Lipopolysaccharide (LPS) is an endotoxin factor present in the cell wall of Gram-negative bacteria and induces various immune responses to infection. Recent studies have reported that LPS induces cellular stress in various cells including oocytes and embryos. Melatonin (N-acetyl-5-methoxytryptamine) is a regulatory hormone of circadian rhythm and a powerful antioxidant. It has been known that melatonin has an effective function in scavenging oxygen free radicals and has been used as an antioxidant to reduce the cytotoxic effects induced by LPS. However, the effect of melatonin on LPS treated early embryonic development has not yet been confirmed. In this study, we cultured mouse embryos in medium supplemented with LPS or/and melatonin up to the blastocyst stage in vitro and then evaluated the developmental rate. As a result of the LPS-treatment, the rate of blastocyst development was significantly reduced compared to the control group in all the LPS groups. Next, in the melatonin only treated group, there was no statistical difference in embryonic development and no toxic effects were observed. And then we found that the treatment of melatonin improved the rates of compaction and blastocyst development of LPS-treated embryos. In addition, we showed that melatonin treatment decreased ROS levels compared to the LPS only treated group. In conclusion, we demonstrated the protective effect of melatonin on the embryonic developmental rate reduced by LPS. These results suggest a direction to improve reproduction loss that may occur due to LPS exposure and bacterial infection through the using of melatonin during in vitro culture.

This study was investigated to test whether the zygote recognized the topoisomerase II beta (TOP2B) mediated DNA fragmentation in epididymal spermatozoa or the nuclease degradation in vas deferens spermatozoa by testing for the presence of gammaH2AX (γH2AX). The γH2AX is phosphorylation of histone protein H2AX on serine 139 occurs at sites flanking DNA double-stranded breaks (DSBs). The presence of γH2AX in the pronuclei of mouse zygotes which were injected with DNA broke epididymal spermatozoa was tested by immunohistochemistry at 5 and 9 h post fertilization, respectively. Paternal pronuclei that arose from epididymal spermatozoa treated with divalent cations did not stain for γH2AX at 5 h. On the other hand, in embryos injected with vas deferences spermatozoa that had been treated with divalent cations, γH2AX was only present in paternal pronuclei, and not the maternal pronuclei at 5 h. Interestingly, both pronuclei stained positively for γH2AX for all treatments and controls at 9 h after sperm injection. In conclusion, the embryos recognize DNA that is damaged by nuclease, but not by TOP2B because H2AX in phosphorylated in paternal pronuclei resulting from spermatozoa treated with fragmented DNA from vas deferens spermatozoa treated with divalent cations, but not from epididymal spermatozoa treated the same way.

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.

To clarify the role of stem cells in hepatocarcinogenesis, CD44 expression was investigated in mouse livers as well as embryonic cell lineages treated with diethylnitrosamine (DEN). Liver tumors induced by DEN were analyzed by immunohistochemisty for CD44. Liver tissues were sampled at 6, 24, and 48 hr after treatment with saline or DEN. Mouse embryonic stem cells (ESCs), hepatic progenitor cells (HPCs), and hepatocyte like cells (HCs), representing 0, 22, and 40 days of differentiation, respectively, were treated with DEN at four doses (0, 1, 5, and 15 mM, respectively) for 24 hr, after which CD44 expression levels were examined by relative quantitative real-time PCR. CD44 expression was weakly detected in tumor cells as well as in some hepatocytes surrounding the tumor cells. However, CD44 expression was not detected in liver tissue treated with DEN at early time points. The CD44 mRNA expression level was significantly different among cells treated with 5 mM DEN at day 22 (P<0.01) as well as 1, 5, and 15 mM DEN at day 40 (P<0.01) compared with control. Taken together, CD44 expression slightly increased in mouse DEN-induced tumors. Furthermore, expression of CD44 in embryonic cell lineages treated with various doses of DEN significantly differed among embryo stem cells and derived hepatic lineage cells. This suggests that CD44 expression may be modulated in the progeny of stem cells during their differentiation toward hepatocytes, and its expression may increase in the tumor stage but not during early carcinogenesis.

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.

