Mitochondrial aggregation caused by cytochalasin B compromises the efficiency and safety of three-parent embryo.

It is widely accepted that cytochalasin B (CB) is required in enucleation of the oocyte in order to stabilize the cytoplasm. However, CB treatment results in the uneven distribution of mitochondria, with aggregation towards the nucleus, which might compromise the efficiency and safety of a three-parent embryo. Here, we demonstrated that CB treatment affected mitochondrial dynamics, spindle morphology and mitochondrial DNA carryover in a concentration-dependent manner. Our results showed that mouse oocytes treated with over 1 µg/ml CB exhibited a more aggregated pattern of mitochondria and diminished filamentous actin expression. Abnormal fission of mitochondria together with changes in spindle morphology increased as CB concentration escalated. Based on the results of mouse experiments, we further revealed the practical value of these findings in human oocytes. Chip-based digital PCR and pyrosequencing revealed that the mitochondrial carryover in reconstituted human embryos was significantly reduced by modifying the concentration of CB from the standard 5 µg/ml to 1 µg/ml before spindle transfer and pronuclear transfer. In conclusion, our findings provide an optimal manipulation for improving the efficiency and safety of mitochondrial replacement therapy.

Preimplantation genetic diagnosis for a carrier with m.3697G > A mitochondrial DNA mutation.

OBJECTIVE: To explore inheritance of the m.3697G > A mitochondrial DNA (mtDNA) mutation and the effectiveness of preimplantation genetic diagnosis (PGD) for the carrier. METHODS: The study encompassed a pedigree of m.3697G > A mtDNA mutation, including one asymptomatic patient who pursued for PGD treatment. Twelve cumulus oocyte complexes (COCs) were collected in the first PGD cycle and 11 COCs in the second cycle. The efficiency of cumulus cells, polar bodies, and trophectoderm (TE) in predicting the m.3697G > A heteroplasmy of embryos was analyzed. RESULTS: From 23 COCs, 20 oocytes were fertilized successfully. On day 5 and 6 post-fertilization, 15 blastocysts were biopsied. The m.3697G > A mutation load of TE biopsies ranged from 15.2 to 100%. In the first cycle, a blastocyst with mutation load of 31.7% and chromosomal mosaicism was transferred, but failed to yield a clinical pregnancy. In the second cycle, a euploid blastocyst with mutation load of 53.9% was transferred, which gave rise to a clinical pregnancy. However, the pregnancy was terminated due to fetal cleft lip and palate. The mutation loads of different tissues (47.7 ± 1.8%) from the induced fetus were comparable to that of the biopsied TE and amniotic fluid cell (49.7%). The mutation load of neither cumulus cells (R2 = 0.02, p = 0.58) nor polar bodies (R2 = 0.33, p = 0.13) correlated with TE mutation load which was regarded as a gold standard. CONCLUSIONS: The m.3697G > A mutation showed a random pattern of inheritance. PGD could be used to reduce the risk of inheritance of a high mutation load. Cumulus cells are not a suitable predictor of blastocyst mutation load.

ATAD3B is a mitophagy receptor mediating clearance of oxidative stress-induced damaged mitochondrial DNA.

