Porcine Embryos Research Articles

Lipofection-Mediated Introduction of CRISPR/Cas9 System into Porcine Oocytes and Embryos.

Simple SummaryLiposome-mediated gene transfer has become an alternative method for establishing a gene targeting framework, and the production of mutant animals may be feasible even in laboratories without specialized equipment. However, whether blastocyst genome editing can be performed by treatment with lipofection reagent, guide RNA, and Cas9, without performing electroporation or microinjection, remains unclear. In this study, we demonstrated that lipofection treatment successfully induced mutation into zygotes during in vitro fertilization and in embryos at the 2- and 4-cell stages. Although liposome-mediated gene editing is a feasible system for use with zona-pellucida-free oocytes/embryos, several challenges must be overcome.Liposome-mediated gene transfer has become an alternative method for establishing a gene targeting framework, and the production of mutant animals may be feasible even in laboratories without specialized equipment. However, how this system functions in mammalian oocytes and embryos remains unclear. The present study was conducted to clarify whether blastocyst genome editing can be performed by treatment with lipofection reagent, guide RNA, and Cas9 for 5 h without using electroporation or microinjection. A mosaic mutation was observed in blastocysts derived from zona pellucida (ZP)-free oocytes following lipofection treatment, regardless of the target genes. When lipofection treatment was performed after in vitro fertilization (IVF), no significant differences in the mutation rates or mutation efficiency were found between blastocysts derived from embryos treated at 24 and 29 h from the start of IVF. Only blastocysts from embryos exposed to lipofection treatment at 29 h after IVF contained biallelic mutant. Furthermore, there were no significant differences in the mutation rates or mutation efficiency between blastocysts derived from embryos at the 2- and 4-cell stages. This suggests that lipofection-mediated gene editing can be performed in ZP-free oocytes and ZP-free embryos; however, other factors affecting the system efficiency should be further investigated.

SIN3A Regulates Porcine Early Embryonic Development by Modulating CCNB1 Expression.

SIN3A is the central scaffold protein of the SIN3/histone deacetylase (HDAC) transcriptional repressor complex. SIN3A participates in the mouse preimplantation development by fine-tuning HDAC1 expression. However, it remains unresolved if this functional significance of SIN3A was conserved in other mammals. Herein, RNA-seq results show a large amount of SIN3A mRNA is present in oocytes and early embryos prior to embryonic genome activation and a low amount thereafter, suggesting a maternal origin of SIN3A in pigs, cattle, mice, and humans. Interestingly, immunofluorescence data show that SIN3A protein level peaks at four-cell stage in pigs compared with morula stage in cattle. SIN3A depletion in early embryos causes a developmental arrest at two-cell stage in pigs but does not affect bovine early embryonic development. In contrast with mouse data, SIN3A depletion results in only a slight decrease and even no difference in HDAC1 expression in porcine and bovine early embryos, respectively. In addition, HDAC1 knockdown does not cause two-cell block but leads to a reduced blastocyst rate. By using unbiased RNA-seq approach, we found that Cyclin B1 (CCNB1) transcript level is dramatically reduced. Moreover, CCNB1 knockdown results in a similar phenotype as SIN3A depletion. Injection of exogenous CCNB1 mRNA into SIN3A-depleted embryos could partly rescue embryonic development to pass two-cell stage. In conclusion, our results indicate SIN3A plays an essential role in porcine early embryonic development, which probably involves the regulation of CCNB1 expression.

In vitro Electroporation in the Presence of CRISPR/Cas9 Reagents as a Safe and Effective Method for Producing Biallelic Knock-Out Porcine Embryos

The production of genetically modified (GM) pigs is considered valuable in biomedical research for the development of model animals for various diseases and pigs with resistance against viral infection. The porcine genome may be modified using several methods, such as somatic cell nuclear transfer (SCNT) using GM cells as the SCNT donor, direct injection of the transgene or the genome editing components (GEC) into fertilized eggs referred to as zygotes, the in vitro electroporation (EP) of the zygotes in the presence of GECs, viral infection using retroviruses, injection of the GECs into the SCNT-treated embryos, and the in vitro EP of the SCNT-treated embryos in the presence of GECs. In our previous study, we administered a cytoplasmic injection of CRISPR/Cas9-based GEC into parthenogenetically-activated porcine oocytes (referred to as parthenotes) and observed that these oocytes comprised a mixture of genome-edited and genome-unedited cells, referred to as the “mosaic”. In contrast, when in vitro EP of the SCNT-treated embryos in the presence of GEC was performed, bi-allelic knock out (KO) of the target gene was detected in most oocytes (82%; 9/11). The production of bi-allelic KO piglets is particularly beneficial for investigating GM domestic animals as it does not require further breeding trials to obtain bi-allelic KO individuals, which would otherwise be a time-consuming and laborious task. In this context, the present study was aimed to confirm the efficiency of in vitro EP in producing bi-allelic KO porcine embryos without multiple breeding trials, for which parthenotes were subjected to EP in the presence of a ribonucleoprotein containing Cas9 protein and single-guide RNA (targeted toward GGTA1). The treated embryos were cultured until they transformed into blastocysts. The genomic DNA isolated from these blastocysts was used for molecular biology analysis to detect the possible insertion and deletion of sequences (indels) at the GGTA1 locus. Among the 32 blastocysts obtained, 21 (66%) were observed to be the bi-allelic KO ones. The remaining embryos either had a normal phenotype (25%; 8/32) or mosaic mutations (9%; 3/32). These findings confirm the efficiency of in vitro EP in producing bi-allelic KO porcine embryos.

