Nuclear Transfer Blastocysts Research Articles

27 THE HISTONE DEACETYLASE INHIBITOR 4-IODO-SUBEROYLANILIDE HYDROXAMIC ACID IMPROVES TOTAL CELL NUMBER IN PIG NUCLEAR TRANSFER BLASTOCYSTS

Treatment of reconstructed pig clones with the histone deacetylase inhibitor (HDACi) Scriptaid immediately after nuclear transfer (NT) and activation results in increased cloning efficiency. Aberrant gene expression examined in NT blastocyst stage embryos is only partially corrected by Scriptaid use; therefore, 2 other HDACi were examined in this study including the class I and II HDACi, suberoylanilide hydroxamic acid (SAHA) and its hydrophobic derivative 4-iodo-SAHA (I-SAHA). Blastocyst rates and total cell numbers were examined across 6 treatment groups (1 μM SAHA, 10 μM SAHA, 1 μM I-SAHA, 10 μM I-SAHA, 0.5 μM Scriptaid and no HDACi treatment). Nuclear transfer was performed on enucleated MII oocytes using 3 different cell lines. Clones were electrically fused and activated, treated with HDACi for 14 to 16 h and cultured to the blastocyst stage in PZM3 under low oxygen tension for 7 days. Blastocyst number was calculated from the total number of fused oocytes. Blastocysts were then fixed in 4% paraformaldehyde and total cell number was determined by Hoechst staining of nuclei. The results from all 3 cell lines were pooled and 782 embryos were examined for blastocyst development from 7 replicates. All statistical analysis was performed by SAS 9.1 and means were separated by least significant difference (P < 0.05). The treatment group 10 μM SAHA had the highest blastocyst rate of 41.9% (n = 124) and was significantly different than no HDACi treatment (29.2%, n = 161; P < 0.003). There was no significant difference in blastocyst rates between 1 μM SAHA, 10 μM SAHA, 1 μM I-SAHA and 0.5 μM Scriptaid with blastocyst rates of 31.6% (n = 168), 41.9% (n = 124) 34.2% (n = 76) and 40.2% (n = 179), respectively (P < 0.05). Treatment with 10 μM I-SAHA significantly decreased development when compared with the other HDACi treatments (17.6%, n = 74, P < 0.05). There was no interaction between treatment and cell line for blastocyst rates (P > 0.45). Total cell number was significantly higher in blastocysts from the 1 μM I-SAHA (37.9, n = 20) treatment group when compared with Scriptaid (29.9, n = 50) and no HDACi treatment (29.4, n = 42; P < 0.04). There were no significant improvements in total cell number between the other concentrations (P > 0.05). Additionally, there was also a significant interaction between cell line used for nuclear transfer and the total cell number (P < 0.002). Two treatments were selected to determine if 10 μM SAHA and 1 μM I-SAHA treatment postnuclear transfer was compatible with term development. Six embryo transfers were performed and 5 recipient pigs became pregnant and developed to term. The results of this study show that treatment with the HDACi, SAHA and I-SAHA postnuclear transfer has the same blastocyst rates as the commonly used HDACi, Scriptaid. Additionally, treatment with 1 μM I-SAHA improves total cell number when compared with Scriptaid or no HDACi treatment. Funding was provided by Food for the 21st Century.

Isolation and culture of embryonic stem-like cells from pig nuclear transfer blastocysts of different days

This study was conducted to establish pig embryonic stem (ES)-like cell lines from nuclear transfer blastocysts. A green fluorescent protein (GFP)-expressing cell line was used as the source of donor cells injected into the enucleated oocytes. Blastocysts were collected at D5 (the fifth day), D7 (the seventh day) and D9 (the ninth day). Differential staining was used to assay the viability and development of blastocysts from the 3 days. The number of inner cell mass (ICM) cells increased from 1.83 ± 0.8 (D5) to 5.37 ± 1.2 (D7) to 7.56 ± 1.5 (D9). The expression profiles of embryonic stem (ES) cell factors (OCT4, SOX2, KLF4 and c-MYC) correlated best with the undifferentiated ES state and were identified by qPCR. The expression of the four factors was increased from D5 to D7, whereas the expression decreased from D7 to D9. We tried to isolate ES-like cells from these embryos. However, ES-like cells from the D7 blastocysts grew slowly and expressed alkaline phosphatase. The cells from the D9 blastocysts grew rapidly but did not express alkaline phosphatase. ES-like cells were not isolated from the D5 blastocysts. These results show that the cells from the D7 embryos are pluripotent but grow slowly. The cells from the D9 embryos grow rapidly but start to lose pluripotency.

