Caspases are a family of cysteine protease enzymes composed of more than 10 members that play essential roles in apoptosis and inflammation. It has been reported that caspases play a critical role in regulating apoptosis at the maternal-conceptus interface in many species. However, the expression and regulation of caspases have not been determined in the endometrium in pigs. Therefore, we analyzed the expression, localization, and regulation of caspases in the endometrium during the estrous cycle and pregnancy in pigs. Real-time RT-PCR analysis showed that caspases were expressed in the endometrium during the estrous cycle and pregnancy. The expression of CASP6, CASP7, and CASP8 during the estrous cycle and CASP3, CASP6, CASP7, and CASP10 changed during pregnancy. Levels of CASP3 mRNA in the endometrium were higher on Day 12 of pregnancy than the estrous cycle and levels of CASP7 mRNAs were highest on Day 15 of estrous cycle and pregnancy. Immunohistochemistry analysis showed that CASP3 protein was localized to endometrial epithelial cells on Days 12 and 15 the estrous cycle and pregnancy, but cleaved CASP3 was localized only to luminal epithelial (LE) cells on Days 12 and 15 of pregnancy in the endometrium. CASP7 protein was localized to endometrial LE cells only on Day 15 of pregnancy. CASP3, CASP6, CASP7, CASP8, and CASP10 mRNAs were detectable in conceptus on D12 and D15 of pregnancy, and chorioallantoic tissues expressed CASP3, CASP6, CASP7, CASP8, CASP8, and CASP10 with increasing levels toward term pregnancy, except CASP3 mRNA. The effect of steroid hormones and interleukin-1βß (IL1B) on CASP3 expression and the effect of interferon-γ(IFNG) on CASP7 expression was determined by endometrial explant cultures and we found that CASP3 expression was increased by IL1B and CASP7 expression was increased by IFNG in a dose-dependent manner. These results showed that caspases were expressed in the endometrium during the estrous cycle and pregnancy in a stage- and/or pregnancy-specific dependent manner and some caspases were regulated by IL1B or IFNG in the endometrial tissues, suggesting that caspase may play an important role in regulating apoptosis for the establishment and maintenance of pregnancy at the maternal-conceptus interface in pigs.

The present study was to assess the in vivo embryo production efficiency using the semen separated according to sex during superovulation in Hanwoo. Seventy Hanwoo donor cows were flushed on day 7 of estrus cycle with same FSH and artificial insemination by the same technicians. Embryos were recovered on 7 days after the third insemination by flushing the uterus with embryo collection medium. KPN semen straws used artificial insemination contained 20 million sperm (total number 60 million per donor). Sex-sorted semen straws contained 4 million sperm (total number 12 million per donor). The results obtained were as follows: No differences were observed in the efficiency of superovulation rates on KPN semen 87%, and sexed semen 100%, respectively. The mean numbers of total embryos are each 12.58 ± 8.31 and 13.25 ± 7.86. The mean numbers of transferable embryos, sexed semen were significantly lower than KPN semen (3.75 ± 1.98 vs. 8.23 ± 6.07, P<0.05). The rates of unfertilized embryos from superovulation using sexed semen were significantly higher than KPN semen (50% vs. 15%, P<0.05). The rate of degenerated 2-cell embryos from sexed and KPN semen was 60.87% and 11.11%, respectively (p<0.05). In conclusion, these results indicate that superovulation using sexed semen was useful, but efficient embryo production was important to reducing the damage caused by the Flowcytometer-based sperm sorting procedure.

Acute kidney injury (AKI) is a disorder that is manifested by a sudden decrease of renal function within several hours, and AKI remains a significant cause that can lead to increasing morbidity and mortality. Although AKI has been extensively studies in animal models, translating the results from animal studies into clinical use has not been successful due to various factors including basal etiology of kidney deficiency and comorbidities and the complexity of this pathology. As a golden parameter, measuring serum creatinine (SCr) and blood urea nitrogen (BUN) has been conventionally used for determining the renal function, however, these biomarkers has been regarded suboptimal to identify renal injuries in early stages. In this study, we attempted to screen other serum biomarkers in early AKI event using cynomolgus monkeys. Two male monkeys, aged 60 months, were subjected to ischemic injury by unilateral clamping of renal pedicles for forty five minutes and then subsequently reperfused; the unclamped kidney was regarded as non-injured controls. Compared with control kidneys, we have found that the concentration of several inflammatory proteins including MCP1, TGFα, GSTα, were higher in the renal vein of injured kidney compared with control side after 24 and 48 hours of AKI. However, changes of serum level of KIM-1, which is one of the most-widely studied marker in rodent studies, were not different after AKI. Our results provide an useful information while developing a novel marker in AKI.