In vitro Electroporation in the Presence of CRISPR/Cas9 Reagents as a Safe and Effective Method for Producing Biallelic Knock-Out Porcine Embryos

The production of genetically modified (GM) pigs is considered valuable in biomedical research for the development of model animals for various diseases and pigs with resistance against viral infection. The porcine genome may be modified using several methods, such as somatic cell nuclear transfer (SCNT) using GM cells as the SCNT donor, direct injection of the transgene or the genome editing components (GEC) into fertilized eggs referred to as zygotes, the in vitro electroporation (EP) of the zygotes in the presence of GECs, viral infection using retroviruses, injection of the GECs into the SCNT-treated embryos, and the in vitro EP of the SCNT-treated embryos in the presence of GECs. In our previous study, we administered a cytoplasmic injection of CRISPR/Cas9-based GEC into parthenogenetically-activated porcine oocytes (referred to as parthenotes) and observed that these oocytes comprised a mixture of genome-edited and genome-unedited cells, referred to as the “mosaic”. In contrast, when in vitro EP of the SCNT-treated embryos in the presence of GEC was performed, bi-allelic knock out (KO) of the target gene was detected in most oocytes (82%; 9/11). The production of bi-allelic KO piglets is particularly beneficial for investigating GM domestic animals as it does not require further breeding trials to obtain bi-allelic KO individuals, which would otherwise be a time-consuming and laborious task. In this context, the present study was aimed to confirm the efficiency of in vitro EP in producing bi-allelic KO porcine embryos without multiple breeding trials, for which parthenotes were subjected to EP in the presence of a ribonucleoprotein containing Cas9 protein and single-guide RNA (targeted toward GGTA1). The treated embryos were cultured until they transformed into blastocysts. The genomic DNA isolated from these blastocysts was used for molecular biology analysis to detect the possible insertion and deletion of sequences (indels) at the GGTA1 locus. Among the 32 blastocysts obtained, 21 (66%) were observed to be the bi-allelic KO ones. The remaining embryos either had a normal phenotype (25%; 8/32) or mosaic mutations (9%; 3/32). These findings confirm the efficiency of in vitro EP in producing bi-allelic KO porcine embryos.

Melatonin protects against defects induced by Enniatin B1 during porcine early embryo development.

Exogenous factors influence embryo development. Enniatin B1 (EB1), one emerging mycotoxin of Fusarium fungi, can cause damage to cells and mouse blastocysts. However, the toxicity of EB1 on porcine embryo development and whether melatonin can eliminate the detrimental effects of EB1 on embryos remain unclear. Here, this work demonstrated that EB1 significantly decreased the cleavage and blastocyst rates and blastocyst cell number of embryos in a dose and time dependent manner. Further study displayed that EB1 obviously destroyed nuclear remodeling dynamics. Importantly, EB1 triggered embryo apoptosis through downregulating the expression of Sod1, Gpx4, Cat and Bcl2l1 while upregulating the transcription of Bax and Caspase3. Moreover, EB1 significantly disrupted the transcription of Dnmt1, Dnmt3a, Tet1 and Tet3, further leading to incomplete DNA demethylation of CenRep, Oct4, Nanog and Sox2, thus, the expression of Eif1a, Oct4, Nanog and Sox2 remarkably decreased. Whereas EB1-exposed embryos were treated with melatonin, these disorders were obviously ameliorated, and the development ability of embryos was also rescued. In conclusion, EB1 exerted detrimental effects on porcine early embryos, while melatonin effectively rescued EB1-mediated defects in embryos. This work provides a novel insight into the improvement of embryo quality and the promotion of human and animal reproduction.

