Optimizing production of in vivo-matured oocytes from superstimulated Holstein cows for in vitro production of embryos using X-sorted sperm.

The present study aimed to establish an efficient system for the production of female embryos from dairy cows by in vitro fertilization (IVF) using X-sorted sperm and in vivo-matured oocytes collected by ovum pick up (OPU). Nonlactating Holstein cows (n = 36) were administered a controlled intravaginal progesterone-releasing (controlled internal drug release) device (d 0), underwent dominant follicle ablation (DFA) or ovulation by administration of 100 µg of GnRH on d 5, and were superstimulated with FSH and PGF2α, following standard procedures. Controlled internal drug release devices were removed on the evening of d 8 or on the morning of d 9, depending on the experiment. For LH surge induction, 200 µg of GnRH was administered on the morning of d 10 (0 h). In experiment 1, the peak (48.1%) of ovulating follicles was detected at 29 to 32 h after GnRH injection (0 h), and the range in the timing of the initiation of ovulation was less by timing from GnRH administration (30.0 ± 2.8h) rather than by timing the onset of estrus (32.7 ± 4.7h). Only 0.9% of total ovulated follicles were recorded before 26 h after GnRH injection. Therefore, OPU was carried out at 26 h and IVF occurred at 30 h after GnRH in experiments 2 and 3. In experiment 2, 83.3 ± 10.8% of oocytes with expanded cumulus cells had extruded the first polar body at 30 h after GnRH injection. The aim of experiment 3 was to compare the effect of either DFA or GnRH-induced LH surge before superstimulation on the efficiency of embryo production by IVF following superstimulation. Progesterone concentrations from d 10 to 12 in the DFA group were lower than those in the GnRH group. A greater proportion of recovered oocytes with expanded cumulus cells from ≥ 8-mm follicles was observed in the DFA group than in the GnRH group (95.9 and 77.4%, respectively). Blastocyst rates in the DFA and GnRH groups (58.0 and 52.8%, respectively) did not differ from those of oocytes collected from nonstimulated OPU and matured in vitro (49.9%). However, the proportion of high-quality blastocysts was higher in the DFA group compared with the GnRH group (54.9 vs. 21.5%). Our results demonstrate that high rates of good-quality blastocysts can be produced by IVF with X-sorted frozen sperm using in vivo-matured oocytes collected by OPU from cows after DFA and superstimulation combined with ovulation induction.

Mobilities of Li(+)-attached butanol isomers in He gas.

Mobilities of Li(+)-attached butanol isomers, (n-BuOH)Li(+), (s-BuOH)Li(+), (i-BuOH)Li(+), and (t-BuOH)Li(+), in helium gas were measured over a range of reduced electric fields (E/N = 25-96 Td) at room temperature. Arrival time measurements accurately identified small differences in the measured mobilities of the isomer ions. At low E/N (≤30 Td, corresponding to a mean collision energy ε≤0.05 eV), (n-BuOH)Li(+) showed a mobility about 1.5% greater than that of the other ions, but at high E/N (≥75 Td, ε≥0.1 eV) its mobility was about 1.1% less.

The association between metabolic parameters and oocyte quality early and late postpartum in Holstein dairy cows.

