Parthenogenetic Porcine Research Articles

Improvement of cloned embryos development by co-culturing with parthenotes: a possible role of exosomes/microvesicles for embryos paracrine communication.

It is well known that embryos cultured in a group can create a microenvironment through secretion of autocrine and paracrine factors that can support and improve the embryos' development when compared to the embryos cultured individually. In this study, we used a co-culture system for paracrine communication between different kinds of embryos. The results showed that co-culture of porcine parthenogenetic (PA) embryos significantly improved the in vitro development of cloned (nuclear transfer, NT) embryos. To reveal the possible mechanism of communication between the two groups, we isolated exosomes/microvesicles (EXs/MVs) from the PA embryos conditioned medium (PA-CM) through differential centrifugation and identified them through transmission electron microscope and immunoflourescence against exosomal/membrane marker CD9. Furthermore, these EXs/MVs were found to contain mRNA of pluripotency genes (Oct4, Sox2, Klf4, c-Myc, and Nanog), and the PKH67-labeled EXs/MVs could be internalized by the NT embryos. The current study demonstrates that cloned embryos' developmental competence can be improved through co-culturing with PA embryos and revealed, for the first time, that in vitro-produced embryos can secrete EXs/MVs as a possible communication tool within their microenvironment. Moreover, it provides a new paradigm for embryo-to-embryo communication in vitro.

Elevated expression of vascular endothelial growth factor (VEGF) 120 in parthenogenetic porcine placentas

Most mammalian parthenogenetic embryos are unable to develop to term due to placental defects, potentially caused by decreased vasculogenesis and angiogenesis of the parthenogenetic placenta. Here we have compared the expression status of vascular endothelial growth factor (VEGF) and angiopoietin family members between normally developing and parthenogenetic porcine placentas. The result showed significantly reduced expression of these genes but elevated expression of VEGF 120 in the parthenogenetic porcine placenta (p < 0.05). We postulate that the abnormal expression levels of VEGF and angiopoietin family members and, especially, the elevated expression of VEGF 120 observed in parthenogenetic porcine placentas are related to the early miscarriage of parthenogenetic embryos in pigs.

Effect of cytochalasin types on the production of heterozygous parthenogenetic porcine embryos and the isolation of putative parthenogenetic embryonic stem cells

Abstract Parthenogenetic embryos have been suggested as an alternative source of embryonic stem cells (ESCs). The present study was undertaken to determine the efficiency with which porcine heterozygous parthenotes could be produced

Effect of human adipose tissue‐derived mesenchymal‐stem‐cell bioactive materials on porcine embryo development

Human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) secrete bioactive materials that are beneficial for tissue repair and regeneration. In this study, we characterized human hAT-MSC bioactive material (hAT-MSC-BM), and examined the effect of hAT-MSC-BM on porcine embryo development. hAT-MSC-BM was enriched with several growth factors and cytokines, including fibroblast growth factor 2 (FGF2), vascular endothelial growth factor A (VEGFA), and interleukin 6 (IL6). Among the various concentrations and days of treatment tested, 10% hAT-MSC-BM treatment beginning on culture Day 4 provided the best environment for the in vitro growth of parthenogenetic porcine embryos. While the addition of 10% fetal bovine serum (FBS) increased the hatching rate and the total cell number of parthenogenetic porcine embryos compared with the control and hAT-MSC culture medium group, the best results were from the group cultured with 10% hAT-MSC-BM. Mitochondrial activity was also higher in the 10% hAT-MSC-BM-treated group. Moreover, the relative mRNA expression levels of development and anti-apoptosis genes were significantly higher in the 10% hAT-MSC-BM-treated group than in control, hAT-MSC culture medium, or 10% FBS groups, whereas the transcript abundance of an apoptosis gene was slightly lower. Treatment with 10% hAT-MSC-BM starting on Day 4 also improved the development rate and the total cell number of in vitro-fertilized embryos. This is the first report on the benefits of hAT-MSC-BM in a porcine embryo in vitro culture system. We conclude that hAT-MSC-BM is a new, alternative supplement that can improve the development of porcine embryos during both parthenogenesis and fertilization in vitro.

