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Summary

Here, we present a protocol to significantly reduce the mitochondrial DNA copy numbers in a bovine oocyte (P < 0.0001). This method utilizes centrifugation and bisection to substantially reduce oocyte mitochondria and may allow for an increased chance of development in the reconstructed interspecies somatic cell nuclear transfer embryos.

Abstract

Interspecies somatic cell nuclear transfer (iSCNT) may be used to rescue endangered species, but two distinct populations of mitochondrial DNA (mtDNA) exist within the reconstructed embryo: one within the recipient ooplasm and one within the donor somatic cell. This mitochondrial heteroplasmy can lead to developmental issues in the embryo and the fetus. Handmade cloning protocols include oocyte bisection, which can be used to decrease the mtDNA copy number, reducing the degree of mitochondrial heteroplasmy in a reconstructed embryo. Centrifugation of denuded, mature bovine oocytes produced a visible mitochondria-dense fraction at one pole of the oocyte. Oocytes' zonae pellucidae were removed by exposure to a pronase solution. Bisection was performed using a microblade to remove the visible mitochondria fraction. qPCR was used to quantify the mtDNA present in DNA samples extracted from whole oocytes and bisected ooplasts, providing a comparison of mtDNA copy numbers before and after bisection. Copy numbers were calculated using cycle threshold values, a standard curve's regression line formula, and a ratio that included the respective sizes of mtDNA PCR products and genomic PCR products. One bovine oocyte had an average mtDNA copy number (± standard deviation) of 137,904 ± 94,768 (n = 38). One mitochondria-depleted ooplast had an average mtDNA copy number of 8,442 ± 13,806 (n = 33). Average mtDNA copies present in a mitochondria-rich ooplast were 79,390 ± 58,526 mtDNA copies (n = 28). The differences between these calculated averages indicate that the centrifugation and subsequent bisection can significantly decrease the mtDNA copy numbers present in the mitochondria-depleted ooplast when compared to the original oocyte (P < 0.0001, determined by one-way ANOVA). The reduction in mtDNA should decrease the degree of mitochondrial heteroplasmy in a reconstructed embryo, possibly fostering standard embryonic and fetal development. Supplementation with mitochondrial extract from the somatic donor cell may also be essential to achieve successful embryonic development.

Introduction

Somatic cell nuclear transfer (SCNT) includes the fusion of an enucleated oocyte from one animal and a somatic cell from an animal of the same species. In most cases, the oocyte and somatic cell originate from the same species, and live birth rates are below 6%1. Some research involves the use of interspecies SCNT (iSCNT), which includes the fusion of a somatic cell and oocyte that originate from two different species. In these studies, live birth rates are even lower than in SCNT-typically less than 1%1. However, iSCNT has the capacity to be used as a method of rescuing endangered species, since somatic cells from these....

Protocol

The following protocol follows the animal care and ethics guidelines provided by Utah State University.

1. Media preparation

  1. Prior to oocyte handling, prepare the following solutions, as described in Table 1: 400 µL of Hyaluronidase Solution, 500 µL of T2 media, 1,020 µL of T20 media, and 800 µL of T10 media.
  2. Divide the T10 media into two wells of a four-well plate, 400 µL per well. Label one well with "M".......

Representative Results

Quantitative PCR (qPCR) results are used to determine the relative quantities of mtDNA present in each ooplast. The described reaction is designed to amplify the 12S region of bovine mtDNA.

If the bisection was successful, the samples from whole oocytes and mitochondria-dense ooplasts will have similar Ct values. The samples from mitochondria-reduced ooplasts will have higher Ct values when compared to the samples from the other two groups. A Ct graph showing s.......

Discussion

Methods previously used to decrease mtDNA copy numbers in oocytes have their respective disadvantages. Micromanipulation-based removal of mitochondria from oocytes decrease mtDNA copy numbers by an average of 64%27. A unique method, previously used for enucleation, involves the use of small diameter Pasteur pipettes and the splitting of a zona pellucida-free oocyte at the border between a microdrop of media and the surrounding mineral oil. Along with the utilization of oocyte centrifugation, this .......

Acknowledgements

The authors wish to thank their colleagues at Utah State University, the Reproductive Science researchers at the San Diego Zoo, and Dr. Rebecca Krisher at Genus PLC.

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Materials

NameCompanyCatalog NumberComments
1.5 mL centrifuge tubesFisher Scientific5408129
60 mm dishSigma-AldrichD8054
CentrifugeEppendorf5424
Cytochalasin BSigma-AldrichC6762
Fetal Bovine SerumSigma-AldrichF2442
M199 MediaSigma-AldrichM4530
Mineral OilSigma-AldrichM8410
Mini CentrifugeSCILOGEXD1008
mtDNA Primer: Forward (12S)GGGCTACATTCTCTACACCAAG
mtDNA Primer: Reverse (12S)GTGCTTCATGGCCTAATTCAAC
NanoDrop SpectrophotometerThermo ScientificND2000
Opthalmic Scalpel with Aluminum HandlePFM Medical207300633Microblade for bisection
Protease/pronaseSigma-AldrichP5147
QIAamp DNA Micro KitQiagen56304
QuantStudio™ 3 - 96-Well 0.2-mLThermoFisherA28567
Search plateFisher ScientificFB0875711A
SYBR Green qPCR Master MixThermoFisherK0221qPCR master mix
Synthetic Oviductal Fluid with HEPES (HSOF)
ThermoPlateTokai HitTPi-SMZSSXHeating stage

References

  1. Loi, P., Modlinski, J. A., Ptak, G. Interspecies somatic cell nuclear transfer: A salvage tool seeking first aid. Theriogenology. 76 (2), 217-228 (2011).
  2. Wani, N. A., Vettical, B. S., Hong, S. B.

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