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Body weight and adiposity are determined by the balance between energy intake, energy expenditure, and nutrient deposition. We have identified differences in appetite-regulating peptides in sheep selectively bred to be either lean or fat, wherein gene expression for orexin and melanin-concentrating hormone are elevated in the lean group. Despite this, the underlying mechanisms leading to differences in body composition in the lean and fat lines remains unknown. We measured postprandial temperature in adipose tissue and muscle to ascertain whether a difference in thermogenesis is associated with the difference in body composition in genetically lean (n = 8) and fat (n = 12) ewes. Body weight was higher (P < 0.01) but percent fat mass was lower (P < 0.001) in the lean group. The percent lean mass was similar in lean and fat groups. Animals received intracerebroventricular cannulae and temperature probes implanted into the retroperitoneal fat and the hind-limb skeletal muscle (vastus lateralis). Animals were meal fed (1100-1600 h) to entrain postprandial thermogenesis. Food intake was similar between lean and fat animals. Postprandial thermogenesis was greater (P < 0.05) in the retroperitoneal adipose tissue of lean animals but not in skeletal muscle. Intracerebroventricular infusion of leptin reduced (P< 0.05) food intake by an equal extent in both groups. Postprandial expression of UCP1 mRNA was greater (P < 0.05) in retroperitoneal fat of lean animals, with similar UCP3 expression in skeletal muscle. Mitochondrial genome sequencing indicated haplotypic clustering in lean and fat animals within both the encoding and nonencoding regions. This demonstrates that differences in body composition may be underpinned by differences in thermogenesis, specifically within adipose tissue. Furthermore, thermogenic differences may be associated with specific mitochondrial DNA haplotypes, suggesting a strong genetic component inherited through the maternal lineage.

Study questionWhat are the effects on fertility of cigarette smoke-induced toxicity on male offspring exposed during the gestational/weaning period?Summary answerMaternal cigarette smoke exposure during the gestational/weaning period causes long-term defects in male offspring fertility.What is known alreadyCigarette smoke is a well-known reproductive toxicant which is particularly harmful to both fetal and neonatal germ cells. However, recent studies suggest a significant portion of young mothers in the developed world still smoke during pregnancy. In the context of male reproductive health, our understanding of the effects of in utero exposure on offspring fertility is limited.Study design, size, durationIn this study, 27 C57BL/6 5-week-old female mice were exposed via the nose-only to cigarette smoke (treatment) or 27 were exposed to room air (control) for 6 weeks before being housed with stud males to produce litters. In the treatment group, smoke exposure continued throughout mating, pregnancy and lactation until weaning of pups at 21 days post birth. Male offspring were examined at post-natal days 3, 6, 12, 21 and 98 (adult).Participants/materials, setting, methodsApproximately 108 maternal smoke-exposed C57BL/6 offspring and controls were examined. Spermatogenesis was examined using testicular histology and apoptosis/DNA damage was assessed using caspase immunohistochemistry and TUNEL. Sertoli cell morphology and fluctuations in the spermatogonial stem cell population were also examined using immunohistochemistry. Microarray and QPCR analysis were performed on adult testes to examine specific long-term transcriptomic alteration as a consequence of maternal smoke exposure. Sperm counts and motility, zona/oolemma binding assays, COMET analysis and mitochondrial genomic sequencing were also performed on spermatozoa obtained from adult treated and control mice. Fertility trials using exposed adult male offspring were also performed.Main results and the role of chanceMaternal cigarette smoke exposure caused increased gonocyte and meiotic spermatocyte apoptosis (P < 0.01) as well as germ cell depletion in the seminiferous tubules of neonatal and juvenile offspring. Aberrant testicular development characterized by abnormal Sertoli and germ cell organization, a depleted spermatogonial stem cell population (P < 0.01), atrophic seminiferous tubules and increased germ cell DNA damage (P < 0.01) persisted in adult offspring 11 weeks after exposure. Microarray analysis of adult offspring testes associated these defects with meiotic germ cell development, sex hormone metabolism, oxidative stress and Sertoli cell signalling. Next generation sequencing also revealed a high mitochondrial DNA mutational load in the testes of adult offspring (P < 0.01). Adult maternal smoke-exposed offspring also had reduced sperm counts with spermatozoa exhibiting morphological abnormalities (P < 0.01), affecting motility and fertilization potential. Odf2, a spermatozoa flagellum component required for coordinated ciliary beating, was also significantly down-regulated (P < 0.01) in maternal smoke-exposed adult offspring, with aberrant localization along the spermatozoa flagellum. Adult maternal smoke-exposed offspring took significantly longer to impregnate control females and had a slight but significant (P < 0.01) reduction in litter size.Limitations, reasons for cautionThis study examined only one species (mouse) using a smoking model which only simulates human cigarette smoke exposure.Wider implications of the findingsThis study represents the first comprehensive animal model of maternal smoking on male offspring reproductive function, suggesting that exposure during the gestational/weaning period causes long-term defects in male offspring fertility. This is due to a compromised spermatogonial stem cell population resulting from gonocyte apoptosis and impaired spermatogenic development. This results in significant germ cell damage and Sertoli cell dysfunction, impacting germ cell number, tubule organization, DNA damage and spermatozoa in adult offspring. This study strengthens the current literature suggesting that maternal exposure impairs male offspring fertility, which is currently debated due to conflicting studies.Study funding/competing interestsThis study was funded by the Australian Research Council, Hunter Medical Research Institute, National Health and Medical Research Council of Australia and the Newcastle Permanent Building Society Charitable Trust. The authors declare no conflict of interest.

