal antibody reacted mainly with a 17-kDa and a 23-kDa compound in the cauda sperm extract. These cauda epididymal 17-and 23-kDa proteins disappeared after orchidectomy, but they reappeared in the same regions after testosterone supplementation, indicating that they were secreted by the epithelium. The fluid and membrane 17-and 23-kDa antigens had a low isoelectric point and were glycosylated. The fluid 17-and 23-kDa proteins had hydrophobic properties: they were highly enriched in the Triton X-114 detergent phase and could be extracted from the cauda epididymal fluid by a chloroform-methanol mixture. These proteins were further purified, and their N-terminal sequences did not match any protein in current databases. A polyclonal antibody against the fluid 17-kDa protein recognized the protein in the cauda epididymal sperm extract and immunolocalized it on the sperm flagellum membrane and at the luminal border of all cells in the cauda epididymal epithelium. These results indicated that secreted glycoproteins with hydrophobic properties could be directly integrated in a specific domain of the sperm plasma membrane.
Epididymal Secretion of a Mouse Sperm Surface Component Recognized by a Monoclonal Antibody
Biology of Reproduction, 1982
We have generated a monoclonal antibody directed against an antigenic determinant appearing on the surface of mouse sperm tails during passage through the epididymis (a determinant that we now term sperm maturation antigen number four [SMA 41). The present study demonstrates that sperm retained in the ductuli efferentes following ligation do not acquire the antigen, suggesting that its appearance is not due to changes intrinsic to the sperm, but that the epididymal environment is required. To examine the role of the epididymis in the appearance of this antigen, sections of unfixed frozen or fixed, paraffin embedded tissue from different regions of the male reproductive tract have been studied by indirect immunofluorescence. Results indicate that the antigen is a secretory product of the epididymal epithelium, produced in a short segment of the distal caput epididymidis. Ligation experiments show that absence of sperm or testicular fluid from the epididymis does not affect production of this antigen. Examination of prepubertal mice indicates that antigen production is age dependent, production beginning in the epididymis of mice between 2 and 4 weeks of age. Indirect immunofluorescence analysis of sections of a variety of tissues and organs shows that the antigen is restricted to sperm and to epithelial cells of the male reproductive tract. Finally, experiments comparing the antibody-induced agglutinability of sperm from the caput epididymidis to that of sperm from the cauda epididymidis gives further evidence that the antigen resides on the sperm surface.
The Biochemical Characterization of Protein De and Its Interaction with Rat Epididymal Sperm
Using traditional column chromatography, Protein DE has been purified from rat epididymides. Affinity, size exclusion, and ion-exchange chromatography were utilized to purify the protein to homogeneity. Protein DE purity was demonstrated using one and two-dimensional electrophoresis. Using the purified sample, an accurate molecular mass of 27,534 Daltons was determined using electrospray-ionization mass spectrometry. After four chromatographic steps, Protein DE was efficiently separated from all detectable epididymal proteins. This report provides the first rapid and reproducible method for purifying protein DE to homogeneity. Using western blot analysis and immunofluorescence, protein D is initially detected in rat epididymal tissue and associated with sperm from the distal caput region. In contrast, when sperm were recovered from the female reproductive tract seven hours after mating, protein D was not detected by western blot, but did display faint immunofluorescence. Additionall...
Purification and identification of sperm surface proteins and changes during epididymal maturation
PROTEOMICS, 2011
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.