Cytoplasmic Transfer Methods for Studying the Segregation of Mitochondrial DNA in Mice.

Heteroplasmic mice represent a valuable tool to study the segregation of different mtDNA haplotypes (mtDNAs with differing alleles) in vivo against a defined nuclear background. We describe two methods for the creation of such models, differing in the resulting initial heteroplasmy levels: (a) transfer of ooplasm and (b) fusion of two blastomeres. These methods result in typical heteroplasmy of 5% and 50% donor mtDNA , respectively. The choice of method depends on the aim of the study. By means of breeding even 100% donor mtDNA can be reached within a few generations.

Transplantation of photoreceptors into the degenerative retina: Current state and future perspectives.

The mammalian retina displays no intrinsic regenerative capacities, therefore retinal degenerative diseases such as age-related macular degeneration (AMD) or retinitis pigmentosa (RP) result in a permanent loss of the light-sensing photoreceptor cells. The degeneration of photoreceptors leads to vision impairment and, in later stages, complete blindness. Several therapeutic strategies have been developed to slow down or prevent further retinal degeneration, however a definitive cure i.e. replacement of the lost photoreceptors, has not yet been established. Cell-based treatment approaches, by means of photoreceptor transplantation, have been studied in pre-clinical animal models over the last three decades. The introduction of pluripotent stem cell-derived retinal organoids represents, in principle, an unlimited source for the generation of transplantable human photoreceptors. However, safety, immunological and reproducibility-related issues regarding the use of such cells still need to be solved. Moreover, the recent finding of cytoplasmic material transfer between donor and host photoreceptors demands reinterpretation of several former transplantation studies. At the same time, material transfer between healthy donor and dysfunctional patient photoreceptors also offers a potential alternative strategy for therapeutic intervention. In this review we discuss the history and current state of photoreceptor transplantation, the techniques used to assess rescue of visual function, the prerequisites for effective transplantation as well as the main roadblocks, including safety and immune response to the graft, that need to be overcome for successful clinical translation of photoreceptor transplantation approaches.

Ooplasmic transfer in human oocytes: efficacy and concerns in assisted reproduction.

BACKGROUND: Ooplasmic transfer (OT) technique or cytoplasmic transfer is an emerging technique with relative success, having a significant status in assisted reproduction. This technique had effectively paved the way to about 30 healthy births worldwide. Though OT has long been invented, proper evaluation of the efficacy and risks associated with this critical technique has not been explored properly until today. This review thereby put emphasis upon the applications, efficacy and adverse effects of OT techniques in human. MAIN BODY: Available reports published between January 1982 and August 2017 has been reviewed and the impact of OT on assisted reproduction was evaluated. The results consisted of an update on the efficacy and concerns of OT, the debate on mitochondrial heteroplasmy, apoptosis, and risk of genetic and epigenetic alteration. SHORT CONCLUSION: The application of OT technique in humans demands more clarity and further development of this technique may successfully prove its utility as an effective treatment for oocyte incompetence.

Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy.

We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells. The fusion of PMVs with mitochondrial deficient Rho0 cells restored mitotic activity under normal culture conditions. Atherosclerosis, type 2 diabetes, Alzheimer's disease, and cancer are age-related diseases that have been reported to be associated with multiple mechanical and functional defects in the cytosol and organelles of a variety of cell types. Bone marrow mesenchymal stem cells (BMSCs) represent a unique cell population from the bone marrow that possess self-renewal capabilities while maintaining their multipotency. The supplementation of senescence cells with young cytoplasm from autologous BMSCs via the fusion of PMVs provides a promising approach to ameliorate or even reverse age-associated phenotypes. This protocol describes how to prepare PMVs from BMSCs via extrusion through a polycarbonate membrane with 3 µm pores, determine the existence of mitochondria and examine the maintenance of membrane potential within PMVs using a confocal microscope, concentrate PMVs by centrifugation, and carry out the in vivo injection of PMVs into the gastrocnemius muscle of mice.