Generating Porcine Chimeras Using Inner Cell Mass Cells and Parthenogenetic Preimplantation Embryos

BackgroundThe development and validation of stem cell therapies using induced pluripotent stem (iPS) cells can be optimized through translational research using pigs as large animal models, because pigs have the closest characteristics to humans among non-primate animals. As the recent investigations have been heading for establishment of the human iPS cells with naïve type characteristics, it is an indispensable challenge to develop naïve type porcine iPS cells. The pluripotency of the porcine iPS cells can be evaluated using their abilities to form chimeras. Here, we describe a simple aggregation method using parthenogenetic host embryos that offers a reliable and effective means of determining the chimera formation ability of pluripotent porcine cells.Methodology/Significant Principal FindingsIn this study, we show that a high yield of chimeric blastocysts can be achieved by aggregating the inner cell mass (ICM) from porcine blastocysts with parthenogenetic porcine embryos. ICMs cultured with morulae or 4–8 cell-stage parthenogenetic embryos derived from in vitro-matured (IVM) oocytes can aggregate to form chimeric blastocysts that can develop into chimeric fetuses after transfer. The rate of production of chimeric blastocysts after aggregation with host morulae (20/24, 83.3%) was similar to that after the injection of ICMs into morulae (24/29, 82.8%). We also found that 4–8 cell-stage embryos could be used; chimeric blastocysts were produced with a similar efficiency (17/26, 65.4%). After transfer into recipients, these blastocysts yielded chimeric fetuses at frequencies of 36.0% and 13.6%, respectively.Conclusion/SignificanceOur findings indicate that the aggregation method using parthenogenetic morulae or 4–8 cell-stage embryos offers a highly reproducible approach for producing chimeric fetuses from porcine pluripotent cells. This method provides a practical and highly accurate system for evaluating pluripotency of undifferentiated cells, such as iPS cells, based on their ability to form chimeras.

Targeted DNA Methylation Analysis by High Throughput Sequencing in Porcine Peri-attachment Embryos

The purpose of this experiment was to implement and evaluate the effectiveness of a next-generation sequencing-based method for DNA methylation analysis in porcine embryonic samples. Fourteen discrete genomic regions were amplified by PCR using bisulfite-converted genomic DNA derived from day 14 in vivo-derived (IVV) and parthenogenetic (PA) porcine embryos as template DNA. Resulting PCR products were subjected to high-throughput sequencing using the Illumina Genome Analyzer IIx platform. The average depth of sequencing coverage was 14,611 for IVV and 17,068 for PA. Quantitative analysis of the methylation profiles of both input samples for each genomic locus showed distinct differences in methylation profiles between IVV and PA samples for six of the target loci, and subtle differences in four loci. It was concluded that high throughput sequencing technologies can be effectively applied to provide a powerful, cost-effective approach to targeted DNA methylation analysis of embryonic and other reproductive tissues.

44 LIMITATIONS OF PORCINE OOPLASM TO REPROGRAM BOVINE SOMATIC CELLS

The oocyte cytoplasm (ooplasm) constitutes a unique mixture of factors that are critical for successful reprogramming of haploid maternal and paternal genomes at fertilization as well as the diploid somatic cell genome after somatic cell nuclear transfer (SCNT). The 1-cell-stage embryos (produced by transfer of bovine fibroblast into porcine enucleated ooplast; interspecies SCNT, iSCNT) were processed at different time points (2, 4, 8, and 12 h post-activation, hpa) for detailed nuclear and nucleolar analysis by transmission electron microscopy (TEM; 4 to 5 embryos per group), and immunofluorescence (4 and 12 hpa) for visualization of nucleolar proteins related to transcription (upstream binding factor, UBF) and processing (fibrillarin; 8 to 9 embryos per group). The parameters of interspecies embryos were compared with porcine parthenogenetic (PA) counterparts (4 to 5 embryos per group for TEM analysis; 8 to 9 embryos per group for immunofluorescence analysis). At all evaluated time points, embryos in both groups displayed pronucleus-like nuclei with abundant euchromatin and characteristic porcine nucleolus precursor body (NPB), indicating maternal origin of nucleolar components. Fibrillarin was in both groups localized into shell-like intranuclear entities surrounding NPB. On the contrary, UBF in PA embryos was at 12 hpa colocalized with fibrillarin, whereas in iSCNT embryos, UBF staining was absent at both time points. Despite the similar morphology and localization of processing factor fibrillarin, striking differences in localization of transcription factor UBF suggest limitation of porcine ooplasm to mediate initial phases of nucleolar remodelling. This work was supported by VEGA 1/0077/11.

