N OCIETY' - the Society for Reproductive Biology

REGULATION OF PROTEIN TYROSINE PHOSPHORYLATION, DURING IN VITRO AGING OFEJACULATED BOVINE SPERMATOZOA.J. Krzyzosiak1,2*, G. McMillan 1 , R. Vishwanath l and P. Molan 2lUvestock Improvement Corporation, Private Bag 3016, Hamilton, New Zealand, 2Department of Biological Sciences,University ofWaikato, Private Bag 3105, Hamilton, New ZealandINTRODUCTIONTyrosine phosphorylation of various sperm proteinsplays a role in regulation of motility, capacitation, andzona pellucida binding of sperm (1). Reactive oxygenspecies promote tyrosine phosphorylation of spermproteins during capacitation (2). The objective was todetermine if changes in protein-tyrosinephosphorylation occur during in vitro storage, and ifthey are affected by the oxygenation state of the storagemedium. The study was extended to determine whetherprotein phosphorylation taking place during storageunder anaerobic and aerobic conditions are reversiblewhen spermatozoa are transferred from oneenvironment to another.METHODSSemen collected from three bulls, was diluted in acommercial diluent (CAPROGEN®) (3), divided andstored under anaerobic or aerobic conditions at ambienttemperature (18-20°C). On day 5 of in vitro storage,one half of the semen aged under anaerobic conditionswas transferred from an anaerobic chamber to anaerobic environment. Similarly, one half of the semenaged under aerobic conditions was transferred to theanaerobic chamber. Proteins were extracted from cellsharvested and washed on days 6, 9, and 12 of storageunder the four conditions. Tyrosine-phosphorylatedproteins were separated by SDS-PAGE and imrnunodetectedon a Western blot with 4G10 antiphosphotyrosineantibody using an ECL detectionsystem.RESULTSWe observed a time dependent increase in tyrosinephosphorylation of number of proteins under anaerobicbut not under aerobic conditions. When sperm agedunder anaerobic conditions were transferred to theaerobic environment, proteins that were phosphorylatedup to this point were dephosphorylated duringsubsequent aerobic incubation. Conversely when sperminitially incubated under aerobic conditions weretransferred to an anaerobic environment, proteins whichdid not display increased protein tyrosinephosphorylation until this point became progressivelyphosphorylated on tyrosine residues (Fig. 1).DISCUSSIONBoth protein tyrosine kinase and phosphatase activitiesare affected by the presence of O 2 in the medium, butthe effect is the opposite of that which is documentedfor sperm undergoing capacitation (4). Changes intyrosine phosphorylation are very slow (12 days) when78compared to typical receptor mediated tyrosinephosphorylation (15 s), or those observed in ejaculatedbovine, sperm incubated under capacitating conditions(4 h) (1).o 6 9 12 6 9 12 6 9 12 6 9 12Fig. 1 Western blot showing tyrosine-phosphorylatedproteinsfrom: semen aged all the time under anaerobicconditions (lanes 2-4), semen aged all the time underaerobic conditions (lanes 5-7), semen transferred onday five from an anaerobic to an aerobic environment(lanes 8-10), semen transferred on day five from anaerobic to an anaerobic environment (lanes 11-13).Storage time (days) is displayed under the blot.In conclusion, an increase in protein tyrosinephosphorylation during ageing is probably differentfrom the one observed during capacitation since, unlikethe latter, it is inhibited in the presence of O 2 and isreversed upon transfer of sperm cells from an anaerobicto aerobic environment. Furthermore, it occurs across amuch longer time span than capacitation. Theidentification of proteins that undergo tyrosinephosphorylation changes during ageing would enable abetter understanding of the role they play in thisprocess.REFERENCESl.Galantino-Homer HL, Visc~nti PE, Kopf GS,BiolofReprod, 1997; 56: 707-719.2.Aitken RJ, Paterson M, Fisher H, Buckingham DW,van Duin M J Cell Sci, 1995; 108: 2017-2025.3.Shannon P, J Dairy Sci, 1965; 48: 1357-1361.4.Aitken R, Buckingham D, Harkiss D, Paterson M,Fisher H, Irvine D, Mol Cell Endocrinol, 1996; 17:83­93.THE EFFECT OF IN VITRO STORAGE ON MITOCHONDRIAL MARKER ENZYME ACTIVITYIN BOVINE SPERMATOZOAtD. V. R. Bullen I . 2 , P. MolanI, and R. Vishwanath 2lUniversity of \Vaikato, Department ofBiological Sciences, Private Bag 3105, Hamilton 2001, New Zealand, 2LivestockImprovement Corporation Ltd., Private Bag 3016, Hamilton 2001, New ZealandINTRODUCTIONBovine sperm lose fertility rapidly during in vitrostorage at room temperature. Motility is alsogradually lost but at a notably lower rate (1). It hasbeen proposed that the more rapid loss of fertilitymay be due to the effects of reactive oxygenspecies (ROS) that may be generated in the courseof respiration.As part of a larger investigation into the effectsof storage in vitro on various biochemicalparameters of sperm mitochondria, the activity ofthree mitochondrial marker enzymes wasmonitored.The enzyme assays described in the literaturehave been applied almost exclusively to somaticmitochondria or purified enzymes (2-4). For thisreason, work was firstly carried out to determinewhether the assays could be reliably applied to anenzyme preparation extracted from spermmitochondria. Then, enzyme activity wasmonitored at regular intervals, throughout a periodof storage.The aim of this study was to determine whetherthe activity of mitochondrial marker enzymes waschanging during the storage period and to assessthe effect of the storage conditions (aerobic oranaerobic) on any changes taking place.METHODSSemen was collected from mature Friesian bullsand diluted in 14G buffer (5). The semen wasstored either aerobically or anaerobically, at roomtemperature (20°C).Sperm extracts were prepared, for use in theenzyme assays, by sonicating sperm samples in anextraction buffer containing 200mM Tris, 2%sodium citrate, and 0.1% Triton X-100. Theenzyme activity of sperm extracts were analysed at0,6, 12, and 20 days after collection.Three enzymes were assayed: citrate synthase(2); cytochrome c oxidase (3); ATP synthetase(4).RESULTSOver a period of 20 days the activity of bothcitrate synthase and ATP synthetase decreasedsignificantly (for both enzymes p < 0.001). Forcitrate synthase the difference in storageatmosphere had a significant effect on activity (p =0.005), with activity decreasing more in theaerobically stored semen than anaerobically stored.The third enzyme, cytochrome c oxidase, showedan increase in activity over time, with thedifference in storage atmosphere causing asignificant difference in activity (p = 0.001; Fig. 1).The increase in cytochrome c oxidase activity wassignificant for sperm stored anaerobically (p