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Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish
Published on: April 22, 2017
Characterization and in vitro control of MPF activity in zebrafish eggs
Kannika Siripattarapravat1, Angela Busta, Juan Pedro Steibel
1Comparative Medicine and Integrative Biology Program-College of Veterinary Medicine, Michigan State University, East Lansing, Michigan 48824, USA.
Abstract:
We describe the characterization of maturation-promoting factor (MPF) in zebrafish eggs and used different defined conditions to maintain its activity in vitro. MPF activity levels are high in freshly ovulated mature eggs and decline rapidly within 5 min after either fertilization or parthenogenetic activation. The MPF activity of eggs matured in vitro declines faster when the eggs are incubated in Hank's culture medium supplemented with 0.5% BSA (H-BSA) than when incubated in Chinook salmon ovarian fluid (CSOF). MPF activity in nonactivated, aged eggs remains high in H-BSA supplemented with 75 microM MG132 or 10 mM caffeine, but neither MG132 nor caffeine can sustain high MPF activity in activated eggs. MG132-treated eggs showed delayed completion of metaphase and extrusion of the second polar body. Nuclear staining of the activated eggs confirmed the correlation between their cell cycle stage and MPF activity at each time point. An embryotoxic effect was found when matured eggs were held in 100 microM of MG132 or 20 mM caffeine for 1 h. Calcium-depleted medium and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid also showed detrimental effects on the embryos. Conversely, nonactivated, aged matured eggs maintained high MPF activity and developmental potential when CSOF was used as a holding medium.
Insights
Maturation-promoting factor (MPF) activity in zebrafish eggs rapidly declines after activation. Chinook salmon ovarian fluid (CSOF) effectively maintains MPF activity and developmental potential in aged, nonactivated eggs.
Area of Science:
- Developmental Biology
- Cell Cycle Regulation
- Zebrafish Reproduction
Background:
- Maturation-promoting factor (MPF) is crucial for cell cycle progression during oocyte maturation.
- Understanding MPF regulation is key to controlling developmental timing in zebrafish.
- MPF activity is known to be transient in activated eggs.
Purpose of the Study:
- To characterize maturation-promoting factor (MPF) activity in zebrafish eggs.
- To investigate methods for maintaining MPF activity in vitro.
- To assess the impact of different conditions on MPF stability and egg development.
Main Methods:
- Zebrafish eggs were matured in vitro.
- MPF activity was measured under various conditions, including different culture media (H-BSA, CSOF) and the addition of specific compounds (MG132, caffeine).
- Egg activation, nuclear staining, and developmental potential were assessed.
Main Results:
- MPF activity was high in freshly ovulated eggs but declined rapidly post-activation.
- Chinook salmon ovarian fluid (CSOF) maintained MPF activity longer than Hank's culture medium with BSA (H-BSA).
- MG132 and caffeine sustained MPF activity in nonactivated, aged eggs but not in activated eggs, with some compounds showing embryotoxicity.
Conclusions:
- MPF activity is highly sensitive to activation and environmental conditions in zebrafish eggs.
- CSOF is a superior medium for preserving MPF activity and developmental potential in aged, nonactivated zebrafish eggs.
- Specific chemical treatments can prolong MPF activity but may have adverse effects on development.