The Effects of 5-Aza-2′- Deoxycytidine and Trichostatin A on Gene Expression and DNA Methylation Status in Cloned Bovine Blastocysts

We previously found that treatment of both donor cells and early cloned embryos with combination of 5-aza-2'-deoxycytidine (5-aza-dC) and trichostatin A (TSA) significantly improve the in vitro and full-term development of nuclear transfer (NT) bovine embryos. To investigate how this treatment improved the epigenetic reprogramming of somatic cell nuclei, we compared the expression levels of DNA methylation-, chromatin structure-, and development-related genes in in vitro fertilized (IVF group), NT (C-NT group), and 5-aza-dC and TSA-treated NT (T-NT group) single blastocyst using quantitative real-time PCR. We also compared the DNA methylation status of satellite I among three groups using bisulfite sequencing analysis and combined bisulfite restriction analysis (COBRA). There were significantly lower levels of DNMT1, DNMT3b, HDAC2, and IGF2 transcripts in T-NT blastocysts than in C-NT blastocysts, whereas the relative abundance of OCT4 and SOX2 mRNA was significantly increased in T-NT blastocysts compared to C-NT blastocysts. In addition, the treatment also reduced the DNA methylation levels of NT blastocysts on satellite I sequence. It is likely that TSA may act synergistically with 5-aza-dC to exert such modifications in gene expression and DNA methylation, subsequently enhancing developmental potential (in vitro and full-term) of treated cloned embryos.

Improving the Quality of Miniature Pig Somatic Cell Nuclear Transfer Blastocysts: Aggregation of SCNT Embryos at the Four‐cell Stage

Miniature pigs share many similar characteristics such as anatomy, physiology and body size with humans and are expected to become important animal models for therapeutic cloning using embryonic stem cells (ESCs) derived by somatic cell nuclear transfer (SCNT). In the present study, we observed that miniature pig SCNT blastocysts possessed a lower total number of nuclei and a lower percentage of POU5F1-positive cells than those possessed by in vitro fertilized (IVF) blastocysts. To overcome these problems, we evaluated the applicability of aggregating miniature pig SCNT embryos at the four-cell stage. We showed that (i) aggregation of two or three miniature pig SCNT embryos at the four-cell stage improves the total number of nuclei and the percentage of POU5F1-positive cells in blastocysts, and (ii) IVF blastocysts with low cell numbers induced by the removal of two blastomeres at the four-cell stage did not exhibit a decrease in the percentage of POU5F1-positive cells. These results suggest that the aggregation of miniature pig SCNT embryos at the four-cell stage can be a useful technique for improving the quality of miniature pig SCNT blastocysts and indicating that improvement in the percentage of POU5F1-positive cells in aggregated SCNT embryos is not simply the consequence of increased cell numbers.

65 SCRIPTAID CORRECTS GENE EXPRESSION OF A FEW ABERRANTLY REPROGRAMMED TRANSCRIPTS IN NUCLEAR TRANSFER PIG BLASTOCYST STAGE EMBRYOS

