MicroRNAs (miRNAs) are approximately 22 nucleotides of small noncoding RNAs that control gene expression at the posttranscriptional level through translational inhibition and destabilization of their target mRNAs. The miRNAs are phylogenetically conserved and have been shown to be instrumental in a wide variety of key biological processes including cell cycle regulation, apoptosis, metabolism, imprinting, and differentiation. Recently, a paper has shown that expression of the miRNA-302/367 cluster expressed abundantly in mouse and human embryonic stem cells (ESCs) can directly reprogram mouse and human somatic cells to induced pluripotent stem cells (iPSCs) efficiently in the absence of any of the four factors, Oct4, Sox2, c-Myc, and Klf4. To apply this efficient method to porcine, we analyzed porcine genomic sequence containing predicted porcine miRNA-302/367 cluster through ENSEMBL database, generated a non-replicative episomal vector system including miRNA-302/367 cluster originated from porcine embryonic fibro-blasts (PEF), and tried to make porcine iPSCs by transfection of the miRNA-302/367 cluster. Colonies expressing EGFP and forming compact shape were found, but they were not established as iPSC lines. Our data in this study show that pig miRNA-302/367 cluster could not satisfy requirement of PEF reprogramming conditions for pluripotency. To make pig iPSC lines by miRNA, further studies on the role of miRNAs in pluripotency and new trials of transfection with conventional reprogramming factors are needed.

The specific genetic modification in porcine somatic cells by gene targeting has been very difficult because of low efficiency of homologous recombination. To improve gene targeting, we designed three kinds of knock-out vectors with α1,3-galactosyltransferase gene (α1,3-GT gene), DT-A/pGT5’/neo/pGT3’, DT-A/NLS/pGT5’/neo/pGT3’ and pGT5’/neo/ pGT3’/NLS. The knock-out vectors consisted of a 4.8-kb fragment as the 5’ recombination arm (pGT5’) and a 1.9-kb fragment as the 3’ recombination arm (pGT3’). We used the neomycin resistance gene (neo) as a positive selectable marker and the diphtheria toxin A (DT-A) gene as a negative selectable marker. These vectors have a neo gene insertion in exon 9 for inactivation of α1,3-GT locus. DT-A/pGT5’/neo/pGT3’ vector contain only positive-nega-tive selection marker with conventional targeting vector. DT-A/NLS/pGT5’/neo/pGT3’ vector contain positive-negative selection marker and NLS sequences in upstream of 5’ recombination arm which enhances nuclear transport of foreign DNA into bovine somatic cells. pGT5’/neo/pGT3’/NLS vector contain only positive selection marker and NLS sequence in downstream of 3’ recombination arm, not contain negative selectable marker. For transfection, linearzed vectors were introduced into porcine ear fibroblasts by electroporation. After 48 hours, the transfected cells were selected with 300 μg/ml G418 during 12 day. The G418-resistant colonies were picked, of which 5 colonies were positive for α1,3-GT gene disruption in 3´ PCR and southern blot screening. Three knock-out somatic cells were obtained from DT-A/NLS/ pGT5’/neo/pGT3’ knock-out vector. Thus, these data indicate that gene targeting vector using nuclear localization signal and negative selection marker improve targeting efficiency in porcine somatic cells.

MYC (v-myelocytomatosis viral oncogene homologue) is a regulator gene that encodes for a nuclear phosphoprotein. Porcine MYC gene was mapped on chromosome SSC 4p13 and is associated with a variety of functions such as cell proliferation and cell growth. MYC expression is coupled to a multitude of physiological processes and is regulated by hormones, growth factors, cytokines, lymphokines, nutritional status, development and differentiation. MYC is also involved in myogenesis, muscle hyperplasia and adipogenesis. In this study, we investigated SNPs in MYC gene and their association with economic traits in Duroc, Landrace and Yorkshire populations. We detected a single point mutation in exon 3 of porcine MYC gene as a change of T to C at 906 base (amino acid position 302, nonsynomous mutation of alanine) in MYC-N domain. MYC mutation (T906C) was significantly associated with age at 90 kg in these breeds, signifying that this mutation can serve as a selection marker for growth traits in pigs.

