Sperm acquire epididymis-derived proteins through epididymosomes.

STUDY QUESTION: Are epididymosomes implicated in protein transfer from the epididymis to spermatozoa? SUMMARY ANSWER: We characterized the contribution of epididymal secretions to the sperm proteome and demonstrated that sperm acquire epididymal proteins through epididymosomes. WHAT IS KNOWN ALREADY: Testicular sperm are immature cells unable to fertilize an oocyte. After leaving the testis, sperm transit along the epididymis to acquire motility and fertilizing abilities. It is well known that marked changes in the sperm proteome profile occur during epididymal maturation. Since the sperm is a transcriptional and translational inert cell, previous studies have shown that sperm incorporate proteins, RNA and lipids from extracellular vesicles (EVs), released by epithelial cells lining the male reproductive tract. STUDY DESIGN, SIZE, DURATION: We examined the contribution of the epididymis to the post-testicular maturation of spermatozoa, via the production of EVs named epididymosomes, released by epididymal epithelial cells. An integrative analysis using both human and mouse data was performed to identify sperm proteins with a potential epididymis-derived origin. Testes and epididymides from adult humans (n = 9) and adult mice (n = 3) were used to experimentally validate the tissue localization of four selected proteins using high-resolution confocal microscopy. Mouse epididymal sperm were co-incubated with carboxyfluorescein succinimidyl ester (CFSE)-labeled epididymosomes (n = 4 mice), and visualized using high-resolution confocal microscopy. PARTICIPANTS/MATERIALS, SETTING, METHODS: Adult (12-week-old) C57BL/CBAF1 wild-type male mice and adult humans were used for validation purposes. Testes and epididymides from both mice and humans were obtained and processed for immunofluorescence. Mouse epididymal sperm and mouse epididymosomes were obtained from the epididymal cauda segment. Fluorescent epididymosomes were obtained after labeling the epididymal vesicles with CFSE dye followed by epididymosome isolation using a density cushion. Immunofluorescence was performed following co-incubation of sperm with epididymosomes in vitro. High-resolution confocal microscopy and 3D image reconstruction were used to visualize protein localization and sperm-epididymosomes interactions. MAIN RESULTS AND THE ROLE OF CHANCE: Through in silico analysis, we first identified 25 sperm proteins with a putative epididymal origin that were conserved in both human and mouse spermatozoa. From those, the epididymal origin of four sperm proteins (SLC27A2, EDDM3B, KRT19 and WFDC8) was validated by high-resolution confocal microscopy. SLC27A2, EDDM3B, KRT19 and WFDC8 were all detected in epithelial cells lining the human and mouse epididymis, and absent from human and mouse seminiferous tubules. We found region-specific expression patterns of these proteins throughout the mouse epididymides. In addition, while EDDM3B, KRT19 and WFDC8 were detected in both epididymal principal and clear cells (CCs), SLC27A2 was exclusively expressed in CCs. Finally, we showed that CFSE-fluorescently labeled epididymosomes interact with sperm in vitro and about 12-36% of the epididymosomes contain the targeted sperm proteins with an epididymal origin. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The human and mouse sample size was limited and our results were descriptive. The analyses of epididymal sperm and epididymosomes were solely performed in the mouse model due to the difficulties in obtaining epididymal luminal fluid human samples. Alternatively, human ejaculated sperm and seminal EVs could not be used because ejaculated sperm have already contacted with the fluids secreted by the male accessory sex glands, and seminal EVs contain other EVs in addition to epididymosomes, such as the abundant prostate-derived EVs. WIDER IMPLICATIONS OF THE FINDINGS: Our findings indicate that epididymosomes are capable of providing spermatozoa with a new set of epididymis-derived proteins that could modulate the sperm proteome and, subsequently, participate in the post-testicular maturation of sperm cells. Additionally, our data provide further evidence of the novel role of epididymal CCs in epididymosome production. Identifying mechanisms by which sperm mature to acquire their fertilization potential would, ultimately, lead to a better understanding of male reproductive health and may help to identify potential therapeutic strategies to improve male infertility. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Spanish Ministry of Economy and Competitiveness (Ministerio de Economía y Competividad; fondos FEDER 'una manera de hacer Europa' PI13/00699 and PI16/00346 to R.O.; and Sara Borrell Postdoctoral Fellowship, Acción Estratégica en Salud, CD17/00109 to J.C.), by National Institutes of Health (grants HD040793 and HD069623 to S.B., grant HD104672-01 to M.A.B.), by the Spanish Ministry of Education, Culture and Sports (Ministerio de Educación, Cultura y Deporte para la Formación de Profesorado Universitario, FPU15/02306 to F.B.), by a Lalor Foundation Fellowship (to F.B. and M.A.B.), by the Government of Catalonia (Generalitat de Catalunya, pla estratègic de recerca i innovació en salut, PERIS 2016-2020, SLT002/16/00337 to M.J.), by Fundació Universitària Agustí Pedro i Pons (to F.B.), and by the American Society for Biochemistry and Molecular Biology (PROLAB Award from ASBMB/IUBMB/PABMB to F.B.). Confocal microscopy and transmission electron microscopy was performed in the Microscopy Core facility of the Massachusetts General Hospital (MGH) Center for Systems Biology/Program in Membrane Biology which receives support from Boston Area Diabetes and Endocrinology Research Center (BADERC) award DK57521 and Center for the Study of Inflammatory Bowel Disease grant DK43351. The Zeiss LSM800 microscope was acquired using an NIH Shared Instrumentation Grant S10-OD-021577-01. The authors have no conflicts of interest to declare.