Mitochondrial DNA (mtDNA) encodes several key components of respiratory chain complexes that produce cellular energy through oxidative phosphorylation. mtDNA is vulnerable to damage under various physiological stresses, especially oxidative stress. mtDNA damage leads to mitochondrial dysfunction, and dysfunctional mitochondria can be removed by mitophagy, an essential process in cellular homeostasis. However, how damaged mtDNA is selectively cleared from the cell, and how damaged mtDNA triggers mitophagy, remain mostly unknown. Here, we identified a novel mitophagy receptor, ATAD3B, which is specifically expressed in primates. ATAD3B contains a LIR motif that binds to LC3 and promotes oxidative stress-induced mitophagy in a PINK1-independent manner, thus promoting the clearance of damaged mtDNA induced by oxidative stress. Under normal conditions, ATAD3B hetero-oligomerizes with ATAD3A, thus promoting the targeting of the C-terminal region of ATAD3B to the mitochondrial intermembrane space. Oxidative stress-induced mtDNA damage or mtDNA depletion reduces ATAD3B-ATAD3A hetero-oligomerization and leads to exposure of the ATAD3B C-terminus at the mitochondrial outer membrane and subsequent recruitment of LC3 for initiating mitophagy. Furthermore, ATAD3B is little expressed in m.3243A > G mutated cells and MELAS patient fibroblasts showing endogenous oxidative stress, and ATAD3B re-expression promotes the clearance of m.3243A > G mutated mtDNA. Our findings uncover a new pathway to selectively remove damaged mtDNA and reveal that increasing ATAD3B activity is a potential therapeutic approach for mitochondrial diseases.

Matching Mitochondrial DNA Haplotypes for Circumventing Tissue-Specific Segregation Bias.

Mitochondrial DNA (mtDNA) segregation associated with donor-recipient mtDNA mismatch in mitochondria replacement therapy leads to unknown risks. Here, to explore whether matching mtDNA haplotypes contributes to ameliorating segregation, we reproduced various degrees of heteroplasmic mice with three single nucleotide polymorphisms to monitor segregation severity. "Segregation" presented in tissues of heteroplasmic mice containing low-level donor mtDNA heteroplasmy, and disappeared as donor mtDNA heteroplasmy levels ascended. Meanwhile, we found that distribution of donor mtDNA among the blastomeres of preimplantation embryos from the heteroplasmic mice shared the same tendency as that in adult tissues. Statistical analysis showed that no selective replication of donor mtDNA occurred during lifespan. Tracking donor mtDNA distribution showed that uneven distribution of donor mtDNA among embryonic blastomeres gradually became even as donor mtDNA heteroplasmy increased, indicating that the "segregation" in tissues was inherited from the uneven distribution. Our finding suggested that donor-recipient mtDNA matching could circumvent segregation in mitochondria replacement therapy.

IL-17 promoted the inhibition of medulloblastoma in mice by splenocyte injection.

BACKGROUND: Interleukin 17 (IL-17) is a proinflammatory cytokine produced by a new subset of activated CD4+ T cells, Th17 cells. We previously showed that increased Th17 cell populations were presented in human medulloblastoma-infiltrating T cells and peripheral blood. In this study, we attempted to address the possible role of Th17 cells in the biologic activity of IL-17 for tumor control. METHODS: We grafted fresh surgically obtained medulloblastoma into syngeneic athymic nude/nude mice. We intrapertonially injected splenocyte and murine IL-17 in mice on the second day. The tumor volume and the life spans of the mice were measured. Meanwhile, the IL-17, IL-6, IL-23, Ccl2, Ccl20 and IFN-gamma expression in the tumors was also examined by real-time PCR, Western blot and enzyme-linked immunosorbent assay. RESULTS: We found that medulloblastoma growth in IL-17-injected mice was significantly inhibited compared to the non-IL-17 treated mice. In contrast to the IL-17 antitumor activity observed in mice injected with splenocytes, we observed that IFN-gamma, IL-6, IL-23, Ccl2, and Ccl20 proteins were significantly increased in tumor tissues of mice injected with IL-17. CONCLUSIONS: These experiments suggest that IL-17 may promote splenocyte antitumor activity in medulloblastoma. We postulate that IL-17's antitumor activity may be related to the increased protein levels of IFN-gamma, IL-6, IL-23, Ccl2, and Ccl20.

Ooplast transfer of triploid pronucleus zygote improve reconstructed human-goat embryonic development.