Blood types in pigs are divided into two types, A and O type. It is important to select and breed O-type pigs that have no hematologic rejection, because the blood type of the donor is an important factor causing immune rejection in xenotransplantation. Therefore, the gene that determines the blood type is GGTA1(glycoprotein, alpha-1,3-galactosyltransferase 1), which generally belongs to the ABO blood group. This study was carried out to develop a simple and accurate method by analyzing the structure of GGTA1 gene which determines blood type. A primer was designed to allow easy identification of the blood type of the target in the first and second deletion regions of intron 7 to distinguish between the A and O genotypes. That is, for the purpose of identifying the blood type using length difference after the PCR, the forward and reverse primers were designed in the highly conservative region. As a result, a pair of primers were prepared and PCR amplification was performed to distinguish three types of genotypes, AA, AO, and OO, using the length difference by electrophoresis. Using the above primers, the parents and their offspring were compared with each other to confirm the correct genetic pattern. And, in four pig breeds, the genes were amplified and the genotype could be correctly identified. In this study, we could diagnose the blood type of AA, AO, OO genotype of pigs by using primer of INDEL region. Especially, since it is possible to diagnose the genotype by the length difference, it is possible to diagnose it quickly and accurately from the gene amplification to the genotype reading

Previous studies have shown that kisspeptin (Kp-10) is expressed in mammalian ovaries; however, the expression and role of Kp-10 in bovine ovarian granulosa cells are still unclear. In this study, we assessed the expression of Kp-10 and its effects on the proliferation and apoptosis of bovine granulosa cells. Immunohistochemical analysis showed that Kp-10 was expressed in the cytoplasm of bovine ovarian granulosa cells. Moreover, MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2- H-tetrazolium bromide) assays showed that 100 nM Kp-10 significantly inhibited the viability of granulosa cells (P<0.01). Flow cytometry analysis showed that Kp-10 could significantly increase accumulation of cells in the G1 phase, decrease accumulation of cells in the S phase, and promote apoptosis in bovine granulosa cells (P<0.05). Additionally, Kp-10 decreased the mRNA levels of Bcl-2, an anti-apoptotic gene; increased the mRNA levels of caspase-3, a pro-apoptotic gene; and increased the mRNA levels of Fas and Fasl, two membrane surface molecule genes (P<0.05). Thus, our findings demonstrated for the first time that Kp-10 inhibited proliferation and promoted apoptosis in bovine ovarian granulosa cells. These findings provide insights into our understanding of the role of Kp-10 in mediating the proliferation of bovine granulosa cells.

Transglutaminase (TGM2) belongs to a family of cross-linking enzymes responsible for catalyzing Ca2+-dependent acyl-transfer reactions between the substrate proteins. TGM2 is a cytosolic protein that has also been observed in the nucleus and can be expressed to the cell surface or extracellular matrix. Despite ubiquitous expression, its functions are poorly understood and still need to be elucidated. Moreover, there is no clear data regarding the role of transglutaminase in mammalian oocytes. So, in this study, we have patterned the transglutaminase 2 (TGM2) and anti-N epsilon gamma glutamyl lysine (AB424) activity in heat stressed mouse oocytes. We have collected mouse oocytes from the (6–9 weeks old) mouse and in vitro matured for 20 h. Immunocytochemistry was performed to checked the transglutaminase 2 (TGM2) and anti-N epsilon gamma glutamyl lysine (AB424) activity after 6 h of heat stress (HS) at 39.1 ℃. Both TGM2 and AB424 expression were significantly (P < 0.05) higher compared to control when oocytes were subjected to HS at 6 h of IVM at 39.1 ℃. Our hypothesis is that TGM2 and AB424 activity may be correlated with the cellular regression and also involvement in apoptosis. We hope that, our study will help to elucidate the normal function of mouse oocyte and also identification of the principal proteins as well as the pathogenic mechanism of altered physiology. These results suggest that the nuclear accumulation of the transglutaminase may play an important role in nuclear remodeling during folliculogenesis and early embryonic development

