Bong-Seok Song

Inheritable histone H4 acetylation of somatic chromatins in cloned embryos

A viable cloned animal indicates that epigenetic status of the differentiated cell nucleus is reprogrammed to an embryonic totipotent state. However, molecular events regarding epigenetic reprogramming of the somatic chromatin are poorly understood. Here we provide new insight that somatic chromatins are refractory to reprogramming of histone acetylation during early development. A low level of acetylated histone H4-lysine 5 (AcH4K5) of the somatic chromatin was sustained at the pronuclear stage. Unlike in vitro fertilized (IVF) embryos, the AcH4K5 level remarkably reduced at the 8-cell stage in cloned bovine embryos. The AcH4K5 status of somatic chromatins transmitted to cloned and even recloned embryos. Differences of AcH4K5 signal intensity were more distinguishable in the metaphase chromosomes between IVF and cloned embryos. Two imprinted genes, Ndn and Xist, were aberrantly expressed in cloned embryos as compared with IVF embryos, which is partly associated with the AcH4K5 signal intensity. Our findings suggest that abnormal epigenetic reprogramming in cloned embryos may be because of a memory mechanism, the epigenetic status itself of somatic chromatins.

Induction of Autophagy Promotes Preattachment Development of Bovine Embryos by Reducing Endoplasmic Reticulum Stress

ABSTRACT The coupling of autophagy and endoplasmic reticulum (ER) stress has been implicated in a variety of biological processes; however, little is known regarding the involvement of the autophagy/ER stress pathway in early embryogenesis or the underlying mechanism(s). Here, we showed that the developmental competence of in vitro-produced (IVP) bovine embryos was highly dependent on the autophagy/ER stress balance. Although relative abundances of autophagy-associated gene transcripts, including LC3, Atg5, and Atg7 transcripts, were high in oocytes and throughout the early stages of preattachment development, extensive autophagosome formation was only detected in fertilized embryos. Using an inducer and inhibitor of autophagy, we showed that transient elevation of autophagic activity during early preattachment development greatly increased the blastocyst development rate, trophectoderm cell numbers, and blastomere survival; these same parameters were reduced by both inhibition and prolonged induction of autophagy. Interestingly, the induction of autophagy reduced ER stress and associated damage, while the developmental defects in autophagy-inhibited embryos were significantly alleviated by ER stress inhibitor treatment, indicating that autophagy is a negative regulator of ER stress in early embryos. Collectively, these results suggest that early embryogenesis of IVP bovine embryos depends on an appropriate balance between autophagy and ER stress. These findings may increase our understanding of important early developmental events by providing compelling evidence concerning the tight association between autophagy and ER stress, and may contribute to the development of strategies for the production of IVP bovine blastocysts with high developmental competence.

Implication of mouse Vps26b-Vps29-Vps35 retromer complex in sortilin trafficking.

The retromer complex, which mediates retrograde transport from endosomes to the trans-Golgi network, is a heteropentameric complex that contains a multifunctional cargo recognition heterotrimer consisted of the vacuolar protein sorting (Vps) subunits Vps26, Vps29, and Vps35. In mammals, there are two different isoforms of Vps26, Vps26a and Vps26b, that localize to the endosome, and to the plasma membrane, respectively. To elucidate the biological significance of the Vps26b isoform, we generated Vps26b knockout mice and studied their molecular, histological, and behavioral phenotypes. We found that the loss of Vps26b results in no significant defects in the behavior, body size, and health of the mice. Vps26b-deficient mice showed a severe reduction of Vps35 protein at cellular level and lacked the Vps26b-Vps29-Vps35 retromer complex, despite the normal presence of the Vps26a-Vps29-Vps35 retromer complex. Relatively, the amount of sortilin was increased approximately 20% in the Vps26b-deficient mice, whereas the sorLA was normal. These results suggest that mouse Vps26b-Vps29-Vps35 retromer complex is implicated in the transport of sortilin from endosomes to the trans-Golgi network (TGN).

Large-scale transcriptome sequencing and gene analyses in the crab-eating macaque (Macaca fascicularis) for biomedical research

BackgroundAs a human replacement, the crab-eating macaque (Macaca fascicularis) is an invaluable non-human primate model for biomedical research, but the lack of genetic information on this primate has represented a significant obstacle for its broader use.ResultsHere, we sequenced the transcriptome of 16 tissues originated from two individuals of crab-eating macaque (male and female), and identified genes to resolve the main obstacles for understanding the biological response of the crab-eating macaque. From 4 million reads with 1.4 billion base sequences, 31,786 isotigs containing genes similar to those of humans, 12,672 novel isotigs, and 348,160 singletons were identified using the GS FLX sequencing method. Approximately 86% of human genes were represented among the genes sequenced in this study. Additionally, 175 tissue-specific transcripts were identified, 81 of which were experimentally validated. In total, 4,314 alternative splicing (AS) events were identified and analyzed. Intriguingly, 10.4% of AS events were associated with transposable element (TE) insertions. Finally, investigation of TE exonization events and evolutionary analysis were conducted, revealing interesting phenomena of human-specific amplified trends in TE exonization events.ConclusionsThis report represents the first large-scale transcriptome sequencing and genetic analyses of M. fascicularis and could contribute to its utility for biomedical research and basic biology.

