Effect of Ex Vivo Model of Porcine Uterus on the Fertilization Ability of Porcine Blastocysts Produced In Vitro

Yongquan Han,Sang-Hwan Hyun 한국동물생명공학회(구 한국동물번식학회) 2018 발생공학 국제심포지엄 및 학술대회 Vol.2018 No.06

The success of in vitro embryo production (IVP) demonstrates that it’s possible to bypass the oviduct throughout early development. However, several studies show that embryos developed in vivo are superior to embryos developed in vitro. Using an ex vivo model of porcine uterus is one of the strategies to investigate fertilization within the oviductal environment. During this study, in vitro matured porcine oocytes (MII) were fertilized with 7.5×107, 15×107 and 30×107 sperm cell for 20 min in porcine uterine ex vivo model. The oocytes were then flushed and performed in vitro culture (IVC) at 39°C for 168 hours under 5% O2, 5% CO2. MII oocytes used for in vitro fertilization (IVF) served as control-1. Before IVF, MII oocytes cultured in porcine uterine ex vivo model for 20 min served as control-2. Within the results, penetration rate, MPN formation, monospermy, polyspermy, and efficiency of fertilization had not shown significant difference between control-1 and control-2 group, respectively. However, penetration rate (treatments: 29.7±4.4, 34.3±3.2, 44.3±7.4 vs. 80.0±1.7), polyspermy (treatments: 5.7±5.7, 9.7±5.8, 8.0±4.0 vs. 33.7±9.5) and efficiency of fertilization (treatments: 23.7±2.3, 29.0±3.6, 35.0±4.6 vs. 43.0±5.8) were significantly decreased in treatment groups compared to control-1 (p<0.05). GSH accumulated levels were significantly decreased in 30×107 sperm cell treated group compared to control-1 (p<0.05) and there was no significant difference in ROS accumulated levels among the groups. For embryo development, the cleavage rate and blastocyst rate had not shown significant difference between control-1 and control-2 group. However, the cleavage rate (treatments: 16.3±2.6, 20.1±2.7, 40.7±13.4 vs. 69.5±6.3, 74.2±3.4) was significantly decreased in treatment groups compared to control-1 and control-2 (p<0.05). And the cleavage rate in the treatment group of 30×107 (40.7±13.4) was significantly higher than the treatment group of 7.5×107 (16.3±2.6) (p<0.05). The blastocyst rate (treatments: 31.7±4.0, 25.7±4.0, 26.7±6.5 vs. 7.2±2.4, 9.9±3.0) was significantly increased in control-1, control-2 and the treatment group of 30×107 compared to 7.5×107 and 15×107 (p<0.05). Therefore, these results suggest that ex vivo model may decrease the penetration rate and efficiency of fertilization by reducing GSH accumulated levels. Cleavage rate and blastocyst rate can be promoted by increasing sperm number during ex vivo fertilization.

Evaluation the Fertilization of Porcine In Vitro Matured Oocyte using by Ex Vivo Model

Yongquan Han,Dibyendu Biswas,Sang-Hwan Hyun 한국동물생명공학회(구 한국동물번식학회) 2017 발생공학 국제심포지엄 및 학술대회 Vol.2017 No.10

Porcine uterine ex vivo model is a useful tool for investigating gamete maturation, activation, fertilization and early embryonic development. This study was to evaluate fertilization ability of in vitro matured (IVM) porcine oocyte (MII) using porcine uterine ex vivo model. IVM-MII oocytes were fertilized with 7.5×107, 15×107 and 30×107 sperm for 20min in porcine uterine ex vivo model. And, the oocytes were then flushed and performed in vitro culture (IVC). IVM-MII oocytes used for in vitro fertilization (IVF) served as control 1. Before IVF, IVM-MII oocytes cultured in porcine uterine ex vivo model for 20min served as control 2. In the results, it was found that the sperm penetration rate in the control 2 (73.0±4.2) was significantly higher than the other treatment group: 7.5×107 (29.7±4.4), 15×107 (34.3±3.2) and 30×107 (44.3±7.4). But there was no significant difference between control 1 and control 2. Monospermic penetration rate in all treatment groups was significantly higher than control groups, and polyspermy rate in all treatment group was also significantly lower than control groups. These results suggested that porcine uterine ex vivo model improved monospermic penetration by reducing sperm penetration ratio. In the further study, it is required to evaluate the embryonic development, GSH and ROS accumulation levels of MII oocytes fertilized using porcine uterine ex vivo model.