As stem cells undergo differentiation, mitochondrial DNA (mtDNA) copy number is strictly regulated in order that specialized cells can generate appropriate levels of adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS) to undertake their specific functions. It is not understood whether tumor-initiating cells regulate their mtDNA in a similar manner or whether mtDNA is essential for tumorigenesis. We show that human neural stem cells (hNSCs) increased their mtDNA content during differentiation in a process that was mediated by a synergistic relationship between the nuclear and mitochondrial genomes and results in increased respiratory capacity. Differentiating multipotent glioblastoma cells failed to match the expansion in mtDNA copy number, patterns of gene expression and increased respiratory capacity observed in hNSCs. Partial depletion of glioblastoma cell mtDNA rescued mtDNA replication events and enhanced cell differentiation. However, prolonged depletion resulted in impaired mtDNA replication, reduced proliferation and induced the expression of early developmental and pro-survival markers including POU class 5 homeobox 1 (OCT4) and sonic hedgehog (SHH). The transfer of glioblastoma cells depleted to varying degrees of their mtDNA content into immunocompromised mice resulted in tumors requiring significantly longer to form compared with non-depleted cells. The number of tumors formed and the time to tumor formation was relative to the degree of mtDNA depletion. The tumors derived from mtDNA depleted glioblastoma cells recovered their mtDNA copy number as part of the tumor formation process. These outcomes demonstrate the importance of mtDNA to the initiation and maintenance of tumorigenesis in glioblastoma multiforme.

Although mitochondrial DNA has been implicated in diseases such as cancer, its role remains to be defined. Using three models of tumorigenesis, namely glioblastoma multiforme, multiple myeloma and osteosarcoma, we show that mitochondrial DNA plays defining roles at early and late tumour progression. Specifically, tumour cells partially or completely depleted of mitochondrial DNA either restored their mitochondrial DNA content or actively recruited mitochondrial DNA, which affected the rate of tumorigenesis. Nevertheless, non-depleted tumour cells modulated mitochondrial DNA copy number at early and late progression in a mitochondrial DNA genotype-specific manner. In glioblastoma multiforme and osteosarcoma, this was coupled with loss and gain of mitochondrial DNA variants. Changes in mitochondrial DNA genotype affected tumour morphology and gene expression patterns at early and late progression. Importantly, this identified a subset of genes that are essential to early progression. Consequently, mitochondrial DNA and commonly expressed early tumour-specific genes provide novel targets against tumorigenesis.

