Porcine Nuclear Transfer Embryos Research Articles

35 Interspecies Somatic Cell Nuclear Transfer Embryos that Form Nucleoli do not Always Activate Mitochondrial Functional Differentiation at the Time of Embryonic Genome Activation

Embryonic genome activation (EGA) is a complex process that needs a good orchestration of all biochemical processes at the time of maternal-to-embryonic transition. Mitochondria are strictly dependent on the nucleus for their correct activity as ~1500 mitochondrial genes have nuclear localisation. The finding of transcriptional activation and accumulation of mRNAs related to mitochondrial biogenesis (Mtango et al. 2008 Reprod. Fertil. Dev. 20, 846-859) around the time of EGA confirmed the role of nucleus in this process. Studying mitochondria behaviour in interspecies somatic cell nuclear transfer (iSCNT) embryos (Lagutina et al. 2010 Reproduction 140, 273-285), we have found that at the time of EGA, mitochondria activation could be demonstrated by JC-1 accumulation. We suggested that comparison of the mean green fluorescence intensity (FI) that corresponds to the fluorescence of the monomeric form of the dye and correlates to relative mitochondrial mass (Mancini et al. 1997) in iSCNT and control nuclear transfer (NT) could serve as a test to assess EGA in iSCNT embryos. The aim of this study was to estimate nuclear-cytoplasmic interaction in iSCNT embryos that formed nucleoli at the time of EGA (Lagutina et al. 2011 Reproduction 141, 453-465) such as embryos derived from bovine oocytes and bovine (control), buffalo or ovine donor nuclei, and from porcine oocytes and porcine (control), horse, or rabbit nuclei. Embryos 72 h after activation were stained with 2 μM JC-1 in SOF-HEPES with 10% FCS at 37°C for 1 h. Images were collected using a fluorescein isothiocyanate (FITC) filter and analysed with Adobe Photoshop Elements 2 (Adobe Systems, San Jose, CA, USA). The data are presented as mean FI of the embryo. To demonstrate the effect of EGA inhibition on mitochondria, bovine and porcine NT embryos were cultured in medium supplemented with 25 μg/mL α-amanitin (AA) from 48 to 72 h after activation. The analyses of mean FI of the embryos showed that ovine and buffalo nuclei were able to support mitochondrial mass accumulation in iSCNT embryos with bovine oocytes equal to control bovine NT embryos (35 ± 11.2; 41.9 ± 14.8; 36.2 ± 7.6, respectively) that was significantly higher than in bovine embryos treated with AA (15.4 ± 4.9; P < 0.05). In the iSCNT embryos composed of porcine oocytes and equine or rabbit nuclei, mean FI values (20 ± 13.4; 18.3 ± 5.5, respectively) were comparable with those in porcine NT embryos treated with AA (16.2 ± 6.2), and were significantly lower than in porcine control (91 ± 47.7; P < 0.05) NT embryos, demonstrating the inability of equine and rabbit nuclei to properly govern the porcine mitochondria mass growth at the time of EGA. In conclusion, nucleolus formation and activation of nuclear encoded mitochondrial genes at the time of EGA cannot serve, per se, as a marker of correct embryonic genome activation in iSCNT embryos, because, in our conditions, no iSCNT embryos developed to blastocyst (Lagutina et al. 2010 Reproduction 140, 273-285). This knowledge about behaviour of different embryo compartments at the time of EGA could extend our understanding of the whole process. This work was funded by Translink (EU FP7 no. 603049) and Xenoislet projects (EU FP7 no. 601827).

27 Quisinostat, a Potent Histone Deacetylase Inhibitor, Regulates the Expression of Pluripotency- and Reprogramming-Related Genes on Somatic Cell Nuclear Transferred Porcine Embryos