The majority of early embryonic mortality in pregnancy occurs during the peri-implantation stage, suggesting that this period is important for conceptus viability and the establishment of pregnancy. Successful establishment of preg-nancy in all mammalian species depends on the orchestrated molecular events that transpire at the conceptus- uterine interface during the peri-implantation period. This maternal-conceptus interaction is especially crucial in pigs because in them non-invasive epitheliochorial placentation occurs, in which the pre-implantation phase is prolonged. During the pre-implantation period, conceptus survival and the establishment of pregnancy are known to depend on the developing conceptus receiving an adequate supply of histotroph, which contains a wide range of nutrients and grow-th factors. Evidence links growth factors including epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), vascular endothelial growth factor (VEGF), and colony-stimulating factor 2 (CSF2) to embryogenesis or implantation in various mammalian species; however, in the case of pig, little is known about such functions of these growth factors, especially their regulatory mechanisms at the maternal-conceptus interface. Our research group has presented evidence for promising growth factors affecting cellular activities of primary porcine trophectoderm (pTr) cells, and we have identified potential intracellular signaling pathways responsible for the activities induced by these factors. Therefore, this review focuses on promising growth factors at the maternal-conceptus interface regulating the develop-ment of the porcine conceptus and playing pivotal roles in implantation events during early pregnancy in pigs.

Transcription activator like effector nucleases (TALENs) are artificial restriction enzymes generated by fusing a TALE DNA binding domain to a DNA cleavage domain which remove and introduce specific genes to produce transgenic animals. To investigate the efficient laboratory techniques for the injection of TALEN mRNA, pEGFP-N1 commercial plasmid were microinjected into porcine parthenogenetic and in vitro fertilization (IVF). In Experiment 1, to investigate injection time, compared 4 different time durations (2 hr, 4 hrs, 6 hrs & 8 hrs) after post activation of parthenogenetic embryos and after 6 hrs of co-incubation with sperms in IVF embryos. There were significant difference (P<0.05) in development to the blastocysts (4.4, 8.9, 3.9, 0.6%), GFP expression in blastocysts (1.3, 5.7, 2.3, 0.0%) which injected after post activation of 4 hrs compared with other 3 groups. IVF embryos after 2 hrs and 4 hrs injected were expressed GFP significantly higher than rest of two groups (P<0.05). In Experiment 2, compared development of 2 different concentrations (20 ng/μl and 50 ng/μl) of EGFP injection. There were significant difference (P<0.05) between two treatments which has higher cleavage (58.8 vs 41.9%), blastocysts development rate (13.0 vs 11.1%) and GFP expressed blastocysts (5.7 vs 0.0%) in 20 ng/μl than the 50 ng/μl in parthenogenetic embryos. In IVF embryos, only 20 ng/μl injected embryos were expressed GFP (4.2%) after 7 days of incubation and 77.3 vs 64.7% of cleavage, 26.4 vs 23.5% development to blastocysts. In Experiment 3, three different volumes (5, 10 and 20 pl) were microinjected into porcine embryos to determine the most appropriate volume. Out of 3 groups, significantly higher development rates of cleavage (68.3, 58.0, 29.3%), blastocysts (11.7, 12.7, 0.5%) and GFP expressed blastocysts (2.9, 7.8, 0.0%) were shown in the 10 pl group (P<0.05). In conclusion, these results imply that 20 ng/μl concentration, 10 pl of volume and injection at 4 hrs after post activation for parthenogenetic and 2∼4 hrs after IVF, 20 ng/μl concentration and 10 pl volume for IVF embryos were more effective microinjection conditions.

