Tyrosine phosphorylation, thiol status, and protein tyrosine phosphatase in rat epididymal spermatozoa

Sperm thiol oxidation and the ability to undergo protein tyrosine phosphorylation are associated with the acquisition of sperm motility and fertilizing ability during passage of spermatozoa through the epididymis. Phosphotyrosine levels in various cells are controlled by tyrosine kinase versus phosphatase, with the latter known to be inhibited by oxidation. In the present paper we examine whether changes in thiol status during sperm maturation affect rat sperm protein phosphotyrosine levels and protein phosphotyrosine phosphatase (PTP) activity. Tyrosine phosphorylation, as demonstrated by immunoblotting (IB), was significantly increased in several sperm tail proteins during maturation in the epididymis. Sperm thiol oxidation with diamide enhanced tail protein phosphorylation; reduction of disulfides with dithiothreitol diminished phosphorylation. In the sperm head, a moderate increase in tyrosine phosphorylation was accompanied by altered localization of phosphotyrosine proteins during maturation. Blocking of thiols and PTP activity with N-ethylmaleimide led to increased tyrosine phosphorylation of protamine in caput sperm heads. Several PTP bands were identified by IB. In the caput spermatozoa, a prominent level of the 50 kDa band was present, whereas in the cauda spermatozoa a very low level of the 50 kDa band was found. PTP activity, measured by using p-nitrophenyl phosphate as a substrate, was significantly higher in the caput spermatozoa (high thiol content) than in the cauda spermatozoa (low thiol content). Our results show that PTP activity is correlated with sperm thiol status and suggest that tyrosine phosphorylation of sperm proteins during sperm maturation is promoted by thiol oxidation and diminished PTP.     

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.     

Changes in rat sperm membrane glycosidase activities and carbohydrate and protein contents associated with epididymal transit

Rat spermatozoa were recovered from the caput, corpus, and cauda epididymides and assayed for glycosidase activity, total nonamino (neutral) carbohydrate, and protein content. The activities of beta-glucosidase, beta-galactosidase, beta-N-acetylglucosaminidase, and beta-N-acetylgalactosaminidase were fluorometrically assayed in spermatozoa and membrane-enriched fractions. Except for beta-glucosidase, the activities of the glycosidases based on protein content were greatest in whole sperm and membrane-enriched fractions obtained from the cauda epididymides. Based on sperm concentration, however, glycosidase activities increased proceeding from the caput to the corpus epididymides, then declined from the corpus to the cauda epididymides. Analyses of nonamino carbohydrate and protein content based on sperm number indicated regional trends similar to those of glycosidase activity. Total nonamino carbohydrate and protein content were highest in corpus sperm, and lowest in cauda sperm. These data indicate major quantitative changes in cell surface carbohydrate as spermatozoa traverse the epididymis. A positive correlation for the membrane-enriched fraction between increasing glycosidase activity and decreasing carbohydrate and protein content suggests that glycosidases may play a significant role in modifying the spermatozoon surface during epididymal transit and maturation.     

Quantitative changes in sperm head morphology during passage through the male excurrent duct system of the rabbit

A fine adjustment of sperm head size and shape occurs during maturation and storage within the male excurrent duct of the rabbit. This remodelling, as judged by morphometric values of area, perimeter, length, width, and shape factors, takes place mostly in passage from the seminiferous tubules of the testis to the distal caput of the epididymis. The dimensions of sperm heads from the distal corpus of the epididymis break the general tendency toward a reduction in size and more elliptical shapes. A period of transport and storage within the epididymal cauda and vas deferens follows in which there are no further changes in sperm head morphometry. It can be concluded that the period immediately following sperm release from the testis is crucial to the final morphological maturation of spermatozoa. Moreover, the fact that changes are detected in the appearance of sperm heads at successive stages of sperm maturation suggests that the dimensions of a particular epididymal spermatozoon may be taken as an approximate indication of its relative maturity. Mol. Reprod. Dev. 51:203–209, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Analyse de la structure chromatinienne du sperme par la cytométrie en flux à l’acridine orange. — intérêt clinique

