Chromosome transfer in mature oocytes

Abstract

In this article, we describe detailed protocols for the isolation and transfer of spindle-chromosomal complexes between mature, metaphase II-arrested oocytes. In brief, the spindle-chromosomal complex is visualized using a polarized microscope and extracted into a membrane-enclosed karyoplast. Chromosomes are then reintroduced into an enucleated recipient egg (cytoplast), derived from another female, by karyoplast-cytoplast membrane fusion. Newly reconstructed oocytes consist of nuclear genetic material from one female and cytoplasmic components, including mitochondria and mitochondrial DNA (mtDNA), from another female. This approach yields developmentally competent oocytes suitable for fertilization and producing embryonic stem cells or healthy offspring. The protocol was initially developed for monkey oocytes but can also be used in other species, including mouse and human oocytes. Potential clinical applications include mitochondrial gene replacement therapy to prevent transmission of mtDNA mutations and treatment of infertility caused by cytoplasmic defects in oocytes. Chromosome transfer between the cohorts of oocytes isolated from two females can be completed within 2 h.

Figure 1

Equipment and pipette setting for chromosome transfer. (a) Micromanipulation station consisting of an inverted microscope, two micromanipulators, microinjectors, spindle imaging and laser systems controlled by a PC. (b) Setting up the holding pipette. The entire line consists of a metal Narishige pipette holder, Teflon tubing and a 20-ml plastic syringe filled with air. Syringe is mounted on a stand. (c) Setting up the enucleation pipette. The entire line consists of a metal Narishige pipette holder, Teflon tubing and a 250-μl Hamilton glass syringe filled with water. The syringe is mounted on the Narishige microinjection system.

Figure 2

Experimental design for serial spindle–chromosomal complex transfer between two cohorts of oocytes. (a) Suggested layout of micromanipulation drops on the manipulation chamber. (b) Recommended experimental plan for serial chromosome transfer between oocytes from two females. Step 1: Isolate a karyoplast from the first (A) oocyte of female 1 and place it next to the cytoplast until the last transfer. Step 2: Isolate a karyoplast from the first (a) oocyte of female 2, briefly soak in HVJ-E and transfer into a perivitelline space of cytoplast A from female 1. Step 3: Isolate and transfer a karyoplast from the second (B) oocyte of female 1 and transfer it into the cytoplast (a) of female 2. Proceed in a similar manner for Steps 4, 5 and 6. During the last step (Step 7), pick up the karyoplast isolated from the oocyte (A) of female 1 and transfer into the last cytoplast (c) of female 2.

Figure 3

Schematic diagram of the chromosome transfer procedure. Chromosome transfer in MII oocytes consists of two chief procedural steps: (a) isolation of the spindle–chromosomal complex into a karyoplast and (b) introduction of the karyoplast into a recipient cytoplast. Step-by-step manipulations of rhesus monkey oocytes can also be found in Supplementary Video.

Figure 4

Experimental examples of good and poor outcomes during chromosome transfer. (a) Optimal (partial) zonal drilling with laser, leaving a thin intact layer. This will prevent cytoplasm leakage during further manipulations. (b) An example of too large a gap in the zona pellucida. (c) Examples of small (desirable, depicted by arrow) and oversized karyoplasts. (d) Cytoplasmic leakage during karyoplast transfer (or ICSI) as a result of a large zona gap. (e) Good contact between karyoplast and cytoplast that is critical for efficient fusion. (f) An example of poor karyoplast–cytoplast contact due to a large pocket in the perivitelline space.