돼지 수정란의 체외 생산 효율성 향상을 위해서는 배발생율과 더불어 고품질의 배를 조기에 선별해야 한다. 체외 배 발생율에 대한 보고는 많지만, 고품질의 배를 선별할 수 있는 기술에 대한 연구는 거의 없었다. 본 연구에서는 돼지 난포란 유래 수정란의 체외배양에 있어서 배반포로의 배 발달과 생존에 미치는 Vitamin K1(vit K1) 첨가 농도, 시기 및 시간의 효과를 검토하였다. 1.0 μM, 3.0 μM 및 6.0 μM vit K1을 배양 1일째 24시간 첨가한 결과, 배반포 발달율이 시험군이 14.5 ± 4.3, 0.0 및 0.0%로써 대조군의 35.5 ± 3.2%에 비하여 유의하게 낮았고(p<0.05), 배반포의 생존율도 대조군이 31.8 ± 2.6%로써 시험군의 22.2 ± 2.9, 0.0 및 0.0%에 비하여 유의하게 높았다(p<0.05). 상기 첨가 농도에서 첨가 시간을 달리한 결과, 1.0 μM 농도에서 6시간 처리군의 배반포 발달율과 생존율이 각각 26.5 ± 2.9% 및 47.2 ± 2.8%로써 가장 높았고 특히, 12시간 처리군보다 유의하게 높았다(p<0.05). 3.0 μM 농도에서는 대조군의 배발달율이 36.4 ± 3.1%로 가장 높았으나, 생존율은 3.0시간 첨가군이 41.7 ± 3.2%로 대조군에 비하여 유의하게 높았다(p<0.05). 6.0 μM 농도에서도 배발달율은 대조군(32.0 ± 2.8%), 생존율은 0.5시간 첨가군(42.9 ± 1.8%)이 가장 높았다. 각각의 vit K1 첨가 농도와 시간을 기준으로 서로 다른 배양 시기에 첨가한 결과, 1.0 μM 6시간 첨가군에서는 배반포 발달율은 배양 4일째 첨가군, 생존율은 배양 2일째 첨가군이 가장 높았다. 한편, 3.0 μM 3.0시간 및 6.0 μM 0.5시간 첨가군에서는 배양 4일째 첨가군의 배반포 발달율(59.5 ± 4.1% 및 50.0 ± 3.6%)과 생존율(72.7 ± 5.4% 및 79.2 ± 4.0%)이 대조군과 다른 시험군에 비하여 유의하게 높았다(p<0.05). 한편, vit K1 첨가에 따른 배반포의 세포 수를 조사한 결과, 첨가군(1.0 μM 6시간 배양 2일째, 3.0 μM 3.0시간 배양 4일째 및 6.0 μM 0.5시간 배양 6일째)이 53.4 ± 5.8, 49.4 ± 3.8 및 51.5 ± 4.5개로써 대조군의 40.2 ± 2.3개에 비하여 유의하게 많았다(p<0.05). 그러나 사멸세포 수는 시험군이 3.2 ± 0.9∼3.7 ± 2.1개로써 대조군의 4.2 ± 1.2개보다 적었으나, 유의차는 없었다. 세포 사멸 유도 유전자인 Bax mRNA 발현은 처리군과 대조군은 비슷하였으나, 세포 사멸 억제 유전자인 Bcl-xL mRNA 발현은 처리군이 대조군보다 높았고 특히, 6.0 μM 0.5시간 배양 4일째 첨가군이 가장 높았다. 이상의 결과로부터 돼지 미성숙 난포란 유래 수정란의 체외 배양에 vit K1의 첨가는 배반포의 생존율과 세포수 증가에 효과적이었다. 그 이유에 대해서는 아직 많은 부분이 밝혀져야 되겠지만, 고품질의 배반포 조기 선발에는 활용이 가능할 것으로 생각된다.