Autocrine or paracrine mediators released by the early embryo are implicated in the support of embryonic development. Their mechanisms and optimal embryo density in the medium, however, are uncertain. This study was conducted to establish the optimal embryo density and culture medium volume in mouse parthenogenetic embryo culture. In experiment 1, culture of parthenogenetirally activated oocytes at a concentration of 2~4 embryos/ uL significantly improved development to the blastoryst stage (72%≤) compared with culture at the lower (0.2~1 embryos㎕, 0~37.5%) and the higher (5~6 embryos㎕, 30~53%) concentration for 120 h when the oocytes were cultured in a 5㎕ drop under mineral oil In experiment 2, the embryos cultured at a concentration of 2~4 embryos㎕ in a 10㎕ drop (81.1%) showed significantly higher blastocyst rates than those in a 5㎕ drop (68.5%). This study optimizes in vitro culture condition by modifying embryo density and the volume of culture medium It may give appropriate level of autocrine and/or paracrine factors to enhance viability and subsequent normal development of mouse parthenogenetic embryos in vitro.

텔로미어란 염색체 말단부에 (TTAGGG)n의 반복 염기서열이 단백질과 결합된 형태를 말하는데 이의 역할은 염색체의 안정성에 본질적으로 작용하여 세포의 노화, 사멸 및 암의 발생과 관련이 있다고 알려져 있다. 반면 텔로머레이스는 telomeric DNA 합성에 직접 관여하는 ribonucleoprotein이다. 본 연구에서는 마우스 염색체의 텔로미어 분포 양상을 제시하고, 초기 배 발생단계별 수정란의 텔로미어 함량과 이들 수정란의 텔로머레이스 활성도를 분석하고자 하였다. 본 분석에는 마우스의 섬유아세포, 생식세포, 정자, 난자 및 1세포기, 2세포기, 4세포기, 8세포기, 상실배와 배반포배의 각 단계별 수정란을 대상으로 하였다. 텔로미어의 양적 분석은 human telomeric DNA probe를 이용한 Q-FISH 방법을 이용하였고, 텔로머레이스 활성도는 TRAP 방법을 이용하였다. 분석 결과 마우스 염색체의 텔로미어는 성 염색체를 포함한 모든 염색체의 앙 말단부에 분포되어 있고, 염색체별 다소의 양적 차이를 보이나 대부분의 염색체에서 q-arm 말단이 p-arm 말단에 비해 높은 텔로미어의 함량을 나타내었다. Q-FISH를 이용한 마우스 초기 배 발생단계별 수정란의 텔로미어의 양적 분석에서 수정 직후 1세포기에서부터 상실배까지 거의 비슷한 텔로미어 함유율을 나타내고 있으나 배반포기에서 월등히 증가된 양상을 나타내었다. TRAP 분석을 이용한 초기배아의 텔로머레이스 활성도는 초기 배 발생 모든 단계에서 이의 활성도를 나타내었으며, 특히 상실배 및 배반포기에서 점진적으로 강한 활성을 보였다. 이상의 분석 결과로부터 마우스의 초기 배 분열단계의 각 세포들에 있어 텔로미어의 함유율과 텔로머레이스 활성도는 높은 상관관계가 있는 것으로 나타났다. 따라서 포유동물의 초기 배자에 있어 텔로미어의 함유율과 텔로머레이스 활성도는 배 발생 및 배자의 세포 분화와 매우 밀접한 관련이 있는 것으로 사료되어 텔로미어의 양적 분석 및 텔로머레이스 활성도 분석은 발생학적 연구를 위한 또 다른 좋은 자료로 생각된다.

배분화과정시 나타나는 변화에 미치는 의 영향을 알아보고자 whole cell voltage clamp 기법, 방사선 등위원소 면역측정법, 그리고 공초점 현미경을 통하여 처리 후 나타나는 전류 변화 및 세포내 농도 변화를 조사하였다. 생쥐의 미성숙 난자는 난소의 난포를 천자하고, 배란난자는 과배란 처리 후 난관에서 회수하였다. 수정란은 과배란 처리 후 수컷 생쥐와 교미를 유도한 후 각각의 단계에 맞는 수정란을 채란하였다. 혈중 의 농도는 심장을 천자하여