Variance of conceptus interferon tau (IFNT), produced by the embryonic trophectoderm, is known as a major conceptus protein that signals the process of maternal recognition of pregnancy in ruminants, essential for the maintenance of early pregnancy. Similar to other IFN genes such as IFNA and IFNB, multiple IFNT genes are present. However, some kinds of IFNT genes actively transcribed and regulated in bovine conceptuses have not been well characterized. In this study, during the course of bovine IFNT gene transcription through the use of next generation sequencer SOLiD3, revealed that among 38 IFN genes registered, only two transcripts, IFNT1 and IFNTc1, were found in conceptuses during early pregnancy. Also, to identify a transcription factor(s) involved in the regulation of IFNT genes, mRNAs for various known transcription factors were investigated by real-time PCR in conceptus tissues, respectively. Furthermore, compared to the IFNT genes, IFNT1 and IFNTc1 had same active levels, which were previously shown to correlate with the appearance of effective antiviral activity. However, the expression levels of these Luc activities differed. Bovine ear fibroblast (EF) cells were cotransfected with luciferase reporter constructs carrying upstream (–631 to -51) promoter regions of IFNT1 or IFNTc1 and various transcription factor expression plasmids, CDX2, AP1(JUN), ETS2 and/or cAMP-response element binding protein (CREB)-binding protein (CREBBP). CDX2, either alone with the other 2 transcription factors, was found to increase luciferase activity approximately 14- and 11-folds, respectively. The degree of transcriptional activation of the IFNTc1 gene was not similar to that IFNT1 gene by AP1, ETS2 or/and CREBBP, expression plasmid. These results suggest that two isoforms of bovine conceptus IFNT genes are regulated differently in conceptuses during early pregnancy.

To test the muscle cell specific gene expression, we examined the ability of human α-skeletal muscle actin (ACTA) promoter or human myoglobin (hMb) promoter to direct the expression of the GFP gene in both muscle and non-muscle cells, respectively. C2C12 cells, a mouse myoblast cell line, provide a powerful model to study skeletal muscle differentiation in vitro. We intended to use this cell line as a model for skeletal muscle-specific gene expression during myogenic differentiation from myoblast to myotubes. We compared marker gene expression profiles of proliferating and differentiated C2C12 cells using RT-PCR and fluorescent microscopy analysis. Also, we found that the expression of PCK1 gene under the control of ACTA promoter was proportionally increased as C2C12 differentiated into myotube form. PCK1 is involved in the regulation of gluconeogenesis. In previous research, transgenic mice with overexpressing PCK1 in skeletal muscle showed a greatly enhanced level of physical activity, which extends well into old age. This is due, in part, to an increased number of mitochondria and a high concentration of triglyceride in their skeletal muscles. These mice also had very little body fat, despite eating 60% more than controls. We also constructed a mesenchymal stem cell line and fetal fibroblast cell line for the experiments aiming to make transgenic animals in which the PCK1 gene is specifically expressed in muscle tissue. Accumulated knowledge of this approach could be applicable to a variety of related biological areas including transgenic animal research, gene function study, anti-aging study, etc. This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Export Promotion Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (316002-5).

Endoplasmic reticulum (ER) stress is well known as a suppressor in osteoblast differentiation and activating transcription factor 3 (ATF3) could be induced by a various extracellular signals including cytokines, hormones, DNA damage. Up to date, although the role of ATF3 have been studied, the function of ATF3 in osteoblast differentiation is still not clear yet. Our study showed that expression level of ATF3 could be incresed by tunicamycin which is ER stress inducer in preosteoblasts. BMPs, which are secreted by osteoblasts, can be important regulators in osteogenic differentiation. The stress-responsive transcription factor ATF3 is a negative regulator of osteoblast differentiation in MC3T3-E1 cells. In this study, we verified that BMP2-stimulated osteoblast differentiation could be inhibited by over-expressed ATF3 through regulating alkaline phosphatase (ALP) expression and activation.