Effects of Activation Methods on DNA Synthesis and Development of Parthenogenetic Porcine Embryos

This study investigated the timing of DNA synthesis and patterns of pronuclear (PN) formation during the first cell cycle, and its influence on developmental competence, velocity and proliferation index of porcine parthenote blastocysts produced by different activation treatments. Oocytes were activated as follows: electrical stimulation (EST), EST combined with 7.5 μg/ml cytochalasin B (EST + CCB), 10 μg/ml cycloheximide (EST + CHX) and 1.9 mm 6-dimethylaminopurine (EST + 6-DMAP) for 3 h. DNA synthesis and PN formation were evaluated using 1 mm 5'bromo-2'deoxy-uridne (BrdU) at 2 h intervals from 1 to 13 h or 5 to 13 h of post-activation (hpa), respectively. In EST, DNA synthesis started at 3 hpa, reached the peak at 11 hpa and decreased at 13 hpa. Treatment with 6-DMAP resulted in an early increase of DNA synthesis at 3 hpa, whereas CCB delayed DNA synthesis for 2 h. In EST and EST + 6-DMAP, most of the eggs showed 1PN, whereas, incidence of 2PN in EST + CCB was higher than 1PN. EST + CHX was observed with 1PN, 2PN and multiple PN. Blastocyst rate in EST + CCB and EST + 6-DMAP were significantly (p<0.05) higher than EST + CHX. But, the developmental velocity was not different among groups. Proliferation index of blastocysts, as indicated the number of blastomere at S-phase of the cell cycle was low in all groups. In conclusion, CCB, CHX and 6-DMAP used for producing porcine parthenogenetic embryos induced different onset of DNA synthesis and PN, but they did not affect the subsequent embryo development.

Sonic Hedgehog improves in vitro development of porcine parthenotes and handmade cloned embryos

This study investigated the expression of Sonic Hedgehog (Shh) signaling pathway and its effect on porcine parthenogenetic (PA) embryo development. The Shh receptor Patched (Ptc1) and co-receptor Smoothened (Smo) were expressed at various stages of PA porcine embryos, at both mRNA and protein levels. Furthermore, the transcriptional activator Gli1 mRNA was first present in the 2-cell stage embryos, and was readily detected at the 4-cell stage and beyond. Culture medium supplemented with 0.5 μg/mL Shh optimized blastocyst rates (58.6 vs. 41.1%; P < 0.05) and the total number of cells per blastocyst (56.4 vs. 45.6 cells; P < 0.05); however, this response was prevented by simultaneous addition of 1 mM cyclopamine (an Shh inhibitor). Moreover, blastocysts that developed in medium containing 0.5 μg/mL Shh had lower apoptotic indices and reduced DNA damage (evaluated by TUNEL and comet assays, respectively). Based on Western-blot analysis, expression of phosphorylated Akt protein in Shh-treated blastocysts was higher than that of the control group (1.22- vs. 0.66-fold, P < 0.05), and less total PARP-1/2 protein was accumulated (0.7-fold, P < 0.05) in treated blastocysts compared to untreated controls. Furthermore, supplementation of Shh (1 μg/mL) also supported development of handmade cloned embryos (50.3 vs. 26.8%; P < 0.05) with reduced apoptotic rates (2.8 vs. 6.3%; P < 0.05). We inferred that the Shh signaling pathway existed in porcine PA embryos and we concluded that Shh supplementation improved the quality and developmental competence of early PA embryos, at least in part, by increasing cell proliferation and reducing apoptosis of the developing embryos.

