Parthenogenetic Porcine Research Articles

NANOG expression in parthenogenetic porcine blastocysts is required for intact lineage specification and pluripotency.

Nanog homeobox (NANOG) is a core transcription factor that contributes to pluripotency along with octamer binding transcription factor-4 (OCT4) and sex determining region-Y box-2 (SOX2). It is an epiblast lineage marker in mammalian pre-implantation embryos and exhibits a species-specific expression pattern. Therefore, it is important to understand the lineage of NANOG, the trophectoderm, and the primitive endoderm in the pig embryo. A loss- and gain-of-function analysis was done to determine the role of NANOG in lineage specification in parthenogenetic porcine blastocysts. We analyzed the relationship between NANOG and pluripotent core transcription factors and other lineage makers. In NANOG-null late blastocysts, OCT4-, SOX2-, and SOX17-positive cells were decreased, whereas GATA binding protein 6 (GATA6)-positive cells were increased. Quantitative real-time polymerase chain reaction revealed that the expression of SOX2 was decreased in NANOG-null blastocysts, whereas that of primitive endoderm makers, except SOX17, was increased. In NANOG-overexpressing blastocysts, caudal type homeobox 2 (CDX2-), SOX17-, and GATA6-positive cells were decreased. The results indicated that the expression of primitive endoderm markers and trophectoderm-related genes was decreased. Taken together, the results demonstrate that NANOG is involved in the epiblast and primitive endoderm differentiation and is essential for maintaining pluripotency within the epiblast.

Loss of Monoallelic Expression of IGF2 in the Adult Liver Via Alternative Promoter Usage and Chromatin Reorganization.

In mammals, genomic imprinting operates via gene silencing mechanisms. Although conservation of the imprinting mechanism at the H19/IGF2 locus has been generally described in pigs, tissue-specific imprinting at the transcript level, monoallelic-to-biallelic conversion, and spatio-temporal chromatin reorganization remain largely uninvestigated. Here, we delineate spatially regulated imprinting of IGF2 transcripts, age-dependent hepatic mono- to biallelic conversion, and reorganization of topologically associating domains at the porcine H19/IGF2 locus for better translation to human and animal research. Whole-genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) of normal and parthenogenetic porcine embryos revealed the paternally hypermethylated H19 differentially methylated region and paternal expression of IGF2. Using a polymorphism-based approach and omics datasets from chromatin immunoprecipitation sequencing (ChIP–seq), whole-genome sequencing (WGS), RNA-seq, and Hi-C, regulation of IGF2 during development was analyzed. Regulatory elements in the liver were distinguished from those in the muscle where the porcine IGF2 transcript was monoallelically expressed. The IGF2 transcript from the liver was biallelically expressed at later developmental stages in both pigs and humans. Chromatin interaction was less frequent in the adult liver compared to the fetal liver and skeletal muscle. The duration of genomic imprinting effects within the H19/IGF2 locus might be reduced in the liver with biallelic conversion through alternative promoter usage and chromatin remodeling. Our integrative omics analyses of genome, epigenome, and transcriptome provided a comprehensive view of imprinting status at the H19/IGF2 cluster.

Effect of Triclosan Exposure on Developmental Competence in Parthenogenetic Porcine Embryo during Preimplantation.

Triclosan (TCS) is included in various healthcare products because of its antimicrobial activity; therefore, many humans are exposed to TCS daily. While detrimental effects of TCS exposure have been reported in various species and cell types, the effects of TCS exposure on early embryonic development are largely unknown. The aim of this study was to determine if TCS exerts toxic effects during early embryonic development using porcine parthenogenetic embryos in vitro. Porcine parthenogenetic embryos were cultured in in vitro culture medium with 50 or 100 µM TCS for 6 days. Developmental parameters including cleavage and blastocyst formation rates, developmental kinetics, and the number of blastomeres were assessed. To determine the toxic effects of TCS, apoptosis, oxidative stress, and mitochondrial dysfunction were assessed. TCS exposure resulted in a significant decrease in 2-cell rate and blastocyst formation rate, as well as number of blastomeres, but not in the cleavage rate. TCS also increased the number of apoptotic blastomeres and the production of reactive oxygen species. Finally, TCS treatment resulted in a diffuse distribution of mitochondria and decreased the mitochondrial membrane potential. Our results showed that TCS exposure impaired porcine early embryonic development by inducing DNA damage, oxidative stress, and mitochondrial dysfunction.

