• Reproductive and Developmental Biology
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• v.29 no.3
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• pp.169-174
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• 2005

This study was comducted to examine the effects of culture medium, and the osmolarity and osmotic change of the culture medium on in vitro maturation of porcine oocytes and developement of porcine parthenogenetic embryos. In Experiment 1, cumulus-oocyte complexes were matured in NCSU-23, mWM and mKRB, respectively, There was no difference in maturation rate($62.1\~71.3\%$) among groups. In Experiment 2, matured oocytes in each medium were activated and cultured for 6 days in the same media. Blastocyst formation rate was higher in NCSU-23($22.9\%$) than those of others($0\~0.6\%$, P<0.05). In Experiment 3, parthenogenetic embryos were cultured for 6 days in NCSU-23 with different osmolarity(300, 280 and 256 mOsmols) adjusted by NaCl. There were no differences in development rates to the blastocyst stage($11.0\~14.4\%$) among groups. In Experiment 4, activated oocytes were cultured for 2 days in NCSU-23 with 300, 280 and 256 mOsmols and then transferred to increased or decreased osmotic condition. Blastocyst formation rate was higher in a group which was transferred from the higher osmotic condition to the lowe. osmotic condition($21.0\%$) than a contrary group( $11.8\%$, (P<0.05). This result shows that the culture medium and the osmolarity of the culture medium affect the development of porcine parthenogenetic embryos, and the change of osmolarity from the higher condition to the lower condition at a certain developmental stage can enhance the development of porcine parthenogenetic embryos.

• Reproductive and Developmental Biology
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• v.28 no.1
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• pp.21-27
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• 2004

This study was conducted to investigate the developmental ability of caprine embryos after somatic cell interspecies nuclear transfer. Recipient bovine and porcine oocytes were obtained from slaughterhouse and were matured in vitro according to established protocols. Donor cells were obtained from an ear-skin biopsy of a caprine, digested with 0.25% trypsin-EDTA in PBS and primary fibroblast cultures were established in TCM-199 with 10% FBS. The matured oocytes were dipped in D-PBS plus 10% FBS ＋ 7.5 $\mu$ g/ml cytochalasin B and 0.05M sucrose. Enucleation were accomplished by aspirating the first polar body and partial cytoplasm which containing metaphase II chromosomes using a micropipette with an out diameter of 20∼30 $\mu$m. A Single donor cell was individually transferred into the perivitelline space of each enucleated oocyte. The reconstructed oocytes were electric fusion with 0.3M mannitol fusion medium. After the electrofusion, embryos were activated by electric stimulation. Interspecies nuclear transfer embryos with bovine cytoplasts were cultured in TCM-199 medium supplemented with 10% FBS including bovine oviduct epithelial cells for 7∼9 day. And porcine cytoplasts were cultured in NCSU-23 medium supplemented with 10% FBS for 6 ∼8 day at $39^{\circ}C, 5% CO_2 $in air. Interspecies nuclear transfer by recipient bovine oocytes were fused with electric length 1.95 kv/cm and 2.10 kv/cm. There was no significant difference between two electric length in fusion rate(47.7 and 44.6%) and in cleavage rate(41.9 and 54.5%). Using electric length 1.95 kv/cm and 2.10 kv/cm in caprine-porcine NT oocytes, there was also no significant difference between two treatments in fusion rate(51.3 and 46.1%) and in cleavage rate(75.0 and 84.9%). The caprine-bovine NT oocytes fusion rate was lower(P＜0.05) in 1 pulse for 60 $\mu$sec(19.3%), than those from 1 pulse for 30 $\mu$sec(50.8%) and 2 pulse for 30 $\mu$sec(31.0%). The cleavage rate was higher(P＜0.05) in 1 pulse for 30 $\mu$sec(53.3%) and 2 pulse for 30 $\mu$sec(50.0%), than in 1 pulse for 60 $\mu$sec(18.2%). The caprine-porcine NT oocytes fusion rate was 48.1% in 1 pulse for 30 $\mu$sec, 45.2% in 2 pulse for 30 $\mu$sec and 48.6% in 1 pulse for 60 $\mu$sec. The cleavage rate was higher(P＜0.05) in 1 pulse for 30 $\mu$sec(78.4%) and 1 pulse for 60 $\mu$sec(79.4%), than in 2 pulse for 30 $\mu$sec(53.6%). In caprine-bovine NT embryos, the developmental rate of morula and blastocyst stage embryos were 22.6% in interspecies nuclear transfer and 30.6% in parthenotes, which was no significant differed. The developmental rate of morula and blastocyst stage embryos with caprine-porcine NT embryos were lower(P＜0.05) in interspecies nuclear transfer(5.1%) than parthenotes(37.4%).

