bluuebunny/crossref_metadata_2025 huggingface dataset:
370 HISTONE PHOSPHORYLATION PATTERNS AND CHROMOSOMAL STABILITY OF CULTURED BOVINE FIBROBLASTS
Authors: Giraldo A. , Lynn J. , Godke R. , Jenkins J. , Bondioli K.
Published: December 2006

The low percentage of cloned offspring produced by nuclear transfer has been attributed to a variety of factors, including aneuploidy of the donor cells. Previous reports indicate that cultured bovine fibroblasts have a significantly higher level of aneuploidy in late passages than in early passages (Giraldo et al. 2005 J. Reprod. Fert. Dev. 17, 167). Phosphorylation of histone H3 at Serine 10 (Ser10) has been shown to be involved in chromosome compaction during cell division.Abnormal phosphorylation of this histone residue during metaphase could lead to abnormal chromosome segregation and extensive chromosome loss during mitosis. Suboptimal culture conditions may lead to abnormal histone H3 phosphorylation (HP) patterns, ultimately inducing missegregation and loss of chromosomes. The objective of the present study was to determine if the high percentage of aneuploid bovine fibroblast cells observed after long-term culture is associated with an abnormal HP pattern. Four bovine fibroblast cell lines were established from 40- to 60-day fetuses. Cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin in 5% CO2 at 37°C and passaged at confluence. Relative levels of HP were determined in three different replicates at population doublings (PD) 2, 10, and 20. Cells were fixed and incubated with an anti-phosphorylated histone H3 (Ser10) antibody, labeled with a secondary antibody, counterstained with propidium iodide, and analyzed for HP fluorescence by flow cytometry. The number of chromosomes was also determined in counts of 800 metaphases. Differences in aneuploidy and HP fluorescence of cells in metaphase among PD were analyzed by χ2 two-way ANOVA, respectively (P < 0.05). The percentages of aneuploid cells in each of the cell lines increased progressively with duration of culture and were elevated from the start. Multinucleated cells were frequently observed after prolonged time in culture in all of the cell lines. The mean phosphorylated histone levels (relative fluorescence intensity) in cells during metaphase were 180.0, 131.5, 174.7, and 157.6 in PD 2; 170.4, 105.72, 145.8, and 152.7 in PD 10; and 274.0, 251.6, 191.4, and 308.3 in PD 20 for the four cell lines, respectively. No difference in HP levels was observed between PD 2 and PD 10. The average of HP during metaphase for the cell lines increased significantly from 160.9 at early passage to 256.3 at late passage (see Table 1). The increase in levels of HP occurred concurrently with the high percentage of aneuploid cells after extended time in culture. These data are consistent with the hypothesis that aneuploid cells observed after long in vitro culture are associated with abnormal HP patterns.

Table 1.
Relative HP fluorescence and aneuploidy at different PD of fetal bovine fibroblasts

This study was supported by Grants in Aid of Research Program (GIAR) from Sigma Xi to A.G.
371 EVALUATION OF ENRICHMENT STRATEGIES FOR PORCINE SPERMATOGONIA BY EXPRESSION OF PROTEIN GENE PRODUCT 9.5, A SPERMATOGONIA-SPECIFIC MARKER IN THE PIG TESTIS
Authors: Luo J. , Megee S. , Rathi R. , Dobrinski I.
Published: December 2006

Transplantation of genetically altered male germ cells is under investigation as a novel route to generate transgenic animal models. Identification and isolation of spermatogonial stem cells are a prerequisite for this strategy. The objectives of this study were to validate a marker for identification of undifferentiated porcine spermatogonia, and to use this marker to develop a practical enrichment strategy for spermatogonia from pig testis. We established that expression of protein gene product (PGP) 9.5 is a spermatogonia-specific marker in porcine testis through analysis of its expression pattern in testis cells, by comparison with the expression of the cell-type specific proteins GATA-4 (expressed in Sertoli cells) or PLZF (expressed in undifferentiated mouse spermatogonia) in seminiferous tubules at different ages, and by comparison of expression levels of PGP 9.5 and the germ cell-specific protein VASA in different cell fractions after differential plating. Using expression of PGP 9.5 as a marker, we characterized enrichment of porcine spermatogonia from two-week-old (2wo) and 10-week-old (10wo) pigs by immunofluorescence either after differential plating only or after velocity sedimentation at unit gravity followed by differential plating. After differential plating with overnight culture to deplete testicular somatic cells that firmly attach to culture dishes, spermatogonia (mean � SEM per 1000 cells) were 5-fold enriched (P < 0.05) in cells remaining in suspension (fraction I) (2wo: 54.0 � 9.1; 10wo: 162.7 � 30.5) and in populations slightly attached to the culture plate (fraction II) (2wo: 92.7 � 8; 10wo: 159.5 � 22.5) compared to the initial samples (2wo: 12.3 � 2.7; 10wo: 27.2 � 2.9). Slightly attached spermatogonia appear to be superior for future experiments due to higher viability (>90%) than spermatogonia remaining in suspension (<50%). Cell populations containing up to 70% spermatogonia with good viability (>80%) were achieved by velocity sedimentation isolation followed by differential plating. These results indicate that expression of PGP 9.5 is a useful marker for identification of undifferentiated porcine germ cells. Simple differential adhesion culture of testis cells harvested from pre-pubertal boars can supply cell populations enriched in spermatogonia for subsequent genetic manipulation and transplantation.
This work was supported by 1 R01 RR17359-01.
372 LASER-ASSISTED LENTIVIRAL TRANSGENESIS IN BOVINE EMBRYOS
Authors: Ewerling S. , Hofmann A. , Klose R. , Rink K. , Brem G. , Pfeifer A. , Wolf E.
Published: December 2006

