Recent studies showed that tight junctions (TJs) integrity and assembly are required for blastocyst development in mouse and pig models. However, the biological functions of TJs associated with embryo implantation and maintenance of pregnancy were not investigated yet. To examine whether disrupted TJs affect further embryo development, we employed RNAi approach and inhibitor treatment. The embryos were injected with Cxadr (Coxsackievirus and adenovirus receptor) siRNA for knock down (KD) and treated with Adam10 (A Disintegrin and Metalloproteinase specific inhibitor 10; GI254023X; SI). We compared blastocyst development and paracellular sealing assay using FITC dextran uptake between control and KD or SI embryos. Finally, we transferred control and Cxadr KD or Adam 10 SI treated blastocyst to uteri of recipients. Cxadr KD and Adam 10 SI showed lower blastocyst development and more permeable to FITC-dextran. Moreover, we observed that half of KD and inhibited embryos failed to maintain pregnancies after the second trimester. Our findings suggested that TJs integrity is required for the maintenance of pregnancy and can be used as a selective marker for the successful application of assisted reproduction technologies.

Enrofloxacin (EFX) is one of the 2nd generation quinolone antibiotics and is now widely used as a broad-spectrum antibiotic for industrial animals. Previous study showed that EFX reduces the cellular metabolic activity of spleen cells and modulates the inflammatory responses. However, little is known about its toxicity on bone marrow (BM) cells. In this study, BM cells were treated with EFX and cellular metabolic activity, cell death, the change of neutrophil (CD11b+Gr1+ cells) proportion, and antigen uptake ability of granulocytes were measured. Compared to the control, EFX-treated cells showed the decrease of cellular metabolic activity, the increase of cell death, and the decreased proportion of neutrophils. In contrast, the antigen uptake ability of granulocytes in BM cells was increased by EFX. These data suggest that EFX has only limited toxicity on BM cells. And also, EFX is safe on BM cells in a range of concentration, 6.25 – 25 μg/mL. This study can provide available data for the safety or toxicity of EFX.

현대 사회에는 과도한 스트레스나 고칼로리의 음식을 섭취하는 습관으로 인하여 탈모 환자가 늘어나는 추세이다. 현재 대표적인 탈모치료제 Minoxidil, Finasteride 등이 있지만 장기간 복용 시 다모증, 성 기능 감소 등의 부작용을 무시할 수 없다. 따라서 본 연구는 발모에 탁월하다고 알려져 있는 안전한 천연시료인 견과류와 해조류의 발모 효과를 확인 하기 위해 실험을 진행하였다. 본 실험에서는 C57BL/6 mouse 등 피부를 제모하여 탈모와 같은 휴지기 상태로 만든 후 견과류의 발모 효과를 알아보고자 견과류군, 해조류군, DW군, DW+왁스군 총 4군으로 나누어 각각의 추출물을 도포해 주었다. 일반 카메라관찰과 고해상도 피부 측정의 육안적 결과에서 견과류 군이 발모의 진행속도가 가장 빠르고 모량이 많았으며, Skin Score 또한 가장 높은 점수를 기록하였다. 조직 학적 분석결과에서 다른 군에 비하여 견과류군의 모낭과 기저세포의 수가 가장 많은 것을 확인하였다. 이러한 모든 결과를 바탕으로 견과류가 C57BL/6 mouse의 발모에 효과가 있는 것으로 판단된다.

