Formation and Dissociation of Sperm Bundles in Monotremes.

Because monotremes are the earliest offshoot of the mammalian lineage, the platypus and short-beaked echidna were studied as model animals to assess the origin and biological significance of adaptations considered unique to therian mammals: epididymal sperm maturation and subsequent capacitation. We show that spermatozoa from both species assemble into bundles of approximately 100 cells during passage through the epididymis and that an epididymal protein-secreted protein, acidic, cysteine-rich (osteonectin; SPARC)-is involved in bundle formation. The bundles persisted during incubation in vitro for at least 1 h under conditions that capacitate therian spermatozoa, and then underwent a time-dependent dissociation to release spermatozoa capable of fertilization. Only after this dissociation could the spermatozoa bind to the perivitelline membrane of a hen's egg, display an altered form of motility reminiscent of hyperactivation, and be induced to undergo an acrosome reaction. It is concluded that the development of sperm bundles in the monotreme epididymis mandates that they require a time-dependent process to be capable of fertilizing an ovum. However, because this functional end point was achieved without overt changes in protein tyrosine phosphorylation (a hallmark of capacitation in therians), it is concluded that the process in monotremes is distinctly different from capacitation in therian mammals.

Epididymal protein markers and fertility.

The last stages of male gamete differentiation occur outside the gonad in a specific environment controlled by the epididymal epithelium. All the fundamental characteristics of a fertile spermatozoon are acquired sequentially during transit through the epididymal tubule. Full understanding of the mechanisms involved in these gamete modifications is a key to understanding and controlling such important stages in male fertility. With the development of new large scale technologies, large amounts of information give hope of identifying the fundamental elements involved in such cellular events and of being able to obtain some markers predictive of male fertility that would be valuable both in human and/or animal reproduction.

Analysis of epididymal sperm maturation by MALDI profiling and top-down mass spectrometry.

The fertilization ability of male gametes is achieved after their transit through the epididymis where important post-gonadal differentiation occurs in different cellular compartments. Most of these maturational modifications occur at the protein level. The epididymal sperm maturation process was investigated using the ICM-MS (Intact Cell MALDI-TOF MS) approach on boar spermatozoa isolated from four different epididymal regions (immature to mature stage). Differential and quantitative MALDI-TOF profiling for whole cells or sub-cellular fractions was combined with targeted top-down MS in order to identify endogenous biomolecules. Using this approach, 172m/z peaks ranging between 2 and 20kDa were found to be modified during maturation of sperm. Using top-down MS, 62m/z were identified corresponding to peptidoforms/proteoforms with post-translational modifications (MS data are available via ProteomeXchange with identifier PXD001303). Many of the endogenous peptides were characterized as N-, C-terminal sequences or internal fragments of proteins presenting specific cleavages, suggesting the presence of sequential protease activities in the spermatozoa. This is the first time that such proteolytic activities could be evidenced for various sperm proteins through quantification of their proteolytic products. ICM-MS/top-down MS thus proved to be a valid approach for peptidome/degradome studies and provided new contributions to understanding of the maturation process of the male gamete involved in the development of male fertility. BIOLOGICAL SIGNIFICANCE: This peptidomic study (i) characterized the peptidome of epididymal spermatozoa from boar (Sus scrofa); (ii) established characteristic molecular phenotypes distinguishing degrees of maturation of spermatozoa during epididymal transit, and (iii) revealed that protease activities were at the origin of numerous peptides from known and unknown proteins involved in sperm maturation and/or fertility processes.

An integrative omics strategy to assess the germ cell secretome and to decipher sertoli-germ cell crosstalk in the Mammalian testis.

Mammalian spermatogenesis, which takes place in complex testicular structures called seminiferous tubules, is a highly specialized process controlled by the integration of juxtacrine, paracrine and endocrine information. Within the seminiferous tubules, the germ cells and Sertoli cells are surrounded by testicular fluid (TF), which probably contains most of the secreted proteins involved in crosstalk between these cells. It has already been established that germ cells can modulate somatic Sertoli cell function through the secretion of diffusible factors. We studied the germ cell secretome, which was previously considered inaccessible, by analyzing the TF collected by microsurgery in an "integrative omics" strategy combining proteomics, transcriptomics, genomics and interactomics data. This approach identified a set of proteins preferentially secreted by Sertoli cells or germ cells. An interaction network analysis revealed complex, interlaced cell-cell dialog between the secretome and membranome of seminiferous cells, mediated via the TF. We then focused on germ cell-secreted candidate proteins, and we identified several potential interacting partners located on the surface of Sertoli cells. Two interactions, APOH/CDC42 and APP/NGFR, were validated in situ, in a proximity ligation assay (PLA). Our results provide new insight into the crosstalk between germ cells and Sertoli cells occurring during spermatogenesis. Our findings also demonstrate that this "integrative omics" strategy is powerful enough for data mining and highlighting meaningful cell-cell communication events between different types of cells in a complex tissue, via a biological fluid. This integrative strategy could be applied more widely, to gain access to secretomes that have proved difficult to study whilst avoiding the limitations of in vitro culture.

