104 3-D VISUALIZATION OF THE FIRST CLEAVAGE AND ABERRATIONS OF BOVINE ZYGOTES BY CONFOCAL LASER SCANNING MICROSCOPY
A. A. Gratao A , A. Beck B , M. Reichenbach E , H. D. Reichenbach D , E. Wolf B C and F. A. Habermann AA Institute of Anatomy, Histology and Embryology, Department of Veterinary Sciences, Ludwig-Maximilians-University Munich, Munich, Bavaria, Germany;
B Chair for Molecular Animal Breeding and Biotechnology, Department of Veterinary Sciences, Ludwig-Maximilians-University Munich, Munich, Bavaria, Germany;
C Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, Ludwig-Maximilians-University Munich, Munich, Bavaria, Germany;
D Institute for Animal Breeding, Bavarian State Research Center for Agriculture, Poing, Bavaria, Germany;
E Bavarian Research Center for Biology of Reproduction, Oberschleissheim, Bavaria, Germany
Reproduction, Fertility and Development 25(1) 199-200 https://doi.org/10.1071/RDv25n1Ab104
Published: 4 December 2012
Abstract
A high proportion of bovine oocytes fertilized in vitro fail to develop beyond the first 4 cleavage cycles. The first mitotic division of the zygote and proper segregation of chromosomes and cytoplasmic components seems to be a particularly delicate task. Notably, zygotes cleaving with a delay of only a few hours seem to have a very low chance of developing to the blastocyst stage. But what exactly goes wrong, how often, and why? To answer such questions we have to visualize in greater detail basic structures and processes such as the sperm aster, DNA replication, migration and apposition of the 2 pronuclei, synchronous chromosome condensation and breakdown of the nuclear envelopes, assembly of the first mitotic spindle and chromosome congression, anaphase, and cytokinesis. Oocytes fertilized in vitro were fixed at different time points around the first cleavage and stained for DNA, Ser10-phosphorylated histone H3, microtubules, and microfilaments. Zygotes were imaged in toto by recording confocal serial sections at 1-µm intervals using a 40× objective (NA = 1.3). Details were recorded with high spatial sampling densities (pixel size 50 × 50 nm, z-step size of 200 nm) close to the Nyquist criterion and restored by maximum likelihood estimation deconvolution using the real point spread function. We present a series of 3-D confocal images captured at different stages of the first cleavage. The images reveal new insights into the formation, structure, and function of the first mitotic spindle and the occurrence of spindle aberrations, irregular chromosome segregation, and abnormal cytokinesis. The microscopic findings guide us to candidate proteins for localization analyses and functional studies based on 3-D fluorescence live-cell imaging of zygotes and early embryos.
This work is supported by the Deutsche Forschungsgemeinschaft (DFG FOR 1041).
