Mitochondrial genotype segregation and effects during mammalian development: applications to biotechnology

Mitochondria are endosymbiotic organelles responsible for energy production in practically every eukaryotic cell. Their uniparental fashion of inheritance, maternally inherited in mammals, and the homogeneity of mitochondrial DNA (mtDNA) within individuals and matrilineages, are biological phenomena that remain unexplained. This paper reviews some of the recent findings on mitochondrial influences on the manner in which embryos develop and how their genotypes are inherited in mammals, with particular emphasis on the genetic "bottleneck" effect. Animal models carrying a mix of mtDNAs (heteroplasmic) have been produced by karyoplast and cytoplast transplantation to analyze the segregation patterns at different stages during embryogenesis, in fetuses and offspring. Comparisons performed between murine and bovine reveal interesting changes in segregation and replication of transplanted mtDNAs. We have recently obtained Bos indicus and Bos taurus fetuses and calves from embryos reconstructed using enucleated polymorphic oocytes of Bos taurus origin. These and other findings on mitochondrial biology will have important implications in determining the cytoplasmic genotype of clones and in the preservation of endangered breeds and species.     

Complete replacement of the mitochondrial genotype in a Bos indicus calf reconstructed by nuclear transfer to a Bos taurus oocyte

Due to the exclusively maternal inheritance of mitochondria, mitochondrial genotypes can be coupled to a particular nuclear genotype by continuous mating of founder females and their female offspring to males of the desired nuclear genotype. However, backcrossing is a gradual procedure that, apart from being lengthy, cannot ascertain that genetic and epigenetic changes will modify the original nuclear genotype. Animal cloning by nuclear transfer using host ooplasm carrying polymorphic mitochondrial genomes allows, among other biotechnology applications, the coupling of nuclear and mitochondrial genotypes of diverse origin within a single generation. Previous attempts to use Bos taurus oocytes as hosts to transfer nuclei from unrelated species led to the development to the blastocyst stage but none supported gestation to term. Our aim in this study was to determine whether B. taurus oocytes support development of nuclei from the closely related B. indicus cattle and to examine the fate of their mitochondrial genotypes throughout development. We show that indicus:taurus reconstructed oocytes develop to the blastocyst stage and produce live offspring after transfer to surrogate cows. We also demonstrate that, in reconstructed embryos, donor cell-derived mitochondria undergo a stringent genetic drift during early development leading, in most cases, to a reduction or complete elimination of B. indicus mtDNA. These results demonstrate that cross-subspecies animal cloning is a viable approach both for matching diverse nuclear and cytoplasmic genes to create novel breeds of cattle and for rescuing closely related endangered cattle.     

Mitochondria transfer into mouse ova by microinjection

A method for mitochondria isolation and interspecific transfer of mitochondria was developed in mice. Mitochondria were isolated from Mus spretus liver samples for microinjection into fertilized ova obtained from superovulated M. musculus domesticus females. Electron microscopic observations of mitochondria preparations used for microinjection demonstrated intact mitochondrial vesicles with little microsomal contamination. Species-specific nested PCR primers complementary to sequence differences in the mitochondrial DNA D-loop region revealed high rates of successful transfer of foreign mitochondria after isolation and injection into zygotes cultured through the blastocyst stage of embryonic development. Of 217 zygotes, 67 survived mitochondria injection and 23 out of 37 zygotes developed were at the blastocyst-stage of embryonic development after 4.5 days of in vitro culture. All 23 of these blastocysts contained detectable levels of foreign mitochondria. These results represent an initial step in developing a model system to study mitochondrial dynamics and development of therapeutic strategies for human metabolic diseases affected by aberrations in mitochondrial function or mutation     

Mitochondrial Genotype Segregation in a Mouse Heteroplasmic Lineage Produced by Embryonic Karyoplast Transplantation