Poor development of the interspecies somatic cell nuclear transfer (iSCNT) embryos was due to nuclear-mitochondrial incompatibility. In humans, it has been known that ooplast transfer (OT) could support normal fertilization, the development of embryos and prevents the transmission of mtDNA disease. To investigate whether OT could support development of the iSCNT embryos, the ooplast of Triploid Pronucleus (3PN) zygote which would be discarded in IVF lab was transferred into the enucleated goat oocytes to construct humanized iSCNT embryos in our study. The results showed the 3PN-OT could significantly improve the early development of humanized iSCNT embryos. The percentage of blastocyst development of OT group was also higher than that of the control group. Interestingly, the morphology of some OT-iSCNT blastocysts was similar to normal human blastocysts in vitro fertilization, while the morphology of iSCNT blastocysts from control group was similar to goat blastocysts. Importantly, the pluripotent marker Oct4 of the OT-iSCNT blastocyst was expressed stronger than that of the control group. These results suggested that 3PN-OT could improve the developmental potency of human iSCNT embryos and would facilitate establishing ESCs from iSCNT blastocysts.

Polar body genome transfer for preventing the transmission of inherited mitochondrial diseases.

Inherited mtDNA diseases transmit maternally and cause severe phenotypes. Currently, there is no effective therapy or genetic screens for these diseases; however, nuclear genome transfer between patients' and healthy eggs to replace mutant mtDNAs holds promises. Considering that a polar body contains few mitochondria and shares the same genomic material as an oocyte, we perform polar body transfer to prevent the transmission of mtDNA variants. We compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Reconstructed embryos support normal fertilization and produce live offspring. Importantly, genetic analysis confirms that the F1 generation from polar body transfer possesses minimal donor mtDNA carryover compared to the F1 generation from other procedures. Moreover, the mtDNA genotype remains stable in F2 progeny after polar body transfer. Our preclinical model demonstrates polar body transfer has great potential to prevent inherited mtDNA diseases.

Neural stem cell transplants improve cognitive function without altering amyloid pathology in an APP/PS1 double transgenic model of Alzheimer's disease.

Neural stem cells (NSCs) are capable of self-renewal and are multipotent. Transplantation of NSCs may represent a promising approach for treating neurodegenerative disorders associated with cognitive decline, such as Alzheimer disease (AD) characterized by extensive loss of neurons. In this study, we investigated the effect of NSC transplantation on cognitive function in the amyloid precursor protein/presenilin-1 (APP/PS1) transgenic mouse, an AD mouse model with age-dependent cognitive deficits. We found that NSCs bilaterally transplanted into hippocampal regions improved spatial learning and memory function in these mice, but did not alter Aß pathology. Immunohistochemical analyses determined that NSCs proliferated, migrated, and differentiated into three neuronal cell types. The improvement in cognitive function was correlated with enhanced long-term potentiation (LTP) and an increase in the neuron expression of proteins related to cognitive function: N-methyl-D-aspartate (NMDA) 2B unit, synaptophysin (SYP), protein kinase C ζ subtypes (PKCζ), tyrosine receptor kinase B (TrkB), and brain-derived neurotrophic factor (BDNF). Taken together, our data indicated that injected NSCs can rescue cognitive deficits in APP/PS1 transgenic mice by replacing neuronal cell types expressing multiple cognition-related proteins that enhance LTP.

Generation of patient-specific induced neuronal cells using a direct reprogramming strategy.

Direct reprogramming of human fibroblasts into functional neurons in vitro by defined factors provides an invaluable resource for regenerative medicine. However, clinical applications must consider the risk of immune rejection, thus patient-specific induced neuronal cells (iNCs) may serve as an ideal source for autologous cell replacement. In this study, we report a robust process for functional neuronal cells from the patients' scalp by lentiviral gene delivery of Ascl1, Myt1l, and Sox2. These three-factor iNCs are similar to human neuronal cells in morphology, surface antigens, gene expression, and electrophysiological characteristics. Our findings might provide a source of patient-specific functional neurons for cell therapy.

Tracking induced pluripotent stem cells-derived neural stem cells in the central nervous system of rats and monkeys.

