Recombinant Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 Decrease Kit Ligand Gene Expression in Canine Ovarian Granulosa Cells

Lili Zhuang,Bo Myeong Lee,Junbo Wu,Eun Do Lee,Chi Sun Yun,Kuk Bin Ji,Ji Hye Lee,Ju Lan Chun,Min Kyu Kim 한국동물생명공학회(구 한국동물번식학회) 2016 발생공학 국제심포지엄 및 학술대회 Vol.2016 No.10

Recombinant Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 Decrease Kit Ligand Gene Expression in Canine Ovarian Granulosa Cells

Lili Zhuang,Bo Myeong Lee,Junbo Wu,Eun Do Lee,Chi Sun Yun,Kuk Bin Ji,Ji Hye Lee,Ju Lan Chun,Min Kyu Kim 한국수정란이식학회 2016 한국수정란이식학회 학술대회 Vol.2016 No.10

Prolonged communication between oocytes and the surrounding somatic cells is one of the unique reproductive physiology in canine. Paracrine Kit ligand (KITL) signaling is a well-known communication between granulosa cells and the oocyte. KITL is a cytokine growth factor secreted by granulosa cells that signals via the c-kit receptor expressed by oocytes. Paracrine factors, including growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), exert their effects by binding with the kinase receptors expressed on the granulosa cells. However, the regulations of GDF9 and BMP15 in the canine KITL expression are currently poorly understood. Therefore, we investigated the effects of GDF9 and BMP15 on the expression of KITL in canine ovarian granulosa cells in vitro. In Annexin V assay recombinant GDF9 and BMP15 did not induce apoptosis in the cultured ovarian granulosa cells. When treated, FSH significantly increased KITL expression, and hCG suppressed its expression. When both FSH and hCG were treated, the expression of KITL was affected by GDF9 and BMP15 in dose and time dependent manner in the luteal granulosa cells. GDF9 (10 ng/mL) significantly decreased KITL expression after12 h. BMP15 (10 ng/mL) significantly also decreased KITL expression after 24 h. Western blot and immunochemistry results indicate that GDF9 activated Smad2/3. After blocking ALK 4/5/7 receptors by SB, GDF9 failed to activate Smad2/3, also BMP15 did not activate Smad1/5/8 after blocking ALK 2/3/6 receptors by DM. So GDF9 exerts its effects via using ALK 4/5/7 receptors to activate SMAD2/3 signaling, and BMP15 binds ALK 2/3/6 receptors to activate SMAD1/5/8 signaling. The expression of KITL was not changed by SB or DM treatment. However, the effect of GDF9 and BMP15, which decreased the expression of KITL, was suppressed by SB or DM treatment. In conclusion, this study provides the first evidence that recombinant GDF9 and BMP15 decrease KITL expression in canine ovarian granulosa cells.

Regulatory Roles of Oocyte Secreted Factors in Canine Cumulus-oocyte Complexes and Granulosa Cells

Lili Zhuang,Dong Eon Kim,Chi Sun Yun,Kuk bin Ji,Eun Ji Lee,Gyeong Yeob Kim,Ji Hye Lee,Min Kyu Kim 한국수정란이식학회 2017 한국수정란이식학회 학술대회 Vol.2017 No.05

Oocyte is the central factor in the bi-directional communication axis in the ovarian follicles. It controls the cumulus or granulosa cells to perform functions which are beneficial for its own development via secreting paracrine growth factors, including GDF9 and BMP15. The aim of this study was to investigate whether the recombinant GDF9 and BMP15 are able to promote meiotic resumption and cumulus expansion of canine COCs during IVM, as well as to demonstrate the actions of GDF9 and BMP15 in regulating the expression of connexin transcripts in the ovarian granulosa cells. As results, GDF9 and BMP15 significantly improved the meiotic resumption rate and cumulus expansion by activating ERK1/2 signaling. Treatments with GDF9 significantly improved the expression of CyclinB1 but inhibited the expression of Cx43 transcripts. In addition, cumulus expansion genes (MAPK1, Ptgs2, Tnfaip6 and Ptx3) were differentially improved by GDF9 and BMP15. In the ovarian granulosa cells, GDF9 suppressed the expression of Cx43 transcripts by binding ALK4/5/7 receptors and activation Smad2/3 signaling, whereas, BMP15 stimulated the expression of Cx43 transcripts by binding ALK2/3/6 receptors and activating Smad1/5/8 signaling. In conclusion, by regulating functions of granulosa/cumulus cells, oocyte has the potential to enhance the growth and maturation of itself.