Identification of a Novel Imprinted Transcript in the Porcine GNAS Complex Locus Using Methylome and Transcriptome of Parthenogenetic Fetuses.

Genomic imprinting in domestic animals contributes to the variance of performance traits. However, research remains to be done on large-scale detection of epigenetic landscape of porcine imprinted loci including the GNAS complex locus. The purpose of this study was to generate porcine parthenogenetic fetuses and comprehensively identify imprinting patterns of the GNAS locus in transcript levels. To this end, both normally fertilized and bimaternal (uniparental) parthenogenetic porcine fetuses were generated, and whole genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) were performed to construct methylome and transcriptome, respectively. Differentially methylated regions (DMRs) between the fetuses were identified through methylome analysis, and parental-origin-specific expression patterns of transcripts were examined with transcriptome. As a result, three major DMRs were identified: paternally methylated Nesp DMR, maternally methylated Nespas-Gnasxl DMR, and maternally methylated Exon1B–Exon1A DMR. Parental-origin-specific expressions of those five DMR-affected transcripts were found, including a novel imprinted transcript, Exon1B, in pigs. In conclusion, using parthenotes, parental-origin-specific imprinting patterns in the porcine GNAS locus was comprehensively identified, and our approach paves the way for the discovery of novel imprinted genes and loci in a genomic context across species.

96 Evaluation of CRISPR/Cas9 alternative delivery in parthenogenetic porcine embryos

Porcine genetic editing is considered promising in biomedical research, particularly for xenotransplantation. However, invitro porcine embryo production is less efficient than in other animal models. Thus, we aimed to perform a rapid optimization essay by producing parthenogenetic porcine embryos to evaluate transgenesis delivery and CRISPR/Cas9 editing efficiency. First, PCX-enhanced green fluorescent protein (EGFP) plasmid was microinjected (30ng μL−1) into a diploid parthenogenic zygote with (lipo+) or without lipofectamine (lipo−), since it has been shown that this transfection reagent improves transgene delivery and increases its expression in bovine embryos (Vichera et al. 2011 Reprod. Domest. Anim. 46, 214-220; https://doi.org/10.1111/j.1439-0531.2010.01642.x). Briefly, invitro-matured oocytes were electrically activated, followed by incubation in synthetic oviductal fluid medium containing 6-dimethylaminopyridine. Embryos were invitro cultured until Day 7, and EGFP-positive (EGFP+) embryos were corroborated at Day 5. Data were analysed using Fisher's exact test. Cleavage rates were no different among groups (EGFP/lipo−: 40%, n=28; EGFP/lipo+: 45%, n=41; control: 41, 3%, n=45; P>0.05). Also, no significant difference in the percentage of EGFP+ embryos was observed between EGFP/lipo+ (31%, n=13) and EGFP/Lipo− (18%, n=5) groups. Although blastocyst rate showed no statistical difference among groups, a lower blastocyst percentage tendency was observed in the EGFP/Lipo+ group compared with the control group (EGFP/Lipo+: 5%, n=2; control: 20%, n=9; P=0.051), suggesting that the presence of lipofectamine and EGFP plasmid may affect embryo development. Next, two guides (single guide (sg) RNA) were designed for the internal regions of GGTA1, CMAH, and VWF target genes, involved in hyperacute rejection and coagulation in xenotransplantation. A liposome-DNA mixture was used: DNA for sgRNA and Cas9, with and without lipofectamine (10× dilution; CRISPR/lipo+, CRISPR/lipo−), diluted to half concentration with 10% polyvinylpyrrolidone, resulting in a final concentration of 20ng ul−1 for all sgRNA and 40ng ul−1 for Cas9. A total of 2 pL of the mixtures was microinjected into the diploid parthenogenetic zygotes which were invitro cultured until Day 7. Genetic editing was corroborated by analysing the presence of a double cut directed by the two sgRNA designed for the target genes, resulting in an amplicon with lower molecular weight compared to the wild-type PCR fragment. No differences in cleavage or blastocyst rates were observed among groups (blastocyst rates: CRISPR/Lipo−: 12%, n=13; CRISPR/Lipo+: 10%, n=7; control: 15%, n=18; P>0.05). Finally, one embryo showed a single deletion for GGTA1 and another showed a double deletion in GGTA1 and VWF genes, out of seven embryos from the CRISPR/Lipo+ group. No gene deletion was confirmed in any of the embryos from the CRISPR/Lipo− group. These results are preliminary data (more experiments are currently being done) suggesting that microinjection of CRISPR/Cas9 with lipofectamine could be used as an alternative delivery system, since it seems to have no impact on porcine embryo development.

