Development of interspecies cloned embryos reconstructed with rabbit (Oryctolagus cuniculus) oocytes and cynomolgus monkey (Macaca fascicularis) fibroblast cell nuclei.

Interspecies somatic cell nuclear transfer (ISCNT) has been proposed as a technique to produce cloned offspring of endangered species as well as to investigate nucleus-cytoplasm interactions in mammalian embryo. However, it is still not known which embryo culture medium is optimal for ISCNT embryos for the nuclear donor or the oocyte recipient. We assessed the effects of the culture medium on the developmental competence of the ISCNT embryos by introducing cynomolgus monkey (Macaca fascicularis) fibroblast nuclei into enucleated rabbit (Oryctolagus cuniculus) oocytes (monkey-rabbit embryo). The monkey-rabbit ISCNT embryos that were cultured in mCMRL-1066 developed to the blastocyst stage, although all monkey-rabbit ISCNT embryos cultured in M199 were arrested by the 4-cell stage. When monkey-rabbit ISCNT and rabbit-rabbit somatic cell nuclear transfer (SCNT) embryos were cultured in mCMRL-1066, the blastocyst cell numbers of the monkey-rabbit ISCNT embryos corresponded to the cell numbers of the control rabbit-rabbit SCNT embryos, which were produced from a rabbit fibroblast nucleus and an enucleated rabbit oocyte. In addition, the presence of mitochondria, which were introduced with monkey fibroblasts into rabbit recipient cytoplasm, was confirmed up to the blastocyst stage by polymerase chain reaction (PCR). This study demonstrated that: (1) rabbit oocytes can reprogramme cynomolgus monkey somatic cell nuclei, and support preimplantation development; (2) monkey-rabbit ISCNT embryos developed well in monkey culture medium at early embryonic developmental stages; (3) the cell number of monkey-rabbit ISCNT embryos is similar to that of rabbit-rabbit SCNT embryos; and (4) the mitochondrial fate of monkey-rabbit ISCNT embryos is heteroplasmic from the time just after injection to the blastocyst stage that has roots in both rabbit oocytes and monkey fibroblasts.

Functional expression of a humanized gene for an omega-3 fatty acid desaturase from scarlet flax in transfected bovine adipocytes and bovine embryos cloned from the cells.

Long-chain n-3 fatty acids can lower the risk of lifestyle-related diseases, therefore, we introduced a plant fatty acid desaturation3 (FAD3) gene into mammalian cells. The FAD3 cDNA was isolated from the immature seeds of scarlet flax and optimized to human high-frequency codon usage for enhancement of its expression levels in mammalian cells (hFAD3). We introduced the gene into bovine muscle satellite cells, which can be differentiated into multilocular adipocytes in vitro. After hFAD3 transfection, the cells were differentiated into adipocytes and their fatty acid composition was analyzed by gas chromatography. The level of alpha-linolenic acid (18:3n-3) in transfected adipocytes increased about ten-fold compared with non-transfected adipocytes. In addition, the levels of docosapentaenoic acid (DPA, 22:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) in transfected adipocytes were significantly higher than those in non-transfected adipocytes. Moreover, we produced bovine cloned embryos from the hFAD3 cells by somatic cell nuclear transfer. Blastocyst rates of hFAD3 clones were the same as the control clones using the non-transfected cells (21% vs 27%, P > 0.05). hFAD3 transcripts were detected in all of the blastocysts. These results demonstrate the functional expression of a plant hFAD3 in mammalian adipocytes, and normal development of cloned embryos carrying the hFAD3 gene.

Deposition of acetylated histones by RNAP II promoter clearance may occur at onset of zygotic gene activation in preimplantation mouse embryos.