일반적으로 세포·조직 및 장기이식 성공 예측은 수여자와 공여자간의 백혈구항원 일치도이고, 불일치 시 심각한 거부 반응을 유발함으로 세포치료제로 사용할 때 우선적으로 백혈구 항원일치도가 고려된다. 그러나 중간엽줄기세포(Mesenchymal Stem Cells, MSCs)는 다른 체세포와 비교하여 상대적으로 낮은 MHC I 항원발현과, 극히 낮은 MHC II 항원을 가지고 있으므로 동종세포치료제로서 주목을 받고 있다. 따라서 본 연구에서는 개 모델에서 MSCs 의 동종세포치료제로서 효능을 예측하기 위해 선행연구로 백혈구 항원(Dog Leukocyte Antigen, DLA)형 및 가계도내 일치도와 유전적다형성(Polymorphism) 을 분석하였다. DLA 분석을 위해 한가계도의 비글(Covance Beagles) 4 두(모견 1 두, 자견 3 두)로 부터 전혈을 채취하고, 밀도구배를 이용하여 백혈구만을 분리 후 DNA들을 각각 추출하였다. DLA 분석은 ClassII 유전자(DLA-DQA, DLA-DQB, DLA-DRB)에서 엑손 2 영역(약 300bp)을 증폭하고 Direct Sequencing 을 통해 밝혀진 염기서열을 NCBI Blast 와 IPD(Immuno Polymorphism Database)를 기반으로 하여 Universal nomenclature 에 따라 유전자형을 판독 하였다. 그 결과 DLA-DQA(022:01/022:01)와 DLA-DQB(107:01/102:01)는 4마리 모두 유전자형이 동일하였으나, DLA-DQB 는 각각 046:01/022:02, 03701/022:02, 00201/022:02, 03701/022:02 로 차이를 보였다. 이 결과를 통해 모견과 자견이 공통적으로 가지는 일배체형(Haplotype)은 DLA–DQA*022:01, DLA-DQB*022:02, DLA-DRB*102:01 이었음을 확인할 수 있었다. 그리고 일부 유전자의 염기서열에서 99% 유사도를 보이는 후보군들이 4 개씩 검색되었는데 이는 단일염기다형성(SNP)에 기인한 유전적다형성(Polymorphism)이 매우 높다는 선행보고들과 유사한 결과를 보였다. 본 실험결과는 향후 DLA 의 일치군과 비 일치군의 개중간엽줄기세포와 말초혈액단핵구세포(PBMC)들의 공배양을 통해 동종세포치료제 연구에 사용될 예정이다. * 본 성과물은 농촌진흥청 반려동물 연구사업(세부과제명 : 반려견에서 DLA 일치하는 줄기세포의 체외 치료능 평가, 세부과제 번호 : PJ013957022018)의 지원에 의해 이루어짐.

Sonic hedgehog (Shh) signaling pathway plays a key role in the development of various vertebrate embryos and remains important in adults. Although Shh signaling pathway has widely been studied in post-implantation stage embryos, only few studies are reported about pre-implantation stage embryos. To investigate the effect of Shh on pre-implantation stage embryos, cyclopamine and purmorphamine were treated to embryos in culture. Cyclopamine acts as an antagonist of the hedgehog signaling because it has a high affinity to Smoothened, a key part of the hedgehog signaling pathway. On the other hand, purmorphamine activate Smoothened and acts as a Shh signaling agonist. The oocytes were collected after superovulation and parthenogenetically activated in Chatot, Ziomek, and Bavister medium (CZB) including 10 mM strontium for 5 hr. The activated oocytes were cultured in potassium simplex optimized medium (KSOM), KSOM with 5 uM of cyclopamine, KSOM with 1 uM of purmorphamine, or KSOM with both 5 uM of cyclopamine and 1 uM of purmorphamine. After 5.5 days in culture, there was no significant difference in blastocyst development among the four experimental groups. However, the hatching rate was increased in the groups containing purmorphamine, and the blastocysts of the purmorphamine-containing groups had higher total cell number than those of other two groups when the cells were counted after Hoechst33342 staining. Quantitative real-time PCR (qRT-PCR) shows the difference of gene expression level which are related to epithelial-mesenchymal transition (EMT). Taken together, this study suggests that the increase of Shh has an effect on the increases of EMT-related genes and hatching rate of pre-implantation stage embryos, and this may improve implantation subsequently.