Effects of epigallocatechin-3-gallate on the developmental competence of parthenogenetic embryos in the pig

This study was conducted to examine the effects of (-)-epigallocatechin gallate (EGCG) supplementation on the developmental competence and quality of parthenogenetic porcine embryos during culture. Parthenogenetic embryos derived from in vitro matured oocytes were cultured for eight days in a modified North Carolina State University (NCSU)-37 solution supplemented with EGCG at different concentrations (0, 1, 5, 10 and 50 µM). Supplementation of 1 and 5 µM EGCG during in vitro culture of embryos showed no significant influence on the rate of cleavage or that of blastocyst formation or on the total cell number and DNA fragmentation indices of blastocysts when compared to those of a control group. However, when 10 and 50 µM EGCG were supplemented into the culture medium, the cleavage rates were significantly lower than those of the other groups. No embryo developed to the blastocyst stage. Results suggest that treatment with low EGCG during in vitro culture has no influence on the developmental competence of porcine embryos but the presence of high concentrations of EGCG is apparently harmful for in vitro development of porcine parthenotes.

387 DERIVATION OF PUTATIVE EMBRYONIC STEM CELLS FROM PORCINE PARTHENOGENETIC BLASTOCYSTS AND THE LOSS OF PARENTAL-SPECIFIC EXPRESSION PATTERNS IN Igf2/H19 GENES

Porcine embryonic stem cells (ESC) can be a useful tool for the production of a transgenic animal and the study of developmental gene regulation. The study of porcine parthenogenetic ESC might also provide advantages in the understanding of changes in human parthenogenetic embryonic stem cells in the culture environment. Because human embryonic stem cells must be maintained stably for therapeutic uses, parthenogenetic porcine embryonic stem cells can give us precious information to help understand human parthenogenetic embryonic stem cells. Three putative porcine embryonic stem cell lines were derived from 99 parthenogenetic embryos. Cumulus-oocyte complexes were collected from prepubertal gilt ovaries and matured in vitro. Diploid parthenogenetic zygotes were produced by electrical activation followed by cytochalasin B treatment to suppress second polar body extrusion. Embryos were cultured to the blastocyst stage. Hatched blastocysts were directly cultured on mitomycin C-inactivated murine embryonic fibroblasts as feeder layers. Primary colonies were formed after 7 days of culture, and the colonies were transferred to new culture dishes 7 days after. They were passsaged every 5 days by physical dissociation, with one colony divided into small clumps and maintained for over 30 passages. These cells morphologically resembled human embryonic stem cells and consistently expressed the markers of pluripotent cells such as alkaline phosphatase, NANOG, OCT-4, SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81. They could be maintained holding the previous characteristics after cryopreservation. Furthermore, we conducted experiments to confirm the expression patterns of the imprinted genes Igf2 and H19 in these ESC and IVF/parthenogenetic blastocysts using quantitative real-time PCR. At the blastocyst stage, the 2 genes were expressed in a parental-specific manner according to their origins in normal fertilized embryos and uniparental embryos. The putative parthenogenetic ESC, on the other hand, showed a high expression of Igf2, the paternally expressed gene, when compared with their blastocyst counterparts. Current work aims to confirm the authenticity of these ESC via teratoma formation in severe combined immunodeficiency mice following injection with these putative parthenogenetic ESC. This work was supported by the BioGreen 21 Program (#20070401034031, #20080401034031), Rural Development Administration, Republic of Korea (HK).

The effects of various antioxidants on the development of parthenogenetic porcine embryos

The major objective of this study was to improve the development rate of parthenogenetic porcine embryos. In this study, the anti-oxidative and anti-apoptotic effects of three antioxidants, β-mercaptoethanol (β-ME), α-tocopherol, and extracellular superoxide dismutase (EC-SOD), were examined on the development of parthenogenetic porcine embryos. The development rate of parthenogenetic porcine embryos to the blastocyst stage was 8.1% for control; 19.1%, 14.6%, and 5.0% for 1, 3, and 5 μM β-ME; 17.2% and 17.5% for 50 and 100 μM α-tocopherol and 12.0% and 4.0% for EC-SOD transgenic mouse embryonic fibroblast (Tg-MEF) and EC-SOD non-transgenic mouse embryonic fibroblast (NTg-MEF) conditioned medium at day 3, respectively. Here, β-ME, α-tocopherol, and EC-SOD Tg-MEF conditioned medium increased the development rate of parthenogenetic porcine embryos to the blastocyst stage (P < 0.05). The average number of total cells and apoptotic cells at the blastocyst was analyzed at the optimal conditions of the three antioxidants. The three antioxidants increased the average number of total cells at the blastocyst, and they decreased apoptotic cells at the blastocyst as compared to control without supplementation (P < 0.05). When the reactive oxygen species levels in two-cell embryos after 1 μM β-ME and 100 μM α-tocopherol treatment were examined, those were lower than control group (P < 0.05). In conclusion, it was found that the three antioxidants, β-mercaptoethanol, α-tocopherol, and EC-SOD Tg-MEF, conditioned medium can play a role as a strong stimulator in the development of parthenogenetic porcine embryos.

