PP2A-B55α, a regulatory subunit of PP2A plays an important roles in regulating cell proliferation and survival. However, the functions for PP2A-B55α in mouse early embryo development is not clear. The objective of present were to investigate the expression patterns and to explore its biological function during mouse early development. Thetranscripts of PP2A-B55α were detected at all developmental stages in mouse embryo and decreased during embryo development. Immunostaining revealed that PP2A-B55α was present in both the nucleus and cytoplasm in early cleavage stage embryos. In the late embryonic development, PP2A-B55α was predominantly located in the cytoplasm. Knockdown (KD) of PP2A-B55α using double strand RNA not affect the proportion of cleaved embryos, but resulted in significantly decreased development to blastocyst stage and reduced total cell number in blastocyst. KD PP2A-B55α is able to induce sustained DNA damage and reduced the transcripts of non-homologous end joining (NHEJ) or homologous recombination (HR) pathways relative genes in mouse early embryo. KD PP2A-B55αcaused apoptosis and increase the transcripts of pro-apoptotic genes in blastocyst. Furthermore, The KDPP2A-B55α showed significantly lower cell proliferating rates (from 5-Bromo-deoxyuridineassayresults) in blastocysts and to talareas of out growth potential was decreased. These observation provide novel in sight into PP2A-B55α expression patterns in mouse early embryos and down-regulation of PP2A-B55α negatively impacted blastocyst development, total cell number, DNA damage, apoptosis, and proliferation and post-hatchingevents.

Humulus japonicus is an ornamental plant in the Cannabaceae family. Although the mode of action of Humulus japonicus is not fully understood, a strong relationship was observed between anti-inflammatory and anticancer in some types of cells. Recent studies also have shown that Humulus japonicus possesses anti-inflammatory activities and may significantly improve antioxidant potential in Raw 264.7 macrophage cells. Thus, the aim of this study was eva-luated the effect of Humulus japonicus extract on sperm motility and subsequent preimplantation developmental com-petence of the bovine embryos. After in vitro maturation, the oocytes with sperms were exposed in in vitro fertilization (IVF) medium supplemented with Humulus japonicus extract (0.01, 0.05, 0.1 μg/mL, respectively) for 1 day. In our results, exposure of IVF medium to Humulus japonicus extract did not affect sperm motility and percentage of pene-trated oocytes but ROS intensity was significantly decreased by 0.01 μg/mL compared with other groups (p< 0.05). Moreover, treatment with 0.01 μg/mL of Humulus japonicus extract was higher the frequency of blastocyst formation than the any other groups (p<0.05). Otherwise, treatment with 0.01 μg/mL of Humulus japonicus extract not increased the total cell number but reduced apoptotic-positive nuclei number. In conclusion, our results indicate that supple-mentation of Humulus japonicus extract in IVF medium may have important implications for improving early embryo-nic development in bovine embryos

The purpose of this study was attempted to new methods in mammalian embryos vitrification. This method was affected to increase of the embryo vitrification efficiency and it would be applied to the field of embryo transfer to recipient by modified loading method of embryo into 0.25 ml plastic straw. The frozen mouse embryos were carried out warmed from two different cell stages (8-cell and blastocyst, respectively) by attachment of an embryo in the vitrification straw (aV) method. All groups were cultured in M-16 medium to determine the development and survivability for 24 h, respectively. Results shown that, the survivability of two different groups were significantly different (94.8% vs. 70.9%). Total cell number was not significantly different the non-frozen blastocyst (99.7 ± 12.4) compared to the post-thaw blastocyst (94.8 ± 15.1). From the 8-cell embryo, total cell number of frozen blastocysts were significantly lower than others groups (74.7 ± 14.6, p<0.05). In the case of cell death analysis, the blastocysts from non-frozen and frozen-thawed 8-cell group were not different (0.0 ± 0.0 vs. 1.9 ± 3.1, p>0.05). However, the apoptotic nuclei of blastocyst were significantly observed the frozen-thawed group (5.4 ± 4.4) compared to non-frozen group (p<0.05). Therefore, this new method of embryos using in-straw dilution and direct transfer into other species would be more simple procedure of embryo transfer rather than step-wise dilution method and cryopreservation vessels, so we can be applied in animal as well as human embryo cryopreservation in further.