Mitochondrial genotypes have been shown to segregate both rapidly and slowly when transmitted to consecutive generations in mammals. Our objective was to develop an animal model to analyze the patterns of mammalian mitochondrial DNA (mtDNA) segregation and transmission in an intraspecific heteroplasmic maternal lineage to investigate the mechanisms controlling these phenomena. Heteroplasmic progeny were obtained from reconstructed blastocysts derived by transplantation of pronuclear-stage karyoplasts to enucleated zygotes with different mtDNA. Although the reconstructed zygotes contained on average 19% mtDNA of karyoplast origin, most progeny contained fewer mtDNA of karyoplast origin and produced exclusively homoplasmic first generation progeny. However, one founder heteroplasmic adult female had elevated tissue heteroplasmy levels, varying from 6% (lung) to 69% (heart), indicating that stringent replicative segregation had occurred during mitotic divisions. First generation progeny from the above female were all heteroplasmic, indicating that, despite a meiotic segregation, they were derived from heteroplasmic founder oocytes. Some second and third generation progeny contained exclusively New Zealand Black/BINJ mtDNA, suggesting, but not confirming, an origin from an homoplasmic oocyte. Moreover, several third to fifth generation individuals maintained mtDNA from both mouse strains, indicating a slow or persistent segregation pattern characterized by diminished tissue and litter variability beyond second generation progeny. Therefore, although some initial lineages appear to segregate rapidly to homoplasmy, within two generations other lineages transmit stable amounts of both mtDNA molecules, supporting a mechanism where mitochondria of different origin may fuse, leading to persistent intraorganellar heteroplasmy.     

Dominant distribution of mitochondrial DNA from recipient oocytes in bovine embryos and offspring after nuclear transfer

In the process of nuclear transfer, heteroplasmic sources of mitochondrial DNA from a donor cell and a recipient oocyte are mixed in the cytoplasm of the reconstituted embryo. The distribution of mitochondrial DNA heteroplasmy in nuclear transfer bovine embryos and resultant offspring was investigated by measuring polymorphism in the displacement loop region of mitochondrial DNA using PCR-mediated single-strand conformation polymorphism. Most offspring (20 of 21 calves) from recipient oocytes of undefined mitochondrial DNA genotypes showed different genotypes from the mitochondrial DNA of donor cells. The single calf that was an exception showed heteroplasmy, including the donor mitochondrial DNA genotype. Six cloned calves were produced from oocytes of a defined mitochondrial DNA genotype. All of these clonal members and various tissues showed only the mitochondrial DNA genotype derived from the oocyte. The mitochondrial DNA from donor cells appeared to be eliminated during early embryonic development; it gradually decreased at the early cleavage stages and was hardly detectable by the blastocyst stage. These results indicate that the genotype of mitochondrial DNA from recipient oocytes may become the dominant category of mitochondrial DNA in calves resulting from nuclear transfer.     

The kinetics of donor cell mtDNA in embryonic and somatic donor cell-derived bovine embryos

The mechanisms controlling the outcome of donor cell-derived mitochondrial DNA (mtDNA) in cloned animals remain largely unknown. This research was designed to investigate the kinetics of somatic and embryonic mtDNA in reconstructed bovine embryos during preimplantation development, as well as in cloned animals. The experiment involved two different procedures of embryo reconstruction and their evaluation at five distinct phases of embryo development to measure the proportion of donor cell mtDNA (Bos indicus), as well as the segregation of this mtDNA during cleavage. The ratio of donor cell (B. indicus) to host oocyte (B. taurus) mtDNA (heteroplasmy) from blastomere(NT-B) and fibroblast(NT-F) reconstructed embryos was estimated using an allele-specific PCR with fluorochrome-stained specific primers in each sampled blastomere, in whole blastocysts, and in the tissues of a fibroblast-derived newborn clone. NT-B zygotes and blastocysts show similar levels of heteroplasmy (11.0% and 14.0%, respectively), despite a significant decrease at the 9-16 cell stage (5.8%; p<0.05). Heteroplasmy levels in NT-F reconstructed zygotes, however, increased from an initial low level (4.7%), to 12.9% (p<0.05) at the 9-16 cell stage. The NT-F blastocysts contained low levels of heteroplasmy (2.2%) and no somatic-derived mtDNA was detected in the gametes or the tissues of the newborn calf cloned. These results suggest that, in contrast to the mtDNA of blastomeres, that of somatic cells either undergoes replication or escapes degradation during cleavage, although it is degraded later after the blastocyst stage or lost during somatic development, as revealed by the lack of donor cell mtDNA at birth.     

The importance of mitochondrial metabolic activity and mitochondrial DNA replication during oocyte maturation in vitro on oocyte quality and subsequent embryo developmental competence