Vitrification of In Vitro Produced Porcine Blastocysts: Influence of Cryoprotectants Toxicity and Embryo Age

Background: Porcine embryos are sensible to all assisted reproduction manipulations, especially the ones that involve cryopreservation. Despite the high cryoprotectant concentrations routinely applied, vitrification is the most effective technique to date. These substances toxicity can also play a negative role in embryo viability. During in vitro porcine embryo production, the speed of development is often unevenly distributed. It is possible that their development speed, affects embryo tolerance to cryoprotectants. This study aimed to evaluate the toxicity of porcine embryos of days 5 or 6 of culture to cryoprotectant agents; as well as to assess embryo survival to vitrification.Material, Methods & Results: Parthenogenetic porcine blastocysts and expanded blastocysts of days 5 and 6 of culture were exposed to toxicity tests (experiments 1 and 2) and vitrification (experiment 3) using different protocols. In the first experiment, three different cryoprotectants were used (Dimethyl sulfoxide - DMSO, Ethylene glycol – EG, and Sucrose - SUC), combined in three different associations (G1: 15% EG + 15% DMSO with 0.5M SUC; G2: 16% EG + 16% DMSO with 0.4M SUC; G3: 18% EG + 18% DMSO with 0.5M SUC). In the fresh Control, embryos of day 6 are more sensible than the ones of day 5, whom showed a lower hatching rate (39.7 vs. 60.8%). After the toxicity (Experiment 1) test, the G1 showed better expansion rates in day 6 (50.0 vs 31.0 and 3.6% for G2 and G3) and higher hatching of day 6 compared to G2 and G3 (23.2, vs. 8.6 and 0.0% for G2 and G3). The fresh non hatched embryos at day 8, derived at day 6, had a lower percentage of cells with cleaved caspase-3 (20.2%) compared with the G1 (30.5%), G2 (31.4%) and G3 (30.5%). The hatched embryos of day 5 from G2 had lower total cell number (TCN) compared with the day 6 hatched embryos, whereas in G1 the TCN was not affected. The second experiment compared EG combined to one of these three extracellular cryoprotectants: Polyvinylpyrrolidone/sucrose/trehalose (respectively groups: PVP, SUC, TRE). The group SUC has raised the best results for day 5 embryos, whereas for day 6 embryos SUC and TRE were both best. The third experiment tested four vitrification protocols, being P1: EG+DMSO+TRE/warming with SUC; P2: EG+DMSO+TRE/warming TRE; P3: EG+TRE/ warming SUC; P4: EG+TRE/warming TRE. The expansion of vitrified day 5 embryos was higher in the P1 (20.0%) in comparison with the other three groups (4.3, 4.3 and 4.4% for P2, P3 and P4, respectively), with no difference for their hatching rates, been it lower comparing to the Control. Day 6 embryos showed no difference in expansion and hatching for the vitrified groups, been them lower than the Control.Discussion: Embryos obtained on day 6 are more sensible than the ones of day 5, fact observed when the embryos were exposed to cryoprotectant solution, as well by the behavior of the no treated Control embryos. The toxicity increases as it does the concentration of intracellular cryoprotectant, where over 16% of the intracellular cryoprotectors already affected the day 6 embryos development. For the day 5 embryos however, 15 or 16% of the intracellular cryoptrotectors, had similar behavior to the embryos. For the extracellular solutions, however, it is variable according the embryos development speed. Indeed, it is necessary to adjust the cryoprotectors to be used to cryopreserve porcine in vitro produced embryos obtained at days 5 and 6 of culture.