During spermatozogenesis, sperm nucleosomal DNA type, linked to histones, is transformed into a special form bound to small basic proteins, the protamines. This process allows sperm nucleus to condense and mature. This maturation process is achieved in the epididymis. When a spermatozoon enters an oocyte, protamines are released and replaced by histones, enabling the sperm nucleus to expand into a male pronucleus. Flow cytometry using acridine orange staining is an objective and quantitative technique well-adapted for the investigation of the chromatin structure at the level of each spermatozoon, as well as the whole ejaculate. This technique allows tracing the young sperm cell nuclei from the diploid stage, through tetraploid until the final haploid stage in mature spermatozoa. It allows also following the sperm maturation process during the epididymal transit. It detects various sperm chromatin condensation defects, hypocondensation, hypercondensation or other aberrations, as well as decondensation defects by using an in vitro assay. These defects may impair sperm fertilizing ability, even after sperm microinjection into the oocyte. Better understanding of sperm chromatin integrity and stability prerequisites might help us improving the quality of various technologies used in assisted medical procreation.     

cSrc is necessary for epididymal development and is incorporated into sperm during epididymal transit

Changes that occur to mammalian sperm upon epididymal transit and maturation render these cells capable of moving progressively and capacitating. Signaling events leading to mammalian sperm capacitation depend on the modulation of proteins by phosphorylation and dephosphorylation cascades. Recent experiments have demonstrated that the Src family of kinases plays an important role in the regulation of these events. However, sperm from cSrc null mice display normal tyrosine phosphorylation associated with capacitation. We report here that, despite normal phosphorylation, sperm from cSrc null mice display a severe reduction in forward motility, and are unable to fertilize in vitro. Histological analysis of seminiferous tubules in the testes, caput and corpus epididymis do not reveal obvious defects. However, the cauda epididymis is significantly smaller, and expression of key transport proteins in the epithelial cells lining this region is reduced in cSrc null mice compared to wild type littermates. Although previously, we and others have shown the presence of cSrc in mature sperm from cauda epididymis, a closer evaluation indicates that this tyrosine kinase is not present in sperm from the caput epididymis, suggesting that this protein is acquired by sperm later during epididymal maturation. Consistent with this observation, cSrc is enriched in vesicles released by the epididymal epithelium known as epididymosomes. Altogether, these observations indicate that cSrc is essential for cauda epididymal development and suggest an essential role of this kinase in epididymal sperm maturation involving cSrc extracellular trafficking.     

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.     

Identification and characterization of P31m, a novel sperm protein in Cynomolgus monkey (Macaca fascicularis)

We have previously described a hamster sperm glycoprotein, P26h, which is implicated in the cascade of events occurring during the interaction between mature spermatozoa and the oocyte's zona pellucida. The P26h is acquired on the acrosomal cap of the spermatozoon during its maturation arising within the epididymis. Lately, using a polyclonal antiserum raised against P26h, a 34 kDa protein, P34H, has been identified on the acrosomal cap of the human spermatozoon. The cloning and sequencing of the cDNA encoding P34H has revealed a 65% similarity between the P34H and P26h amino acid sequences. Considering that P26h shows total immunocontraceptive properties in the hamster, it is of relevant importance to have an animal model phylogenetically closer to the human. Using the Cynomolgus monkey, we searched for a protein autologous to the human P34H. A 31 kDa protein, the P31m, localized on the acrosomal cap of the monkey spermatozoon has been identified by a Western blot analysis and by immunohistochemical techniques using an anti-hamster P26h antiserum. Northern blot analysis showed increasing high levels of the P31m mRNA through the epididymis and at lower levels in the testis. In situ hybridization showed the presence of the P31m mRNA in the principal cells of the epididymis. The cloning and sequencing of the cDNA encoding the P31m showed a high homology of 97% identity between the P31m and P34H nucleotidic sequences. This study clearly demonstrates that the monkey P31m is the homologous protein of the hamster P26h and of the human P34H. Mol. Reprod. Dev. 59: 431-441, 2001.     