The low efficiency of somatic cell nuclear transfer (SCNT) has been attributed mostly to inefficient epigenetic reprogramming. Recently, various histone deacetylase inhibitors (HDACi) were used to improve developmental competence of SCNT embryos in several species. However, limited information is available on the effects of quisinostat (JNJ-26481585, JNJ), a second-generation HDACi with high cellular potency towards Class I and II histone deacetylases. Based on our previous study, among various concentrations, treatment with 100 nM JNJ could improve embryo development into blastocysts compared with the control (23.50 ± 1.30 v. 13.97 ± 1.37; P < 0.05). Thus, in the present study, treatment with 100 nM JNJ was used for further investigation into the relative expression of genes related to pluripotency and reprogramming in order to assess the quality of pre-implantation embryos cultured in media with JNJ using quantitative real-time PCR. Porcine fibroblasts isolated from kidney of adult pigs from passage 6 to 8 were used as nuclear donor cells for SCNT. After SCNT, embryos were cultured with or without 100 nM JNJ during the first 24 h of in vitro culture, and blastocysts from each experimental group were collected and kept at –80°C until analysis. Total RNAs were extracted, and transcribed into cDNA before amplification. Then, the relative expression of development-related (Oct4, Sox2, and Nanog), histone acetylation-related (HDAC1, HDAC2, and HDAC3) and DNA methylation-related (DNMT1, DNMT3a, and DNMT3b) genes between the control and 100 nM JNJ groups were compared. All experiments were repeated 4 times and results were analysed by independent t-test using SPSS 17.0K (SPSS Inc., Chicago, IL, USA). Treatment with 100 nM JNJ showed significant increases in the expression Oct4, Sox2, and Nanog compared with the control (P < 0.05). Moreover, there was significantly lower expression of HDAC1, HDAC2, HDAC3, DNMT1, DNMT3a, and DNMT3b in the 100 nM JNJ treatment than in the control (P < 0.05). These expression results moderately illustrated more active transcriptional factors, stable maintenance of embryonic pluripotency, and lesser activity of histone acetylation and DNA methylation enzymes, enhancing the blastocyst formation rate in the treatment group. In conclusion, we suggest that improvement of the in vitro developmental competence of porcine SCNT embryos might be related to positive regulations of JNJ on the expression levels of genes related to pluripotency and reprogramming. This study was supported by the NRF (#2015R1C1A2A01054373; 2016M3A9B6903410), Research Institute for Veterinary Science and the BK21 PLUS Program.

A Comparison of m-Carboxycinnamic Acid Bishydroxamide with Trichostatin A as Histone Deacetylase Inhibitor on the Developmental Competence of Somatic Cell Nuclear Transfer Porcine Embryos

The cloning efficiency is extremely low despite successful somatic cell nuclear transfer (SCNT) method producing cloned animals in several mammals. In general, faulty epigenetic modifications underlying the incomplete reprogramming of donor cell nuclei after SCNT mainly results in low cloning efficiency. The nuclear reprogramming process involves epigenetic modifications, such as DNA demethylation and histone acetylation, which may be an important factor in enhancing the cloning efficiency. Recently, the histone deacetylase inhibitors (HDACi), such as trichosatin A (TSA) and m-carboxycinnamic acid bishydroxamide (CBHA), to increase histone acetylation have been used to improve the developmental competence of SCNT embryos. Therefore, we compared the effects of TSA with CBHA on the in vitro developmental competence and pluripotency-related gene expression (Oct4, Nanog and Sox2) in porcine cloned blastocysts and histone acetylation pattern (H3K9ac). The porcine cloned embryos were treated with a 50nM concentration of TSA and 100μM concentration of CBHA during the in vitro early culture (10h) after cell fusion and then were assessed to cleavage rate, development to the blastocyst stage and pluripotency-related gene expression in NT blastocyst also, level of histone acetylation in zygote, 2cell, 4cell stage. As results, Although NT, TSA and CBHA treated NT embryos were not different between all groups for cleavage rates, the developmental competence to the blastocyst stage was significantly increased in CBHA treated embryos (22.7%) compared to that of normal NT and TSA treated NT embryos (8.1% and 15.4%)(p<0.05). In addition, all of pluripotent transcription factors (Oct4, Nanog and Sox2) were expressed in the CBHA treated NT embryos, however, Sox2 and Oct4 were expressed in TSA treated NT embryos and expression pattern of CBHA treated NT embryos is particularly similar to that of IVF embryos. Also, CBHA treated NT embryos were increased in level of histone acetylation (H3K9ac) at the zygote, 2-cell, 4-cell stage compared to those of NT and TSA treated NT embryos. In conclusion, the treatment of CBHA as a histone deacetylase inhibitor significantly increased the developmental competence of porcine NT embryos and pluripotency-related gene expressions(Oct4, Nanog and Sox2) in NT blastocysts and level of histone acetylation (H3K9ac).

