Epididymal functions and their hormonal regulation

The epididymis is a complex organ which maintains a specific intraluminal environment thought to be important for effecting sperm maturation in proximal regions and sperm storage in distal regions of the duct. The composition of the internal milieu is achieved both by transport between blood and lumen (and vice versa) and by synthesis and secretion into the lumen. Several low-molecular weight organic molecules achieve high concentration in the epididymal lumen, but their functions in the events of sperm maturation and storage still remain unclear. Metabolic processes occurring within epididymal tissue and the absorptive and secretory activity of the epididymal epithelium are regulated by androgens. The synthesis of some, but not all, secretory proteins is also androgen-dependent. In addition to androgens, other hormones and local testicular factors may influence epididymal function. There is now increasing evidence that epididymal-specific and androgen-dependent secretory proteins play a fundamental role in modifying the surface characteristics of sperm in preparation for the events of fertilization.     

Association of the protein D and protein E forms of rat CRISP1 with epididymal sperm

Cysteine-rich secretory protein 1 (CRISP1) is a secretory glycoprotein produced by the rat epididymal epithelium in two forms, referred to as proteins D and E. CRISP1 has been implicated in sperm-egg fusion and has been shown to suppress capacitation in rat sperm. Several studies have suggested that CRISP1 associates transiently with the sperm surface, whereas others have shown that at least a portion of CRISP1 persists on the surface. In the present study, we demonstrate that protein D associates transiently with the sperm surface in a concentration-dependent manner, exhibiting saturable binding to both caput and cauda sperm in a concentration range that is consistent with its capacitation-inhibiting activity. In contrast, protein E persists on the sperm surface after all exogenous protein D has been dissociated. Comparison of caput and cauda sperm reveal that protein E becomes bound to the sperm in the cauda epididymidis. We show that protein E associates with caput sperm, which do not normally have it on their surfaces, in vitro in a time- and temperature-dependent manner. These studies demonstrate that most CRISP1 interacts with sperm transiently, possibly with a specific receptor on the sperm surface, consistent with its action in suppressing capacitation during epididymal storage of sperm. These studies also confirm a tightly bound population of protein E that could act in the female tract.     

Immunochemical localization and association with spermatozoa of androgen-regulated proteins of MR 24000 secreted by the mouse epididymis

A polyclonal monospecific immune serum was raised against androgen-regulated proteins with Mr 24000 secreted by the mouse caput epididymidis. Sections of frozen tissues from the different regions of the epididymis have been studied by indirect immuno- fluorescence. Results indicate that the antigens are secretory proteins produced by the epithelial cells of the caput epididymidis, essentially in the medial and distal segments. Accumulation of the antigens was observed in the lumen of the caput and the corpus epididymal duct. Subsequently, their association with the sperm surface occurred and persisted down to the cauda epididymidis.     

Immunochemical localization and association with spermatozoa of androgen-regulated proteins of MR 24000 secreted by the mouse epididymis

A polyclonal monospecific immune serum was raised against androgen-regulated proteins with Mr 24000 secreted by the mouse caput epididymidis (6). Sections of frozen tissues from the different regions of the epididymis have been studied by indirect immuno- fluorescence. Results indicate that the antigens are secretory proteins produced by the epithelial cells of the caput epididymidis, essentially in the medial and distal segments. Accumulation of the antigens was observed in the lumen of the caput and the corpus epididymal duct. Subsequently, their association with the sperm surface occurred and persisted down to the cauda epididymidis.     

Changes in lectin-binding features of ram sperm surfaces associated with epididymal maturation and ejaculation

