Proceedings of the 5th International Symposium on EQUINE ...

Havemeyer Foundation Monograph Series No. 3ORGANISATION OF THE CYTOSKELETON DURINGIN VITRO MATURATION OF HORSE OOCYTESJ. L.Tremoleda, E. J. Schoevers*, T. A. E. Stout, B. Colenbrander andM. M. Bevers*Department <strong>of</strong> Equine Sciences, *Department <strong>of</strong> Farm Animal Health, Faculty <strong>of</strong> Veterinary Medicine,Utrecht University, Utrecht, The Ne<strong>the</strong>rlandsINTRODUCTIONMeiotic maturation is a complex process duringwhich <strong>the</strong> oocyte must undergo a series <strong>of</strong> nuclearand cytoplasmic changes in order to produce aviable, fertilisable and developmentally competentovum (Albertini et al. 1993). This process involves<strong>the</strong> breakdown <strong>of</strong> <strong>the</strong> germinal vesicle andreorganisation and segregation <strong>of</strong> <strong>the</strong>chromosomes with formation <strong>of</strong> <strong>the</strong> meioticstructures and fur<strong>the</strong>r extrusion <strong>of</strong> <strong>the</strong> polar body.These changes are associated with a completereorganisation <strong>of</strong> <strong>the</strong> cytoskeleton <strong>of</strong> <strong>the</strong> oocytewhich in o<strong>the</strong>r species has been described in terms<strong>of</strong> changes in <strong>the</strong> distribution <strong>of</strong> <strong>the</strong> microtubulesand micr<strong>of</strong>ilaments (mouse: Messinger et al. 1991;pig: Kim et al. 1996; man: Kim et al. 1998).Despite this important role in oocyte development,little information is available with regard to <strong>the</strong>cytoskeletal changes that take place during <strong>the</strong>meiotic maturation <strong>of</strong> equine oocytes. The aim <strong>of</strong>this study was to examine <strong>the</strong> changes in <strong>the</strong>distribution <strong>of</strong> microtubules and micr<strong>of</strong>ilamentsand <strong>the</strong> relationship <strong>of</strong> <strong>the</strong>se cytoskeletal elementsto chromatin configuration, during in vitromaturation <strong>of</strong> horse oocytes.MATERIALAND METHODSCumulus oocyte complexes (COCs) wererecovered from <strong>the</strong> ovaries <strong>of</strong> slaughtered maresby aspirating follicles smaller than 30 mm indiameter. Once recovered, COCs were washed inHEPES-buffered Tyrodes medium containing0.1% polyvinylalcohol and 0.2% BSA and <strong>the</strong>nevaluated under a stereomicroscope. Only oocyteswith a complete, compact, multilayered cumulusinvestment were selected for culture. Theseoocytes were incubated in M199 mediumsupplemented with 10% FCS, 0.01 units/mlporcine FSH and 0.01 units/ml equine LH at 39ºCin a humidified atmosphere <strong>of</strong> 5% CO 2 in air. After0, 12, 24 and 36 h <strong>of</strong> culture, COCs were denudedby vortexing in a calcium-free 0.25% solution <strong>of</strong>trypsin in EBSS. The oocytes were <strong>the</strong>n washed inPBS and permeabilised, for 1 h at 39ºC, usingmedium M, a glycerol-based microtubulestabilisingsolution (Simerly and Schatten 1993).Next, <strong>the</strong> oocytes were fixed for 30 min in 2%paraformaldehyde in PBS at room temperature and<strong>the</strong>y were <strong>the</strong>n maintained at 4ºC for 2–5 daysprior staining. With regard to <strong>the</strong> stainingtechniques employed, first <strong>the</strong> microtubules werelabelled by incubating fixed oocytes for 90 min at37ºC with a monoclonal anti-tubulin antibody(Sigma) diluted 1:250 in PBS. After incubation,<strong>the</strong> oocytes were washed several times in PBScontaining 0.1% BSA (Sigma) and <strong>the</strong>n incubatedfor 1 h in a blocking solution (Simerly andSchatten 1993). Then <strong>the</strong> oocytes were exposed toa secondary antibody conjugated totetramethylrhodamine isothiocyanate (TRITC) for1 h at 37ºC. Once <strong>the</strong> microtubules had been thuslabelled, <strong>the</strong> oocytes were incubated for 1 h withAlexa Fluor 488 phalloidin to enable detection <strong>of</strong><strong>the</strong> micr<strong>of</strong>ilaments and for 15 min with TO-PRO 3(Molecular Probes) to allow visualisation <strong>of</strong> <strong>the</strong>DNA. Finally <strong>the</strong> stained oocytes were mountedon glass microscope slides with an antifadesuspension. The oocytes were examined using alaser scanning confocal microscope, equippedwith a krypto-argon ion laser which was able tosimultaneously excite TRITC for <strong>the</strong> visualisation<strong>of</strong> <strong>the</strong> microtubules, Alexa Fluor 488 for <strong>the</strong>micr<strong>of</strong>ilaments, and TO-PRO 3 for <strong>the</strong> DNA,respectively. The images were recorded digitally19