The objective of this was to study the association between metabolic parameters and oocyte quality in postpartum lactating dairy cows as assessed by oocyte morphology and development after fertilization and culture in vitro. Holstein-Friesian spring-calving cows were used (n = 16, parity 3.0 ± 0.36, weight at calving 611 ± 16.2 kg, previous 305-d milk yield 6,454.0 ± 276.4 kg). Bodyweight (BW) and body condition score were recorded at approximately 2 wk before expected calving date, at calving, and then weekly until the end of the experiment (approximately 80 d postpartum). Blood plasma samples were collected weekly, starting 2 wk before the expected calving date and continuing until the end of the experiment and were analyzed for nonesterified fatty acids (NEFA), ß-hydroxybutyrate (BHBA), insulin, insulin-like growth factor-I, and glucose. Transvaginal oocyte recovery was carried out twice weekly on each cow for a period of approximately 12 wk starting 14 d after calving until approximately 80 d postpartum. A linear decrease in BW was observed from calving (d 0) to d 28, after which it remained stable. Body condition score decreased from 14 d precalving, reaching a nadir at approximately d 35 to 42, after which it increased to the end of the period. Nonesterified fatty acid concentrations were significantly elevated from the week before calving until d 42 postcalving, whereas BHBA concentration was significantly elevated from calving to d 49 postcalving. Insulin-like growth factor-I concentration dramatically decreased from d -14 to a nadir on d 7. A significant increase in glucose concentration occurred from d -7 to d 0, followed by a precipitous decrease to d 7. Based on the metabolic profiles (particularly NEFA and BHBA concentrations), data from d 0 to 42 postpartum (period 1) were compared with corresponding data from d 42 to 80 (period 2). Apart from body condition score, all of the physiological parameters measured (milk yield, BW, and blood metabolites) differed significantly between the 2 periods. In particular, insulin-like growth factor-I, insulin, and glucose concentrations were higher post-d 42, whereas BHBA and NEFA were lower compared with pre-d 42 postpartum. The number of oocytes recovered per session and oocyte quality grade did not differ between periods. Positive associations of follicles aspirated and insulin, BHBA, and NEFA were detected. The number of oocytes recovered was positively associated with milk yield, BW, and glucose and NEFA concentrations. The number of cleaved oocytes was positively associated with BW and NEFA concentration. In conclusion, the data do not provide evidence of an effect of lactation-induced metabolic stress on oocyte developmental competence in the postpartum dairy cow assessed in terms of morphological quality and ability to develop following in vitro fertilization.

A mutation in the signal recognition particle 7S RNA of the yeast Yarrowia lipolytica preferentially affects synthesis of the alkaline extracellular protease: in vivo evidence for translational arrest.

Replacement of the signal recognition particle (SRP) 7S gene (SCR1) on a replicating plasmid with scr1-1 (G to A at 129 and A to T at 131 in the consensus sequence -GNAR- in the loop of domain III) resulted in temperature sensitivity for growth of cells in which both chromosomal SRP 7S RNA genes were deleted. Pulse-chase immunoprecipitation experiments were done after a shift to non-permissive temperature using the major secreted protein the alkaline extracellular protease (AEP) as a reporter molecule. No untranslocated AEP precursor was detected in a strain with scr1-1 on a plasmid, but the amount of the largest AEP precursor (55 kD) immunoprecipitated as a percentage of total protein synthesized was reduced 68% compared to an isogenic strain with SCR1 on the plasmid. The possibility that an untranslocated precursor was synthesized but not detected because of instability was largely eliminated by detection of a 53-kD untranslocated precursor of a mutated AEP (P17M; methionine replaced proline in the second position of the pro-peptide) which chased to the 55-kD translocated AEP precursor. Thus, SRP has a role in the biosynthesis of AEP. Possibly, the scr1-1 mutation does not affect signal recognition or translational arrest but instead results in maintenance of translational arrest of AEP synthesis. The results also suggest that AEP can be translocated in vivo either co-translationally in which SRP is at least involved in biosynthesis or posttranslationally without SRP involvement.

Prognostic impact of residual lateral lymph node metastasis after neoadjuvant (chemo)radiotherapy in patients with advanced low rectal cancer.

Background: There is a lack of large studies focusing on the prognostic significance of lateral lymph node (LLN) metastasis following LLN dissection (LLND) in rectal cancer. The aim of this study was to evaluate the prognostic impact of LLN metastases on survival of patients with advanced low rectal cancer. Methods: Consecutive patients with locally advanced, but not metastatic, extraperitoneal rectal cancer treated with neoadjuvant (chemo)radiotherapy plus total mesorectal excision between 2004 and 2015 were included in the study. LLND was performed when pretreatment imaging documented enlarged LLNs (7 mm or greater in size). Localization of nodal metastases and long-term outcomes were analysed. Kaplan-Meier analysis was used to compare the survival of patients with ypN0 disease with that of patients with mesorectal ypN+/LLN- status and patients with positive LLNs. The Cox proportional hazards model was used to evaluate predictors of disease-free survival (DFS) and local recurrence. Results: A total of 613 patients were included in the study; LLND was performed in 212 patients (34·6 per cent) and 57 (9·3 per cent) had LLN metastasis. Patients with LLN metastasis had improved DFS and local recurrence cumulative incidence rates compared with patients with mesorectal ypN2+/LLN- disease (DFS: P = 0·014; local recurrence: P = 0·006). Although the DFS rate of patients with LLN metastasis was worse than that of patients with ypN0 disease (P < 0·001), the cumulative incidence of local recurrence was similar (P = 0·491). In multivariable analysis, residual LLN metastasis was not an independent predictor of worse DFS or local recurrence. Conclusion: LLN metastasis is not an independent predictor of local recurrence or survival. Survival of patients presenting with LLN metastasis after (chemo)radiotherapy was intermediate between that of patients with ypN0 status and those with mesorectal ypN2 positivity.