We investigated the contribution of phosphorylated RNA polymerase II (RNAP II) and dynamic epigenetic changes to the onset of minor zygotic gene activation (ZGA). Using immunofluorescence staining, we observed that the nuclear localization of RNAP II was initiated by 6 hours post insemination (hpi), whereas RNAP II phosphorylated at serine residue 5 of the carboxyl-terminal domain (CTD) was localized by 9 hpi, and then RNAP II phosphorylated at serine residue 2 of the CTD was localized in the nucleus of embryos by 12 hpi. In a transient gene expression assay using a plasmid reporter gene (pß-actin/luciferase+/SV40) injected during 6-9 hpi into the male pronucleus, the luciferase+ gene was actively transcribed and translated by 13 and 15 hpi, respectively, indicating that a transcriptionally silent state persisted for at least 4 hours after injection. We found that the methylation status in the chicken ß-actin promoter region of the plasmid reporter gene may not be associated with the transcriptionally silent state before minor ZGA. Exposure to trichostatin A did not induce premature expression of the silent reporter gene injected into 1-cell embryos containing histone deacetylase activity and did not affect the amount of luciferase produced per embryo. Acetylated histone H3 lysine 9/14 and acetylated histone H4 lysine 12 and 16 were enriched preferentially in the injected reporter gene at least until 13 hpi, which coincided with the transcriptionally active state. Taken together, these results suggest that deposition of selectively acetylated histones onto the chromatin of 1-cell embryos functions together with transcriptional elongation by RNAP II and that this sequential chromatin remodeling is involved in the molecular mechanism associated with the onset of minor ZGA in the preimplantation mouse embryo.

Inhibition of the ubiquitin-proteasome system leads to delay of the onset of ZGA gene expression.

In mammalian oocytes, the ubiquitin-proteasome system (UPS) is suggested to play important roles in oocyte meiosis resumption, spindle assembly, polar body emission and pronuclear formation by regulating cyclin B1 degradation. However, little is known about the direct relationship between zygotic gene activation (ZGA) and degradation of maternal proteins. Here, we investigated the role of the UPS in the onset of ZGA in early mouse embryos. First, we found degradation of cyclin B1 protein in fertilized oocytes at 1 hpi by western blot analysis and used these oocytes throughout this study. Subsequently, we determined optimal experimental conditions for transient inhibition of proteasomal activity by specific and reversible proteasomal inhibitor MG132 in the G1 phase of the first cell cycle. Under the selected optimal conditions, we subjected transient MG132-treated embryos to reverse transcription (RT)-PCR analysis of expression of four ZGA genes, i.e., the hsp70.1, MuERV-L, eif-1a and zscan4d genes. As a result, we found that onset of expression of the four examined ZGA genes was delayed in both normally developed 2-cell embryos and arrested 1-cell embryos. Our results indicate that proteasomal degradation of proteins by the UPS plays a pivotal role in the molecular mechanisms of ZGA in early mouse embryos.

Abnormal DNA methylation of the Oct-4 enhancer region in cloned mouse embryos.

Oct-4 is essential for normal embryonic development, and abnormal Oct-4 expression in cloned embryos contributes to cloning inefficiency. However, the causes of abnormal Oct-4 expression in cloned embryos are not well understood. As DNA methylation in regulatory regions is known to control transcriptional activity, we investigated the methylation status of three transcriptional regulatory regions of the Oct-4 gene in cloned mouse embryos--the distal enhancer (DE), the proximal enhancer (PE), and the promoter regions. We also investigated the level of Oct-4 gene expression in cloned embryos. Immunochemistry revealed that 85% of cloned blastocysts expressed Oct-4 in both trophectoderm and inner cell mass cells. DNA methylation analysis revealed that the PE region methylation was greater in cloned morulae than in normal morulae. However, the same region was less methylated in cloned blastocysts than in normal blastocysts. We found abnormal expression of de novo methyltransferase 3b in cloned blastocysts. These results indicate that cloned embryos have aberrant DNA methylation in the CpG sites of the PE region of Oct-4, and this may contribute directly to abnormal expression of this gene in cloned embryos.

Somatic cell nuclear transfer: infinite reproduction of a unique diploid genome.

In mammals, a diploid genome of an individual following fertilization of an egg and a spermatozoon is unique and irreproducible. This implies that the generated unique diploid genome is doomed with the individual ending. Even as cultured cells from the individual, they cannot normally proliferate in perpetuity because of the "Hayflick limit". However, Dolly, the sheep cloned from an adult mammary gland cell, changes this scenario. Somatic cell nuclear transfer (SCNT) enables us to produce offspring without germ cells, that is, to "passage" a unique diploid genome. Animal cloning has also proven to be a powerful research tool for reprogramming in many mammals, notably mouse and cow. The mechanism underlying reprogramming, however, remains largely unknown and, animal cloning has been inefficient as a result. More momentously, in addition to abortion and fetal mortality, some cloned animals display possible premature aging phenotypes including early death and short telomere lengths. Under these inauspicious conditions, is it really possible for SCNT to preserve a diploid genome? Delightfully, in mouse and recently in primate, using SCNT we can produce nuclear transfer ES cells (ntES) more efficiently, which can preserve the eternal lifespan for the "passage" of a unique diploid genome. Further, new somatic cloning technique using histone-deacetylase inhibitors has been developed which can significantly increase the previous cloning rates two to six times. Here, we introduce SCNT and its value as a preservation tool for a diploid genome while reviewing aging of cloned animals on cellular and individual levels.