In porcine embryo culture, one of reactive oxygen species (ROS) is harmful factors that are made during in vitro culture. To decrease the detrimental effect of ROS on embryo development, superoxide dismutase, catalase and glutathione peroxidase could be used in the embryo culture. Out of these antioxidants, 7,8-dihydroxyflavone (7,8-DHF) was reported its antioxidant effects to prevent the glutamine-triggered apoptosis. Therefore, this study was performed to investigate the most appropriate concentration of 7,8-DHF in porcine embryonic development. For that, 5 different concentration (0, 0.1, 0.5, 1, 2 uM) of 7,8-DHF was supplemented in the porcine IVM media and then maturation and blastocyst formation rates were compared among 5 groups. In maturation rates of porcine oocytes, significant higher maturation rates was shown in the 1.0 uM group compared with another 4 groups (83.3 ± 2.1 vs. 80.7 ± 1.4, 79.8 ± 1.4, 78.3 ± 1.2, 79.4 ± 1.6), respectively (P<0.05). In the embryo culture, 1.0 uM group also showed the significant higher cleavage rates (76.8 ± 3.1 vs. 62.1 ± 5.0, 65.7 ± 4.0, 68.6 ± 3.7, 64.6 ± 4.0%) and blastocyst formation rates - (39.6 ± 4.0% vs. 28.6 ± 3.3, 31.1 ± 3.9, 29.3 ± 2.5, 39.6 ± 4.0, 26.4 ± 3.2%), respectively (P<0.05). There was no significant difference among 5 groups in the cell number of blastocyst (P<0.05). In conclusion, supplement of 1.0 uM of 7,8-DHF was effective to increase the porcine embryonic development competence as antioxidant to ROS.

본 연구는 돼지 정자의 동결 건조 시 동결 보호제인 trehalose의 효과와 동결 건조 시간과 동결 건조 후 저장 기간에 따라 정자의 생존성과 체외 성숙 난자 내 동결 건조 정자를 직접 주입한 후 전핵 형성율, 난할율 그리고 배발달 성적을 조사하였다. 동결 건조 후 정자의 생존율은 trehalose 무첨가구에 비해 trehalose를 첨가한 처리구에서 높은 생존율을 보였으며, 75 mM의 trehalose를 첨가하여 동결 건조한 정자들의 생존율이 가장 높은 것으로 나타났다. 또한, 동결 건조 후 저장 기간이 길어질수록 생존율이 낮아지는 경향이었다. 체외 성숙 난자 내 동결 건조 정자를 직접 주입 후 전핵 형성율은 trehalose 첨가구에서 유의적으로 높았으며(p<0.05), 난할율과 배발달 성적도 trehalose 첨가구에서 유의적으로 높았고(p<0.05), 정자의 동결 건조 시간이 짧을수록 높은 난할율과 배발달율을 보였다.

The present study assessed the effect of FSH and LH on oocyte meiotic, cytoplasmic maturation and on the expression level and polyadenylation status of several maternal genes. Cumulus-oocyte complexes were cultured in the presence of FSH, LH, or the combination of FSH and LH. Significant cumulus expansion and nuclear maturation was observed upon exposure to FSH alone and to the combination of FSH and LH. The combination of FSH and LH during entire IVM increased the mRNA level of four maternal genes, C-mos, Cyclin B1, Gdf9 and Bmp15, at 28 h. Supplemented with FSH or LH significantly enhanced the polyadenylation of Gdf9 and Bmp15; and altered the expression level of Gdf9 and Bmp15. Following parthenogenesis, the exposure of oocytes to combination of FSH and LH during IVM significantly increased cleavage rate, blastocyst formation rate and total cell number, and decreased apoptosis. In addition, FSH and LH down-regulated the autophagy gene Atg6 and upregulated the apoptosis gene Bcl-xL at the mRNA level in blastocysts. These data suggest that the FSH and LH enhance meiotic and cytoplasmic maturation, possibly through the regulation of maternal gene expression and polyadenylation. Overall, we show here that FSH and LH inhibit apoptosis and autophagy and improve parthenogenetic embryo competence and development.

