Production of Transgenic Male Piglet as Research Model for Alzheimer's Disease

Mi-Ryung Park,Kyong-Woon Kim,Jae-Seok Woo,Seongsoo Hwang,In-Sul Hwang1,Tae-Uk Kwak,Ji-Hyun Lim,Se-Pil Park 한국동물생명공학회(구 한국동물번식학회) 2017 발생공학 국제심포지엄 및 학술대회 Vol.2017 No.10

Alzheimer’s disease (AD) is a progressive neurodegenerative disease associated with memory loss and cognitive impairments. An AD transgenic (Tg) pig model would be useful for preclinical testing of therapeutic agents. In this study, we report the use of somatic cell nuclear transfer (SCNT) to produce transgenic pigs over-expressing the human AD related genes (APP, APPswedish, Presenilin 1, and Tau). Transgenic embryos were generated by SCNT of from ear fibroblasts expressing AD genes. A total of 1808 (average 258) SCNT embryos were transferred into 7 recipients. Pregnancy was successfully maintained in one recipient. We obtained 1 cloned male piglet from a surrogate gilts and the weight of piglet was 935 g. But, the male piglet died two days after birth. The piglet expressed AD related genes by PCR and western blot analysis. Transgenes were expressed in multiple tissues, and at especially high levels in brain. AD Tg pig might be very useful for studying the disease and for testing new therapeutics in preclinical studies of human AD.

Effect of a short-term in vitro exposure time on the production of in vitro produced piglets

In-Sul Hwang,Dae-Jin Kwon,Tae-Uk Kwak,Joo-Young Lee,Nam-Woong Hyung,Hyeon Yang,Keon Bong Oh,Sun-A Ock,Eung-Woo Park,Gi-Sun Im,Seongsoo Hwang 한국수정란이식학회 2016 한국동물생명공학회지 Vol.31 No.2

Although piglets have been delivered by embryo transfer (ET) with in vitro produced (IVP) embryos and blastocysts, a success rate has still remained lower level. Unlike mouse, human, and bovine, it is difficult to a production of piglets by in vitro fertilization (IVF) because of an inappropriate in vitro culture (IVC) system in pig. Therefore, the present study was conducted to investigate whether minimized exposure time in IVC can improve the pregnancy and delivery rates of piglets. Immediately after IVM, the oocytes were denuded and co-incubated with freshly ejaculated boar semen for 3.5 to 4 hours at 38.5 ˚C under 5% CO2 in air. To avoid long-term exposure to in vitro state, we emitted IVC step after IVF. After that the presumptive zygotes were transferred into both oviducts of the surrogate on the same day or 1 day after the onset of estrus. Pregnancy was diagnosed on day 28 after ET and then was checked regularly every month by ultrasound examination. The 3 out of 4 surrogates were determined as pregnant (75%) and a total of 5 piglets (2 females and 3 males) were delivered at 118.3 ± 2.5 days of pregnancy period. In conclusion, a short-term exposure time may be an important factor in the production of IVP-derived piglets. It can be apply to the in vitro production system of transgenic pig by IVF, cloning, and pronuclear microinjection methods.

Fertility preservation in pig using ovarian tissues by vitrification method

In-Sul Hwang 한국동물생명공학회(구 한국동물번식학회) 2022 Journal of Animal Reproduction and Biotechnology Vol.37 No.2

Cryopreservation of porcine ovarian tissue by vitrification method is a promising approach to preserve genetic materials for future use. However, information is not enough and technology still remains in a challenge stage in pig. Therefore, the objective of present study was to determine possibility of vitrification method to cryopreserve porcine ovarian tissue and to confirm an occurrence of cryoinjuries. Briefly, cryoinjuries and apoptosis patterns in vitrified-warmed ovarian tissue were examined by histological evaluation and TUNEL assay respectively. In results, a damaged morphology of oocytes was detected among groups and the rate was significantly (p < 0.05) lower in vitrification group (25.8%) than freezing control group (67.7%), while fresh control group (6.6%) showed significantly (p < 0.05) lower than both groups. In addition, cryoinjury that form a wave pattern of tissues around follicles was found in the frozen control group, but not in the fresh control group as well as in the vitrification group. Apoptotic cells in follicle was observed only in freezing control group while no apoptotic cell was found in both fresh control and vitrification. Similarly, apoptotic patterns of tissues not in follicle were comparable between fresh control and vitrification groups while freezing control group showed increased tendency. Conclusively, it was confirmed that vitrification method has a prevention effect against cryoinjury and this method could be an alternative approach for cryopreservation of genetic material in pigs. Further study is needed to examine the viability of oocytes derived from vitrified-warmed ovarian tissue.