Effect of Knockout Serum Replacement During Postwarming Recovery Culture on the Development and Quality of Vitrified Parthenogenetic Porcine Blastocysts.

The postwarming recovery culture, as one of the steps in cryopreservation process, is directly correlated with the survival and quality of embryos. Generally, recovery medium includes undefined serum or serum components that may cause the instability of results and other problems. The objective of this study was to evaluate the effect of knockout serum replacement (KSR) as a substitute for serum during recovery culture on the development and quality of vitrified parthenogenetic porcine blastocysts. Fetal bovine serum (FBS) was used as a positive control. The expanded blastocysts on day 5 were vitrified by the Cryotop method, and recovered with 10% (v/v) KSR or 10% (v/v) FBS for 48 hours after warming. Survival and hatching rates of vitrified blastocysts were significantly increased by KSR or FBS supplementation. The vitrified blastocysts recovered in KSR or FBS exhibited significantly decreased percentages of membrane damage and apoptosis, and increased total cells. Addition of KSR or FBS during recovery culture significantly reduced reactive oxygen species levels, and improved mitochondrial activity and adenosine triphosphates content in the vitrified blastocysts. Vitrification did not affect the gene expression of PCNA, CDX2, and CPT1, but significantly increased mRNA levels of POU5F1 and uPA. KSR added to the recovery medium significantly upregulated mRNA levels of PCNA and CPT1, and downregulated POU5F1 mRNA levels. The expression levels of PCNA, CDX2, CPT1, and uPA in vitrified blastocysts were significantly upregulated by addition of FBS to recovery medium. Moreover, the BAX: BCL2L1 ratio was similar between fresh and vitrified blastocysts, and KSR or FBS supplementation had no effect on the value. In conclusion, our data showed that KSR supplementation during recovery culture can improve the development and quality of vitrified parthenogenetic porcine blastocysts. These findings provide a useful reference that KSR could be used to replace FBS as a defined serum supplement for recovery culture of vitrified blastocysts.

Erratum to : Effect of Endoplasmic Reticulum (ER) Stress Inhibitor Treatment during Parthenogenetic Activation on the Apoptosis and In Vitro Development of Parthenogenetic Porcine Embryos

[This corrects the article DOI: 10.12717/DR.2018.22.3.235.].

Effects of PRDM14 Silencing on Parthenogenetically Activated Porcine Embryos.

PRDM14, a member of the PRDM family protein, plays an important role in the regulation of epigenetic reprogramming. Knockdown of Prdm14 in germ cells can lead to female and male subfertility, and the function of PRDM14 appears to be conserved across mammalian species. Thus, we analyzed the expression of Prdm14 in parthenogenetic embryos at all stages of preimplantation and then evaluated the effect of Prdm14 downregulation on porcine parthenogenetic embryonic development. We found that Prdm14 transcripts are highly expressed in the metaphase II (MII) oocyte, and their level gradually increased from the 2-cell to 8-cell stage and slightly declined at the blastocyst stage during the development of parthenogenetic porcine embryos. Prdm14 was knocked down in oocytes at the MII stage by the injection of siRNA to assess the role of this protein in epigenetic remodeling and embryo development. Prdm14 knockdown significantly decreased the cleavage and blastocyst rates without changing the total cell number of the blastocysts. In addition, the expression levels of the antiapoptotic gene BCL-2 and the pluripotency-related genes OCT4 and SOX2 were also significantly decreased. These results showed that PRDM14 could affect embryonic development through regulating the expression levels of the pluripotency and antiapoptotic genes during the development of parthenogenetically activated porcine embryos.