Biotechnologies for cloning animals and in vitro embryo production have the potential to produce biomedical models for various researches. Especially, pigs are a suitable model for xenotransplantation, transgenic production and various areas of reproductive research due to its physiological similarities to human. However, utilization of in vitro-produced embryos for transfer remains limited. Despite improvement over past few decades, obstacles associated with the production of good quality embryos in vitro still exist which limit the efficiency of cloning. One of major problems includes improper in vitro maturation (IVM) and culture (IVC). Oxidative stress caused from in vitro culture conditions contributes to inadequate IVM and IVC which leads to poor developmental competence of oocytes, failure of fertilization and embryo development. To reduce the oxidative stress, various antioxidants have been used to IVM and IVC system. However, limited information is available on the effects of resveratrol on livestock reproductions. Resveratrol is a polyphenolic natural product and well known as an antioxidant in foods and beverages (e.g. in grapes and red wine). Resveratrol is known to be cardioprotective, anticarcinogenic, anti-inflammatory, antioxidant and antiapoptotic. This paper will review the effects of resveratrol on in vitro maturation of oocytes and embryo development.

The objective of this study was to investigate the effects of NEAA and leptin supplemented to in vitro culture medium on the developmental competence of porcine embryos after intracytoplasmic sperm injection (ICSI), and to modify the culture condition to improve the quality and the development of ICSI-derived porcine embryos in vitro. After ICSI, the putative zygotes were then cultured in PZM-3 medium with/without NEAA or leptin. The proportion of embryos that developed to the blastocyst stage significantly increased when 1% NEAA (24.62%) was added to the medium compared with 2% NEAA and no NEAA group (17.24% and 20.24%, respectively, p<0.05). The effect of different concentration of leptin (0, 10, 100, 500 ng/ml) was evaluated on the development of porcine ICSI embryos cultured in vitro. In case of blastocyst formation, 100 ng/ml group (27.05%) showed significantly higher rate than 10, 500 ng/ml, and control group (23.45%, 17.99%, and 19.68%, respectively, p<0.05). We also evaluated the effects of different NEAA and leptin treatment time on the development of porcine embryos after ICSI. Among groups of embryos cultured in the presence of NEAA or leptin for whole 7 days (D 1-7), first 4 days (D 1-4), the subsequent 3 days (D 5-7), both NEAA (27.13%, 21.17 %, and 17.56%, respectively, p<0.05) and leptin (25.60%, 20.61%, and 16.53%, respectively, p<0.05) showed that supplementation for whole 7 days significantly increased the blastocyst formation rate compared with the other groups of D1-4 and D5-7. We further evaluated the combination effect of 1% NEAA and 100 ng/ml leptin compared with the effect of each supplementation with 1% NEAA or 100 ng/ml leptin or no supplementation on development of embryos. For blastocyst formation, combination group of NEAA and leptin (24.78%) showed significantly higher rate than other three groups (18.37%, 20.44 %, and 13.27%, respectively, p<0.05). We further evaluated the expression of proapoptosis genes such as BAX and BAK and anti-apoptosis genes, BCL-XL and BCL-2 in blastocysts cultured in the presence of 100 ng/ml leptin. RT-PCR analysis revealed that leptin supplementation significantly decreased the expression of pro-apoptosis genes as well as increased the expression of anti-apoptosis genes. These results of present study demonstrate that NEAA and leptin could improve the in vitro development of ICSI- derived porcine embryos with optimal concentration of each reagent. Furthermore, the optimal culture condition could increase the quality of ICSI-derived embryos in vitro.