Body weight and adiposity are determined by the balance between energy intake, energy expenditure, and nutrient deposition. We have identified differences in appetite-regulating peptides in sheep selectively bred to be either lean or fat, wherein gene expression for orexin and melanin-concentrating hormone are elevated in the lean group. Despite this, the underlying mechanisms leading to differences in body composition in the lean and fat lines remains unknown. We measured postprandial temperature in adipose tissue and muscle to ascertain whether a difference in thermogenesis is associated with the difference in body composition in genetically lean (n = 8) and fat (n = 12) ewes. Body weight was higher (P0.01) but percent fat mass was lower (P0.001) in the lean group. The percent lean mass was similar in lean and fat groups. Animals received intracerebroventricular cannulae and temperature probes implanted into the retroperitoneal fat and the hind-limb skeletal muscle (vastus lateralis). Animals were meal fed (1100-1600 h) to entrain postprandial thermogenesis. Food intake was similar between lean and fat animals. Postprandial thermogenesis was greater (P0.05) in the retroperitoneal adipose tissue of lean animals but not in skeletal muscle. Intracerebroventricular infusion of leptin reduced (P0.05) food intake by an equal extent in both groups. Postprandial expression of UCP1 mRNA was greater (P0.05) in retroperitoneal fat of lean animals, with similar UCP3 expression in skeletal muscle. Mitochondrial genome sequencing indicated haplotypic clustering in lean and fat animals within both the encoding and nonencoding regions. This demonstrates that differences in body composition may be underpinned by differences in thermogenesis, specifically within adipose tissue. Furthermore, thermogenic differences may be associated with specific mitochondrial DNA haplotypes, suggesting a strong genetic component inherited through the maternal lineage.

Mitochondria are the organelles responsible for producing the majority of a cell's ATP and also play an essential role in gamete maturation and embryo development. ATP production within the mitochondria is dependent on proteins encoded by both the nuclear and the mitochondrial genomes, therefore co-ordination between the two genomes is vital for cell survival. To assist with this co-ordination, cells normally contain only one type of mitochondrial DNA (mtDNA) termed homoplasmy. Occasionally, however, two or more types of mtDNA are present termed heteroplasmy. This can result from a combination of mutant and wild-type mtDNA molecules or from a combination of wild-type mtDNA variants. As heteroplasmy can result in mitochondrial disease, various mechanisms exist in the natural fertilization process to ensure the maternal-only transmission of mtDNA and the maintenance of homoplasmy in future generations. However, there is now an increasing use of invasive oocyte reconstruction protocols, which tend to bypass mechanisms for the maintenance of homoplasmy, potentially resulting in the transmission of either form of mtDNA heteroplasmy. Indeed, heteroplasmy caused by combinations of wild-type variants has been reported following cytoplasmic transfer (CT) in the human and following nuclear transfer (NT) in various animal species. Other techniques, such as germinal vesicle transfer and pronuclei transfer, have been proposed as methods of preventing transmission of mitochondrial diseases to future generations. However, resulting embryos and offspring may contain mtDNA heteroplasmy, which itself could result in mitochondrial disease. It is therefore essential that uniparental transmission of mtDNA is ensured before these techniques are used therapeutically.

Mitochondria, among other functions, generate energy in the form of ATP. The chondrial genome, located within each mitochondrion, encodes some of the polypeptides associated with the electron transfer chain (ETC) and ATP production. Transcription and replication of mitochondrial DNA (mtDNA) is dependent upon the import of transcription and replication factors encoded by the nucleus. Certain point mutations and large-scale deletions to mtDNA can be either severely debilitating or lethal. The transmission and inheritance of mtDNA [not readable: see to offspring is strictly regulated and specific to each species. In many mammalian systems, paternal mtDNA is eliminated very early during embryonic development. However, it is possible that the paternal molecule could be extruded to those cells destined to become trophoblasts and may act as a regulator of embryonic cell fate. Furthermore, the increasing use of more sophisticated assisted reproductive techniques has led to the incorporation of extraneous mtDNA in both the reconstructed oocyte and embryo with transmission to the offspring at varying degrees.