Ram sperm, isolated from the caput, corpus, and cauda epididymidis, plus ejaculated cells were washed free of loosely bound components and tested for their ability to bind fluorescein-conjugated lectins (Con A, SBA, RCA, PNA, ECA and WGA) as assessed by epiluminescent-fluorescence light microscopy and flow cytometry. Detailed preliminary studies established an appropriate lectin-to-sperm ratio and incubation conditions for quantitative comparisons of sperm cell types and permitted a detailed analysis of both the amount of lectin bound as well as its distribution on the various aspects of the cell surface. Con A (mannose positive) bound weakly over the entire surface, with little change associated with maturation in the male tract. SBA (N-acetylgalactosamine positive) bound moderately strongly to caput sperm, with an emphasis on the apical ridge portion of the cell; during epididymal transit this binding was greatly diminished and was regained upon ejaculation. RCA, PNA, and ECA (galactose positive) gave generally equivalent results, where initially strong binding to the entire sperm surface decreased (over all parts of the surface except the anterior head) during epididymal maturation, with no change associated with ejaculation. WGA (sialic acid positive) binding initially was weak, but increased with epididymal transit and ejaculation. In vitro incubations with beta-galactosidase and neuraminidase confirmed the assignments given above. These data, when coupled with previous reports describing the heterogeneous distribution of proteins and lipids and changes in their distribution associated with epididymal maturation, serve to quantitatively describe changes in those aspects of the cell surface that are probably responsible for the acquisition of the capacity of the sperm to bind successfully to the oocyte.     

Changes in sperm surface membrane and luminal protein fluid content during epididymal transit in the boar

The surface membrane protein of boar sperm and the proteins in the fluid surrounding the gametes were analyzed during epididymal transit. The present study demonstrated that sequential dramatic changes occur in protein composition of the sperm membrane and epididymal fluid during epididymal transit. The maturation process of the boar sperm surface was characterized by a complex sequential evolution of the composition and orientation of macromolecules in the sperm membrane. Epididymal maturation resulted in the progressive disappearance of most of the surface testicular compounds, which were either renewed or masked by new permanent or transient low molecular weight polypeptides on the boar sperm surface membrane. In the fluid surrounding the spermatozoa, composition of the luminal proteins was altered throughout the epididymal transit and several new compounds were characterized. Very few proteins were correlated either with blood plasma or sperm surface compounds.     

Maturational changes of the CD52-like epididymal glycoprotein on cynomolgus monkey sperm and their apparent reversal in capacitation conditions

A major epididymal secretory protein in men has a colinear cDNA sequence with lymphocyte CD52, a sialylated glycoprotein. Immunostaining and flow cytometric detection of cynomolgus monkey sperm CD52 during epididymal maturation showed increases from 20 to 85% stained sperm from the caput to the corpus with staining intensities doubled. Freshly prepared cauda sperm showed only 10% staining while they markedly increased in percentage and intensity of staining upon incubation at 37 degrees C under capacitating conditions, but not at 4 degrees C. Western blotting of proteins from fresh cauda sperm revealed no less antigen than corpus sperm. Staining of ejaculated sperm exhibited similar increases during incubation. Further washing with a high salt medium before staining to remove any electrostatically-bound molecules masking the antigen showed no effect. Incubation-induced increases in antigen binding were accelerated by the addition of neuraminidase (0.25 and 0.5 U/ml), but not affected by the sialyl residue-rich fetuin (5 mg/ml) competing for any endogenous neuraminidase. There were no concomitant decreases in the staining of sialic acid residues during capacitation-incubation. These findings suggest a cryptic antigen epitope site as a consequence of sperm maturation and subsequent re-exposure under capacitation conditions, but not due to the removal of sialic acid residues by endogenous neuraminidase. Involvement of endogenous proteases was also ruled out, as incubation in the presence of protease inhibitors did not hinder the increases but resulted in a dose-dependent enhancement in staining, suggesting some protease-sensitive unmasking process. In conclusion, the monkey epididymal secreted CD52 on sperm underwent changes in antigenic characteristics during sperm maturation which were reversed under capacitation conditions.     

Effects of caput ligation on rat sperm and epididymis: protein thiols and fertilizing ability.