Developmental Competence of Bovine Early Embryos Depends on the Coupled Response between Oxidative and Endoplasmic Reticulum Stress

ABSTRACT The stress produced by the coupling of reactive oxygen species (ROS) and endoplasmic reticulum (ER) has been explored extensively, but little is known regarding their roles in the early development of mammalian embryos. Here, we demonstrated that the early development of in vitro-produced (IVP) bovine embryos was governed by the cooperative action between ROS and ER stress. Compared with the tension produced by 5% O2, 20% O2 significantly decreased the blastocyst formation rate and cell survival, which was accompanied by increases in ROS and in levels of sXBP-1 transcript, which is an ER stress indicator. In addition, treatment with glutathione (GSH), a ROS scavenger, decreased ROS levels, which resulted in increased blastocyst formation and cell survival rates. Importantly, levels of sXBP-1 and ER stress-associated transcripts were reduced by GSH treatment in developing bovine embryos. Consistent with this observation, tauroursodeoxycholate (TUDCA), an ER stress inhibitor, improved blastocyst developmental rate, trophectoderm proportion, and cell survival. Moreover, ROS and sXBP-1 transcript levels were markedly decreased by supplementation with TUDCA, suggesting a possible mechanism governing the mutual regulation between ROS and ER stress. Interestingly, knockdown of XBP-1 transcripts resulted in both elevation of ROS and decrease of antioxidant transcripts, which ultimately reduced in vitro developmental competence of bovine embryos. Based on these results, in vitro developmental competence of IVP bovine embryos was highly dependent on the coupled response between oxidative and ER stresses. These results increase our understanding of the mechanism(s) governing early embryonic development and may improve strategies for the generation of IVP embryos with high developmental competence.

Tauroursodeoxycholic Acid Enhances the Pre-Implantation Embryo Development by Reducing Apoptosis in Pigs

Apoptosis is an important determinant of the normal development of pre-implantation embryos in vitro. Recently, endoplasmic reticulum (ER) stress-mediated apoptosis has been extensively investigated in a wide variety of diseases. Efficient functioning of the ER is essential for most cellular activities and survival. Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, has been reported to attenuate ER stress-mediated cell death by interrupting the classic pathways of apoptosis. Therefore, in this study, the anti-apoptotic effect of TUDCA on ER stress-induced apoptosis was examined in pre-implantation pig embryos. Also, tunicamycin was used to investigate the effects of ER stress on pig embryo development. After in vitro maturation and fertilization, presumptive pig embryos were cultured in NCSU-23 medium supplemented with TUDCA or TM for 6 days at 39 °C, 5% CO(2) in air. All data were analysed using one-way anova and Duncans multiple range test in the statistical analysis system (SAS). In addition, we also determined the optimal TM and TUDCA concentrations. Samples were treated with TM at concentrations of 0, 1, 2 or 5 μm and with TUDCA at concentrations of 0, 100, 200 or 300 μm. When TM was used during in vitro culture, only 8.2% (8/97) of the embryos developed to the blastocyst stage when the treatment concentration was 1 μm compared with 27.4% (28/102) of the embryos in the control group (p < 0.05). In contrast, the frequency of blastocyst formation and the number of cells were higher when treated with 200 μm TUDCA compared with the control group (32.8% and 39.5 vs 22.2% and 35.6, p < 0.05). Moreover, the developmental rate to the blastocyst stage embryo in the group treated with TM and TUDCA was not significantly different from that of the control group (17.8%, 26/142 vs 24.9%, 36/145). Furthermore, the blastocyst cell number was enhanced (31.9 vs 36.9) and apoptosis reduced (TUNEL-positive nuclei number, 6.0 vs 3.2) by TUDCA treatment in pig embryos. In the real-time quantitative RT-PCR analysis, the expression of anti-apoptotic Bcl-XL gene was shown to be increased in the blastocyst stage because of TUDCA treatment, whereas expression of pro-apoptotic Bax was decreased. In addition, we also found that TUDCA decreased the rate of TM-induced apoptosis in the pre-implantation stage. Taken together, our results indicate that TUDCA improves the developmental competence of pig embryos by modulating ER stress-induced apoptosis during the pre-implantation stage.

Inactivated Sendai-virus-mediated fusion improves early development of cloned bovine embryos by avoiding endoplasmic-reticulum-stress-associated apoptosis

Somatic cell nuclear transfer (SCNT) is a powerful tool, not only for producing cloned animals, but also in revealing various early developmental events. However, relatively little is known regarding the biological events and underlying mechanism(s) directly associated with early development of SCNT embryos. Here, we show that production of high-quality bovine SCNT blastocysts is dependent on the method used for fusion and the associated reduction in endoplasmic reticulum (ER) stress. During fusion between the donor cell and the enucleated oocyte, electrofusion triggers spontaneous oocyte activation, accompanied by an increase in intracellular Ca(2+) and improper nuclear remodelling. These events can be greatly reduced by the use of Sendai virus (SV)-mediated fusion. Moreover, SV-SCNT improves the blastulation rate and blastocyst quality, defined by the number and ratio of inner cell mass and trophectoderm cells in each blastocyst, in comparison with electrofusion-mediated SCNT (E-SCNT). Interestingly, expression of ER-stress-associated genes and blastomere apoptosis were significantly increased in E-SCNT embryos. These increases could be reversed by inhibition of ER stress or by using the SV-mediated fusion method. Collectively, these results indicate that SV-mediated fusion improves the developmental competence and quality of SCNT blastocysts, by reducing ER-stress-associated apoptosis.

