title: Cleaved PARP-1, an Apoptotic Marker, can be Detected in Ram Spermatozoa.
abstract: The presence of apoptotic features in spermatozoa has been related to lower quality and functional impairment. Members of the poly-ADP-ribose polymerases (PARP) familyare involved in both DNA repair and apoptosis, playing important roles in spermatogenesis. Poly-ADP-ribose polymerase can be cleaved by caspases, and the presence of its cleavage product (cPARP) in spermatozoa has been related to chromatin remodelling during spermatogenesis and to the activation of apoptotic pathways. There are no reports on immunodetection of cPARP in ram spermatozoa; thus, we have tested a commercially available antibody for this purpose. cPARP was microscopically detected in the acrosomal ridge of some spermatozoa (indirect immunofluorescence). A preliminary study was carried out by flow cytometry (direct immunofluorescence, FITC). Ram semen was extended in TALP and incubated for 4 h with apoptosis inducers staurosporine (10 μm) or betulinic acid (200 μm). Both inducers and incubation caused a significant increase in cPARP spermatozoa (0 h, control: 21.4±3.3%, inducers: 44.3±1.4%; 4 h, control: 44.3±2.4%, inducers: 53.3±1.4%). In a second experiment, we compared the sperm fractions after density gradient separation (pellet and interface). The pellet yielded a slightly lower proportion of cPARP spermatozoa (28.5±1.2% vs 36.2±2.0% in the interface; p < 0.001), and a 12-h incubation increased cPARP similarly in both fractions (p < 0.001). cPARP seems to be an early marker of apoptosis in ram semen, although its presence in untreated samples was weakly related to worse quality (pellet/interface). We suggest to study the relationship of PARP and cPARP levels with between-male differences on sperm fertility.
authors.affiliation: Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Veterinaria, Instituto Universitario de Investigación en Ciencias Ambientales de Aragón, Universidad de Zaragoza, Zaragoza, Spain.
authors.forename: A
authors.initials: A
authors.lastname: Casao
authors.affiliation: INDEGSAL, University of León, León, Spain.|Department of Molecular Biology, University of León, León, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Mata-Campuzano
authors.affiliation: INDEGSAL, University of León, León, Spain.|Department of Molecular Biology, University of León, León, Spain.
authors.forename: L
authors.initials: L
authors.lastname: Ordás
authors.affiliation: Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Veterinaria, Instituto Universitario de Investigación en Ciencias Ambientales de Aragón, Universidad de Zaragoza, Zaragoza, Spain.
authors.forename: J A
authors.initials: JA
authors.lastname: Cebrián-Pérez
authors.affiliation: Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Veterinaria, Instituto Universitario de Investigación en Ciencias Ambientales de Aragón, Universidad de Zaragoza, Zaragoza, Spain.
authors.forename: T
authors.initials: T
authors.lastname: Muiño-Blanco
authors.affiliation: INDEGSAL, University of León, León, Spain.|Department of Molecular Biology, University of León, León, Spain.
authors.forename: F
authors.identifier: http://orcid.org/0000-0003-2987-4302
authors.initials: F
authors.lastname: Martínez-Pastor
mesh_terms: D000177:Acrosome / Q000201:enzymology; D000818:Animals; D017209:Apoptosis; D015415:Biomarkers; D053148:Caspase 3 / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D004789:Enzyme Activation; D005434:Flow Cytometry / Q000662:veterinary; D019084:Fluorescent Antibody Technique, Indirect / Q000662:veterinary; D008297:Male; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000032:analysis* / Q000378:metabolism*; D012756:Sheep; D013091:Spermatogenesis; D013094:Spermatozoa / Q000201:enzymology* / Q000502:physiology
chemical_list: D015415:Biomarkers; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D053148:Caspase 3; D020169:Caspases