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

It has been widely accepted that mammalian spermatozoa are infertile when they leave the testes and require a period of maturation in both the epididymis and the female reproductive tract before acquiring the ability to fertilize an oocyte. However, the necessity for such a complex process of posttesticular sperm maturation appears to be unique to mammals because it is well established that these processes do not directly influence the fertilizing ability of the spermatozoa of birds, reptiles, and other lower vertebrates. Because of their key evolutionary position and form of reproduction, we contend that monotremes (platypus and echidna) provide a unique model for resolving why these processes are necessary. In the present review, we examine evidence that the epididymal maturation of monotreme spermatozoa is far less complex than in other mammals. However, a unique feature of the monotreme epididymis lies in its ability to promote the formation of elaborate sperm bundles that serve to greatly enhance the cells' motility. It is suggested that this intriguing cooperative strategy used by monotreme sperm represents an early form of epididymal maturation that appears to have been elaborated upon during the evolution of higher mammals, possibly as an adaptation for sperm competition.

Once shed from their fostering Sertoli cells, spermatozoa leave the testis and are transported passively by seminiferous fluid through the rete testis. Then, these immature cells enter the complex efferent duct system that is joined to the unique and convoluted epididymal duct. This epididymal duct, lined by a continuous layer of epithelial cells joined by tight junctions, is a tube several meters long (up to 60 m in domestic mammals) and forms an organ that is classically subdivided into 3 major anatomical regions: the head/caput, the corpus/body, and the tail/cauda. Spermatozoa travel throughout the duct for several days to weeks, depending on the species, and may be stored for even longer periods in the cauda part of the epididymis and vas deferens. During their journey the proportion of potentially "mature" spermatozoa increases, but it is only when they reach the cauda epididymidis that almost all spermatozoa have acquired their natural fertilizing ability, which involves progressive motility, the ability to undergo the postejaculatory events (capacitation and hyperactivation), and the capacity to recognize and to bind to the oocyte investments and egg plasma membrane. Recent secretomic, proteomic, and transcriptomic studies have provided new information on the functions and the regionalization of the epididymis and revealed some insights into the complexity of epididymal fluid. Among genes and proteins highly expressed by this tissue, many have roles related to sperm protection (such as oxidation), but a large number of new compounds related to innate immunity have also been discovered. This review will focus on possible new control mechanisms that these studies have suggested for this tissue.

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., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The epididymis plays a major role in the acquisition of sperm fertility. In order to shed light on specific features of epididymal function in mammalian species, we characterized the luminal proteins (luminal proteome) and secreted proteins (secretome) in the bovine epididymis. We identified 172 different luminal proteins in 9 distinct epididymal regions. The concentration and secretory activity of luminal proteins were quantified throughout the epididymis. Among the most abundant secreted proteins, we found lipocalin 5, (LCN5), NADP(+)dependent prostaglandin dehydrogenase (PTGDS), Niemann-Pick disease type C2 protein (NPC2), glutathione peroxidase type 5 (GPX 5), clusterin (CLU), hexosaminidase B (HEXB) and galactosidase (GLB1), each of which is released in distinct epididymal regions. Gelsolin, (GSN) previously not described in mammalian epididymal fluid, appeared to be a major protein secreted exclusively in the distal region of the bovine epididymis, where fully mature spermatozoa are stored. Although the major epididymal proteins are conserved between mammalian species, this study highlights the specificity and mechanisms of protein processing of epididymal secretion in the bull. In addition, this study provides a major insight into the sequential changes occurring in the sperm environment while gaining fertilizing capacity and could provide new information for the future identification of potential fertility markers., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The present review examines whether monotremes may help to resolve three questions relating to sperm production in mammals: why the testes descend into a scrotum in most mammals, why spermatozoa are infertile when they leave the testes and require a period of maturation in the specific milieu provided by the epididymides, and why ejaculated spermatozoa cannot immediately fertilise an ovum until they undergo capacitation within the female reproductive tract. Comparisons of monotremes with other mammals indicate that there is a need for considerable work on monotremes. It is hypothesised that testicular descent should be related to epididymal differentiation. Spermatozoa and ova from both groups share many of the proteins that are thought to be involved in gamete interaction, and although epididymal sperm maturation is significant it is probably less complex in monotremes than in other mammals. However, the monotreme epididymis is unique in forming spermatozoa into bundles of 100 with greatly enhanced motility compared with individual spermatozoa. Bundle formation involves a highly organised interaction with epididymal proteins, and the bundles persist during incubation in vitro, except in specialised medium, in which spermatozoa separate after 2-3 h incubation. It is suggested that this represents an early form of capacitation.

