Seung-Bin Yoon

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.

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.

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.

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.

Valproic acid enhances early development of bovine somatic cell nuclear transfer embryos by alleviating endoplasmic reticulum stress

Despite the positive roles of histone deacetylase inhibitors in somatic cell nuclear transfer (SCNT), few studies have evaluated valproic acid (VPA) and its associated developmental events. Thus, the present study was conducted to elucidate the effect of VPA on the early development of bovine SCNT embryos and the underlying mechanisms of action. The histone acetylation level of SCNT embryos was successfully restored by VPA, with optimal results obtained by treatment with 3mM VPA for 24h. Importantly, the increases in blastocyst formation rate and inner cell mass and trophectoderm cell numbers were not different between the VPA and trichostatin A treatment groups, whereas cell survival was notably improved by VPA, indicating the improvement of developmental competence of SCNT embryos by VPA. Interestingly, VPA markedly reduced the transcript levels of endoplasmic reticulum (ER) stress markers, including sXBP-1 and CHOP. In contrast, the levels of GRP78/BiP, an ER stress-alleviating transcript, were significantly increased by VPA. Furthermore, VPA greatly reduced cell apoptosis in SCNT blastocysts, which was further evidenced by the increased levels of the anti-apoptotic transcript Bcl-xL and decreased level of the pro-apoptotic transcript Bax. Collectively, these results suggest that VPA enhances the developmental competence of bovine SCNT embryos by alleviating ER stress and its associated developmental damage.

Supplementation with estradiol-17β improves porcine oocyte maturation and subsequent embryo development

Metaphase II oocyte production was significantly increased by treatment with E(2) during the first half of the total in vitro maturation (IVM) period, which was further evidenced by an increase in monospermic fertilization, blastocyst formation, or blastomere viability of IVF- or somatic cell nuclear transfer-derived embryos. Thus, we concluded that transient E(2) supplementation could improve the IVM rate and subsequent developmental competence in pigs.

Efficient Production of Transgenic Mice by Intracytoplasmic Injection of Streptolysin-O-Treated Spermatozoa

Many methods for efficient production of transgenic animals for biomedical research have been developed. Despite great improvements in transgenesis rates resulting from the use of intracytoplasmic sperm injection (ICSI), the ICSI‐based sperm‐mediated gene‐transfer (iSMGT) technique is still not optimal in terms of sperm permeabilization efficiency and subsequent development. Here, we demonstrate that streptolysin‐O (SLO) can efficiently permeabilize mouse spermatozoa, leading to improved developmental competence and high transgenesis rates in iSMGT embryos and pups. In particular, the most efficient production of iSMGT‐transgenic embryos resulted from pretreatment with 5 U/ml SLO for 30 min and co‐incubation with 1.0 ng/µl of an EGFP expression vector. By incubating spermatozoa with Cy‐3‐labelled DNA, we found that fluorescence intensity was prominently detected in the head region of SLO‐treated spermatozoa. In addition, blastocyst development rate and blastomere survival were greatly improved by iSMGT using SLO‐treated spermatozoa (iSMGT‐SLO) as compared to freeze‐thawed spermatozoa. Consistent with this, a high proportion of transgenic offspring was obtained by iSMGT‐SLO after transfer into foster mothers, reaching 10.6% of the number of oocytes used (42.3% among pups). Together with successful germline transmission of transgenes in all founders analyzed, our data strongly suggest that SLO makes spermatozoa amenable to exogenous DNA uptake, and that the iSMGT‐SLO technique is an efficient method for production of transgenic animals for biomedical research. Mol. Reprod. Dev. 80: 233–241, 2013.