Scriptaid is a histone deacetylase inhibitor (HDACi) that can increase cloning efficiency. The objective of this study was to identify aberrantly reprogrammed transcripts by performing transcriptional profiling between in vivo (IVV), nuclear transfer (NT) blastocyst stage embryos and the donor cell line (cells). This was followed by measuring HDAC activity (Epigentek) in zygotes and by real-time PCR on a selected subset of genes at the blastocyst stage to determine if Scriptaid treatment (NTS) corrected the aberrant gene expression. NTS embryos were treated with 500 nM Scriptaid for 14 h after activation. NT and NTS embryos were transferred into gilts on Day 0 or 1 of oestrus and collected 6 days later by uterine flush. IVV samples were collected on Day 8 of gestation. 3 pools of 10 to 15 embryos and cells were collected for each treatment and analysed twice. For transcriptional profiling, total RNA was isolated by using Trizol (Invitrogen, Carlsbad, CA, USA), amplified by using an Ovation Ribo-SPIA linear amplification kit (Nugen), labelled with Cy5 and compared to reference labelled with Cy3. Lowess normalization and analysis was performed in Genespring 7.3.1. ANOVA was performed with the Benjamini and Hochberg False Discovery Rate. Transcripts that were different between IVV and NT (P ≤ 0.20) and significantly different from the donor cell line (P ≤ 0.05) were classified as being aberrantly reprogrammed. This comparison resulted in 119 under- and 60 over-compensated transcripts. Functional annotation classification was performed in DAVID and identified under-compensated pathways (oxidative phosphorylation and protein biosynthesis) and over-compensated pathways (chromatin packaging/remodelling and protein complex assembly). Fourteen transcripts were chosen for real-time PCR validation and evaluation of the effect of Scriptaid. Relative gene expression was compared between IVV, NT, NTS, and cells by the comparative Ct method with SYBR Green Supermix (Bio-Rad) and statistical analysis was performed in SAS 9.1 (SAS Institute Inc., Cary, NC, USA) by using a least significant difference test (P ≤ 0.05). NTS embryos had 3 transcripts returning to the same level as IVV (H3F3A, CAPG, and SEPT7). The level of the majority of the transcripts (8/14) was not affected by NTS treatment, e.g. histone deacetylase SIRT1 and H1 histone, member 0 (H1F0). However, Scriptaid treatment caused COX5A to be further over compensated in NTS with expression levels higher than IVV and NT. 2 transcripts had expression levels that were lower in NTS compared to both IVV and NT including GPD1L and EIF3E. Scriptaid treatment significantly affected gene expression in 6 of the 14 transcripts evaluated. Scriptaid treatment of the reconstructed zygotes did not affect the majority of the transcripts when measured at the blastocyst stage. HDAC activity was significantly reduced in NTS compared to NT 1-cell stage embryos (P ≤ 0.038). While Scriptaid reduced HDAC activity, it returned only a few genes to normal IVV levels. This project was supported in part by the USDA NRI (2006-35203-17282) and Food for the 21st Century.

The Effect of TSA Treatment on Gene Expressions in Porcine Cloned Embryos During Preimplantation Development.

It has been proposed that nuclear reprogramming in somatic cell nuclear transferred (SCNT) embryos may be enhanced by after treatment with epigenetic modifiers, such as TSA, scriptaid, or sodium butyrate. The present study was conducted using experiments involving quantitative real-time PCR in order to determine the effect of TSA on the expression pattern of seven genes (OCT4, ID1, H19, NNAT, PEG1, CYTOKERATIN8 and 18) in nuclear transfer (NT) blastocysts treated with/without TSA and in vivo-derived blastocysts. The results showed that the pluripotent genes, OCT4 and ID1, were transcribed at a low level in NT blastocysts than these of in vivo counterparts. There appeared to be a slightly higher expression of these genes in NT blastocysts with TSA treatment compared with untreated ones, but this was not significant. However, the imprinting genes, H19 and NNAT, tended to be highly expressed in both NT blastocysts with and without TSA treatment than in in vivo blastocysts. PEG1 transcripts were nearly equally expressed in all samples. As a trophoblast marker genes, cytokeratin 8 and 18, were transcribed at a low level in NT blastocysts than these of in vivo counterparts. Moreover, these genes were expressed at a similar level in TSA treated and untreated NT blastocysts. Our findings revealed that transcriptional activity of the tested genes, except for PEG1, became high or low in NT blastocysts when compared with in vivo blastocysts. These result indicated these was the differences in gene expression patterns between in vivo and cloned blastocysts, regardless of TSA treatment. Nevertheless, the several genes in NT blastocysts after TSA treatment showed a slight tendency towards the expression patterns of in vivo blastocysts. (poster)

