Da-Yuan Chen

Interspecies Implantation and Mitochondria Fate of Panda-Rabbit Cloned Embryos

Abstract Somatic cell nuclei of giant pandas can dedifferentiate in enucleated rabbit ooplasm, and the reconstructed eggs can develop to blastocysts. In order to observe whether these interspecies cloned embryos can implant in the uterus of an animal other than the panda, we transferred approximately 2300 panda-rabbit cloned embryos into 100 synchronized rabbit recipients, and none became pregnant. In another approach, we cotransferred both panda-rabbit and cat-rabbit interspecies cloned embryos into the oviducts of 21 cat recipients. Fourteen recipients exhibited estrus within 35 days; five recipients exhibited estrus 43–48 days after embryo transfer; and the other two recipients died of pneumonia, one of which was found to be pregnant with six early fetuses when an autopsy was performed. Microsatellite DNA analysis of these early fetuses confirmed that two were from giant panda-rabbit cloned embryos. The results demonstrated that panda-rabbit cloned embryos can implant in the uterus of a third species, the domestic cat. By using mitochondrial-specific probes of panda and rabbit, we found that mitochondria from both panda somatic cells and rabbit ooplasm coexisted in early blastocysts, but mitochondria from rabbit ooplasm decreased, and those from panda donor cells dominated in early fetuses after implantation. Our results reveal that mitochondria from donor cells may substitute those from recipient oocytes in postimplanted, interspecies cloned embryos.

Trichostatin A (TSA) improves the development of rabbit‐rabbit intraspecies cloned embryos, but not rabbit‐human interspecies cloned embryos

The interspecies somatic cell nuclear transfer (iSCNT) technique for therapeutic cloning gives great promise for treatment of many human diseases. However, the incomplete nuclear reprogramming and the low blastocyst rate of iSCNT are still big problems. Herein, we observed the effect of TSA on the development of rabbit–rabbit intraspecies and rabbit–human interspecies cloned embryos. After treatment with TSA for 6 hr during activation, we found that the blastocyst rate of rabbit–rabbit cloned embryos was more than two times higher than that of untreated embryos; however, the blastocyst rate of TSA‐treated rabbit–human interspecies cloned embryos decreased. We also found evident time‐dependent histone deacetylation‐reacetylation changes in rabbit–rabbit cloned embryos, but not in rabbit–human cloned embryos from fusion to 6 hr after activation. Our results suggest that TSA‐treatment does not improve blastocyst development of rabbit–human iSCNT embryos and that abnormal histone deacetylation‐reacetylation changes in iSCNT embryos may account for their poor blastocyst development. Developmental Dynamics 237:640–648, 2008.

Polo-Like Kinase-1 Is a Pivotal Regulator of Microtubule Assembly During Mouse Oocyte Meiotic Maturation, Fertilization, and Early Embryonic Mitosis

Abstract Polo-like kinases (Plks) are a family of serine/threonine protein kinases that have been activated through phosphorylation. The activity of these kinases has been shown to be required for regulating multiple stages of mitotic progression in somatic cells. In this experiment, the changes in Plk1 expression were detected in mouse oocytes through Western blotting. The subcellular localization of Plk1 during oocyte meiotic maturation, fertilization, and early cleavage as well as after antibody microinjection or microtubule assembly disturbance was studied by confocal microscopy. The quantity of Plk1 protein remained stable during meiotic maturation and decreased gradually after fertilization. Plk1 was localized to the spindle poles of both meiotic and mitotic spindles at the early M phase and then translocated to the middle region. At anaphase and telophase, Plk1 was concentrated at the midbody of cytoplasmic cleavages. Plk1 was concentrated between the male and female pronuclei after fertilization. Plk1 disappeared at the spindle region when microtubule formation was inhibited by colchicine or staurosporine, while it was concentrated as several dots in the cytoplasm after taxol treatment. Plk1 antibody injection decreased the germinal vesicle breakdown rate and distorted MI spindle organization. Our results indicate that Plk1 is a pivotal regulator of microtubule organization during mouse oocyte meiosis, fertilization, and cleavage and that its functions may be regulated by other kinases, such as staurosporine-sensitive kinases.