Adiponectin Improves In Vitro Development of Cloned Porcine Embryos by Reducing Endoplasmic Reticulum Stress and Apoptosis.

Simple SummarySuccessful attenuation of endoplasmic reticulum (ER) stress signaling has a beneficial outcome in in vitro embryonal improvement. We evaluated the effect of adiponectin during in vitro culture in porcine embryos derived from parthenogenetic activation and somatic cell nuclear transfer (SCNT). We found that 15 and 30 μg/mL adiponectin treatment significantly improved cleavage rates, blastocyst formation rates, and total cell number (TCN) of blastocysts derived from parthenogenetic activation and reduced the expression levels of XBP1. In SCNT embryos, the cleavage rate, blastocyst formation rate, and TCN of blastocysts were significantly improved by 15 μg/mL adiponectin treatment compared to the control. In addition, the 15 μg/mL adiponectin treatment reduced the levels of XBP1 expression and ER stress-related genes, increased expression levels of pluripotency-related genes, and decreased apoptosis-related gene expression. Comprehensively, treatment with 15 μg/mL adiponectin enhanced the in vitro developmental capacity of early-stage SCNT porcine embryos by reducing ER stress and apoptosis.The main factor of embryonic demise is endoplasmic reticulum (ER) stress. Successful attenuation of ER stress results in an improvement in embryo development. We studied the impact of adiponectin in the in vitro culture (IVC) of porcine embryos derived from parthenogenetic activation and somatic cell nuclear transfer (SCNT). The first experiment revealed that 15 and 30 μg/mL adiponectin treatments improved cleavage, blastocyst rates, and total cell number (TCN) of parthenogenetic embryos and reduced the expression of XBP1 compared to the 5 μg/mL adiponectin treatment and control groups (p < 0.05). The second experiment showed that cleavage rate, blastocyst formation rate, and TCN of blastocysts were improved in the 15 μg/mL adiponectin treatment group compared with the control group, with significantly reduced XBP1 expression in ≥4-cell stage SCNT embryos and blastocysts (p < 0.05). Treatment with 15 μg/mL adiponectin significantly improved the expression of XBP1 and reduced the expression of ER stress-related genes (uXBP1, sXBP1, PTPN1, and ATF4), increased the expression levels of pluripotency-related genes (Nanog and SOX2), and decreased apoptosis-related gene expression (Caspase-3). These results suggest that 15 μg/mL adiponectin enhanced the in vitro developmental capacity of early-stage SCNT porcine embryos by reducing ER stress and apoptosis.

Lycopene Improves In Vitro Development of Porcine Embryos by Reducing Oxidative Stress and Apoptosis

In vitro culture (IVC) for porcine embryo development is inferior compared to in vivo development because oxidative stress can be induced by the production of excessive reactive oxygen species (ROS) under high oxygen tension in the in vitro environment. To overcome this problem, we investigated the effect of lycopene, an antioxidant carotenoid, on developmental competence and the mechanisms involved in mitochondria-dependent apoptosis pathways in porcine embryos. In vitro fertilized (IVF) embryos were cultured in IVC medium supplemented with 0, 0.02, 0.05, 0.1, or 0.2 μM lycopene. The results indicate that 0.1 μM lycopene significantly increased the rate of blastocyst formation and the total cell numbers, including trophectoderm cell numbers, on Day In terms of mitochondria-dependent apoptosis, IVF embryos treated with 0.1 μM lycopene exhibited significantly decreased levels of ROS, increased mitochondrial membrane potential, and decreased expression of cytochrome c on Days 2 and Furthermore, 0.1 μM lycopene significantly decreased the number and percentage of caspase 3-positive and apoptotic cells in Day-6 blastocysts. In addition, Day-2 embryos and Day-6 blastocysts treated with 0.1 μM lycopene showed significantly reduced mRNA expression related to antioxidant enzymes (SOD1, SOD2, CATALASE) and apoptosis (BAX/BCL2L1 ratio). These results indicate that lycopene supplementation during the entire period of IVC enhanced embryonic development in pigs by regulating oxidative stress and mitochondria-dependent apoptosis.

Induction of autophagy protects against extreme hypoxia-induced damage in porcine embryo.