폐경은 여성비만의 중요한 원인이다. 본 연구는 폐경여성의 동물모델인 난소절제 암컷 쥐에서 몸무게와 혈청 속 지질 성분의 조절에 대한 제니스테인의 농도 의존적 영향을 수영운동과 비교함으로써, 비만 조절에 대한 제니스테인의 효과적인 농도를 조사하였다. 난소절제 암컷 쥐는 대조군, 수영 운동군, 제니스테인 농도별(0.005%, 0.05%, 0.1% wt/wt) 처리군으로 나누고, 모든 쥐는 고지방식 사료를 8주 동안 섭취하였다. 고지방식 사료를 섭취한 대조군에 비해 수영운동을 실시한 군과 제니스테인이 농도별로 처리된 군 모두 몸무게, 백색지방조직의 무게, 혈청 속 지질 성분 농도 및 간조직의 지질 성분 축적이 감소되었다. 이러한 몸무게, 백색지방조직의 무게, 혈청 속 지질 성분 농도 및 간조직의 지질 성분 축적에 대한 제니스테인의 감소효과는 제니스테인 처리농도에 의존적이었고 제니스테인 농도 0.1%에서 가장 효과적이었으며 1시간 수영운동을 실시한 경우와 유사한 효과를 나타내었다. 본 연구결과들은 난소가 절제된 암컷 쥐에서 적정농도의 제니스테인 처리는 비만개선에 대해 수영운동과 유사한 효과를 나타낸다는 것을 제시한다. 제니스테인 보충제 식이의 섭취는 페경기 여성의 비만예방에 도움을 줄 것이다.

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.

Nelumbo nucifera Gaertn has been usedas a traditional remedy and food source in South Korea. It promotes gastrointestinal function and controls blood pressures. Nelumbo nucifera Gaertn water extracts supplement at 5, 10, 50, 100, 250, 500, 1,000 μg/mL after a 48 h pre-treatment with the mitogen (ConA or LPS) increased the mouse splenocytes proliferation. Water extract supplement also increased the cytokine production (IL-1β, TNF-α and IFN-γ), measured by a cytokine ELISA kit. For the result of in vitro study, the proliferation of splenocytes and cytokine production activated by peritoneal macrophages increased when water extracts were supplemented in the range of 50~500 μL/mL concentration. Specifically, the levels of the splenocytes proliferation, IL-1β, TNF-α and IFN-γ were the highest at 250 μL/mL concentration. This in vitro study suggestedthat supplementation with Nelumbo nucifera Gaertn water extracts may enhance the immune function by regulating the splenocyte proliferation and enhancing the cytokine production activating macrophage in vitro.

Soy isoflavones have been reported to possess many physiological activities such as antioxidant activity and inhibition of cancer cell proliferation. This study investigated the photoprotective effects of soybean extract in human fibroblast cell line and hairless mice model. Human fibroblast was treated with soybean extract before and after ultraviolet B (UVB; 290-302 nm) irradiation. In the soybean extract treated group, the cells showed better resistance to ultraviolet (UV) than control group. The amount of type I collagen recovered from the soybean treated group was higher than the vehicle group exposed to UV-induced damage. Moreover, increased expression of metalloproteinases-1 as a result of UV irradiation was suppressed by the soybean extract. Female mice were orally administered soybean extract and irradiated with UVB light for 8 weeks. The effects of the soybean extract on the skin appearance, collagen deposition and epidermal thickness in the UV-damaged mouse skin were analyzed using histopathological methods. In soybean extract treated group, the skin had a better morphology than that of the control group. Furthermore, the amount of type I collagen was increased and overexpression of MMP-1 was reduced in the soybean extract group compared to vehicle group. Additionally, up-regulation of pro-inflammatory cytokines induced by UV irradiation was suppressed by dietary soybean extract treatment. It appears that soybean extract had a photoprotective effect, including anti-aging and anti-inflammatory effect, from UV-induced damage in not only human fibroblast, but also hairless mice. We confirmed that these effects were possibly due to promotion of collagen synthesis and inhibition of MMP-1 expression.