Tyrphostin A9 improves blastocyst development in porcine embryos through induction of dynamin-related protein 1-dependent mitochondrial fission

Mitochondrial dynamics are associated with the development of porcine embryos. However, little is known about the effects of mitochondrial dynamics-related genes (Drp1 and pDrp1-Ser616) on early porcine embryo development. Here, we investigated the effect of Drp1-dependent mitochondrial fission signaling on the development of porcine embryos using the mitochondrial fission inducer, tyrphostin A9 (TA9). We determined that TA9 (1μM) treated embryos were increased the mitochondrial functions, blastocyst development rate and quality, as well as decreased mitochondria-specific superoxide and mitochondrial apoptosis. Thus, TA9-induced appropriate mitochondrial fission improved the developmental competence via maintenance of a balance in mitochondrial dynamics in porcine embryo.

Native plants ( Phellodendron amurense and Humulus japonicus ) extracts act as antioxidants to support developmental competence of bovine blastocysts

Objective Phellodendron amurense (P. amurense) and Humulus japonicus (H. japonicus) are closely involved in anti-oxidative response and increasing antioxidant enzymes activities. However, the effects of their extracts on development of preimplantation bovine embryos have not been investigated. Therefore, we investigated the effects of P. amurense and H. japonicus extracts on developmental competence and quality of preimplantation bovine embryos. Methods After in vitro fertilization, bovine embryos were cultured for 7 days in Charles Rosenkrans amino acid medium supplemented with P. amurense (0.01 μg/mL) and H. japonicus (0.01 μg/mL). The effect of this supplementation during in vitro culture on development competence and antioxidant was investigated. Results We observed that the blastocysts rate was significantly increased (p<0.05) in P. amurense (28.9%±2.9%), H. japonicus (30.9%±1.5%), and a mixture of P. amurense and H. japonicus (34.8%± 2.1%) treated groups compared with the control group (25.4%±1.6%). We next confirmed that the intracellular levels of reactive oxygen species (ROS) were significantly decreased (p<0.01) in P. amurense and/or H. japonicus extract treated groups when compared with the control group. Our results also showed that expression of cleaved caspase-3 and apoptotic cells of blastocysts were significantly decreased (p<0.05) in bovine blastocysts derived from both P. amurense and H. japonicus extract treated embryos. Conclusion These results suggest that proper treatment with P. amurense and H. japonicus extracts in the development of preimplantation bovine embryos improves the quality of blastocysts, which may be related to the reduction of ROS level and apoptosis.

Iloprost supports early development of in vitro-produced porcine embryos through activation of the phosphatidylinositol 3-kinase/AKT signalling pathway

Despite evidence of the presence of prostaglandin (PG) I2 in mammalian oviducts, its role in early development of in vitro-produced (IVP) embryos is largely unknown. Thus, in the present study we examined the effects of iloprost, a PGI2 analogue, on the in vitro developmental competence of early porcine embryos and the underlying mechanism(s). To examine the effects of iloprost on the development rate of IVF embryos, iloprost was added to the in vitro culture (IVC) medium and cultured for 6 days. Supplementation of the IVC medium with iloprost significantly improved developmental parameters, such as blastocyst formation rate, the trophectoderm:inner cell mass ratio and cell survival in IVF and parthenogenetically activated (PA) embryos. In addition, post-blastulation development into the expanded blastocyst stage was improved in iloprost-treated groups compared with controls. Interestingly, the phosphatidylinositol 3-kinase (PI3K)/AKT signalling pathway was significantly activated by iloprost supplementation in a concentration-dependent manner (10-1000nM), and the beneficial effects of iloprost on the early development of porcine IVF and PA embryos was completely ablated by treatment with 2.5μM wortmannin, a PI3K/AKT signalling inhibitor. Importantly, expression of the PI3K/AKT signalling pathway was significantly reduced in somatic cell nuclear transfer (SCNT) compared with IVF embryos, and iloprost supported the early development of SCNT embryos, as was the case for IVF and PA embryos, suggesting a consistent effect of iloprost on the IVC of IVP porcine embryos. Together, these results indicate that iloprost can be a useful IVC supplement for production of IVP early porcine embryos with high developmental competence.