Class
Article
College
College of Agriculture and Applied Sciences
Department
Animal, Dairy, and Veterinary Sciences Department
Faculty Mentor
Irina Polejaeva
Presentation Type
Poster Presentation
Abstract
Mitochondrial heteroplasmy, the presence of more than one population of mitochondrial DNA (mtDNA) in one cell, can cause metabolic issues and reduced ATP synthesis, even at relatively low levels. The presence of this condition in somatic cell nuclear transfer (SCNT) embryos likely contributes to reduced prenatal and postnatal viability: the majority of the oocyte’s mitochondria is still present at the time of nuclear transfer, as well as through any further embryonic development post-fusion and activation. Though the somatic cell also contributes its mitochondria (which can successfully communicate with its nucleus), the oocyte’s mitochondria compose a greater proportion of the total amount of mitochondria within the embryo. As an attempt to reduce the incidence of mitochondrial heteroplasmy in these SCNT embryos, we are using handmade cloning techniques to remove a large fraction of the oocyte’s mitochondria prior to fusion with the somatic cell. The oocytes (matured in IVM media for 21 hours post-collection from abattoir-derived ovaries) are stripped of their cumulus cells and centrifuged at an optimized relative centrifugal force (15000g for 12 minutes). The centrifugation forces the mitochondria to one end of the oocyte, creating a fraction that is visible via microscopy. Oocytes are then bisected to remove as much of this mitochondrial fraction as possible. Two mitochondria-reduced ooplasts (oocyte halves) are fused around a single somatic cell before the embryo is chemically activated in ionomycin (5 μM) for 4.5 minutes, then in cycloheximide (10 μg/ml) and 6-diethylaminopurine (2 mM) for 4 hours. The embryo is cultured in SOF-aa media in a 5% O2, 5% CO2 incubator set at 39.5˚C in a well-of-the-well (WOW) system. The zona pellucida is removed using pronase (5 mg/ml) prior to bisection, so WOW culture prevents the embryos from sticking together, while being close enough to one another to allow for inter-embryonic communication. Thus far, multiple embryos constructed using this method have reached the blastocyst stage in vitro, indicating that this could be a viable method of reducing the incidence of mitochondrial heteroplasmy in SCNT embryos. Presentation Time: Wednesday, 9-10 a.m. Zoom link: https://usu-edu.zoom.us/j/81298203941?pwd=WXZkRjhqdlZNTVlidXk3UnB1K2VtUT09
Location
Logan, UT
Start Date
4-12-2021 12:00 AM
Included in
Using Handmade Cloning to Reduce Oocyte Mitochondria in Reconstructed Embryos
Logan, UT
Mitochondrial heteroplasmy, the presence of more than one population of mitochondrial DNA (mtDNA) in one cell, can cause metabolic issues and reduced ATP synthesis, even at relatively low levels. The presence of this condition in somatic cell nuclear transfer (SCNT) embryos likely contributes to reduced prenatal and postnatal viability: the majority of the oocyte’s mitochondria is still present at the time of nuclear transfer, as well as through any further embryonic development post-fusion and activation. Though the somatic cell also contributes its mitochondria (which can successfully communicate with its nucleus), the oocyte’s mitochondria compose a greater proportion of the total amount of mitochondria within the embryo. As an attempt to reduce the incidence of mitochondrial heteroplasmy in these SCNT embryos, we are using handmade cloning techniques to remove a large fraction of the oocyte’s mitochondria prior to fusion with the somatic cell. The oocytes (matured in IVM media for 21 hours post-collection from abattoir-derived ovaries) are stripped of their cumulus cells and centrifuged at an optimized relative centrifugal force (15000g for 12 minutes). The centrifugation forces the mitochondria to one end of the oocyte, creating a fraction that is visible via microscopy. Oocytes are then bisected to remove as much of this mitochondrial fraction as possible. Two mitochondria-reduced ooplasts (oocyte halves) are fused around a single somatic cell before the embryo is chemically activated in ionomycin (5 μM) for 4.5 minutes, then in cycloheximide (10 μg/ml) and 6-diethylaminopurine (2 mM) for 4 hours. The embryo is cultured in SOF-aa media in a 5% O2, 5% CO2 incubator set at 39.5˚C in a well-of-the-well (WOW) system. The zona pellucida is removed using pronase (5 mg/ml) prior to bisection, so WOW culture prevents the embryos from sticking together, while being close enough to one another to allow for inter-embryonic communication. Thus far, multiple embryos constructed using this method have reached the blastocyst stage in vitro, indicating that this could be a viable method of reducing the incidence of mitochondrial heteroplasmy in SCNT embryos. Presentation Time: Wednesday, 9-10 a.m. Zoom link: https://usu-edu.zoom.us/j/81298203941?pwd=WXZkRjhqdlZNTVlidXk3UnB1K2VtUT09