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.5
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• pp.643-652
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• 2017

Objective: Prostaglandins (PGs) function in various reproductive processes, including luteolysis, maternal pregnancy recognition, conceptus development, and parturition. Our earlier study has shown that PG transporters ATP-binding cassette, subfamily C, member 4 (ABCC4) and solute carrier organic anion transporter family, member 2A1 (SLCO2A1) are expressed in the uterine endometrium in pigs. Since several other PG transporters such as ABCC1, ABCC9, SLCO4C1, and SLCO5A1 are known to be present in the uterine endometrium, this study investigated the expression of these PG transporters in the porcine uterine endometrium and placenta. Methods: Uterine endometrial tissues were obtained from gilts on day (D) 12 and D15 of the estrous cycle and days 12, 15, 30, 60, 90, and 114 of pregnancy. Results: ABCC1, ABCC9, SLCO4C1, and SLCO5A1 mRNAs were expressed in the uterine endometrium, and levels of expression changed during the estrous cycle and pregnancy. Expression of ABCC1 and ABCC9 mRNAs was localized mainly to luminal and glandular epithelial cells in the uterine endometrium, and chorionic epithelial cells during pregnancy. Conceptuses during early pregnancy and chorioallantoic tissues from mid to late pregnancy also expressed these PG transporters. $Estradiol-17{\beta}$ increased the expression of ABCC1 and SLCO5A1, but not ABCC9 and SLCO4C1 mRNAs and increasing doses of $interleukin-1{\beta}$ induced the expression of ABCC9, SLCO4C1, and SLCO5A1 mRNAs in endometrial explant tissues. Conclusion: These data showed that several PG transporters such as ABCC1, ABCC9, SLCO4C1, and SLCO5A1 were expressed at the maternal-conceptus interface, suggesting that these PG transporters may play an important role in the establishment and maintenance of pregnancy by regulating PG transport in the uterine endometrium and placenta in pigs.

• Journal of Life Science
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• v.20 no.5
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• pp.697-702
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• 2010

Ten farrow-finish farms participated in this seromonitoring that was conducted to investigate the porcine brucellosis situation in Korea. In total, eight (80.0%) of the 10 farms and 139 (24.0%) of 578 pigs tested showed a positive response in the Rose Bengal test (RBT). Seroprevalence levels were determined using RBT according to age; 35 (14.6%) of 239 piglets, 36 (31.3%) of 115 growing pigs, and 68 (30.4%) of 224 finishing pigs and sows were positive, respectively. All positive samples in RBT were tested with the tube agglutination test (TAT) and competitive ELISA (C-ELISA), simultaneously. Although 48 samples came up positive in the TAT, all samples tested with C-ELISA were negative. Among 26 rectal swab samples from the TAT positive-pigs, Yersinia enterocolitica O:9 was isolated from seven samples (26.9%). Therefore, we speculated that the positive reaction of RBT and TAT in this study might be induced by the serologically cross-reacting bacteria with Brucella abortus.

