CENTRIFUGATION OF BOVINE OOCYTES FOR NUCLEAR MICROMANIPULATION AND SPERM MICROINJECTION

The reproductive biotechnologies of intracytoplasmic sperm microinject ion, nuclear transfer and DNA microinjection require the visualization of cytoplasmic components and nuclei of oocytes and early embryonic c ells, Bovine oocytes were matured and fertilized in vitro, and then ce ntrifuged at the germinal vesicle, metaphase If and pronuclear stages and at syngamy, These (n = 536) were examined using light and transmis sion electron microscopy, The organelles stratified in five distinct z ones in a consistent pattern in both oocytes and zygotes, though relat ive fractions changed in organelle composition after fertilization. Th ese comprised a centripetal lipid zone, below which was a vesicular zo ne, then a supra-equatorial band of smooth endoplasmic reticulum (SER) , a clear zone and a centrifugal mitochondrial zone, Cortical granules were located peripherally, single or clumped together, in the clear a nd mitochondrial zones, The nuclei were usually found associated with the SER or Golgi membranes, and chromatin was clumped at one pole with in the nucleus, The maturation spindles were often located beneath the oolemma in all zones, while the first mitotic spindle was usually loc ated in the clear zone, Some of the oocytes were activated by centrifu gation and completed maturation.