Despite of the immense breakthroughs of induced pluripotent stem cells (iPSCs), clinical application of iPSCs and their derivates remains hampered by a lack of definitive in vivo studies. Here, we attempted to track iPSCs-derived neural stem cells (NSCs) in the rodent and primate central nervous system (CNS) and explore their therapeutic viability for stem cell replacement in traumatic brain injury (TBI) rats and monkeys with spinal cord injury (SCI). Superparamagnetic iron oxide (SPIO) particles were used to label iPSCs-derived NSCs in vitro. Labeled NSCs were implanted into TBI rats and SCI monkeys 1 week after injury, and then imaged using gradient reflection echo (GRE) sequence by 3.0T magnetic resonance imaging (MRI) scanner. MRI analysis was performed at 1, 7, 14, 21, and 30 days, respectively, following cell transplantation. Pronounced hypointense signals were initially detected at the cell injection sites in rats and monkeys and were later found to extend progressively to the lesion regions, demonstrating that iPSCs-derived NSCs could migrate to the lesion area from the primary sites. The therapeutic efficacy of iPSCs-derived NSCs was examined concomitantly through functional recovery tests of the animals. In this study, we tracked iPSCs-derived NSCs migration in the CNS of TBI rats and SCI monkeys in vivo for the first time. Functional recovery tests showed obvious motor function improvement in transplanted animals. These data provide the necessary foundation for future clinical application of iPSCs for CNS injury.

MicroRNAs in regulation of pluripotency and somatic cell reprogramming: small molecule with big impact.

MicroRNAs (miRNAs), a group of small non-coding RNAs, have emerged as significant modulators in the establishment and generation of pluripotency, a developmental process that consists of complex cell-fate arrangements. The finding of embryonic stem cell (ESC) cycle-specific miRNAs reveals an important regulation scheme of pluripotency. Subsequent studies showed the ESC-enriched or ESC-depleted miRNAs can regulate induced pluripotent stem cells(iPSC). Moreover, miRNA profiling of iPSC and ESC may distinguish them from one another and facilitate the complex of regulatory network. The accumulative effects of miRNA action enable using miRNA alone to generate iPSCs. Despite the robustness of iPSC studies, further investigations are needed since miRNA may have more impact on induced pluripotency, and the roles of miRNAs in somatic cell nuclear transfer (SCNT), another approach toward cellular reprogramming, remains unclear. This point-of-view article will discuss miRNAs and their impact on the normal and induced pluripotency, as well as bring new insights on somatic cell reprogramming.

Early exercise affects mitochondrial transcription factors expression after cerebral ischemia in rats.

Increasing evidence shows that exercise training is neuroprotective after stroke, but the underlying mechanisms are unknown. To clarify this critical issue, the current study investigated the effects of early treadmill exercise on the expression of mitochondrial biogenesis factors. Adult rats were subjected to ischemia induced by middle cerebral artery occlusion followed by reperfusion. Expression of two genes critical for transcriptional regulation of mitochondrial biogenesis, peroxisome proliferator-activated receptor coactivator-1 (PGC-1) and nuclear respiratory factor-1 (NRF-1), were examined by RT-PCR after five days of exercise starting at 24 h after ischemia. Mitochondrial protein cytochrome C oxidase subunit IV (COX IV) was detected by Western blot. Neurological status and cerebral infarct volume were evaluated as indices of brain damage. Treadmill training increased levels of PGC-1 and NRF-1 mRNA, indicating that exercise promotes rehabilitation after ischemia via regulation of mitochondrial biogenesis.

BDNF promotes EGF-induced proliferation and migration of human fetal neural stem/progenitor cells via the PI3K/Akt pathway.