Data in support of peptidomic analysis of spermatozoa during epididymal maturation.

The final differentiation of the male germ cell occurs in the epididymal duct where the spermatozoa develop the ability to be motile and fertilize an ovum. Understanding of these biological processes is the key to understanding and controlling male fertility. Comparative studies between several epididymal maturation states could be an informative approach to finding sperm modifications related to maturation and fertility. Here we show the data from differential peptidomic/proteomic analyses on spermatozoa isolated from 4 epididymal regions (immature to mature stage) using a profiling approach based on MALDI-TOF mass spectrometry and, combined to top-down MS in order to identify peptidoforms and proteoforms. By this way, 172m/z peaks ranging between 2 and 20 kDa were found to be modified during maturation of sperm. A total of 62m/z were identified corresponding to 32 different molecular species. The interpretation of these data can be found in the research article published by Labas and colleagues in the Journal of Proteomics in 2014 [1].

New insights into epididymal function in relation to sperm maturation.

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.

The contribution of proteomics to understanding epididymal maturation of mammalian spermatozoa.

The acquisition of the ability of the male gamete to fertilize an ovum is the result of numerous and sequential steps of differentiation of spermatozoa that occur as they transit from the testis to the end of the epididymal tubule. The post gonadal sperm modifications are mostly related to motility, egg binding, and penetration processes. As the activity of the epididymis and its luminal fluid composition are believed to be directly related to 'sperm maturation', a review on epididymal proteins is presented. Comparative studies have shown that the epididymal activities are species specific. Nevertheless, for all mammalian species studied, similarities exist in the sequential proteomic changes of the luminal composition of the epididymal tubule and proteins on the sperm surface. The potential roles of these modifications are discussed.

Epididymal protein Rnase10 is required for post-testicular sperm maturation and male fertility.

Eutherian spermatozoa are dependent on the environment of the proximal epididymis to complete their maturation; however, no specific epididymal factors that mediate this process have so far been identified. Here, we show that targeted disruption of the novel gene Rnase10 encoding a secreted proximal epididymal protein in the mouse results in a binding defect in spermatozoa and their inability to pass through the uterotubal junction in the female. The failure to gain the site of fertilization in the knockout spermatozoa is associated with a gradual loss of ADAM3 and ADAM6 proteins during epididymal transit. In the distal epididymis, these spermatozoa appear to lack calcium-dependent associations with the immobilizing glutinous extracellular material and are released as single, vigorously motile cells that display no tendency for head-to-head agglutination and lack affinity to the oviductal epithelium. In sperm-egg binding assay, they are unable to establish a tenacious association with the zona pellucida, yet they are capable of fertilization. Furthermore, these sperm show accelerated capacitation resulting in an overall in vitro fertilizing ability superior to that of wild-type sperm. We conclude that the physiological role of sperm adhesiveness is in the mechanism of restricted sperm entry into the oviduct rather than in sperm-egg interaction.

Inactivation of AMPKα1 induces asthenozoospermia and alters spermatozoa morphology.

AMP-activated protein kinase (AMPK), a key regulator of cellular energy homeostasis, is present in metabolic tissues (muscle and liver) and has been identified as a modulator of the female reproductive functions. However, its function in the testis has not yet been clearly defined. We have investigated the potential role of AMPK in male reproduction by using transgenic mice lacking the activity of AMPK catalytic subunit α1 gene [α1AMPK knockout (KO)]. In the testis, the α1AMPK subunit is expressed in germ cells and also in somatic cells (Sertoli and Leydig cells). α1AMPK KO male mice show a decrease in fertility, despite no clear alteration in the testis morphology or sperm production. However, in α1AMPK(-/-) mice, we demonstrate that spermatozoa have structural abnormalities and are less motile than in control mice. These spermatozoa alterations are associated with a 50% decrease in mitochondrial activity, a 60% decrease in basal oxygen consumption, and morphological defects. The α1AMPK KO male mice had high androgen levels associated with a 5- and 3-fold increase in intratesticular cholesterol and testosterone concentrations, respectively. High concentrations of proteins involved in steroid production (3ß-hydroxysteroid dehydrogenase, cytochrome steroid 17 alpha-hydroxylase/17,20 lysate, and steroidogenic acute regulatory protein) were also detected in α1AMPK(-/-) testes. In the pituitary, the LH and FSH concentrations tended to be lower in α1AMPK(-/-) male mice, probably due to the negative feedback of the high testosterone levels. These results suggest that total α1AMPK deficiency in male mice affects androgen production and quality of spermatozoa, leading to a decrease in fertility.