To profile the proteome in porcine plasma, blood samples were collected from adult male barrows and those plasma were retrieved. For the depletion or pre-fractionation of high-abundance proteins, plasma samples were treated with commercial kits. Then, protein profiling was initiated using one and two-dimensional electrophoresis. Proteins were spotted and then identified by MALDI-TOF-TOF and LC-MS-MS. In the results, more than forty six proteins were identified and the reference map was constructed. The pre-treatment for the removal of high-abundance proteins caused the changes in 2-DE images and some of the proteins were newly uncovered after the most of high abundant proteins were removed. However, it is expected for further steps necessary to identify more low-abundance proteins that may contain potential bio-markers.

Muscle satellite cell (SC) is responsible for postnatal muscle growth, repair, and regeneration. Satellite cell is an im-portant source of multi-potent stem cell process and differentiation into adipogenic, myogenic, and osteoblastogenic. The objective of this study was to identify alter of transcriptome during differentiation in porcine satellite cell and to elevated transcriptome at different stages of postnatal development to gain insight into the differences in differ-entiated PSC. We used RNA-seq technique to investigate the transcriptomes during differentiation in pig muscle. Sequence reads were obtained from Illumina HiSeq2000. Differentially expressed genes (DEG) were detected by EdgeR. Gene ontology (GO) terms are powerful tool for unification among representation genes or products. In study of GO biological terms, functional annotation clustering involved in cell cycle, apoptosis, extracellular matrix, phosphoryla- tion, proteolysis, and cell signaling in differences stage. Taken together, these results would be contributed to a better understanding of muscle biology and processes underlying differentiation. Our results suggest that the source of DEGs could be better understanding of the mechanism of muscle differentiation and transdifferentiation.

Satellite cells were derived from muscular tissue in postnatal pig. Satellite cell is an important to growth and development in animal tissues or organs. However, the progress underlying induced differentiation is not clear. The aim of this study was to evaluate the morphologic and the transcriptome changes in porcine satellite cell (PSC) treated with insulin, rosiglitazone, or dexamethasone respectively. PSC was obtained from postnatal muscle tissue. In study 1, for study the effect of insulin and FBS on the differentiated satellite cells, cells were cultured at absence or presence of insulin treated with FBS. Total RNA was extracted for determining the expression levels of myo-genic PAX3, PAX7, Myf5, MyoD, and myogenin genes by real-time PCR. Myogenic genes decreased expression levels of mRNA in treated with insulin. In study 2, in order to clarify the relationship between rosiglitazone and lipid in differentiated satellite cells, we further examined the effect of FBS on lipid accumulation in the presence or absence of the rosiglitazone and lipid. Significant differences were observed between rosiglitazone and lipid by FBS. The mRNA of FABP4 and PPARγ increased in rosiglitazone treatment. In study 3, we examined the effect of dexame-thasone on osteogenic differentiation in PSC. The mRNA was increased osteoblasotgenic ALP and ON genes treated with dexamethasone in 2% FBS. Dexamethasone induces osteoblastogenesis in differentiated PSC. Taken together, in differentiated PSCs, FABP4 and PPARγ increased to rosiglitazone. Whereas, no differences to FBS and lipid. These results were not comparable with previous reports. Our results suggest that adipogenic, myogenic, and osteoblasto-genic could be isolated from porcine skeletal muscle, and identify culture conditions which optimize proliferation and differentiation formation of PSC.