Effect of porcine uterus as <i>ex vivo</i> model of fertilizing ability and gene expression pattern on blastocysts

Han, Yongquan,Biswas, Dibyendu,Yoon, Junchul David,Jeon, Yubyeol,Hyun, Sang Hwan Elsevier 2019 Theriogenology Vol.129 No.-

<P><B>Abstract</B></P> <P>The success of <I>in vitro</I> embryo production demonstrates that the oviduct can be bypassed during early embryonic development. Using an <I>ex vivo</I> model of porcine uterus is one of the strategies used to investigate fertilization within the oviductal environment. In this study, <I>in vitro</I>-matured porcine oocytes (MII) were fertilized with 7.5 × 10<SUP>7</SUP>, 15 × 10<SUP>7</SUP>, or 30 × 10<SUP>7</SUP> sperm cells for 20 min in the oviduct of a porcine uterine <I>ex vivo</I> model. MII oocytes used for <I>in vitro</I> fertilization (IVF) served as control 1; those cultured in the oviduct of the <I>ex vivo</I> model for 20 min before IVF served as control 2. In present study, the penetration rate, polyspermy, and fertilization efficiency, and accumulated reactive oxygen species (ROS) levels in the treatment groups were significantly decreased compared to those in the control 1 group. During embryonic development, the cleavage rates in the treatment groups were significantly lower than those in the control groups. The cleavage rate in the 30 × 10<SUP>7</SUP> sperm cell-treated group was higher than that in the 7.5 × 10<SUP>7</SUP> sperm cell-treated group. The blastocyst formation rate in control 1 and 2, and 30 × 10<SUP>7</SUP> sperm cell-treated groups increased compared to that in the 7.5 and 15 × 10<SUP>7</SUP> sperm cell-treated groups. <I>PCNA</I>, <I>HSP70.2</I>, and <I>GLUT1</I> were upregulated in the treatment groups and <I>POU5F1</I>, <I>BAX</I>, <I>GPX1</I> were upregulated in the treatment and control 2 groups, compared to the control 1 group. These results suggest that an <I>ex vivo</I> model may decrease the penetration rate and fertilization efficiency by increasing the accumulated ROS levels and inducing the expression of apoptosis- and stress-related genes. However, the model improved the monospermy rate and expression of embryo developmental competence genes. This is the first study that evaluates the effect of an <I>ex vivo</I> model of porcine uterus on fertilization parameters, and the development of porcine embryos.</P> <P><B>Highlights</B></P> <P> <UL> <LI> An <I>ex vivo</I> model may decrease the penetration rate and fertilization efficiency. </LI> <LI> The model improved the monospermy rate and expression of embryo developmental competence genes. </LI> <LI> The <I>ex vivo</I> model increase the accumulated ROS levels and expression of apoptosis- and stress-related genes in embryos. </LI> </UL> </P>

Efficient and Secure Identity-Based Public Auditing for Dynamic Outsourced Data with Proxy

( Haiyang Yu ),( Yongquan Cai ),( Shanshan Kong ),( Zhenhu Ning ),( Fei Xue ),( Han Zhong ) 한국인터넷정보학회 2017 KSII Transactions on Internet and Information Syst Vol.11 No.10

Cloud storage becomes a new trend that more and more users move their data to cloud storage servers (CSSs). To ensure the security of cloud storage, many cloud auditing schemes are proposed to check the integrity of users’ cloud data. However, most of them are based on public key infrastructure, which leads to complex certificates management and verification. Besides, most existing auditing schemes are inefficient when user uploads a large amount of data or a third party auditor (TPA) performs auditing for multiple users’ data on different CSSs. To overcome these problems, in this paper, we propose an efficient and secure auditing scheme based on identity-based cryptography. To relieve user’s computation burden, we introduce a proxy, which is delegated to generate and upload homomorphic verifiable tags for user. We extend our auditing scheme to support auditing for dynamic data operations. We further extend it to support batch auditing in multiple users and multiple CSSs setting, which is practical and efficient in large scale cloud storage system. Extensive security analysis shows that our scheme is provably secure in random oracle model. Performance analysis demonstrates that our scheme is highly efficient, especially reducing the computation cost of proxy and TPA.