Elucidating Adsorption Mechanisms of Phthalate Esters upon Carbon Nanotubes/Graphene and Natural Organic Acid Competitive Effects in Water by DFT and MD Calculations

Zhuang Wang,Se Wang,MINDONG CHEN,Defu Xu,Lili Tang,Degao Wang 대한화학회 2015 Bulletin of the Korean Chemical Society Vol.36 No.6

Simulations at multiple levels were performed to investigate the aqueous adsorption of phthalate esters (PAEs) on carbon nanoparticles and to find the competitive effect of a low molecular weight natural organic acid (benzoic acid) on the adsorption process. Six PAEs of varying alkyl side chain lengths and three carbon-based nanomaterials including a single-walled carbon nanotube (SWNT), double-walled carbon nanotube (DWNT), and graphene (G) were studied. Results showed that the adsorption energies calculated using density functional theory increase with increasing length of the PAE alkyl chain. G exhibits higher adsorption capacity for the PAEs than SWNT and DWNT. The absolute adsorption energies of these systems also display a positive linear correlation with the hydrophobicity of the PAE molecules. Molecular dynamics simulations indicate that the presence of neutral/anionic benzoic acid in water alleviates the PAE adsorption. Furthermore, anionic benzoic acid exerts more impact on the PAE adsorption than the neutral form.

Impacts of C60-Ionic Liquids (ILs) Interactions and IL Alkyl Chain Length on C60 Dispersion Behavior: Insights at the Molecular Level

Zhuang Wang,Lili Tang,Degao Wang 대한화학회 2014 Bulletin of the Korean Chemical Society Vol.35 No.9

Mechanisms underlying the impacts of interactions between carbon nanoparticles (CNPs) and ionic liquids (ILs) on the physicochemical behavior of CNPs need to be more full worked out. This manuscript describes a theoretical investigation at multiple levels on the interactions of fullerene C60 with 21 imidazolium-based ILs of varying alkyl side chain lengths and anionic types and their impacts on C60 dispersion behavior. Results show that π-cation interaction contributed to mechanism of the C60-IL interaction more than π-anion interaction. The calculated interaction energy (EINT) indicates that C60 can form stable complex with each IL molecule. Moreover, the direction of charge transfer occurred from IL to C60 during the C60-IL interaction. Quantitative models were developed to evaluate the self-diffusion coefficient of C60 (Dfullerene) in bulk ILs. Three interpretative molecular descriptors (heat of formation, EINT, and charge) that describe the C60-IL interactions and the alkyl side chain length were found to be determinants affecting Dfullerene.

Bioprospecting of Novel and Bioactive Metabolites from Endophytic Fungi Isolated from Rubber Tree Ficus elastica Leaves

( Zhuang Ding ),( Tao Tao ),( Lili Wang ),( Yanna Zhao ),( Huiming Huang ),( Demeng Zhang ),( Min Liu ),( Zhengping Wang ),( Jun Han ) 한국미생물생명공학회(구 한국산업미생물학회) 2019 Journal of microbiology and biotechnology Vol.29 No.5

Endophytic fungi are an important component of plant microbiota, and have the excellent capacity for producing a broad variety of bioactive metabolites. These bioactive metabolites not only affect the survival of the host plant, but also provide valuable lead compounds for novel drug discovery. In this study, forty-two endophytic filamentous fungi were isolated from Ficus elastica leaves, and further identified as seven individual taxa by ITS-rDNA sequencing. The antimicrobial activity of these endophytic fungi was evaluated against five pathogenic microorganisms. Two strains, Fes1711 (Penicillium funiculosum) and Fes1712 (Trichoderma harzianum), displayed broad-spectrum bioactivities. Our following study emphasizes the isolation, identification and bioactivity testing of chemical metabolites produced by T. harzianum Fes1712. Two new isocoumarin derivatives (1 and 2), together with three known compounds (3-5) were isolated, and their structures were elucidated using NMR and MS. Compounds 1 and 2 exhibited inhibitory activity against Escherichia coli. Our findings reveal that endophytic fungi from the rubber tree F. elastica leaves exhibit unique characteristics and are potential producers of novel natural bioactive products.