Muscular satellite cell (SC), which is stem cell of postnatal pig, is an important for study of differentiation into adipogenesis, myogenesis, and osteoblastogenesis. In this study, we isolated and examined from pig muscle tissue to determine capacity in proliferate, differentiate, and expression of various genes. Porcine satellite cells (PSC) were isolated from semimembranosus (SM) muscles of 90∼100 days old pigs according to standard conditions. The cell proliferation increased in multi-potent cell by Masson’s, oil red O, and Alizarin red staining respectively. We per-formed the expression levels of differentiation related genes using real-time PCR. We found that the differentiation into adipocyte increased expression levels of both fatty acid binding protein 4 (FABP4) and peroxisome proliferator- acti-vated receptor gamma (PPARγ) genes (p<0.01). Myocyte increased the expression levels of the myosin heavy chain (MHC), myogenic factor 5 (Myf5), myogenic regulatory factor (MyoD), and Myogenic factor 4 (myogenin) (p<0.01). Osteo-blast increased the expression levels of alkaline phosphatase (ALP) (p<0.01). Finally, porcine satellite cells were indu-ced to differentiate towards adipogenic, myogenic, and osteoblastogenic lineages. Our results suggest that muscle satellite cell in porcine may influence cell fate. Understanding the progression of PSC may lead to improved strat-egies for augmenting meat quality.

핵이식(NT) 기술을 이용하여 여러 동물 종에서 성공적으로 복제산자가 보고되고 있지만, 아직까지 비효율적인 기술로 남아있다. 본 연구에서는 돼지 체세포 복제 생산 효율성을 증진시키기 위한 방안으로 수핵난자의 품질에 초점을 맞추어 Brilliant cresyl blue (BCB) 염색을 통하여 발육능이 우수한 미성숙 난자를 선발하고, 난자의 감수분열 재개에 관여하는 단백질 합성을 비특이적으로 억제하는 cycloheximide (CHXM)을 이용하여 돼지 난자의 감수분열 재개를 억제시켜 난자의 성숙 동기화를 유도하였다. 또한 핵초기화에 밀접한 영향을 주는 핵막붕괴(NEBD)와 조기염색체응축 (PCC)을 유도하는 MPF의 활성화를 높이기 위하여 단백질 phosphatase 억제제인 caffeine을 첨가하여 수핵난자의 품질을 향상시키고자 하였다. 실험 방법으로는 13 mM BCB 첨가된 배양액에 90분 동안 미성숙난자를 배양하여 BCB 용액의 착색 여부를 구분하여 선발하고, 5 ㎍/ml CHXM를 체외 성숙액에 첨가하여 난자성숙 동기화를 유도하였다. 또한 탈핵 후 탈핵난자를 caffeine을 처리하여 세포주기 관련 단백질의 활성화를 인위적으로 조절하여 체세포복제 수핵난자로 사용하였다. 실험 결과로서 BCB 염색 돼지 미성숙 난자를 대조구와 비교할 때 제2 감수분열 중기(MII)에 도달하는 체외성숙율과 단위발생란의 배반포기까지의 체외 발육율이 유의적으로 증가하는 것이 관찰되었다. 또한 미성숙 돼지 난자의 초기 성숙 (12∼16시간)에 CHXM를 처리하였더니 난자 감수분열 재개가 억제되어 GV기에 핵 성숙이 정지되어 동기화가 유도되었다. GV기에 세포주기 동기화된 난자들은 CHXM를 제거하였을 때 난자 성숙의 진행속도도 일치하는 것이 관찰되었다. 이런 결과는 가장 적합한 탈핵시기인 제1 감수분열 후/말기(AI/TI)에 난자들이 다수 분포하여 대조구에 비하여 높은 탈핵율 (87.9%)을 얻을 수 있었다 (P < 0.05). 덧붙여 5 mM의 caffeine을 돼지 난자에 12시간 처리하였을 때 난자 MPF의 활성화가 증가하는 것이 관찰되었지만 (P < 0.05), 10 mM caffeine 농도를 처리하였을 때 MPF의 활성화가 오히려 감소되어 단위발생란의 배반포기까지의 체외발육에도 악영향을 주는 것이 관찰되었다.