Enhancement of histone acetylation by trichostatin A during in vitro fertilization of bovine oocytes affects cell number of the inner cell mass of the resulting blastocysts.

Histone acetylation is one of the major mechanisms of epigenetic reprogramming of gamete genomes after fertilization to establish a totipotent state for normal development. In the present study, the effects of trichostatin A (TSA), an inhibitor of histone deacetylase, during in vitro fertilization (IVF) of bovine oocytes on subsequent embryonic development were investigated. Cumulus-enclosed oocytes obtained from slaughterhouse bovine ovaries were matured in vitro and subjected to IVF in a defined medium supplemented with 0 (control), 5, 50, and 500 nM TSA for 18 h. After IVF, presumptive zygotes were cultured in modified synthetic oviductal fluid (mSOF) medium until 168 h postinsemination (hpi). Some oocytes were immunostained using antibody specific for histone H4-acetylated lysine 5 at 10 hpi. Cleavage, blastocyst development and cell number of inner cell mass (ICM) and trophectoderm (TE) of blastocysts were assessed. TSA treatment enhanced histone acetylation that was prominent in decondensed sperm nuclei. TSA did not affect the postfertilization cleavage, blastocyst rates, and TE cell number. However, it significantly enhanced ICM cell number (p < 0.05). These results indicate that TSA treatment during IVF of bovine oocytes does not affect blastocyst development but alters the cell number of ICM, suggesting that overriding epigenetic modification of the genome during fertilization has a carryover effect on cell proliferation and differentiation in preimplantation embryos. Thus, further environmental quality controls in assisted reproductive technologies are needed in terms of factors which affect chromatin remodelling.

Recovery of cell nuclei from 15,000 years old mammoth tissues and its injection into mouse enucleated matured oocytes.

Here, we report the recovery of cell nuclei from 14,000-15,000 years old mammoth tissues and the injection of those nuclei into mouse enucleated matured oocytes by somatic cell nuclear transfer (SCNT). From both skin and muscle tissues, cell nucleus-like structures were successfully recovered. Those nuclei were then injected into enucleated oocytes and more than half of the oocytes were able to survive. Injected nuclei were not taken apart and remained its nuclear structure. Those oocytes did not show disappearance of nuclear membrane or premature chromosome condensation (PCC) at 1 hour after injection and did not form pronuclear-like structures at 7 hours after injection. As half of the oocytes injected with nuclei derived from frozen-thawed mouse bone marrow cells were able to form pronuclear-like structures, it might be possible to promote the cell cycle of nuclei from ancient animal tissues by suitable pre-treatment in SCNT. This is the first report of SCNT with nuclei derived from mammoth tissues.

Signs of biological activities of 28,000-year-old mammoth nuclei in mouse oocytes visualized by live-cell imaging.

The 28,000-year-old remains of a woolly mammoth, named 'Yuka', were found in Siberian permafrost. Here we recovered the less-damaged nucleus-like structures from the remains and visualised their dynamics in living mouse oocytes after nuclear transfer. Proteomic analyses demonstrated the presence of nuclear components in the remains. Nucleus-like structures found in the tissue homogenate were histone- and lamin-positive by immunostaining. In the reconstructed oocytes, the mammoth nuclei showed the spindle assembly, histone incorporation and partial nuclear formation; however, the full activation of nuclei for cleavage was not confirmed. DNA damage levels, which varied among the nuclei, were comparable to those of frozen-thawed mouse sperm and were reduced in some reconstructed oocytes. Our work provides a platform to evaluate the biological activities of nuclei in extinct animal species.

Cis-acting elements (E-box and NBE) in the promoter region of three maternal genes (Histone H1oo, Nucleoplasmin 2, and Zygote Arrest 1) are required for oocyte-specific gene expression in the mouse.