Lack of effects of ooplasm transfer on early development of interspecies somatic cell nuclear transfer bison embryos.

BACKGROUND: Successful development of iSCNT (interspecies somatic cell nuclear transfer) embryos depends on complex interactions between ooplasmic and nuclear components, which can be compromised by genetic divergence. Transfer of ooplasm matching the genetic background of the somatic cell in iSCNT embryos is a valuable tool to study the degree of incompatibilities between nuclear and ooplasmic components. This study investigated the effects of ooplasm transfer (OT) on cattle (Bos taurus) and plains bison (Bison bison bison) embryos produced by iSCNT and supplemented with or without ooplasm from cattle or plains bison oocytes. RESULTS: Embryos in all groups were analysed for developmental competence that included cleavage rates, ATP content, and expression of nuclear- and mitochondrial- encoded genes at 8-16 cell stage. Interestingly, no significant differences were observed in embryo development, ATP content, and expression of nuclear respiratory factor 2 (NRF2), mitochondrial transcription factor A (TFAM) and mitochondrial subunit 2 of cytochrome c oxidase (mt-COX2) among groups. Thus, although OT did not result in any detrimental effects on the reconstructed embryos due to invasive manipulation, significant benefits of OT were not observed up to the 8-16 cell stage. CONCLUSIONS: This study showed that a viable technique for OT + SCNT is possible, however, further understanding of the effects of OT on blastocyst development is necessary.

Assisted reproductive technologies: advances in medical practice or human subject research?

Although human subject research is regulated by federal agencies, the differences between research and innovative clinical practice are often blurred. Research and innovative practices share the similar goals of obtaining additional knowledge and improving medical treatment. Research, however, is more specifically defined as "a systematic investigation, including research development, testing and evaluation, designed to develop or contribute to generalizable knowledge." Aprocedure consistent with this definition is subject to distinct federal regulations and other ethical procedural safeguards. When unregulated innovative practices, not neatly fitting within this definition of research, are first implemented, safeguards do not necessarily exist because use of these procedures is primarily guided by individual physician judgment. Recognizing that the application of innovative advancements in ART may very well benefit numerous prospective infertile patients and may initially appear to be safe and effective, these new and novel procedures may be associated with yet unknown long-term risks and safety concerns unless more formal scientific study is conducted to support efficacy and safety.

Spindle assembly checkpoint-related meiotic defect in oocytes from LT/Sv mice has cytoplasmic origin and diminishes in older females.

The spindle assembly checkpoint (SAC) ensures proper segregation of chromosomes by delaying anaphase onset until all kinetochores are properly attached to the spindle microtubules. Oocytes from the mouse strain LT/Sv arrest at the first meiotic metaphase (MI) due to, as reported recently, enormously prolonged activity of the SAC. We compared the dynamics of cyclin B1-GFP degradation, the process which is a measure of the SAC activity, in chromosomal and achromosomal halves of LT/Sv oocytes. In chromosome-containing oocyte halves arrested at MI, cyclin B1-GFP was not degraded indicating active SAC. However, in the halves lacking chromosomes, which is a condition precluding the SAC function, degradation always occurred confirming that MI arrest in LT/Sv oocytes is SAC dependent. Transferring the germinal vesicle (GV) from LT/Sv oocytes into the enucleated oocytes from wild-type mice resulted in the progression through meiosis one, indicating that a SAC-activating defect in LT/Sv oocytes is cytoplasmic, yet can be rescued by foreign cytoplasm. These results may help to define the etiology of the human infertility related to the oocyte MI arrest, indicating the involvement of the SAC as likely candidate, and point to GV transfer as the possible therapy. Finally, we found that majority of oocytes isolated from old LT/Sv mice complete the first meiosis. Reciprocal transfers of the GV between the oocytes from young and old LT/Sv females suggest that the factor(s) responsible for the reversal of the phenotype in oocytes from old mice is located both in the GV and in the cytoplasm.