Cloning efficiency following ES cell nuclear transfer is influenced by the methylation state of the donor nucleus altered by mutation of DNA methyltransferase 3a and 3b

The epigenetic state of donor cells plays a vital role in the nuclear reprogramming and chromatin remodeling of cloned embryos. In this study we investigated the effect of DNA methylation state of donor cells on the development of mouse embryos reconstructed with embryonic stem (ES) cell nuclei. Our results confirmed that deletion of the DNA methyltransferase 3a (Dnmt3a) and DNA methyltransferase 3b (Dnmt3b) distinctly decreases the level of DNA methylation in ES cells. In contrast to wild type ES cells (J1), Dnmt3a − / − 3b − / − (DKO) and Dnmt3b − / − (3bKO) donor cells significantly elevated the percentage of embryonic stem cell nuclear transfer (ECNT) morula, blastocysts and postimplantation embryos (P < 0.05). However, the efficiency of establishment of NT-ES cell lines derived from DKO reconstructed blastocysts was not improved, and the expression pattern of OCT4 and CDX2 in cloned blastocysts and postimplantation embryos was not altered either. Our results suggest that the DNA methylation state of the donor nucleus is an important factor in regulation of the donor nuclear reprogramming.

Establishment and characterization of embryonic stem-like cells from porcine somatic cell nuclear transfer blastocysts

This study was aimed to establish embryonic stem (ES)-like cells from blastocysts derived from somatic cell nuclear transfer (SCNT) in pig. Somatic cells isolated from both day-30 fetus and neonatal cloned piglet were used for donor cells. A total of 60 blastocysts (46 and 14 derived from fetal and neonatal fibroblast donor cells, respectively) were seeded onto a mitotically inactive mouse embryonic fibroblast (MEF) monolayer and two ES-like cell lines, one from each donor cell type, were established. They remained undifferentiated over more than 52 (fetal fibroblast-derived) and 48 (neonatal fibroblast-derived) passages, while retaining alkaline phosphatase activity and reactivity with ES specific markers Oct-4, stage-specific embryonic antigen-1 (SSEA-1), SSEA-4, TRA-1-60 and TRA-1-81. These ES-like cells maintained normal diploid karyotype throughout subculture and successfully differentiated into embryoid bodies that expressed three germ layer-specific genes (ectoderm: beta-III tubulin; endoderm: amylase; and mesoderm: enolase) after culture in leukemia inhibitory factor-free medium. Microsatellite analysis confirmed that they were genetically identical to its donor cells. Combined with gene targeting, our results may contribute to developing an efficient method for producing transgenic pigs for various purposes.

Establishment of trophoblast stem cell lines from somatic cell nuclear-transferred embryos

Placental abnormalities occur frequently in cloned animals. Here, we attempted to isolate trophoblast stem (TS) cells from mouse blastocysts produced by somatic cell nuclear transfer (NT) at the blastocyst stage (NT blastocysts). Despite the predicted deficiency of the trophoblast cell lineage, we succeeded in isolating cell colonies with typical morphology of TS cells and cell lines from the NT blastocysts (ntTS cell lines) with efficiency as high as that from native blastocysts. The established 10 ntTS cell lines could be maintained in the undifferentiated state and induced to differentiate into several trophoblast subtypes in vitro. A comprehensive analysis of the transcriptional and epigenetic traits demonstrated that ntTS cells were indistinguishable from control TS cells. In addition, ntTS cells contributed exclusively to the placenta and survived until term in chimeras, indicating that ntTS cells have developmental potential as stem cells. Taken together, our data show that NT blastocysts contain cells that can produce TS cells in culture, suggesting that proper commitment to the trophoblast cell lineage in NT embryos occurs by the blastocyst stage.