Environmental conditions during early mammalian embryo development are critical and some adaptational phenomena are observed. However, the mechanisms underlying them remain largely masked. Previously, we reported that AQP5 expression is modified by the environmental condition without losing the developmental potency. In this study, AQP11 was examined instead. To compare expression pattern between in vivo and in vitro, we conducted quantitative RT-PCR and analyzed localization of the AQP11 by whole mount immunofluorescence. When the fertilized embryos were developed in the maternal tracts, the level of Aqp11 transcripts was decreased dramatically until 2-cell stage. Its level increased after 2-cell stage and peaked at 4-cell stage, but decreased again dramatically until morula stage. Its transcript level increased again at blastocyst stage. In contrast, the levels of Aqp11 transcript in embryos cultured in vitro were as follows. The patterns of expression were similar but the overall levels were low compared with those of embryos grown in the maternal tracts. AQP11 proteins were localized in submembrane cytoplasm of embryos collected from maternal reproductive tracts. The immune-reactive signals were detected in both trophectoderm and inner cell mass. However, its localization was altered in in vitro culture condition. It was localized mainly in the plasma membrane of the blastocysts contacting with external environment. The present study suggests that early stage embryo can develop successfully by themselves adapting to their environmental condition through modulation of the expression level and localization of specific genes like AQP11.

Adaptive development of early stage embryo is well established and recently it is explored that the mammalian embryos also have adaptive ability to the stressful environment. However, the mechanisms are largely unknown. In this study, to evaluate the possible role of aquaporin in early embryo developmental adaptation, the expression of aquaporin (AQP) 5 gene which is detected during early development were examined by the environmental condition. To compare expression patterns between in vivo and in vitro, we conducted quantitative RT-PCR and analyzed localization of the AQP5 by whole mount immunofluorescence. At in vivo condition, Aqp5 expressed in oocyte and in all the stages of preimplantation embryo. It showed peak at 2-cell stage and decreased continuously until morula stage. At in vitro condition, Aqp5 expression pattern was similar with in vivo embryos. It expressed both at embryonic genome activation phase and second mid-preimplantation gene activation phase, but the fold changes were modified between in vivo embryos and in vitro embryos. During in vivo development, AQP5 was mainly localized in apical membrane of blastomeres of 4-cell and 8-cell stage embryos, and then it was localized in cytoplasm. However, the main localization area of AQP5 was dramatically shifted after 8-cell stage from cytoplasm to nucleus by in vitro development. Those results explore the modification of Aqp5 expression levels and location of its final products by in vitro culture. It suggests that expression of Aqp5 and the roles of AQP5 in homeostasis can be modulated by in vitro culture, and that early stage embryos can develop successfully by themselves adapting to their condition through modulation of the specific gene expression and localization.

이온 비의존적으로 작동하는 포도당 수송체 (glucose transporter 1, Glut1)는 생쥐 배아의 세포막을 경계로 포도당을 수송하는 주요통로이다. 성장인자 가운데 insulin-like growth factor-I (IGF-I)은 생쥐배아에서 포도당의 유입을 증가시키는 것으로 알려져있으나 이러한 효과가 IGF-I 의한 Glut1의 전사조절 효과에 기인한 것인지는 알려져 있지 않다. 본 연구는 포도당과 IGF-I 생쥐의 착상전 배아 발생과 G

Phospholipase C (PLC)는 다양한 세포주에서 세포내 신호전달에 중요한 역할을 한다고 알려져 있으나, 생쥐 난자성숙 과정과 착성전 배아발생 과정에서 PLC의 역할과 발현은 아직 연구된 바 없다. 본 연구에서는 난자성숙과 착상전 배아발생 과정에서 생쥐의 PLC β1과 γ1의 유전자 발현을 조사하기 위하여 한 개의 난자 혹은 배아에서 추출된 total RNA를 사용하여 경쟁적 RT-PCR 방법으로 mRNA를 정량하였다. PL