We examined the promoter activities of three mouse maternal genes (H1oo, Npm2, and Zar1) in oocytes and pre-implantation embryos, and examined the promoters for cis-acting elements of 5'-flanking region to obtain the best promoter for inducing oocyte-specific gene expression. For the assay, we injected firefly luciferase gene constructs under the control of the promoters into the oocytes and embryos. Each promoter region showed transcriptional activity in oocytes, but not in fertilized embryos. Deletion analysis showed that a putative E-box region at position -72 of the H1oo promoter and at the -180 of the Npm2 promoter were required for basal transcriptional activity in oocytes. Moreover, a putative NBE motif (NOBOX DNA binding elements) (-1796) was shown to enhance basal transcriptional activity of the Npm2 promoter. Thus, the E-box and/or NBE may be key regulatory regions for the expression of the examined maternal genes (H1oo and Npm2) in growing mouse oocytes.

A mouse and embryonic stem cell derived from a single embryo.

Embryonic stem cells (ESCs) are a good material for the study of mammalian development, production of genetically modified animals, and drug discovery because they proliferate infinitely while maintaining a multilineage differentiation potency and a normal karyotype. However, ethical considerations limit the use of human embryos for the establishment of ESCs. Recently, ESCs have been produced from blastomeres divided by biopsy in mice and humans. The method is expected to be less controversial because it does not destroy the embryo. However, no one has yet produced both a pup and an ESC from a single embryo. Here, we describe the production of individual/ESC pairs from each of three embryos out of 20 attempts, and is thus considered efficient. Blastomere-derived ESC could differentiate some types of tissues and contribute to chimera mouse. These results show that each blastomere at two-cell stage possesses pluripotency and separated blastomeres maintain viability to develop to a pup or pluripotent ESC.

Primate embryonic stem cells proceed to early gametogenesis in vitro.

Embryonic stem cells (ESCs) of nonhuman primates are important for research into human gametogenesis because of similarities between the embryos and fetuses of nonhuman primates and those of humans. Recently, the formation of germ cells from mouse ESCs in vitro has been reported. In this study, we established cynomolgus monkey ES cell lines (cyESCs) and attempted to induce their differentiation into germ cells to obtain further information on the development of primate germ cells by observing the markers specific to germ cells. Three cyESCs were newly established and confirmed to be pluripotent. When the cells are induced to differentiate, the transcripts of Vasa and some meiotic markers were expressed. VASA protein accumulated in differentiated cell clumps and VASA-positive cells gathered in clumps as the number of differentiation days increased. In the later stages, VASA-positive clumps coexpressed OCT-4, suggesting that these cells might correspond to early gonocytes at the postmigration stage. Furthermore, meiosis-specific gene expression was also observed. These results demonstrate that cyESCs can differentiate to developing germ cells such as primordial germ cells (PGCs) or more developed gonocytes in our differentiation systems, and may be a suitable model for studying the mechanisms of primate germ cell development.

Mouse zygote-specific proteasome assembly chaperone important for maternal-to-zygotic transition.

During the maternal-to-zygotic transition (MZT), maternal proteins in oocytes are degraded by the ubiquitin-proteasome system (UPS), and new proteins are synthesized from the zygotic genome. However, the specific mechanisms underlying the UPS at the MZT are not well understood. We identified a molecule named zygote-specific proteasome assembly chaperone (ZPAC) that is specifically expressed in mouse gonads, and expression of ZPAC was transiently increased at the mouse MZT. ZPAC formed a complex with Ump1 and associated with precursor forms of 20S proteasomes. Transcription of ZPAC genes was also under the control of an autoregulatory feedback mechanism for the compensation of reduced proteasome activity similar to Ump1 and 20S proteasome subunit gene expression. Knockdown of ZPAC in early embryos caused a significant reduction of proteasome activity and decrease in Ump1 and mature proteasomes, leading to accumulation of proteins that need to be degraded at the MZT and early developmental arrest. Therefore, a unique proteasome assembly pathway mediated by ZPAC is important for progression of the mouse MZT.

Donor cells at the G1 phase enhance homogeneous gene expression among blastomeres in bovine somatic cell nuclear transfer embryos.