The battle to prevent the transmission of mitochondrial DNA disease: Is karyoplast transfer the answer?

Air quality data gathered from five nonurban sites in China over a 12-month period from August 1994 to August 1995, along with meteorological observations from the same region and period, are used to identify and characterize a nonurban ozone (O3) pollution episode in China. Because of the influence of the Asian Monsoonal Circulation, high O3 concentrations were not observed at the nonurban sites during the summer months. However, enhanced O3 concentrations were observed during the other seasons, especially the fall and early winter. A more detailed inspection of the O3 data during the period from October 15, 1994, to January 15, 1995, indicated the occurrence of a multipleday episode in late October/early November when high O3 concentrations were observed at all four monitoring sites located in eastern China. Meteorological conditions during the episode were characterized by the presence of a strong and stationary high-pressure ridge over eastern China; synoptic conditions quite similar to those observed during regional O3 pollution episodes over the United States, Canada, and Europe. An updated version of the Regional Acid Deposition Model (RADM) driven by meteorological fields derived from the Regional Climate Model (RegCM) and spatially disaggregated anthropogenic emissions prepared by the Chinese Academy of Meteorological Sciences is used to simulate 3 months of the observed O3 data from China. Comparisons between observations and model calculations indicate that the model is able to reproduce some of the key features of the O3 distribution and its relationship to the concentration of one primary pollutant (i.e., sulfur dioxide) provided the comparison is made using averaging times of several days or more. However, simulation of day-to-day variations in O3 at a given site was poorly correlated with observations. Model simulations suggest that peak O3 concentrations during this episode would respond to changes in NOx and VOC emissions in a spatially inhomogeneous manner. In general, rural areas in southern China tend to be NOx-limited, but rural areas in northern China tend to be VOC-limited. The Yangtze Delta region, where the highest O3 concentrations were observed and predicted to occur, was found to be transitional between VOC and NOx limitation.

Newly available data from non-urban locations in China along with regional model simulations suggest that ground-level ozone may be sufficiently high to affect China's winter wheat production. As non-urban ozone increases with industrialization, its effects on crops could hinder efforts to meet increasing food demands in the coming decades, in China.

A global three-dimensional chemical transport model is used to investigate seasonal variations of anthropogenic sulfur in the troposphere. Particular emphasis is placed on detailed comparisons of the modeled surface sulfur dioxide (SO2) and sulfate (SO4) concentrations and sulfate wet deposition fluxes with measurements from the Eulerian Model Evaluation Field Study (EMEFS) and Cooperative Program for Monitoring and Evaluation of the Long Range Transmission of Air Pollutants in Europe (EMEP) field programs in North America and Europe, respectively. Initial comparisons of model results with measurements reveal a systematic tendency of the model to overestimate SO2 concentrations and underestimate SO4 concentrations while producing a reasonable fit to measured wet deposition fluxes. Through a series of sensitivity tests we find that the addition of a nonphotochemical pathway for converting SO2 to SO4 in the boundary layer with a pseudo first-order rate of constant of 1–2×10−6 s−1 provides the most reasonable method of bringing the model results into better agreement with the EMEFS and EMEP data sets. We propose that this additional pathway may be related to heterogeneous reactions between SO2 and atmospheric aerosols that typically are not included in models of the atmospheric sulfur cycle. Despite the vastly improved simulation of surface SO2 and SO4 when this hypothetical heterogeneous oxidation pathway is included, the model is unable to simultaneously simulate the large seasonal cycle in surface SO4 observed over eastern North America and the almost total absence of a seasonal cycle in surface SO4 over Europe. The seasonal cycles in model-predicted column SO4 burdens are similar, but not identical, to those for surface SO4 because of regional differences in transport, free tropospheric oxidation, and in-cloud removal. We find that the summer-to-winter ratio in column SO4 is larger over eastern North America than it is over Europe; however, both are larger than that for eastern Asia, where wintertime column SO4 is predicted to exceed summertime column SO4.

