metalloproPhotoprotective Effects of Soybean Extract against UV-Induced Damage in Human Fibroblast and Hairless Mouse Model

Young-Chang Cho,Jae-Bok Han,Sang-Ik Park 사단법인 한국동물생명공학회 2019 한국동물생명공학회지 Vol.34 No.1

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.

에이코사펜타인산이 돼지난포란의 체외 성숙에 미치는 영향

김강식,박흠대 사단법인 한국동물생명공학회 2019 한국동물생명공학회지 Vol.34 No.3

Among fatty acid families, the polyunsaturated fatty acids were demonstrated to be mediators in various reproductive processes as precursor of steroid hormone (via cholesterol) and prostaglandins (via arachidonic acid), and in the last decade, major research was focused on the effects of omega-6 and especially omega-3 fatty acid. Eicosapentaenoic acid, the longest members of omega-3 fatty acid family, can be produced by a series of desaturation and elongation reactions from shorter member such as α-Linolenic acid. However, very few studies have provided detailed descriptions of Eicosapentaenoic acid effects and mechanisms of action in mammalian oocytes. The purpose of this study was to evaluate the effect of Eicosapentaenoic acid supplementation on in vitro maturation and developmental potential of porcine oocytes. Various concentrations of Eicosapentaenoic acid was added into in vitro maturation medium, and we evaluated the degree of cumulus expansion, nuclear maturation rate, blastocysts quality, and levels of prostaglandin E2, 17β-estradiol, progesterone in the spent medium. High doses (100 mM) of Eicosapentaenoic acid supplementation significantly inhibited cumulus expansion and oocyte nuclear maturation, and prostaglandin E2 synthesis also significantly decreased compared with other groups (p < 0.05). Supplementation of 50 mM Eicosapentaenoic acid showed higher quality blastocysts in terms of high cell numbers and low apoptosis when compared with other groups (p < 0.05), and synthesis ratio of E2/P4 also significantly increased compared with control group (p < 0.05). However, Supplementation of 100 mM Eicosapentaenoic acid showed high apoptosis when compared with other groups (p < 0.05), and synthesis ratio of 17b-estradiol/progesterone also significantly decreased compared with control group (p < 0.05). Our results indicated that supplementation with appropriate levels of Eicosapentaenoic acid beneficially affects the change of hormone synthesis for controlling oocyte maturation, leading to improved embryo quality. However, high doses of Eicosapentaenoic acid treatment results in detrimental effects.

