br Mitochondrial replacement therapy Because of

Mitochondrial replacement therapy Because of the uniparental, and usually maternal mode of mitochondrial inheritance diseases caused by mitochondria dysfunction are propagated from mother to offspring in a “dominant” manner. Mitochondrial replacement therapy, which involves transfer of a maternal nucleus from a mother with abnormal mitochondria (hereafter donor) to the enucleated oocyte or egg of a woman with healthy mitochondria (hereafter recipient oocyte or egg), recently emerged as a promising strategy to prevent transmission of devastating mitochondrial DNA diseases and infertility (Fig. 1). The procedure, when successful, results in embryos, referred to as three parent embryos, with the healthy mitochondria of the recipient egg and the nuclear genome of the donor mother, that are ideally free of diseased mitochondria. However, evidence shows that contaminating donor mitochondria accompany the nucleus during the transfer procedure (Tachibana et al., 2013; Lee et al., 2012; Yamada et al., 2016), raising questions about whether metforman or selection of mitochondria in subsequent generations will lead to biased enrichment of the donor mitochondria over time. This is a potential concern for individuals generated by mitochondrial replacement therapy if critical tissues become diseased due to enrichment for the “contaminating” mitochondria. As discussed below, this potential drift toward diseased donor mitochondria is a potentially more significant issue for female embryos that are generated by mitochondrial replacement therapy because their mitochondria will be transmitted to the next generation. Maternal-effect genes are those genes expressed by the mother that are transcribed in the oocyte to produce all of the RNAs and proteins that will be needed throughout oogenesis and into early embryogenesis. Prior to the meiotic divisions, oocytes become transcriptionally silenced so the late stage oocytes and early embryos must utilize the maternally provided RNAs and proteins for their development. Like these maternal-effect genes, mitochondria are maternally inherited so the products of the mitochondrial genome at this stage can be considered within the scope of maternal-effect processes. If the bottleneck mechanism involves matching nuclear and mitochondrial genomes, then presumably this selection would be carried out in early oocytes by components encoded by the nuclear and mitochondrial genomes. Because the transfer procedure to generate a three parent embryo is performed relatively late in oogenesis, at stages after potential mitochondrial bottleneck selection and amplification during oocyte growth that will be discussed later in this review, mitochondria transferred at this late stage would have already passed the earlier selection and be identified as maternal, whether marked or unmarked, as so far ubiquitination or similar marks have only been reported for paternal mitochondria. Therefore, no biased elimination would be expected. Thereafter, the early embryo environment might be receptive to the mitochondria of both mothers despite the apparent mismatch between the mitochondrial and nuclear genomes. This is plausible because although the embryo has the nucleus of another oocyte, the embryo for a period of time expresses the products of its original nuclear genome – the persisting maternal RNA and protein products that were produced at stages prior to the transfer, and upon genome activation the RNA and protein products produced by the new donor nuclear genome. Moreover, in human embryos paternal mitochondria linger until around the time of genome activation, acquisition of pluripotency and the first fate decisions (Ankel-Simons and Cummins, 1996), indicating that prior to this time the mechanisms to clear “foreign” mitochondria might not be as robust. The early cleavages of the blastula are reductive, generating smaller cells with each nuclear replication and division cycle. During this period of rapid division, no replication of mitochondrial DNA has been detected in several vertebrates examined (Lee et al., 2012; Otten et al., 2016; Piko and Matsumoto, 1976); thus, despite differences in onset between species each cleavage prior to zygotic genome activation and acquisition of pluripotency would generate cells with less mitochondrial DNA per cell. This period without mitochondrial replication has been identified as a genetic bottleneck in primates based on examination of heteroplasmic embryos (Lee et al., 2012). In that work, prior to 8-cell stage, cells were heteroplasmic, thereafter, specific tissues of the fetuses at later stages showed evidence of homoplasmy attributed to tissue-specific differences in mitochondrial copy number at stages after DNA replication resumes (Lee et al., 2012). This genetic bottleneck in embryogenesis corresponds to the period when the maternal products are degraded and are replaced by the products of the zygotic genome. The progeny of mitochondrial replacement therapy (MRT) would be subject to this bottleneck, and if cell-type specific selection favors the contaminating mitochondria then the individual will suffer from mitochondrial disease in those tissues. In the recent work from Yamada and colleagues, such enrichment of contaminating, albeit healthy mitochondria in their case, was observed in cardiac and connective tissues of embryos and human stem cell lines generated by mitochondrial replacement therapy (Yamada et al., 2016).