When mammalian spermatozoa exit the testis, they show a highly specialized morphology; however, they are not yet able to carry out their task: to fertilize an oocyte. This property, that includes the acquisition of motility and the ability to recognize and to fuse with the oocyte investments, is gained only after a transit through the epididymis during which the spermatozoa from the testis travel to the vas deferens. The exact molecular mechanisms that turn these cells into fertile gametes still remain mysterious, but surface-modifying events occurring in response to the external media are key steps in this process. Our laboratory has established cartographies of secreted (secretomes) and present proteins (proteomes) in the epididymal fluid of different mammals and have shown the regionalized variations in these fluid proteins along the epididymis. We have found that the main secreted proteins are common in different species and that enzymatic activities, capable of controlling the sperm surface changes, are present in the fluid. Our studies also indicate that the epididymal fluid is more complex than previously thought; it contains both soluble and particulate compartments such as exosome-like vesicles (epididymosomes) and certainly specific glycolipid-protein micelles. Understanding how these different compartments interplay to modify sperm components during their transit will be a necessary step if one wants to control and to ameliorate sperm quality and to obtain valuable fertility markers helpful to establish a male fertility based genetic selection.

In this study, we purified the first member of a new ribonuclease (RNase) A family from fluid of the proximal caput of the boar epididymis. This protein, named "Train A," is the most abundant compound secreted in the anterior part of the boar epididymis. After 2D electrophoresis, it is characterized by more than 10 isoforms ranging in size from 26 to 33 kDa and pI from 5 to 8.5. Several tryptic peptides were N-terminal sequenced, and an antiserum against one of these peptides was obtained. The protein was immunolocalized in the epididymal epithelium of the proximal caput, especially in the Golgi zone and the apical cytoplasm of the principal cells. In the lumen, spermatozoa were negative but droplets of reaction product were observed within the lumen. Full lengths of Train A cDNA were obtained from a lambdagt11 boar caput epididymis library and sequenced. The deduced protein is composed of 213 amino acids, including a 23-amino acid peptide signal and a potential N-glycosylation site. The mRNA of this protein has been retrieved and partially sequenced in the bull, horse, and ram, and homologous cDNA is found in databanks for the rat, mouse, and human. All the sequences are highly conserved between species. This protein and its mRNA are male-specific and exclusively expressed in the proximal caput of the epididymis, the only site where they have been found. Train A presents an RNase A family motif in its sequence. The RNase A family is a group of several short proteins (20-14 kDa) with greater and lesser degrees of ribonucleolytic activity and with supposed different roles in vivo. However, the presence of a long-conserved N-terminal specific sequence and the absence of RNase catalytic site for Train A indicate that Train A protein is a member of a new family of RNase A.

The final stages of sperm differentiation occur outside the gonad and are not under the genomic control of germ cells. Only sequential interactions with the medium surrounding the sperm are believed to induce the final steps of spermatogenesis. The epididymis, a long tubule with very active secretory and reabsorption functions, is able to create sequential changes in the composition of luminal fluid throughout its length. The chronologies of the changes, which occur on/in the sperm with those in their surrounding environment, are described. Correlations between the highly regionalized epididymal activities and sperm characteristics linked to their survival and fertility potential are presented in this review., (Copyright 2003 Wiley-Liss, Inc.)

This review considers the role of proteins secreted by the epididymis on post-testicular sperm maturation and storage. Two-dimensional gels show that 150 to 200 proteins are secreted into the epididymal lumen. Most are secreted in relatively small amounts; in rams, for example, fewer than ten contribute 90% of the total secretion and only two contribute 52% of the total protein secreted. Most of the proteins are confined to specific regions of the epididymis. The changing pattern of protein secretion along the epididymis corresponds to change in surface protein on spermatozoa, but no epididymal proteins have been identified that appear to be directly involved in modifying the sperm membrane. Most of the major proteins that have been identified seem to be playing a homeostatic role in maintaining the epididymal milieu for spermatozoa.

A specific 135-kDa 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-kDa protein were established. A clone, B-11, reacting both with epididymal fluid and with sperm plasma membranes was selected and used in this study. Immunoblotting analysis showed that B-11 reacted only with a 135-kDa protein among epididymal fluid proteins. In contrast, B-11 did not recognize a similar 135-kDa sperm protein but did strongly react with a 27-kDa protein among sperm membrane proteins, extracted by NP-40 in the presence of protease inhibitors. B-11 also reacted only with a 27-kDa protein fragment among trypsin digests of the 135-kDa epididymal protein. The 135-kDa protein was first detected, by ELISA 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 kDa 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-kDa protein was distributed all over the equatorial region of the acrosome of less than 10% 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.