에스트로겐이 생쥐 초기배의 $\textrm{Ca}^{2+}$ 전류에 미치는 영향

강다원,신용원,김은심,홍성근,한재희 사단법인 한국동물생명공학회 2001 한국동물생명공학회지 Vol.16 No.2

배분화과정시 나타나는 $Ca^{2+}$ 변화에 미치는 $E_2$의 영향을 알아보고자 whole cell voltage clamp 기법, 방사선 등위원소 면역측정법, 그리고 공초점 현미경을 통하여 $E_2$처리 후 나타나는 $Ca^{2+}$ 전류 변화 및 세포내 $Ca^{2+}$ 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 $E_2$의 농도는 심장을 천자하여 혈액을 채취한 후 배발달 단계와 호르몬 처리 시간이 일치하는 혈액만을 사용하였다. 본 실험의 결과를 요약하면 다음과 같다. 1. $E_2$처리시 미성숙난자의 제 1극체 형성률 (성숙의 지표)은 $E_2$를 처리하지 않은 난자(83% : 83/100)보다 $E_2$를 처리한 난자 (94%, 94/100)에서 유의적 (P<0.05)으로 높게 나타났다. 2. $E_2$를 처리하였을 때 $Ca^{2+}$ 내향전류의 변화는 -10 mV에서 -1.23$\pm$0.01 nA (n=15)에서 -1.50$\pm$0.03 nA (n=15)로 122% 상승함으로써 유의한 (P<0.05) 변화를 보였다. 3. $E_2$를 처리하지 않은 난자 및 수정란을 1로 한 후 $E_2$를 처리한 난자 및 수정란의 변화를 상대적인 값으로 표시하였다. $E_2$처리한 난자는 1.22$\pm$0.17 (n=10), $E_2$처리한 전핵배는 1.20$\pm$0.14 (n=10), $E_2$처리한 2세포기배는 1.07$\pm$0.01 (n=10), 4세포기배는 1.05$\pm$0.09 (n=10)를 나타냄으로써 수정란의 단계마다 $E_2$의 반응 결과가 차이가 남을 알 수 있었다. 4. $E_2$농도 곡선에서 PMSG 처리 후 $E_2$의 혈중농도는 계속적인 상승을 보이다가 배란시기에 최고치를 나타내었으며, 배란 후 다시 감소하여 8세포기에서는 급격한 감소현상이 나타났다. 이후 다시 상실기를 거쳐 배반포기 임신기간동안 $E_2$의 농도가 상승하였다. 5. $E_2$처리 후 세포내 $Ca^{2+}$ 농도변화의 결과로, $E_2$를 처리하지 않은 난자들의 세포내 $Ca^{2+}$ 농도는 836.4$\pm$131.2 (n=10), $E_2$를 처리한 난자들은 1736.4$\pm$192.0 (n=10)로써 유의한 (P<0.05) 차이를 보였다. 이상의 결과로부터 $E_2$처리에 의한 세포내 $Ca^{2+}$ 농도 상승은 $E_2$가 $Ca^{2+}$ 통로를 자극함으로써 세포바깥의 $Ca^{2+}$이 세포안으로 이동하여 나타나는 변화로 생각된다. Steroid hormones control the expression of many cellular regulators, and a role thor estrogen in mouse oocytes has been well documented. The preovulatory $E_2$increment is generally accepted as the endocrine process regulating induction of in vivo oocyte maturation To address whether the activity of the T-type $Ca^{2+}$ channel is altered by 17 beta-estradiol ( $E_2$), we examined the actions of $E_2$on the calcium channel of mouse oocytes and early embryos. Oocrtes were collected from the oviduct of mice treated with pregnant mare's serum gonadotropin (PMSG) and human choronic gonadotropin (hCG). Whole cell voltage clamp technique and confocal microscopy were used to examine that $E_2$increase intracellular $Ca^{2+}$ concentration ([C $a^{2+}$]$_{i}$ ) via voltage dependent $Ca^{2+}$ channel (VDC) and estrogen receptor (FSR), and $E_2$concentration by the use of radioimmunoassay (RIA) were examined in mouse. The results obtained were as follows: The peak of $Ca^{2+}$ current induced by $E_2$increased 122% to 1.50$\pm$0.03 nA from 1.23$\pm$0.21 nA (n=15) in the presence of 5 mM extracellular $Ca^{2+}$ concentration ([C $a^{2+}$]$_{o}$ ). The increased $Ca^{2+}$ current was temporally associated with $Ca^{2+}$ transients. The intracellular $Ca^{2+}$ level increased 207%~30 s following the addition of 1${\mu}{\textrm}{m}$ $E_2$(relative fluorescence intensity: 836.4$\pm$131.2 for control, n=10, 1736.4$\pm$192.0 in the presence of $E_2$, n=10). $E_2$increased amplitude of $Ca^{2+}$ current and [C $a^{2+}$]$_{i}$ . $E_2$-induced $Ca^{2+}$ current and $E_2$concentration in blood were showed difference on the stage of embryo. These results suggest that $E_2$modulate $Ca^{2+}$ channel to increase $Ca^{2+}$ influx.$Ca^{2+}$ influx.

Reversible Effects of Exogenous GM3 on Meiotic Maturation and Cumulus Cells Expansion of Porcine Cumulus-oocyte Complexes

