title: Penicillamine prevents ram sperm agglutination in media that support capacitation.
abstract: Ram spermatozoa are difficult to capacitate in vitro. Here we describe a further complication, the unreported phenomenon of head-to-head agglutination of ram spermatozoa following dilution in the capacitation medium Tyrodes plus albumin, lactate and pyruvate (TALP). Sperm agglutination is immediate, specific and persistent and is not associated with a loss of motility. Agglutination impedes in vitro sperm handling and analysis. So the objectives of this study were to investigate the cause of sperm agglutination and potential agents which may reduce agglutination. The percentage of non-agglutinated, motile spermatozoa increased when bicarbonate was omitted from complete TALP suggesting that bicarbonate ions stimulate the agglutination process. d-penicillamine (PEN), a nucleophilic thiol, was highly effective at reducing agglutination. The inclusion of 250 μM PEN in TALP reduced the incidence of motile, agglutinated spermatozoa from 76.7 ± 2.7% to 2.8 ± 1.4%. It was then assessed if PEN (1 mM) could be included in existing ram sperm capacitation protocols (TALP +1 mM dibutyryl cAMP, caffeine and theophylline) to produce spermatozoa that were simultaneously capacitated and non-agglutinated. This protocol resulted in a sperm population which displayed high levels of tyrosine phosphorylated proteins and lipid disordered membranes (merocyanine-540) while remaining motile, viable, acrosome-intact and non-agglutinated. In summary, PEN (1 mM) can be included in ram sperm capacitation protocols to reduce sperm agglutination and allow for the in vitro assessment of ram sperm capacitation.
authors.forename: T
authors.initials: T
authors.lastname: Leahy
authors.forename: J P
authors.initials: JP
authors.lastname: Rickard
authors.affiliation: Faculty of Veterinary ScienceUniversity of Sydney, Sydney, New South Wales 2006, AustraliaDiscipline of Biological SciencesFaculty of Science and IT and Faculty of Health and Medicine, University of Newcastle, Callaghan, New South Wales 2308, Australia.
authors.forename: R J
authors.initials: RJ
authors.lastname: Aitken
authors.forename: S P
authors.initials: SP
authors.lastname: de Graaf
mesh_terms: D000418:Albumins / Q000494:pharmacology; D000818:Animals; D003470:Culture Media; D007552:Isotonic Solutions / Q000494:pharmacology; D019344:Lactic Acid / Q000494:pharmacology; D008297:Male; D019188:Organ Preservation Solutions / Q000737:chemistry / Q000494:pharmacology; D010396:Penicillamine / Q000494:pharmacology*; D019289:Pyruvic Acid / Q000494:pharmacology; D055101:Semen Analysis / Q000662:veterinary; D012662:Semen Preservation / Q000379:methods* / Q000662:veterinary; D012756:Sheep; D013073:Sperm Agglutination / Q000187:drug effects*; D013075:Sperm Capacitation / Q000187:drug effects*
chemical_list: D000418:Albumins; D003470:Culture Media; D007552:Isotonic Solutions; D019188:Organ Preservation Solutions; C037692:Tyrode's solution; D019344:Lactic Acid; D019289:Pyruvic Acid; D010396:Penicillamine