Neurogenesis is a complex process, which contributes to the ability of the adult brain to function normally and adapt to diseases. Epidermal growth factor (EGF) is known to play an important role in neurogenesis; however, the underlying mechanism is still unclear. Here, we hypothesized that brain-derived neurotrophic factor (BDNF) can enhance the effect of EGF on neurogenesis. Using in vitro cell culture of aborted human fetal brain tissues, we investigated proliferation and migration of neural stem/progenitor cells (NSPCs) after treatment with EGF and different concentrations of BDNF. EGF stimulated proliferation and migration of NSPCs, and this effect was significantly enhanced by co-incubation with BDNF. In the NSPCs treated with 50 ng/mL BDNF, BrdU incorporation was significantly increased (from 7.91% to 17.07%), as compared with that in the control. Moreover, the number of migrating cells was at least 2-fold higher than that in the control. Furthermore, phosphorylation of Akt-1 was increased by BDNF treatment, as well. By contrast, the enhancing effect of BDNF on EGF-induced proliferation and migration of NSPCs were abolished by an inhibitor of PI3K, LY294002. These findings suggest that BDNF promotes EGF-induced proliferation and migration of NSPC through the PI3K/Akt pathway, providing significant insights into not only the mechanism underlying EGF-induced neurogenesis but also potential neuronal replacement strategies to treat brain damage.

N-terminal pro-B-type natriuretic peptide in patients with isolated traumatic brain injury: a prospective cohort study.

BACKGROUND: The role of brain natriuretic peptide (BNP) after traumatic brain injury (TBI) remains unclear, and its relationship with hyponatremia is still controversial. The aim of this study is to investigate the secretion pattern of N-terminal (NT)-proBNP in patients with TBI and to assess the relationship between NT-proBNP, sodium balance, and intracranial pressure (ICP). METHODS: We measured serum NT-proBNP levels of 84 patients with isolated TBI on a daily basis from day 1 to day 14 after injury. RESULTS: In average, the peak of BNP level was measured at 703.9 pg/mL±179.1 pg/mL on day 3 after injury, which was correlated to the severity of TBI. Among patients with severe TBI, plasma NT-proBNP concentrations in patients with hyponatremia were statistically higher than those without hyponatremia (p<0.05). In the hyponatremic group, the plasma NT-proBNP increased to a peak of 1001.16 pg/mL±131.52 pg/mL within 48 hours after injury and maintained at a high level for 3 days. In the normonatremic group, the plasma NT-proBNP reached a peak of 826.43 pg/mL±337.43 pg/mL on day 5 and quickly decreased thereafter. In addition, we found plasma NT-proBNP concentrations in patients with ICP>15 mm Hg were significantly higher than those in patients with ICP≤15 mm Hg (p<0.01). CONCLUSIONS: This study provides evidence that BNP plasma concentrations increase rapidly after TBI. Plasma BNP concentrations are correlated with hyponatremia in severe TBI patients but not in mild and moderate TBI patients. Furthermore, patients with elevated ICP have a higher serum BNP level in first 4 days after injury.

Detection of neural stem cells function in rats with traumatic brain injury by manganese-enhanced magnetic resonance imaging.

BACKGROUND: Previously we had successfully tracked adult human neural stem cells (NSCs) labeled with superparamagnetic iron oxide particles (SPIOs) in host human brain after transplantation in vivo non-invasively by magnetic resonance imaging (MRI). However, the function of the transplanted NSCs could not be evaluated by the method. In the study, we applied manganese-enhanced MRI (ME-MRI) to detect NSCs function after implantation in brain of rats with traumatic brain injury (TBI) in vivo. METHODS: Totally 40 TBI rats were randomly divided into 4 groups with 10 rats in each group. In group 1, the TBI rats did not receive NSCs transplantation. MnCl2·4H2O was intravenously injected, hyperosmolar mannitol was delivered to disrupt rightside blood brain barrier, and its contralateral forepaw was electrically stimulated. In group 2, the TBI rats received NSCs (labeled with SPIO) transplantation, and the ME-MRI procedure was same to group 1. In group 3, the TBI rats received NSCs (labeled with SPIO) transplantation, and the ME-MRI procedure was same to group 1, but diltiazem was introduced during the electrical stimulation period. In group 4, the TBI rats received phosphate buffered saline (PBS) injection, and the ME-MRI procedure was same to group 1. RESULTS: Hyperintense signals were detected by ME-MRI in the cortex areas associated with somatosensory in TBI rats of group 2. These signals, which could not be induced in TBI rats of groups 1 and 4, disappeared when diltiazem was introduced in TBI rats of group 3. CONCLUSION: In this initial study, we mapped implanted NSCs activity and its functional participation within local brain area in TBI rats by ME-MRI technique, paving the way for further pre-clinical research.

