Sperm ubiquitination in epididymal feline semen

Ubiquitin is a 8.5-kDa peptide that tags other proteins for proteasomal degradation. It has been proposed that ubiquitination might be responsible for the elimination of defective spermatozoa during transit through the epididymis in humans and cattle, but its exact biological function in seminal plasma has not yet been clarified. In the domestic cat (Felis catus), the percentage of immature, unviable, and abnormal spermatozoa decreases during the epididymal transit, indicating the existence of a mechanism that removes defective spermatozoa. Magnetic cell separation techniques, based on the use of magnetic beads coated with anti-ubiquitin antibodies, may allow the selective capture of ubiquitinated spermatozoa from semen, thus contributing to the identification of a potential correlation between semen quality and ubiquitination process. Moreover, the selective identification of all the ubiquitinated proteins in different epididymal regions could give a better understanding of the ubiquitin role in feline sperm maturation. The aims of this study were as follows: (1) to verify the possibility of separating ubiquitinated spermatozoa with magnetic ubiquitin beads and identify the morphological and acrosomal differences between whole sample and unbound gametes, (2) to characterize all the ubiquitinated proteins in spermatozoa retrieved in the three epididymal regions by a proteomic approach. The data indicated the presence of ubiquitinated proteins in cat epididymal semen. However, a correlation between abnormal and ubiquitinated spermatozoa has not been found, and ubiquitin cannot be considered as a biomarker of quality of epididymal feline spermatozoa. To the author's knowledge, this is the first identification of all the ubiquitinated proteins of cat spermatozoa collected from different epididymal regions. The proteomic pattern allows a further characterization of cat epididymal semen and represents a contribute to a better understanding of the ubiquitin role in feline sperm maturation.     

Le transit epididymo-deferentiel

The epididymis and vas deferens constitute not only a simple conduit for sperm transport but also play an important physiological role in the development of sperm fertilizing ability. The epithelial compartment plays a major functional role in determining the biochemical composition of the luminal fluid in which the spermatozoa undergo a series of structural, biochemical and metabolic changes. During epididymal transit spermatozoa acquire their capacity for motility and also their ability to attach and bind to the zona pellucida and fertilize the oocyte. In man, sperm maturation may occur in the extreme proximal region of the epididymis. The regulation of epididymal and vasa deferential function, as well as sperm maturation, are under androgenic control     

Creatine phosphokinase in domestic cat epididymal spermatozoa

Mammalian spermatozoa that have not completed final testicular sperm maturation have residual cytoplasm and increased creatine phosphokinase (CK) content. This study determined: (1) if CK could be detected by immunostaining cat spermatozoa from the caput, corpus, and cauda epididymis, (2) fluctuations in the proportions of spermatozoa with mature or immature CK-staining patterns during epididymal sperm transit, and (3) how well sperm maturity (as determined by a CK marker) correlated with testicular or epididymal dysfunctions associated with morphological sperm abnormalities. One epididymis was collected from each of 37 cats after orchiectomy and processed immediately to allow sperm morphology evaluations on a 'regional' basis. Sperm released from the contralateral epididymis were evaluated for motility, sperm membrane integrity, and immunostaining with CK-B antibodies. Proportions of spermatozoa with malformed or detached heads, proximal droplets and acrosomal or midpiece abnormalities decreased (P < 0.05) from the caput to the cauda epididymis. In contrast, proportions of spermatozoa that were motile, membrane-intact or with flagellar abnormalities or distal droplets increased (P < 0.05) from the caput to cauda region. Percentages of spermatozoa with an immature CK-staining pattern also decreased (P < 0.05) with epididymal transit (which differs from that reported for the human and stallion). There was no correlation (P > 0.05) between sperm morphology and the CK-staining patterns. In summary, the results reveal that some specific sperm malformations in the domestic cat are of testicular origin, whereas others develop during epididymal transit.     