Relationship between imprinted expression and ploidy in parthenogenetic porcine blastocysts

Relationship between imprinted expression and ploidy in parthenogenetic porcine blastocysts

Effects of chemical activation and season on birth efficiency of cloned pigs

The effects of chemical activation on birth efficiency of cloned pigs were studied by investigating the developmental process from porcine oocyte activation to birth of cloned pigs. Three different activation methods were used: (i) Electroporation (Ele); (ii) Ele followed by incubation with 6-dimethylaminopurine (6-DMAP); and (iii) Ele followed by a treatment with cycloheximide (CHX). In experiment 1, the rates of cleavage, developmental rates and cell number of porcine parthenogenetic (PA) embryos were investigated in the three treatment groups. In experiment 2, NT embryos produced by the three different activation treatments were compared for the rates of cleavage, development and cell number. Finally, the effects of Ele and Ele+CHX activation methods on birth efficiency of cloned pigs were compared. The activated oocytes treated by combination activation generally showed a higher (P<0.05) blastocyst rate and produced more expanded blastocysts than oocytes activated with Ele. The rates of cleavage and total cell number of parthenotes were not significantly different. Parthenogenetic embryos activated with 6-DMAP developed into blastocyst and expanded blastocyst stages at a significantly (P<0.05) higher rate than those treated with Ele, but the developmental capability was dramatically decreased in NT embryos. With the CHX activation method, the NT embryo blastocyst rate was substantially (P<0.05) increased although the production of expanded blastocysts was not significantly different from that by the other two methods. The birth rate of cloned pigs increased in the CHX group, though the rate was not significantly different from Ele. The effects of season on developmental rate of the porcine PA embryos and birth rate of cloned pigs were also examined in our study. Porcine oocytes collected in the spring had higher developmental capabilities than those collected in the winter. However, no difference in birth rate of the cloned pigs was found between the oocytes collected in the two seasons. The results obtained from PA and NT embryos, following different activation methods, were inconsistent, suggesting that activation mechanisms are dissimilar in PA and NT embryos. Although the chemical activation in our study leads to an elevation of the blastocyst rate, it does not improve the oocyte's molecular programming and so does not significantly improve the efficiency of producing cloned pig births.

Relationship between Developmental Ability and Cell Number of Day 2 Porcine Embryos Produced by Parthenogenesis or Somatic Cell Nuclear Transfer

In vitro produced porcine embryos have potential application in reproductive biotechnology. However, their development potential has been very low. This study evaluated the in vitro developmental ability and quality of cloned and parthenogenetic porcine embryos having 2-4 cells or 5-8 cells on Day 2 of in vitro culture. Analysis of results showed that 2 to 4 cell embryos had higher ability to form blastocysts than 5 to 8 cell embryos (p<0.05). Blastocysts produced from culture of 2 to 4 cell embryos also contained higher cell numbers and had lower BAX:BCLxL transcript ratio than those produced from 5 to 8 cell embryos (p<0.05), thereby suggesting 2 to 4 cell embryos have higher development potential. Further investigation revealed that 5 to 8 cell embryos had higher incidence (100±0.0%) of blastomeric fragmentation than 2 to 4 cell embryos (15.2±5.5% for parthenogenetic and 27.7±7.1% for cloned embryos). This suggests that low development potential of 5 to 8 cell embryos was associated with blastomeric fragmentation. In conclusion, we have shown that morphological selection of embryos based on cell number on Day 2 of in vitro culture could offer a practical and valuable non-invasive means to select good quality porcine embryos.