김진우,박효진,정재민,양슬기,김민지,김인수,제갈호근,구덕본 사단법인 한국동물생명공학회 2018 한국동물생명공학회지 Vol.33 No.4

Ganglioside GM3 is known as an inhibition factor of cell differentiation and proliferation via inhibition of epidermal growth factor receptor (EGFR) phosphorylation. Our previous study showed that the exogenous ganglioside GM3 reduced the meiotic maturation of porcine oocytes and induced apoptosis at 44 h of in vitro maturation (IVM). However, the role of ganglioside GM3 in the relationship between EGFR signaling and apoptosis during porcine oocyte maturation has not yet been studied. First, porcine cumulus-oocyte complexes (COCs) were cultured in the NCSU-23 medium with exogenous ganglioside GM3 according to maturation periods (non-treated, only IVM I: 0 - 22 h, only IVM II: 22 - 44 h and IVM I & II: 0 - 44 h). We confirmed that the proportion of germinal vesicle breakdown (GVBD) increased significantly in the IVM I treated group than in the control group. We also confirmed that the meiotic maturation until M II stage and polar body formation decreased significantly in the only IVM I treated group. Cumulus cell expansion and mRNA levels of the expansion-related factors (HAS2, TNFAIP6 and PTX3) decreased significantly in the IVM I treated group than in the control group. Protein levels of EGFR, p-EGFR, ERK1/2, and p-ERK1/2 decreased significantly in the GM3-treated groups, during the IVM I period. In addition, cellular apoptosis, determined using TUNEL assay, and protein levels of Cleaved caspase 3, were increased significantly in the GM3-treated COCs during the IVM I period. Based on these results, ganglioside GM3 exposure of porcine COCs during the IVM I period reduced meiotic maturation and cumulus cell expansion via inhibition of EGFR activity in pigs.

Potential Endocrine Disrupting Effects of Phthalates in In Vitro and In Vivo Models

Tien-Thanh Nguyen,정의만,양현,현상환,최경철,정의배 사단법인 한국동물생명공학회 2010 한국동물생명공학회지 Vol.25 No.4

Thousands of new chemicals have been introduced to environment during last decades. Many of them and common consumer products have been shown to be the endocrine disrupting chemicals. One such chemical group is the phthalates, used in soft poly vinyl chloride (PVC) material and in a huge number of consumer products. The prevalence of these modern chemicals have a remarkable increase. Approximately 3.5 million tons of the main phthalate, di-(2-ethylhexyl) phthalate (DEHP), are produced annually worldwide and indeed, DEHP is considered a ubiquitous environmental contaminant. It has been demonstrated that high doses of phthalate can adversely affect adult and developing animals. In this review, we critically discuss the conclusions of recently original research papers and provide an overview of studies on reproductive disrupting effects of phthalates. In addition, we review the reproductive toxicity data of phthalates in some in vitro research and in both male and female reproductive systems in experimental and domestic animals. Finally, we point out some critical issues that should be addressed in order to clarify the implication of phthalates for human reproduction.

Distribution and Potential Toxicological Effects of 2,2’,4,4’-tetrabromodiphenyl Ether (BDE-47) as a Endocrine Disrupting Chemical in Human and Animals

정의만,양현,안범수,이근식,현상환,최경철,정의배 사단법인 한국동물생명공학회 2011 한국동물생명공학회지 Vol.26 No.4

Polybrominated diphenyl ethers (PBDEs) are a class of “brominated” (bromine containing) man-made chemicals used as flame retardant additives in plastics, foams, and textiles. PBDEs are found in various environmental contaminants in air, soil, sediment, and water, and 209 individual forms (congeners) of PBDE exist. Among these,2,2',4,4'-tetrabromodiphenyl ether (BDE-47) is the dominant congener found in the environment. Exposure to BDE-47is now worldwide, and levels of BDE-47 have been detected in the blood of animals, including humans. BDE-47 can adversely affect the developmental system in both humans and animals. BDEs have structural similarities to polychlorinated biphenyls and thyroid hormones. However, recent studies have shown that BDEs may act as hormonal disrupting chemicals with detrimental effects. Therefore, a reliable assessment of BDE-47 toxicological action is required to understand the detrimental impacts of BDE-47 on human health. In this review, we overview recent studies on the distribution and potential toxicological effects of BDE-47 in humans and animals

항산화 효소가 첨가된 Percoll에 의해 분리한 돼지 정액의 동결-융해 능력

이경진,이상희,주선호,김유진,양진우,이연주,황보용,이승형,이승태,이은송,박춘근 사단법인 한국동물생명공학회 2015 한국동물생명공학회지 Vol.30 No.3

The objective of this study was to evaluate the efficiency of sperm cryosurvival in boar sperm separated by Percoll containing antioxidant enzymes. The boar semen was collected into a pre-warmed (37℃) thermos bottle by gloved-hand method and was separated by 65% Percoll with superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) before freezing. The frozen sperm was thawed at 38.5℃ for 45 sec in water-bath for sperm characteristic analysis. The sperm were estimated with SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction, Rhodamine123/PI double staining for mitochondrial integrity and were analyzed using flow cytometry. In results, sperm viability, acrosome reaction and mitochondrial integrity were improved in separated sperm groups compared with unseparated sperm by Percoll (UP) group. Especially, viability was significantly higher in sperm separated by Percoll containing 400 IU CAT group compared with other groups (P<0.05). And acrosome reaction was decreased in sperm separated by Percoll with 300 IU SOD, 400 IU CAT and 0.5 mM GSH groups compared with other groups, however, there were no significantly difference mitochondrial integrity among sperm separated by Percoll with antioxidant enzymes. In conclusion, we suggest that use of Percoll containing antioxidant enzymes for sperm separation will be beneficial for sperm cryopreservation in pigs.