Development changes occurring in the lipids of ram epididymal spermatozoa plasma membrane

Ram spermatozoa were obtained from different regions (caput, corpus, and cauda) of the epididymis and their plasma membrane was removed using a nitrogen cavitation treatment (750 psi, 10 min equilibration at 4 degrees C). Membrane was recovered after sucrose gradient centrifugation and identified using 125I-succinylated concanavalin A (125I-succConA) as a surface marker. Based on fluorescein isothiocyanate-succConA (FITC-succConA) labeling and electron microscopy, cavitation removed plasma membrane from the anterior sperm head in the area overlying the acrosome. Cholesterol was the major sterol in plasma membrane, with desmosterol present in sperm entering the epididymis (caput sperm) but negligible in sperm after epididymal transit (cauda sperm). Ethanolamine and choline phosphoglycerides represented 70-80% of membrane phospholipids, with the ethanolamine fraction decreasing relative to choline phosphoglycerides during epididymal transit. The molar ratio of cholesterol to phospholipid increased in the plasma membrane during maturation. The bulk phospholipid-bound fatty acids consisted primarily of palmitoyl acyl groups (16:0) in caput sperm and docosahexaenoyl acyl groups (22:6) in cauda sperm. The choline phosphoglyceride fraction was purified and analyzed. It consisted of a mixture of ether acyl glycero-3-phosphocholine and diacyl phosphoglyceride, with the dominant acyl residue, at all stages of epididymal maturation, being 22:6 throughout epididymal transit. The significance of these findings relative to acquisition of fertilization capacity by sperm during epididymal maturation is discussed.     

Characteristics of donkey spermatozoa along the length of the epididymis

In mammals, the epididymis has numerous interrelated functions including absorptive and secretory activity that affect luminal environment and cell membrane, and the maturation and storage of sperm. Spermatozoa acquire their motility and fertilizing ability during their passage through the epididymis and the motility of epididymal spermatozoa should be a balance between the maturation of flagellum and the inhibition of the flagellar machinery. In this study maturational change in sperm characteristics were evaluated in the epididymis of donkey. Spermatozoa collected from four portions of the epididymis (head, cranial corpus, caudal corpus, tail) were compared before and after ejaculation for viability, mitochondrial activity, kinetic parameters, and morphology. A significant increase in the mitochondrial activity along the epididymis was reported, suggesting a possible involvement in the motion mechanism. This should be corroborated by the significant correlation between mitochondrial activity and the total and progressive motility and the increase in velocities of spermatozoa recorded by computer-assisted sperm analysis. The percentage of most of the abnormal spermatozoa were similar in all tracts, with a great variability between jackasses. Only the bent midpiece percentage decreased significantly along epididymis. A significant increase in the percentage of distal cytoplasmic droplets (DCD), and a simultaneous decrease in the proximal cytoplasmic droplets (PCD), was found. The DCD fell down after ejaculation suggesting the late loss of the cytoplasmic residual (DCD) in the donkey, as hypothesized in the stallion. Because the prevalence of PCD were similar in both tail epididymal and ejaculated spermatozoa, a defect of the maturative process in the PCD sperm should be speculated.     

Sperm malformations throughout the boar epididymal duct

Taking into account the importance of the sperm epididymal maturation process, and the consequential changes in the spermatozoa, we studied eight different sperm malformations in the caput, corpus, and cauda regions of the epididymis of healthy and sexually mature Landrace boars in order to determine the origin of these sperm abnormalities. Epididymal sperm characteristics were examined using light microscopy, scanning and transmission electron microscopy. The incidence of each type of malformation investigated was established after counts of 10 000 spermatozoa in each of the three epididymal regions. The different sperm malformations studied were: (1) spermatozoa with tail folded at the connecting piece; (2) spermatozoa with tail folded at the midpiece; (3) spermatozoa with tail folded at the Jensen's ring; (4) spermatozoa with tail folded at the principal piece; (5) coiled tail spermatozoa; (6) spermatozoa with two fused tails; (7) macrocephaly; and (8) microcephaly. The count performed in each epididymal region indicated that, whereas significant differences (P ≤ 0.01) existed between the frequencies of some types of sperm malformations and the epididymal region from where the sperm originate, other sperm malformations were more uniformly distributed along the epididymal duct. Among the eight different sperm malformations studied, three were found to be of secondary origin: spermatozoa with tail folded at the Jensen's ring (originated in the epididymal cauda); spermatozoa with coiled tail; and spermatozoa with two fused tails (originated in the epididymal corpus). Knowing the origin of spermatozoa abnormalities will assist research into the study of infertility and reproductive pathology.     