The success rate of bovine somatic cell nuclear transfer (SCNT) embryos to full term has been reported to be higher with G1 cells than with G0 cells. To better understand the reason for this, we analyzed the kinetics of luminescence activity in bovine SCNT embryos from G0 and G1 cells carrying a luciferase gene under the control of the ß-actin promoter during early embryonic development. At 60-h postfusion, when bovine embryonic gene activation (EGA) begins, the luminescence activity was higher in G1-SCNT embryos than G0-SCNT embryos. Moreover, half of the G1-SCNT embryos exhibited homogeneous luminescence among the blastomeres, whereas more than half of the G0-SCNT embryos exhibited mosaic luminescence. To characterize the differential luminescence pattern in SCNT embryos, the expressions of several endogenous genes and the level of DNA methylation were determined in all blastomeres of SCNT embryos with or without luminescence. The expressions of several development-related genes (H2AFZ, GJA1, and BAX) and level of DNA methylation of the SCNT embryos with luminescence were the same as those of normal embryos produced by in vitro fertilization. A higher success rate in G1-SCNT embryos is thought to contribute to homogeneous expression among all blastomeres at EGA.

Expression analysis of circadian genes in oocytes and preimplantation embryos of cattle and rabbits.

We previously showed that circadian genes clock, bmal1, cry1, cry2, per1, and per2 are expressed and function as maternal mRNA regulating events in the oocytes and preimplantation embryos of mice. Recent evidence indicates however that either or both expression profiles of circadian genes in some tissues, and transcript sequences of circadian genes, differ to generate the physiological differences between diurnal and nocturnal species. We therefore investigated the expression profiles of circadian genes in oocytes and preimplantation embryos of species other than mice, namely cattle and rabbits, representing diurnal and nocturnal species, respectively, and determined the protein sequences of circadian genes in these species. Quantitative real-time PCR revealed that all circadian genes considered in this study were present in the oocytes and preimplantation embryos of both species, and the transcript amounts of clock, cry1 and per1 contained in oocytes were significantly higher than in preimplantation embryos of both species. The transcripts of clock, cry1, and per1 of cattle and rabbits were determined by primer walking, and functional domains in the estimated amino acid sequences were compared between cattle and rabbits and with those of humans and mice. The sequences of clock, cry1, and per1 in cattle and rabbits closely resembled those in mice (85-100% homologies), and no difference based on diurnality or nocturnality was observed. These findings suggest that circadian genes in the oocytes and preimplantation embryos of mammals fulfill the same functions across species as maternal mRNA.

Resurrection of a bull by cloning from organs frozen without cryoprotectant in a -80 degrees c freezer for a decade.

Frozen animal tissues without cryoprotectant have been thought to be inappropriate for use as a nuclear donor for somatic cell nuclear transfer (SCNT). We report the cloning of a bull using cells retrieved from testicles that had been taken from a dead animal and frozen without cryoprotectant in a -80 degrees C freezer for 10 years. We obtained live cells from defrosted pieces of the spermatic cords of frozen testicles. The cells proliferated actively in culture and were apparently normal. We transferred 16 SCNT embryos from these cells into 16 synchronized recipient animals. We obtained five pregnancies and four cloned calves developed to term. Our results indicate that complete genome sets are maintained in mammalian organs even after long-term frozen-storage without cryoprotectant, and that live clones can be produced from the recovered cells.

Proteomic analysis of the mouse ovary in response to two gonadotropins, follicle-stimulating hormone and luteinizing hormone.

Functional and structural changes in the mammalian ovary are coordinately regulated by the pituitary glycoprotein hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), leading to follicular development, ovulation and transformation of follicles into corpus lutea. To investigate protein profiles during these processes of the mouse ovarian cycle, we applied combined methods (two-dimensional gel electrophoresis [2-DE] for separation and visualization of proteins plus matrix laser desorption/ionization time-of-flight mass spectrometry [MALDI-TOF/MS] analysis for protein identification) for comparative proteomic analysis using immature mice at 3 weeks of age. Protein profiles were obtained from proteins extracted from intact ovaries that had been collected from pregnant mare serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG)-primed immature mice at 0 (no PMSG), 24 and 48 h post PMSG, as well as at 10 and 20 h post hCG. The results showed that 1028 common protein spots were found in representative gels that had been separated in the 3 to 11 pH range and the 15-200 kDa range, 253 protein spots (24.6%) of which were differentially expressed (p<0.05) during the mouse ovarian cycle. Of these 253 protein spots, 99 were identified by MALDI-TOF/MS. This comparative proteomic approach to identifying proteins that were potentially involved in the complex process of the ovarian cycle could contribute to our understanding of the molecular basis of functional and structural changes in the ovary in response to gonadotropins. Furthermore, the interesting ovarian proteins identified in this study may eventually serve as diagnostic biomarker candidates of ovarian function.