18 DEVELOPMENTAL COMPETENCE OF CLONED PORCINE EMBRYOS PRODUCED WITH DIFFERENT CLONING PROCEDURES

Two nuclear transfer (NT) techniques are routinely used to produce cloned animals, traditional cloning (TC) and handmade cloning (HMC). The TC embryos keep their zona and can be transferred at early stages, whereas HMC embryos are zona-free and must be cultured to the morula/blastocyst stage before transfer. Some studies have shown that in vitro culture reduces embryo development and quality, but it is not known whether embryos produced by TC or HMC differ because of the NT method or the in vitro culture. Therefore, we investigated the developmental competence and histone acetylation (H3K18ac) of porcine NT embryos produced by TC and HMC with (Day 5 and 6) or without (Day 0) in vitro culture. Nuclear transfer experiments were performed on same day (Day 0), using same batch of porcine oocytes and donor cells and same in vitro culture conditions. Cloning procedures were previously described (TC : Cloning Stem Cells 10 : 355; HMC : Zygote 20 : 61). Parthenogenetically activated embryos (PA) were used as control of activation and culture conditions. Embryos from all groups were collected for immunostaining of H3K18ac on Days 0, 5, and 6. The normalized H3K18ac level was calculated as previously described (Epigenetics 6 : 177). Cell numbers per blastocyst in each group were counted on Days 5 and 6. The cleavage rate (Day 2) and blastocyst rates (Days 5 and 6) between groups were analysed by Chi-squared test, whereas cell number per blastocysts and H3K18ac level between groups and days were analysed by ANOVA (SAS version 9.2; SAS Institute Inc., Cary, NC, USA). Cleavage rate of HMC embryos was lower than that of TC embryos, but blastocyst rate and cell number per blastocyst were higher in the HMC group compared with TC (Table 1). Differences of H3K18ac level between HMC, TC, and PA groups were only observed on Day 6 but not on Day 0 or Day 5. Within HMC and TC groups, there was no difference in H3K18ac level between Day 0 and Day 5, but the level was lower on Day 6 compared with Day 5 in the HMC group, whereas the TC group displayed the opposite pattern. In conclusion, NT embryos produced by HMC show higher blastocyst rate and cell number per blastocyst compared with TC embryos. Both in vitro culture and the NT method result in differences of the normalized H3K18ac levels. Further study is needed to investigate putative differences between NT embryos produced by HMC and TC compared to in vivo embryos also after transfer to recipients. Table 1.Cleavage and blastocyst rate, cell numbers, and normalized H3K18ac level for handmade cloning (HMC), traditional cloning (TC), and parthenogenetically activated (PA) embryos1

Effects of Trichostatin A on In Vitro Development of Porcine Parthenogenetic and Nuclear Transfer Embryos

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

Effects of trichostatinAon histone acetylation and methylation characteristics in early porcine embryos after somatic cell nuclear transfer

Until now, the efficiency of animal cloning by somatic cell nuclear transfer (SCNT) has remained low. Efforts to improve cloning efficiency have demonstrated a positive role of trichostatin A (TSA), an inhibitor of deacetylases, on the development of nuclear transfer (NT) embryos in many species. Here, we report the effects of TSA on pre-implantation development of porcine NT embryos. Our results showed that treatment of reconstructed porcine embryos with 50 nmol/L TSA for 24 h after activation significantly improved the production of blastocysts (P < 0.05), while treating donor cells with the same solution resulted in increases in cleavage rates and blastomere numbers (P < 0.05). However, TSA treatment of both donor cells and SCNT embryos did not improve blastocyst production, nor did it increase blastomere numbers. Using indirect immunofluorescence, we found that TSA treatment of NT embryos could improve the reprogramming of histone acetylation at lysine 9 of histone 3 (H3K9) and affect nuclear swelling of transferred nuclei. However, no apparent effect of TSA treatment on H3K9 dimethylation (H3K9me2) was observed. These findings suggest a positive effect of TSA treatment (either treating NT embryos or donor cells) on the development of porcine NT embryos, which is achieved by improving epigenetic reprogramming.