Iloprost, a prostacyclin analogue, stimulates meiotic maturation and early embryonic development in pigs

Oviduct fluid contains various cytokines and growth factors that enhance the embryo development during the preimplantation period. In hatched embryos, prostacyclin (PGI(2)) improves implantation, but its role during oocyte maturation and early embryo development remains contentious. Therefore, in the present study, we examined the effects of a PGI(2) analogue (iloprost) on meiotic maturation and early embryonic development in pigs, as well on the structural integrity, mitochondrial membrane potential and apoptosis in blastocysts. First, meiotic maturation in pig oocytes was examined in the presence of increasing concentrations of iloprost (1, 5 and 10 muM). After IVM, a higher proportion of iloprost-treated compared with untreated oocytes was in MII (90.0% v. 65.7%, respectively; P < 0.05). In addition, protein kinase A activity increased in iloprost-treated oocytes, indicating increased intracellular cAMP concentrations. After 22 h iloprost treatment (44 h total incubation time), western blotting demonstrated increased expression of extracellular signal-regulated kinase (ERK) 1/2, phosphorylated (p-) ERK1/2, cAMP response element-binding protein (CREB), p-CREB and cyclo-oxygenase-2, indicating activation of the mitogen-activated protein kinase and PGI(2) pathways. In addition, the frequency of polyspermy decreased in iloprost-treated oocytes (19.9%) compared with control (35.8%), whereas the rate of blastocyst formation increased (P < 0.05). Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) showed that the number of nuclei containing fragmented DNA at the blastocyst stage decreased in the iloprost-treated group compared with control (1.2% v. 3.6%, respectively). In conclusion, iloprost appears to play a direct role in porcine oocyte maturation by enhancing blastocyst structure and survival.

Induction of autophagy during in vitro maturation improves the nuclear and cytoplasmic maturation of porcine oocytes

While a critical role of autophagy in mammalian early embryogenesis has been demonstrated, few studies have been conducted regarding the role of autophagy in in vitro maturation (IVM) of immature oocytes. In the present study we investigated the effect of rapamycin, a chemical autophagy inducer, on the nuclear and cytoplasmic maturation of porcine oocytes. Rapamycin treatment led to increased expression of LC3-II, an autophagy marker. Compared with the control group, as well as the 5 and 10nM rapamycin treatment groups, the rate of MII oocyte production was higher in the 1nM rapamycin treatment group, indicating improvement in nuclear maturation. In the analyses of cytoplasmic maturation, we found that the level of p34(cdc2), a cytoplasmic maturation marker, and the monospermic fertilisation rate were higher in the 1nM rapamycin treatment group than in the other groups. Moreover, the beneficial effect of 1nM rapamycin on cytoplasmic maturation of MII oocytes was further evidenced by increases in blastocyst formation rate, total cell number and cell survival. In the blastocyst embryos, anti-apoptotic Bcl-xL transcript levels were elevated in the 1nM rapamycin-treated group, whereas pro-apoptotic Bax transcript levels were decreased. Collectively, these results suggest that induction of autophagy during IVM contributes to enhancement of the nuclear and cytoplasmic maturation of porcine oocytes.

Prostacyclin stimulates embryonic development via regulation of the cAMP response element-binding protein–cyclo-oxygenase-2 signalling pathway in cattle

Prostacyclin (PGI(2)) in oviducal fluid is synthesised from arachidonic acid by cyclo-oxygenase (COX) and prostacyclin synthetase and enhances the implantation and live birth potential of mouse embryos. In the present study, we investigated the developmental competence of bovine embryos by examining the effects of the PGI(2) analogue iloprost on blastocyst development, quality and COX-2 expression during IVF and somatic cell nuclear transfer (SCNT). Bovine IVF and SCNT embryos were cultured in CR1-aa medium supplemented with 0.3% bovine serum albumin in either the presence or absence of 1 mum iloprost at 38.5 degrees C and 5% CO(2). After 3 days of culture, cleaved embryos were cultured for 4 days in the same medium supplemented with 10% fetal bovine serum. For both IVF and SCNT embryos, iloprost improved the blastocyst developmental rate and cell numbers. In the presence of iloprost, the proportion of expanded blastocysts was significantly higher among the IVF embryos and fewer apoptotic cell nuclei were observed. Expression of COX-2 mRNA and protein, evaluated using real-time polymerase chain reaction and immunoblotting, respectively, was increased in the presence of iloprost. These results suggest that PGI(2) improves the developmental competence of embryos via regulation of the cAMP response element-binding protein-COX-2 signalling pathway in cattle.