Early alteration of the self-renewal/differentiation threshold in trophoblast stem cells derived from mouse embryos after nuclear transfer

Development after nuclear transfer (NT) is subjected to defects originating from both the epiblast and the trophoblast parts of the conceptus and is always accompanied by placentomegaly at term. Here we have investigated the origin of the reprogramming errors affecting the trophoblast lineage in mouse NT embryos. We show that trophoblast stem (TS) cells can be derived from NT embryos (ntTS cells) and used as an experimental in vitro model of trophoblast proliferation and differentiation. Strikingly, TS derivation is more efficient from NT embryos than from controls and ntTS cells exhibit a growth advantage over control TS cells under self-renewal conditions. While epiblast-produced growth factors Fgf4 and Activin exert a fine-tuned control on the balance between self-renewal and differentiation of control TS cells, ntTS cells exhibit a reduced dependency upon their micro-environment. Since the supply of growth factors is known do decrease at the onset of placental formation in vivo we propose that TS cells in NT embryos continue to self-renew during a longer period of time than in fertilized embryo. The resulting increased pool of progenitors could contribute to the enlarged extra-embryonic region observed in the early trophoblast of in vivo grown mouse NT blastocysts that results in placentomegaly.

Global gene expression analysis of bovine somatic cell nuclear transfer blastocysts and cotyledons

Low developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos is a universal problem. Abnormal placentation has been commonly reported in SCNT pregnancies from a number of species. The present study employed Affymetrix bovine expression microarrays to examine global gene expression patterns of SCNT and in vivo produced (AI) blastocysts as well as cotyledons from day-70 SCNT and AI pregnancies. SCNT and AI embryos and cotyledons were analyzed for differential expression. Also in an attempt to establish a link between abnormal gene expression patterns in early embryos and cotyledons, differentially expressed genes were compared between the two studies. Microarray analysis yielded a list of 28 genes differentially expressed between SCNT and AI blastocysts and 19 differentially expressed cotyledon genes. None of the differentially expressed genes were common to both groups, although major histocompatibility complex I (MHCI) was significant in the embryo data and approached significance in the cotyledon data. This is the first study to report global gene expression patterns in bovine AI and SCNT cotyledons. The embryonic gene expression data reported here adds to a growing body of data that indicates the common occurrence of aberrant gene expression in early SCNT embryos.

Transgene Expression of Green Fluorescent Protein and Germ Line Transmission in Cloned Pigs Derived fromIn VitroTransfected Adult Fibroblasts

The pig represents the xenogeneic donor of choice for future organ transplantation in humans for anatomical and physiological reasons. However, to bypass several immunological barriers, strong and stable human genes expression must occur in the pig's organs. In this study we created transgenic pigs using in vitro transfection of cultured cells combined with somatic cell nuclear transfer (SCNT) to evaluate the ubiquitous transgene expression driven by pCAGGS vector in presence of different selectors. pCAGGS confirmed to be a very effective vector for ubiquitous transgene expression, irrespective of the selector that was used. Green fluorescent protein (GFP) expression observed in transfected fibroblasts was also maintained after nuclear transfer, through pre- and postimplantation development, at birth and during adulthood. Germ line transmission without silencing of the transgene was demonstrated. The ubiquitous expression of GFP was clearly confirmed in several tissues including endothelial cells, thus making it a suitable vector for the expression of multiple genes relevant to xenotransplantation where tissue specificity is not required. Finally cotransfection of green and red fluorescence protein transgenes was performed in fibroblasts and after nuclear transfer blastocysts expressing both fluorescent proteins were obtained.

Comparative pluripotency analysis of mouse embryonic stem cells derived from wild-type and infertile hermaphrodite somatic cell nuclear transfer blastocysts

Therapeutic cloning, whereby embryonic stem cells (ESCs) are derived from patient-specific cloned blastocysts via somatic cell nuclear transfer (SCNT), holds great promise for treating many human diseases using regenerative medicine. Teratoma formation and germline transmission have been used to confirm the pluripotency of mouse stem cells, but human embryonic stem cells (hESCs) have not been proven to be fully pluripotent owing to the ethical impossibility of testing for germ line transmission, which would be the strongest evidence for full pluripotency. Therefore, formation of differentiated cells from the three somatic germ layers within a teratoma is taken as the best indicator of pluripotency in hESC lines. The possibility that these lines lack full multi-or pluripotency has not yet been evaluated. In this study, we established 16 mouse ESC lines, including 3 genetically defective nuclear transfer-ESC (ntESC) lines derived from SCNT blastocysts of infertile hermaphrodite F1 mice and 13 ntESC lines derived from SCNT blastocysts of normal F1 mice. We found that the defective ntESCs expressed all in vitro markers of pluripotency and could form teratomas that included derivatives from all three germ layers, but could not be transmitted via the germ line, in contrast with normal ntESCs. Our results indicate that teratoma formation assays with hESCs might be an insufficient standard to assess full pluripotency, although they do define multipotency to some degree. More rigorous standards are required to assess the safety of hESCs for therapeutic cloning.