Mitochondrial metabolic capacity and DNA replication have both been shown to affect oocyte quality, but it is unclear which one is more critical. In this study, immature oocytes were treated with FCCP or ddC to independently inhibit the respective mitochondrial metabolic capacity or DNA replication of oocytes during in vitro maturation. To differentiate their roles, we evaluated various parameters related to oocyte maturation (germinal vesicle break down and nuclear maturation), quality (spindle formation, chromosome alignment, and mitochondrial distribution pattern), fertilization capability, and subsequent embryo developmental competence (blastocyst formation and cell number of blastocyst). Inhibition of mitochondrial metabolic capacity with FCCP resulted in a reduced percent of oocytes with nuclear maturation; normal spindle formation and chromosome alignment; evenly distributed mitochondria; and an ability to form blastocysts. Inhibition of mtDNA replication with ddC has no detectable effect on oocyte maturation and mitochondrial distribution, although high-dose ddC increased the percent of oocytes showing abnormal spindle formation and chromosome alignment. ddC did, however, reduce blastocyst formation significantly. Neither FCCP nor ddC exposure had an effect on the rate of fertilization. These findings suggest that the effects associated with lower mitochondrial DNA copy number do not coincide with the effects seen with reduced mitochondrial metabolic activity in oocytes. Inhibiting mitochondrial metabolic activity during oocyte maturation has a negative impact on oocyte maturation and subsequent embryo developmental competence. A reduction in mitochondrial DNA copy number, on the other hand, mainly affects embryonic development potential, but has little effect on oocyte maturation and in vitro fertilization.     

Effects of Glucose Repression on the Transmission and Recombination of Mitochondrial Genes in Yeast (SACCHAROMYCES CEREVISIAE)

Matings of a number of Saccharomyces cerevisiae stocks give different output ratios of mitochondrial genotypes depending on whether the cells are glucose-repressed or derepressed. The effects of glucose repression are independent of cellular mating type and mitochondrial genotype, and take place at least in part after zygotes are formed. An explanation is proposed in terms of changes in the relative numbers of mitochondrial DNA molecules contributed by the a and α parents, modified by selective replication or destruction of molecules inside the zygote.     

Persistence of human mitochondrial DNA throughout the development to the blastocyst of mouse zygotes microinjected with human mitochondria

The conditions for transfer of human mitochondria into fertilised mouse ova were elaborated. Species-specific primers were designed to discriminate human mitochondrial DNA (mtDNA) and the endogenous mtDNA in the preimplantation embryos. Human mitochondria isolated from the HepG2 cell line were microinjected into murine zygotes, and the latter cultured for 96 h to the blastocyst stage. The polymerase chain reaction allowed the detection of human mtDNA at every stage of embryo cleavage. In some cases a clear disparity in distribution of human mtDNA among blastomeres was evident.     

Development of bovine-ovine interspecies cloned embryos and mitochondria segregation in blastomeres during preimplantation

The objective of the study was to investigate interspecies somatic cell nuclear transfer (iSCNT) embryonic potential and mitochondrial DNA (mtDNA) segregation during preimplantation development. We generated bovine-ovine reconstructed embryos via iSCNT using bovine oocytes as recipient cytoplasm and ovine fetal fibroblast as donor cells. Chromosome composition, the total cell number of blastocyst and embryonic morphology were analyzed. In addition, mtDNA copy numbers both from donor cell and recipient cytoplasm were assessed by real-time PCR in individual blastocysts and blastomeres from 1- to 16-cell stage embryos. The results indicated the following: (1) cell nuclei of ovine fetal fibroblasts can dedifferentiate in enucleated bovine ooplasm, and the reconstructed embryos can develop to blastocysts. (2) 66% of iSCNT embryos had the same number of chromosome as that of donor cell, and the total cell number of iSCNT blastocysts was comparable to that of sheep parthenogenetic blastocysts. (3) RT-PCR analysis in individual blastomeres revealed that the ratio of donor cell mtDNA: recipient cytoplasm mtDNA remained constant (1%) from the one- to eight-cell stage. However, the ratio decreased from 0.6% at the 16-cell stage to 0.1% at the blastocyst stage. (4) Both donor cell- and recipient cytoplasm-derived mitochondria distributed unequally in blastomeres with progression of cell mitotic division. Considerable unequal mitochondrial segregation occurred between blastomeres from the same iSCNT embryos.     

Mouse Zygotes Respond to Severe Sperm DNA Damage by Delaying Paternal DNA Replication and Embryonic Development