Prevention of mitochondrial disease inheritance by assisted reproductive technologies: prospects and challenges.

BACKGROUND: Mitochondrial diseases are caused by the mutations in both nuclear and mitochondrial DNA (mtDNA) and the treatment options for patients who have mitochondrial disease are rather limited. Mitochondrial DNA is transmitted maternally and does not follow a Mendelian pattern of inheritance. Since reliable and predictable detection of mitochondrial disorders in embryos and oocytes is unattainable at present, an alternative approach to this problem has emerged as partial or complete replacement of mutated mtDNA with the wild-type mtDNA through embryo manipulations. Currently available methods to achieve this goal are germinal vesicle transfer (GVT), metaphase chromosome transfer (CT), pronuclear transfer (PNT) and ooplasmic transfer (OT). SCOPE OF REVIEW: We summarize the state of the art regarding these technologies and discuss the implications of recent advances in the field for clinical practice. MAJOR CONCLUSIONS: CT, PNT and GVT techniques hold promise to prevent transmission of mutant mtDNA through ARTs. However, it is clear that mtDNA heteroplasmy in oocytes, embryos and offspring produced by these methods remains as a legitimate concern. GENERAL SIGNIFICANCE: New approaches to eliminate transmission of mutant mtDNA certainly need to be explored in order to bring the promise of clinical application for the treatment of mitochondrial disorders. This article is part of a Special Issue entitled Biochemistry of Mitochondria, Life and Intervention 2010.

Improvement of porcine oocytes with low developmental ability after fusion of cytoplasmic fragments prepared by serial centrifugation.

We have shown in pigs that oocytes denuded of cumulus cells at 24 h of in vitro maturation culture and subsequently matured for a total of 46 h (DO24 oocytes) have lower cytoplasmic maturity than those matured with cumulus cells for 46 h and then denuded (DO46 oocytes). In the present study, DO24 zona-free oocytes were fused with one (1C) or two (2C) cytoplasmic fragments produced by serial centrifugation ("centri-fusion") of DO46 oocytes (DO24+1C and DO24+2C oocytes, respectively). Groups of (1) DO46 (a control), (2) DO24, (3) DO24+1C and (4) DO24+2C oocytes were partheno-activated by an electrical pulse or fertilized in vitro and subsequently cultured for 6 days. In the fused groups, female pronucleus (FPN) formation rates were higher than that in the DO24 group after parthenogenetic activation (PA); however, the blastocyst rates were intermediate between those of the control and DO24 groups. After in vitro fertilization, the male pronucleus (MPN) formation rates in the fused groups were similar to that in the control group and higher than that in the DO24 group; the normal fertilization rate in the DO24+2C group was higher than that in the DO24 group and similar to that in the control group, resulting in significantly higher blastocyst rates in the DO24+2C and control groups than that in the DO24 group. These results suggest that centri-fusion using ooplasm from fully matured DO46 oocytes can offer a potentially novel approach for restoration of cytoplasmic maturity to oocytes with low developmental ability and subsequent improvement of fertilization and developmental competence.

Fixing oocytes? A bovine model provides new hope.

In a previous issue of Reproductive BioMedicine Online, Chiaratti and co-workers presented a bovine model for ooplasmic transfer, which demonstrated a positive effect on early development. Developmental deficits resulting from artificial treatment of recipient eggs with a toxic compound were ameliorated by the addition of small volumes of healthy donor cytoplasm. This model provides an important advance in the understanding of ooplasmic effects in early development and addresses issues about the prior human trials in this area.

Ooplast-mediated developmental rescue of bovine oocytes exposed to ethidium bromide.