Fates of donor and recipient mitochondrial DNA during generation of interspecies SCNT-derived human ES-like cells.

To investigate nuclear donor and cytoplast recipient mitochondria fate and their effects on generation of interspecies somatic cell nuclear transfer (iSCNT)-derived human embryonic stem (ES)-like cells, iSCNT embryos were reconstructed between enucleated goat oocytes and human neural stem cells (hNSCs). A total of 10.74% cleaved embryos (13/121) developed to blastocyst stage. One typical primary ES-like (tpES-like) colony and two nontypical primary ES-like (non-tpES-like) colonies designated as non-tpES-like cell-1 and non-tpES-like cell-2, respectively, were obtained from the inner cell masses of iSCNT blastocysts. The tpES-like cells expressed ESC markers. Both human and goat mtDNA could be detected in the embryos at 2-8-, 16-32-cell, and blastocyst stages, and in tpES-like colony and two non-tpES-like colonies. Human mtDNA copies per cell from embryos at two- to eight-cell stage to the three colonies maintain almost its original level, whereas 2.88 x 10(5) goat mtDNA copies per oocyte decreased to 10.8 copies per tpES-like cell, 493 copies per non-tpES-like cell-1, and 77.6 copies per non-tpES-like cell-2, resulting in 43.75% (8.4/19.2), 1.24% (6.2/499), and 14.63% (13.3/90.9) mtDNA content in tpES-like cell, non-tpES-like cell-1, and non-tpES-like cell-2 was that of nuclear donor, respectively. Human-specific Tfam and Polg mRNA could be detected in cells of the three colonies. However, tpES-like colony failed to be passaged. The mRNA level of CoxIV encoded by nuclear donor in tpES-like cell was higher than that in non-tpES-like cell, but significantly lower than that of human ESC, suggesting proper nuclear-cytoplasmic communication would not be established in tpES-like cells. Thus, the data suggest that (1) goat oocytes could reprogram human neural stem cells (hNSCs) into embryonic state and further support the inner cell mass (ICM) of iSCNT blastocyst to form tpES-like colony; (2) nuclear donor mtDNA could be replicated and maintain its original level during the reduction of recipient mitochondrial DNA copies, (3) nuclear-cytoplasmic communication and recipient mtDNA copies might affect the derivation of iSCNT-derived ES-like cells.

Close relatedness between exotic nucleus and cytoplast can improve the postimplantation development rate of cloned intersubspecies embryos.

To improve intersubspecies cloning efficiency, this paper provides six kinds of SCNT embryos with different nuclear-cytoplasmic relatedness to compare the relatedness on the cloning efficiency. Three kinds of SCNT embryos with different relatedness are produced by using Boer goat fibroblast cells as nuclear donors and oocytes of Sannen goat, crossbred F1 (Sannen goat x Boer goat) and Boer goat as cytoplast recipients. Four kinds of SCNT embryos with different relatedness are produced by using Sannen goat oocytes as recipients and fibroblast cells of Boer goat, crossbred F2 (crossbred F1 x Boer goat), crossbred F1, and Sannen goat as nuclear donors. Results show that no obvious differences were observed for preimplantation development of these SCNT embryos. However, different nuclear-cytoplasmic relatedness resulted in obvious differences for postimplantation development of these SCNT embryos. The relatedness is complementary: improving either cytoplasmic compatibility relatedness to nucleus or nuclear relatedness to cytoplast could reduce the gestation loss rate, and increase the birth rate of cloned intersubspecies embryos significantly. But a further amelioration of the relatedness did not improve the postimplantation development in direct proportion. These results suggested that close nuclear-cytoplasmic relatedness can improve the postimplantation development rate of cloned intersubspecies embryos.