Mouse zygotes do not activate apoptosis in response to DNA damage. We previously reported a unique form of inducible sperm DNA damage termed sperm chromatin fragmentation (SCF). SCF mirrors some aspects of somatic cell apoptosis in that the DNA degradation is mediated by reversible double strand breaks caused by topoisomerase 2B (TOP2B) followed by irreversible DNA degradation by a nuclease(s). Here, we created zygotes using spermatozoa induced to undergo SCF (SCF zygotes) and tested how they responded to moderate and severe paternal DNA damage during the first cell cycle. We found that the TUNEL assay was not sensitive enough to identify the breaks caused by SCF in zygotes in either case. However, paternal pronuclei in both groups stained positively for γH2AX, a marker for DNA damage, at 5 hrs after fertilization, just before DNA synthesis, while the maternal pronuclei were negative. We also found that both pronuclei in SCF zygotes with moderate DNA damage replicated normally, but paternal pronuclei in the SCF zygotes with severe DNA damage delayed the initiation of DNA replication by up to 12 hrs even though the maternal pronuclei had no discernable delay. Chromosomal analysis of both groups confirmed that the paternal DNA was degraded after S-phase while the maternal pronuclei formed normal chromosomes. The DNA replication delay caused a marked retardation in progression to the 2-cell stage, and a large portion of the embryos arrested at the G2/M border, suggesting that this is an important checkpoint in zygotic development. Those embryos that progressed through the G2/M border died at later stages and none developed to the blastocyst stage. Our data demonstrate that the zygote responds to sperm DNA damage through a non-apoptotic mechanism that acts by slowing paternal DNA replication and ultimately leads to arrest in embryonic development.     

Mitochondrial organization in prepubertal goat oocytes during in vitro maturation and fertilization

The aim of this study was to evaluate mitochondrial distribution during in vitro maturation (at 0, 15, 20, and 27 hr of IVM) and fertilization of prepubertal goat oocytes compared to mitochondrial distribution of ovulated and in vitro fertilized oocytes from adult goats. Oocytes from prepubertal goats were recovered from a slaughterhouse and were matured in M199 with hormones and serum for 27 hr. Ovulated oocytes were collected from gonadotrophin-treated Murciana goats. Frozen-thawed spermatozoa were selected by centrifugation in Percoll gradient and were capacitated in DMH with 20% steer serum for 1 hr. Ovulated and IVM-oocytes were inseminated in DMH medium with steer serum and calcium lactate for 20 hr. Oocytes and presumptive zygotes were stained with Mitotraker Green FM and observed under a confocal laser scanning microscope. Ultrastructural morphology of oocytes and presumptive zygotes were analyzed by transmission electron microscopy (TEM). Prepubertal goat oocytes at germinal vesicle stage (GV) presented mitochondria localized in the cortical and perinuclear region. IVM-oocytes at metaphase II presented mitochondria peripheral polarized to the region opposite were the metaphase spindle is positioned and within the polar body. Ovulated oocytes presented peripheral mitochondria distribution and mitochondrial aggregation around the MII spindle. At 20 hr post-insemination, mitochondria were distributed around the two synchronous pronuclei (2PN rpar; in zygotes ovulated oocytes whereas in prepubertal 2PN-zygotes mitochondria presented a peripheral polarized distribution. Images by TEM detected that immature prepubertal goat oocytes that are less electrodense and present fewer cristae than in vitro matured prepubertal goat oocytes; these are characterized by being associated to swollen vesicles. Mol. Reprod. Dev. 73: 617-626, 2006 (c) 2006 Wiley-Liss, Inc.     

Mitochondrial dynamics and aging: Mitochondrial interaction preventing individuals from expression of respiratory deficiency caused by mutant mtDNA

In mammalian cells, there is an extensive and continuous exchange of mitochondrial DNA (mtDNA) and its products between mitochondria. This mitochondrial complementation prevents individuals from expression of respiration deficiency caused by mutant mtDNAs. Thus, the presence of mitochondrial complementation does not support the generally accepted mitochondrial theory of aging, which proposes that accumulation of somatic mutations in mtDNA is responsible for age-associated mitochondrial dysfunction. Moreover, the presence of mitochondrial complementation enables gene therapy for mitochondrial diseases using nuclear transplantation of zygotes.     

Maternal Diet-Induced Obesity Alters Mitochondrial Activity and Redox Status in Mouse Oocytes and Zygotes

The negative impact of obesity on reproductive success is well documented but the stages at which development of the conceptus is compromised and the mechanisms responsible for the developmental failure still remain unclear. Recent findings suggest that mitochondria may be a contributing factor. However to date no studies have directly addressed the consequences of maternal obesity on mitochondria in early embryogenesis.Using an established murine model of maternal diet induced obesity and a live cell dynamic fluorescence imaging techniques coupled with molecular biology we have investigated the underlying mechanisms of obesity-induced reduced fertility. Our study is the first to show that maternal obesity prior to conception is associated with altered mitochondria in mouse oocytes and zygotes. Specifically, maternal diet-induced obesity in mice led to an increase in mitochondrial potential, mitochondrial DNA content and biogenesis. Generation of reactive oxygen species (ROS) was raised while glutathione was depleted and the redox state became more oxidised, suggestive of oxidative stress. These altered mitochondrial properties were associated with significant developmental impairment as shown by the increased number of obese mothers who failed to support blastocyst formation compared to lean dams. We propose that compromised oocyte and early embryo mitochondrial metabolism, resulting from excessive nutrient exposure prior to and during conception, may underlie poor reproductive outcomes frequently reported in obese women.     

