Discussion:
Lipid microdomains are small, organized structures made of cholesterol and sphingolipids in the cell membrane of a cell (Pike, 2003). Isolation of lipid microdomains can occur with sucrose density gradients and ultracentrifugation. Because the components of lipid microdomains are less dense, they remain “floating” on the extracellular leaf of the plasma membrane. Fractions 1 – 4 of a western blot are the lowest densities of the sucrose gradients and are thought to be where lipid microdomain reside because those fractions are the least dense and capture the lower weight proteins. This is such because the top four fractions contain the most caveolin and flotillin of the fractions as seen in our results.
Since caveolin and flotillin are associated with lipid microdomains, the top four fractions are more likely the cell membrane. Fractions 5 – 10 are considered where the heavier proteins reside and can be thought of cytosolic proteins. If the addition of fish oils enriched with omega-3 fatty acids do affect lipid microdomains, then we should see a shift of caveolin and flotillin to the bulk lipids. If that shift occurs, then we can postulate that fish oils with omega-3 fatty acids do disrupt lipid microdomains.
In controls, caveolin and flotillin are found mostly in fractions 1 to 4 of the sucrose gradient. After adding fish oils which contain omega-3 fatty acids, caveolin and flotillin were no longer located in fractions 1 – 4 and seemed to have shifted to fractions 5 – 10. We postulate that this happens because the fatty acid is pushing the proteins, cholesterol, and sphingolipids of the lipid microdomain elsewhere to make room for itself in the system. This causes a dispersal of the lipid microdomains and therefore causes the system to become unorganized. When the lipid microdomain is no longer organized, the apoptotic hormone PGF2α cannot dock to its receptors and signal the cell’s death and regression of the CL. This is a controversy, though, due to the size of the microdomain. They are so small (5 nm – 500 nm) that we cannot isolate just one alone, even with the assistance of a microscope. Since we cannot see or isolate them, lipid microdomains are rather difficult to work with and investigating if disruption of lipid microdomains occurs (Calder, 2007).
With the dispersion of plasma membranes and possibly lipid microdomains, researchers have been able to inhibit known cellular signaling pathways in many different cell lines. In our experiment, we are looking at inhibiting the apoptotic PGF2α cell signaling pathway. This inhibition of PGF2α pathway might be suggested by our findings resulting in a shift of the known proteins associated with lipid microdomains. Thus, allowing the CL and the bovine embryo more time to establish and maintain a healthy pregnancy to full term. Omega-3 fatty acids in fish oils might be a novel and inexpensive approach to improve bovine maternal recognition of pregnancy from our conclusions and warrant further investigation.
In conclusion, a shift in the proteins caveolin and flotillin from fractions 1-4 to fractions 5-10 was seen and this was postulated as due to the omega-3 fatty acids incorporating into the lipid microdomains. This shift of the proteins associated with lipid microdomains support our hypothesis. Due to these results, it is reasonable to continue further experiments with fish oils containing omega-3 fatty acids and lipid microdomains on the bovine CL. If we can find a novel and inexpensive method to attenuate PGF2α and inhibit CL regression through fish oils, then the pregnant cow may have more time be able to identify the embryos interferon-t signal and maternal recognition of pregnancy would be accepted. With improvement of bovine reproduction, a reduction of cost to the U.S. beef and dairy industries can occur and these savings ultimately follow through to the consumer.
Since caveolin and flotillin are associated with lipid microdomains, the top four fractions are more likely the cell membrane. Fractions 5 – 10 are considered where the heavier proteins reside and can be thought of cytosolic proteins. If the addition of fish oils enriched with omega-3 fatty acids do affect lipid microdomains, then we should see a shift of caveolin and flotillin to the bulk lipids. If that shift occurs, then we can postulate that fish oils with omega-3 fatty acids do disrupt lipid microdomains.
In controls, caveolin and flotillin are found mostly in fractions 1 to 4 of the sucrose gradient. After adding fish oils which contain omega-3 fatty acids, caveolin and flotillin were no longer located in fractions 1 – 4 and seemed to have shifted to fractions 5 – 10. We postulate that this happens because the fatty acid is pushing the proteins, cholesterol, and sphingolipids of the lipid microdomain elsewhere to make room for itself in the system. This causes a dispersal of the lipid microdomains and therefore causes the system to become unorganized. When the lipid microdomain is no longer organized, the apoptotic hormone PGF2α cannot dock to its receptors and signal the cell’s death and regression of the CL. This is a controversy, though, due to the size of the microdomain. They are so small (5 nm – 500 nm) that we cannot isolate just one alone, even with the assistance of a microscope. Since we cannot see or isolate them, lipid microdomains are rather difficult to work with and investigating if disruption of lipid microdomains occurs (Calder, 2007).
With the dispersion of plasma membranes and possibly lipid microdomains, researchers have been able to inhibit known cellular signaling pathways in many different cell lines. In our experiment, we are looking at inhibiting the apoptotic PGF2α cell signaling pathway. This inhibition of PGF2α pathway might be suggested by our findings resulting in a shift of the known proteins associated with lipid microdomains. Thus, allowing the CL and the bovine embryo more time to establish and maintain a healthy pregnancy to full term. Omega-3 fatty acids in fish oils might be a novel and inexpensive approach to improve bovine maternal recognition of pregnancy from our conclusions and warrant further investigation.
In conclusion, a shift in the proteins caveolin and flotillin from fractions 1-4 to fractions 5-10 was seen and this was postulated as due to the omega-3 fatty acids incorporating into the lipid microdomains. This shift of the proteins associated with lipid microdomains support our hypothesis. Due to these results, it is reasonable to continue further experiments with fish oils containing omega-3 fatty acids and lipid microdomains on the bovine CL. If we can find a novel and inexpensive method to attenuate PGF2α and inhibit CL regression through fish oils, then the pregnant cow may have more time be able to identify the embryos interferon-t signal and maternal recognition of pregnancy would be accepted. With improvement of bovine reproduction, a reduction of cost to the U.S. beef and dairy industries can occur and these savings ultimately follow through to the consumer.