Manipulating the Mitochondrial Genome To Enhance Cattle Embryo Development

Abstract

The mixing of mitochondrial DNA (mtDNA) from the donor cell and the recipient oocyte in embryos and offspring derived from somatic cell nuclear transfer (SCNT) compromises genetic integrity and affects embryo development. We set out to generate SCNT embryos that inherited their mtDNA from the recipient oocyte only, as is the case following natural conception. While SCNT blastocysts produced from Holstein (Bos taurus) fibroblasts were depleted of their mtDNA, and oocytes derived from Angus (Bos taurus) cattle possessed oocyte mtDNA only, the coexistence of donor cell and oocyte mtDNA resulted in blastocysts derived from nondepleted cells. Moreover, the use of the reprogramming agent, Trichostatin A (TSA), further improved the development of embryos derived from depleted cells. RNA-seq analysis highlighted 35 differentially expressed genes from the comparison between blastocysts generated from nondepleted cells and blastocysts from depleted cells, both in the presence of TSA. The only differences between these two sets of embryos were the presence of donor cell mtDNA, and a significantly higher mtDNA copy number for embryos derived from nondepleted cells. Furthermore, the use of TSA on embryos derived from depleted cells positively modulated the expression of CLDN8, TMEM38A, and FREM1, which affect embryonic development. In conclusion, SCNT embryos produced by mtDNA depleted donor cells have the same potential to develop to the blastocyst stage without the presumed damaging effect resulting from the mixture of donor and recipient mtDNA.

Additional mitochondrial DNA influences the interactions between the nuclear and mitochondrial genomes in a bovine embryo model of nuclear transfer

“…Our previous report showed that depleting mtDNA from donor cells prior to SCNT can prevent the transmission of donor cell mtDNA to the embryo as these embryos harboured only oocyte mtDNA. Furthermore, the gene expression patterns of embryos produced from depleted cells were different to those of nondepleted cells 35 . Collectively, this resulted in an improvement to SCNT.…”

“…Moreover, we compared expression patterns of miNT blastocysts derived from depleted cells cultured in the absence and presence of TSA to SCNT blastocysts derived from depleted cells cultured in the presence of TSA, which we assumed to be the gold standard, as we have previously demonstrated for SCNT embryos 35 . For the comparison between miNT blastocysts derived from depleted cells cultured in the absence of TSA and SCNT blastocysts derived from depleted cells cultured in the presence of TSA, 454 out of the 894 DEGs were upregulated (FDR < 0.05, Supplementary File 2 ).…”

“…Donor cell mtDNA can be eliminated prior to intraspecies SCNT by using mtDNA depletion agents, such as ethidium bromide or 2′,3′-dideoxycytidine (ddC). This approach ensures that the resultant embryo transmits recipient oocyte-only mtDNA 33 – 35 and, thus, mimics the patterns of mtDNA inheritance consistent with the less invasive assisted reproductive technologies, such as in vitro fertilisation 36 , and natural conception 2 . ddC is an effective depletion agent as it directly interferes with mtDNA replication 37 and does not affect the integrity of the cell’s chromosomal DNA as the depletion process takes place 35 .…”

“…This approach ensures that the resultant embryo transmits recipient oocyte-only mtDNA 33 – 35 and, thus, mimics the patterns of mtDNA inheritance consistent with the less invasive assisted reproductive technologies, such as in vitro fertilisation 36 , and natural conception 2 . ddC is an effective depletion agent as it directly interferes with mtDNA replication 37 and does not affect the integrity of the cell’s chromosomal DNA as the depletion process takes place 35 . However, for interspecies SCNT, which uses a donor cell and an oocyte from two different species, and is widely used to rescue endangered species 38 – 44 , there may be benefits in retaining donor cell mtDNA.…”

Applications of omics and nanotechnology to improve pig embryo production in vitro

“…In cattle, in an attempt to mimic the natural conception process, mitochondrial DNA (mtDNA) was depleted from donor cells, and the effects of TSA during embryo culture were evaluated. TSA treatment improved blastocyst formation of SCNT embryos originating from mtDNA-depleted donor cells to rates observed for embryos derived from nondepleted cells (Srirattana & John, 2017). However, supplementation with mtDNA in depleted donor cells enhanced reprogramming of SCNT embryos when compared to the co-treatment with TSA or TSA alone (Srirattana & John, 2018).…”