Effects of 5-aza-2’-deoxyctidine on the development of porcine parthenogenetic and nuclear transfer embryos

The current study was to investigate whether embryo or fetal fibroblast cells treated with 5-aza-2’-deoxyctidine (5-aza-dC) have a positive effect on the in vitro development of porcine parthenogenetic and cloned embryos. To this end, porcine fetal fibroblast cells were treated with different concentrations (5 nM, 50 nM and 500 nM) of 5-aza-dC for different exposure times (1, 6 and 20 hours), the results showed that DNA methylation in PRE-1 SINE region was gradually reduced over time in cells treated with 5-aza-dC. To determine the effect of 5-aza-dC on in vitro development of porcine activated oocytes, the parthenogenetic embryo was treated with 5-aza- dC. Notably, treatment with 5 nM 5-aza-dC for 1 hour led to a significant improvement in blastocyst development, compared with the control group. The effects of donor cell treatment with 5-aza-dC on porcine cloned embryos development were further examined by treating fetal fibroblast cells with various concentrations (5 nM, 50 nM and 500 nM) of 5-aza-dC for different exposure times (1, 6 and 20 hours). Exposure of cells in 5 nM 5-aza-dC for 1 - 20 hours led to a significant improvement in the percentage of developed blastocysts, while treatment with 500 nM 5-aza-dC did not affect blastocyst development, compared to untreated controls. These findings indicate that treatment of fetal fibro-blast cells with relatively low concentrations of 5-aza-dC for short exposure times improves subsequent blastocyst development of porcine cloned embryos.

54 EXPRESSION PATTERN OF DNMT1 AND DNMT3a GENES DURING INTERGENERIC SOMATIC CELL NUCLEAR TRANSFER EMBRYO DEVELOPMENT

One of the most-discussed reasons for developmental incompetence of embryos constructed by the cloning procedure is inadequate reprogramming of the transferred nucleus to a state equivalent to that of an early embryonic nucleus. Previous studies have shown species-dependent expression patterns of DNA methyltransferase (DNMT) genes in mammalian oocytes and preimplantation embryos, and also a correlation between incomplete DNA methylation and the lack of NT success in mammals. In the present study, the expression pattern of DNMT1 and DNTM3a genes at the 2-cell and 4-cell stages of bovine versus porcine intergeneric nuclear transfer (iSCNT) embryos was observed by reverse-transcriptase (RT) PCR. All pools were done in triplicate and contained 10 iSCNT embryos. The species-specific primers for DNMT1 and DNMT3a genes were designed for determination of de novo synthesis of epigenetic enzymes. As positive controls, porcine and bovine parthenogenetic embryos were used. Gene transcription for bovine DNMT1 (bDNMT1) and DNMT3a (bDNMT3a) was not observed in 2- and 4-cell stage embryos generated by bovine fibroblast transfer into the porcine ooplasm; however, using primers for pig DNMT1 (pDNMT1) and DNMT3a (pDNMT3a), positive results were obtained. In the 2- and 4-cell-stage embryos constructed using porcine fibroblast and bovine ooplasm, only the bovine-specific primers showed positive signals. Based on the different timing of major genome activation during embryonic development in bovine and porcine embryos, the strong influence of ooplasm on introduced fibroblast was expected. Despite the mRNA presence of DNMT1 and DNMT3a enzymes of oocyte origin, de novo transcription of somatic DNMT1 and DNMT3a genes was not detected and iSCNT embryos did not develop beyond the 4-cell stage. These results strongly suggest species-specific and maternally driven regulation of epigenetic reprogramming during early embryogenesis. This work was supported by VEGA 1/0077/11.

143 EFFECTS OF BOAR SEMINAL PLASMA IN IN VITRO CULTURE OF PORCINE EMBRYOS

Seminal plasma consisting of carbohydrates, proteins, and lipids not only serves as a nutritive and protective medium for sperm cells but also play a pivotal role in inducing the tolerance to pre-existing immune cells as well as improving the intra-uterine conditions for implantation of fertilized embryos (Guerin et al. 2009 Hum. Reprod. Update 15, 517–535). However, the effects of seminal plasma in in vitro culture of fertilized embryos are unknown. In the present study, the seminal plasma was separated from the second fraction of a normal farm boar (n = 1) by centrifugation and filtered seminal plasma was stored at –30°C until use. In a preliminary experiment, the optimal activity of seminal plasma was evaluated by incubating the embryos for different time intervals. To investigate the developmental rates, electrically (EA) (triplicates, n = 490) or chemically (CA) (quintuplicates, n = 599) activated 2-day-old porcine embryos were incubated for 3 h in PZM-5 medium (Funakoshi Co., Tokyo, Japan, Catalog no. IFP0410P) containing 0% (EA: n = 122 and CA: n = 152), 0.1% (EA: n = 123 and CA: n = 148), 0.5% (EA: n = 122 and CA: n = 150), or 1% (EA: n = 123 and CA: n = 149) seminal plasma. Similarly, the developmental rate of chemically activated 2-day-old somatic cell nuclear transferred porcine embryos (quadruplicates, n = 239) was studied after incubation with 0% (n = 119) or 0.1% (n = 120) seminal plasma for 3 h. A significant difference was noticed only in the rate of blastocyst formation in the chemically activated embryos treated with 0.1% seminal plasma (31.7 v. 24.8% in the 0% group, ANOVA; P &lt; 0.05; Prism5, GraphPad Software Inc., La Jolla, CA, USA). None of the treatments showed a significant effect on the cleavage rate and cell numbers of blastocysts. In conclusion, the seminal plasma did not show any harmful effect on early embryos development. Furthermore, the seminal plasma (0.1%) improved the rate of blastocyst formation among the chemically activated nuclear transferred embryos. The results of this preliminary study suggest that the addition of seminal plasma during embryo transfer could increase the rate of pregnancy in pig. This study was supported by MKE (#10033839-2012-21), IPET (#311011-05-1-SB010), the Research Institute for Veterinary Science, and TS Corporation.