Antecedentes: No existen en la literatura grandes estudios dirigidos a investigar la importancia pronóstica de las metástasis en los ganglios linfáticos laterales (lateral lymph nodes, LLN) después de la disección de los mismos (LLN dissection, LLND) en pacientes con cáncer de recto. El objetivo de este estudio fue evaluar el impacto pronóstico de las metástasis en los LLN sobre la supervivencia de los pacientes con cáncer de recto. Métodos: Se analizaron 613 pacientes consecutivos con cáncer de recto localmente avanzado extraperitoneal y no metastásico tratados con (quimio)radioterapia neoadyuvante seguida de resección total del mesorrecto (total mesorectal excision, TME) entre 2004 y 2015. Se realizó una LLND cuando el estudio mediante pruebas de imagen previo el tratamiento mostró LLN aumentados de tamaño ≥ 7 mm. Se analizó la localización de las metástasis ganglionares y los resultados a largo plazo. El análisis de supervivencia se realizó mediante el método de Kaplan­Meier para comparar las supervivencias de los pacientes ypN0 frente a los pacientes ypN con positividad mesorrectal/LLN negativos y frente a los pacientes LLN positivos. Se utilizó el modelo de riesgo proporcional de Cox para evaluar los factores predictivos de supervivencia libre de enfermedad y de recidiva local. Resultados: Se realizó una LLND en 212 (34,6%) pacientes, y 57 (9,3%) pacientes presentaban metástasis en los LLN. Los pacientes con metástasis en los LLN presentaron mejores curvas de incidencia acumulada de recidiva local y de supervivencia libre de enfermedad en comparación con los pacientes con ganglios mesorrectales ypN2 positivos/LLN negativos (respectivamente, P = 0,0135 y P = 0,0060). Aunque la curva de la supervivencia libre de enfermedad de los pacientes con metástasis en los LLN fue peor que la de los pacientes ypN0 (P < 0,0001), la incidencia acumulada de recidiva local fue similar (P = 0,4905). En el análisis multivariable, la metástasis residual en los LLN no fue un factor predictivo independiente de peor supervivencia libre de enfermedad ni de recidiva local. Conclusión: Las metástasis en los LLN no es un factor predictivo independiente de recidiva local o supervivencia. Los pacientes que presentaron metástasis en los LLN después de (quimio)radioterapia mostraron características de supervivencia intermedias entre ypN0 y pacientes con ganglios mesorrectales ypN2 positivos.

Production of monozygotic twin calves using the blastomere separation technique and Well of the Well culture system.

The present study was conducted to establish a simple and efficient method of producing monozygotic twin calves using the blastomere separation technique. To produce monozygotic twin embryos from zona-free two- and eight-cell embryos, blastomeres were separated mechanically by pipetting to form two demi-embryos; each single blastomere from the two-cell embryo and tetra-blastomeres from the eight-cell embryo were cultured in vitro using the Well of the Well culture system (WOW). This culture system supported the successful arrangement of blastomeres, resulting in their subsequent aggregation to form a demi-embryo developing to the blastocyst stage without a zona pellucida. There was no significant difference in the development to the blastocyst stage between blastomeres separated from eight-cell (72.0%) and two-cell (62.0%) embryos. The production rates of the monozygotic pair blastocysts and transferable paired blastocysts for demi-embryos obtained from eight-cell embryos (64.0 and 45.0%, respectively) were higher than those for demi-embryos obtained from two-cell embryos (49.0 and 31.0%, P<0.05). The separated demi-embryos obtained from eight-cell embryos produced by IVM/IVF of oocytes collected by ovum pick-up (OPU) from elite cows and cultured in wells tended to have a higher pregnancy rate (78.9% vs. 57.1%) and similar monozygotic twinning rate (40.0% vs. 33.3%) compared with monozygotic twin blastocysts obtained by the conventional bisection of in vivo derived blastocysts. In conclusion, producing twins by separation of blastomeres in OPU-IVF embryos, followed by the WOW culture system, yielded viable monozygotic demi-embryos, resulting in high rates of pregnancy and twinning rates after embryo transfer.