Impact of epididymal maturation on the tyrosine phosphorylation patterns exhibited by rat spermatozoa

As mammalian spermatozoa migrate through the epididymis, they acquire functionality characterized by the potential to express coordinated movement and the competence to undergo capacitation. The mechanisms by which spermatozoa gain the ability to capacitate during epididymal transit are poorly understood. The purpose of this study was to investigate the impact of epididymal maturation on the signal transduction pathways regulating tyrosine phosphorylation, because this process is thought to be central to the attainment of a capacitated state and expression of hyperactivated motility. Western blot and immunocytochemical analyses demonstrated that epididymal maturation in vivo is associated with a progressive loss of phosphotyrosine residues from the sperm head. As cells pass from the caput to the cauda epididymis, tyrosine phosphorylation becomes confined to a narrow band at the posterior margin of the acrosomal vesicle. Epididymal maturation of rat spermatozoa was also associated with an acquired competence to respond to high levels of intracellular cAMP by phosphorylating tyrosine residues on the sperm tail. Immature caput spermatozoa were incapable of exhibiting this response, despite the apparent availability of cAMP and protein kinase A. These findings help to clarify the biochemical changes associated with the functional maturation of spermatozoa during epididymal transit.     

Morphology and motility of spermatozoa from different regions of the epididymal duct in the domestic cat

Spermatozoa undergo important maturational changes as they pass through the epididymal duct. Some domestic cats and many species of wild felids have high proportions of abnormal spermatozoa in their ejaculates. The epididymis has been shown to be able to remove certain abnormal sperm forms in some species while other sperm abnormalities originate in the epididymis. So far, it has not been shown how the epididymis affects sperm morphology in the domestic cat. Therefore, motility and sperm morphology were studied in spermatozoa from the efferent ducts and from the 6 regions of the epididymal duct. There were significant decreases in the proportions of spermatozoa with abnormalities of the sperm head, acrosomal defects, acrosomal abnormalities and in the proportion of midpiece abnormalities. In contrast, there was a small but significant increase in the proportion of spermatozoa with abnormalities of the tail. Spermatozoa acquired the capacity for motility in Region 4, where the cytoplasmic droplet also moved from a proximal to a distal position, indicating that important maturational changes take place in this region. The results of this study demonstrate that the proportions of sperm abnormalities originating in the testes decrease during epididymal transport, while some sperm tail abnormalities may actually originate in the epididymis.     

Tyrosine phosphorylation as evidence of epididymal cauda participation in the sperm maturation process of Corynorhinus mexicanus bat

The bat Corynorhinus mexicanus provides an interesting experimental model for the study of epididymal sperm maturation because after spermatogenesis and the regression of the testes, this bat stores sperm in the epididymal cauda for several months. Earlier research conducted by our group suggested that sperm maturation in this species must be completed in the caudal region of the epididymis. One of the major signal transduction events during sperm maturation is the tyrosine phosphorylation of sperm proteins. The aim of the present study was to comparatively evaluate tyrosine phosphorylation in spermatozoa obtained from the caput, corpus and cauda of the epididymis during the sperm storage period. The maturation status of the sperm was determined by the percentage of capacitation and tyrosine phosphorylation in sperm obtained from the epididymis. The highest proportion of tyrosine phosphorylation was registered after the sperm had reached the cauda epididymis during the middle of the storage period. In conclusion, in Corynorhinus mexicanus and most likely in other chiropteran species with an asynchronous male reproductive pattern, epididymal sperm maturation ends in the caudal region of the epididymis and is related to the time that the sperm remains in the epididymis before mating activity.     

Characterization of fibronectin as a marker for human epididymal sperm maturation.