• Korean Journal of Veterinary Service
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• v.27 no.3
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• pp.273-280
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• 2004

The studies were performed for the PRRS antigen and antibody detection from breeding farms, artificial insemination(AI) center and growing farms in Jeonbuk province. 1. Specific PRRS primers were successfully amplified ORF6 617bp and ORF7 448 bp on agarose gel. 2. RT-PCR method has been establish by commercial kit and the thermal cycler program consisted of 30 cycles: $95^{\circ}C$ for 30 sec, $45^{\circ}C$ for 30 sec, and $72^{\circ}C$ for 45 sec. 3. The results of PRRS antibody test by ELISA method in AI centers were $6.6\%,\;53.3\%$ and breeding farms $65\%,\;65\%\;and\;38.7\%$, respectively. The serological positive of the antibody in gilt higher than sow. 4. The sero-positive of the PRRS antibody showed average $21\%$ in domestic farms, $56.2\%$ in breeding farms, and $29.9\%$ in AI center.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.9
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• pp.1254-1262
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• 2014

Lymphoid enhancer binding factor 1 (LEF-1) is a member of the T-cell specific factor (TCF) family, which plays a key role in the development of breast endothelial cells. Moreover, LEF-1 gene has been identified as a candidate gene for teat number trait. In the present study, we detected two novel mutations (NC_010450.3:g. 99514A>G, 119846C>T) by DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism in exon 4 and intron 9 of LEF-1 in Guanzhong Black, Hanjiang Black, Bamei and Large White pigs. Furthermore, we analyzed the association between the genetic variations with teat number trait in these breeds. The 99514A>G mutation showed an extremely significant statistical relevance between different genotypes and teat number trait in Guanzhong (p<0.001) and Large White (p = 0.002), and significant relevance in Hanjiang (p = 0.017); the 119846C>T mutation suggested significant association in Guanzhong Black pigs (p = 0.042) and Large White pigs (p = 0.003). The individuals with "AG" or "GG" genotype displayed more teat numbers than those with "AA"; the individuals with "TC" or "CC" genotype showed more teat numbers than those with "TT". Our findings suggested that the 99514A>G and 119846C>T mutations of LEF-1 affected porcine teat number trait and could be used in breeding strategies to accelerate porcine teat number trait improvement of indigenous pigs breeds through molecular marker assisted selection.

• Reproductive and Developmental Biology
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• v.37 no.4
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• pp.269-279
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• 2013

Low efficiency of somatic cell nuclear transfer (SCNT) is attributed to incomplete reprogramming of transfered nuclei into oocytes. Trichostatin A (TSA), histone deacetylase inhibitor and 5-aza-2'deoxycytidine (5-aza-dC), DNA methylation inhibitor has been used to enhance nuclear reprogramming following SCNT. However, it was not known molecular mechanism by which TSA and 5-aza-dC improve preimplantation embryo and fetal development following SCNT. The present study investigates embryo viability and gene expression of cloned porcine preimplantation embryos in the presence and absence of TSA and 5-aza-dC as compared to embryos produced by parthenogenetic activation. Our results indicated that TSA treatment significantly improved development. However 5-aza-dC did not improve development. Presence of TSA and 5-aza-dC significantly improved total cell number, and also decreased the apoptotic and autophagic index. Three apoptotic-related genes, Bak, Bcl-xL, and Caspase 3 (Casp3), and three autophagic-related genes, ATG6, ATG8, and lysosomal-associated membrane protein 2 (LAMP2), were measured by real time RT-PCR. TSA and 5-aza-dC treatment resulted in high expression of anti-apoptotic gene Bcl-xL and low pro-apoptotic gene Bak expression compared to untreated NT embryos or parthenotes. Furthermore, LC3 protein expression was lower in NT-TSA and NT-5-aza-dC embryos than those of NT and parthenotes. In addition, TSA and 5-aza-dC treated embryos displayed a global acetylated histone H3 at lysine 9 and methylated DNA H3 at lysine 9 profile similar to the parthenogenetic blastocysts. Finally, we determined that several DNA methyltransferase genes Dnmt1, Dnmt3a and Dnmt3b. NT blastocysts showed higher levels Dnmt1 than those of the TSA and 5-aza-dC blastocysts. Dnmt3a is lower in 5-aza-dC than NT, NTTSA and parthenotes. However, Dnmt3b is higher in 5-aza-dC than NT and NTTSA. These results suggest that TSA and 5-aza-dC positively regulates nuclear reprogramming which result in modulation of apoptosis and autophagy related gene expression and then reduce apoptosis and autophagy. In addition, TSA and 5-aza-dC affects the acetylated and methylated status of the H3K9.