Lentiviral vectors have been shown to be a powerful tool to create transgenic livestock (Hofmann et al. 2003 EMBO Rep. 4, 1054-1060; Hofmann et al. 2004 Biol. Reprod. 71, 405-409). Due to their inability to pass the zona pellucida (ZP) by themselves, viral particles were delivered by subzonal microinjection of oocytes or zygotes. Here we show that an artificial opening of the ZP, generated by an infrared 1.48 �m microsurgical diode laser (OCTAX Laser Shot", kindly provided by MTG, Altdorf, Germany) and subsequent culture of oocytes in virus suspension, was sufficient to allow virus integration and expression of eGFP. The ZP of denuded in vitro-matured bovine oocytes was microdrilled with 2 to 4 laser shots of 3.0-ms pulse length each to create an opening of at least 40 �m. Oocytes were transferred to microdroplets (20 �L of Fert Talp) containing the virus suspension (lentiviral vector containing the eGFP reporter gene under the control of the human phosphoglycerate-kinase 1 promoter). After incubation for 2 or 4 h (5% CO2, maximum humidity, 39�C), oocytes were washed thoroughly in fresh droplets of medium to remove excessive virus. Sperm was added (0.5 � 106/mL) for 8 h. In vitro culture of embryos (5% CO2, 5% O2, maximum humidity, 39�C) was continued in culture medium (SOF) under oil for up to 8 days. Cleavage rate and number of blastocysts were determined on Days 2 and 8, respectively. Expression of eGFP was analyzed on Day 8 by fluorescence microscopy using an eGFP-specific filter with UV light source under an inverted microscope. In total, 267 oocytes were microdrilled. The range of optimal virus concentration was analyzed by titration. Seven different concentrations in a range of 1.25 � 106 to 5 � 107 IU/mL were used at an incubation time of 4 h. In one experiment using a high virus concentration, the incubation time was reduced to 2 h. Control groups were treated equally except for addition of virus. All experiments resulted in blastocysts showing eGFP expression in both the trophoblast and the inner cell mass. In four experiments we used aliquots of the same virus preparation (1.25 � 107 IU/mL) for infection. The cleavage rate ranged from 50 to 70%, and blastocyst rate from 15 to 35%. In three experiments showing good to average embryo development, 25 to 83% of the resulting blastocysts expressed eGFP, indicating efficient lentiviral transduction of oocytes following laser-assisted zona microdrilling. Studies directly comparing zona microdrilling and subzonal injection with regard to the efficacy of lentiviral transgenesis and the incidence of polyspermy are underway.
376 VECTOR-MEDIATED GENE TRANSFER INTO RHESUS MACAQUE EMBRYOS
Authors: Kubisch H. M. , Gagliardi C. , Romero D. G. , Bunnell B. A. , Ratterree M. S.
Published: December 2006