Fertilization involves adhesive interactions between gametes similar to those mediated by fibronectin (FN) in other cellular systems. Fibronectin has been found on the equatorial segment of ejaculated human serum. As sperm capacity to interact with the oocyte is acquired during epididymal transit, the possible participation of FN in human sperm maturation was studied. The presence of FN in both epididymal sperm and fluid was demonstrated by the detection of a major component of 220 kD in immunoblot studies using anti-FN antisera. The concentration of FN in soluble tissue extracts of epididymis was determined by enzyme-linked immunosorbent assay (ELISA). A gradual increase along the length of the organ, averaging 12-fold from proximal caput to distal corpus, was detected. Immunocytochemistry assays indicated that the number of spermatozoa with immunoreactive FN over the equatorial segment increased from 18% in caput to 64% in distal corpus epididymis. Immunoprecipitation of medium from epididymal explants culture with anti-FN antiserum demonstrated the de novo synthesis of FN in vitro. The greater number of FN-positive sperm coincident with FN accumulation in distal regions of the epididymis supports the role of FN in sperm maturation.     

Maturation of spermatozoa in the epididymis of the Chinese hamster

Chinese hamster spermatozoa gain their ability to move when they descend from the testis to the distal part of the caput epididymis, but it is not until they enter the corpus epididymis that they become capable of fertilizing eggs. The maturation of the spermatozoa proceeds as they further descend the tract and perhaps continues even in the vas deferens. During transit between the distal caput and proximal cauda epididymides, small membrane-limited vesicles (and tubules) appear on the plasma membrane over the acrosomes of the spermatozoa. The number of vesicles appearing on the sperm brane reaches a maximum when the spermatozoa are in the proximal cauda epididymis. It declines sharply in the distal cauda epididymis. Spermatozoa in the vas deferens are free of the vesicles. The origin, chemical nature, and functional role of the vesicles that appear on the sperm surface during epididymal transit must be the subject of further investigation.     

Identification of a hamster epididymal region-specific secretory glycoprotein that binds nonviable spermatozoa

Even though the epididymis produces an environment promoting sperm maturation and viability, some sperm do not survive transit through the epididymal tubule. Mechanisms that segregate the epididymal epithelium and/or the viable sperm population from degenerating spermatozoa are poorly understood. We report here the identification and characterization of HEP64, a 64-kDa glycoprotein secreted by principal cells of the corpus and proximal cauda epididymidis of the hamster that specifically binds to and coats dead/dying spermatozoa. The HEP64 monomer contains approximately 12 kDa carbohydrate and, following chemical deglycosylation, migrates as a approximately 52-kDa polypeptide. Both soluble (luminal fluid) and sperm-associated HEP64 are assembled into disulfide-linked high molecular weight oligomers that migrate as a doublet band of 260/280 kDa by nonreducing SDS-PAGE. In the epididymal lumen, HEP64 is concentrated into focal accumulations containing aggregates of structurally abnormal or degenerating spermatozoa, and examination of sperm suspensions reveals that HEP64 forms a shroudlike coating surrounding abnormal spermatozoa. The HEP64 glycoprotein firmly binds degenerating spermatozoa and is not released by either nonionic detergent or high salt extraction. Electron microscopic immunocytochemistry demonstrates that HEP64 localized to an amorphous coating surrounding the abnormal spermatozoa. The potential mechanisms by which this epididymal secretory protein binds dead spermatozoa as well as its possible functions in the sperm storage function of the cauda epididymidis are discussed.     

Differences between epididymal and ejaculated sperm characteristics in donkey

Spermatozoa acquire their motility and fertilizing ability during their passage through the epididymal canal. In the epididymal caput and corpus spermatozoa undergo several biochemical and metabolic changes while the cauda of the epididymis should be considered as the primarily site for storage of the spermatozoa. In the horse spermatozoa from cauda epididymis were collected and frozen, and the fertility of semen assessed. However, no studies have detailed semen characteristics of spermatozoa collected from the cauda epididymis in the jackass. In this study sperm characteristics of spermatozoa in the cauda epididymis of the donkey was reported and a comparison with ejaculated spermatozoal characteristics was performed. Samples from 10 Martina Franca jackasses were collected and analyzed for viability (Propidium iodide/Sybr-14? fluorescent stain), mitochondrial activity (Mitotraker? fluorescent stain), objective motility characteristics (by Computer Assisted Sperm Analyzer - CASA) and morphology. A higher viability and mitochondrial activity in the cauda epididymis samples were reported in this paper. Samples reported in this paper were identified and the percentage of total and progressive spermatozoa was comparable, but trajectories were more rapid (higher VCL) with less progressiveness (higher ALH and lower STR and LIN) in the cauda epididymis. Sperm morphology showed a pronounced variability between jackasses, with comparable values for all morphological subclasses. In this study the loss of the distal cytoplasmic droplets happen close to or after ejaculation because the percentage fell to nearly 0% after ejaculation. As suggested for bulls, the presence of a similar percentage in sperm with proximal cytoplasmic droplet in epididymal and ejaculated semen is likely to indicate a failure in the maturation process.     