The localization of protein carboxyl-methylase in sperm tails

Protein carboxyl-methylase (PCM), an enzyme known to be involved in exocytotic secretion and chemotaxis, has been studied in rat and rabbit spermatozoa. PCM activity and its substrate methyl acceptor protein(s) (MAP) were demonstrated in the supernate after solubilization of the sperm cell membrane by detergent (Triton X-100). A protein methylesterase that hydrolyzes methyl ester bonds created by PCM was demonstrated in rabbit but not in rat spermatozoa. This enzyme was not solubilized by nonionic detergent. The specific activities of PCM in rat spermatozoa from caput and cauda epididymis were similar and lower than that found in testis. By contrast, MAP substrates were low in testis and increased in parallel with sperm maturation in the epididymis. Multiple MAP were demonstrated in spermatozoa by polyacrylamide gel electrophoresis. The pattern of these proteins was similar in spermatozoa from different portions of the reproductive tract. Fractionation of heads and tails of rat spermatozoa on sucrose gradients indicated that PCM was found exclusively in the tail fraction, whereas MAP was detected both in head and tail fractions. The presence of all the components of the protein carboxyl-methylation system in spermatozoa and the localization of PCM and some of its substrates in the sperm tail are consistent with their involvement in sperm cell motility.     

Temporal expression and localization of protein farnesyltransferase during spermiogenesis and posttesticular sperm maturation in the hamster

Spermiogenesis and posttesticular sperm maturation in the epididymis are distinct developmental processes that result in a polarized spermatozoon possessing a plasma membrane partitioned into segment-specific domains of distinct composition and function. The mechanisms that specify the distribution of intracellular organelles and target proteins to restricted membrane domains are not well understood. In this study we examined the expression pattern and distribution of protein farnesyltransferase (FTase) in hamster spermatids and epididymal spermatozoa to determine if protein lipidation may represent a potential mechanism to regulate protein association with specific organelles or the plasma membrane. Round spermatids exhibited only weak immunostaining with antibody against the β-subunit of FTase, whereas elongating spermatids exhibited a high level of FTase expression that was segregated to the cytoplasmic lobe surrounding the anterior flagellum. Although FTase was released with the residual body, mature spermatids retained FTase within the midpiece and cytoplasmic droplet. In epididymal spermatozoa, FTase remained associated with the cytoplasmic droplet during its migration to the midpiece-principal piece junction; following release of the cytoplasmic droplet, no immunodetectable FTase was noted in the midpiece segment. Immunoblotting demonstrated the presence of both the α and β subunits of FTase in sperm lysates. The temporal expression pattern and restricted distribution of FTase in spermatids and epididymal spermatozoa suggest a potential role in regulating protein association with specific organelles and/or membrane domains of the mature spermatozoon. Mol. Reprod. Dev. 48:71–76, 1997. © 1997 Wiley-Liss, Inc.     

Maturation-dependent modification of the protein phosphorylation profile of isolated goat sperm plasma membrane.

Highly purified plasma membranes, isolated by an aqueous two-phase polymer method from goat epididymal spermatozoa, were found to possess a kinase activity that causes phosphorylation of serine and threonine residues of several endogenous plasma membrane proteins. Cyclic AMP, cyclic GMP, Ca(2+)-calmodulin, phosphatidylserine-diolein, polyamines and heparin had no appreciable effect on this kinase. Autoradiographic analysis showed that the profile of the phosphorylation of membrane proteins by this endogenous cAMP-independent protein kinase underwent marked modulation during the transit of spermatozoa through the epididymis. In caput sperm plasma membrane, 18, 21, 43, 52, 74 and 90 kDa proteins were phosphorylated, whereas, in the corpus and cauda epididymal spermatozoa, a differential phosphorylation pattern was observed with respect to the 90, 74, 21 and 18 kDa proteins. The rate of phosphorylation of the 74 kDa protein decreased markedly during the early phase of sperm maturation (caput to distal corpus epididymides) whereas there was little change in kinase activity in sperm plasma membrane. In contrast, the rates of phosphorylation of the 18 and 21 kDa proteins increased during the terminal phase (distal corpus to distal cauda epididymides) of sperm maturity, although the kinase activity of membrane decreased significantly during this phase. The modulation of the phosphorylated states of these specific membrane proteins may play an important role in the maturation of epididymal spermatozoa.     