The level of carnitine and acetylcarnitine in spermatozoa of boar epididymal origin and of human ejaculates was demonstrated. In the epididymal fluid of boars, the concentration of carnitine (nmol/mg protein) began to increase from 20 in the distal caput to rise progressively to 700 in the distal cauda. By contrast, the carnitine content of spermatozoa only started to increase in the proximal cauda where the concentration of carnitine in the fluid was 200-300 nmol/mg protein, then gradually increased in spermatozoa from more distal sites. The increase in the acetylcarnitine content of spermatozoa paralleled that of the carnitine amount, represented 50% of total carnitine (carnitine + acetylcarnitine) and coincided with the acquisition of progressive motility. In two populations of human seminal spermatozoa selected by migration and characterised by a very large difference in their percentage of progressively motile cells, higher carnitine and acetylcarnitine contents (40%) were found in migrated spermatozoa compared to the residual population. These results suggest that accumulation of carnitine and its metabolite may be an important factor in the acquisition and the maintenance of progressive motility. Measurement of acetylcarnitine content of human seminal spermatozoa could be used as a marker of epididymal maturation.

Mammalian spermatozoa acquire the ability to move forward during epididymal maturation and the corpus occurs as a critical region where all the patterns of movements can be observed: immature, 'erratic' and mature. The absence of coordination of the flagellar movement of the transitory forms provides a difference in the initiation sites and the waves propagation between the two sides of the axonema. The mechanisms that may be required to activate selectively the tubulin-dynein system on the two sides of the flagellum during the initiation of the movement of immature sperm in mammals are unknown.

To study successive surface changes of maturing ram spermatozoa, we determined the 125I-labeling patterns of testicular spermatozoa and of spermatozoa from 10 consecutive regions of the epididymis. Overall, three phases of cell surface transformations are distinguishable: Phase I occurs in the caput epididymidis and it is characterized by a series of rapid surface changes. The most striking surface transformations occur during transport of spermatozoa from the testis into the proximal caput epididymidis. All major surface components in the zones 78 to 115 kd disappear or are lost from the surface of testicular spermatozoa. Concurrently, several low molecular weight components (17 to 65 kd) appear or become increasingly accessible to 125I. Phase II represents a period of relative quiescence which is confined to the corpus epididymidis. Phase III takes place in the cauda epididymidis where several existing (97, 65 and 41 kd) and new (24 kd) proteins become the predominant features of the sperm cell surface. Electrophoretic analyses of luminal fluid proteins from corresponding regions of the testis and epididymis also show that the most striking changes occur between the rete testis and the proximal caput epididymidis. No rete testis fluid (RTF) components are detectable in luminal fluid of the proximal caput epididymidis. In the epididymis, however, fluid proteins are more persistent than sperm surface components. Several major fluid components (i.e., 95, 76, 21.5, 19.5 and 16 kd) persist throughout the epididymis. Other fluid proteins are of a more transient nature as, for example, a 25 kd molecular weight component (regions E1 through E6) or the 180, 62, 37 and 32 kd components in regions E4 to E10, the 270, 115 and 105 kd proteins in regions E6 to E10 and the 360, 145, 125 and 62 kd molecular weight components in regions E7 to E10. No direct relationships could be established between intrinsic surface components and exogenous fluid proteins from corresponding regions of the testis and epididymis. These results demonstrate a much greater complexity of sequential surface transformation in maturing epididymal spermatozoa than was predictable from our earlier studies of testicular and ejaculated spermatozoa. Apparently, ram spermatozoa must undergo extensive surface renovations in the caput epididymidis before the surface protein pattern typical of mature spermatozoa slowly develops.

A more sensitive assay for protein methylesterase (PME) was developed. The new assay measures the methanol formed from hydrolysis of protein-methyl esters instead of methyl esters remaining on proteins. The formation of methanol is linear with time and enzyme concentration up to 20% of substrate hydrolysis. With this assay, we have detected very low PME activity in rat spermatozoa from cauda epididymidis. However, PME activity in spermatozoa from caput epididymidis was 10-fold higher. In a more detailed study on bull spermatozoa during epididymal maturation, we observed that PME activity was low in testicular spermatozoa, increased in spermatozoa from caput epididymidis to a maximum of 1.29 pmol/mg protein, and progressively decreased to a very low level (0.06 pmol/mg protein) in spermatozoa from cauda epididymidis. The drop in PME activity during epididymal transit parallels the progressive acquisition of motility and fertilizing capacity by spermatozoa.

Spermatozoa were collected from the rete testis of conscious boars, from the cauda epididymidis by retro-flushing, and by ejaculation. Testicular spermatozoa showed no progressive motility, and that of ejaculated was greater than that of epididymal spermatozoa. Glycolysis and respiration of testicular spermatozoa, while lower than that of the more mature cells, were only slightly affected by the incubation conditions. Epididymal spermatozoa converted 83% of the glucose they utilized to CO2 or lactate, but testicular cells converted only 35% to these metabolites. Synthesis of lipid was greatest by testicular spermatozoa. With the more mature cells hyperosmolar conditions depressed CO2 production, but increased lactate production, and these changes were greater for ejaculated than for epididymal spermatozoa. Glycolysis plus respiration of these cells was related to their motility. These results were interpreted as showing increasing motility, glycolysis and respiration with maturation, but also decreased synthetic capacity and increased sensitivity to the environment.