In situ localization of mitochondrial DNA replication in intact mammalian cells

Nearly all of the known activities required for mitochondrial DNA (mtDNA) replication and expression are nuclear-encoded gene products, necessitating communication between these two physically distinct intracellular compartments. A significant amount of both general and specific biochemical information about mtDNA replication in mammalian cells has been known for almost two decades. Early studies achieved selective incorporation of the thymidine analog 5-Bromo-2-deoxy-Uridine (BrdU) into mtDNA of thymidine kinase-deficient (TK[-]) cells. We have revisited this approach from a cellular perspective to determine whether there exist spatiotemporal constraints on mtDNA replication. Laser-scanning confocal microscopy was used to selectively detect mtDNA synthesis in situ in cultured mammalian cells using an immunocytochemical double-labeling approach to visualize the incorporation of BrdU into mtDNA of dye-labeled mitochondria. In situ detection of BrdU-incorporated mtDNA was feasible after a minimum of 1- 2 h treatment with BrdU, consistent with previous biochemical studies that determined the time required for completion of a round of mtDNA replication. Interestingly, the pattern of BrdU incorporation into the mtDNA of cultured mammalian cells consistently radiated outward from a perinuclear position, suggesting that mtDNA replication first occurs in the vicinity of nuclear-provided materials. Newly replicated mtDNA then appears to rapidly distribute throughout the dynamic cellular mitochondrial network.     

The effect of zygotic bud position on the transmission of mitochondrial genes in Saccharomyces cerevisiae.

Summary Segregation of mitochondrial genomes in yeast zygotes has been investigated by partial pedigree analysis of crosses involving the markers cap, ery, oli1 and par. The results demonstrate that the segregation pattern of markers is non-random during the first zygote generation and is directly related to slow mixing of the zygote cytoplasm. We have observed that a first bud may be formed at the center or either end of the dumbbell-shaped zygote. Cytoplasmic mixing is particularly slow in those zygotes producing first end buds.Clones derived from first end buds are usually pure (or nearly so) for a parental genotype and so detectable recombination of mitochondrial markers is reduced in these zygotes. Cells derived from a zygote after removal of a first end bud are predominantly of the other parental genotype. This observation suggests that a large fraction of the available segregating units enters each first bud and illustrates one means of obtaining complete segregation (even in multi-factor crosses) at the first generation. First center buds generally receive mitochondrial markers from both parents and the recombination frequency in such clones (and the clones derived from isolated first center buds) is significantly higher than in similar clones from zygotes with first end buds. Therefore, the distribution of first bud positions within a population of zygotes can influence the recombination frequency between mitochondrial loci. The delay in cytoplasmic mixing in combination with certain patterns of zygotic budding can distort the relationship between input of mitochondrial genomes and the output of a cross.The phage analogy model of yeast mitochondrial genetics has been re-examined in light of these data. The assumption of rapid panmixis is not supported by the data from any of the crosses analyzed here. Since panmixis is most closely approximated in zygotes with first center buds, crosses with predominantly zygotes of that type may be the ones where the model is most applicable.     

A potential role for triglyceride as an energy source during bovine oocyte maturation and early embryo development

The potential role of endogenous triglyceride in bovine oocyte maturation and preimplantation development has been investigated. Bovine immature oocytes were recovered from abattoir-derived ovaries, matured and fertilised in vitro and the zygotes grown to the blastocyst stage in SOFaaBSA. Methyl palmoxirate (MP) blocks the oxidation of fatty acids by inhibiting mitochondrial carnitine palmitoyltransferase A. The development of zygotes exposed to MP during oocyte maturation, and of zygotes exposed to MP during embryo culture has been assessed in terms of oxygen consumption by oocytes and embryos during a 4-6 hr incubation period in the presence of MP and as blastocyst formation and cell number. Immature oocytes exposed to MP during maturation had reduced capacity to form blastocysts after fertilisation; the same effect was apparent, but to a lesser extent, in zygotes exposed to MP during embryo development. Oxygen consumption values of oocytes and blastocysts in the absence of exogenous substrates were similar to those in control medium containing nutrients. MP-inhibited oxygen consumption of immature oocytes, mature oocytes, cleavage stages embryos and blastocysts by 64, 45, 12 and 13%, respectively. The data are consistent with a role for triglyceride as a key energy source during bovine oocyte maturation and potentially, during preimplantation embryo development.