Glycocalyx characterisation and glycoprotein expression of Sus domesticus epididymal sperm surface samples.

The sperm surface is covered with a dense coating of carbohydrate-rich molecules. Many of these molecules are involved in the acquisition of fertilising ability. In the present study, eight lectins (i.e. Arachis hypogae (peanut) agglutinin (PNA), Lens culimaris (lentil) agglutinin-A (LCA), Pisum sativum (pea) agglutin (PSA), Triticum vulgari (wheat) germ agglutinin (WGA), Helix pomatia agglutinin (HPA), Phaseolus vulgaris (red kidney bean) leucoagglutinin (PHA-L), Glycine max (soybean) agglutinin (SBA) and Ulex europaeus agglutinin I (UEA-I)) were investigated to identify changes in the nature and localisation of glycoproteins in boar spermatozoa migrating along the epididymal duct. Complementary procedures included measurement of global lectin binding over the surface of the viable sperm population by flow cytometry, analysis of lectin localisation on the membrane of individual spermatozoa using fluorescence microscopy and the electrophoretic characterisation of the major sperm surface glycoprotein receptors involved in lectin binding. A significant increase was found in sperm galactose, glucose/mannose and N-acetyl-d-glucosamine residues distally in the epididymis. Moreover, the sperm head, cytoplasmic droplet and midpiece were recognised by most of the lectins tested, whereas only HPA and WGA bound to the principal piece and end piece of the sperm tail. Fourteen sperm surface proteins were observed with different patterns of lectin expression between epididymal regions. The sperm glycocalyx modifications observed in the present study provide an insight into the molecular modifications associated with epididymal maturation, which may be correlated with the degree of maturation of ejaculated spermatozoa.

Proteomic analysis of mare follicular fluid during late follicle development.

BACKGROUND: Follicular fluid accumulates into the antrum of follicle from the early stage of follicle development. Studies on its components may contribute to a better understanding of the mechanisms underlying follicular development and oocyte quality. With this objective, we performed a proteomic analysis of mare follicular fluid. First, we hypothesized that proteins in follicular fluid may differ from those in the serum, and also may change during follicle development. Second, we used four different approaches of Immunodepletion and one enrichment method, in order to overcome the masking effect of high-abundance proteins present in the follicular fluid, and to identify those present in lower abundance. Finally, we compared our results with previous studies performed in mono-ovulant (human) and poly-ovulant (porcine and canine) species in an attempt to identify common and/or species-specific proteins. METHODS: Follicular fluid samples were collected from ovaries at three different stages of follicle development (early dominant, late dominant and preovulatory). Blood samples were also collected at each time. The proteomic analysis was carried out on crude, depleted and enriched follicular fluid by 2D-PAGE, 1D-PAGE and mass spectrometry. RESULTS: Total of 459 protein spots were visualized by 2D-PAGE of crude mare follicular fluid, with no difference among the three physiological stages. Thirty proteins were observed as differentially expressed between serum and follicular fluid. Enrichment method was found to be the most powerful method for detection and identification of low-abundance proteins from follicular fluid. Actually, we were able to identify 18 proteins in the crude follicular fluid, and as many as 113 in the enriched follicular fluid. Inhibins and a few other proteins involved in reproduction could only be identified after enrichment of follicular fluid, demonstrating the power of the method used. The comparison of proteins found in mare follicular fluid with proteins previously identified in human, porcine and canine follicular fluids, led to the identification of 12 common proteins and of several species-specific proteins. CONCLUSIONS: This study provides the first description of mare follicular fluid proteome during the late follicle development stages. We identified several proteins from crude, depleted and enriched follicular fluid. Our results demonstrate that the enrichment method, combined with 2D-PAGE and mass spectrometry, can be successfully used to visualize and further identify the low-abundance proteins in the follicular fluid.

Expression, immunolocalization and processing of fertilins ADAM-1 and ADAM-2 in the boar (Sus domesticus) spermatozoa during epididymal maturation.