MEK1/2 Regulates Microtubule Organization, Spindle Pole Tethering and Asymmetric Division During Mouse Oocyte Meiotic Maturation

It is well known that MAPK plays pivotal roles in oocyte maturation, but the function of MEK (MAPK kinase) remains unknown. We have studied the expression, subcellular localization and functional roles of MEK during meiotic maturation of mouse oocytes. We found that MEK1/2 phoshorylation (p-MEK1/2, indicative of MEK activation) was low in GV (germinal vesicle) stage, increased 2h after GVBD (germinal vesicle breakdown), and reached the maximum at metaphase II. Secondly, we found that P-MEK1/2 was restricted in the GV prior to GVBD. In prometaphase I and metaphase I, p-MEK1/2 was mainly associated with the spindle, especially with the spindle poles. At anaphase I and telophase I, p-MEK1/2 became diffusely distributed in the region between the separating chromosomes, and then became associated with the midbody. The association of p-MEK1/2 with spindle poles was further confirmed by its colocalization with the centrosomal proteins, γ-tubulin and NuMA. Thirdly, we have investigated the possible functional role of MEK1/2 activation by intravenous administration and intrabursal injection of a specific MEK inhibitor, U0126, and by microinjection of MEK siRNA into oocytes. All these manipulations cause disorganized spindle poles and spindle structure, misaligned chromosomes and larger than normal polar bodies. Our results suggest that MEK1/2 may function as a centrosomal protein and may have roles in microtubule organization, spindle pole tethering and asymmetric division during mouse oocyte maturation.

Protein Kinase C and Mitogen-Activated Protein Kinase Cascade in Mouse Cumulus Cells: Cross Talk and Effect on Meiotic Resumption of Oocyte

Abstract Protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in cumulus cells are involved in FSH-induced meiotic resumption of cumulus-enclosed oocytes (CEOs), but their regulation and cross talk are unknown. The present experiments were designed to investigate 1) the possible involvement of MAPK cascade in PKC-induced meiotic resumption; 2) the regulation of PKC on MAPK activity in FSH-induced oocyte maturation; and 3) the pattern of PKC and MAPK function in induced meiotic resumption of mouse oocytes. PKC activators, phorbol 12-myristate 13-acetate (PMA) and 1-oleoyl-2-acetyl-sn-glycerol (OAG), induced the meiotic resumption of CEOs and activation of MAPK in cumulus cells, whereas this effect could be abolished by PKC inhibitors, calphostin C and chelerythrine, or MEK inhibitor U0126. These results suggest that PKC might induce the meiotic reinitiation of CEOs by activating MAPK in cumulus cells. Both PKC inhibitors and U0126 inhibited the FSH-induced germinal vesicle breakdown (GVBD) of oocytes and MAPK activation in cumulus cells, suggesting that PKC and MAPK are involved in FSH-induced GVBD of mouse CEOs. Protein synthesis inhibitor cycloheximide (CHX) inhibited FSH- or PMA-induced oocyte meiotic resumption, but not the MAPK activation in cumulus cells. FSH and PKC activators induced the GVBD in denuded oocytes cocultured with cumulus cells in hypoxanthine (HX)-supplemented medium, and this effect could be reversed by U0126. Thus, when activated by FSH and PKC, MAPK may stimulate the synthesis of specific proteins in cumulus cells followed by secretion of an unknown positive factor that is capable of inducing GVBD in oocytes.

Changes in Histone Acetylation During Postovulatory Aging of Mouse Oocyte

Abstract Because some animals and human beings potentially engage in sexual activity at any day of the menstrual cycle, this may cause fertilization of postovulatory aged oocytes, which result in decreased potential of embryo development and longevity of offspring. To investigate the involvement of histone acetylation in the function of postovulatory aging, we examined the changes of histone acetylation by immunostaining with specific antibodies against various acetylated lysines on histones H3 and H4. We found that the acetylation levels of lysine 14 on histone H3 and lysines 8 and 12 on histone H4 in mouse oocytes were gradually increased during in vivo and in vitro postovulatory aging. Furthermore, the acetylation levels on these sites were markedly decreased or increased when the process of postovulatory aging was artificially delayed or accelerated, respectively. These results indicated that the gradual acetylation on some lysines of histones H3 and H4 is one of the phenomena in the process of postovulatory aging. Moreover, raising the level of histone acetylation by trichostatin A can accelerate the progression of postovulatory aging, suggesting that alteration of the acetylation on histones H3 and H4 can affect the progression of postovulatory aging in mouse oocytes.

The Histone Demethylase JMJD2C Is Stage-Specifically Expressed in Preimplantation Mouse Embryos and Is Required for Embryonic Development

Abstract Epigenetic modifications play a pivotal role in embryonic development by dynamically regulating DNA methylation and chromatin modifications. Although recent studies have shown that core histone methylation is reversible, very few studies have investigated the functions of the newly discovered histone demethylases during embryonic development. In the present study, we investigated the expression characteristics and function of JMJD2C, a histone demethylase that belongs to the JmjC-domain-containing histone demethylases, during preimplantation embryonic development of the mouse. We found that JMJD2C is stage-specifically expressed during preimplantation development, with the highest activity being observed from the two-cell to the eight-cell stage. Depletion of JMJD2C in metaphase II oocytes followed by parthenogenetic activation causes a developmental arrest before the blastocyst stage. Moreover, consistent with a previous finding in embryonic stem (ES) cells, depletion of JMJD2C causes a significant down-regulation of the pluripotency gene Nanog in embryos. However, contrary to a previous report in ES cells, we observed that other pluripotency genes, Pou5f1 and Sox2, are also significantly down-regulated in JMJD2C-depleted embryos. Furthermore, the depletion of JMJD2C in early embryos also caused significant down-regulation of the Myc and Klf4 genes, which are associated with cell proliferation. Our data suggest that the deregulation of these critical genes synergistically causes the developmental defects observed in JMJD2C-depleted embryos.