For a long time, it was perceived that the mitochondrial genome (mtDNA) was primarily associated with energy production, as it encodes key genes associated with the electron transfer chain, which produces the vast majority of cellular ATP through oxidative phosphorylation. However, the mitochondrial genome is taking an ever-increasingly important role is cellular differentiation, cell fate and, thus, cell function. Previously, we have shown that copy number of the maternally inherited mitochondrial genome is strictly regulated during development. Indeed, oocytes, embryos and stem cells regulate their mtDNA copy number so that, once cells commit to a specific lineage, they increase their mtDNA copy number in a cell-specific manner.1, 2, 3 This enables cells to meet their needs for ATP in order that they can support their specialized functional requirements. For example, neurons have a high requirement for ATP generated through OXPHOS to generate action potentials and mediate neurotransmitter activity. Indeed, in undifferentiated cells, the control of mtDNA copy number is a tightly regulated process as these cells establish the mtDNA set point, which is defined as a small number of mtDNA copies per cell that remains constant until cells initiate differentiation.1, 2 Cells then use this population of mtDNA as a template for replication as differentiation ensues. However, cancer cells fail to undergo these transformations as they are unable to mediate mtDNA replication.4 Increasing evidence now points to the regulation of mtDNA, either through the number of mtDNA copies5 or specific mutations within mtDNA,6 as key factors associated with the onset of tumorigenesis. In our paper published in Cell Death Discovery, we have used three experimental models to show that depletion of mtDNA directly affects the initiation and progression of tumors in vivo.7 In each of the three different malignant tumor models, glioblastoma multiforme, multiple myeloma and osteosarcoma, cells with extensively depleted levels of mtDNA had compromised initiation or a significant delay in the onset of tumorigenesis or complete failure to establish tumorigenesis. We further demonstrated that, in those tumors arising from extensively mtDNA-depleted cells, mtDNA was recovered as part of the process leading to the onset of tumorigenesis. This is achieved by the extensively depleted cells undergoing DNA demethylation of the nuclear encoded mtDNA-specific polymerase (POLGA),3 which is otherwise hypermethylated, and enhances the replication of mtDNA. Furthermore, cells unable to replenish their own mtDNA recruited mtDNA from the surrounding tissues. The acquisition of mtDNA by tumor cells has been previously demonstrated by Tan et al.,8 whereby they showed that mtDNA was acquired from the surrounding cells of the same species. Our study, however, shows that this can also occur between species, namely human cells acquiring mouse mtDNA but this likely serves primarily to mediate the initiation of tumorigenesis (Figure 1). Indeed, the human mtDNA replication factors are unable to regulate the replication of mtDNA from a more divergent species such as mouse and with only limited efficiency in closely related non-human primates, such as the chimpanzee. These findings suggest that mtDNA could be a potential therapeutic target for the treatment of various cancers but, in order to avoid relapse of the disease, continuous depletion of mtDNA might be necessary until all tumor