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

Equine Embryo TransferTABLE 1: Changes in nuclear stage during IVM <strong>of</strong> equine oocytes*Time (h) Number <strong>of</strong> GV Prometaphase M-I M-II Degeneratein culture oocytes (%) (%) (%) (%) (%)0 50 35(70) - - - 15 (30)12 4811(23) 20(42) 2 (4) 1 (2) 14 (29)24 49 1 (2) - 14 (28) 17 (35) 17 (35)36 54 1 (2) - 5 (9) 23 (43) 24 (46)* GV= germinal vesicle; M-I= metaphase –I; MII= metaphase –IIand archived on an erasable magnetic opticaldiskette.RESULTSIn total, 201 oocytes were analysed using <strong>the</strong>CLSM and Table 1 shows <strong>the</strong> number <strong>of</strong> oocytesexamined at <strong>the</strong> different times during in vitromaturation. At <strong>the</strong> onset <strong>of</strong> culture, most <strong>of</strong> <strong>the</strong>oocytes (70%) were in <strong>the</strong> germinal vesicle stage,shown as diffuse chromatin pattern localisedwithin an organelle free area in <strong>the</strong> ooplasm. Atthis stage <strong>of</strong> development, micr<strong>of</strong>ilaments andweakly stained microtubules, were distributedthroughout <strong>the</strong> ooplasma. After 12 h <strong>of</strong> IVM, <strong>the</strong>largest proportion <strong>of</strong> oocytes was inpromethaphase (42%) and individualchromosomes were visible as aggregated dots <strong>of</strong>already condensed chromatin, around which <strong>the</strong>microtubules had concentrated. By contrast, <strong>the</strong>micr<strong>of</strong>ilaments were observed more near <strong>the</strong>cortical region <strong>of</strong> <strong>the</strong> oocyte. After 24 h <strong>of</strong> IVM,<strong>the</strong> oocytes were predominantly in Metaphase I(28%) or Metaphase II (35%) and by 36 h an evengreater proportion had reached Metaphase II(43%). In Metaphase I oocytes, <strong>the</strong> microtubuleswere seen to have organised into elongated asterswhich formed <strong>the</strong> meiotic spindle supporting <strong>the</strong>already aligned chromosomes. In Metaphase II,<strong>the</strong> spindle was observed as a symmetrical, barrelshapedstructure with 2 anastral poles and it wasnow located in <strong>the</strong> periphery <strong>of</strong> <strong>the</strong> cytoplasm with<strong>the</strong> chromosomes aligned along <strong>the</strong> metaphaseplate. Microtubules were only ever detected as<strong>the</strong>se elongated asters in <strong>the</strong> spindle and <strong>the</strong>y werenot detected in any o<strong>the</strong>r areas <strong>of</strong> <strong>the</strong> cytoplasm.During both Metaphases I and II, micr<strong>of</strong>ilamentswere concentrated in <strong>the</strong> oocyte cortex, andespecially micr<strong>of</strong>ilament-rich domains were foundoverlying <strong>the</strong> meiotic spindle, and also around <strong>the</strong>area <strong>of</strong> <strong>the</strong> polar body formation and subsequentlyextrusion. Labelling consistent with <strong>the</strong> presence<strong>of</strong> micr<strong>of</strong>ilament labelling was also detectedwithin <strong>the</strong> zona pellucida <strong>of</strong> <strong>the</strong> evaluated oocytesto varying degrees <strong>of</strong> intensity. A high proportion<strong>of</strong> oocytes were (30% at 0 h), or became (46% at36 h), degenerate during maturation (Table 1) asevidenced by <strong>the</strong>ir aberrant chromatin andcytoskeletal patterns. In <strong>the</strong>se degenerate oocytes,<strong>the</strong> DNA was <strong>of</strong>ten not visible at all or was visibleonly as hairlike strands or scattered small dropswhile <strong>the</strong> microtubules and micr<strong>of</strong>ilaments weredistributed in clusters <strong>of</strong> ei<strong>the</strong>r one or both,scattered throughout <strong>the</strong> ooplasma.DISCUSSIONThe present study enables <strong>the</strong> first description <strong>of</strong>cytoskeletal organisation, and its relationship tochromatin configuration, during <strong>the</strong> process <strong>of</strong> invitro maturation <strong>of</strong> horse oocytes. In summary, weshowed that <strong>the</strong> distribution <strong>of</strong> bothmicr<strong>of</strong>ilaments and microtubules, <strong>the</strong> 2 majorcytoskeletal components <strong>of</strong> a mammalian ovum,change in parallel with <strong>the</strong> process <strong>of</strong>chromosomal alignment and segregation during<strong>the</strong> meiotic maturation process. After <strong>the</strong> germinalvesicle breakdown, <strong>the</strong> microtubules coalesced t<strong>of</strong>orm <strong>the</strong> spindle apparatus and <strong>the</strong>reafter played aclear role in chromosomal segregation andformation <strong>of</strong> <strong>the</strong> first polar body. The aggregationand accumulation <strong>of</strong> micr<strong>of</strong>ilaments in <strong>the</strong> oocytecortex, initially distributed throughout <strong>the</strong>ooplasm, may suggest that <strong>the</strong>y may play asignificant role in <strong>the</strong> migration <strong>of</strong> o<strong>the</strong>r organellesduring cytoplasmic maturation, a range <strong>of</strong>processes that appears to be critical in enabling anoocyte to achieve full developmental competence.During this study, we found that a largeproportion <strong>of</strong> oocytes were (30% at <strong>the</strong> onset <strong>of</strong>maturation), or became (46% after 36 h),degenerate during maturation in vitro. The20