Bub1 prevents chromosome misalignment and precocious anaphase during mouse oocyte meiosis

In mitosis the checkpoint proteins ensure faithful chromosome segregation by delaying onset of anaphase until all sister chromatids align at the metaphase plate of the bipolar spindle correctly. In the present study we blocked the function of Bub1 during meiosis by microinjecting anti-Bub1 specific antibody into cytoplasm of mouse oocytes, and found that depletion of Bub1 induced evident cyclin B degradation and precocious anaphase onset. Bub1 suppression also overrode the checkpoint-dependent cell cycle arrest provoked by a low dosage of nocodazole. Furthermore, Bub1 depletion induced a significantly higher percentage of oocytes with misaligned chromosomes. In addition, we depicted the localization dynamics of Bub1 in response to spindle damage and its relationship with microtubules and chromosomes, providing further evidence for Bub1’s role as a spindle checkpoint protein. Our data suggest that Bub1 is a critical spindle checkpoint protein that regulates accurate chromosome alignment and homolog disjunction in mammalian oocyte meiosis.

Histone deacetylation is required for orderly meiosis.

Histone acetylation is associated with a diversity of chromatin-related processes in mitosis. However, its roles in mammalian oocyte meiosis are largely unknown. In the present study, we first investigated in detail the acetylation changes during porcine oocyte maturation using a panel of antibodies specific for the critical acetylated forms of histone H3 and H4, and showed meiosis stage-dependent and lysine residue-specific patterns of histone acetylation. By using trichostatin A (TSA), a general inhibitor of histone deacetylases (HDACs), we further determined that selective inhibition of histone deacetylation (thereby maintaining hyperacetylation) delayed the onset of germinal vesicle breakdown and produced a high frequency of lagging chromosomes or chromatin bridges at anaphase and telophase I (AT-I), suggesting that histone deacetylation is required for orderly meiotic resumption and accurate chromosome segregation in porcine oocytes. In addition, we examined the localization and expression of HDAC1 by performing immunofluorescence and immunoblotting analysis. The results showed that subcellular translocation, expression level and phosphorylated modification of HDAC1 were temporally regulated and likely to co-participate in the establishment of histone acetylation profiles in oocyte meiosis.

Ubiquitin-proteasome pathway modulates mouse oocyte meiotic maturation and fertilization via regulation of MAPK cascade and cyclin B1 degradation.

Degradation of proteins mediated by ubiquitin-proteasome pathway (UPP) plays important roles in the regulation of eukaryotic cell cycle. In this study, the functional roles and regulatory mechanisms of UPP in mouse oocyte meiotic maturation, fertilization, and early embryonic cleavage were studied by drug-treatment, Western blot, antibody microinjection, and confocal microscopy. The meiotic resumption of both cumulus-enclosed oocytes and denuded oocytes was stimulated by two potent, reversible, and cell-permeable proteasome inhibitors, ALLN and MG-132. The metaphase I spindle assembly was prevented, and the distribution of ubiquitin, cyclin B1, and polo-like kinase 1 (Plk1) was also distorted. When UPP was inhibited, mitogen-activated protein kinase (MAPK)/p90rsk phosphorylation was not affected, but the cyclin B1 degradation that occurs during normal metaphase-anaphase transition was not observed. During oocyte activation, the emission of second polar body (PB2) and the pronuclear formation were inhibited by ALLN or MG-132. In oocytes microinjected with ubiquitin antibodies, PB2 emission and pronuclear formation were also inhibited after in vitro fertilization. The expression of cyclin B1 and the phosphorylation of MAPK/p90rsk could still be detected in ALLN or MG-132-treated oocytes even at 8 h after parthenogenetic activation or insemination, which may account for the inhibition of PB2 emission and pronuclear formation. We also for the first time investigated the subcellular localization of ubiquitin protein at different stages of oocyte and early embryo development. Ubiquitin protein was accumulated in the germinal vesicle (GV), the region between the separating homologous chromosomes, the midbody, the pronuclei, and the region between the separating sister chromatids. In conclusion, our results suggest that the UPP plays important roles in oocyte meiosis resumption, spindle assembly, polar body emission, and pronuclear formation, probably by regulating cyclin B1 degradation and MAPK/p90rsk phosphorylation.