In porcine production, porcine litter size is a quantitative trait and its heritability is especially low. So it is necessary to identify porcine reproductive gene and protein. The establishment of pregnancy requires performance of a receptive endometrium and ovary. The endometrium and ovary go through transformations in response to physiological changes initiated by local factors including ovarian hormones and uterine environment that make it for possible pregnancy. The endometrium and ovary secrete a wide array of growth factors, cytokines and proteins. Based on these background, we analyzed the endometrial tissue protein of porcine and would find out biomarker proteins related to porcine litter size. We sorted the two groups according to litter size of porcine: a small litter size group (SLSG) (n=2) and a large litter size group (LLSG) (n=2). The porcine endometrial tissue and ovary samples were preprocessed for proteomic analysis. In order to comparison, samples of each 2mg endometrium protein and ovary protein were separated form pI and molecular weight in the same conditions by applying a pH 3.0-10.0 IPG gels for the first dimension and then 8-16% SDS-PAGE gel for the second dimension. After proteins were visualized by staining with Commassie brilliant blue (CBB), image analysis was performed with Image Master detect variations in protein spots between large litter size group and small litter size group endometrium. And then differential proteins were identified using MALDI-TOF analysis. The master images of 2-DE gel images obtained from 2mg samples of large litter size group and small litter size group endometrial proteins at pH 3.0-10.0 revealed more than 400 protein spots in pH 3.0-10.0 range. When we analyzed the levels of expression of proteins that protein spots appeared more than 1.5-fold difference in endometrial tissue from porcine. In comparison of SLSG(small litter size group) with LLSG(large litter size group), a total of 18 protein spots differentially expressed on porcine endometrial tissue 2-DE gels, among which 9 spots were up-regulated proteins as retinol dehydrogenase 16-like isoform 1, Acrosin-binding protein, alpha-N-acetylgalactosaminidase. phosphoglycerate kinase 2, Acrosin-binding protein in LLSG. And 8 spots were up-regulated proteins as phosphoglycerate kinase 2, prenylcysteine oxidase in SLSG.

The embryonic genome activation (EGA) is genetically activated states that embryos make the materials such as growth factors for using themselves. EGA is various because they have many materials, different site, different stage, also different species. At this time, transcription factors are expressed. Transcription factors bind to specific DNA region, and regulate the gene expression. Thus, we check the expression of transcription factors, we can know that embryo development is very well or not. The development stages of embryos are basically the stages from fertilization to blastocyst. So, we check the embryos oocyte to blastocyst. In our experiments, we focus the early developmental transcription factors such as Cdx2, Oct4, Sox2, Nanog and E-Cadherin. Above antibody factors showed different expression sites, and there were many differentiated parts from other animal species. In addition, we compared the SCNT and parthenogenetic activation (PA) because these are same methods using electrical activation among the embryo production methods. Our results showed not only similar patterns but also different patterns between pig and mouse. Therefore, we have to investigate that different patterns of transcription factors play a role in pigs, and why occur.

Prolactin is an anterior pituitary hormone involved in various physiological phenomenon including reproduction. The prolactin receptor (PRLR) is detected in diverse tissues such as brain, ovary, placenta and uterus in several mammalian species. A total of 227 pigs [Korean native pigs (KNP) 27; Landrace pigs 29; Korean native pigs x Landrace F1 91; Nanchuckmacdon pigs 80] were used to investigate the allele frequency difference of the prolactin receptor (PRLR) gene among the four pig lines. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with Alu I restriction enzyme was used to determine the genotypes of PRLR. Frequencies of PRLR alleles among the four different pig lines were not significantly different (Chi-square=3.94, DF=3, P=0.27). A total of 40 Nanchuckmacdon pigs were used to investigate the effect of the prolactin receptor (PRLR) gene on total number of piglets born (TNB), number of piglets of alive (NBA) using general linear model implemented in MINITAB software. For TNB, the AB genotype had higher genotypic value (10.61) than the values of AA (9.83) and BB (10.30). Likewise, the AB genotype had higher genotypic value (8.96) than the values of AA (8.18) and BB (8.90) for NBA. However, these associations of the PRLR gene with TNB and NBA were not statistically significant. In conclusion, it is necessary to increase the sample size for investigating the effect of the PRLR gene on TNB and NBA in pigs.