Epigenetic modification of fetal fibroblasts improves developmental competency and gene expression in porcine cloned embryos

Treatment of somatic cells with DNA methylation and histone deacetylation inhibitors has been hypothesized to improve the potential reprogramming after nuclear transfer (NT). The objective of this study was to investigate the developmental competence and gene expression during the porcine preimplantation development of in vitro fertilized (IVF) and embryos cloned with porcine fetal fibroblasts (pFF) (pFF-NT), and pFF treated by 0.5 μM 5-azacytidine (5-azaC) (pFF+5-azaC-NT) or 1.0 mM sodium butyrate (NaB) (pFF+NaB-NT). IVF embryos had significantly (P < 0.05) higher blastocyst rates (27.7 ± 2.6 %) and total cell numbers (46.7 ± 3.9). However, NT embryos from pFF+5-azaC and pFF+NaB showed enhanced developmental potential with significantly (P < 0.05) higher rates of blastocysts (21.3 ± 2.9 % and 22.4 ± 1.7 %, respectively) than those from pFF (15.1 ± 2.5 %). Further, NT embryos from pFF+5-azaC and pFF+NaB (33.8 ± 4.1 and 35.7 ± 5.2, respectively) had significantly (P < 0.05) higher total cell numbers than those from pFF (24.6 ± 3.5). Differential expression pattern of genes involved in DNA methylation (DNA methyltransferases- DNMT1, DNMT2, DNMT3A and DNMT3B), histone acetylation (histone acetyltransferase 1- HAT1) and histone deacetylation (histone deacetylases- HDAC1, HDAC2 and HDAC3) were observed in NT embryos when compared to IVF counterparts. However, the relative expressions of genes in pFF+5-azaC-NT and pFF+NaB-NT groups were largely comparable to those of IVF embryos than pFF-NT embryos. In conclusion, modification of the epigenetic status by reducing DNA methylation or enhancing histone acetylation levels in pFF improved the developmental rates, total cell number and the transcription profile of porcine NT embryos. Thus, somatic cells with relatively hypomethylated or hyperacetylated genome may enhance reprogramming efficiency in porcine NT.

Identification and characterization of an oocyte factor required for development of porcine nuclear transfer embryos

Nuclear reprogramming of differentiated cells can be induced by oocyte factors. Despite numerous attempts, these factors and mechanisms responsible for successful reprogramming remain elusive. Here, we identify one such factor, necessary for the development of nuclear transfer embryos, using porcine oocyte extracts in which some reprogramming events are recapitulated. After incubating somatic nuclei in oocyte extracts from the metaphase II stage, the oocyte proteins that were specifically and abundantly incorporated into the nuclei were identified by mass spectrometry. Among 25 identified proteins, we especially focused on a multifunctional protein, DJ-1. DJ-1 is present at a high concentration in oocytes from the germinal vesicle stage until embryos at the four-cell stage. Inhibition of DJ-1 function compromises the development of nuclear transfer embryos but not that of fertilized embryos. Microarray analysis of nuclear transfer embryos in which DJ-1 function is inhibited shows perturbed expression of P53 pathway components. In addition, embryonic arrest of nuclear transfer embryos injected with anti-DJ-1 antibody is rescued by P53 inhibition. We conclude that DJ-1 is an oocyte factor that is required for development of nuclear transfer embryos. This study presents a means for identifying natural reprogramming factors in mammalian oocytes and a unique insight into the mechanisms underlying reprogramming by nuclear transfer.