Goat MII ooplasts support preimplantation development of embryos cloned from other species.

The preimplantation development competences of somatic cell nuclear transfer (SCNT) embryos reconstructed with enuleated goat (Capra hircus) Metaphase II (MII) oocytes matured in vivo and whole cells derived from adult fibroblasts of several mammalian species (goat, boer goat, bovine, tahr, panda) and human patient were evaluated. Results obtained from our experiments revealed that these reconstructed SCNT embryos could complete preimplantation development to form blastocysts. The fusion rate and blastocyst rate of intra-species SCNT embryos (Capra hircus as control) was 78.67 (557/708); 56.29% (264/469), that of sub-species or inter-species SCNT embryos were: boer goat 78.18% (541/692); 33.90% (40/118), bovine 70.53% (146/207); 22.52% (25/111), tahr 53.51% (61/114); 5.26% (3/570), panda 79.82% (1159/1452); 8.35% (75/898) and human 68.76% (317/461); 5.41% (16/296), respectively. It is concluded that (1) there are no relationships between fusion rate and relativeness of the recipient cytoplasm to nucleus donor cells, (2) cytoplast of the goat MII oocyte can support the preimplantation development of SCNT embryos reconstructed with nucleus from other species, (3) the blastocyst rate of close relative inter-species SCNT embryos is higher than that of distant relative inter-species SCNT embryos.

Different intervals of ovum pick-up affect the competence of oocytes to support the preimplantation development of cloned bovine embryos.

The objective of this study was to determine the effect of different frequencies of transvaginal ovum pick-up (OPU) on the quantity of recovered cumulus oocyte complexes (COCs) and subsequently the competence of matured oocytes to support the preimplantation development of cloned bovine embryos. The COCs were aspirated from the ovaries of 6 Chinese Holstein cows by transvaginal follicle aspiration twice a week (every 3 or 4 days) (Group I), every 5 days (Group II), once a week (every 7 days) (Group III), every 10 days (Group IV), and once every 2 weeks (every 14 days) (Group V). The developmental stages of the follicles were confirmed by the diameter of the dominant follicle (DF) and harvested COCs, and the dynamics of the follicular wave were clarified. In addition, extrusions of the first polar body (PB I) from the oocytes were observed at different time intervals after the initiation of in vitro maturation (IVM) to identify the appropriate culture time window for somatic cell nuclear transfer. Matured oocytes were used to produce cloned bovine embryos that were subsequently cultured in the goat oviduct. After 7 days, the embryos were flushed out, and the developmental rates of the blastocysts were compared among the five groups. The results showed that the aspirations of all follicles >or=3 mm in diameter (D1) induced and synchronized the dynamics of the follicular wave, and the subordinate follicles became atretic after 4 days (D5). Another follicular wave started between D7 and D10, and atresia in the subordinate follicles in the second follicular wave began on D14. The timing of meiotic progression (from the initiation of IVM to the extrusion of PB I) in the oocytes obtained by OPU was later than that of the oocytes obtained from the abattoir. Between 20 and 24 hr after the initiation of IVM, 20% of the oocytes extruded their PB I. Further, 80% (520/650) of the harvested COCs were arrested at metaphase II (MII) by 22 hr of the initiation of IVM and were used as cytoplast donors. The rates of development of the reconstituted embryos to the blastocyst stage were 23.1% (Group I), 15.0% (Group II), 10.9% (Group III), 4.9% (Group IV), and 29.0% (Group V). The results indicate that the developmental potential of follicles from the same living donors were different when different intervals of OPU were adopted and early atretic follicles from the second follicular wave had higher competence to support the early development of cloned bovine embryos.