Mammalian spermatozoa mature while passing through the epididymis. Maturation is accompanied by thiol oxidation to disulfides. In rats, sperm become motile and fertile in the cauda. We have previously demonstrated that rat caput sperm contain mostly thiols and that upon passage from the corpus to the cauda epididymidis, sperm protein thiols are oxidized. The present work was undertaken to study the role of the regions of the epididymis in sperm maturation as reflected in the thiol status, fertility, and motility of the spermatozoa. The distal caput epididymidis of mature albino rats was ligated on one side. After 5 days, sperm were isolated from the ligated caput and from caput and cauda of the control side. Thiol groups in sperm, epididymal luminal fluid (EF), and epididymal tissue were labeled using the fluorescent thiol-labeling agent monobromobimane. After ligation, changes were observed in a) sperm proteins, sperm nuclear proteins, and epididymal fluid by electrophoresis; b) epididymal tissues by histochemistry; c) progressive motility by phase microscopy; and d) fertilizing ability after insemination into uteri of immature females. We found that after ligation, caput sperm thiols, especially protamine thiols, are oxidized, rendering them similar to mature sperm isolated from the cauda epididymidis. Spermatozoa from ligated caput epididymidis gain progressive motility and partial fertilizing ability. Morphology of epithelial cells of ligated caput is similar to that of cauda cells. However, other changes in caput EF and epithelium induced by ligation render the ligated caput epididymidis different from either control caput or cauda. Hence, sperm thiol oxidation, along with the development of fertilizing ability, can occur in sperm without necessity for sperm transit through the corpus and cauda epididymidis.     

Molecular and functional characterization of the rabbit epididymal secretory protein 52, REP52

As part of a systematic study of rabbit epididymal proteins involved in sperm maturation, we have identified and characterized a novel glycoprotein (rabbit epididymal secretory protein 52 [REP52]) of 52 kDa. REP52 is synthesized and secreted in a tissue-specific manner by the mid (region 6) and distal (region 7) corpus epididymidis and associates weakly with the sperm surface overlying the principal piece of the tail. Sequencing of cloned REP52 cDNA demonstrated that this protein represents a novel member of the highly conserved fibronectin type II (FN2) module protein family. The protein appears related but not homologous to ungulate seminal plasma proteins and is the first known example to be identified as a rabbit epididymal secretory protein. Consistent with other members of this protein family, REP52 possessed a high level of sequence identity within the FN2 module-encoding domains, but a highly variable N-terminal sequence that failed to show significant homology with published sequences. By analogy with evidence from studies of the ungulate seminal plasma proteins it is hypothesized that the tandemly arranged FN2 modules could facilitate the association of REP52 with sperm phosphatidylcholine residues on the outer leaflet of the sperm tail. It is also considered likely that these domains represent key elements for the function of this novel protein, a conclusion supported by the fact that antisera raised against the REP52 protein blocked in vitro fertilization in a concentration-dependent fashion.     

Epididymal cell secretory activities and the role of proteins in boar sperm maturation

The final stages of sperm differentiation occur outside the gonad, in the epididymal tubule. These last maturation steps, essential to the quality of spermatozoa, are not under the genomic control of the germ cells. A series of sequential interactions with the epididymal fluid, mostly specific proteins present in the lumen of different regions, are believed to induce the final steps of sperm maturation. In order to provide the luminal changes required for this maturation to occur, the epithelium may resort to two basic mechanisms: absorption and secretion. Far from being a uniform channel, the epididymal duct is a canal with highly specialized regional differentiation of its epithelial ultrastructure and its secretory and absorptive functions. This review focuses on the ultrastructural characteristic of the epithelial cells, their specific secretory activity according to the epididymal regions and their eventual role in sperm maturation of the boar. The chronology of the changes that occur in and on the sperm and in the surrounding environment are described. Relationships between the highly regionalized epididymal activities, sperm characteristics linked to their survival and fertility potential are also presented in this review.     

Mouse cysteine-rich secretory protein 4 (CRISP4): a member of the Crisp family exclusively expressed in the epididymis in an androgen-dependent manner

The final maturation of spermatozoa produced in the testis takes place during their passage through the epididymis. In this process, the proteins secreted into the epididymal lumen along with changes in the pH and salt composition of the epididymal fluid cause several biochemical changes and remodeling of the sperm plasma membrane. The Crisp family is a group of cysteine-rich secretory proteins that previously consisted of three members, one of which-CRISP1-is an epididymal protein shown to attach to the sperm surface in the epididymal lumen and to inhibit gamete membrane fusion. In the present paper, we introduce a new member of the Crisp protein family, CRISP4. The new gene was discovered through in silico analysis of the epididymal expressed sequence tag library deposited in the UniGene database. The peptide sequence of CRISP4 has a signal sequence suggesting that it is secreted into the epididymal lumen and might thus interact with sperm. Unlike the other members of the family, Crisp4 is located on chromosome 1 in a cluster of genes encoding for cysteine-rich proteins. Crisp4 is expressed in the mouse exclusively in epithelial cells of the epididymis in an androgen-dependent manner, and the expression of the gene starts at puberty along with the onset of sperm maturation. The identified murine CRISP4 peptide has high homology with human CRISP1, and the homology is higher than that between murine and human CRISP1, suggesting that CRISP4 represents the mouse counterpart of human CRISP1 and could have similar effects on sperm membrane as mouse and human CRISP1.     