Fertilin alpha (ADAM-1) and beta (ADAM-2) are integral membrane proteins of the ADAM family that form a fertilin complex involved in key steps of the sperm-oocyte membrane interaction. In the present work, we analyzed the presence of ADAM-1 and ADAM-2 mRNAs, the spermatozoa proteins' processing and their sub-cellular localization in epididymal samples from adult boars. ADAM-1 and ADAM-2 mRNAs were highly produced in the testis, but also in the vas efferens and the epididymis. On immunoblots of sperm extracts, ADAM-1 subunit appeared as a main reactive band of ~50-55 kDa corresponding to occurrence of different isoforms throughout the epididymal duct, especially in the corpus region where isoforms ranged from acidic to basic pI. In contrast, ADAM-2 was detected as several bands of ~90 kDa, ~75 kDa, ~50-55 kDa and ~40 kDa. The intensity of high molecular mass bands decreased progressively in the distal corpus where lower bands were also transiently observed, and only the ~40 kDa was observed in the cauda. The presence of bands of different molecular weights likely results from a proteolytic processing occurring mainly in the testis for ADAM-1, and also throughout the caput epididymis for ADAM-2. Immunolocalization showed that fertilin migrates from the acrosomal region to the acrosomal ridge during the sperm transit from the distal corpus to the proximal cauda. This migration is accompanied by an important change in the extractability of a part of ADAM-1 from the sperm membrane. This suggests that the fertilin surface migration may be triggered by the biochemical changes induced by the epididymal post-translational processing of both ADAM1 and ADAM-2. Different patterns of fertilin immunolocalization then define several populations of spermatozoa in the cauda epididymis. Characterization of such fertilin complex maturation patterns is an important step to develop fertility markers based on epididymal maturation of surface membrane proteins in domestic mammals.

The epididymal transcriptome and proteome provide some insights into new epididymal regulations.

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.

Purification and identification of sperm surface proteins and changes during epididymal maturation.

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.

Monotremes provide a key to understanding the evolutionary significance of epididymal sperm maturation.

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.

Identification of luminal and secreted proteins in bull epididymis.

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.

Testicular descent, sperm maturation and capacitation. Lessons from our most distant relatives, the monotremes.

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.

New proteins identified in epididymal fluid from the platypus (Ornithorhynchus anatinus).

The platypus epididymal proteome is being studied because epididymal proteins are essential for male fertility in mammals and it is considered that knowledge of the epididymal proteome in an early mammal would be informative in assessing the convergence and divergence of proteins that are important in the function of the mammalian epididymis. Few of the epididymal proteins that have been identified in eutherian mammals were found in platypus caudal epididymal fluid, and the major epididymal proteins in the platypus (PXN-FBPL, SPARC and E-OR20) have never been identified in the epididymis of any other mammal.

The adult boar testicular and epididymal transcriptomes.

BACKGROUND: Mammalians gamete production takes place in the testis but when they exit this organ, although spermatozoa have acquired a specialized and distinct morphology, they are immotile and infertile. It is only after their travel in the epididymis that sperm gain their motility and fertility. Epididymis is a crescent shaped organ adjacent to the testis that can be divided in three gross morphological regions, head (caput), body (corpus) and tail (cauda). It contains a long and unique convoluted tubule connected to the testis via the efferent ducts and finished by joining the vas deferens in its caudal part. RESULTS: In this study, the testis, the efferent ducts (vas efferens, VE), nine distinct successive epididymal segments and the deferent duct (vas deferens, VD) of four adult boars of known fertility were isolated and their mRNA extracted. The gene expression of each of these samples was analyzed using a pig generic 9 K nylon microarray (AGENAE program; GEO accession number: GPL3729) spotted with 8931 clones derived from normalized cDNA banks from different pig tissues including testis and epididymis. Differentially expressed transcripts were obtained with moderated t-tests and F-tests and two data clustering algorithms based either on partitioning around medoid (top down PAM) or hierarchical clustering (bottom up HCL) were combined for class discovery and gene expression analysis. Tissue clustering defined seven transcriptomic units: testis, vas efferens and five epididymal transcriptomic units. Meanwhile transcripts formed only four clusters related to the tissues. We have then used a specific statistical method to sort out genes specifically over-expressed (markers) in testis, VE or in each of the five transcriptomic units of the epididymis (including VD). The specific regional expression of some of these genes was further validated by PCR and Q-PCR. We also searched for specific pathways and functions using available gene ontology information. CONCLUSION: This study described for the first time the complete transcriptomes of the testis, the epididymis, the vas efferens and the vas deferens on the same species. It described new genes or genes not yet reported over-expressed in these boar tissues, as well as new control mechanisms. It emphasizes and fulfilled the gap between studies done in rodents and human, and provides tools that will be useful for further studies on the biochemical processes responsible for the formation and maintain of the epididymal regionalization and the development of a fertile spermatozoa.

Mammalian epididymal proteome.

In all mammalian species, the final differentiation of the male germ cell occurs in the epididymal duct where the spermatozoa develop the ability to be motile and fertilize an ovum. Understanding of these biological processes is the key to understanding and controlling male fertility. Comparative studies between several mammals could be an informative approach to finding common sperm modifications which are not species-specific. The new global biological approaches such as transcriptomes and proteomes provide considerable information which can be used for such comparative approaches. This report summarizes our proteomic studies of the epididymis of several mammals, including humans.