Spermine reduces reactive oxygen species levels and decreases cryocapacitation in canine sperm cryopreservation

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>

Despite the donor's age, human adipose-derived stem cells enhance the maturation and development rates of porcine oocytes in a co-culture system

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>

Clinical assessment after human adipose stem cell transplantation into dogs

이석희,Erif M.N. Setyawan,Yoo-Bin Choi,Jeong Chan Ra,Sung Keun Kang,Byeong Chun Lee,김지은 대한수의학회 2018 Journal of Veterinary Science Vol.19 No.3

Adipose tissue-derived stem cell (ASCs) are an attractive source of stem cells with therapeutic applicability in various fields for regenerating damaged tissues because of their stemness characteristics. However, little has reported on evaluating adverse responses caused by human ASC therapy. Therefore, in the present study, a clinical assessment after human ASC transplantation into dogs was undertaken. A total of 12 healthy male dogs were selected and divided into four groups: saline infusion, saline bolus, ASC infusion, and ASC bolus groups. Physical assessment and blood analysis were performed following ASC transplantation, and the concentrations of angiogenic factors, and pro- and anti-inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA). There were no adverse vital sign responses among the dogs. Blood analyses revealed no remarkable complete blood count or serum chemistry results. ELISA results for angiogenic and anti-inflammatory factors including matrix metalloproteinase 9 (MMP9), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and interleukin-10 (IL-10) were significantly higher in the two ASCs groups than in the controls. In conclusion, this study demonstrated that transplantation of human ASCs produced no adverse effects and could be used safely in dogs. In addition, human ASCs could be involved in modulating secretions of angiogenic factors including MMP9, VEGF, bFGF, and HGF and anti-inflammatory factor IL-10.

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.

Potential of polylactic-co-glycolic acid (PLGA) for delivery Jembrana disease DNA vaccine Model (pEGFP-C1-tat)

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.

Iodixanol supplementation during sperm cryopreservation improves protamine level and reduces reactive oxygen species of canine sperm

Abdillah, Dimas A.,Setyawan, Erif M. N.,Oh, Hyun Ju,Ra, Kihae,Lee, Seok Hee,Kim, Min Jung,Lee, Byeong Chun The Korean Society of Veterinary Science 2019 Journal of Veterinary Science Vol.20 No.1

<P>The objective of this study was to analyze the protective effects of iodixanol on dog spermatozoa during cryopreservation. The optimal concentration of iodixanol, 1.5%, was determined using fresh spermatozoa and was applied in the following experiments. The 1.5% iodixanol group showed significantly increased sperm motility from that in the control (<I>p</I> < 0.05). Lower mitochondrial reactive oxygen species (ROS) modulator (<I>ROMO1</I>) and pro-apoptotic gene (<I>BAX</I>) expressions, together with higher expressions of protamine-2 (<I>PRM2</I>), protamine-3 (<I>PRM3</I>), anti-apoptotic B-cell lymphoma-2 (<I>BCL2</I>), and sperm acrosome associated-3 (<I>SPACA3</I>) genes were detected in the iodixanol-treated group. In addition, decreased protamine deficiency and cryocapacitation were observed in the treatment group. Our results show that supplementation with 1.5% iodixanol is ideal for reducing production of ROS and preventing detrimental effects during the canine sperm cryopreservation process, effects manifested as increased motility and reduced cryocapacitation in frozen-thawed spermatozoa.</P>

Determination of gold and palladium in environmental samples by FAAS after dispersive liquid–liquid microextraction pretreatment

Cos¸ kun O¨ zdemir,S¸ erife Sac¸macı,SenolKartal,Mustafa Sac¸macı 한국공업화학회 2014 Journal of Industrial and Engineering Chemistry Vol.20 No.6

A new dispersive liquid–liquid microextraction (DLLME) method is proposed for rapid separation,simultaneous extraction and preconcentration of gold and palladium at ultra trace amounts. Theextraction of the analytes was performed in the presence of 5-[(E)-(2,6-diaminopyridine-3-yl)diazenyl]-1,3,4-thiadiazole-2-thiol (DAT) as chelating agent. Chloroform and acetone were used as extraction anddispersive solvents, respectively. The variables affecting the complexation and extraction conditionswere optimized. The calibration curves were linear in the range of 1.1–85 and 0.9–124 mg L-1 with thedetection limits of 0.4 and 0.6 mg L-1, and with the enrichment factors of 94 and 113 for Au and Pd,respectively. The precision (RSD%) was better than 2.4%. The accuracy of the method was verified byanalysing the certified standard reference material (CDN–PGMS-10). The results show that thedispersive liquid–liquid microextraction pretreatment is a sensitive, rapid, simple and safe method forthe separation/preconcentration of gold and palladium.

A simple dispersive liquid–liquid microextraction method for determination of Ag(I) by flame atomic absorption spectrometry

Teslima Das¸ bas¸ ı,S¸ erife Sac¸macı,Ahmet U¨ lgen,SenolKartal 한국공업화학회 2015 Journal of Industrial and Engineering Chemistry Vol.28 No.-

A simple and efficient dispersive liquid–liquid microextraction (DLLME) procedure was optimized forthe determination of Ag(I) by flame atomic absorption spectrometry (FAAS). In this method, 8-hydroxyquinoline was used as a chelating reagent for Ag(I) and resulted complex was extracted inchloroform, while ethanol is used as disperser solvent. The effective parameters on the recovery of Ag(I),such as the pH of sample, the type and volume of extraction and disperser solvents, the amount of 8-hydroxyquinoline reagent, extraction time, the effect of temperature and interfering ions of samplesolution were investigated. The calibration graph of this procedure was linear in the range of 0.02–0.4 mg L 1 with detection limit of 2.0 mg L 1 (n = 21). The relative standard deviation (RSD) was 4% andthe preconcentration factor was 65. Good recovery (%) values were obtained changing between 95 and102%. The certified reference material (CWW-TM-D waste water) was used to verify the accuracy of thedeveloped method (recovery, 96%). The proposed method was applied for determination of silver indifferent water samples, anode slime and cream samples.

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.

Ten years progress from “Snuppy”

Hyun Ju Oh,Min Jung Kim,Geon A Kim,Young Kwang Jo,Erif M. N. Setyawan,Seok Hee Lee,Jin Choi,Hye jin Kim,Fibrianto Yuda,Sung Keun Kang,Jung Chan Ra,So Gun Hong,Jung Eun Park,Ok Jae Koo,Min Kyu Kim,Cheo 대한수의학회 2015 대한수의학회 학술대회발표집 Vol.2015 No.-