title: Estrous sheep serum enables in vitro capacitation of ram spermatozoa while preventing caspase activation.
abstract: Estrous sheep serum (ESS) is considered the most efficient agent for in vitro capacitation of ram spermatozoa. We have explored the relationship between caspase activation and capacitation in ram. Semen samples from 17 rams were cryopreserved. In vivo fertility was evaluated after intrauterine artificial insemination. Samples were submitted to four treatments: control, ESS (10%), caspase inhibitor (Z-VAD-FMK), and estrous ewe serum plus caspase inhibitor (I + E). Sperm samples were incubated for 30 minutes at 38.5 °C and 5% CO2 and analyzed with flow cytometry for mitochondrial membrane potential (MitoTracker deep red), sperm viability and apoptosis-like changes (YO-PRO-1/propidium iodide), acrosomal status (peanut agglutinin-fluorescein isothiocyanate), membrane fluidity (merocyanine 540), and caspase activity (Vybrant FAM kits for polycaspases, caspase-8, and caspases 3-7). Estrous sheep serum induced changes compatible with capacitation, doubling the proportion of viable spermatozoa with increased merocyanine 540 and increasing YO-PRO-1(+) and acrosome-reacted spermatozoa (P < 0.05). Incubation increased the proportion of spermatozoa with activated caspases (P < 0.05), which was abolished by the treatments. We detected a simultaneous decrease in the proportion of the viable and caspase(-) spermatozoa after the incubation, which was prevented by the presence of estrous ewe serum (P < 0.05). The analysis of caspases 3/7 and 8 resulted in less marked differences. Fertility was positively related to viability and inactivated caspases and negatively to viable-capacitated spermatozoa and active caspases. In vitro induction of capacitation in thawed ram spermatozoa by using ESS suggests a downregulation in apoptotic pathways. However, males with the lowest fertility showed parameters similar to high-fertility males, suggesting that other factors were involved apart from capacitation and/or caspase activation.
authors.affiliation: SaBio IREC (CSIC-UCLM-JCCM), Campus Universitario, Albacete, Spain.
authors.forename: E
authors.initials: E
authors.lastname: Del Olmo
authors.affiliation: SaBio IREC (CSIC-UCLM-JCCM), Campus Universitario, Albacete, Spain.
authors.forename: O
authors.initials: O
authors.lastname: García-Álvarez
authors.affiliation: SaBio IREC (CSIC-UCLM-JCCM), Campus Universitario, Albacete, Spain.
authors.forename: A
authors.initials: A
authors.lastname: Maroto-Morales
authors.affiliation: Regional Center of Animal Selection and Reproduction (CERSYRA) JCCM, Valdepeñas, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Ramón
authors.affiliation: Regional Center of Animal Selection and Reproduction (CERSYRA) JCCM, Valdepeñas, Spain.
authors.forename: P
authors.initials: P
authors.lastname: Jiménez-Rabadán
authors.affiliation: SaBio IREC (CSIC-UCLM-JCCM), Campus Universitario, Albacete, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Iniesta-Cuerda
authors.affiliation: SaBio IREC (CSIC-UCLM-JCCM), Campus Universitario, Albacete, Spain.
authors.forename: L
authors.initials: L
authors.lastname: Anel-Lopez
authors.affiliation: Department of Molecular Biology, University of León, León, Spain; Institute for Animal Health and Cattle Development (INDEGSAL), University of León, León, Spain.
authors.forename: F
authors.initials: F
authors.lastname: Martinez-Pastor
authors.affiliation: SaBio IREC (CSIC-UCLM-JCCM), Campus Universitario, Albacete, Spain.
authors.forename: A J
authors.initials: AJ
authors.lastname: Soler
authors.affiliation: SaBio IREC (CSIC-UCLM-JCCM), Campus Universitario, Albacete, Spain.
authors.forename: J J
authors.initials: JJ
authors.lastname: Garde
authors.affiliation: SaBio IREC (CSIC-UCLM-JCCM), Campus Universitario, Albacete, Spain; Faculty of Pharmacy (UCLM), Albacete, Spain. Electronic address: mrocio.fernandez@uclm.es.
authors.forename: M R
authors.initials: MR
authors.lastname: Fernández-Santos
mesh_terms: D000818:Animals; D017209:Apoptosis; D020169:Caspases / Q000502:physiology*; D015925:Cryopreservation / Q000662:veterinary; D004789:Enzyme Activation / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004971:Estrus / Q000097:blood*; D005260:Female; D005298:Fertility / Q000502:physiology; D007315:Insemination, Artificial / Q000662:veterinary; D008297:Male; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary; D012756:Sheep / Q000097:blood*; D013075:Sperm Capacitation / Q000502:physiology*; D013094:Spermatozoa / Q000502:physiology / Q000648:ultrastructure
chemical_list: D004791:Enzyme Inhibitors; D020169:Caspases
keywords: Apoptosis; Capacitation; Estrous sheep serum; Ram