Identification of post-translational modifications that occur during sperm maturation using difference in two-dimensional gel electrophoresis

Difference in two-dimensional (2-D) gel electrophoresis (DIGE) is a novel method for analyzing up to three samples in one 2-D gel and using the information gained to study post-translational modifications of proteins. We describe the use of DIGE to isolate and characterize those proteins that undergo processing in spermatozoa as they transit the epididymal tract. We find up to 60 protein spots are significantly modified as sperm traverse the epididymis. In this article, we report eight unambiguous protein identifications and demonstrate that one protein, the beta-subunit of the mitochondrial F1-ATPase, is serine-phosphorylated as sperm undergo epididymal maturation. We suggest that phosphorylation of this particular protein in a cAMP-dependent manner may contribute to the mechanisms by which motility is conferred upon spermatozoa.     

Characterization of maturation-dependent extrinsic proteins of the rat sperm surface

Mammalian spermatozoa must mature in the epididymis before they can fertilize an egg. It is known that modification of the protein composition of the sperm surface is an important part of the maturation process. In this paper, we present data on two related glycoproteins that can be extracted from mature but not immature spermatozoa. Cell surface radioiodination has shown that these proteins are on the sperm surface, and immunofluorescence microscopy, by use of monospecific antibodies to the proteins, has indicated that their localization is restricted to the periacrosomal region of the sperm head. We have also shown that in vitro, these proteins will bind to the identical region of immature sperm. Immunohistochemical localization of the proteins in the epididymis shows that they are produced and secreted by the cauda region. The significance of the addition of these proteins to the sperm surface in both maturation and fertilization is discussed.     

Association study and expression analysis of CD9 as candidate gene for boar sperm quality and fertility traits

Cluster-of-differentiation antigen 9 (CD9) gene expressed in the male germ line stem cells is crucial for sperm-egg fusion, and was therefore selected as candidate gene for boar semen quality. The association of CD9 with boar sperm quality and fertility trait was analyzed using a total of 340 boars both from purebred Pietrain and Pietrain×Hampshire crosses. A single nucleotide polymorphism (g.358A>T) in intron 6 was significantly associated with sperm motility (MOT) (P<0.001), plasma droplet rate (PDR) (P<0.001) and abnormal spermatozoa rate (ASR) (P<0.01). Boars were divided into two groups with group 1 (G-I) boars having a higher SCON and SMOT, lower SVOL (sperm volume) and group 2 (G-II) having a lower SCON and SMOT, higher SVOL. The mRNA and protein expression levels were evaluated in reproductive, non-reproductive tissues and spermatozoa from G-I and G-II animals by using quantitative real-time PCR and western blotting. When both reproductive and non-reproductive tissues were examined, highest mRNA was expressed in prostate gland, then in the body of the epididymis, vas deferens and tail of the epididymis. In case of reproductive tissues, CD9 expression was higher in tissues and spermatozoa collected from G-I boars than those collected from G-II boars. The mRNA expression was significantly different (P<0.05) in body of epididymis from G-I and G-II boars. The CD9 protein expression results from western blot were coincided with the results of qRT-PCR. Moreover, CD9 protein localization in Leydig cells, Sertoli cells, epithelial cells and spermatozoa was remarkable which indicated the important role of CD9 in spermatogenesis process. By using mRNA and protein expression profiles, it could be shown that CD9 plays a crucial role during sperm development, especially within the epididymis where the maturation of the sperm, a key process for the sperm quality and motility takes place. These results will improve the understanding of the functions of the CD9 in spermatogenesis within the reproductive tracts and will shed light on CD9 as a candidate gene in the selection of good sperm quality boars.     

Regulation of epididymal function and sperm maturation--endocrine approach to fertility control in male.

The structural and functional integrity of the epididymis, the acquisition of fertilizing ability by spermatozoa and their viability within the epididymis are androgen dependent phenomena. Although the precise mechanism by which sperm maturation and viability in the epididymis are brought about by androgen are not clearly understood, it is generally held that specific epididymal secretions produced under the influence of androgen affect these events. Though the spermatozoa appear to remain viable in a low androgen environment, sperm maturation requires a relatively high androgen environment. Against this background the potentiality of antiandrogens as extragonadal antifertility agents has been discussed. Studies with steroidal and nonsteroidal antiandrogens have revealed that in adult animals the secretory activity of the epididymis, as evidenced by the level of glycerylphosphorylcholine, either remains unaffected or is stimulated under their influence. These studies have further indicated that the extragonadal antifertility action of antiandrogens will depend upon their ability to (1) lower the testicular androgen synthesis and/or androgen binding protein, which possibly serves as a carrier of androgen from the testis to epididymis; (2) to lower local androgen synthesis as a result of reduced levels of circulating androgen, and (3) to inhibit 5 alpha-reduction of testosterone to dihydrotestosterone and/or to inhibit androgen binding to receptors. Success in the rational development of new antifertility agents for male which will act by controlling epididymal function will depend upon a clear understanding of the factors that regulate epididymal secretion and the role of epididymal secretions in sperm maturation and survival.     

