Computer-aided meiotic maturation assay (CAMMA) of zebrafish (danio rerio) oocytes in vitro.
We have developed a new technique called Computer-Aided Meiotic Maturation Assay (CAMMA) for imaging large arrays of zebrafish oocytes and automatically collecting image files at regular intervals during meiotic maturation. This novel method uses a transparency scanner interfaced to a computer with macro programming that automatically scans and archives the image files. Images are stacked and analyzed with ImageJ to quantify changes in optical density characteristic of zebrafish oocyte maturation. Major advantages of CAMMA include (1) ability to image very large arrays of oocytes and follow individual cells over time, (2) simultaneously image many treatment groups, (3) digitized images may be stacked, animated, and analyzed in programs such as ImageJ, NIH-Image, or ScionImage, and (4) CAMMA system is inexpensive, costing less than most microscopes used in traditional assays. We have used CAMMA to determine the dose response and time course of oocyte maturation induced by 17alpha-hydroxyprogesterone (HP). Maximal decrease in optical density occurs around 5 hr after 0.1 micro g/ml HP (28.5 degrees C), approximately 3 hr after germinal vesicle migration (GVM) and dissolution (GVD). In addition to changes in optical density, GVD is accompanied by streaming of ooplasm to the animal pole to form a blastodisc. These dynamic changes are readily visualized by animating image stacks from CAMMA; thus, CAMMA provides a valuable source of time-lapse movies for those studying zebrafish oocyte maturation. The oocyte clearing documented by CAMMA is correlated to changes in size distribution of major yolk proteins upon SDS-PAGE, and, this in turn, is related to increased cyclin B(1) protein.
Lessman
Charles A
CA
The University of Memphis, Department of Biology, Memphis, TN 38152-3540, USA. Clessman@memphis.edu
Nathani
Ravikanth
R
Uddin
Rafique
R
Walker
Jamie
J
Liu
Jianxiong
J
D000818:Animals; D020732:Cytogenetic Analysis / Q000379:methods*; D005260:Female; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008540:Meiosis; D009865:Oocytes / Q000166:cytology*; D046888:Tissue Array Analysis / Q000379:methods*; D015027:Zebrafish / Q000254:growth & development*