Expression and functional analyses of circadian genes in mouse oocytes and preimplantation embryos: Cry1 is involved in the meiotic process independently of circadian clock regulation.

In mammals, circadian genes, Clock, Arntl (also known as Bmal1), Cry1, Cry2, Per1, Per2, and Per3, are rhythmically transcribed every 24 h in almost all organs and tissues to tick the circadian clock. However, their expression and function in oocytes and preimplantation embryos have not been investigated. In this study we found that the circadian clock may stop in mouse oocytes and preimplantation embryos. Real-time PCR analysis revealed the presence of transcripts of these genes in both oocytes and preimplantation embryos; however, their amounts did not oscillate every 24 h in one- to four-cell and blastocyst-stage embryos. Moreover, immunofluorescence analyses revealed that CLOCK, ARNTL, and CRY1 were localized similarly in the nuclei of germinal vesicle (GV) oocytes and one-cell- to four-cell-stage embryos. Because CRY1 is known to interact with the CLOCK-ARNTL complex to suppress transcription-promoting activity of the complex for genes such as Wee1, Cry2, Per1, Per2, and Per3 in cells having the ticking circadian clock, we hypothesized that if the circadian clock functions in GV oocytes and one-cell- to four-cell-stage embryos, CLOCK, ARNTL, and CRY1 might suppress the transcription of these genes in GV oocytes and one-cell- to 4-cell-stage embryos as well. As a result, knockdown of CRY1 in GV oocytes by RNA interference did not affect the transcription levels of Wee1, Cry2, Per1, Per2, and Per3, but it reduced maturation ability. Thus, it seems that circadian genes are not involved in circadian clock regulation in mouse oocytes and preimplantation embryos but are involved in physiologies, such as meiosis.

Identification of ZAG1, a novel protein expressed in mouse preimplantation, and its putative roles in zygotic genome activation.

We isolated a mouse cDNA, zag1 (zygotic gene activation-associated gene 1), that has an open reading frame of 1,728-bp encoding a protein of 66.2 kDa including both a bipartite nuclear targeting sequence and a P-loop motif containing nucleoside triphosphate hydrolase motifs. Northern blot analysis of mouse tissues showed that zag1 was widely expressed but was especially prominent in the ovary and testis. RT-PCR analysis of in vitro fertilized embryos showed that the abundance of zag1 transcripts in oocytes decreased after fertilization, and zag1 mRNA was detected at 15 h post insemination (hpi) in fertilized embryos indicating that the gene was expressed at the start of zygotic gene activation at the mouse 1-cell stage. The nuclear-localization of ZAG1 protein in mouse preimplantation embryos at 15 hpi was confirmed by both subcellular analysis of enhanced green fluorescent protein (EGFP)-tagged ZAG1 and immunocytochemical analysis with anti-ZAG1 antibody. Subsequently, using yeast two-hybrid screening, we identified U2 small nuclear ribonucleoprotein B (U2B"), which is associated with pre-mRNA splicing, as a putative interacting partner of ZAG1 protein. Furthermore, knockdown of zag1 expression by an antisense DNA plasmid induced arrest and/or delay of embryonic development in injected 1-cell embryos. These results suggest that ZAG1 may be closely associated with zygotic gene expression in mouse preimplantation embryos.

Mitochondrial dysfunction of in vitro grown rabbit oocytes results in preimplantation embryo arrest after activation.

To clarify the mechanism that impairs development of in vitro grown (IVG) oocytes, we assessed whether the developmental disability of IVG oocytes is caused by cytoplasmic dysfunction. First, we assessed the cleavage of nuclear-substituted oocytes cultured in vitro. The nuclei, but not the cytoplasm, of the IVG oocytes were able to support subsequent cleavage after artificial activation. The mitochondrial activity of the oocytes increased as the follicles grew. However, the mitochondrial activity of the IVG oocytes was significantly lower than that of ovulated oocytes and oocytes recovered from follicles with diameters of more than 300 microm. Furthermore, the expression levels of mitochondrial transcriptional factor A (TFAM) in the oocytes increased in a similar manner. However, the expression levels of TFAM in the IVG oocytes was significantly lower than that of ovulated oocytes and oocytes recovered from follicles with diameters of more than 300 microm. Taken together, these results indicate that the low developmental competence of IVG oocytes is caused by a cytoplasm deficiency due to low mitochondrial activity.