The Leading Blastomere of the 2-Cell Stage Parthenogenetic Porcine Embryo Contributes to the Abembryonic Part First

Polarity formation in preimplantation embryos is controversial. To investigate the embryonic-abembryonic axis in the pig, porcine parthenotes were used to prevent the topological change caused by polyspermy as well as to avoid the influences of sperm entry position. For lineage tracing, DiI, a fluorescence dye, was injected into only a blastomere of the 2-cell stage embryos. If the first blastomere to divide was labeled, the embryo was included in the leading group, and while all others were included in the lagging group. In 60.5% of the blastocysts in the lagging group, the progeny of the labeled blastomeres formed the inner cell mass (ICM) and adjacent trophectoderm (TE) hemisphere; 62.1% of the blastocysts in the leading group had progeny of the labeled blastomeres distributed only to the TE (opposite of ICM). The rest of the lagging and leading groups showed random distributions. Unlike murine parthenotes, biased mitochondrial distribution was also found in porcine parthenotes (38.1%). Our findings indicate that the ;leading' blastomere of the 2-cell porcine parthenote forms the distal TE (abembryonic) and that the 'lagging' blastomere forms the remaining portion of the blastocyst, including the ICM (embryonic). Biased distribution of mitochondria in each 2-cell blastomere may contribute partly to this event.

4 THE HISTONE METHYLTRANSFERASE Suv39h2 ADOPTS A NUCLEAR LOCALIZATION DURING CLEAVAGE DEVELOPMENT IN PARTHENOGENETIC PORCINE EMBRYOS

Successful cleavage development of mammalian embryos requires precise activation and repression of transcription. Covalent modifications to histone proteins, such as methylation and acetylation, play a key role in transcriptional regulation. In particular, dimethylation of the lysine 9 residue of histone protein H3 (H3K9) results in gene silencing and heterochromatin formation. Our lab has previously shown that transcripts encoding the five histone methyltransferases known to methylate H3K9 (Suv39h1, Suv39h2, ESET, G9a, and EHMT1) are present in different amounts during oocyte maturation and cleavage development. Specifically, Suv39h2 is in the greatest abundance in GV and metaphase II stage oocytes and is also present throughout cleavage development. The aim of this study was to determine the localization of Suv39h2 protein in the GV-stage oocyte and pronuclear, 2 cell, and 4 cell stage parthenogenetic porcine embryos. We hypothesized that Suv39h2 protein would localize to the nucleus based on its high transcript abundance throughout cleavage development. To test this hypothesis, we performed a microinjection experiment in which mRNA encoding a porcine Suv39h2-GFP fusion protein was injected into metaphase II porcine oocytes. Porcine oocytes were matured in a defined medium (TCM-199 supplemented with 0.1% PVA, 0.069 mg mL–1 cysteine, 10 ng mL–1 EGF, 0.5 IU mL–1 LH and FSH) for 42 to 44 h at 39°C in 5% CO2, then denuded of cumulus cells just before microinjection. Two separate treatment groups were microinjected intracytoplasmically with 1 μg μL–1 GFP or Suv39h2-GFP mRNA, respectively. Microinjection was performed using a FemtoJet microinjector (Eppendorf, Hamburg, Germany). The treatment groups and non-injected controls were electroactivated independently and cultured in PZM medium supplemented with 3 mg mL–1 BSA for 12 (pronuclear), 24 (2 cell), or 48 (4 cell) hours at 39°C in 5% CO2, depending on desired stage of development. Before visualization under UV light, embryos were stained with Hoechst 33342 for 15 minutes. Oocytes and embryos were analyzed for GFP expression at the GV, pronuclear, 2 and 4 cell stages of development using epifluorescence microscopy. Two to four biological replicates were performed for each stage of embryo development. We found that Suv39h2-GFP protein showed nuclear localization in most GV-stage oocytes (n = 11/14) and pronuclear (n = 17/17), 2-cell (n = 34/36), and 4-cell (n = 9/9) stage embryos. Chi square analysis revealed this pattern to be different from that observed in embryos injected with GFP mRNA, where GFP did not display nuclear localization at any stage of development (n = 12; P &lt; 0.05). These results indicate that Suv39h2 is localized in the nucleus of oocytes and cleaved embryos, which suggests that this histone methyltransferase plays an important role in methylating H3K9.

