Genetic diversity and inferred dispersal history of the Schlegel's Japanese Gecko (Gekko japonicus) in Northeast Asia based on population genetic analyses and paleo-species distribution modelling.

To understand the genetic diversity and dispersal history of Schlegel's Japanese gecko (Gekko japonicus), we performed genetic analyses and paleo-species distributional modelling. For the genetic analysis, we analyzed mitochondrial DNA (mtDNA) (cytochrome b [Cytb] and NADH dehydrogenase 2 [ND2]) and seven microsatellite loci of 353 individuals from 11 populations (2 east coast China, 4 west and central coast Japan and 5 Korea). For the paleo-species distribution modelling, we used 432 occurrence data points (125 China, 291 Japan and 16 Korea) over the Pleistocene and Holocene. China is inferred to be the source population, which had higher genetic diversity (mtDNA) and more private alleles (mtDNA) compared to Japanese and Korean populations. Differences between the three counties were very small in the mtDNA haplotype network despite some genetic structure among the three countries. Microsatellite analysis inferred that genetic exchange has actively occurred among the Chinese, Japanese and Korean populations. Suitable habitats in Japan should have been plentiful by the mid-Holocene, but have only recently become available in Korea. These results suggest that dispersal of G. japonicus occurred after the Holocene warming from the east coast of China to the west and central coasts of Japan and Korea, and gene flow is actively occurring among the three countries.

Mitochondria-targeted DsRed2 protein expression during the early stage of bovine somatic cell nuclear transfer embryo development.

Somatic cell nuclear transfer (SCNT) has been widely used as an efficient tool in biomedical research for the generation of transgenic animals from somatic cells with genetic modifications. Although remarkable advances in SCNT techniques have been reported in a variety of mammals, the cloning efficiency in domestic animals is still low due to the developmental defects of SCNT embryos. In particular, recent evidence has revealed that mitochondrial dysfunction is detected during the early development of SCNT embryos. However, there have been relatively few or no studies regarding the development of a system for evaluating mitochondrial behavior or dynamics. For the first time, in mitochondria of bovine SCNT embryos, we developed a method for the visualization of mitochondria and expression of fluorescence proteins. To express red fluorescence in mitochondria of cloned embryos, bovine ear skin fibroblasts, nuclear donor, were stably transfected with a vector carrying mitochondria-targeting DsRed2 gene tagged with V5 epitope (mito-DsRed2-V5 tag) using lentivirus-mediated gene transfer because of its ability to integrate in the cell genome and the potential for long-term transgene expression in the transduced cells and their dividing cells. From western blotting analysis of V5 tag protein using mitochondrial fraction and confocal microscopy of red fluorescence using SCNT embryos, we found that the mitochondrial expression of the mito-DsRed2 protein was detected until the blastocyst stage. In addition, according to image analysis, it may be suggested possible use of the system for visualization of mitochondrial localization and evaluation of mitochondrial behaviors or dynamics in early development of bovine SCNT embryos.

Exogenous γ-tocotrienol promotes preimplantation development and improves the quality of porcine embryos.

γ-tocotrienol (GTT), an isomer of vitamin E, has been the subject of increasing interest due to its strong anti-oxidant effects. Therefore, in this study, the effects of GTT on blastocyst development, expression levels of reactive oxygen species (ROS) and apoptotic index were investigated in preimplantation porcine embryos. After in vitro maturation and fertilisation, porcine embryos were cultured for 6 days in porcine zygote medium 3 supplemented with or without GTT (200µM) under oxidative stress conditions (200µM hydrogen peroxide (H2O2)). Blastocyst development was significantly improved in the GTT-treated group when compared with the H2O2-treated group (P<0.05). Subsequent evaluation of the intracellular levels of ROS and numbers of apoptotic nuclei in GTT-treated blastocysts revealed that ROS levels of GTT-treated porcine blastocysts were decreased (P<0.05) and the numbers of apoptotic nuclei were reduced by GTT treatment in porcine embryos. Moreover, the total cell numbers of blastocysts were significantly increased in the GTT-treated group relative to the untreated group under H2O2-induced oxidative stress (P<0.05). The expression levels of apoptosis-related genes (BCL-XL, BAX) in GTT-treated blastocysts were then investigated using real-time reverse transcription polymerase chain reaction. Expression of the anti-apoptotic BCL-XL gene was shown to be increased in the GTT-treated blastocyst group, whereas expression of the pro-apoptotic BAX gene was decreased. Taken together, these results suggest that GTT (200µM) under H2O2-induced oxidative stress, thereby improving the developmental competence of porcine embryos via modulation of intracellular levels of ROS and the apoptotic index during the preimplantation stage.

Mdivi-1, mitochondrial fission inhibitor, impairs developmental competence and mitochondrial function of embryos and cells in pigs.