In the mammalian female reproductive tract, physiological oxygen tension is lower than that of the atmosphere. Therefore, to mimic in vivo conditions during in vitro culture (IVC) of mammalian early embryos, 5% oxygen has been extensively used instead of 20%. However, the potential effect of hypoxia on the yield of early embryos with high developmental competence remains unknown or controversial, especially in pigs. In the present study, we examined the effects of low oxygen tension under different oxygen tension levels on early developmental competence of parthenogenetically activated (PA) and in vitro-fertilized (IVF) porcine embryos. Unlike the 5% and 20% oxygen groups, exposure of PA embryos to 1% oxygen tension, especially in early-phase IVC (0-2 days), greatly decreased several developmental competence parameters including blastocyst formation rate, blastocyst size, total cell number, inner cell mass (ICM) to trophectoderm (TE) ratio, and cellular survival rate. In contrast, 1% oxygen tension did not affect developmental parameters during the middle (2-4 days) and late phases (4-6 days) of IVC. Interestingly, induction of autophagy by rapamycin treatment markedly restored the developmental parameters of PA and IVF embryos cultured with 1% oxygen tension during early-phase IVC, to meet the levels of the other groups. Together, these results suggest that the early development of porcine embryos depends on crosstalk between oxygen tension and autophagy. Future studies of this relationship should explore the developmental events governing early embryonic development to produce embryos with high developmental competence in vitro.

MicroRNA-210 Regulates Endoplasmic Reticulum Stress and Apoptosis in Porcine Embryos.

Simple SummaryThe purpose of this study was to explore the effect of miR-210 on in vitro embryo development, mRNA expression related endoplasmic reticulum (ER) stress. Treatment with a miR-210-inhibitor significantly improved in vitro embryo development and total blastocyst cell number (TCN). Furthermore, miR-210-inhibitor treatment downregulated ER stress and apoptosis-related gene expression, while simultaneously improving embryo capacity. In contrast, a miR-210-mimic decreased in vitro embryo development, TCN, upregulated ER stress and apoptosis genes, and concomitantly impaired embryo quality. Therefore, we suggest that miR-210 plays an important role in porcine in vitro embryo development.Endoplasmic reticulum (ER) stress can be triggered during in vitro embryo production and is a major obstacle to embryo survival. MicroRNA (miR)-210 is associated with cellular adaptation to cellular stress and inflammation. An experiment was conducted to understand the effects of miR-210 on in vitro embryo development, ER stress, and apoptosis; to achieve this, miR-210 was microinjected into parthenogenetically activated embryos. Our results revealed that miR-210 inhibition significantly enhanced the cleavage rate, blastocyst formation rate, and total cell number (TCN) of blastocysts, and reduced expression levels of XBP1 (p < 0.05). miR-210 inhibition greatly reduced the expression of ER stress-related genes (uXBP1, sXBP1, ATF4, and PTPN1) and Caspase 3 and increased the levels of NANOG and SOX2 (p < 0.05). A miR-210-mimic significantly decreased the cleavage, blastocyst rate, TCN, and expression levels of XBP1 compared with other groups (p < 0.05). The miR-210-mimic impaired the expression levels of uXBP1, sXBP1, ATF4, PTPN1, and Caspase 3 and decreased the expression of NANOG and SOX2 (p < 0.05). In conclusion, miR-210 plays an essential role in porcine in vitro embryo development. Therefore, we suggest that miR-210 inhibition could alleviate ER stress and reduce apoptosis to support the enhancement of in vitro embryo production.

Transcriptome and epigenome analysis of porcine embryos from non-esterified fatty acid-exposed oocytes

Increasing evidence indicates that maternal malnutrition leads to decreased female fertility and dysregulated metabolic homeostasis in offspring. High levels of non-esterified fatty acids (NEFAs) in follicular fluid were reported to be involved in these maternal nutritional effects, but the mechanisms remain unclear. This study explored the mechanisms of action of abnormal NEFA levels during porcine oocyte in vitro maturation (IVM) on early embryo development (blastocysts) using phenotypic, transcriptomic, and epigenetic analysis. The oocytes were treated during IVM with, in addition to the 1% (v/v) porcine follicular fluid in the control group, a combination of 468 μmol/L palmitic acid, 194 μmol/L stearic acid, and 534 μmol/L oleic acid supplemented to North Carolina State University-23 (NCSU-23) maturation medium to achieve a high level of NEFAs. After IVM, oocytes were in vitro fertilized and then cultured in regular conditions for blastocysts. Expanded blastocysts were collected to complete transcriptomic and epigenetic analysis. Macroscopically, high level of NEFAs impaired embryo development by reducing the blastocyst rate. Analysis of the transcriptome revealed that pathways related to inflammation, apoptosis, metabolism, and oxidative stress were the most affected. Moreover, DNA methylation data demonstrated differentially methylated regions in genes related to cellular metabolism and inflammation processes. Therefore, our conclusion is that high level of NEFAs during IVM might affect porcine early embryo development by diminishing blastocyst rate and altering gene expression, especially at the metabolism and cell status levels, which could further decrease the embryo quality.

