16 Use of a hand-made cloning protocol to reduce oocyte mitochondria

A significant issue in interspecies somatic cell nuclear transfer (iSCNT) is mitochondrial heteroplasmy. Two distinct populations (one within the recipient ooplasm and one within the donor somatic cell) exist within the reconstructed embryo, which can lead to developmental issues in the embryo and the fetus. Hand-made cloning protocols include oocyte bisection, which can be used to decrease the mitochondrial DNA (mtDNA) copy number in the oocyte, therefore reducing the incidence of mitochondrial heteroplasmy in the reconstructed embryo. This study determines whether a significant number of mtDNA copies can be removed from a bisected oocyte, which would allow this protocol to reduce heteroplasmy. Denuded mature bovine oocytes were centrifuged at 15,000 × g for 12 min to produce a visible mitochondria-dense fraction at one end of the oocyte. Oocytes’ zonae pellucidae were removed by exposure to a pronase solution (5 mg mL⁻¹). Bisection was performed using a microblade to remove the visible mitochondria fraction. Quantitative PCR (qPCR) was used to quantify the mtDNA present in whole oocytes and bisected ooplasts, providing a comparison of mtDNA copy numbers before and after bisection. DNA was extracted from somatic cells, oocytes, and ooplasts using the manufacturer protocols (QIAamp DNA Micro Kit; Qiagen). Before oocyte and ooplast mtDNA quantification, a standard curve (efficiency = 93%, R² = 0.97) was created using the qPCR results of DNA extracted from bovine somatic cell dilutions. Subsequent qPCR runs included DNA extracted from whole oocytes and bisected ooplasts, which contained either the majority or the minority of the oocyte’s mitochondria. Copy numbers were calculated using cycle threshold values, the standard curve’s regression line formula, and a ratio that included the respective sizes of mtDNA PCR products (12S rRNA gene) and genomic PCR products (GAPDH). The average (±SD) mtDNA copy number in one bovine oocyte (n = 10) was 45 565 ± 37 169. The average mtDNA copy number in an ooplast with mitochondrial numbers purposely reduced was 8396 ± 13 287 (n = 28). An ooplast containing the majority of the oocyte’s mitochondria had an average of 74 653 ± 58 877 mtDNA copies (n = 26). These results indicate that the bisection of a bovine oocyte following centrifugation can significantly decrease the mtDNA copy numbers present in the ooplast compared with the original oocyte (P < 0.0001, determined by one-way ANOVA) and with the ooplast with a concentrated portion of mitochondria (P < 0.0001, determined by one-way ANOVA). This modification would decrease the incidence of mitochondrial heteroplasmy in the reconstructed embryo, promoting proper embryonic and fetal development. Supplementation with mitochondrial extract from the somatic cell donor cell may be essential to achieve embryonic development.