To date, there are no protocols optimized to the effective separation of spermatogonial stem cells (SSCs) from testicular cells derived from mouse testes, thus hindering studies based on mouse SSCs. In this study, we aimed to determine the most efficient purification method for the isolation of SSCs from mouse testes among previously described techniques. Isolation of SSCs from testicular cells derived from mouse testes was conducted using four different techniques: differential plating (DP), magnetic-activated cell sorting (MACS) post-DP, MACS, and positive and negative selection double MACS. DP was performed for 1, 2, 4, 8, or 16 h, and MACS was performed using EpCAM (MACSEpCAM), Thy1 (MACSThy1), or GFR α1 (MACSGFRα1) antibodies. The purification efficiency of each method was analyzed by measuring the percentage of cells that stained positively for alkaline phosphatase. DP for 8 h, MACSThy1 post-DP for 8 h, MACSGFRα1, positive selection double MACSGFRα1/EpCAM, and negative selection double MACSGFRα1/α-SMA were identified as the optimal protocols for isolation of SSCs from mouse testicular cells. Comparison of the purification efficiencies of the optimized isolation protocols showed that, numerically, the highest purification efficiency was obtained using MACSGFRα1. Overall, our results indicate that MACSGFRα1 is an appropriate purification technique for the isolation of SSCs from mouse testicular cells.

Disulfiram is a drug used to treat alcohol dependence. Recent studies have shown that disulfiram also has anti-cancer effects. Considering that many anti-cancer agents have side effects, including immunosuppression, it is important to check if disulfiram has some cytotoxicity to immune cells. In this study, mouse spleen cells were treated with disulfiram and the metabolic activity was measured. Disulfiram increased the cell death of spleen cells according to annexin V-FITC/PI staining analysis. In addition, disulfiram decreased the mitochondrial membrane potential of spleen cells. The toxicity of disulfiram was concentration dependent. Interestingly, disulfiram affected the population of lymphocytes and the subset of spleen cells was altered. This study provides clinicians and researchers with valuable information regarding the toxicity of disulfiram to mouse spleen cells, particularly lymphocytes.

Mitochondria is energy generating organelle. It synthesizes ATP, which is the essential energy source of many cellular processes. During producing energy, some redox centres leak electrons to oxygen and it is contributory to the reactive oxygen species. Besides, mitochondria have significant functions in metabolism, calcium homeostasis, and fatty acid oxidation. Also mitochondria has importance to the breakdown of the ovarian follicles and could be factor determining oocyte of quality adversely. Increasing evidence shows that the number of mitochondria affect oocyte of developmental competence and maturation detrimentally during aging. Oocyte is the mitochondria-rich cell and enable the organelle to have competence for fertilization and early embryonic development. Occurrence of blastomere depends on distribution change of mitochondria which present in the egg. Lonicera caerulea treatment inhibited ovarian mitochondrial oxidative damage by suppressing mitochondrial reactive oxygen species (mROS) generation, decreasing apoptosis, controlling disintegration of mitochondrial membrane potential and conserving respiratory chain complex activities. The purpose of this study is to identify if mouse accepting treatment with L. caerulea could counter age-induced sterility and ovarian mitochondrial OS in a model organism of ovarian ageing.

Lonicera Caerulea(Honey berry) has been used in medical treatment in Russia, Japan, China and Korea. It has high level of vitamin C and polyphenolics. Polyphenolics can improve anti-inflammatory effect and prevent cancer, diabetes mellitus type 2. Also, Vitamin C is a representative anti- oxidant. however, It is still unknown what effect it will have on the oxidation stress of the reproductive system. In previous studies, ROS can be produced when it is exposed to heat stress and has negative effect on sperm's maturation, capacitation, hyperactivation, acrosome reaction and fusion of egg and sperm. Therefore, The purpose of this study is to investigate the antioxidant effects of L.caerulea on the sperm and egg cells of mice. At first, it conducted using ICR mouse(n=20) during 4 weeks. There are four groups of mouse(n=5 per group). Also, L.caerulea was taken by oral gavage. Group Ⅰ(control) kept at 23℃~27℃ and administer D.W(0.5ml/day), Likewise, Group Ⅱ(HB) kept at room temperature but gave HB(0.5ml/day), Group Ⅲ (HB+HS) received heat stress (40℃) using hyperthermia induction chamber and gave HB at same dose. and Group Ⅳ(HS) exposed heat stress only. Mainly, we showed degree of gene expression using Western blot in SOD, HSP 70, 17β-HSD and Real time PCR. It can find correlation between intracellular activity like steroid hormone, apoptosis under ROS and antioxidant activity of L.caerulea.