Oocyte activation procedures and influence of serum on porcine oocyte maturation and subsequent parthenogenetic and nuclear transfer embryo development

The viability of SCNT embryos is poor, with an extremely low cloned piglet production rate. In the present work, we studied the effect of three activation protocols based on ionomycin treatment (5 microM ionomycin for 5 min and incubated in 2 mM 6-DMAP for 3.5 h) or electric stimuli (two square wave electrical DC pulses of 1.2 kV/cm for 30 micros) combined or not with 6-DMAP on parthenogenetic embryo development. Oocytes activated by ionomycin plus 6-DMAP showed lower cleavage (47.2 vs. 78.5-81.5; p < 0.05) and blastocyst rates (11.3 vs. 29.2-32.1; p < 0.05) than those activated by electrical and electrical plus 6-DMAP treatments. Also, we studied the effect of addition of serum to maturation medium (0% vs. 10%) on nuclear maturation and further parthenogenetic and SCNT embryo development. We observed in the parthenogenetic embryos that cleavage rates in the serum-free group were significantly higher than in the serum-supplemented group (81.8 vs. 69.6% respectively; p < 0.05), although these differences were not detected in blastocyst rates or blastocyst nuclei numbers. Regarding SCNT embryos, no significant differences were observed in cleavage or blastocyst rates between different experimental groups of SCNT embryos. In conclusion, electrical pulse followed or not by 6-DMAP was found to be an efficient procedure to artificially activate MII porcine oocytes. Moreover, the addition of serum to oocyte maturation media did not seem to improve parthenogenetic or SCNT porcine embryo development.

Incidence of apoptosis in parthenogenetic porcine embryos generated by using protein kinase or protein synthesis inhibitors

In mammals, embryonic development can be artificially initiated by activating the oocyte using a number of methods. In the present research, we investigated whether butyrolactone I and cycloheximide, two chemicals frequently used in combined oocyte activation protocols, have any detrimental effect on programmed cell death in the developing porcine embryo. Parthenogenetic porcine blastocysts were generated by the following methods: (1) electroporation followed by blocking the activity of specific protein kinases with butyrolactone I; (2) electroporation followed by inhibition of protein synthesis using cycloheximide; and (3) electroporation only (control). The viability of the embryos was evaluated by monitoring the frequency of cells with early and late signs of programmed cell death. There was no difference between the embryos in terms of blastocyst formation (ranging from 27.5 ± 3.8 and 33.3 ± 3.5%) and total nuclear number (ranging from 23.5 ± 1.1 and 31.8 ± 4.4). In addition, the occurrence of apoptosis was also similar in the three experimental groups. The proportion of cells with active caspase-9 (a sign of early apoptosis) in the blastocysts produced by the different activation methods was between 19.4 ± 1.9 and 23.0 ± 2.4%. The annexin V assay revealed that phosphatidylserine flip (another early apoptotic event) took place in 24.4 ± 2.1 to 32.3 ± 2.4% of the blastomeres. Finally DNA fragmentation, a sign of late stage apoptosis determined by the TUNEL assay, occurred in 8.5 ± 2.4 to 10.1 ± 3.0% of the cells. The results indicate that temporary inhibition of specific protein kinases or protein synthesis does not increase the onset of apoptosis in parthenogenetic porcine blastocysts.

Kinetin enhances in vitro development of parthenogenetic and nuclear transfer porcine embryos

Culture conditions affect the development of mammalian embryos in vitro. Kinetin belongs to the family of N(6)-substituted adenine derivates and promotes cell division, synthesis of DNA repair enzymes, superoxide dismutase activity, and ribosomal RNA transcription. We investigated the effects of kinetin on in vitro development of parthenogenetic and nuclear transfer (NT) porcine embryos. These embryos were cultured with or without kinetin in either BSA- or polyvinyl alcohol-containing medium for 7 days. mRNA expression of three developmentally important genes, HSP70, Glut-1, and poly[A] polymerase in NT embryos was analyzed. Regardless of kinetin supplementation, the proportion of blastocysts and blastocyst cells were not significantly different in parthenogenetic embryos. However, kinetin supplementation increased expansion and hatching rates in all groups. In somatic cell NT embryos, kinetin increased the proportion of embryos developed to blastocysts from 7.5% to 15.4% in medium supplemented with PVA. However, gene expression levels of HSP70, poly[A] polymerase and Glut-1 mRNA were not significantly different in NT blastocysts. The present study indicates that kinetin not only improves blastocyst expansion and cell number of parthenogenetic porcine embryos but also enhances NT porcine embryo development in a completely defined culture condition in vitro.