Mitochondria are highly dynamic organelles that undergo constant fusion/fission as well as activities orchestrated by large dynamin-related GTPases. These dynamic mitochondrial processes influence mitochondrial morphology, size and function. Therefore, this study was conducted to evaluate the effects of mitochondrial fission inhibitor, mdivi-1, on developmental competence and mitochondrial function of porcine embryos and primary cells. Presumptive porcine embryos were cultured in PZM-3 medium supplemented with mdivi-1 (0, 10 and 50 µM) for 6 days. Porcine fibroblast cells were cultured in growth medium with mdivi-1 (0 and 50 µM) for 2 days. Our results showed that the rate of blastocyst production and cell growth in the mdivi-1 (50 µM) treated group was lower than that of the control group (P < 0.05). Moreover, loss of mitochondrial membrane potential in the mdivi-1 (50 µM) treated group was increased relative to the control group (P < 0.05). Subsequent evaluation revealed that the intracellular levels of reactive oxygen species (ROS) and the apoptotic index were increased by mdivi-1 (50 µM) treatment (P < 0.05). Finally, the expression of mitochondrial fission-related protein (Drp 1) was lower in the embryos and cells in the mdivi-1-treated group than the control group. Taken together, these results indicate that mdivi-1 treatment may inhibit developmental competence and mitochondrial function in porcine embryos and primary cells.

Characterization of pig sperm hyaluronidase and improvement of the digestibility of cumulus cell mass by recombinant pSPAM1 hyaluronidase in an in vitro fertilization assay.

Although sperm hyaluronidase is thought to play an important role in mammalian fertilization, the molecular function underlying these steps remains largely unknown. In mouse models, sperm-specific SPAM1 and HYAL5 hyaluronidase are believed to function in both sperm penetration of the cumulus matrix and sperm-ZP binding. However, gene-targeting studies for SPAM1 or HYAL5 show that hyaluronidases are not essential for fertilization, despite the fact that exogenous hyaluronidase can disrupt the cumulus matrix. Therefore, to evaluate whether sperm hyaluronidase is essential for mammalian fertilization, it is necessary to generate HYAL5/SPAM1 double-knockout mice. However, generating double-knockout mice is very difficult because these two genes exist on the same chromosome. Recently, investigators have begun to employ the pig model system to study human disease due to its similarities to human anatomy and physiology. In this study, we confirmed that pig SPAM1 exists as a single copy gene on chromosome 18 and is specifically expressed in the testis. In addition, we expressed recombinant pig SPAM1 in human embryonic kidney 293 cells and showed that these enzymes possess hyaluronidase activity. We also demonstrated that a polyclonal antibody against pig sperm hyaluronidase inhibits sperm-egg interactions in an in vitro fertilization (IVF) assay. Our results suggest that pig SPAM1 may play a critical role in pig fertilization and that recombinant SPAM1 can disperse the oocyte-cumulus complex in an IVF assay.

A cathepsin B inhibitor, E-64, improves the preimplantation development of bovine somatic cell nuclear transfer embryos.

Bovine somatic cell nuclear transfer (SCNT) is an important and powerful tool for basic research and biomedical and agricultural applications, however, the efficiency of SCNT has remained extremely low. In this study, we investigated the effects of cathepsin B inhibitor (E-64) supplementation of culture medium on in vitro development of bovine SCNT embryos. We initially used three concentrations of E-64 (0.1, 0.5, 1.0 µm), among which 0.5 µm resulted in the highest rate of blastocysts production after in vitro fertilization (IVF), and was therefore used for further experiments. Blastocyst development of SCNT embryos in the E-64 treatment group also increased relative to the control. Moreover, the cryosurvival rates of IVF and SCNT blastocysts were increased in E-64 treatment groups when compared with the control. On the other hand, we found that IVF and SCNT blastocysts derived from E-64-treated groups had increased total cell numbers and decreased apoptotic nuclei. Furthermore, assessment of the expression of apoptosis-related genes (Bax and Bcl-xL) in bovine IVF and SCNT blastocysts treated with E-64 by real-time RT-PCR analysis revealed suppressed expression of the pro-apoptotic gene Bax and stimulated expression of the anti-apoptotic gene Bcl-xL. Taken together, these finding indicate that addition of E-64 to embryo culture medium may have important implications for improving developmental competence and preimplantation quality in bovine IVF and SCNT embryos.

Forced collapse of the blastocoel enhances survival of cryotop vitrified bovine hatching/hatched blastocysts derived from in vitro fertilization and somatic cell nuclear transfer.

Freezing of bovine blastocysts has been widely used to improve the feasibility of cattle production by the embryo transfer technique. However, the low survival of vitrified-warmed embryos and their further development are crucial problems. Particularly, the production of offspring in vitrified-warmed bovine hatching/hatched blastocysts derived from in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) is very low. Thus, we examined the effects of forced blastocoel collapse (FBC) before vitrification of bovine IVF- and SCNT-derived hatching/hatched embryos on the survival rate and apoptosis index after warming. Under optimal conditions, the overall survival rates in vitrified-warmed bovine IVF- and SCNT-derived hatching/hatched blastocysts were higher in FBC groups than in non-FBC groups (p<0.05). The total cell numbers of vitrified-warmed hatching/hatched blastocysts were higher in FBC groups than in non-FBC groups (p<0.05). Otherwise, the number of apoptotic positive cells of vitrified-warmed hatching/hatched blastocysts was lower in FBC groups than in non-FBC groups (p<0.05). Taken together, these findings suggest that forced collapse of the blastocoel using a pulled Pasteur pipette is an effective pretreatment technique for vitrification of bovine IVF- and SCNT-derived hatching/hatched blastocysts.