Effect of Endoplasmic Reticulum (ER) Stress Inhibitor Treatment during Parthenogenetic Activation on the Apoptosis and In Vitro Development of Parthenogenetic Porcine Embryos.

We investigate the effect of endoplasmic reticulum (ER) stress inhibitor treatment during parthenogenetic activation of oocytes on the ER stress generation, apoptosis, and in vitro development of parthenogenetic porcine embryos. Porcine in vitro matured oocytes were activated by 1) electric stimulus (E) or 2) E+10 μM Ca-ionophore (A23187) treatment (EC). Oocytes were then treated by ER stress inhibitors such as salubrinal (200 nM) and tauroursodeoxychloic acid (TUDCA, 100 μM) for 3 h prior to in vitro culture. Parthenogenetic embryos were sampled to analyze ER stress and apoptosis at the 1-cell and blastocyst stages. The x-box binding protein 1 (Xbp1) mRNA and ER stress-associated genes were analyzed by RT-PCR or RT-qPCR. Apoptotic gene expression was analyzed by RT-PCR. At the 1-cell stage, although no difference was observed in Xbp1 splicing among treatments, BiP transcription level in the E group was significantly reduced by salubrinal treatment, and GRP94 and ATF4 transcription levels in EC group were significantly reduced by all treatments (p<0.05) compared to control. In the EC group, both apoptotic genes were reduced by ER stress inhibitor treatments compared to control (p<0.05) except Caspase-3 gene by TUDCA treatment. These results suggest that the treatment of ER stress inhibitor during parthenogenetic activation can reduce ER stress, and thereby reduce apoptosis and promote in vitro development of porcine parthenogenetic embryos.

C-Phycocyanin protects against mitochondrial dysfunction and oxidative stress in parthenogenetic porcine embryos

C-Phycocyanin (CP) is a biliprotein enriched in blue-green algae that is known to possess antioxidant, anti-apoptosis, anti-inflammatory, and radical-scavenging properties in somatic cells. However, the protective effect of CP on porcine embryo developmental competence in vitro remains unclear. In the present study, we investigated the effect of CP on the development of early porcine embryos as well as its underlying mechanisms. Different concentrations of CP (2, 5, 8, 10 μg/mL) were added to porcine zygote medium 5 during in vitro culture. The results showed that 5 μg/mL CP significantly increased blastocyst formation and hatching rate. Blastocyst formation and quality were significantly increased in the 50 μM H2O2 treatment group following 5 μg/mL CP addition. CP prevented the H2O2-induced compromise of mitochondrial membrane potential, release of cytochrome c from the mitochondria, and reactive oxygen species generation. Furthermore, apoptosis, DNA damage level, and autophagy in the blastocysts were attenuated by supplementation of CP in the H2O2-induced oxidative injury group compared to in controls. These results suggest that CP has beneficial effects on the development of porcine parthenotes by attenuating mitochondrial dysfunction and oxidative stress.

ID: 1066 Effects of biotin supplementation on the in vitro maturation of oocytes and the development of parthenogenetic diploid porcine embryos

In mammals, Biotin serves a coenzyme in the metabolism of glucose, amino acids, and fatty acids. Biotin deficiency causes decreased rates of cell proliferation, disfunction in germ cells and fetal development. This study was carried out to determine the influence of Biotin supplementation to invitro maturation medium on the development of porcine oocyte and embryos. Biotin (0.0, 1.0, 10.0, 100.0 mg/l, respectively) was added into the oocyte maturation medium, the quality of mature oocytes was evaluated after 42h culturing. The parthenogenetic diploid embryos were produced by using electro-activation system, the quality of embryos was noted at 1-4 cells stage. The results showed that, Biotin can enhance the formation of the first polar body at the concentration of 10 mg/l, it can also improve the activation efficiency of parthenogenetic diploid embryos at the preimplantation stage from 2-4 cells. Therefore, the supplementation of 10mg/l Biotin to the in-vitro maturation medium has a beneficial effect on the parthenogenetic diploid embryos development in the pig.