Intracellular precursors and secretion of alkaline extracellular protease of Yarrowia lipolytica.

Processing and secretion of the alkaline extracellular protease (AEP) from the yeast Yarrowia lipolytica was studied by pulse-chase and immunoprecipitation experiments. Over half of newly synthesized AEP was secreted by 6 min. Over 99% of AEP activity which was external to the cytoplasmic membrane was located in the supernatant medium. Polypeptides of 55, 52, 44, 36, and 32 kilodaltons (55K, 52K, 44K, 36K, and 32K polypeptides) were immunoprecipitated from [3H]leucine-labeled cell extracts by rabbit antibodies raised against mature, secreted AEP (32K polypeptide). Experiments with tunicamycin and endoglycosidase H indicated that the 55K, 52K, and 44K polypeptides contained about 2 kilodaltons of N-linked oligosaccharide and that the 36K and 32K polypeptides contained none. Results of pulse-chase experiments did not fit a simple precursor-product relationship of 55K----52K----44K----36K----32K. In fact, maximum labeling intensity of the 52K polypeptide occurred later than for the 44K and 36K polypeptides. Secretion of polypeptides of 19 and 20 kilodaltons derived from the proregion of AEP indicated that one major processing pathway was 55K----52K----32K. The gene coding for AEP (XPR2) was cloned and sequenced. The sequence and the immunoprecipitation results suggest that AEP is originally synthesized with an additional preproI-proII-proIII amino-terminal region. Processing definitely involves cleavage(s) after pairs of basic amino acids and the addition of one N-linked oligosaccharide. Signal peptidase cleavage, dipeptidyl aminopeptidase cleavages, and at least one additional proteolytic cleavage may also be involved.

Energy metabolism after ischemic preconditioning in streptozotocin-induced diabetic rat hearts.

OBJECTIVES: The aim of this study was to compare the cardioprotective effects of preconditioning in hearts from streptozotocin-induced diabetic rats with its effects in normal rat hearts. BACKGROUND: The protective effect of ischemic preconditioning against myocardial ischemia may come from improved energy balance. However, it is not known whether preconditioning can also afford protection to diabetic hearts. METHODS: Isolated perfused rat hearts were either subjected (preconditioned group) or not subjected (control group) to preconditioning before 30 min of sustained ischemia and 30 min of reperfusion. Preconditioning was achieved with two cycles of 5 min of ischemia followed by 5 min of reperfusion. RESULTS: In the preconditioned groups of both normal and diabetic rats, left ventricular developed pressure, high energy phosphates, mitochondrial adenosine triphosphatase and adenine nucleotide translocase activities were significantly preserved after ischemia-reperfusion; cumulative creatine kinase release was smaller during reperfusion; and myocardial lactate content was significantly lower after sustained ischemia. However, cumulative creatine kinase release was less in the preconditioned group of diabetic rats than in the preconditioned group of normal rats. Under ischemic conditions, more glycolytic metabolites were produced in the diabetic rats (control group) than in the normal rats, and preconditioning inhibited these metabolic changes to a similar extent in both groups. CONCLUSIONS: The present study demonstrates that in both normal and diabetic rats, preservation of mitochondrial oxidative phosphorylation and inhibition of glycolysis during ischemia can contribute to preconditioning-induced cardioprotection. Furthermore, our data suggest that diabetic myocardium may benefit more from preconditioning than normal myocardium, possibly as a result of the reduced production of glycolytic metabolites during sustained ischemia and the concomitant attenuation of intracellular acidosis.

Cytokine-induced nitric oxide production inhibits mitochondrial energy production and impairs contractile function in rat cardiac myocytes.