Impacts of C₆₀-Ionic Liquids (ILs) Interactions and IL Alkyl Chain Length on C₆₀ Dispersion Behavior: Insights at the Molecular Level

Wang, Zhuang,Tang, Lili,Wang, Degao Korean Chemical Society 2014 Bulletin of the Korean Chemical Society Vol.35 No.9

Mechanisms underlying the impacts of interactions between carbon nanoparticles (CNPs) and ionic liquids (ILs) on the physicochemical behavior of CNPs need to be more full worked out. This manuscript describes a theoretical investigation at multiple levels on the interactions of fullerene $C_{60}$ with 21 imidazolium-based ILs of varying alkyl side chain lengths and anionic types and their impacts on $C_{60}$ dispersion behavior. Results show that ${\pi}$-cation interaction contributed to mechanism of the $C_{60}$-IL interaction more than ${\pi}$-anion interaction. The calculated interaction energy ($E_{INT}$) indicates that $C_{60}$ can form stable complex with each IL molecule. Moreover, the direction of charge transfer occurred from IL to $C_{60}$ during the $C_{60}$-IL interaction. Quantitative models were developed to evaluate the self-diffusion coefficient of $C_{60}$ ($D_{fullerene}$) in bulk ILs. Three interpretative molecular descriptors (heat of formation, $E_{INT}$, and charge) that describe the $C_{60}$-IL interactions and the alkyl side chain length were found to be determinants affecting $D_{fullerene}$.

Inhibition of deubiquitinases alters gamete ubiquitination states and sperm-oocyte binding ability in pigs

Wang, Yang,Zhuang, Lili,Chen, Xuan,Xu, Man,Li, Zuochen,Jin, Yi Elsevier 2017 Animal reproduction science Vol.187 No.-

<P><B>Abstract</B></P> <P>This study was undertaken to investigate the dynamics of protein ubiquitination in pig gametes and their micro-environments, as well as to explore the action of deubiquitinases (DUBs) in sperm-oocyte binding. Protein ubiquitination states were evaluated by in the ejaculated sperm, seminal plasma, epididymal sperm, oocytes, zona pellucida (ZP) and follicular fluid (FF) by western blotting. Different concentrations of PR-619, a non-selective inhibitor of DUBs, were used to treat oocytes during <I>in vitro</I> maturation (IVM), the maturation rate, amount of ubiquitinated ZP proteins, and ZP solubility were assessed. The PR-619 was also used to treat sperms during capacitation, then the ubiquitinated amounts of acrosin inhibitor (AI) proteins were evaluated. The number of sperm attached to the ZP of each oocyte was subsequently determined after gamete co-incubation. The study indicates the existence of ubiquitinated proteins (76kDa) in sperm, seminal plasma, oocytes, and follicular fluid (FF). The amount of ubiquitinated ZP proteins changed as growth of follicles progressed. Treatment with PR-619 at 10 and 15μM concentrations during IVM reduced the maturation rate of pig oocytes (<I>P</I> <0.05), while treatments with 10μM of PR-619 extended the ZP dissolution time (<I>P </I><0.05). Treatment with PR-619 enhanced AI ubiquitination and improved amounts of 30-kDa ubiquitinated proteins (<I>P </I><0.05). Treatment with PR-619 at the 10μM dose effectively reduced the number of sperm attached to per oocyte (<I>P<</I> 0.05). Ubiquitinated proteins were present in gametes and their micro-environments. The DUBs were important in regulating pig gamete ubiquitination and sperm-oocyte binding.</P>

