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Nugraha Setyawan, Erif Maha,Oh, Hyun Ju,Kim, Min Jung,Kim, Geon A.,Lee, Seok Hee,Choi, Yoo Bin,Ra, Kihae,Lee, Byeong Chun Elsevier 2018 Theriogenology Vol.115 No.-
<P><B>Abstract</B></P> <P>The paracrine interactions between cumulus-oocyte complexes (COCs) and follicular somatic cells during <I>in vitro</I> maturation (IVM) were investigated. To optimize IVM conditions, many studies have applied exogenous growth factors and cell feeding/co-culture systems using various cell types to replicate the natural follicular microenvironment during IVM. A potential candidate as cell feeders is adipose-derived stem cells (ASCs) which secrete high levels of growth factors that have roles in oocyte maturation. However, the cell donor's age should be considered because biological aging also occurs in stem cells. In the present study, the contributions of ASCs from young and old donors on an IVM co-culture system were analyzed by comparing the oocyte maturation rate, cumulus expansion index, preimplantation development after parthenogenetic activation (PA), and expression of growth factor signaling genes related to oocyte maturation in ASCs, oocytes and cumulus cells under the same culture conditions. Our study demonstrated that the confluence, viability and cell size of ASCs between young and old donors were not significantly different and only the Fibroblast Growth Factor 2 (<I>FGF2</I>) signaling gene showed higher expression in ASCs from young donors. The oocyte maturation rate in the young donor group (87.8 ± 1.2%) was significantly higher than in the old donor (81.1 ± 2.1%) and control (73.8 ± 2.1%) groups. After IVM, most gene expression levels in oocytes and cumulus cells in the co-culture groups were higher than in the control but the apoptotic ratios were reduced. The blastocyst development rates were not different between the young and old donor groups (23.9 ± 1.3% and 20.7 ± 0.8%, respectively) but the percentages were higher in both groups compared to the control group (16.4 ± 1.2%). A similar pattern was also found for blastocyst total cell numbers in that the young donor group (87.5 ± 5.2 cells) was not different than the old donor group (77.5 ± 3.4 cells) but both groups exhibited higher number of cells compared with the control group (57.9 ± 6.0 cells, p < .05). Our study strongly suggested that the co-culture IVM system with ASCs greatly improved the maturation and development rates of porcine oocytes. Moreover, ASCs from young donors more effectively supported porcine oocyte maturation than those from old donors although this difference did not translate into improved developmental competence.</P> <P><B>Highlights</B></P> <P> <UL> <LI> IVM with co-culture strategy improved the effectivity of oocyte maturation. </LI> <LI> The confluence, viability and cell size between young and old donor were similar. </LI> <LI> The FGF2 signalling gene showed higher expression in young ASCs donor. </LI> <LI> Maturation rate in young donor was the highest than old donor and control groups. </LI> <LI> Co-culture IVM system up regulated most of signalling genes in oocytes and cumulus. </LI> </UL> </P>
Setyawan, Erif Maha Nugraha,Kim, Min Jung,Oh, Hyun Ju,Kim, Geon A.,Jo, Young Kwang,Lee, Seok Hee,Choi, Yoo Bin,Lee, Byeong Chun Elsevier 2016 Biochemical and biophysical research communication Vol.479 No.4
<P><B>Abstract</B></P> <P>The objective of this study was to determine the ability of spermine to act as an antioxidant in scavenging reactive oxygen species (ROS), maintaining sperm function and decreasing cryocapacitation after cryopreservation. Although motility did not increase with spermine treatment, values for membrane integrity were significantly increased (P < 0.05). Higher percentages of linearity and straightness with a lower amplitude of lateral head displacement (ALH) indicated that spermine inhibits hyperactivation. Concentrations of intracellular and extracellular ROS were decreased in the treatment group (P < 0.05). Higher expression of an anti-apoptotic gene (<I>Bcl</I>-2) and lower expression of a pro-apoptotic gene (<I>Bax</I>), together with decreased expression of the mitochondrial ROS modulator <I>ROMO</I>1, DNA repair due to oxidative damage (<I>OGG</I>1), spermine synthase (<I>SMS</I>), NADPH oxidase associated with motility (<I>NOX</I>5) and spermine amino oxidase (<I>SMOX</I>), all showed that 5.0 mM spermine treatment was beneficial to spermatozoa. Furthermore, the proportion of live spermatozoa with intact acrosomes after thawing in the treatment group was higher than in the control. After incubation in canine capacitating medium, numbers of live capacitated spermatozoa with reacted acrosomes were higher than in the control. Our results indicate that 5.0 mM spermine is an optimal concentration for maintaining sperm function, reducing ROS production, preventing apoptosis and adverse effects of cryocapacitation during canine sperm cryopreservation.