cells have been targeted. Figure 1 Although depletion of mtDNA copy number shows reduced tumor formation, mtDNA-depleted tumor cells can either replenish their own mtDNA or acquire mtDNA from the surrounding cells, in order to restore impaired tumorigenicity. This is further exemplified ... We further identified specific changes in mtDNA variants in tumors derived from mtDNA-depleted cells. MtDNA variants differ amongst cancer types, but they were also modulated at different stages of tumor progression in two models with decreases at early progression and increases at late progression in our glioblastoma and osteosarcoma models but an overall reduction in our multiple myeloma model at late progression, suggesting that this is cancer type-dependent. We further discovered that the mtDNA-encoded complex I gene ND6 is a hotspot for mtDNA variants in tumorigenesis. One plausible explanation is that ND6 is the only protein-coding gene on the light strand of mtDNA and the last gene to be replicated, increasing the possibility of proofreading errors. Other studies have suggested that higher numbers of mtDNA variants in cancers are likely to contribute to the disease phenotype and the development of cancer.6, 9 Moreover, a previous study demonstrated that mtDNA haplotypes can influence mRNA expression of pluripotent factors in murine pluripotent stem cells,10 implying an influence from the mitochondrial genome on the nucleus and phenotype. Could this effect be a potential therapeutic approach for cancers, for instance, by replacing mtDNA carrying pathogenic mutations with the wild-type mtDNA? We found that repopulation with wild-type mtDNA from human neural stem cells in the extensively mtDNA-depleted osteosarcoma cells, however, did not prevent tumorigenesis. Instead, it restored the tumorigenicity to levels similar to that of undepleted osteosarcoma cells and those repopulated with mtDNA from human glioblastoma cells (Figure 1). This provides further support for the crucial role that mtDNA has in the establishment of tumorigenesis. Nevertheless, further work is required with wild-type mtDNA from cell types derived from the same lineage to clarify the impact that mtDNA variants have on tumor development. We also extended our investigations to the transcriptomic landscape, where we repeatedly observed altered gene expression related to hepatic fibrosis, angiogenesis and RhoGDI signaling pathways in tumors with depleted mtDNA. Moreover, we observed that the repopulation of mtDNA partially restored the expression of numerous genes involved in these pathways. Using in-depth transcriptomic analysis of altered expression in tumors at multiple stages of development, we were able to detect and isolate genes that affect post-translational modification and osteoclastogenesis. These findings emphasize the effects that the mitochondrial genome has on the nuclear genome and provides a new approach to detect specific markers for different types of cancers, especially as certain mtDNA haplotypes are more predisposed to cancer.6, 11, 12 Overall, this study contributes to our understanding of the role that mtDNA plays in tumorigenesis. Most importantly, these results demonstrate that, although the chromosomal genome regulates the transcription and replication of the mitochondrial genome and contributes largely to disease phenotypes, mtDNA influences the initiation and progression of tumorigenesis. Consequently, mtDNA is a potential therapeutic candidate.