64 VALPROIC ACID ENHANCES IN VITRO DEVELOPMENT OF SOMATIC CELL NUCLEAR TRANSFER EMBRYOS IN PIGS

Epigenetic modification influences reprogramming and subsequent development of somatic cell nuclear transfer embryos. Such modification includes an increase of histone acetylation and a decrease of DNA methylation in transferred donor nuclei. Histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA) and valproic acid (VPA) have been known to maintain high cellular levels of histone acetylation. Hence, the treatment of HDACi to NT embryos may increase efficiency of cloning. Indeed, TSA treatment has significantly enhanced the developmental competence of nuclear transfer embryos in several species including pigs (Zhang et al. 2007 Cloning Stem Cells 9, 357-363; Li et al. 2008 Theriogenology 70, 800-808). Valproic acid, another type of HDACi, has often been used to assist reprogramming of somatic cells into induced pluripotent stem cells in mice. In the present study, we tested the potency of VPA compared with TSA on the enhancement of in vitro development in porcine nuclear transfer embryos. Reconstructed embryos were produced by transferring nuclei of adult ear skin fibroblasts into enucleated oocytes. After electrical activation, these embryos were cultured in PZM-3 containing no HDACi (control), 5 mM VPA, or 50 nM TSA for 24 h, and another 5 days thereafter without HDACi. At least 3 replicates were conducted for the following experiments. The rates of cleavage were not different among the VPA, TSA, and control groups. However, the rate of blastocyst development was significantly higher (P &lt; 0.05) in embryos treated with VPA than in those treated with TSA and without HDACi (125/306, 40.8% v. 94/313, 30.0% v. 80/329, 24.3%). Differential staining of inner cell mass (ICM) and trophectoderm (TE) also supported the beneficial effect of VPA treatment in NT embryos. Compared with the control group, the number of TE cells was significantly increased (P &lt; 0.05) in the VPA and TSA treatment groups (79.3 ± 7.4 v. 74.6 ± 9.2 v. 40.0 ± 6.7). Moreover, VPA treatment significantly increased (P &lt; 0.05) the number of ICM cells compared with the control (15.6 ± 1.7 v. 10.8 ± 2.6), whereas no differences were observed between the TSA treatment and control group (12.9 ± 3.0 v. 10.8 ± 2.6). The present study demonstrates that VPA enhances in vitro development of nuclear transfer embryos, in particular by an increase of blastocyst formation and the number of ICM cells, suggesting that VPA may be more potent than TSA in supporting developmental competence of cloned embryos. However, long-term effects of different HDACi in the development of nuclear transfer embryos, including any adverse outcome from destabilizing epigenetic condition, remain to be determined by further in vivo embryo transfer studies.

Porcine nuclear transfer using somatic donor cells altered to express male germ cell function

Recent studies reported that the direct transformation of one differentiated somatic cell type into another is possible. In the present study, we were able to modulate the cell fate of somatic cells to take on male germ cell function by introducing cell extracts derived from porcine testis tissue. Fibroblasts were treated with streptolysin O, which reversibly permeabilises the plasma membrane, and incubated with testis extracts. Our results showed that the testis extracts (TE) could activate expression of male germ cell-specific genes, implying that TE can provide regulatory components required for altering the cell fate of fibroblasts. Male germ cell function was sustained for more than 10 days after the introduction of TE. In addition, a single TE-treated cell was injected directly into the cytoplasm of in vitro-matured porcine oocytes. The rate of blastocyst formation was significantly higher in the TE-treated nuclear donor cell group than in the control cell group. The expression level of Nanog, Sox9 and Eomes was drastically increased when altered cells were used as donor nuclei. Our results suggest that TE can be used to alter the cell fate of fibroblasts to express male germ cell function and improve the developmental efficiency of the nuclear transfer porcine embryos.