Changes in plasma membrane glycoproteins of rat spermatozoa during maturation in the epididymis

Glycoproteins on the plasma membrane of testicular and cauda epididymidal spermatozoa have been labeled with galactose oxidase/NaB [3H]4 and sodium metaperiodate/NaB[3H]4, followed by analysis on SDS polyacrylamide gels. The major glycoprotein labeling on testicular spermatozoa has a molecular weight 110,000 whereas on cauda epididymidal spermatozoa greater than 90% of the radio-label is incorporated into proteins of molecular weight 32,000. These 32,000-mol wt X proteins are homologous with proteins of similar molecular weight purified from the epididymal secretion and which have been shown previously to be synthesized in the caput epididymidis under hormonal control. Immunofluorescence revealed that the 32,000-mol wt proteins are present on the flagellum of mature but not immature spermatozoa and that they have a patchy distribution suggesting that they are mobile within the plane of the membrane. The membrane-bound 32,000-mol wt proteins possess hydrophobic domains as revealed by charge-shift electrophoresis and they also label with a lipophilic photoaffinity probe suggesting that they are in contact with the lipid bilayer. The evidence indicates that there is a considerable reorganization of the molecular structure of the plasma membrane of spermatozoa during maturation in the epididymis and that some of the changes are brought about by a direct interaction with epididymal secretory proteins.     

Évolution de la composante lipidique de la membrane plasmique des spermatozoïdes durant la maturation épididymaire

One aspect of mammalian post-testicular sperm maturation is the progressive change in their plasma membrane lipid composition. These modifications in lipids allow sperm cells to fuse with oocytes during fertilization. A significant share of these sperm lipid changes occurs during their descent through the epididymal tubule. It then continues within the female genital tract during the capacitation process, an essential prerequisite for acrosomic reaction and hence fertilization. This review presents what is known concerning the sperm plasma membrane lipid changes during epididymal maturation in various mammalian models. In the first section, after a brief presentation of the classic eukaryotic cell plasma membrane lipid organization, the emphasis is on the particularities of sperm plasma membrane lipids. The second section presents the different changes occurring in the three major classes of lipids (i.e. phospholipids, sterols and fatty acids) during the sperm’s epididymal descent. The final section briefly describes the mechanisms by which these lipid changes might happen in the epididymal lumen environment. The role played by lipid-rich vesicles secreted by the epididymal epithelium via apocrine secretory processes is highlighted.     

Localization of epididymal secretory proteins on rat spermatozoa

Spermatozoa from the testis and cauda epididymidis of the rat were surface labelled with radioactive iodide. Detergent extracts of radioiodinated spermatozoa immunoprecipitated with antisera against specific epididymal proteins, followed by polyacrylamide gel electrophoresis, revealed two proteins (D and E of Mr 27 000 and 28 000, respectively) which became associated with spermatozoa during epididymal transit. These proteins were observed by immunofluorescence microscopy to be located over a restricted area of the head surface. Proteins with similar molecular weight were labelled on spermatozoa from the cauda epididymidis, but not from the testis, by reaction with sodium boro[3H]hydride in the presence of galactose oxidase. However, failure to immunoprecipitate with antibodies to Proteins D and E and non-coincident migration on two-dimensional gel electrophoresis established the non-identity of these proteins. Compared with Proteins D and E, two other major epididymal secretory proteins (Proteins B and C of Mr 16 000) associated with spermatozoa to a relatively minor extent during epididymal transit.     