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Spermine improved kinematic parameters and membrane integrity of frozen sperm. </LI> <LI> Spermine decreased intracellular and extracellular ROS during cryopreservation. </LI> <LI> Nitro Blue Tetrazolium followed by ELISA was used for measuring ROS level. </LI> <LI> Spermine reduced cryocapacitation during canine sperm cryopreservation. </LI> <LI> Spermine increased the number of capacitated sperm after CCM incubation. </LI> </UL> </P>
Vorinostat Induces Cellular Senescence in Fibroblasts Derived from Young and Aged Dogs
김민정,오현주,Erif Maha Nugraha Setyawan,최유빈,이석희,이병천 한국임상수의학회 2017 한국임상수의학회지 Vol.34 No.1
Although HDACIs affect ubiquitously expressed histone deacetylase and increase cellular senescence, therehas been little study on the effect of age on treatment with HDACIs. Accordingly, the purpose of this study wasto compare cellular senescence status and vorinostat-induced senescence in fibroblasts derived from aged dogs comparedto young dogs. Skin tissues were taken from young (1-year-old) and aged (7-year-old) male dogs, and fibroblasts werecultured without (control) or with 10 uM of vorinostat for 24 hr. Beta-galactosidase activity was assessed, and realtimepolymerase chain reaction and western blotting were performed to analyze the expression levels of transcriptsand proteins related to cellular senescence. Beta-galactosidase activity was higher in aged dogs compared to youngdogs in the control group, and was increased by vorinostat treatment. Expression of p21, p53 and p16 transcripts washigher in the aged than in the young group, and all transcripts were affected by vorinostat in both young and agedgroups. Western blot results showed lower H3K9 acetylation in the aged dogs compared to the young dogs, and theacetylation was increased by vorinostat treatment in both groups. However, there was no significant difference betweenthe transcript or protein alterations induced by vorinostat.
Lalu Unsunnidhal,Raden Wasito,Erif Maha Nugraha Setyawan,Ziana Warsani,Asmarani Kusumawati 대한수의학회 2021 Journal of Veterinary Science Vol.22 No.6
Background: The development of a vaccine for Jembrana disease is needed to prevent losses in Indonesia's Bali cattle industry. A DNA vaccine model (pEGFP-C1-tat) that requires a functional delivery system will be developed. Polylactic-co-glycolic acid (PLGA) may have potential as a delivery system for the vaccine model. Objectives: This study aims to evaluate the in vitro potential of PLGA as a delivery system for pEGFP-C1-tat. Methods: Consensus and codon optimization for the tat gene was completed using a bioinformatic method, and the product was inserted into a pEGFP-C1 vector. Cloning of the pEGFP-C1-tat was successfully performed, and polymerase chain reaction (PCR) and restriction analysis confirmed DNA isolation. PLGA-pEGFP-C1-tat solutions were prepared for encapsulated formulation testing, physicochemical characterization, stability testing with DNase I, and cytotoxicity testing. The PLGA-pEGFP-C1-tat solutions were transfected in HeLa cells, and gene expression was observed by fluorescent microscopy and real-time PCR. Results: The successful acquisition of transformant bacteria was confirmed by PCR. The PLGA:DNA:polyvinyl alcohol ratio formulation with optimal encapsulation was 4%:0.5%:2%, physicochemical characterization of PLGA revealed a polydispersity index value of 0.246, a particle size of 925 nm, and a zeta potential value of −2.31 mV. PLGA succeeded in protecting pEGFP-C1-tat from enzymatic degradation, and the percentage viability from the cytotoxicity test of PLGA-pEGFP-C1-tat was 98.03%. The PLGA-pEGFP-C1-tat demonstrated luminescence of the EGFP-tat fusion protein and mRNA transcription was detected. Conclusions: PLGA has good potential as a delivery system for pEGFP-C1-tat.