The aim of this study was to investigate the role of Src homology 2-containing phosphotyrosine phosphatase SHP2 in intricate signaling network invoked by oocyte to achieve cytoplasmic maturation and also blastocyst development. Activation of SHP2 regulates multicellular differentiation, proliferation and survival through numerous signal pathways. The most prominent pathway is RAS/PI3K and p-AKT signaling cascade, as a result mitogenic effect become enhanced. Oocytes were cultured in cisplatin an anticancer drug, but selective activator of SHP2 and our grouping were SOF medium alone, SOF + EGF, SOF + CISPLATIN 0.3 μM, and SOF + EGF + CISPLATIN 0.3 μM. We evaluated that EGF neutralizes the apoptotic effect of cisplatin as well as maintain the high expression of SHP2, as a result blastocyst development become boosted up. We also found that inhibition of SHP2 with its specific inhibitor PHPS1 5 μM decreases the blastocyst development and neutralizes growth factors effect. The developmental ability and quality of bovine embryos were determined by assessing their cell number, gene expression, immunofluorescence, and immunoblot. The differences in embryo development between experimental groups were analyzed by one-way ANOVA. Our results show that SHP2 have significant effect on MAP kinase pathways which expand the cumulus cells during oocyte maturation and blastocyst development as compare to inhibition of SHP2 with PHPS1. SHP2 not only transduce the signaling of epidermal growth factor but it also has a role in signal transduction of FGF and IGF. The expression of ERK, PI3K/p-AKT and mTOR was increased with EGF, but with the treatment of SHP2 inhibitor the expression of these genes become drop done. So we can conclude from these results that SHP2 is important for oocyte maturation as well as for blastocyst development.

Estrogen receptor 2 (ESR2) is involved in oestrogen-related apoptosis in cell cycle spermatogenesis but their effects have not yet confirmed in pig. Therefore, this study was performed to investigatetheir association with semen motility and kinematic characteristics. DNA samples from 126 Duroc pigs with records of sperm motility and kinematic characteristics [Total motile spermatozoa (MOT), Curvilinear velocity(VCL), Straight-line velocity(VSL), the ratio between VSL and VCL(LIN), Amplitude of Lateral Head displacement(ALH)] were used in present study. A single nucleotide polymorphism (g.35547A>G) was associated with MOT, VCL, VAP and ALH in Duroc population (p < 0.05). Therefore, we suggest that the porcine ESR2 may be used as a molecular marker for Duroc boar semen quality, although its functional effect was not clear yet. These results will improve the understanding of the functions of the ESR2 in spermatogenesis within the reproductive tracts, and will shed light on ESR2 as a candidate gene in the selection of good sperm quality boars.

Cryopreservation of boar semen is continually researched in reproductive technologies and genetic resource banking in breed conservation. For evaluating the boar semen quality, sperm motility (MOT) is an important parameter because the movement of spermatozoa indicates active metabolism, membrane integrity and fertilizing capacity. Recently, polymorphisms reported to be significant association with sperm MOT. This study was conducted to evaluate the SNP in the coding region of ESR1 (g.672C>T inexon 1) as a positional controlling for motility and kinematic characteristics of post-thawed boar semen. To results,The g.672C>T was significantly associated with frozen semen motility and kinematic characteristics. g.158 T>C SNP was high significantly associated with MOT, VCL, VSL and VAP Also, the SNP was low significantly associated with ALH.Therefore, we suggest that theSNP in the coding region of ESR1 (g.672C>T in exon 1) may be used as a molecular marker for Duroc boar Post-thawed semen quality.