Previous studies have shown that Lonicera caerulea has a chemical protective effect. Phenolic and vitamin C contained in Lonicera caerulea prevent cancer, diabetes and cardiovascular disease, lower blood pressure and delay the aging process. However, the antioxidant mechanism of male reproductive system to heat stress is still unknown. Male reproductive system is very sensitive to heat. When scrotum temperature increase, oxidative stress can occur. Oxidative stress affects sperm motility and spermatogenesis, resulting in infertility. Therefore, we investigated the antioxidant effect of L. Caerulea in male genitalia by inducing oxidative stress by artificially exposing the testicles to heat at 42 ° C. The experiment was performed by dividing the ICR mouse into four groups. Each group is n = 5. Control group (C) and heat stress group (HS) were oral gavage administered D.W. Honey berry group (HB) and Honeyberry / heat stress group (HB + HS) were oral gavage administered honey berry (250mg / kg / day). HS groups (n=5, per n=5) received heat stress by exposing their lower bodies in the water bath at 42℃ for 30 minutes. We confirmed that there was a significant difference in the motility, morphology and the number of sperms using CASA(computer-assisted semen analysis). Lipid peroxidation assay results showed heat causes oxidative stress in serum. This study is conducting to investigate the antioxidant effect of L. Caerulea. Histologically analyzed the testicular form of each group, the activity level of heat shock protein and the level of reactive oxygen species were measured by Western blot and the level of catalase and HSP-90 was examined by RT-PCR analysis. Thus, studies of testicular morphology, sperm kinetics, hormone levels, heat shock protein expression and antioxidant enzymes under heat stress have shown that L. Caerulea ingestion has Anti-oxidant and thermal protective activity on the testis by heat damage.

Mammalian fetal ovaries contains numerous primordial germ cells, however fewer ones can yield mature oocytes due to apoptosis and follicle atresia. Successful in vitro reconstitution of primordial germ cells has recently had a significant effect in the field of assisted reproductive technologies. However, the regulatory mechanisms underlying oogenesis remain unknown and recapitulation of oogenesis in vitro remains unachieved. Therefore, development of methods for obtaining mature oocytes by culturing the fetal ovaries in vitro could contribute to clarify these mechanisms. We adapt an in vitro system for culturing mouse fetal ovaries that support successful follicle assembly and improve oocyte growth and maturation. Ovarian tissues from 12.5 days postcoitum (dpc) fetal mice were cultured in vitro and the matured oocytes were differentiated from primordial germ cells after a 31 days culture period. Our results demonstrate that mouse fetal germ cells are able to form primordial follicles with artificial ovarian cells, and that oocytes within the growing follicles are able to mature normally in vitro. Taken together, this in vitro culture system is expected to aid in the development of new strategies to identify the reasons behind failure of follicle assembly and offer a platform for innovative research into preservation of female germ cells and conservation of endangered species.

Transglutaminase (TGM2) belongs to a family of cross-linking enzymes responsible for catalyzing Ca2+-dependent acyl-transfer reactions between the substrate proteins. TGM2 is a cytosolic protein that has also been observed in the nucleus and can be expressed to the cell surface or extracellular matrix. Despite ubiquitous expression, its functions are poorly understood and still need to be elucidated. Moreover, there is no clear data regarding the role of transglutaminase in mammalian oocytes. So, in this study, we have patterned the transglutaminase 2 (TGM2) and anti-N epsilon gamma glutamyl lysine (AB424) activity in heat stressed mouse oocytes. We have collected mouse oocytes from the (6–9 weeks old) mouse and in vitro matured for 20 h. Immunocytochemistry was performed to checked the transglutaminase 2 (TGM2) and anti-N epsilon gamma glutamyl lysine (AB424) activity after 6 h of heat stress (HS) at 39.1 ℃. Both TGM2 and AB424 expression were significantly (P < 0.05) higher compared to control when oocytes were subjected to HS at 6 h of IVM at 39.1 ℃. Our hypothesis is that TGM2 and AB424 activity may be correlated with the cellular regression and also involvement in apoptosis. We hope that, our study will help to elucidate the normal function of mouse oocyte and also identification of the principal proteins as well as the pathogenic mechanism of altered physiology. These results suggest that the nuclear accumulation of the transglutaminase may play an important role in nuclear remodeling during folliculogenesis and early embryonic development