Junctional adhesion molecule A: expression in the murine epididymal tract and accessory organs and acquisition by maturing sperm.

STUDY QUESTION: Is junctional adhesion molecule A (JAM-A), a sperm protein essential for normal motility, expressed in the murine post-testicular pathway and involved in sperm maturation? SUMMARY ANSWER: JAM-A is present in the prostate and seminal vesicles and in all three regions of the epididymis where it is secreted in epididymosomes in the luminal fluid and can be delivered to sperm in vitro. WHAT IS KNOWN ALREADY: JAM-A shares with the plasma membrane Ca2+ATPase 4 (PMCA4, the major Ca2+ efflux pump in murine sperm) a common interacting partner, CASK (Ca2+/CaM-dependent serine kinase). JAM-A, like PMCA4, plays a role in Ca2+ regulation, since deletion of Jam-A results in significantly elevated intracellular Ca2+ levels and reduced sperm motility. Recently, PMCA4 was reported to be expressed in the epididymis and along with CASK was shown to be in a complex on epididymosomes where it was transferred to sperm. Because of the association of JAM-A with CASK in sperm and because of the presence of PMCA4 and CASK in the epididymis, the present study was performed to determine whether JAM-A is expressed in the epididymis and delivered to sperm during their maturation. STUDY DESIGN, SIZE, DURATION: The epididymides, prostate and seminal vesicles were collected from sexually mature C57BL/6J and Institute for Cancer Research mice and antibodies specific for JAM-A and Ser285 -phosphorylated JAM-A (pJAM-A) were used for the analysis. Tissues, sperm and epididymal luminal fluid (ELF) were studied. Epididymosomes were also isolated for study. Caput and caudal sperm were co-incubated with ELF individually to determine their abilities to acquire JAM-A in vitro. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sections of all three regions of the epididymis were subjected to indirect immunofluorescence analysis. Epididymal tissues, fluid, sperm, prostate and seminal vesicle tissues were analyzed for JAM-A and/or pJAM-A via western blotting analysis. The relative amounts of JAM-A and pJAM-A among epididymal tissues, ELF and sperm were detected by western blot via quantification of band intensities. Epididymosomes were isolated by ultracentrifugation of the ELF after it was clarified to remove cells and tissue fragments, and the proteins western blotted for JAM-A and pJAM-A, and exosomal biochemical markers. FACS analysis was used to quantify the amount of JAM-A present on caput and caudal sperm, as well as the amount of JAM-A acquired in vitro after their co-incubation with ELF. MAIN RESULTS AND THE ROLE OF CHANCE: Western blots revealed that JAM-A is expressed in all three regions of the epididymis, the prostate and seminal vesicles. As confirmed by indirect immunofluorescence, a western blot showed that JAM-A has a higher expression in the corpus and caudal regions, where it is significantly (P < 0.01) more abundant than in the caput. Both JAM-A and Ser285-phosphorylated JAM-A (pJAM-A) are secreted into the ELF where it is highest in the distal regions. In the ELF, both JAM-A and pJAM-A were detected in epididymosomes. Western blotting of sperm proteins showed a significant (P < 0.01) increase of JAM-A and pJAM-A in caudal, compared with caput, sperm. Flow-cytometric analysis confirmed the increase in JAM-A in caudal sperm where it was 1.4-fold higher than in caput ones. Co-incubation of caput and caudal sperm with ELF demonstrated ~2.3- and ~1.3-fold increases, respectively, in JAM-A levels indicating that epididymosomes transfer more JAM-A to caput sperm that are less saturated with the protein than caudal ones. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: First, although the ELF was clarified prior to ultracentrifugation for epididymosome isolation, we cannot rule out contamination of the epididymosomal proteins by those from epididymal epithelial cells. Second, the JAM-A detected in the prostate and seminal vesicles might not necessarily be secreted from those organs and may only be present within the tissues, where it would be unable to impact sperm in the ejaculate. WIDER IMPLICATIONS OF THE FINDINGS: Although performed in the mouse the study has implications for humans, as the highly conserved JAM-A is a signaling protein in human sperm. There is physiological significance to the finding that JAM-A, which regulates sperm motility and intracellular Ca2+, exists in elevated levels in the cauda where sperm gain motility and fertilizing ability. The study suggests that the acquisition of JAM-A in the epididymal tract is involved in the mechanism by which sperm gain their motility during epididymal maturation. This increased understanding of sperm physiology is important for aspects of ART. STUDY FUNDING AND COMPETING INTEREST(S): The work was supported by NIH-RO3HD073523 and NIH-5P20RR015588 grants to P.A.M.-D. The authors declare there are no conflicts of interests.