Differential acetylation of histone H4 lysine during development of in vitro fertilized, cloned and parthenogenetically activated bovine embryos

The oocyte is remarkable in its ability to remodel parental genomes following fertilization and to reprogram somatic nuclei after nuclear transfer (NT). To characterise the patterns of histone H4 acetylation and DNA methylation during development of bovine gametogenesis and embryogenesis, specific antibodies for histone H4 acetylated at lysine 5 (K5), K8, K12 and K16 residues and for methylated cytosine of CpG dinucleotides were used. Oocytes and sperm lacked the staining for histone acetylation, when DNA methylation staining was intense. In IVF zygotes, both pronuclei were transiently hyper-acetylated. However, the male pronucleus was faster in acquiring acetylated histones, and concurrently it was rapidly demethylated. Both pronuclei were equally acetylated during the S to G2-phase transition, while methylation staining was only still observed in the female pronucleus. In parthenogenetically activated oocytes, acetylation of the female pronucleus was enriched faster, while DNA remained methylated. A transient de-acetylation was observed in NT embryos reconstructed using a non-activated ooplast of a metaphase second arrested oocyte. Remarkably, the intensity of acetylation staining of most H4 lysine residues peaked at the 8-cell stage in IVF embryos, which coincided with zygotic genome activation and with lowest DNA methylation staining. At the blastocyst stage, trophectodermal cells of IVF and parthenogenetic embryos generally demonstrated more intense staining for most acetylated H4 lysine, whilst ICM cells stained very weakly. In contrast methylation of the DNA stained more intensely in ICM. NT blastocysts showed differential acetylation of blastomeres but not methylation. The inverse association of histone lysine acetylation and DNA methylation at different vital embryo stages suggests a mechanistically significant relationship. The complexities of these epigenetic interactions are discussed.

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.

The mRNA expression of brain-derived neurotrophic factor in oocytes and embryos and its effects on the development of early embryos in cattle

The aims of the study were to measure the mRNA expression of brain-derived neurotrophic factor (BDNF) in bovine oocytes and early embryos derived from in vitro fertilization (IVF), parthenogenetic activation (PA) and nuclear transfer (NT), and to investigate the effects of BDNF on the development of IVF and parthenogenetic embryos. Bovine oocytes matured in vitro for 22 h were in vitro fertilized or parthenogenetic activated. By reverse transcription-PCR and quantitative real-time PCR, we found that germinal vesicle (GV) oocytes, metaphase II (MII) oocytes, 4-cell and 8-cell embryos, morulae, and blastocysts were all shown to express mRNA for BDNF. The mRNA levels for BDNF gene were different in bovine oocytes and IVF embryos at different stages (P < 0.01), with the highest expression in MII oocytes and the lowest expression in 8-cell embryos. The mRNA for BDNF was highly expressed in the PA and IVF blastocysts compared to the NT blastocysts (P < 0.01). Supplementation of culture media with BDNF at the concentration of 40 μg/l caused a significant increase in the rates of in vitro-fertilized blastocyst formation (P < 0.05) and parthenogenetic blastocyst formation (P < 0.05). However, the rate of oocyte cleavage in BDNF groups was not significantly different from that in the BDNF-free control (P > 0.05) after IVF or PA. We have also investigated the effects of BDNF on the growth of granulosa cells, which were used for co-culture of bovine early embryos. The results revealed that supplementation of culture media with 20 μg/l BDNF promoted the growth of granulosa cells (P < 0.01). Taken together, these results provided evidence for the role of neurotrophins in promoting early embryonic development as well as in the growth of granulosa cells by the co-culture system, indicating that BDNF can directly or indirectly promote bovine early embryo development.