The CRISPR/Cas9 system is widely applied in genome engineering due to its simplicity and versatility. Although this has revolutionized genome-editing technology, knock-in animal generation via homology directed repair (HDR) is not as efficient as non-homologous end-joining DNA-repair-dependent knockout. Although its double-strand break activity may vary, Cas9 derived from Streptococcus pyogenens allows robust design of single-guide RNAs (sgRNAs) within the target sequence; However, prescreening for different sgRNA activities delays the process of transgenic animal generation. To overcome this limitation, multiple sets of different sgRNAs were examined for their knock-in efficiency. We discovered profound advantages associated with single-stranded oligo-donor-mediated HDR processes using overlapping sgRNAs (sharing at least five base pairs of the target sites) as compared with using non-overlapping sgRNAs for knock-in mouse generation. Studies utilizing cell lines revealed shorter sequence deletions near target mutations using overlapping sgRNAs as compared with those observed using non-overlapping sgRNAs, which may favor the HDR process. Using this simple method, we successfully generated several transgenic mouse lines harboring loxP insertions or single-nucleotide substitutions with a highly efficiency of 18~38%. Our results demonstrate a simple and efficient method for generating transgenic animals harboring foreign-sequence knock-ins or short-nucleotide substitutions by the use of overlapping sgRNAs.

Although there are several methods for establishment of stem cell line, most of them has critical limit such as, ethical problem and infectious concern. Accordingly, we investigated the cell fusion technique as a new tool to establish a stem cell line. We cultured mouse embryonic stem cell (ESC) and somatic cells. Then, these two type cells were fused by electro cell fusion that consist of three steps (AC→DC→AC). The fused cells were individually transferred into a 96-well plate and cultured in ESC culture medium for 6 ~ 7 days. Newly formed colonies were evaluated several analysis methods like morphology, alkaline phosphatase (AP) activities, expression of pluripotency marker genes and proteins, and karyotyping. The fusion efficiency from the ESC and somatic cell into colony formation was about 0.3 ~ 0.5 %. The electro cell fused (EF) new stem cell colonies (EF-SC1 ~ 4) were indicated normally round-shape morphology similarly to ESC colonies and each colonies were expressed green fluorescent protein that having somatic cells. Also, all EF-SC groups were highly expressed AP activity and pluripotency marker proteins, POU5f1, NANOG, SOX-2 and SSEA-1. In the transcription levels, all EF-SC groups were significantly higher level of expression in Pou5f1 and Nanog compared to donor cells (ESC and somatic cell) (p<0.05). In particular, the level of Pou5f1 expression was about 2-folds higher in EF-SC2 and EF-SC3 groups than in control and EF-SC1 groups (p<0.05). Also, the level of Nanog expression was very significantly higher in EF-SC2 group (3.5-folds) compared to control ESC group, and the expression levels among treatment groups were variable (ESC<EF-SC1<EF-SC4<EF-SC3<EF-SC2, p<0.05). In karyotype analysis, the results of EF-SC2 and EF-SC3 were presented the same that of ESC, while that of EF-SC1 and EF-SC4 shown aneuploid mutation in chromosome 8. Taken together, these results demonstrate that electro cell fusion technique can be used as a new method to establish of stem cell lines.