The use of pituitary adenylate cyclase-activating polypeptide in the pre-maturation system improves in vitro developmental competence from small follicles of porcine oocytes

박규미,김규준,Minghui Jin,Yongquan Han,소경하,현상환 아세아·태평양축산학회 2019 Animal Bioscience Vol.32 No.12

Objective: We investigated how pituitary adenylate cyclase-activating polypeptide (PACAP) affects embryonic development during pre-in vitro maturation (pre-IVM) using porcine oocytes isolated from small follicles. Methods: We divided the follicles into the experimental groups by size (SF, small follicles; MF, medium follicles) and treated with and without PACAP and cultured for 18 hours (Pre-SF[-]PACAP; without PACAP, Pre-SF[+]PACAP; with PACAP) before undergoing IVM. The gene expression related to extracellular matrix formation (amphiregulin, epiregulin, and hyaluronan synthase 2 [HAS2]) and apoptosis (Bcl-2-associated X [BAX], B-cell lymphoma 2, and cysteine-aspartic acid protease 3) was investigated after maturation. The impact on developmental competence was assessed by the cleavage and blastocyst rate and total cell number of blastocysts in embryos generated from parthenogenesis (PA) and in vitro fertilization (IVF). Results: Cleavage rates in the Pre-SF(+)PACAP after PA were significantly higher than SF and Pre-SF(-)PACAP (p<0.05). The cleavage rates between MF and Pre- SF(+)PACAP groups yielded no notable differences after IVF. Pre-SF(+)PACAP displayed the higher rate of blastocyst formation and greater total cell number than SF and Pre-SF(-)PACAP (p<0.05). Cumulus cells showed significant upregulation of HAS2 mRNA in the Pre-SF(+)PACAP compared to the SF (p<0.05). In comparison to other groups, the Pre-SF(+)PACAP group displayed a downregulation in mRNA expression of BAX in matured oocytes (p<0.05). Conclusion: The PACAP treatment during pre-IVM improved the developmental potential of porcine oocytes derived from SF by regulating cumulus expansion and apoptosis of oocytes.

Lysophosphatidic acid increases <i>in vitro</i> maturation efficiency via uPA-uPAR signaling pathway in cumulus cells

Hwang, Seon-Ung,Kim, Kyu-Jun,Kim, Eunhye,Yoon, Junchul David,Park, Kyu Mi,Jin, Minghui,Han, Yongquan,Kim, Mirae,Lee, Gabsang,Hyun, Sang-Hwan Elsevier 2018 Theriogenology Vol.113 No.-

<P><B>Abstract</B></P> <P>Lysophosphatidic acid (LPA) is a phospholipid-derived signaling molecule with biological activities, such as stimulating cell proliferation, differentiation and migration. In the present study, we examined the effect of LPA on porcine oocytes during <I>in vitro</I> maturation (IVM) and subsequent embryonic development following parthenogenetic activation (PA) and <I>in vitro</I> fertilization (IVF). During IVM, the maturation medium was supplemented with various concentrations of LPA (0, 10, 30, and 60 μM). After 42 h of IVM, the 30 μM LPA-treated group showed a significant (<I>P</I> <<I>0.05</I>) increase in nuclear maturation and intracellular glutathione (GSH) levels compared with the other groups. The 30 μM LPA-treated group exhibited a significant decrease in intracellular reactive oxygen species (ROS) levels compared with the other groups. In PA, the 30 μM LPA-treated group had significantly higher cleavage (CL) and blastocyst (BL) rates compared with those of the other LPA-treated groups. In IVF, the 30 μM LPA-treated group had significantly higher CL and BL rates than the other LPA-treated groups. The expression of the developmental competence gene (proliferating cell nuclear antigen<I>, PCNA</I>) in the oocytes and cumulus cells of the individuals in the 30 μM LPA-treated group was significantly increased compared with the control group. In addition, the specific expression of urokinase Plasminogen Activator (uPA) and uPA Receptor (<I>uPAR</I>) in cumulus cells was significantly increased in the 30 μM LPA-treated group. The western blotting results revealed that LPA improves the activities of p38 mitogen-activated protein kinase (MAPK) and epidermal growth factor (EGF) by enhanced phosphorylation. In conclusion, treatment with 30 μM LPA during IVM promotes enhances the EGF-EGFR signaling pathway, resulting in cumulus cell expansion. And then, this treatment improves the developmental potential of PA and IVF porcine embryos by enhancing nuclear and cytoplasmic maturation and reducing ROS.</P>