Pluripotent blastomeres of mammalian pre-implantation embryos and embryonic stem cells (ESCs) are characterized by limited oxidative capacity and great reliance on anaerobic respiration. Early pre-implantation embryos and undifferentiated ESCs possess small and immature mitochondria located around the nucleus, have low oxygen consumption and express high levels of glycolytic enzymes. However, as embryonic cells and ESCs lose pluripotency and commit to a specific cell fate, the expression of mtDNA transcription and replication factors is upregulated and the number of mitochondria and mtDNA copies/cell increases. Moreover, upon cellular differentiation, mitochondria acquire an elongated morphology with swollen cristae and dense matrices, migrate into wider cytoplasmic areas and increase the levels of oxygen consumption and ATP production as a result of the activation of the more efficient, aerobic metabolism. Since pluripotency seems to be associated with anaerobic metabolism and a poorly developed mitochondrial network and differentiation leads to activation of mitochondrial biogenesis according to the metabolic requirements of the specific cell type, it is hypothesized that reprogramming of somatic cells towards a pluripotent state, by somatic cell nuclear transfer (SCNT), transcription-induced pluripotency or creation of pluripotent cell hybrids, requires acquisition of mitochondrial properties characteristic of pluripotent blastomeres and ESCs.

Mitochondrial DNA (mtDNA) copy number is strictly regulated during differentiation so that cells with a high requirement for ATP generated through oxidative phosphorylation have high mtDNA copy number, whereas those with a low requirement have few copies. Using immunoprecipitation of DNA methylation on 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), which distinguish between de novo DNA methylation and demethylation, respectively, we set out to determine whether DNA methylation at exon 2 of the human mtDNA-specific polymerase (DNA polymerase gamma A (POLGA)) regulates cell-specific mtDNA copy number in highly proliferative and terminally differentiated cells. Highly proliferative cancer and pluripotent and multipotent cells possessed low mtDNA copy number and were highly methylated at exon 2 of POLGA in contrast to post-mitotic cells. Unlike neural stem cells, cancer cells were unable to differentiate and remained extensively DNA methylated at exon 2 of POLGA. However, mtDNA depletion of cancer cells reduced DNA methylation at exon 2 of POLGA as they replenished mtDNA to form tumours in mice. Glioblastoma cells treated with the DNA demethylation agent 5-azacytidine over 28 days of astrocyte-induced differentiation demethylated exon 2 of POLGA leading to increased mtDNA copy number and expression of the astrocyte endpoint marker glial fibrillary acidic protein (GFAP). However, the demethylation agent vitamin C (VitC) was unable to sustain increased mtDNA copy number and differentiation, as was the case when VitC was withdrawn after short-term treatment. These data demonstrate that DNA demethylation of POLGA is an essential regulator of mtDNA copy number and cellular fate and that cancer cells are only able to modulate DNA methylation of POLGA and mtDNA copy number in the presence of a DNA demethylation agent that inhibits de novo methyltransferase 1 activity.

The life cycle of many cell types can hinge on the presence of death factors that can control programmed cell death. The Fas-mediated apoptotic pathway has been implicated in controlling apoptosis during spermatogenesis in a number of mammalian species. In the human, the presence of nuclear DNA damage in ejaculated spermatozoa has pointed to a possible role for apoptosis during spermatogenesis. The presence of other molecular markers of apoptosis has, however, not been shown. More importantly, differences in these markers have not been investigated in men with normal and abnormal sperm parameters. In this study we examine for the presence of the cell surface protein Fas in ejaculated human spermatozoa. Ejaculated spermatozoa (55 samples) were labeled with anti-human Fas antibody and the number of spermatozoa displaying Fas were counted using a fluorescence-activated cell sorter (FACS). In 30/31 (96.8%) normal males (>20 million sperm per milliliter), less than 10% of the spermatozoa were Fas positive. In contrast, 14/24 (58.3%) oligozoospermic samples (

Interspecies somatic cell nuclear transfer (iSCNT) offers significant opportunities to analyze and understand nuclear-cytoplasmic interactions. Using a murine-porcine interspecies model, we investigated the importance of nuclear-cytoplasmic compatibility, specifically mitochondrial DNA (mtDNA), on successful development. Transfer of somatic murine fetal fibroblasts into enucleated porcine oocytes resulted in extremely low blastocyst rates (0.4%); increased DNA strand breaks; deficient nuclear pore complex arrangements and increased aberrant karyokinesis than observed in porcine-porcine SCNT embryos. Using allele specific-PCR analysis, murine mtDNA was detected at ever-decreasing levels to the blastocyst stage, with peak levels being 0.14 ± 0.055% in 2-cell embryos. Furthermore, these embryos reduced total mtDNA copy number during preimplantation development in a manner similar to porcine embryos. Injecting mouse embryonic stem cell extract and mitochondria along with the murine donor cell into a mitochondria depleted porcine oocyte, increased blastocyst zona pellucida thinning and blastocyst rates significantly (0.4% vs 3.42%) compared to the non-supplemented iSCNT group. They also had significantly more murine mtDNA at the 2-cell stage than the non-supplemented embryos, which was maintained throughout preimplantation development. At later stages of preimplantation development, they possessed 48.00% ± 17.38% murine mtDNA and exhibited a mtDNA copy number profile similar to murine embryos. Overall, these data demonstrate that the addition of species compatible cytoplasmic factors and mitochondrial DNA improve developmental competence of iSCNT embryos.