Comparative analysis of human reproductive proteomes identifies candidate proteins of sperm maturation

Male reproductive proteomes provide basis for studying gene products and its involvement or regulation in sperm physiology. Here, a comparative study between these proteomes was performed to find potential proteins and functions associated with human sperm maturation. Seven reproductive proteomes associated with human sperm physiology were integrated. Gene ontology analysis were performed using DAVID and Panther tools to determine enriched functions. Total of 270 proteins overlapped between epididymal, prostatic milieu and sperm proteome were thought to be candidate proteins involved in sperm maturation, and they showed enriched functions of proteasomal protein catabolic process and protein folding. 34 epididymal milieu proteins and 274 prostatic milieu proteins were contributed to the composition of seminal fluids proteome. Literatures have confirmed the involvements in sperm maturation of many of these proteins The spatial expressions of 24 epididymal milieu proteins involved in chaperone and antioxidant activity were authenticated by real-time RT-PCR. These proteins may serve as candidate molecules for future studies of sperm maturation and male infertility.     

Glycoproteins: a variable factor in surface transformation of ram spermatozoa during epididymal transit

To determine sequential surface glycoprotein changes in ram spermatozoa during epididymal maturation, labeling procedures were used that were specific for galactosyl, galactosaminyl, and sialyl residues. Spermatozoa and fluids were collected from the rete testis through surgically inserted catheters or flushed from the lumen of selected regions of the epididymis: i.e., caput, proximal and distal corpus, and cauda epididymidis. Ejaculated spermatozoa were collected by electrical stimulation. Electrophorectic analysis of galactose (GAO)-sodium boro[3H]hydride (NaB3H4)-treated spermatozoa revealed a sharp overall decrease in carbohydrate residue labeling during sperm transport through the efferent ducts and caput epididymidis, whereas several high molecular weight components in the 600K to 250K zone persisted throughout epididymal transit. Preincubation of spermatozoa with neuraminidase (NEUA) exposed galactose residues that had not been labeled with GAO alone (i.e., 97K, 43K, 24K) in both cauda epididymal and ejaculated spermatozoa. Treatment with sodium metaperiodate-NaB3H4 labeled many of the surface components displayed by NEUA-GAO-treated spermatozoa and revealed an overall shift in sialyl residue labeling from high molecular weight components in immature testicular spermatozoa to low molecular weight components in mature cells. The labeling procedures applied allowed only a qualitative interpretation of the results and they presumably represent the minimum possible changes. Nonetheless, our results demonstrate that glycoproteins are a major factor in surface transformations of ram spermatozoa in the epididymis, especially during the initial stages of maturation.     

Biochemical and binding characteristics of boar epididymal fluid proteins

During the passage through the epididymis, testicular spermatozoa are directly exposed to epididymal fluid and undergo maturation. Proteins and glycoproteins of epididymal fluid may be adsorbed on the sperm surface and participate in the sperm maturation process, potentially in sperm capacitation, gamete recognition, binding and fusion. In present study, we separated proteins from boar epididymal fluid and tested their binding abilities. Boar epididymal fluid proteins were separated by size exclusion chromatography and by high-performance liquid chromatography with reverse phase (RP HPLC). The protein fractions were characterized by SDS-electrophoresis and the electrophoretic separated proteins after transfer to nitrocellulose membranes were tested for the interaction with biotin-labeled ligands: glycoproteins of zona pellucida (ZP), hyaluronic acid and heparin. Simultaneously, changes in the interaction of epididymal spermatozoa with biotin-labeled ligands after pre-incubation with epididymal fluid fractions were studied on microtiter plates by the ELBA (enzyme-linked binding assay) test. The affinity of some low-molecular-mass epididymal proteins (12-17 kDa and 23 kDa) to heparin and hyaluronic acid suggests their binding ability to oviductal proteoglycans of the porcine oviduct and a possible role during sperm capacitation. Epididymal proteins of 12-18 kDa interacted with ZP glycoproteins. One of them was identified as Crisp3-like protein. The method using microtiter plates showed the ability of epididymal fluid fractions to change the interaction of the epididymal sperm surface with biotin-labeled ligands (ZP glycoproteins, hyaluronic acid and heparin). These findings indicate that some epididymal fluid proteins are bound to the sperm surface during epididymal maturation and might play a role in the sperm capacitation or the sperm-zona pellucida binding.