Effect of a short-term in vitro exposure time on the production of in vitro produced piglets

Hwang, In-Sul,Kwon, Dae-Jin,Kwak, Tae-Uk,Lee, Joo-Young,Hyung, Nam-Woong,Yang, Hyeon,Oh, Keon Bong,Ock, Sun-A,Park, Eung-Woo,Im, Gi-Sun,Hwang, Seongsoo The Korean Society of Embryo Transfer 2016 한국동물생명공학회지 Vol.31 No.2

Although piglets have been delivered by embryo transfer (ET) with in vitro produced (IVP) embryos and blastocysts, a success rate has still remained lower level. Unlike mouse, human, and bovine, it is difficult to a production of piglets by in vitro fertilization (IVF) because of an inappropriate in vitro culture (IVC) system in pig. Therefore, the present study was conducted to investigate whether minimized exposure time in IVC can improve the pregnancy and delivery rates of piglets. Immediately after IVM, the oocytes were denuded and co-incubated with freshly ejaculated boar semen for 3.5 to 4 hours at $38.5^{\circ}C$ under 5% $CO_2$ in air. To avoid long-term exposure to in vitro state, we emitted IVC step after IVF. After that the presumptive zygotes were transferred into both oviducts of the surrogate on the same day or 1 day after the onset of estrus. Pregnancy was diagnosed on day 28 after ET and then was checked regularly every month by ultrasound examination. The 3 out of 4 surrogates were determined as pregnant (75%) and a total of 5 piglets (2 females and 3 males) were delivered at $118.3{\pm}2.5$ days of pregnancy period. In conclusion, a short-term exposure time may be an important factor in the production of IVP-derived piglets. It can be apply to the in vitro production system of transgenic pig by IVF, cloning, and pronuclear microinjection methods.

Effect of Cytochalasin B Treatment on the improvement of Survival Rate in Vitrified Pig Oocytes

In-Sul Hwang,Mi-Ryung Park,Tae-Uk Kwak,Sang-Hyun Park,Ji-Hyun Lim,Seongsoo Hwang,Sung Woo Kim 한국수정란이식학회 2018 한국수정란이식학회 학술대회 Vol.2018 No.11

To improve survival rates of vitrified pig oocytes, the treatment of cytoskeletal stabilizer on an appropriate time is one of the possible approaches. However, the exact treatment timing and effect of cytoskeletal stabilizer such as cytochalasin B (CB) is not well known during oocyte vitrification procedures. Thus, the present study was conducted to determine optimal treatment timing of CB during vitrification and warming procedures. In experiment 1, the survival rates of the post-warming pig oocytes were analyzed by fluorescein diacetate (FDA) assay with 4 classifications. In results, post-warming oocytes showed significantly (p<0.05) decreased number of alive oocytes (31.8% vs. 86.4%) compared to fresh control. In detail, the significant difference (p<0.05) was found only in strong fluorescence (18.2% vs. 70.5%) not in intermediate fluorescence groups (13.6% vs. 15.9%). In experiment 2, CB was treated before (CB-Vitri) and after (Vitri-CB) vitrification. In results, group of Vitri-CB showed significantly (p<0.05) higher (91.6%) survival rates compared to group of CB-Vitri (83.7%), significantly (p<0.05) and comparable with group of Vitri Control (88.7%) by morphological inspection. In FDA assay results, group of Vitri-CB showed significantly (p<0.05) higher (44.2%) survival rates compared to groups of CB-Vitri (36.7%) and Vitri Control (35.1%). In conclusion, the increased survival rates of post-warming pig oocyte treated with Vitri-CB method are firstly described here. The main finding of present study is that the CB treatment during recovery could be helpful to refresh the post-warming pig oocyte resulting its improved survival rates.