The Biological and Functional Significance of the Sperm Acrosome and Acrosomal Enzymes in Mammalian Fertilization

The mammalian spermatozoon undergoes continuous modifications during spermatogenesis, maturation in the epididymis, and capacitation in the female reproductive tract. Only the capacitated spermatozoa are capable of binding the zona-intact egg and undergoing the acrosome reaction. The fertilization process is a net result of multiple molecular events which enable ejaculated spermatozoa to recognize and bind to the egg's extracellular coat, the zona pellucida (ZP). Sperm–egg interaction is a species-specific event which is initiated by the recognition and binding of complementary molecule(s) present on sperm plasma membrane (receptor) and the surface of the ZP (ligand). This is a carbohydrate-mediated event which initiates a signal transduction cascade resulting in the exocytosis of acrosomal contents. This step is believed to be a prerequisite which enables the acrosome reacted spermatozoa to penetrate the ZP and fertilize the egg. This review focuses on the formation and contents of the sperm acrosome as well as the mechanisms underlying the induction of the acrosome reaction. Special emphasis has been laid on the synthesis, processing, substrate specificity, and mechanism of action of the acid glycohydrolases present within the acrosome. The hydrolytic action of glycohydrolases and proteases released at the site of sperm-zona binding, along with the enhanced thrust generated by the hyperactivated beat pattern of the bound spermatozoon, are important factors regulating the penetration of ZP. We have discussed the most recent studies which have attempted to explain signal transduction pathways leading to the acrosomal exocytosis.     

Lactate dehydrogenase activity of rat epididymis and spermatozoa: effect of constant light

During its passage through the epididymis, the gamete undergoes a process of "maturation" leading to the acquisition of its fertilizing ability. The epididymis displays regional variations in the morphology and metabolic properties of its epithelium which are relevant for the progressive development of mature sperm characteristics. The epididymis has spontaneous peristaltic contractions and receives sympathetic innervation that is modulated by melatonin, a hormone synthesized and released by the pineal gland. Constant lighting disrupts melatonin synthesis and secretion. We have studied the effect of constant light on lactate dehydrogenase (LDH; EC 1.1.1.27) and its isozyme C4 activities and protein content in whole epididymis, epididymal tissue and in spermatozoa from caput and cauda segments. Animals were exposed from birth to an illumination schedule of 14 h light:10 h dark (group L:D). At 60 days of age one group of animals was submitted to constant light over 50 days (group L:L). In order to test the fertilizing ability, the rats of each group were mated with soliciting estrous females. The percentage of pregnancies in females mated with males maintained in L:L was remarkably lower than those in females mated with males maintained in the L:D photoperiod (44% and 88% respectively). Constant light increased protein concentration and LDH activity in caput as well as in cauda of total epididymis. On the contrary, in epididymal tissue, the protein content decreased in both epididymal sections compared with controls. When enzymatic activity was expressed in Units per spermatozoa, constant light induced a significant reduction of total LDH and LDHC4 in caput and cauda spermatozoa while LDH activity of epididymal tissue was not affected. In spite of the decrease in LDH per sperm cell when rats were exposed to constant light, in total epididymis (epididymis tissue plus sperm cells content) and in spermatozoa, values of enzyme activities expressed per weight unit were higher than those of controls. This is explained by the increase in the amount of stored spermatozoa, both in caput and cauda, produced by exposure of animals to constant light. Our results confirm that in rats, chronic exposure to constant light promotes a reduction of fertilizing ability and indicates that continuous lighting reduces the total LDH and LDHC4 activities, possibly due to moderate aging of spermatozoa within the duct by lengthening of the sperm transit through the epididymis.     

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.