Interspecies somatic cell nuclear transfer (iSCNT) offers significant opportunities for the derivation of embryonic stem cells (ESCs) that can model diseases. In this study, we investigated the development of murine-porcine iSCNT embryos and compared these to outcomes associated with porcine-porcine SCNT embryos. Enucleation was performed on in vitro-matured porcine oocytes using the Oosight spindle viewer system (CRI, UK). Either murine embryonic fibroblasts or porcine cumulus cells were injected into porcine ooplasm using a piezo-electric micromanipulator. After being exposed to the porcine ooplasm for 1 h, the reconstructed oocytes were activated by a combination of A23187 and 6-DMAP for 3.5 h. For the murine-porcine embryos (n = 9375), we used a serial culture method, namely porcine zygote medium (PZM) for 48 h followed by incubation with KSOM for another 5 days. We found that the majority of murine-porcine embryos (73.58%) arrested at the 4- to 8-cell stage, whilst a very small minority progressed through to blastocyst (0.064%). Porcine-porcine SCNT reconstructed oocytes (n = 142) cultured in PZM achieved a 15% blastocyst development rate. In the murine-porcine embryos, 10/44 embryos had chromosomal DNA loss associated with apoptosis, as determined by immunocytochemistry using the H2A.X antibody. We also found that at later stages, morula to blastocyst, the mouse-to-pig reconstructed embryos did not exhibit murine-specific Oct-4 expression patterns. Furthermore, murine mitochondrial DNA tended to be eliminated rather than preserved by the murine nuclei, which could render any subsequently derived ESCs dysfunctional due to 2 diverse genomes contributing genes to the electron transfer chain.

Mammalian mtDNA is approximately 16.6kb in size. It has 37 genes, 13 of which encode protein subunits of the oxidative phosphorylation (OXPHOS) system, the major ATP-generating pathway of the cell. Normally, mammals inherit a single mtDNA genome (homoplasmy) from their mother. Somatic cell nuclear transfer (SCNT) violates this strict maternal, homoplasmic inheritance of mtDNA as cytoplasm is transferred along with the nucleus, which often results in an oocyte harboring both donor and recipient mtDNA genomes (heteroplasmy). This been previously reported (reviewed St. John JC 2002 Theriogenology 57, 109–123). To overcome the problem of donor mtDNA transmission, we have developed and characterized mtDNA-depleted C. hircus (goat) cells for use as donors in SCNT. C. hircus primary foetal fibroblast cells were established in culture and depleted of their mtDNA by supplementing their growth medium with a low concentration, 50ngmL−1, of ethidium bromide (EthBr). Conventional PCR, using a series of primers designed specifically for goat mtDNA, was used to screen for the presence of mtDNA during the EthBr treatment. In addition, mitochondrial organization, activity and morphology in the cells were analyzed using the mitochondrial specific fluoroprobe JC1. mtDNA-encoded and mitochondrial transcription factor A (mtTFA) transcript levels were analysed using RTPCR. Furthermore, both mtDNA depleted and non-depleted cells were characterised using immunocytochemistry to detect the expression of specific protein subunits of the OXPHOS system. Progressive mtDNA depletion was observed, using conventional PCR, in cells treated for 3 to 25 days with EthBr, while 42 days of culture resulted in complete depletion. RTPCR showed a progressive reduction followed by complete elimination of the mtDNA-encoded ND1, ND2, ND3, COX I and mtTFA transcripts. In addition, the expression of mtDNA-encoded protein subunits, e.g. COXI, of the OXPHOS system were reduced following mtDNA depletion whereas the expression of nuclear-DNA encoded protein subunits, e.g. COXVic, were unaltered. We hypothesize that the elimination of mtDNA and mtDNA transcripts from the donor cells will facilitate normal mtDNA replication and transcription in SCNT embryos, thus maintaining the strict unimaternal transmission of mtDNA to the offspring. Consequently, genetically identical offspring will be generated which have identical nuclear and mitochondrial DNA content, assuming oocytes from the same ovary are used. This technique is important for the generation of offspring for the livestock industry and animal models for the analysis of single gene disorders as well as the propagation of endangered species.