Development and Preservation of Avian Sperm.

Terminally differentiated avian sperm consist of a head which male genetic material locates and flagellum that provides the motive force to propel them towards the fertilization site. The apical end of the sperm head accommodates a secretory vesicle, called an acrosome, that undergoes acrosome reaction releasing proteolytic content to penetrate the peri-vitelline membrane of an egg. Transcriptionally and translationally inactive, sperm need to rely on these distinct compartments in which different functions are preassembled, in order to achieve the goal of "fertilization". How are these complex structures with high functionality formed? Spermatogenesis is divided into an early stage in which diploid spermatogonia is proliferated into round spermatids thorough mitotic and meiotic divisions, and a late stage in which round spermatids are transformed into sperm though nuclear condensation and elongation of the sperm head, and formation of accessory structures. Recently, it was reported in aves that morphologically differentiated sperm undergo post-testicular maturation during passage through the male genital tract, suggesting that a similar system to mammals might be involved in the acquisition of fertilizing ability in avian sperm. Investigation for mechanisms underlying how sperm regulate their functions which are necessary to achieve fertilization is important for developing reproductive biotechnology in aves, because cryopreservation of poultry sperm is still not reliable for use in commercial production or for the preservation of genetic resources. In this review, we firstly provide an update on avian spermatogenesis, and then discuss the uniqueness of structure and functions of avian sperm, highlighting differences from mammalian sperm. Lastly, we discuss the molecular mechanism and current techniques of cryopreservation for avian sperm.

Proteomics: A valuable approach to elucidate spermatozoa post -testicular maturation in the endangered Acipenseridae family.

Proteomics techniques, such as two-dimensional polyacrylamide gel electrophoresis, mass spectrometry, and differential gel electrophoresis, have been extensively used to describe the protein composition of male gametes in different animals, mainly mammals. They have also provided a deeper understanding of protein functions involved in sperm processes, as in processes that in humans lead to male infertility. However, few studies focus on fish sperm proteomics and even fewer have tried to explore the proteomic profile of Sturgeon spermatozoa. Sturgeon is an endangered, ancient group of fish species exploited mostly for caviar. In this fish group, a part of the process that leads to final functional maturation of spermatozoa so as to have the capability to activate eggs during the fertilization process. This process has a broad similarity to post-testicular maturation in mammals; where spermatozoa leaving the testes must be mixed with seminal fluid along the transit through the Wolffian ducts to modify its surface membrane protein composition, leading to axonemal and acrosomal competence. The aim of this study was to review the current literature on various proteomic techniques, their usefulness in separating, identifying and studying the proteome composition of the fish spermatozoon, as well as their potential applications in studying the post-testicular maturation process in Sturgeon. Such understanding could lead to development of more sophisticated aquaculture techniques, favorable for sturgeon reproduction.

Sperm maturation in sturgeon (Actinopterygii, Acipenseriformes): A review.

The morphology of the reproductive system of acipenseriform fishes is quite different from that of teleostean species, but an associated unique physiological difference in male sturgeons was not discovered until recently; sperm of sturgeons passes through the kidneys then via Wolffian ducts into the environment rather that emptying directly through seminal ducts. The mixing of sperm with excretory products has been found to be a requisite for the capacity to be activated (maturation step) instead of being deleterious. In the current review we summarize results of studies performed in our laboratory on physiological processes involved in sturgeon sperm maturation, namely changes in: 1) ionic environment; 2) sensitivity of spermatozoa to calcium ions (Ca2+); 3) antioxidant enzymes and proteolytic activities; and 4) content in macroergic phosphates arising during this maturation process. We also discuss taxa-specific aspects of sturgeon sperm maturation in relation to hormonal regulation of spermiation, and the unusual features of sturgeon sperm maturation relative to using testicular sturgeon sperm in aquaculture.