• BMB Reports
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• v.38 no.2
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• pp.225-231
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• 2005

Acid-labile subunit (ALS) is a component of the 150-kDa insulin-like growth factor-binding protein-3 (IGFBP-3) complex, which, by sequestering the majority of IGFs-I and -II and thereby prolonging the half-life of them in plasma, serves as a circulating reservoir of IGFs in mammalian species. A pGEX-2T plasmid and a baculovirus expression constructs harboring a coding sequence for glutathione-S transferase (GST)-porcine ALS (pALS) fusion protein were expressed in BL21(DE3) E. coli and Sf9 insect cells, respectively. The expressed protein was purified by glutathione or Ni-NTN affinity chromatography, followed by cleavage of the fusion protein using Factor Xa. In addition, pALS and hIGFBP-3 were also produced in small amounts in the Xenopus oocyte expression system which does not require any purification procedure. A 65-kDa pALS polypeptide was obtained following the prokaryotic expression and the enzymatic digestion, but biochemical characterization of this polypeptide was precluded because of an extremely low expression efficiency. The baculovirus-as well as Xenopus-expressed pALS exhibited the expected molecular mass of 85 kDa which was reduced into 75 and 65 kDa following deglycosylation of Asn-linked carbohydrates by Endo-F glycosidase, indicating that the expressed pALS was properly glycosylated. Moreover, irrespective of the source of pALS, the recombinant pALS and hIGFBP-3 formed a 130-kDa binary complex which could be immunoprecipitated by anti-hIGFBP-3 antibodies. Collectively, results indicate that an authentic pALS protein can be produced by the current expression systems.

• Journal of Embryo Transfer
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• v.20 no.3
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• pp.207-215
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• 2005

본 연구는 돼지 난포란의 동결보존 후 생존성과 난자의 활성화 처리에 따른 체외발생율과 이를 이용한 핵 이식배의 체외발생율을 조사하였다. 활성화 처리된 배는 $5\%$ FBS가 첨가된 NCSU 23 배양액으로 $38.5^{\circ}C$, $5\%\;CO_2$와 $95\%$ air의 조건으로 배양하였다. 1. 난포란을 EDS와 $5\%$ PVP로 동결 후 $10\%$ FBS가 첨가된 NCSU 23 배양액으로 $0{\~}10$시간 배양했을 때 체외발생율은 $36.0\%$로서 대조군인 비동결 난포란의 체외발생율 $46.0\%$에 비해 낮았다. 2. Ethanol과 cyclojexamide로 처리 후 42 및 46시간 배양한 배의 분할율은 각각 $33.3\%$, $36.0\%$ 및 $27.1\%$, $30.0\%$로서 대조군의 $8.8\%$, $11.4\%$에 비해 높게 나타났다. 3. 동결 및 비동결 난포란을 이용한 핵이식 배의 융합율과 발생율 간에는 유의한 차이가 없었다. 4. Ethanol과 cyclojexamide로 활성화 처리한 난자를 이용하여 재구축한 핵 이식배의 발생율은 $2.8\%$, $5.3\%$ 및 $1.5\%$, $2.9\%$로서 대조군의 $0.0\%$, $0.0\%$에 비해 높은 발생율을 나타냈다.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.13 no.8
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• pp.3532-3536
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• 2012

Understanding of the maternal transcriptome increased to elucidate the underlying molecular mechanism of normal oocyte maturation, which depends on a precise sequence of changes in maternal genes expression. Previous reports that the translational potential of a maternal mRNA is generally determined by the length of the poly(A) tail, and deadenylation is usually the first sign of mRNA degradation. However, in vitro cultured system has the underlying molecular mechanisms remain unclear. We determined whether the role of molecular basis, four important maternal genes, C-mos, cyclin-B1 (regulatory subunit of MPF), BMP15 and GDF9, were selected for detection of their precise mRNA expression patterns by real-time PCR and for determination of their polyadenylation status by poly(A) tail PCR during oocyte maturation. In the present study. the abnormal expression of maternal mRNAs prior to zygotic genome activation, which results in suppression of the corresponding protein level, may be responsible for, at least in part, a profound defect in further embryonic development. Reasonable expression of maternal gene is crucial for proper oocyte maturation and further embryonic development.