Effect of Cytochalasin B Treatment on the Improvement of Survival Rate in Vitrified Pig Oocyte

Hwang, In-Sul,Park, Mi-Ryung,Kwak, Tae-Uk,Park, Sang-Hyun,Lim, Ji-Hyun,Kim, Sung Woo,Hwang, Seongsoo The Korean Society of Developmental Biology 2018 발생과 생식 Vol.22 No.3

To improve survival rates of vitrified pig oocytes, the treatment of cytoskeletal stabilizer on an appropriate time is one of the possible approaches. However, the exact treatment timing and effect of cytoskeletal stabilizer such as cytochalasin B (CB) is not well known during oocyte vitrification procedures. Thus, the present study was conducted to determine optimal treatment timing of CB during vitrification and warming procedures. In experiment 1, the survival rates of the postwarming pig oocytes were analyzed by fluorescein diacetate (FDA) assays with 4 classifications. In results, post-warming oocytes showed significantly (p<0.05) decreased number of alive oocytes (31.8% vs. 86.4%) compared to fresh control. In detail, the significant difference (p<0.05) was found only in strong fluorescence (18.2% vs. 70.5%) not in intermediate fluorescence groups (13.6% vs. 15.9%). In experiment 2, CB was treated before (CB-Vitri) and after (Vitri-CB) vitrification. In results, group of Vitri-CB showed significantly (p<0.05) higher (91.6%) survival rates compared to group of CB-Vitri (83.7%), significantly (p<0.05) and comparable with group of Vitri Control (88.7%) by morphological inspection. In FDA assay results, group of Vitri-CB showed significantly (p<0.05) higher (44.2%) survival rates compared to groups of CB-Vitri (36.7%) and Vitri Control (35.1%). In conclusion, the increased survival rates of post-warming pig oocyte treated with Vitri-CB method are firstly described here. The main finding of present study is that the CB treatment during recovery could be helpful to refresh the post-warming pig oocyte resulting its improved survival rates.

Rescue of vitrified-warmed bovine oocytes with rho-associated coiled-coil kinase inhibitor.

Hwang, In-Sul,Hara, Hiromasa,Chung, Hak-Jae,Hirabayashi, Masumi,Hochi, Shinichi Society for the Study of Reproduction [etc.] 2013 BIOLOGY OF REPRODUCTION Vol.89 No.2

<P>Cryotolerance of matured bovine oocytes is not fully practical even though a promising vitrification procedure with a ultrarapid cooling rate was applied. The present study was conducted to investigate whether recovery culture of vitrified-warmed bovine oocytes with an inhibitor (Y-27632) of Rho-associated coiled-coil kinase (ROCK) can improve the developmental potential after in vitro fertilization (IVF) and in vitro culture. Immediately after warming, almost all oocytes appeared to be morphological normal. Treatment of the postwarming oocytes with 10 μM Y-27632 for 2 h resulted in the significantly higher oocyte survival rate before IVF as well as higher cleavage rate and blastocyst formation rate. Quality analysis of the resultant blastocysts in terms of total cell number and apoptotic cell ratio also showed the positive effect of the Y-27632 treatment. Time-dependent change in mitochondrial activity of the vitrified-warmed oocytes was not influenced by ROCK inhibition during the period of recovery culture. However, the ability of ooplasm to support single-aster formation was improved by the ROCK inhibition. Thus, inhibition of ROCK activity in vitrified-warmed bovine oocytes during a short-term recovery culture can lead to higher developmental competence, probably due to decreased apoptosis and normalized function of the microtubule-organizing center.</P>

Increased Apoptosis in Pig Parthenogenetic Fetuses Resulting Fetal Degeneration during Early Gestation Period

In-Sul Hwang,Seung-Chan Lee,Tae-Uk Kwak,Mi-Ryung Park,Sun-A Ock,Keon Bong Oh,Jae-Seok Woo,Gi-Sun Im,Seongsoo Hwang 한국동물생명공학회(구 한국동물번식학회) 2017 발생공학 국제심포지엄 및 학술대회 Vol.2017 No.10