Investigation of the DJ-1 Function in the Higher Mammals

Eun Young Kim,Lili Zhuang,Dong Ern Kim,Chi Sun Yun,Eun Ji Lee,Keun Jung Kim,Jae am Lim,Jun Jong Baek,Seung Bum Lim,Min Kyu Kim 한국동물생명공학회(구 한국동물번식학회) 2017 Reproductive & Developmental Biology(Supplement) Vol.41 No.2

The DJ-1 was found to be the first case that an oncogene also play a role for causative gene of neurodegenerative diseases. The DJ-1 is a multi-functional protein that play important role in tissue and participates in a number of intracellular signaling pathways. Especially, DJ-1 protein has a cellular protective role and promoted cell survival under an oxidative stress. However, the cellular protective mechanism of DJ-1 is not fully understand, and we needs to be further study their functions in canine. In the present study, we investigated the protective role of DJ-1 against induced oxidative stress, cell viability, cell proliferation assay, cellular apoptosis detection analysis, intracellular ROS were measured appropriately. The results showed that DJ-1 overexpressed cells were up-regulated cell viability under oxidative stress conditions, whereas loss of DJ-1 cells were down-regulated the cell survival activity. Additionally, overexpression of DJ-1 cells increased cell resistance to oxidative stress and inhibited the elevation of cell death and cellular ROS induced apoptosis. Moreover, DJ-1 overexpressed cells was increased mitochondrial functions by using confocal microscopy with MitoTracker staining. On the contrary to this, DJ-1 null cells show defective cellular protection and mitochondria activity against oxidative stress conditions. The data indicate that canine DJ-1 protein enhances the cellular survival activity and promote cell survival activity under the oxidative stress by attenuates cellular apoptosis and ROS generation.

L-carnitine Enhances Bovine Oocyte Quality and the Developmental Competence of Somatic Cell Nuclear Transfer Embryos

Junbo Wu,Kang Sun Park,Lili Zhuang,Dong Eon Kim,Eun Do Lee,Chi Sun Yun,Eun Ji Lee,Beom Sik Kim,Min Kyu Kim 한국수정란이식학회 2017 한국수정란이식학회 학술대회 Vol.2017 No.05

Somatic cell nuclear transfer (SCNT) is the technique which generates embryos by transferring diploid nucleus into an enucleated oocyte, it has produced specific animals successfully in a variety of species. However, the developmental capacity of SCNT embryos is still relatively lower than that of embryos produced in vivo. Oocyte is a kind of lipid rich cells, its quality limits the efficiency of embryo production. L-carnitine is a co-enzyme facilitating the transportation of long chain fatty acids across the inner mitochondria membrane where fatty acids are used for generating adenosine triphosphate (ATP) via beta-oxidation. It also has antioxidant actions which may protect mitochondrial membranes and DNA against damage induced by reactive oxygen species (ROS). Whether L-carnitine is functional in bovine SCNT embryos are unknown. Therefore, the objective of this study was to examine the effects of L-carnitine on oocyte maturation and developmental competence of subsequent SCNT embryos. L-carnitine was supplemented during IVM, then intracellular ROS and GSH levels, mitochondrial activity, gene expression of COCs were analyzed at the end of IVM. SCNT embryos were produced subsequently, apoptosis detection and gene expression evaluation were performed in blastocysts. In the results, treatments with 1.5 mM and 3 mM L-carnitine significantly improved maturation rates (P<0.05). Treatments with 3 mM L-carnitine effectively induced improvement in nuclear maturation, intracellular GSH levels and mitochondrial activity, as well as a reduction in intracellular ROS levels (P<0.05). mRNA levels of CPT1A, ACAA1, ACAA2, AREG, EREG, SOD1, GPX4, GLUT1 and CDC2 transcripts were effectively up-regulated by 3 mM L-carnitine treatments (P < 0.05). Similarly, 3mM L-carnitine induced an increase in blastocyst developmental rates and an improvement in blastocyst quality (P<0.05). Our study indicates that L-carnitine treatment during IVM improves oocyte nuclear maturation and subsequent SCNT embryo development.