Sow longevity is important for efficient and profitable pig farming. Recently, there has been an increasing interest in social genetic effect (SGE) of pigs on stress-tolerance and behavior. The present study aimed to estimate genetic correlations among average daily gain (ADG), stayability (STAY), and number of piglets born alive at the first parity (NBA1) in Korean Yorkshire pigs, using the SGE model. The phenotypic records of ADG and reproductive traits of 33,120 and 11,654 pigs, respectively, were evaluated. The direct effect on ADG had a significantly negative genetic relationship with STAY, whereas the social effect on ADG had a neutral genetic relationship. In addition, the genetic correlation between the social effects on ADG and NBA1 tended to be positive, unlike the direct effects. The genetic correlation of the total effect on ADG with that of STAY was negative but non-significant, owing to the social effect. These results suggested that total genetic effect on growth in the SGE model might reduce the negative effect on sow longevity owing to the growth potential of pigs. We recommend including social effects as selection criteria in breeding programs to obtain satisfactory genetic changes in both growth and longevity.

The porcine zygotic genome activation occurs along with global epigenetic remolding at the 4-cell stage. The histone acetylation, regulating DNA transcription, replication and so on, requires adequate acetyl-CoA. Acetyl-CoA produced by translocated pyruvate dehydrogenase in the nucleus of mammalian cells has been reported, which is commonly considered locating in the mitochondria. To find out whether the nuclear pyruvate dehydrogenase regulating the histone acetylation by controlling generation of acetyl-CoA, a multiple sgRNAs-CRISPR/Cas9 targeting strategy was employed to generate a pyruvate dehydrogenase E1 alpha1 (Pdha1) knockout (KO) parthenogenetic embryo model. Results showed that the targeting efficiency of Pdha1 reached more than 90%. Hence, this model was used in the subsequent experiments. Furthermore, a translocation of Pdha1 during zygotic genome activation was found by immunofluorescent staining and was significantly inhibited by Pdha1 KO. Meanwhile, the 8-cell stage embryo rate significantly decreased after 72 h (24.19% vs 12.53%, control vs Pdha1 KO), indicating a 4-cell arrest. In addition, the nuclear histone acetylation level significantly decreased when Pdha1 was KO. To determine whether the zygotic genome transcription was affected, the qPCR was performed and showed that the mRNA level of Eif1A, Acly, Sqle and Pdha1 all dropped significantly in the Pdha1 KO group compared to the control. In conclusion, the translocated Pdha1 generates acetyl-CoA for histone acetylation inside the nucleus of porcine embryos, which promotes the zygotic genome activation of porcine embryos.

The transcription factor POU5F1, also known as OCT4 plays critical roles in maintaining pluripotency during early mammalian embryonic development and in embryonic stem cells. It is important to establish an OCT4 promoter region-based reporter system to study pluripotency. However, there is still a lack of information about the porcine OCT4 upstream region. To improve our understanding of the porcine OCT4 regulatory region, we identified conserved regions in the porcine OCT4 promoter upstream region by sequence-based comparative analysis using various mammalian genome sequences. The similarity of nucleotide sequences in the 5' upstream region was low among mammalian species. However, the OCT4 promoter and four regulatory regions, including distal and proximal enhancer elements, had high similarity. The putative transcription factor binding sites in the Oct4 5' upstream region nucleotide sequences from mice and pigs also differed. Some of these genes are related to pluripotency, and further research will allow us to better understand the differences in species-specific pluripotency. Next, a functional analysis of the porcine OCT4 promoter region was conducted. Luciferase reporter assay results indicated that the porcine OCT4 distal enhancer and proximal enhancer were highly activated in mouse embryonic stem cells and embryonic carcinoma cells, respectively. Similar to OCT4 upstream-based reporter systems derived from other species, the porcine OCT4 upstream region-based reporter constructs showed exclusive expression patterns depending on the state of pluripotency. This work provides basic information about the porcine OCT4 upstream region and various porcine OCT4 fluorescence reporter constructs, which can be applied to study species-specific pluripotency in early embryo development and the establishment of embryonic stem cells in pigs. This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education (NRF-2017R1D1A1B03032256).