Supplementation of fetal bovine serum increased the quality of in vitro fertilized porcine embryo.

Objective:The present study aimed to explain the effect of fetal bovine serum (FBS) on the in vitro production of porcine embryos and the molecular effects of FBS on the growing of porcine embryos.Materials and Methods:Immature porcine oocytes were matured and fertilized in vitro. The resulting zygotes were cultured in porcine zygotic medium-3- until day 7 and FBS was added on day 4. Without FBS, it was treated as a control group. Quantitative real-time PCR and 2′,7′-dichloro-dihydro-fluorescein diacetate (H2DCFDA) molecular staining techniques were used to detect the expression patterns of apoptosis-associated genes and the accumulation of reactive oxygen species (ROS), respectively. Paired student’s t-test was used by GraphPad Prism statistical software.Results:FBS supplementation boosted blastocyst (BL) development and total cell count per BL substantially (p < 0.05). However, hatching and hatched BLs also increased in the FBS-treated group compared to the control. We also found that ROS accumulation in FBS-treated embryos was significantly reduced (p < 0.05) compared to the control group. The expression of the anti-apoptotic gene BCL-2 was significantly increased in FBS-treated BLs, but the pro-apoptotic gene, caspase-3 expression, was significantly reduced in FBS-treated BLs.Conclusion:Our results suggest that FBS supplementation in porcine culture media could increase porcine embryo production by decreasing ROS accumulation and increasing the anti-apoptotic gene expression in developing BLs.

42 Disruption of endogenous SOX2 during porcine embryo development using the CRIPSR/Cas9 system

The lineage specification of the pre-implantation embryo is important to understand the developmental process, but it remains unclear because the expression of lineage-specific genes is distinct among species. Pigs have genetic and physiological traits similar to humans; however, there are differences in gene expression during the pre-implantation stage. To select a candidate gene that affects the formation of the inner cell mass (ICM) in porcine embryo, we conducted preliminary experiments. First, we measured the expression level of candidate genes for lineage specification in parthenogenetic-activated embryos. The expression of pluripotent genes peaked on Day 3 and thereafter decreased gradually. Next, we conducted immunocytochemistry. OCT4 was expressed in all cells in morula and Day 5 blastocyst, but some Day 7 blastocysts expressed OCT4 in both ICM and trophectoderm (TE), whereas others expressed OCT4 only in ICM. NANOG was not observed in the morula stage, whereas SOX2 was located in a restricted area. To examine the effect of SOX2 in ICM formation, we injected plasmid expressing Cas9 and guide (g)RNA using Lipofectamine for efficient transgene expression at the 2-cell stage to increase viability by inducing mosaicism. The expression of enhanced green fluorescent protein (EGFP) contained in the plasmid confirmed that the plasmid was operating normally. In SOX2-knockout (KO) early blastocysts, the numbers of total cells and SOX2- and NANOG-positive cells were greatly decreased, while OCT4 was expressed in most cells. As in early blastocysts, SOX2-KO late blastocysts had fewer cells expressing SOX2, NANOG, and SOX17 than control. To identify the transcriptional consequences of SOX2 reduction, we performed quantitative PCR analysis on non-injected and PX458-gRNA injected blastocysts. Injection of PX458-gRNA resulted in downregulation of NANOG, SOX17, and SMAD7, but not SOX2 and OCT4. Furthermore, proliferation-associated genes were downregulated in injected blastocysts. In conclusion, SOX2-targeted porcine embryos showed blastocoel formation, the inner cell mass formed poorly, and embryos have inefficient cells. Also, the depletion of SOX2 in porcine blastocysts downregulated pluripotent genes and proliferation genes. This work was supported by the BK21 Plus Program, the National Research Foundation of Korea (NRF) grant funded by the Korea government (NRF-2019R1C1C1004514), the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through the Development of High Value-Added Food Technology Program funded by the Ministry of Agriculture, Food, and Rural Affairs (MAFRA; 118042-03-3-HD020).

Roles of cell differentiation factors in preimplantation development of domestic animals.

In mammalian embryos, the first visible differentiation event is the segregation of the inner cell mass (ICM) and trophectoderm (TE) during the transition from the morula to the blastocyst stage. The ICM, which is attached to the inside of the TE, develop into the fetus and extraembryonic tissues, while the TE, which is a single layer surrounding the fluid-filled cavity called the blastocoel, will provide extraembryonic structures such as the placenta. ICM/TE differentiation is regulated by the interaction between various transcriptional factors. However, little information is available on the segregation of the ICM and TE lineages in preimplantation embryos of domestic animals, such as cattle and pigs. This review focuses on the roles of cell differentiation factors that regulate the ICM/TE segregation of preimplantation bovine and porcine embryos. Understanding the mechanism of cell differentiation in early embryos is necessary to improve the in vitro production systems for bovine and porcine embryos.