DNA methylation-mediated silencing of FLT1 in parthenogenetic porcine placentas

Studies show that perturbed expression of vascular endothelial growth factor (VEGF) family genes is related to abnormal development of parthenogenetic (PA) placenta. In this study, the methylation status of VEGF family genes were compared between PA and normal placentas using bisulfite sequencing PCR (BSP). Results showed no significant difference in the methylation of VEGF-A differentially methylated region (DMR), placental growth factor (PIGF) DMR, and kinase insert domain receptor (KDR) DMR, whereas FMS-like tyrosine kinase 1 (FLT1) DMR was hypermethylated in PA placentas. These results suggested that abnormal methylation of FLT1 DMR might trigger the developmental failure of porcine PA placentas.

ID: 1087 Effects of oocytes collection and electro-activation protocols on maturation and preimplantation development of parthenogenetic diploid porcine embryos

Advances in porcine embryos production have been impaired by the in vitro maturation (IVM) system and in vitro developments (IVD) are still not optimized. The present study was undertaken to determine the effects of different oocytes collection methods (aspiration and dissection) on oocytes maturation and combine with the different electro-activation protocols on in vitro pre-implantation development of parthenogenetic diploid porcine embryos. The results showed that dissection method was significantly higher both in quantity (87.23%, p &lt;0.05) of matured oocytes and in the rate of oocytes with very good quality (53.00%) compared with aspiration method (62.00%, 30.22%; respectively). We further accessed the quality of matured oocytes competence via parthenogenetic embryo development. Using four electro-activation protocols, we found that the electro-activation condition had no effect on the development of embryos to the 2-cell, 4-cell and 8-cell stages. However, the oocytes activated by the 60V/cm amplitude of pulse group had less (41.33%; p &lt;0.05) development at the morula stage than 90V/cm, 100V/cm and 150V/cm amplitude of pulse groups (73.99%, 67.62%, 72.55%; respectively). The embryos develop to blastocyst stage was highest in the group induced by 90V/cm electro pulse was 71.76% compared to 100V/cm, 150V/cm, and 60V/cm (62.86%, 62.05%, and 37.63%, p&lt;0.001, respectively). We conclude that the combining of dissection method for porcine oocytes collection and electro-activation with the amplitude of pulses 90V/cm can enhance both the activation success and development competition in in vitro pre- implantation parthenogenetic diploid porcine embryos.

Endoplasmic Reticulum (ER) Stress and Apoptosis in Parthenogenetic Porcine Embryos following Different Combination of Activation Methods

Endoplasmic Reticulum (ER) Stress and Apoptosis in Parthenogenetic Porcine Embryos following Different Combination of Activation Methods

