Research on feline in vitro maturation and the cell cycle

Abstract

The feline meiotic cycle is poorly understood. In order to elucidate the events occurring during meiosis in the cat oocyte, a study of the levels of Maturation Promoting Factor (MPF) and MAP Kinase (MAPK), enzymes thought to be necessary to induce oocyte maturation, is important. However, before MPF levels can be studied, a method for maturing feline oocytes in vitro efficiently must be perfected. Furthermore, the information gathered from this study will be beneficial for research on the conservation of endangered feline species and for other reproductive techniques. This study focused on identifying an efficient method of maturing feline oocytes in vitro and observing MPF and MAPK levels at different stages of maturation and after activation in the feline oocyte. A study of the comparative efficiencies of different maturation media was conducted using feline oocytes collected from spayed feline tracts. Three different previously published maturation medium protocols were chosen and compared to determine which produced the highest rate of maturation. As a control, oocytes were put into the base maturation medium without the addition of hormones. The oocytes were incubated for 24 hours in humidified 5% CO2 in air. The oocytes were scored to determine the stage of meiosis they had achieved--germinal vesicle stage, metaphase I, or metaphase II. Only oocytes reaching the metaphase II stage were considered matured. The results of this experiment showed that increasing hormone concentrations in the culture medium increases the rate of feline oocyte maturation. To study the cell cycle and MPF and MAPK activity during meiosis, oocytes from the germinal vesicle, metaphase I, and metaphase II stages of meiosis were collected after maturation in the medium determined to be most efficient, as well as activated oocytes. Activated oocytes were obtained by activating mature oocytes with an ionomycin/cycloheximide treatment. Levels of MPF and MAPK activity at different stages in the feline cell cycle were determined by SDS-PAGE after MPF and MAPK reactions. The results of this study show that MPF and MAPK activities are low at the germinal vesicle stage of meiosis and high at both metaphase I and metaphase II in the feline oocyte. Furthermore, the activity of MPF and MAPK decreases after activation until the cell starts mitotic divisions when the levels start to rise again.