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Novel Proteomic Profiling of Epididymal Extracellular Vesicles in the Domestic Cat Reveals Proteins Related to Sequential Sperm Maturation with Differences Observed between Normospermic and Teratospermic Individuals

2020; Elsevier BV; Volume: 19; Issue: 12 Linguagem: Inglês

10.1074/mcp.ra120.002251

ISSN

1535-9484

Autores

Tricia Rowlison, Timothy P. Cleland, Mary Ann Ottinger, Pierre Comizzoli,

Tópico(s)

Reproductive System and Pregnancy

Resumo

Extracellular vesicles (EVs) secreted by the epididymal epithelium transfer to spermatozoa key proteins that are essential in promoting motility and subsequent fertilization success. Using the domestic cat model, the objectives were to (1) characterize and compare protein content of EVs between segments of the epididymis, and (2) compare EV protein compositions between normo- and teratospermic individuals (producing >60% of abnormal spermatozoa). Epididymal EVs from adult cats were isolated and assessed via liquid chromatography tandem MS. Both male types shared 3008 proteins in total, with 98 and 20 EV proteins unique to normospermic and teratospermic males, respectively. Expression levels of several proteins changed between epididymal segments in both male types. Several proteins in both groups were related to sperm motility (e.g. hexokinase 1, adenylate kinase isoenzyme) and zona pellucida or oolemma binding (e.g. disintegrin and metalloproteinase domain proteins, zona binding proteins 1 and 2). Interestingly, seven cauda-derived EV proteins trended downward in teratospermic compared with normospermic males, which may relate to poor sperm quality. Collective results revealed, for the first time, EV proteins related to sequential sperm maturation with differences observed between normospermic and teratospermic individuals. Extracellular vesicles (EVs) secreted by the epididymal epithelium transfer to spermatozoa key proteins that are essential in promoting motility and subsequent fertilization success. Using the domestic cat model, the objectives were to (1) characterize and compare protein content of EVs between segments of the epididymis, and (2) compare EV protein compositions between normo- and teratospermic individuals (producing >60% of abnormal spermatozoa). Epididymal EVs from adult cats were isolated and assessed via liquid chromatography tandem MS. Both male types shared 3008 proteins in total, with 98 and 20 EV proteins unique to normospermic and teratospermic males, respectively. Expression levels of several proteins changed between epididymal segments in both male types. Several proteins in both groups were related to sperm motility (e.g. hexokinase 1, adenylate kinase isoenzyme) and zona pellucida or oolemma binding (e.g. disintegrin and metalloproteinase domain proteins, zona binding proteins 1 and 2). Interestingly, seven cauda-derived EV proteins trended downward in teratospermic compared with normospermic males, which may relate to poor sperm quality. Collective results revealed, for the first time, EV proteins related to sequential sperm maturation with differences observed between normospermic and teratospermic individuals. Mammalian sperm cells undergo structural modifications throughout epididymal transit. This includes sperm membrane changes as well as addition of key surface proteins which are critical in sperm progression through the cumulus cells, binding and crossing of the zona pellucida, and finally binding and fuzing with the oolemma (1Dacheux J.-L. Dacheux F. New insights into epididymal function in relation to sperm maturation.Reproduction. 2014; 147: R27-R42Crossref PubMed Scopus (197) Google Scholar, 2Martin-DeLeon P.A. Epididymal SPAM1 and its impact on sperm function.Mol., and Cell Endo. 2006; 250: 114-121Crossref PubMed Scopus (64) Google Scholar, 3Sullivan R. Saez F. 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Classical protein secretion by the epididymal epithelium is achieved through the merocrine pathway in which proteins contain signal sequences and are secreted individually from the epithelium (1Dacheux J.-L. Dacheux F. New insights into epididymal function in relation to sperm maturation.Reproduction. 2014; 147: R27-R42Crossref PubMed Scopus (197) Google Scholar, 13Nicander L. Malmqvist M. Ultrastructural observations suggesting merocrine secretion in the initial segment of the mammalian epididymis.Tissue Cell. 1977; 184: 487-490Google Scholar). Once secreted into the luminal fluid, proteins bind with the sperm surface before being incorporated into the cell. Another secretion pathway has been identified in which proteins without a signal sequence are secreted within membranous vesicles termed, extracellular vesicles (EV), ranging in size from approximately 30–300 nm (3Sullivan R. Saez F. 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While not as severe, these secondary abnormalities still greatly diminish sperm quality and overall fertility. Comparing EV protein content of teratospermic males to the normospermic baseline will further elucidate the underlying basis of teratospermia, which is rampant in the 38 species of felids that are listed as threatened or endangered as reported by the International Union for Conservation of Nature (39IUCN Red List of Threatened Species, Version 2020.1.http://www.iucnredlist.orgGoogle Scholar). The domestic cat serves as an ideal model because it is a close relative of wild felids. Furthermore, studies in the domestic cat have improved our basic understanding of human reproductive physiology, including potential physiological sources of teratospermia (40Wildt D.E. Comizzoli P. Pukazhenthi B. Songsasen N. Lessons from biodiversity—the value of nontraditional species to advance reproductive science, conservation, and human health.Mol. Reprod, Devel. 