Mitochondrial reactive oxygen species regulate mitochondrial biogenesis in porcine embryos.

The number of mitochondria in blastocysts is a potential marker of embryo quality. However, the molecular mechanisms governing the mitochondrial number in embryos are unclear. This study was conducted to investigate the effect of reduced mitochondrial reactive oxygen species (ROS) levels on mitochondrial biogenesis in porcine embryos. Oocytes were collected from gilt ovaries and activated to generate over 4 cell-stage embryos at day 2 after activation. These embryos were cultured in media containing either 0.1 μM MitoTEMPOL (MitoT), 0.5 μM Mitoquinol (MitoQ), or vehicle (ethanol) for 5 days to determine the rate of development to the blastocyst stage. The mitochondrial number in blastocysts was evaluated by real-time polymerase chain reaction (PCR). Five days after activation, the embryos (early morula stage) were subjected to immunostaining to determine the expression levels of NRF2 in the nucleus. In addition, the expression levels of PGC1α and TFAM in the embryos were examined by reverse transcription PCR. One day of incubation with the antioxidants reduced the ROS content in the embryos but did not affect the rate of development to the blastocyst stage. Blastocysts developed in medium containing MitoT had lower mitochondrial DNA copy numbers and ATP content, whereas MitoQ showed similar but insignificantly trends. Treatment of embryos with either MitoT or MitoQ decreased the expression levels of NRF2 in the nucleus and levels of PGC1α and TFAM. These findings indicate that reductions in mitochondrial ROS levels are associated with low mitochondrial biogenesis in embryos.

4 Aryl hydrocarbon receptor targets are upregulated in porcine blastocyst-stage embryos that were cultured invitro: A transcriptional analysis

Invitro-produced (IVP) porcine embryos are developmentally delayed compared with those derived invivo. Thus, efforts have been made to modify the current medium for IVP of porcine embryos in an attempt to shift the abundance of target transcripts towards the invivo level. The objective of the current study was to identify differences in mRNA abundance that may account for reduced developmental competence in IVP embryos and to determine whether alterations to maturation and culture media directed the transcriptional profiles of IVP embryos towards an invivo state. Following AI of gilts, an oviduct and tip of the uterine horn were flushed on Day 2 to recover 4-cell stage embryos; these were cultured for 4 days in MU3, generating IVM and invitro-cultured (IVC) blastocyst-stage embryos. On Day 6, the gilts were killed, and the contralateral horns were flushed to obtain invivo-derived (IVV) blastocyst-stage embryos. The third group of blastocyst-stage embryos, referred to as invitro-matured and cultured (IVMC), were created by aspirating cumulus–oocyte complexes from slaughterhouse-derived ovaries, maturing and fertilizing invitro, and culturing for 6 days in MU3. Total RNA was extracted from pools of 10 blastocyst-stage embryos with 3 replicates per group. First- and second-strand cDNA was synthesised and sequenced by using the Illumina platform (Illumina Inc.). After removal of adapters and reads that mapped to porcine rRNA genes and the PhiX genome, reads were mapped to the Sus scrofa genome by using STAR (version 2.7.1a) with default options. Pairwise comparisons were performed to test for differential expression of genes by using the Bioconductor package DESEqn 2. Transcripts were differentially abundant (false discovery rate &amp;lt;0.05) between IVV and IVC embryos (2,450), between IVV and IVMC embryos (3,045), and between IVC and IVMC embryos (262). Pathways related to cell cycle were downregulated in IVC and IVMC compared with IVV embryos, and pathways related to amino acid transport in metabolism were upregulated in IVC and IVMC compared with IVV embryos. Of particular interest, message for cytochrome P450 family 1 subfamily A member 1 (CYP1A1) was only present in the IVC (956 reads) and IVMC (381 reads) embryos but not in the IVV embryos (0 reads). The aryl hydrocarbon receptor (AHR) promotes transcription of CYP1A1, which encodes a monooxygenase involved in xenobiotic metabolism. Moreover, abundance of 12 other AHR targets was increased in IVC and IVMC embryos (false discovery rate &amp;lt;0.05) compared with IVV embryos. Thus, production of porcine embryos invitro may activate AHR, resulting in altered transcriptional profiles and reduced competence. This research was funded by USDA-NIFA (2019-67011-29543).