Ooplasm transfer has been used successfully to treat infertility in women with ooplasmic insufficiency and has culminated in the birth of healthy babies. To investigate whether mitochondrial dysfunction is a factor in ooplasmic insufficiency, bovine oocytes were exposed to ethidium bromide, an inhibitor of mitochondrial DNA replication and transcription, during in-vitro maturation (IVM). Exposure of immature oocytes to ethidium bromide for 24h during IVM hampered meiotic resumption and the migration of cortical granules. However, a briefer treatment with ethidium bromide during the last 4h of IVM led to partial arrest of preimplantation development without affecting oocyte maturation. Ooplasm transfer was then performed to rescue the oocytes with impaired development. In spite of this developmental hindrance, transfer of normal ooplasm into ethidium bromide-treated oocytes resulted in a complete rescue of embryonic development and the birth of heteroplasmic calves. Although this study unable to determine whether developmental rescue occurred exclusively through introduction of unaffected mitochondria into ethidium bromide-damaged oocytes, e.g. ethidium bromide may also affect other ooplasm components, these results clearly demonstrate that ooplasm transfer can completely rescue developmentally compromised oocytes, supporting the potential use of ooplasm transfer in therapeutic applications.

Therapeutic treatments of mtDNA diseases at the earliest stages of human development.

More than 150 pathogenic mitochondrial DNA (mtDNA) mutations associated with a range of illnesses have been described in humans. These mutations are carried by one in 400 people and their inheritance is exclusively maternal. Currently there is no method to prevent mtDNA diseases, which highlights the need for strategies to predict their transmission. Here we outline the scientific background and unique difficulties in understanding the transmission of mtDNA diseases, explaining why their management has lagged so far behind the genetics revolution. Moreover, both current and future management options, including cytoplasmic and nuclear transfer, are also discussed.

Pronounced segregation of donor mitochondria introduced by bovine ooplasmic transfer to the female germ-line.

Ooplasmic transfer (OT) has been used in basic mouse research for studying the segregation of mtDNA, as well as in human assisted reproduction for improving embryo development in cases of persistent developmental failure. Using cattle as a large-animal model, we demonstrate that the moderate amount of mitochondria introduced by OT is transmitted to the offspring's oocytes; e.g., modifies the germ line. The donor mtDNA was detectable in 25% and 65% of oocytes collected from two females. Its high variation in heteroplasmic oocytes, ranging from 1.1% to 33.5% and from 0.4% to 15.5%, can be explained by random genetic drift in the female germ line. Centrifugation-mediated enrichment of mitochondria in the pole zone of the recipient zygote's ooplasm and its substitution by donor ooplasm led to elevated proportions of donor mtDNA in reconstructed zygotes compared with zygotes produced by standard OT (23.6% +/- 9.6% versus 12.1% +/- 4.5%; P < 0.0001). We also characterized the proliferation of mitochondria from the OT parents-the recipient zygote (Bos primigenius taurus type) and the donor ooplasm (B. primigenius indicus type). Regression analysis performed for 57 tissue samples collected from the seven OT fetuses at different points during fetal development found a decreasing proportion of donor mtDNA (r(2) = 0.78). This indicates a preferred proliferation of recipient taurine mitochondria in the context of the nuclear genotype of the OT recipient expressing a B. primigenius indicus phenotype.

Effects of ooplasm transfer on paternal genome function in mice.