2010; 77: 397-409Crossref PubMed Scopus (91) Google Scholar). The objectives of the study were to (1) characterize and compare protein content of EVs between segments of the cat epididymis, and (2) compare EV protein compositions between normo- and teratospermic adults. All chemicals and reagents were purchased from Sigma Company (St. Louis, MO), unless otherwise stated. The study did not require the approval of the Animal Care and Use Committee of the Smithsonian Conservation Biology Institute because cat testes were collected at local veterinary clinics as byproducts from owner-requested routine orchiectomies. Adult (>1 year) domestic cat testis samples were supplied by local veterinary clinics following routine orchiectomy (n = 20 male tracts total). Tracts were transported and stored in Phosphate Buffered Saline (PBS) at 4 °C until processing within a 24 h period. Epididymal tissues were then removed from the rest of the testis in PBS using a scalpel blade until further processing. A small sample of spermatozoa was isolated by making one 3 mm incision into the distal end of the cauda segment (41Axnér E. Linde-Forsberg C. Einarsson S. Morphology and motility of spermatozoa from different regions of the epididymal duct in the domestic cat.Theriogenology. 1999; 52: 767-778Crossref PubMed Scopus (60) Google Scholar). The sample was then fixed by diluting 1:1 with 4% paraformaldehyde (PFA) and stained with Coomassie Brilliant Blue R-250 (0.1% Coomassie Brilliant Blue R-250, 50% methanol, and 10% glacial acetic acid), then mounted with Permount™ Mounting Medium (Fisher Scientific, Pittsburgh, PA). A proportion of sperm cells were recorded, analyzing 200 cells total per each individual male. Individuals were considered teratospermic if ≥ 60% of cauda sperm cells had a morphological abnormality (34Howard J. Brown J.L. Bush M. Wildt D.E. Teratospermic and normospermic domestic cats: ejaculate traits, pituitary-gonadal hormones, and improvement of spermatozoal motility and morphology after swim-up processing.Andrology. 1990; 11: 204-215Google Scholar). To first assess overall abnormalities in this sample type, sperm cells were isolated from the cauda segment of individuals until a sample size of n = 5 normospermic males, and n = 7 teratospermic males were attained, and the different morphological abnormalities recorded. It should be noted that the prevalence of teratospermia in the samples used in this study does not represent the overall prevalence of the general domestic cat population. A separate set of samples were then assessed for the collection of EVs (n = 4 normospermic males, and n = 4 teratospermic males). EV samples were subsequently collected within an hour to reduce sample loss and protein degradation. Following identification of whether a sample was normospermic or teratospermic, entire epididymides were separated into the different, consecutive, segments (caput, corpus, cauda) (41Axnér E. Linde-Forsberg C. Einarsson S. Morphology and motility of spermatozoa from different regions of the epididymal duct in the domestic cat.Theriogenology. 1999; 52: 767-778Crossref PubMed Scopus (60) Google Scholar) and further minced using a scalpel blade in PBS (n = 4 normospermic males, and n = 4 teratospermic males). Luminal fluid was allowed to seep for 5–10 min before collected into microcentrifuge tubes. Cell debris was discarded from the supernatant by a series of centrifugations at 700 × g for 10 min and 3000 × g for 10 min at room temperature, with the supernatant transferred to a new microcentrifuge tube following each centrifugation. The EV fraction was isolated from the remaining luminal fluid by ultracentrifugation at 100,000 × g for 2 h at 4 °C and re-suspended in fresh PBS (Beckman Coulter Optima l-90K, SW 55 Ti rotor, 3.5 ml polycarbonate tubes catalog number: 349622, filled to 3 ml each, with full dynamic braking to 0 rpm, Kadj = 88). Aliquots of EV samples were then stored at −20 °C. Samples were processed, and analyzed via MS (described below) within one month's time. Successful isolation of EVs was previously confirmed via observations performed using a transmission electron microscope (Zeiss 10 CA Transmission Electron Microscope) at the University of Maryland Laboratory for Biological Ultrastructure (26Rowlison T. Ottinger M.A. Comizzoli P. Key factors enhancing sperm fertilizing ability are transferred from the epididymis to the spermatozoa via EVs in the domestic cat model.Assist. Reprod. Genetics. 2018; 35: 221-228Crossref PubMed Scopus (24) Google Scholar). All relevant data of collection may be found in the EV-TRACK knowledgebase EV-TRACK ID: EV200074, https://evtrack.org/. It should be noted that this study is using the operative term, Extracellular Vesicle (EV), in accordance to the guidelines set forth by the International Society for Extracellular Vesicles (17Théry C. Witwer K.W. Aikawa E. Alcaraz M.J. Anderson J.D. Andriantsitohaina R. Antoniou A. Arab T. Archer F. Atkin-Smith G.K. Ayre D.C. Bach J.-M. Bachurski D. Baharvand H. Balaj L. Baldacchino S. Bauer N.N. Baxter A.A. Bebawy M. Beckham C. Bedina Zavec A. Benmoussa A. Berardi A.C. Bergese P. Bielska E. Blenkiron C. Bobis-Wozowicz S. Boilard E. Boireau W. Bongiovanni A. Borràs F.E. Bosch S. Boulanger C.M. Breakefield X. Breglio A.M. Brennan M.Á. Brigstock D.R. Brisson A. Broekman M.L. Bromberg J.F. Bryl-Górecka P. Buch S. Buck A.H. Burger D. Busatto S. Buschmann D. Bussolati B. Buzás E.I. Byrd J.B. Camussi G. Carter D.R. Caruso S. Chamley L.W. Chang Y.-T. Chen C. Chen S. Cheng L. Chin A.R. Clayton A. Clerici S.P. Cocks A. Cocucci E. Coffey R.J. Cordeiro-da-Silva A. Couch Y. Coumans F.A. Coyle B. Crescitelli R. Criado M.F. D'Souza-Schorey C. Das S. Datta Chaudhuri A. de Candia P. De Santana E.F

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