Streptolysin-O Treatment of Fetal Fibroblasts Improves Cell Fusion and In Vitro Development of Porcine Nuclear Transfer Embryos

Streptolysin O (SLO) is a known bacterial protein that forms very large pores in the plasma membranes of mammalian cells. SLO has been used in the delivery of proteins into living cells following permeabilization. The objective of this study was to investigate the effects of SLO treatment in donor cells that generate pores in the plasma membrane during in vitro development of porcine reconstructed embryos. In experiment 1, fetal fibroblast cells were trypsinized, treated with SLO for 0, 30, 50 and 70 min and then their fusion rates and developmental capabilities were tested following reconstruction. SLO treatment for 50 min produced a higher blastocyst formation rate compared with the other treatments. In experiment 2, fetal fibroblasts were treated with 200 ng/ml SLO for 50 min at the confluent or single-cell stages of the nuclear transfer protocol (i.e., before and after trypsinization, respectively), and the in vitro development of the resulting porcine reconstructed embryos was investigated over 6 days in culture. Oocytes receiving cells treated with SLO post-trypsinization showed higher fusion and blastocyst formation rates compared with those receiving untreated cells. Collectively, these findings show that SLO-mediated permeabilization of porcine fetal fibroblast cells appears to improve the fusion rates and in vitro development of porcine reconstructed embryos.

94 NUCLEAR-CYTOPLASMIC INTERACTION AND DEVELOPMENT OF CLONED EMBRYOS

Although cloned canine offspring have been produced, the efficiency of nuclear transfer (NT) is still far from expectations. The most important factor interfering with the success of NT is the shortage of canine matured oocytes. In the present study, the nuclear-cytoplasmic interaction and in vitro development of porcine oocytes cloned with canine fetal fibroblasts (cFF) and canine mesenchymal stem cells (cMSC) were assessed by examining the developmental rate, embryonic morphology, total cell number, canine genome expression, and in vivo development following the transfer of iNT embryos into the oviducts of surrogate mother. Isolated cMSC from bone marrow were characterized by early transcription factors (Oct3/4, Sox2, and Nanog) and by differentiation into adipogenic, osteogenic, and neuronal cells under controlled in vitro conditions. Porcine cumulus–oocyte complexes (COC) were matured in vitro as described by Kim YS et al. (2005 Mol. Reprod. Dev. 70, 308–313). Porcine NT embryos were used for the control group. Canine MSC showed their characteristic property of attaching to plastic culture flasks and forming a monolayer of spindle-shaped cells. Adherent cells showed moderate expression of Oct3/4, Sox2, and Nanog protein by immunostaining. Following osteogenic cell induction, cMSC transformed into mineralized nodules by von Kossa staining. Oil red O staining revealed that MSC produced lipid droplets after incubation in adipogenic media. Neuronal-like cells with multipolar, round cell bodies organized into a network-like structure were confirmed by immunostaining of neuronal markers (NFM). The tissue-specific mRNA levels were confirmed after differentiation by RT-PCR (osteogenic cell, Osteocalcin; adipogenic cell, PPAR γ; neuronal cell, NFM). The control group blastocysts (porcine oocytes-pMSC) exhibited significantly (P &lt; 0.05) greater development and total cell numbers of than blastocysts of interspecies groups (porcine oocytes-cFF and cMSC). The canine genome GAPDH was detected in cFF, cMSC, and interspecies NT embryos using PCR, whereas there was no detection in porcine oocytes and the negative control. Most of the embryos arrested at the 8- to 16-cell stage and only 5% of embryos developed to blastocyst stage. The interspecies-NT embryos (average 35 embryos/recipient dog) were surgically transferred into the oviducts of 3 recipient dogs. Two of them showed exterior signs of implantation; i.e. a slight thickening of the uterus and small pea-sized lumps. No pregnancies were detected in the recipients after ovariohysterectomy surgery. These results demonstrate the potential of cMSC isolated from bone marrow to differentiate into mesoderm (osteogenic and adipogenic cell) and ectoderm (neuronal cell)-like cells under specific induction conditions. Although interspecies NT embryos could not implant in the canine uterine wall, the results provided valuable information on canine MSC and interspecies NT. This work was supported by Grant No. 20070301034040 from Bio-Organ, Republic of Korea.