Factors Affecting the Development of Embryos Produced by Nuclear Transfer

이준희,Keith H. S. Campbell 사단법인 한국동물생명공학회 2012 한국동물생명공학회지 Vol.27 No.4

The development of embryos reconstructed by nuclear transfer is dependent upon numerous factors including the type of recipient cell, method of enucleation, the type of donor cell, method of reconstruction, activation, the cell cycle stage of both the donor nucleus and the recipient cytoplasm and the method of culture of the reconstructed embryos. Many of these points which have been reviewed extensively elsewhere (Sun and Moor, 1995; Colman, 1999; Oback and Wells, 2002; Renard et al., 2002; Galli et al., 2003b), here we will concentrate on main area, the production of suitable cytoplast and nuclear donor, nuclear-cytoplasmic coordination, oocyte activation, culture of reconstructed embryos, and the effects that this may have on development

Exogenous Nitric Oxide Donation During In Vitro Maturation Improves Embryonic Development after Parthenogenesis and Somatic Cell Nuclear Transfer in Pigs

ELAHI MD FAZLE,신혜지,이주형,이승태,이근식,이은송 사단법인 한국동물생명공학회 2018 한국동물생명공학회지 Vol.33 No.4

Nitric oxide (NO) has an important role in oocyte maturation and embryonic development in mammals. This study examined the effect of exogenous NO donor S-nitroso-N-acetylpenicillamine (SNAP) in a maturation medium on meiotic progression and embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) in pigs. When oocytes were exposed to 0.1 μM SNAP for first 22 h of in vitro maturation (IVM) in Experiment 1, SNAP significantly improved blastocyst development in both defined and standard follicular fluid-supplemented media compared to untreated control (48.4 vs. 31.7-42.5%). SNAP treatment significantly arrested meiotic progression of oocytes at the germinal vesicle stage at 11 h of IVM (61.2 vs. 38.7%). However, there was no effect on meiotic progression at 22 h of IVM (Experiment 2). In Experiment 3, when oocytes were treated with SNAP at 0.001, 0.1 and 10 μM during the first 22 h of IVM to determine a suitable concentration, 0.1 μM SNAP (54.2%) exhibited a higher blastocyst formation than 0 and 10 μM SNAP (36.6 and 36.6%, respectively). Time-dependent effect of SNAP treatment was evaluated in Experiment 4. It was observed that SNAP treatment for the first 22 h of IVM significantly increased blastocyst formation compared to no treatment (57.1% vs. 46.2%). Antioxidant effect of SNAP was compared with that of cysteine. SNAP treatment significantly improved embryonic development to the blastocyst stage (49.1-51.5% vs. 34.4-37.5%) irrespective of the presence or absence of cysteine (Experiment 5). Moreover, SNAP significantly increased glutathione (GSH) content and inversely decreased the reactive oxygen species (ROS) level and mitochondrial oxidative activity in IVM oocytes. SNAP treatment during IVM showed a stimulating effect on in vitro development of SCNT embryos (Experiment 7). These results demonstrates that SNAP improves developmental competence of PA and SCNT embryos probably by maintaining the redox homeostasis through increasing GSH content and mitochondrial quality and decreasing ROS in IVM oocytes.

Principle of Emulsion PCR and Its Applications in Biotechnology

채창훈 사단법인 한국동물생명공학회 2019 한국동물생명공학회지 Vol.34 No.4

Emulsion polymerase chain reaction (PCR) is performed on compartmentalized DNA, allowing a large number of PCR reactions to be carried out in parallel. Emulsion PCR has unique advantages in DNA amplification. It can be applied in many molecular biological assays, especially those requiring highly sensitive and specific DNA amplification. This review discusses the principle of emulsion PCR and its applications in biotechnology. Related technologies are also discussed.