Comparative Gene Expression Analysis of Bovine Nuclear-Transferred Embryos with Different Developmental Potential by cDNA Microarray and Real-Time PCR to Determine Genes That Might Reflect Calf Normality

Placental abnormalities are the main factor in the high incidence of somatic cell clone abnormalities. The expression of several trophoblast cell-specific molecules is enhanced during gestational days 7 to 14. To determine the possible genes whose expression patterns might reflect calf normality, we first compared the gene expression profiles on day 15 between in vitro-fertilized (IVF) embryos and two types of somatic cell nuclear-transferred embryos with either a high (FNT) or low (CNT) incidence of neonatal abnormalities using a cDNA microarray containing 16 of 21 placenta-specific genes developed from tissues collected across gestation. To identify significant genes from the screening of day 15 embryos, genes with a less than two-fold difference in expression between IVF and CNT embryos, and those with a greater than two-fold difference between IVF and FNT and between CNT and FNT were considered to contribute to clone abnormalities. These two comparisons revealed 18 down-regulated and 18 upregulated genes of the 1722 genes examined. We then examined the expression levels of 10 genes with known functions in eight-cell and blastocyst-stage embryos by real-time PCR. The mRNA expression pattern of interferon (IFN)-tau, a trophectoderm-related gene, differed between IVF, CNT, and FNT eight-cell embryos; few or none of the IVF or CNT eight-cell embryos expressed IFN-tau mRNA, but all eight-cell FNT embryos expressed IFN-tau. IFN-tau mRNA expression was significantly higher in IVF blastocysts, however, than in nuclear-transferred blastocysts. Average IFN-tau mRNA expression in FNT blastocysts was not different from that in CNT blastocysts, due to one CNT blastocyst with high expression. The precise relation between early expression of IFN-tau mRNA and inferior developmental potential in cloned embryos should be examined further.

Comparison of the interferon‐tau expression from primary trophectoderm outgrowths derived from IVP, NT, and parthenogenote bovine blastocysts

The expression of interferon-tau (IFN-tau) is essential for bovine embryo survival in the uterus. An evaluation of IFN-tau production from somatic cell nuclear transfer (NT)-embryo-derived primary trophectoderm cultures in comparison to trophectoderm cultured from parthenogenote (P) and in vitro matured, fertilized, and cultured (IVP) bovine embryos was performed. In Experiment 1, the success/failure ratio for primary trophectoderm colony formation was similar for IVP and NT blastocysts [IVP = 155/29 (84%); NT 104/25 (81%)], but was decreased (P = .05) for P blastocysts [54/43 (56%)]. Most trophectoderm colonies reached diameters of at least 1 cm within 3-4 weeks, and at this time, 72 hr conditioned cell culture medium was measured for IFN-tau concentration by antiviral activity assay. The amount of IFN-tau produced by IVP-outgrowths [4311 IU/mL (n = 155)] was greater (P < .05) than that from NT- [626 IU/mL (n = 104)] and P - [1595 IU/mL (n = 54)] derived trophectoderm. Differential expression of IFN-tau was confirmed by immunoblotting. In Experiment 2, colony formation was again similar for IVP and NT blastocysts [IVP = 70/5 (93%); NT 67/1 (99%)] and less (P < .05) for P blastocysts [65/27 (70%)]. Analysis of trophectoderm colony size after 23 days in culture showed a similar relationship with P-derived colonies being significantly smaller in comparison to IVP and NT colonies. A differential expression of IFN-tau was also observed again, but this time as measured over time in culture. Maximal IFN-tau production was found at day-14 of primary culture and diminished to a minimum by the 23rd day.