OBJECTIVES: The present study examined whether nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) can directly inhibit aerobic energy metabolism and impair cell function in interleukin (IL)-1beta,-stimulated cardiac myocytes. BACKGROUND: Recent reports have indicated that excessive production of NO induced by cytokines can disrupt cellular energy balance through the inhibition of mitochondrial respiration in a variety of cells. However, it is still largely uncertain whether the NO-induced energy depletion affects myocardial contractility. METHODS: Primary cultures of rat neonatal cardiac myocytes were prepared, and NO2-/NO3- (NOx) in the culture media was measured using Griess reagent. RESULTS: Treatment with IL-1beta (10 ng/ml) increased myocyte production of NOx in a time-dependent manner. The myocytes showed a concomitant significant increase in glucose consumption, a marked increase in lactate production, and a significant decrease in cellular ATP (adenosine 5'-triphosphate). These metabolic changes were blocked by co-incubation with N(G)-monomethyl-L-arginine (L-NMMA), an inhibitor of NO synthesis. Sodium nitroprusside (SNP), a NO donor, induced similar metabolic changes in a dose-dependent manner, but 8-bromo-cyclic guanosine 3',5'-monophosphate (8-bromo-cGMP), a cGMP donor, had no effect on these parameters. The activities of the mitochondrial iron-sulfur enzymes, NADH-CoQreductase and succinate-CoQreductase, but not oligomycin-sensitive ATPase, were significantly inhibited in the IL-1beta, or SNP-treated myocytes. Both IL-1beta and SNP significantly elevated maximum diastolic potential, reduced peak calcium current (I(Ca)), and lowered contractility in the myocytes. KT5823, an inhibitor of cGMP-dependent protein kinase, did not block the electrophysiological and contractility effects. CONCLUSIONS: These data suggest that IL-1beta-induced NO production in cardiac myocytes lowers energy production and myocardial contractility through a direct attack on the mitochondria, rather than through cGMP-mediated pathways.

Effects of glibenclamide and nicorandil in post-ischaemic contractile dysfunction of perfused hearts in normotensive and spontaneously hypertensive rats.

OBJECTIVE: We have demonstrated previously that nicorandil, an ATP-sensitive potassium channel opener, improved post-ischaemic contractile dysfunction of perfused hearts in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats dose-dependently. This study aimed to characterize the effect of glibenclamide, an ATP-sensitive potassium channel blocker, and nicorandil in post-ischaemic contractile dysfunction of SHR and WKY rats. METHODS: The perfused hearts were subjected to 30 min of global ischaemia and then 30 min of reperfusion. Administration of 10 or 50 mumol/l glibenclamide or of a combination of glibenclamide and 300 mumol/l nicorandil was performed for 10 min before the ischaemia. The left ventricular developed pressure and end-diastolic pressure were measured. RESULTS: Postischaemic contractile function was better in WKY rats than it was in SHR. Neither glibenclamide nor a combination of glibenclamide and nicorandil influenced the postischaemic contractile function or increased the incidence of reperfusion arrhythmias. The recoveries of coronary flow and heart rate after reperfusion were poor and the incidence of reperfusion arrhythmias was low in SHR. CONCLUSIONS: These results suggest that nicorandil improves postischaemic contractile dysfunction via a mechanism involving ATP-sensitive potassium channel opening both in SHR and in WKY rats. The hypertensive hearts were more susceptible to cardiac reperfusion dysfunction, compared with normal hearts.

Counterattack of re-emerging tuberculosis after 38 years.

The recent tuberculosis prevalence rates in Japan have been among the highest in industrialised countries. In 1997 the number of newly identified tuberculosis patients in Japan increased for the first time in 38 years. There are many underlying reasons for this resurgence, including public ignorance of the threat, epidemiological factors, insufficient medical countermeasures, and inadequate public health measures against tuberculosis. Improving the situation will require urgent scientific and political action.

A comparative study of rural clinics in remote islands and inland areas.

The social and professional isolation of physicians remains an important issue in rural areas. However, few studies have investigated the involvement of geographic factors in the isolation. This study investigates rural public clinics in inland and remote island locations and attempts to objectively compare the isolation of these physicians. A mailed questionnaire was sent to rural clinics where graduate physicians from Jichi Medical School were working in 1994 and 1995. Among the 198 clinics with one or more full-time physicians, 185 (93 percent) responded to the inquiry. Geographic and demographic factors of the communities were compared between 43 clinics located in remote islands and the other 142 rural inland clinics. Rural clinics in remote islands have smaller subject populations, fewer part-time physicians, a longer journey to the nearest city, and a longer distance and travel time to the base hospital than rural inland clinics. Physicians in remote island clinics had less medical training and are more isolated than other physicians. More than half of the clinic physicians in remote islands have no regular training schedule, in contrast to less than a quarter of the inland clinic physicians. Almost all clinics (97.7%) in remote islands do not have a part-time physician, whereas about 20 percent of the rural inland clinics do. Physicians in remote island clinics are more socially and professionally isolated than those in inland clinics. Strategies to reduce these problems should be given priority in rural health policy and measures tailored to rural clinics in remote islands.