101 TOWARDS OPTIMAL IN VITRO CULTURE CONDITIONS FOR PIG-MONKEY AGGREGATION CHIMERAS

Induced pluripotent stem cells (iPSCs) are promising for developing novel cell-based medical treatments. One potential use of iPSCs is for xenotransplantation via production of chimeric organisms. The generation of organs originated from the iPSCs of one individual in a foreign organism could allow the production of immune compatible organ transplants. One of the major problems down this road is to define the different nutrient needs of chimeric embryos (i.e. iPSCs and the host embryos). Here, we evaluated different media for supporting development of chimeric embryos consisting of parthenogenetic porcine embryos and iPS cells, either from pig or nonhuman primate, to identify the optimal medium conditions for pig-monkey aggregation chimeras. First, we cultured 3-day-old porcine parthenogenetic embryos in porcine zygote medium (PZM), iPSC-medium, and mixtures of the two media to identify the most suitable culture conditions. Three-day-old parthenogenetic embryos developed poorly in pure iPSC-medium (6.3% blastocyst rate), but grew in a mixture of PZM and iPSC medium. The best results were achieved with PZM and PZM with 10% or 25% iPSC medium (38.8% and 30% blastocyst rates, respectively). Next, we checked aggregation results of chimeric embryos produced with two different iPSC lines in the respective media (PZM, PZM+10% iPSC medium, PZM+25% iPSC medium). The porcine iPSC line (piPSC) and cynomolgus monkey iPSC line (ciPSC) carry fluorescent markers (piPSC: GFP, ciPSC: venus), thus facilitating detection of integration into the host embryos. After aggregation of iPS cells between two Day-3 parthenogenetic embryos (sandwich technique), blastocyst rates at Day 6 were determined (see Table 1). While culture in PZM allowed for the highest blastocyst rate (&gt;40%), the iPS cell participation was very low (0–11.1%). Cultivation in PZM+25% iPS medium was compatible with a high rate of embryo-iPSC chimeras (50–100%), but with a low blastocyst rate (7.7–16.7%). Possibly, the iPS cells proliferate more rapidly than blastomeres and the embryo is overgrown. The best results were obtained in medium mixture PZM+10% iPSC medium (25–40% blastocyst rate with 66.7–100% iPS cell participation). These results show that supplementation of the basic culture medium with 10% iPSC medium yields high blastocyst rates for chimeric embryos, and also ensures much higher iPS cell participation. This improves the use of 3-day-old embryos in aggregation chimeras. Table 1.Development of parthenogenetic porcine embryo aggregates with porcine (p) or cynomolgus monkey (c) iPS cells

GlutaMAX prolongs the shelf life of the culture medium for porcine parthenotes

In vitro porcine embryo production systems have been established and well characterized. However, the efficiency of embryo development during IVC is still very low. In the present study, we have investigated the development of parthenogenetic porcine embryos in the well-known PZM-5 medium for porcine embryos, which was modified by replacing glutamine with the GlutaMAX supplement. We revealed that blastocyst apoptosis was significantly lower in the presence of GlutaMAX, which reduced the release of mitochondrial cytochrome c. Furthermore, the expression of apoptosis genes was significantly lower during GlutaMAX treatment (P < 0.05). The modified medium was also examined for the eventual loss of its efficacy in the presence of GlutaMAX. Three, 6, and 12 months after medium preparation, blastocyst formation in the GlutaMAX-supplemented medium was significantly higher than the number of blastocysts in the medium containing glutamine. After a long period of storage, ammonia concentration was significantly increased in the glutamine medium, whereas it was not statistically different in the GlutaMAX medium. Elevated ammonia concentrations reduced the mitochondrial membrane potential and ATP content of blastocysts in the glutamine medium. These results demonstrate that GlutaMAX can reduce blastocyst apoptosis via inhibition of the cytochrome c pathway and significantly extend the shelf life of the culture medium to at least 1 year.

Isoform-specific imprinting of the MEST gene in porcine parthenogenetic fetuses

Aberrant expression of imprinted genes is the main reason for developmental retardation in mammalian parthenogenetic fetuses. Mesoderm specific transcript (MEST) is a maternally imprinted gene that is linked to cancer and is necessary for normal early embryonic development. Tissue and isoform-specific imprinting of MEST have been identified in humans and mice, but have not yet been identified in pigs. In this study, the three isoforms of porcine MEST were identified using the GenBank and Ensembl sequence databases. Then, we determined MEST isoform-specific mRNA expression and methylation levels by quantitative real-time PCR (qRT-PCR) and bisulfite sequencing PCR analysis (BSP) between porcine parthenogenetic (PA) and control (Con) fetuses, respectively. Altogether, our results demonstrated that the expression of MEST-1A and MEST-1B has no evident differences between PA and Con groups; however, there is no expression of MEST-1C in PA fetuses. In addition, the results of BSP showed that the hypermethylation of exon 1c of MEST-1C was observed in PA samples. Thus, we suggested that MEST-1C was isoform-specific imprinting, which may be contributed to differential methylation status of exon 1c in porcine fetuses.

