Centrifugation of bovine oocytes for nuclear micromanipulation and sperm microinjection

Abstract

The reproductive biotechnologies of intracytoplasmic sperm microinjection, nuclear transfer and DNA microinjection require the visualization of cytoplasmic components and nuclei of oocytes and early embryonic cells. Bovine oocytes were matured and fertilized in vitro, and then centrifuged at the germinal vesicle, metaphase II and pronuclear stages and at syngamy. These (n = 536) were examined using light and transmission electron microscopy. The organelles stratified in five distinct zones in a consistent pattern in both oocytes and zygotes, though relative fractions changed in organelle composition after fertilization. These comprised a centripetal lipid zone, below which was a vesicular zone, 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 and mitochondrial zones. The nuclei were usually found associated with the SER or Golgi membranes, and chromatin was clumped at one pole within the nucleus. The maturation spindles were often located beneath the oolemma in all zones, while the first mitotic spindle was usually located in the clear zone. Some of the oocytes were activated by centrifugation and completed maturation.