Xenotransplantation of pig organs into primates results in fatal damage, referred as hyperacute rejection (HAR), and acute humoral xenograft rejection (AHXR), to the organ graft mediated by antibodies pre-existing and newly-producing in primates against their cognate pig antigens. Functional ablation of α1,3-galactosyltransferase (Gal-T KO) of pig which is an enzyme involved in synthesis of Gala1-3Galb1-4GlcNAc-R antigen is essentially required to prevent HAR. Moreover, additional genetic modification under Gal-T KO background for enforced expression of human complement regulatory proteins which can inhibits complement activation is known to effectively imped HAR and AHXR. In this study, we constructed a membrane cofactor protein (MCP) expression cassette under control of human EF1α promoter. This cassette was inserted between homologous recombination regions corresponding to Gal-T locus. Subsequently this vector was introduced into ear skin fibroblasts of female pig by nucleofection. We were able to obtained 40 clones by neomycin selection and 4 clones among them were identified as clones targeted into Gal-T locus of MCP expression cassette by long-range PCR. Real time RT-PCR was shown to down-regulation of Gal-T expression. From these results, we demonstrated human EF1α promoter could induce efficient expression of MCP on cell surface of fibroblasts of female pig.

Pyracantha is a genus of thorny evergreen large shrubs in the family of Rosaceae, with common names Firethorn or Pyracantha. It's extract has also been used in cosmetics as a skin-whitening agent and functioning through tyrosinase inhibition. Recent studies have shown that pyracantha extract possesses antioxidant activities and may significantly improve lipoprotein metabolism in rats. Although the mode of action of Pyracantha extract is not fully understood, a strong relationship was observed between antioxidant and apoptosis in some types of cells. Thus, the aim of this study was to evaluated the effect of pyracantha extract on blastocysts formation and their quality of the porcine parthenogenetic embryos. After parthenogenetic activation by chemicals, presumptive porcine parthenogenetic embryos were cultured in PZM-3 medium supplemented with extracts of pyracantha leaf, stalk and root for 6 day (1, 5 and 10 μg/ml, respectively). In our results, the frequency of blastocyst formation in pyracantha root extract (5 μg/ml) treated group had increased that of other groups. Furthermore, blastocysts derived from pyracantha root extract (5 μg/ml) treated group had increased the total cell numbers and reduced apoptotic index. Blastocyst development was significantly improved in the pyracantha root extract (5 μg/ml) treated group when compared with the H2O2 treated group (p<0.05). Subsequent evaluation of the intracellular levels of ROS in pyracantha root extract (5 μg/ml) treated groups under H2O2 induced oxidative stress were decreased (p<0.05). In conclusion, our results indicate that treatment of pyracantha root extract may improve in vitro development of porcine parthenogenetic embryos through its antioxidative and antiapoptotic effects.

The objective of this study was to examine the effects of high concentrations of glucose on porcine parthenotes developing in vitro. Addition of 55 mM glucose to the culture medium of embryos at the four-cell-stage significantly inhibited blastocyst formation, resulting in fewer cells in blastocyst-stage embryos and increased levels of apoptosis and autophagy compared to control. Quantitative reverse transcriptase (RT) PCR analysis revealed that the expression of pro-apoptotic genes (Caspase 3, Bax and Bak) and autophagy genes (Atg6 and Atg8/Lc3) were increased significantly by the addition of 55 mM glucose to the culture medium compared to control. MitoTracker Green fluorescence revealed a decrease in the overall mitochondrial mass compared to control. However, the addition of 55 mM glucose had no effect on mRNA expression of the nuclear DNA-encoded mitochondrial-related genes, cytochrome oxidase (Cox) 5a, Cox5b and Cox6b1. These results suggest that hyperglycemia reduced the mitochondrial content of porcine embryos developing in vitro and that this may hinder embryonic development to the blastocyst stage and embryo quality by increasing apoptosis and autophagy in these embryos.