139 EFFICIENT CONDITIONS OF CYTOPLASMIC MICROINJECTION OF FOREIGN DNA TO GENERATE PORCINE TRANSGENIC EMBRYOS

To establish the efficient cytoplasmic microinjection system in the porcine embryos, pEGFP-N1 plasmid were microinjected into porcine parthenogenetic (PA) and in vitro-fertilized (IVF) embryos to investigate the optimal injection time, volume, and concentration. In experiment 1, to investigate the optimal injection time, development rates were compared among groups of 4 different time durations (2, 4, 6, and 8 hours) in the PA and IVF embryos with time point after activation and sperm removal, respectively. There were no significant differences (P &lt; 0.05) between the 4 groups regarding the cleavage rates. However, there were significant differences (P &lt; 0.05) in development to the blastocysts (4.4, 8.9, 3.9, 0.6%) and GFP expression in blastocysts (1.3, 5.7, 2.3, 0.0%), which was injected after postactivation of 4 hours compared with another 3 groups. The IVF embryos injected after 2 and 4 hours expressed GFP significantly higher than the other two groups, which injected at 6 and 8 hours (P &lt; 0.05). In experiment 2, EGFP-N1 with 2 different concentrations (20 and 50 ng μL–1) was injected in the PA and IVF embryos to investigate the optimal concentration. In PA embryos, there were significant differences in 20 ng μL–1-injected embryos, which had higher cleavage (58.8 v. 41.9%) than blastocysts (13.0 v. 11.1%) and GFP expression rates (P &lt; 0.05). In IVF embryos, GFP were expressed only in 20 ng μL–1 embryos, GFP (4.2%) in the blastocysts showed no significant difference in the cleavage (77.3 v. 64.7%) and blastocyst rates (26.4 v. 23.5%). 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 &lt; 0.05). In conclusion, these results imply that a 20 ng μL–1 concentration, 10 pL of volume, injection 4 hours after activation for PA embryos, as well as injection 2 and 4 hours after sperm removal, a 20 ng μL–1 concentration, and 10 pL of volume for IVF embryos were more effective cytoplasmic microinjection conditions.

Amino acid supplementation affects imprinted gene transcription patterns in parthenogenetic porcine blastocysts.

To determine whether exogenous amino acids affect gene transcription patterns in parthenogenetic porcine embryos, we investigated the effects of amino acid mixtures in culture medium. Parthenogenetic embryos were cultured in PZM3 medium under four experimental conditions: 1) control (no amino acids except L-glutamine and taurine); 2) nonessential amino acids (NEAA); 3) essential amino acids (EAA); and 4) NEAA and EAA. The rate of development of embryos to the four-cell stage was not affected by treatment. However, fewer (P<0.05) embryos cultured with EAA (12.8%) reached the blastocyst stage as compared with the control group (25.6%) and NEAA group (30.3%). Based on these findings, we identified genes with altered expression in parthenogenetic embryos exposed to medium with or without EAAs. The results indicated that EAA influenced gene expression patterns, particularly those of imprinted genes (e.g., H19, IGF2R, PEG1, XIST). However, NEAAs did not affect impaired imprinted gene expressions induced by EAA. The results also showed that mechanistic target of rapamycin (MTOR) mRNA expression was significantly increased by EAA alone as compared with control cultures, and that the combined treatment with NEAA and EAA did not differ significantly from those of control cultures. Our results revealed that gene transcription levels in porcine embryos changed differentially depending on the presence of EAA or NEAA. However, the changes in the H19 mRNA observed in the parthenogenetic blastocysts expression level was not related to the DNA methylation status in the IGF2/H19 domain. The addition of exogenous amino acid mixtures affected not only early embryonic development, but also gene transcription levels, particularly those of imprinted genes. However, this study did not reveal how amino acids affect expression of imprinted genes under the culture conditions used. Further studies are thus required to fully evaluate how amino acids affect transcriptional regulation in porcine embryos.