BACKGROUND: The ooplasm plays a central role in forming the paternal pronucleus, and subsequently in regulating the expression of paternally inherited chromosomes. Previous studies in mice have revealed genetic differences in paternal genome processing by ooplasm of different genotypes. Ooplasm donation coupled to intracytoplasmic sperm injection (ICSI) has been used in human assisted reproductive technology (ART). This procedure exposes the developing paternal pronucleus to 'foreign' ooplasm, which may direct aberrant epigenetic processing. The potential effects of the foreign ooplasm on epigenetic information in the paternal pronucleus are unknown; however, some human progeny from ooplasm donation procedures display abnormalities. METHODS: In this study, we employed inter-genotype ooplasm transfer followed by ICSI using two mouse strains, C57BL/6 and DBA/2, to explore the influence of foreign ooplasm on paternal pronucleus function. In order to assay for effects on the paternal genome without masking effects of the maternal genome, we examined ooplasm effects in diploid androgenones, which are produced by pronuclear transfer to contain exclusively two paternal sets of chromosomes, in combination with ICSI. RESULTS: There was no significant effect of intra-strain ooplasm transfer among androgenones made with either C57BL/6 or DBA/2 oocytes. There was a significant negative effect on androgenone blastocyst development with inter-genotype transfer (10% volume) of DBA/2 ooplasm to C57BL/6 oocytes (P < 0.05). The reciprocal inter-genotype ooplasm transfer had no significant effect. CONCLUSIONS: Thus, inter-genotype ooplasm transfer in conjunction with ICSI can alter the function of the paternal genome. However, the effect of foreign ooplasm is restricted to a negative effect, with no evidence of a positive effect. This study provides important new information about the possible consequences of ooplasm donation in human ART.

Effects of ooplasm manipulation on DNA methylation and growth of progeny in mice.

New techniques to boost male and female fertility are being pioneered at a rapid pace in fertility clinics to increase the efficiency of assisted reproduction methods in couples in which natural conception has not been achieved. This study investigates the possible epigenetic effects of ooplasm manipulation methods on postnatal growth and development using a mouse genetic model, with particular emphasis on the possible effects of intergenotype manipulations. We performed interstrain and control intrastrain maternal pronuclear transfers, metaphase-II spindle transfers, and ooplasm transfer between C57BL/6 and DBA/2 mice, and found no major, long-term growth defects or epigenetic abnormalities, in either males or females, associated with intergenotype transfers. Ooplasm transfer itself was associated with reduced viability, and additional subtle effects of ooplasm strain of origin were observed. Both inter- and intrastrain ooplasm transfer were associated with subtle, transient effects on growth early in life. We also performed inter- and intrastrain germinal vesicle transfers (GVTs). Interstrain GVT females, but not males, had significantly lower body weights at birth and thereafter compared with the intrastrain GVT and non-GVT controls. No GVT-associated changes were observed in DNA methylation of the Mup1, Rasgrf1, H19, Snrpn, or Peg3 genes, nor any difference in expression of the imprinted Rasgrf1, Igf2r, or Mest genes. These results indicate that some ooplasm manipulation procedures may exert subtle effects on growth early in life, while intergenotype GVT can result in significant growth deficiencies after birth.

Ooplasmic and nuclear transfer to prevent mitochondrial DNA disorders: conceptual and normative issues.

UNLABELLED: BACKGROUND; Mitochondrial DNA (mtDNA) disorders are an important cause of human diseases. In view of the limitations of prenatal diagnosis and preimplantation genetic diagnoses, alternatives such as ooplasmic transfer (OT) and nuclear transfer (NT) have been proposed to prevent the transmission of mtDNA mutations. Both OT and NT are radical in the sense that they do not entail genetic selection, but genetic intervention to correct the genetic cause of the disease. METHODS: After interviews with experts in the field, the relevant literature was searched and analyzed. Bioethical issues were divided into conceptual and normative points. RESULTS: OT is the transfer of normal mitochondria to a carrier's oocyte containing mutant mtDNA. In case of NT, a donated oocyte is enucleated and replaced with the nuclear DNA from a woman carrying a mtDNA mutation. NT can be performed both before and after in vitro fertilization, respectively, with the nucleus of an unfertilized oocyte, with the pronuclei of the zygote, or with the nucleus of a blastomere of an embryo. Conceptual questions regard whether these techniques amount to germ-line modification and human cloning. Normative questions concern, among others, the significance of intervening in the mtDNA, the implications of having 'three genetic parents', the ethics of oocyte donation and the health and safety risks for children conceived as a result of one of these techniques. CONCLUSIONS: Further interdisciplinary debate and research is needed to determine whether a clinical application of OT and NT can be morally justified, and if so, under which conditions.