SOMATIC CELL NUCLEAR TRANSFER USING DONOR CELLS SELECTED FOR DNA METHYLTRANSFERASE 1 (DNMT1) EXPRESSION

Bovine nuclear transfer (NT) embryos and fetuses often display abnormalities that are not observed in their in vivo produced counterparts, and are attributed to large offspring syndrome. Gene expression is altered in NT embryos and is thought to be due to inadequate reprogramming of the somatic cell nucleus. It is known that the embryonic genome must be rapidly demethylated, but often does not occur normally in NT embryos. This may be due to the introduction of Dnmt1s into the NT embryo, the enzyme responsible for maintaining DNA methylation (DM) in somatic cells, but which is never found in early embryos. Chemicals, such as 5azaC, have been used to artificially reduce DM, but have proven problematic in NT. We previously showed that culture without chemicals can be used, with cell type and time in culture affecting expression of Dnmt1 in donor cells. Here we tested the effects of using donor cells selected for Dnmt1 expression (low vs. high) as a means to reduce DM in NT embryos. Cell lines were grown to passage 4, 8 and 12, diluted out and frozen in individual use aliquots for later use. An aliquot of each cell line was taken for RNA isolation, and Dnmt1 expression analysis using Real-time RTPCR. Two cell lines with the same genotype, but at different passages were selected as having higher (HD) and lower Dnmt1 (LD) expression for use in NT experiments. Cumulus oocyte complexes were retrieved by slashing 2–8mm follicles on ovaries obtained from the abattoir, and were matured for 17–19h. Metaphase II oocytes (n=874) were selected for polar body extrusion and were enucleated for use in NT. Cells were transferred from either HD or LD groups, and fused. Fused couplets were activated with ionomycin and DMAP using standard protocols, and cultured within treatment in KSOM to d7 (activation=0). Global DM was evaluated in NT (LD and HD) and in vitro produced (IVP) control embryos (2-cell, 8-cell and blastocyst stages, 10/treatment and stage) by immunocytochemistry using 5MeC antibody (Calbiochem) and a FITC-conjugated secondary antibody (Santa Cruz). IgG and PBS served as negative controls to show antibody specificity. Pixel data were collected using AxioVision software (Zeiss) and were analyzed by LSMeans ANOVA using SAS, and significance was deemed p<0.05. There was significantly less DM in 2-cell IVP when compared to either LD or HD NT embryos (p<0.05). By the 8-cell stage, NT produced with LD had lower DM than 8- cell HD NT (p<0.05), but was still higher than 8-cell IVP (p<0.05). NT blastocysts produced with LD had DM similar to IVP (p>0.05), and both were lower than HD NT blastocysts (p<0.05). These data indicate that cell lines selected for reduced Dnmt1 expression can reduce global DM in the developing NT embryo. However, DM was not reduced to the levels found in IVP embryos at either the 2- or the 8-cell stages. Future efforts will need to determine if these reductions are adequate and allow for normal development in vivo, or if additional efforts will need to be made to further mimic normal embryonic reprogramming events. This project was supported by National Research Initiative Competitive Grant no. 2006- 35203-16620 from the USDA Cooperative State Research, Education, and Extension Service. (platform) (platform)

The effects of time of first cleavage, developmental stage, and delipidation of nuclear-transferred porcine blastocysts on survival following vitrification

The effect of removing cytoplasmic lipid droplets (delipidation) at the 2-cell and developmental stages on the survival of porcine somatic cell nuclear-transferred blastocysts developed from the enucleated oocytes receiving somatic cells from kidney of an adult female after cryopreservation was examined. Vitrification was performed using the Cryoloop method with a small volume of medium (0.5 μl). To select 2-cell embryos with a high potential to develop into blastocysts, the relationship between the timing of the first cleavage and the developmental potential was examined. The potential of nuclear-transferred oocytes to develop into blastocysts in the intermediate-cleavage group (20–24 h after activation, 25%) was slightly or significantly ( P < 0.05) higher than that in fast-cleavage (<20 h after activation, 13%) and slow-cleavage groups (>24 h after activation, 5%). Most non-delipidated blastocysts did not survive after thawing (0% for early-stage and 9% for advanced-stage blastocysts), but the survival rate of delipidated blastocysts 48 h after culture (54% and 72%, respectively) was not significantly different from that of non-vitrified blastocysts (80% and 92%, respectively). The survival rate of advanced-stage blastocysts after vitrification was slightly higher than that of early-stage blastocysts. The present study demonstrates that somatic cell nuclear-transferred porcine blastocysts developed from embryos selected at the 2-cell stage can be preserved by vitrification with a small volume of medium if the lipid droplets of the embryos are first removed.