97 Function of species-specific OCT4 reporter system during porcine embryo development

The present study examined the activity and function of pig OCT4 (POU5F1) enhancer in porcine early embryo development. OCT4 is one of the master regulators for the pluripotency of early mammalian embryonic development and embryonic stem cells. It has two regulatory elements: distal enhancer (DE) and proximal enhancer (PE) in the upstream regulatory region. They are activated under different conditions such as naïve and primed. It is known that two enhancers are activated to produce OCT4 simultaneously or sequentially depending on the state of pluripotency. Many porcine OCT4 upstream region-based reporter systems have been reported because this is a necessary part of studying porcine-specific pluripotency. However, the porcine-specific OCT4 reporter system has never been transfected and functionally tested during porcine early embryo development. We performed functional tests of the previously established porcine-specific OCT4 reporter system in the early embryo development stage. Porcine embryos were micro-injected with the pOCT4-ΔPE-eGFP (DE-GFP) containing a distal enhancer and core promoter and pOCT4-ΔDE-DsRed2 (PE-RFP) containing a proximal enhancer and core promoter. They were cultured in PZM-3 at 39°C in a humidified atmosphere, 5% CO2, and 5% O2 for 168h. Fifty of the 100 embryos were injected with OCT4 reporter system as treatment groups and the other 50 were not injected (control groups). We analysed mRNA and protein expression of GFP and RFP using quantitative real-time PCR and confocal microscopy by developmental stages. The introduced reporter system could function beginning with the 4-cell stage. The expression of GFP and RFP was observed simultaneously in the early embryonic development stage up to blastocyst, indicating that porcine OCT4 was produced by distal and proximal enhancers, unlike mouse Oct4 expression, which was only controlled by the distal enhancer during early embryo development. Therefore, the mechanisms and functions of distal and proximal enhancers of porcine OCT4 were different from those of the mouse. This is similar to results of the previous experiment using porcine-induced pluripotent stem cells, which suggest that porcine OCT4 is expressed by two enhancers and in a stage-specific manner during reprogramming and that pigs do not use only one enhancer in pluripotent states, but two enhancers at the same time, and there is only a difference in degree. This work showed that the porcine OCT4 reporter system enables non-destructive analysis during early embryo development and it could be applied to study species-specific pluripotency and to help the establishment of naïve embryonic stem cells in the pig. This work was supported by the BK21 Plus Program, the National Research Foundation of Korea (NRF) grant funded by the Korea government (NRF-2019R1C1C1004514), the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through the Development of High Value-Added Food Technology Program funded by the Ministry of Agriculture, Food, and Rural Affairs (MAFRA; 118042-03-3-HD020).

Introduction of a point mutation in the KRAS gene of in vitro fertilized porcine zygotes via electroporation of the CRISPR/Cas9 system with single-stranded oligodeoxynucleotides.

This study aimed to investigate the efficiency of KRAS gene editing via CRISPR/Cas9 delivery by electroporation and analyzed the effects of the non-homologous end-joining pathway inhibitor Scr7 and single-stranded oligodeoxynucleotide (ssODN) homology arm length on introducing a point mutation in KRAS. Various concentrations (0-2µM) of Scr7 were evaluated; all concentrations of Scr7 including 0µM resulted in the generation of blastocysts with a point mutation and the wild-type sequence or indels. No significant differences in the blastocyst formation rates of electroporated zygotes were observed among ssODN homology arm lengths, irrespective of the gRNA (gRNA1 and gRNA2). The proportion of blastocysts carrying a point mutation with or without the wild-type sequence and indels was significantly higher in the ssODN20 group (i.e., the group with a ssODN homology arm of 20bp) than in the ssODN60 group (gRNA1: 25.7% vs. 5.4% and gRNA2: 45.5% vs. 5.9%, p<.05). In conclusion, the CRISPR/Cas9 delivery with ssODN via electroporation is feasible for the generation of point mutations in porcine embryos. Further studies are required to improve the efficiency and accuracy of the homology-directed repair.

Meta-analysis of melatonin treatment and porcine somatic cell nuclear transfer embryo development.

Porcine somatic cell nuclear transfer (SCNT) plays an important role in many areas of research. However, the low efficiency of SCNT in porcine embryos limits its applications. Porcine embryos contain high concentrations of lipid, which makes them vulnerable to oxidative stress. Some studies have used melatonin to reduce reactive oxygen species damage. At present there are many reports concerning the effect of exogenous melatonin on porcine SCNT. Some studies suggest that the addition of melatonin can increase the number of blastocyst cells, while others indicate that melatonin can reduce the number of blastocyst cells. Therefore, a meta-analysis was carried out to resolve the contradiction. In this study, a total of 63 articles from the past 30 years were analyzed, and six papers were finally selected. Through the analysis, it was found that the blastocyst rate was increased by adding exogenous melatonin. Melatonin had no effect on cleavage rate or the number of blastocyst cells, but did decrease the number of apoptotic cells. This result is crucial for future research on embryo implantation.