Lectin-binding sites on the plasma membranes of rabbit spermatozoa: Changes in surface receptors during epididymal maturation and after ejaculation

Modifications in rabbit sperm plasma membranes during epididymal passage and after ejaculation were investigated by used of three lectins: concanavalin A (Con A); Ricinus communis I (RCA(I)); and wheat germ agglutinin (WGA). During sperm passage from caput to cauda epididymis, agglutination by WGA drastically decreased, and agglutination by RCA(I) slightly decreased, although agglutination by Con A remained approximately unchanged. After ejaculation, spermatozoa were agglutinated to a similar degree or slightly less by Con A, WGA, and RCA(I), compared to cauda epididymal spermatozoa. Ultrastructural examination of sperm lectin-binding sites with ferritin- lectin conjugates revealed differences in the densities of lectin receptors in various sperm regions, and changes in the same regions during epididymal passage and after ejaculation. Ferritin-RCA(I) showed abrupt changes in lectin site densities between acrosomal and postacrosomal regions of sperm heads. The relative amounts of ferritin-RCA(I) bound to heads of caput epididymal or ejaculated spermatozoa. Tail regions were labeled by ferritin RCA(I) almost equally on caput and cauda epididymal spermatozoa, but the middle-piece region of ejaculated spermatozoa was slightly more densely labeled than the principal-piece region, and these two regions on ejaculated spermatozoa were labeled less than on caput and cuada epididymal spermatozoa. Ferritin-WGA densely labeled the acrosomal region of caput epididymal spermatozoa, although labeling of cauda epidiymal spermatozoa was relatively sparse except in the apical area of the acrosomal region. Ejaculated spermatozoa bound only a few molecules of ferritin-WGA, even at the highest conjugate concentrations used. Caput epididymal, but not cauda epididymal or ejaculated spermatozoa, bound ferritin-WGA in the tail regions. Dramatic differences in labeling densities during epididymal passage and after ejaculation were not found with ferritin-Con A.     

Changes in distribution of phosphomannosyl receptors during maturation of rat spermatozoa

The aim of the present work was to study the distribution of the cation-independent (CI) and cation-dependent (CD) mannose-6-phosphate receptors (MPRs) in spermatozoa obtained from either rete testis or three regions of rat epididymis. We observed that both receptors underwent changes in distribution as spermatozoa passed from rete testis to cauda epididymis. CI-MPR was concentrated in the dorsal region of the head in rete testis sperm and that this labeling extended to the equatorial segment of epididymal spermatozoa. CD-MPR, however, changed from a dorsal distribution in rete testis, caput, and corpus to a double labeling on the dorsal and ventral regions in cauda spermatozoa. The percentages of spermatozoa that showed staining for either CI-MPR or CD-MPR increased from rete testis to epididymis. The observed changes were probably the result of a redistribution during transit rather than an unmasking of receptors. The fluorescence corresponding to CD-MPR and CI-MPR on the dorsal region disappeared when caudal spermatozoa underwent the acrosomal reaction. Receptors were localized on the plasmalemma of spermatozoa, as observed by immunoelectron microscopy. Changes in distribution may be related to a maturation process, which suggests new roles for the phosphomannosyl receptors.     

Human sperm proteins from testicular and epididymal origin that participate in fertilization: modulation of sperm binding to zona-free hamster oocytes, using monoclonal antibodies.