Impact of assisted reproductive technologies: a mitochondrial perspective of cytoplasmic transplantation.

Many of the assisted reproductive techniques associated with maternal aging, disease states, or implantation failure aim to correct poor developmental capacity. These techniques are highly invasive and require the exchange of nuclear or cytoplasmic material from a donor oocyte to compensate for deficiencies inherent in the affected individual. These techniques are based on the assumption that the cytoplasm of the donor oocyte can effectively substitute the necessary component(s) to enable development to proceed. Several studies have attempted to inject cytoplasm from "normal" (young) donors, into aged eggs, again assuming that beneficial components of the cytoplasm are transferred to restore developmental capacity. These invasive assisted reproduction technology (ART) procedures aim to eliminate chromosomal abnormalities, improve the quality of oocytes deficient in some important cytoplasmic factors necessary for maturation and/or subsequent development, and eliminate maternally inherited diseases (particularly mitochondrial myopathies). However, in order to develop such ART, understanding the processes involving mitochondrial DNA replication and transcription is imperative, as asynchrony between mitochondrial and nuclear genomes may cause problems in mitochondrial function, localization, and biogenesis.

Ooplasmic transfer: problems and prospects.

Cytoplasmic transfer between human oocytes, which represents a complete cytoplasmic exchange, has been performed recently as a means to improve the outcome of assisted reproduction and becomes a hotspot of researches. Many studies have indicated that mitochondria in the oocytoplasm obviously affect fertilization of the oocytes and early embryo development. However, ooplasmic transfer can lead to mitochondrial DNA heteroplasmy and the prospect of mitochondrial heteroplasmy and its potential problems necessitate further studies. The authors reviews the ooplasmic transfer, the relation between ooplasm and fertilization and embryo development, and the mitochondrial heteroplasmy. The authors also propose a new theory of "reverse cloning technique".

Injection of somatic cell cytoplasm into oocytes before intracytoplasmic sperm injection impairs full-term development and increases placental weight in mice.

This study investigated the effects on fertilized embryo development of somatic cytoplasm after its injection into intact mouse oocytes. Mature oocytes collected from female B6D2F1 mice were injected with cumulus cell cytoplasm of different volumes and from different mouse strains (B6D2F1, ICR, and C57BL/6), or with embryonic cytoplasm. After culture for 1 h, B6D2F1 sperm were injected into those oocytes by intracytoplasmic sperm injection (ICSI). The oocytes were examined for pre- and postimplantation developmental competence. Increases in the volume of the somatic cytoplasm from onefold to fourfold resulted in an impairment of blastocyst development and full-term development (28% and 7%, respectively, vs. 96% and 63%, respectively, in the control group; P < 0.01). An increase in the volume of somatic cytoplasm reduced the expression of POU5F1 (more commonly known as OCT4) in expanded blastocysts. The frequency of embryos that developed to the blastocyst stage did not differ when B6D2F1 or ICR somatic cytoplasm was injected, but injection of C57BL/6 somatic cytoplasm induced a two-cell block in embryo development. Injection of the cytoplasm from fertilized embryos did not reduce the frequency of embryos attaining full-term development. Interestingly, somatic cytoplasm significantly increased the placental weight of ICSI embryos, even the injection of onefold cytoplasm (0.20 +/- 0.02 [n = 32] vs. 0.12 +/- 0.02 in the control group [n = 87]; P < 0.01). These findings indicate that the injection of somatic cytoplasm into oocytes before ICSI causes a decrease in preimplantation development, clearly impairs full-term development, and causes placental overgrowth in fertilized embryos. To our knowledge, placental overgrowth phenotypes are only caused by interspecies hybridization and cloning, and in genetically modified mice. Here, we report for the first time that somatic cytoplasm causes abnormal placentas in fertilized embryos. This study suggests that somatic cell cytoplasmic material is one cause of the low rate of full-term development in cloned mammals.