64 SIN3 transcription regulator family member A regulates porcine early embryonic development by modulating CCNB1 expression

SIN3 transcription regulator family member A (SIN3A) is the central scaffold protein of the SIN3/HDAC (histone deacetylase) transcriptional repressor complex. We previously found that SIN3A participates in the mouse pre-implantation development by finetuning HDAC1 expression. However, it remains unresolved whether this functional significance of SIN3A is conserved in other mammals. The objective of this work was thus to characterise the expression profiles and the functional role of SIN3A in pre-implantation development using non-rodent animal models. RNA sequencing results show that a large amount of SIN3A mRNA is present in oocytes and early embryos before embryonic genome activation and a low amount thereafter, suggesting a maternal origin of SIN3A in all species examined. Interestingly, immunofluorescence data show that SIN3A protein level peaks at the 4-cell stage in pigs compared with the morula stage in cattle, suggesting a differential role of SIN3A among species. To explore the function of SIN3A in early embryonic development, we used a short interfering (si)RNA-mediated knockdown approach in porcine parthenogenetic activated (PA) embryos. Immunocytochemical analysis showed that SIN3A levels were diminished ∼80% compared with nonspecific siRNA (NC) injected control (n=3). To monitor the developmental potential of embryo depleted of SIN3A, we injected SIN3A-siRNA into MII stage oocytes, followed by parthenogenetic activation, and percent cleavage and blastocyst formation were recorded. We found that SIN3A knockdown (KD) did not affect the cleavage rate (NC vs. KD, 83.63±3.63% vs. 80.08±4.66%, n=5), but significantly reduced blastocyst rate compared with the NC group (NC vs. KD, 36.64±4.28% vs. 6.33±3.12%, n=5). Specifically, SIN3A depletion in early embryos causes developmental arrest at 2-cell stage in pigs but does not affect early embryonic development in bovines. In contrast with mouse data, SIN3A depletion results in only a slight decrease and even no difference in HDAC1 expression in porcine and bovine early embryos, respectively. In addition, HDAC1 knockdown does not cause 2-cell block but leads to a reduced blastocyst rate, suggesting that the effect of SIN3A depletion on porcine early embryos is independent of HDAC1. RNA-Seq analysis was used to compare the global transcript content between NC and KD 2-cell embryos. A total of 23 genes (14 upregulated and 9 downregulated) had undergone significant changes. Interestingly, cyclin B1 (CCNB1) ranked second among downregulated genes. To test whether knockdown of CCNB1 would display a similar phenotype in porcine early embryos, we injected CCNB1-siRNA into pronuclear stage. CCNB1 KD resulted in a similar phenotype as SIN3A depletion. Injection of exogenous CCNB1 mRNA into SIN3A-depleted embryos could partly rescue embryonic development. In conclusion, our results indicate SIN3A plays an essential role in porcine early embryonic development, probably involving the regulation of CCNB1 expression. This work was funded by National Natural Science Foundation of China, the Anhui Provincial Natural Science Foundation and China Postdoctoral Science Foundation.

Growth Arrest Specific Gene 6 Improved in vitro Maturation of Oocytes and the Development Potential of Porcine Embryo after Parthenogenetic Activation

In this study, expression pattern-and function of growth arrest specific gene 6 (Gas6) during porcine oocyte maturation and early embryonic development were systemically investigated. The expression of Gas6 in the process of follicular development was studied by immunohistochemical staining, immunofluorescence, QRT-PCR and western blot. Immunohistochemical staining results showed that Gas6 was expressed in the oocytes, especially the nucleus, from the primary to preovulatory stages. The further immunofluorescence results showed that Gas6 was expressed thoughout the in vitro matured porcine oocytes, and the staining signal decreased in the first 12 h but then increased during the process of oocyte maturation. QRT-PCR analysis revealed that Gas6 mRNA was continuously expressed during the process of oocyte maturation, disappeared after parthenogenetic activation, re-appeared and elevated in the blastocysts stage. The following western blot analysis revealed a similar expression pattern with QRT-PCR and immunofluorescence. In conclusion, the expression pattern of Gas6 gene is closely related to oocytes maturation and embryo development. Gas6 may play a crucial role in the process of oocytes maturation and improve the development potential of early embryos by promoting the cytoplasmic maturation.