36 TREATMENT OF PORCINE FETAL FIBROBLASTS WITH DNA METHYLATION INHIBITORS OR HISTONE DEACETYLATION INHIBITORS IMPROVES DEVELOPMENT OF NUCLEAR TRANSFER EMBRYOS

Epigenetic status of the genome of a donor nucleus has an important effect on the developmental potential of cloned embryos produced by somatic cell nuclear transfer (SCNT). DNA methylation inhibitors [such as 5-aza-2′-deoxyctidine (5-aza-dC), zebularine, and RG108] and histone deacetylase inhibitors [such as trichostatin A (TSA), sodium butyrate (NaBu), and scriptaid (SCR)] have been widely used for the alteration of the levels of the epigenetic modification of somatic cells. This study was designed to investigate the DNA methylation status of porcine fetal fibroblast cells treated with TSA or 5-aza-dC and to determine whether treatments with DNA methylation inhibitors or histone deacetylation inhibitors could improve the in vitro development of porcine reconstructed embryos. When the levels of DNA methylation in the PRE-1 sequence (repeat sequence in a euchromatic region) were examined by bisulfite sequencing following treatment of porcine fetal fibroblast cells with TSA or 5-aza-dC for 1 h, DNA methylation was decreased in 5-nm or 50-nm concentrations even if they were not significantly different. To evaluate the effect of DNA methylation inhibitors and histone deacetylation inhibitors on development of porcine nuclear transfer embryos, porcine fetal fibroblast cells were treated with 5 nm of 5-aza-dC, zebularine, or RG108 for 1 h, or with 50 nm of TSA, NaBu, or SCR for 1 h, or treated with both 50 nm TSA and 5 nm 5-aza-dC for 1 h before NT. The reconstructed embryos were electrically fused and cultured in PZM-3 for 6 days. Developmental rates of the reconstructed embryos from donor cells treated with 5-aza-dC, zebularine, or RG-108 to blastocysts significantly increased compared to the control group (21.4, 23.3, and 22.1 v. 12.3%). Blastocyst rates of the reconstructed embryos from donor cells treated with TSA, SCR, and NaBu also were significantly improved compared to the control group (30.0, 23.9, and 22.4 v. 14.5%), and TSA treatment was the highest in blastocyst rates among the treated groups. However, the development rate to the blastocyst stage was not affected when the combination of TSA and 5-aza-dC was treated. In conclusion, treatment of donor cells with DNA methylation inhibitors or histone deacetylase inhibitors improved the subsequent blastocyst development of porcine reconstructed embryos even though combined treatment with both inhibitors had no beneficial effect.

Gene Expression Analysis of Nuclear Transfer Porcine Embryos Using Somatic Donor Nuclei That Express Male Germ Cell Function.

Gene Expression Analysis of Nuclear Transfer Porcine Embryos Using Somatic Donor Nuclei That Express Male Germ Cell Function.

Longer Exposure of Porcine Nuclear Transfer Embryos to Cycloheximide Increases Apoptosis Rate in Blastocysts.

Longer Exposure of Porcine Nuclear Transfer Embryos to Cycloheximide Increases Apoptosis Rate in Blastocysts.

Effects of Insulin, Transferrin, and Selenium Complex (ITS) on the Development of Porcine Nuclear Transfer Embryos.

Effects of Insulin, Transferrin, and Selenium Complex (ITS) on the Development of Porcine Nuclear Transfer Embryos.

Control of nuclear remodelling and subsequent in vitro development and methylation status of porcine nuclear transfer embryos

We attempted to control the nuclear remodelling of somatic cell nuclear transfer embryos (NTs) and examined their subsequent development and DNA methylation patterns in pigs. Porcine foetal fibroblasts were fused to enucleated oocytes treated with either 5 mM caffeine for 2.5 h or 0.5 mM vanadate for 0.5 h. After activation, NTs were cultured in vitro for 6 days to examine their development. The nuclear remodelling type of the reconstituted embryos was evaluated 1 h after fusion. Methylated DNA of in vitro-fertilised (IVF) embryos and NTs at various developmental stages and of donor cells was detected using a 5-methylcytosine (5-MeC) antibody. Caffeine-treated NTs induced premature chromosome condensation at a high rate (P<0.05), whereas most vanadate-treated NTs formed a pronucleus-like structure. Although cleavage rates to the two-cell stage did not differ among groups, delayed cleavage was observed in the vanadate-treated group. The blastocyst formation rate was significantly reduced by vanadate treatment compared with caffeine-treated and non-treated (control) NT groups (P<0.05). The apoptotic cell index of NT blastocysts was lower in the caffeine-treated group than in other groups (P<0.05). The methylation patterns were similar among NTs, but more hypermethylated DNA was observed at the four-cell stage of control and vanadate-treated NTs when compared with that in IVF embryos (P<0.05). Thus, the nuclear remodelling type controlled by caffeine or vanadate treatment can affect in vitro development and the methylation status of NTs in relation to nuclear reprogramming.