Use of Bisection to Reduce Mitochondrial DNA in the Bovine Oocyte

The protocol significantly reduces the mitochondrial DNA copy number in the oocyte. That might be beneficial for intraspecies cloning. This method reduces mitochondrial DNA copy numbers in bovine oocytes by over 90%which could reduce the incidence of mitochondrial heteroplasmy in cloned embryos.

Laura Adams, a PhD student from my lab, will demonstrate the procedure. To begin, using a pipette, collect the selected mature oocytes from the HSOF drop, and place them into the 1.5 milliliter tube containing 50 microliters of the HSOF CB solution. Centrifuge the oocytes at 15, 000 times G for 12 minutes.

While the oocytes are being centrifuged, prepare the bisection plate. For this, start by making a pattern on the lid of a 60 millimeter Petri dish using 20 microliters per drop, and fully cover the drops with mineral oil. Using a thin tipped marker, mark the lines underneath the dish.

Cover the dish with an opaque lid until centrifugation is complete to prevent osmolarity changes of microdrops. As soon as centrifugation is complete, use a 200 microliter pipette to collect the oocytes within the solution, and move them into an empty portion of a new search plate with four, 400 microliter HSOF drops, before washing the oocytes through HSOF drops. Turn the heating stage on to 38.5 degrees Celsius.

Use a mouth pipette to move the centrifuged oocytes from the HSOF drop to the top left T2 drop of the bisection plate. Then wash the oocytes through the next three drops on the top row. Deposit the oocytes in one of the pronase drops, assuring there is little to no contact between them.

Observe the oocytes until there is clear deformation of the zonae pellucidae. Once a single oocyte zona pellucida has become deformed, move that oocyte to the neighboring T2 drop. Repeat as additional oocytes become deformed, until all oocytes have been removed from the pronase and placed in the T2 drop.

Observe the oocytes until only a thin layer of the zona pellucida remains. Wash the oocytes through the next three drops within the row, then deposit them in vertical lines within the CBT20 drops. Begin with fewer oocytes in the vertical lines, and increase the number of oocytes per drop as bisection skills progress.

Focus on the first, leftmost CBT20 drop that contains oocytes, and use a mouth pipette to rotate the oocytes so that the mitochondria-dense portion of each oocyte is either facing toward, or away from the microscope's arm. Rest the tip of the microblade to the left of the topmost oocyte, in line with the space directly above the mitochondria-dense portion. Keeping the tip of the blade in the same place, carefully lower the blade to cut all the way through the oocyte.

Ensure that the mitochondria-dense ooplast and mitochondria-reduced ooplast are of the approximately same size, and the blade is bisecting in the clearest segment of the centrifuged oocyte. When lifting the blade, ensure that the same line is maintained, and that the tip of the blade remains in the same place, then gently lift the tip from the plate. Repeat bisection steps for all oocytes within the first bisection drop.

If oocytes are present in additional drops, orient and bisect the remaining oocytes. Using a mouth pipette, collect mitochondria-reduced ooplasts from the first bisection drop. Place them in the left hand T20 drop located in the bottom row of the plate.

Repeat for all remaining bisection drops. Using a mouth pipette, collect mitochondria dense ooplasts from the first bisection drop. Place them in the right hand T20 drop located in the bottom row of the plate.

Repeat for all remaining bisection drops. Retrieve the T10 four-well dish from the incubator. Using a mouth pipette, move all mitochondria-reduced ooplasts to the well labeled MR, and move the mitochondria-dense ooplasts to the well labeled M.Place the four-well plate back into the carbon dioxide-controlled incubator.

Allow ooplasts to rest for at least 30 minutes prior to quantification of mtDNA. For a successful bisection, the samples from whole oocytes and mitochondria-dense ooplasts have similar CT values. The samples from mitochondria-reduced ooplasts have higher CT values when compared to the samples from the other two groups.

For an unsuccessful bisection, the bisection does not reduce the mtDNA content effectively in the mitochondria-reduced ooplasts. Following the production of a standard curve and the use of the provided mtDNA copy number formulas, an average oocyte mtDNA reduction of 93.88%has been achieved using this protocol. Correctly orienting each oocyte in the bisection drop, precisely bisecting each oocyte, and accurately sorting the ooplasts, are all critical steps to take in order to obtain successful results.

Two ooplasts can be fused with one somatic cell. The fused triplets can then be chemically activated and cultured as reconstructed embryos.