Developmental potential of cloned embryos is related closely to epigenetic modification of somatic cell genome. The present study was to investigate the effects of applying histone deacetylation inhibitor, trichostatin A (TSA) to activated porcine embryos on subsequent development of porcine parthenogenetic and nuclear transfer embryos. Electrically activated oocytes were treated with 5 nM TSA for different exposure times (0, 1, 2 and 4 hr) and then the activated embryos were cultured for 7 days. The reconstructed embryos were treated with different concentrations of 0, 5, 10 and 25 nM TSA for 1 hr. Also 5 nM TSA was tested with different exposure times of 0, 0.5, 1, 2 and 4 hr. And fetal fibroblast cells were treated with 50 nM TSA for 1, 2 or 4 hr and with 5 nM TSA for 1 hr. Cumulus-free oocytes were enucleated and reconstructed by TSA-treated donor cells and electrically fused and cultured for 6 days. In parthenogenetic activation experiments, 5 nM TSA treatment for 1 hr significantly improved the percentage of blastocyst developmental rates than the other groups. Total cell number of blastocysts in 1 hr group was significantly higher than other groups or control. Similarly, blastocyst developmental rates of porcine NT embryos following 5 nM TSA treatment for 1 hr were highest. And the reconstructed embryos from donor cells treated by 50 nM TSA for 1 hr improved the percentage of blastocyst developmental rates than the control group. In conclusion, TSA treatment could improve the subsequent blastocyst development of porcine parthenogenetic and nuclear transfer embryos.

The present study was conducted to examine the effect of antioxidant treatment during parthenogenetic activation procedure on the reactive oxygen species (ROS) levels and in vitro development of porcine parthenogenetic embryos. Porcine in vitro matured oocytes were activated by a combination of electric stimulus and 2 mM 6- dimethylaminopurine (6-DAMP) before in vitro culture. During the activation period, oocytes were treated with 50 μM β-mercaptoethanol (β-ME), 100 μM L-ascorbic acid (Vit. C) or 100 μM L-glutathione (GSH). To examine the ROS level, porcine parthenogenetic embryos were stained in 10 μM dichlorohydrofluorescein diacetate (H2DCFDA) dye 20 h after culture, examined under a fluorescence microscope, and the fluorescence intensity (pixels) were analyzed in each embryo. The parthenogenetic embryos were cultured for 6 days to evaluate the in vitro development. The apoptosis was measured by TUNEL assay. The H2O2 levels of parthenogenetic embryos were significantly lower in antioxidant treatment groups (26.9±1.6～29.1±1.3 pixels/embryo, p<0.05) compared to control (33.2±1.7 pixels/embryo). The development rate to the blastocyst stage was increased in antioxidant treatment groups (32.0～32.5%) compared to control (26.9%, p<0.05), although, there was no difference in apoptosis among groups. The result suggests that antioxidant treatment during parthenogenetic activation procedure can inhibit the ROS generation and enhance the in vitro development of porcine parthenogenetic embryos.

Although assisted reproductive technology is very useful to develop novel and therapeutic biomaterials for reproduction, research on molecular mechanism of folliculogenesis in pig is not clear. Therefore, the alteration of gene expression during follicular development in pigs was examined in this study. The expression of folliculogenesis-related genes was quantified in preantral (250～300 μm) and antral (>300 μm in diameter) follicles, and overall gene expression was evaluated by a genome-wide microarray. The microarray results showed that 219 genes were differentially expressed, and of those, 10 and 22 known genes showed higher and less expression at the preantral stage than at antral stages, respectively. Among them, the expression of NR0B1, PPARG, GATA4, and ANXA2 genes related to folliculogenesis was validated by quantitative real-time PCR analysis. The expression of PPARG and GATA4 genes were increased at antral stages, but a significantly stage-specific increase (p<0.05) was only detected in annexin A2 (ANXA2) in antral-stage follicles. The expression of NR0B1 genes was increased at preantral stage and these patterns of gene expression were comparable to the results obtained by microarray analysis. We propose that the systematical regulation of genes supporting specific follicle stage should be employed for improved in-vitro folliculognesis.