In order to identify human sperm surface proteins involved in the gamete recognition process, mouse monoclonal antibodies were directed against human spermatozoa and screened with live spermatozoa by enzyme-linked immunosorbent assay (ELISA). Immunoperoxidase staining of human testis showed the early presence of four corresponding proteins on germinal cells, while six were detected primarily in testis fluid. The presence of 17 proteins was evidenced in the epididymis. Eight were detected with a decreasing gradient from the beginning to the end of the organ, including vasa efferentia for three of them. The other nine were observed in only one defined segment, usually the caput epididymis, which was found to be the most active region. Comparison of spermatozoa patterns from testis, vasa efferentia, and the three regions of epididymis pointed out a progressive coating. By contrast, three antibodies displayed a migration of spermatozoa surface domains in the course of epididymal transit. Six antibodies were found to inhibit human spermatozoa adherence to zona-free hamster oocytes, while nine promoted it. Molecular weights of antigens corresponding to nine of the antibodies ranged from 11 to 215 kDa. No correlation could be established with previously described human proteins. These observations emphasize the role of epididymis in human sperm maturation.     

Immunodissection of sperm surface modifications during epididymal maturation

Mammalian spermatozoa undergo changes in morphology, composition, and function during transit through the epididymis. These changes correlate with acquisition by sperm of the ability to fertilize ova. It has been found that sperm from the cauda epididymidis, but not those from the caput epididymidis, are able to bind to the zona pellucida. This would imply a modification in sperm surface characteristics. Biochemical and immunological studies have demonstrated changes in sperm surface composition during epididymal maturation. These changes involve addition of epididymal secretory products to the sperm surface, loss or alteration of existing sperm surface molecules, and possibly the unmasking of preexisting molecules or epitopes. Several laboratories have studied the epididymal secretory proteins in the rat, but a consensus has not been reached on the identification, characterization, source, and sperm surface association of these proteins. Monoclonal antibodies are beginning to be used to characterize sperm surface components and sperm maturation antigens. They are proving to be valuable tools for the dissection of epididymal maturation when used in conjunction with biochemical and physiological approaches.     

Intra-acrosomal 155,000 dalton protein increases the antigenicity during mouse sperm maturation in the epididymis: A study using a monoclonal antibody MC101

We found an intra-acrosomal antigen of about 155,000 daltons (155 kDa) in a survey using the monoclonal antibody MC101 raised against mouse cauda epididymal spermatozoa. Morphological studies by means of indirect immunofluorescence and immunogold electron microscopy localized the antigen to the cortex region of the anterior acrosome. Avidin biotin complex immunocytochemistry initially demonstrated a faint signal at the anterior acrosome in the testis spermatozoa that increased in intensity as the sperm moved toward the distal epididymis. This incremental immunoreactivity was also confirmed by immunoblotting following one-dimensional SDS-PAGE. The 155 kDa protein band was immunostained, and it was much more intense in the cauda epididymal than in the caput and corpus epididymal spermatozoa. Only a trace or no immunostain was evident in the caput or testis spermatozoa. The antigen localization did not change during passage through the epididymis, being confined at the cortex region of the anterior acrosome. The epididymal epithelial cells were not immunostained. These findings suggested that the 155 kDa protein is biochemically modified, further implying that the biochemical alteration of intra-acrosomal material is involved in sperm maturation in the epididymis. © 1995 wiley-Liss, Inc.     

Epididymis as a target for contraception

Advantage of using a vaccine based on sperm antigens is that it can be used both in males and females as individuals who have antisperm antibodies are usually infertile but otherwise healthy. Several sperm specific antigens identified as prospective candidates for immunocontraception are of testicular origin. For the purpose of immunocontraception it may be desirable not to disrupt spermatogenesis and testicular function. Concept of post testicular maturation of spermatozoa has been very well established. During post testicular voyage spermatozoa undergo a series of complex and sequential events which transforms the immature immotile spermatozoa into mature sperm. Acquisition of functional maturity is necessary for progressive motility, zona pellucida recognition culminating in sperm egg binding. Importance of epididymal maturation is highlighted by the fact that high percentage of male infertility in human originates from the malfunction of the epididymis. The epididymis has also shown to be involved in sperm storage and provides an adequate environment for final maturation of the sperm. It provides a conducive microenvironment by virtue of which the spermatozoa are protected during the storage. In view of this it is imperative that more attention needs to be focused on epididymal antigens. The information obtained will enable us to identify epididymal antigens relevant to fertility and also help in infertility diagnosis.