[Clinical significance of diffuse vasoconstriction of coronary arteries--a study using hyperventilation thallium-201 myocardial imaging].

Hyperventilation Thallium-201 imaging was evaluated for the examination of the existence of ischemia in the cases of diffuse vasoconstriction under the ergonovine maleate provocative test for coronary artery. Transient myocardial perfusion defect (PD) was demonstrated in 14 patients with ergonovine induced vasospasm (group S), and 13 of these patients also demonstrated redistribution (RD) (92.4%). In 14 patients with diffuse vasoconstriction (group D), nine demonstrated PD, and all of them revealed RD. On the other hand, only one of ten (10%) patients demonstrated PD and RD in a group of patients without spasm or diffuse vasoconstriction (group N). In addition, the left ventricular myocardium was divided into nine segments on a SPECT image, and the mean minimum washout rate (WOR) of each segment was evaluated. These values were compared with the percent change of the lung/heart ratio between early and delayed images (delta L/H%). Both the mean minimum WOR mean and delta L/H% of group D were significantly smaller than that of group N (p < 0.001), and only approximated to group S. Thus, the possibility of myocardial ischemia of diffuse vasoconstrictive coronary artery is implicated and such patients are supposed to be treated medically as vasospastic angina.

Time-of-flight secondary ion mass spectrometry with transmission of energetic primary cluster ions through foil targets.

We developed time-of-flight (TOF) secondary ion (SI) mass spectrometry that provides informative SI ion mass spectra without needing a sophisticated ion beam pulsing system. In the newly developed spectrometry, energetic large cluster ions with energies of the order of sub MeV or greater are used as primary ions. Because their impacts on the target surface produce high yields of SIs, the resulting SI mass spectra are informative. In addition, the start signals necessary for timing information on primary ion incidence are provided by the detection signals of particles emitted from the rear surface of foil targets upon transmission of the primary ions. This configuration allows us to obtain positive and negative TOF SI mass spectra without pulsing system, which requires precise control of the primary ions to give the spectra with good mass resolution. We also successfully applied the TOF SI mass spectrometry with energetic cluster ion impacts to the chemical structure characterization of organic thin film targets.

Note: Absolute detection efficiency of a tapered microchannel plate for Ne⁺ ions.

The absolute detection efficiency of a tapered microchannel plate with an open-area ratio of 90% was measured for Ne(+) with energies up to 5 keV, and comparison with the results for Xe(+) was made. As in the case of Xe(+), the maximum detection efficiency was 90%. The energy dependence of the efficiency curves normalized with respect to the open-area ratios was examined based on the scaling law proposed previously.

Direct human mitochondrial transfer: a novel concept based on the endosymbiotic theory.

Mitochondria play an essential role in eukaryotes, and mitochondrial dysfunction is implicated in several diseases. Therefore, intercellular mitochondrial transfer has been proposed as a mechanism for cell-based therapy. In addition, internalization of isolated mitochondria cells by simple coincubation was reported to improve mitochondrial function in the recipient cells. However, substantial evidence for internalization of isolated mitochondria is still lacking, and its precise mechanism remains elusive. We tested whether enriched mitochondria can be internalized into cultured human cells by simple coincubation using fluorescence microscopy and flow cytometry. Mitochondria were isolated from endometrial gland-derived mesenchymal cells (EMCs) or EMCs stably expressing mitochondrial-targeted red fluorescent protein (EMCs-DsRed-mito), and enriched by anti-mitochondrial antibody-conjugated microbeads. They were coincubated with isogeneic EMCs stably expressing green fluorescent protein (GFP). Live fluorescence imaging clearly showed that DsRed-labeled mitochondria accumulated in the cytoplasm of EMCs stably expressing GFP around the nucleus. Flow cytometry confirmed the presence of a distinct population of GFP and DsRed double-positive cells within the recipient cells. In addition, transfer efficiency depended on mitochondrial concentration, indicating that human cells may possess the inherent ability to internalize mitochondria. Therefore, this study supports the application of direct transfer of isogeneic mitochondria as a novel approach for the treatment of diseases associated with mitochondrial dysfunction.