Parthenogenetic embryo without contribution of paternal genome would be terminated during early gestation period in pigs. Therefore, the present study was designed to analyze the characteristics of parthenogenetic fetuses to identify the possible causes for fetal degeneration during early gestation period. In brief, the pig parthenogenetic fetuses were produced by embryo transfer of parthenogenetic embryos into surrogates. Then, the pig parthenogenetic fetuses were recovered at day 26 and 35 of gestation and conducted to analyze histological characteristics. In results, the parthenogenetic pig fetuses were generated and recovered at day 26 and 35 successfully. The size of parthenogenetic fetuses (n=15, 1.5 cm) recovered at day 26 of gestation were significantly smaller than normal fetuses (n=18, 2.0 cm) while the weights were comparable (0.36 vs. 0.51 g). The size and weight of parthenogenetic fetuses (n=9, 1.8 cm, 0.47 g) recovered at day 35 of gestation were significantly lower than normal fetuses (n=3, 3.67 cm, 3.71 g). The paraffin-embedded sections from parthenogenetic fetus at day 26 showed normal formation of major organs while parthenogenetic fetus at day 35 showed terminated and degenerated formation of major organs. Additionally, the apoptotic cell number of parthenogenetic fetuses (4.4) recovered at day 35 of gestation were significantly (p<0.05) higher than normal fetuses (2.0) while the result of day 26 of gestation showed comparable (1.8 vs. 2.0). Conclusively, our results can suggest that increased apoptosis in fetal tissues also leads to abnormal development of parthenogenetic pig fetus resulting delayed development until day 26 and then termination and degeneration from day 36.

Analysis of Semen Parameters in α1,3-Galactosyltransferase^-/- Boars

Hwang, In-Sul,Lee, Seung-Chan,Kim, Sung Woo,Kwon, Dae-Jin,Park, Mi-Ryung,Yang, Hyeon,Oh, Keon Bong,Ock, Sun-A,Woo, Jae-Seok,Im, Gi-Sun,Hwang, Seongsoo The Korean Society of Embryo Transfer 2017 한국동물생명공학회지 Vol.32 No.2

It is very difficult to get the information about semen quality analysis in transgenic pigs because of limited numbers and research facilities. Therefore, in the present study, we analyzed the semen quality of transgenic boars generated for xenotransplantation research. Briefly, the semen samples were collected from 5 homozygous ${\alpha}1,3$-Galactosyltransferase knock-out ($GalT^{-/-}$) transgenic boars and immediately transported to the laboratory. These semen samples were decupled with DPBS and conducted to analyze semen parameters by a computer-assisted semen analysis (CASA) system. The boar semen were examined all 12 parameters such as total motility (TM), curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), and hyperactivated (HYP), etc. In results, among the 5 $GalT^{-/-}$ boars, three boars (#134, 144, and 170) showed normal range of semen parameters, but #199 and 171 boars showed abnormal ranges of semen parameters according to standard ranges of semen parameters. Unfortunately, #171 boar showed azoospermia symptom with rare sperm counts in the original semen. Conclusively, assessment of semen parameters by CASA system is useful to pre-screening of reproductively healthy boar prior to natural mating and artificial insemination for multiplication and breeding.

Characterization of α-Gal Epitope in Cells and Tissues from Homozygous α-1,3-Galactosyltransferase Knockout Pigs

In-Sul Hwang,Dae-Jin Kwon,Tae-Uk Kwak,Keon Bong Oh,Sun-A Ock,Hak-Jae Chung,Gi-Sun Im,Seongsoo Hwang 한국동물번식학회 2015 Reproductive & developmental biology Vol.39 No.4

To overcome the hyperacute immune rejection during pig-to-non-human primates xenotranasplantation, we have produced and bred α-1,3-galactosyltransferase knock-out (GalT —/—) pigs. In this study, the somatic cells and tissues from the GalT —/— pigs were characterized by an analysis of the expression of Galα-1,3-Gal (α-Gal) epitope. Briefly, ear fibroblast cell lines of 19 homozygous GalT —/— pigs were established and cryopreserved. The expression of α-Gal epitope in the cells was measured by fluorescence activated cell sorter (FACS) analysis using BS-I-B4 lectin. Also, the homozygous (GalT —/—) cells and tissues samples were immunostained with BS-I-B4 lectin for analysis of α-Gal epitope expression. The results showed that the expression of α-Gal epitope in GalT —/— cells (0.2 %) were significantly (p< 0.05) down-regulated to the range of cynomolgus monkey fibroblast (0.2 %) cells compared to heterozygous (GalT —/+) (9.3 %) and wild type (GalT +/+) (93.7 %) fibroblast cells. In the immunostaining results, while the expression of α-Gal epitope was detected a partly in GalT —/+ cells and mostly in GalT +/+ cells, it was almost not detected in the GalT —/— cells. Also, immunostaining results from various tissues of the GalT —/— pig showed that the expression of α-Gal epitope was not detectable, whereas various tissues from GalT +/+ pig showed a strong expression of α-Gal epitope. Our results demonstrated that α-Gal epitope expressions from GalT —/— pigs were successfully knocked out to prevent hyperacute immune rejection for further study of xenotransplantation.