Recovery of In Vivo Matured Oocytes from a Bitch with Hydrometra
김민정,조영광,강상철,오현주,김지은,Erif Maha Nugraha Setyawan,최유빈,이석희,김현일,이병천 한국임상수의학회 2015 한국임상수의학회지 Vol.32 No.6
One year old mixed-breed bitch was examined to retrieve in vivo matured oocytes. Laparotomy was performed 72 hr after ovulation determined by serum progesterone concentration, and abnormally enlarged left uterus horn was found. Both ovaries had eight corpus lutea, and a total 16 in vivo matured oocytes having perivitelline space within 25 μm, polar body, and metaphase II nucleus were recovered by flushing oviducts. This is the first study to confirm in vivo maturation of oocytes from a bitch with hydrometra, which suggests that oocytes recovered from canids with reproductive disease could be valuable sources for assisted reproductive technologies.
Kim, Min Jung,Oh, Hyun Ju,Choi, Yoo Bin,Lee, Sanghoon,Setyawan, Erif Maha Nugraha,Lee, Seok Hee,Lee, Seung Hoon,Hur, Tai Young,Lee, Byeong Chun 家畜繁殖硏究所 2018 Journal of Reproduction and Development Vol.64 No.3
<P> This study was conducted to investigate whether the treatment of dog to pig interspecies somatic cell nuclear transfer (iSCNT) embryos with a histone deacetylase inhibitor, to improve nuclear reprogramming, can be applied to dog SCNT embryos. The dog to pig iSCNT embryos were cultured in fresh porcine zygote medium-5 (PZM-5) with 0, 1, or 10 μM suberoylanilide hydroxamic acid (SAHA) for 6 h, then transferred to PZM-5 without SAHA. Although there were no significant differences in cleavage rates, the rates of 5-8-cell stage embryo development were significantly higher in the 10 μM group (19.5 ± 0.8%) compared to the 0 μM groups (13.4 ± 0.8%). Acetylation of H3K9 was also significantly higher in embryos beyond the 4-cell stage in the 10 μM group compared to the 0 or 1 μM groups. Treatment with 10 μM SAHA for 6 h was chosen for application to dog SCNT. Dog cloned embryos with 0 or 10 μM SAHA were transferred to recipients. However, there were no significant differences in pregnancy and delivery rates between the two groups. Therefore, it can be concluded that although porcine oocytes support nuclear reprogramming of dog fibroblasts, treatment with a histone deacetylase inhibitor that supports nuclear reprogramming in dog to pig iSCNT embryos was not sufficient for reprogramming in dog SCNT embryos. </P>
CHOI, Yoo Bin,KIM, Geon A,OH, Hyun Ju,KIM, Min Jung,JO, Young Kwang,SETYAWAN, Erif Maha Nugraha,LEE, Seok Hee,LEE, Byeong Chun The Japanese Society of Veterinary Science 2016 The Journal of veterinary medical science Vol.78 No.2
<P>Somatic cell nuclear transfer is a useful tool to maintain genetic information of animals. The Gyeongju Donggyeong dog is a breed registered as natural monument in Korea. The unique feature of the Donggyeong dog is its tail, as the Donggyeong dog can be classified as either short tailed or tailless. The aim of this study was to preserve the Donggyeong dog’s unique feature by cloning. Fibroblasts were obtained from a short-tailed Donggyeong dog. <I>In vivo</I> matured oocytes were enucleated, microinjected with a donor cell and fused electrically. Reconstructed embryos were transferred to six recipient dogs. One surrogate became pregnant, and one short-tailed Donggyeong dog was delivered. This study demonstrated that the phenotype of the Donggyeong dog could be conserved by somatic cell nuclear transfer.</P>