A series of experiments was performed to assess the suitability of various viral vectors for transformation of rhesus macaque (Macaca mulatta) embryos. Viral vectors included the adenovirus-associated virus (AAV) containing a CMV-EGFP transgene and a lentivirus (FUGW) carrying the green fluorescent protein (GFP) gene from either the jellyfish or a humanized version from renilla linked to an ubiquitin promoter. Embryos were generated by in vitro fertilization of oocytes retrieved by laparoscopy from superovulated females. Resulting zygotes were injected under the zona pellucida (ZP) with varying viral concentrations. Embryos were subsequently cultured for 5 days and thereafter analyzed by epifluorescence microscopy. A total of 62 zygotes were injected with one of three vectors AAV2 (25), AAV2.1 (15), or AAV2.7 (22). Of these 22, 9 and 16, respectively, reached the blastocyst and morula stage. There was no difference in the percentage of embryos expressing GFP between vectors (24, 53.2, and 36.4%, respectively). However, all of the positive embryos proved to be mosaics. In a second set of experiments, FUGW was injected under the ZP of 155 embryos. Of these, 76 received virus carrying renilla GFP, while the remaining 79 were injected with virus carrying the jellyfish GFP. Following injection with renilla, 52 reached the blastocyst/morula stage on Day 7, while 43 containing jellyfish GFP proceeded to this stage. Expression of jellyfish GFP could be seen in 65% of the embryos of which 35.6% were mosaics, whereas renilla GFP was found in only 15.8% of the embryos, although none of these were mosaic. To determine whether the mosaic expression was caused by transgene silencing, three mosaic embryos were dissociated on Day 3 and 10; 10 and 8 blastomeres, respectively, were obtained. Analysis by PCR showed all but one blastomere carrying the vector. Similarly, the presence of the vector was identified by PCR in 17 of 19 non-expressing embryos injected with renilla. These results show that AAV and lentivirus can transform rhesus embryos, which can subsequently continue in development. However, identification of positive embryos by epifluorescence alone may not be sufficient.
Funding was provided by NIH/NCRR grant RR000164-13.
38 FULL TERM DEVELOPMENT IN A COW CARRYING A NUCLEAR TRANSFER EMBRYO DERIVED FROM FIBROBLASTS AND OOCYTES OF ITS OWN CLONE
Authors: Heyman Y. , Lebourhis D. , Chavatte-Palmer P. , Richard C. , Renard J. P. , Vignon X.
Published: December 2006

Cloned animals are a unique model for studying the role of maternal immune response in failure of pregnancies often reported in bovine somatic cloning. Major histocompatibility complex class 1 (MHC1) has been shown to be down-regulated in placentomes during pregnancy, and abnormal MHC1 expression by the fetal membranes has been suggested to be the cause of pregnancy failure in bovine clones. We designed this study to investigate whether pregnancy losses would be decreased in cloned cows transferred with their genetically identical conceptus. Bovine fibroblasts were established from a skin biopsy on an adult Holstein cow from which a clone of 12 females had previously been derived. Five heifers of this set of clones were used for providing oocytes through (OPU) after stimulation, and seven were used as recipient heifers. Fibroblasts from this genotype were thawed, grown to confluency, and then used as the source of donor cells for NT. Recipient oocytes were matured in vitro before enucleation at 20–22 h post maturation (hpm). Embryos were reconstructed by fusion with donor cells at 23–24 hpm and activated in 10 µg/mL cycloheximide and 5 µg/mL cytochalasin B for 5 h after fusion, and then co-cultured on Vero cells for 7 days in microdrops of B2 medium. A control group of NT embryos was produced simultaneously with the same cell line and unrelated oocytes from slaughterhouse ovaries. Embryos were transferred into synchronous recipients (one blastocyst/recipient). The pregnancies were followed by ultrasonography. The reconstructed embryos fused at the same rate in both groups of oocytes, but the cleavage rate was significantly lower in the clone group (Table 1). The reason for this low rate is unclear. However, pregnancy rates after transfer of NT blastocysts were similar up to term between the related and non-related groups of transplanted embryos. These results suggest that the maintenance of pregnancy in bovine is not linked to genetic disparity between fetus and dam. More investigations are required to elucidate why the in vitro development was so low, and whether pregnancies established in cows with their genetically identical conceptus are less prone to abortion or physiopathological developments.

Table 1.
Development of NT embryos with clone or unrelated cell/oocyte/ recipient genotype
380 COMPARISON OF TRANSGENE EXPRESSIONS BY ICSI AND PRONUCLEAR MICROINJECTION IN MURINE AND PORCINE EMBRYOS
Authors: Saito H. , Kawano H.-O. , Kurome M. , Tomii R. , Ueno S. , Hiruma K. , Nagashima H.
Published: December 2006

Intracytoplasmic sperm injection (ICSI) of DNA-binding sperm produces transgenic offspring as effectively as pronuclear microinjection (PNM). A significant difference in these two methods is that DNA is introduced into MII oocytes during ICSI, which is likely to allow earlier gene integration compared to PNM. This leads us to hypothesize that ICSI reduces the chance of development of a mosaic embryo, a mixture of transgene-positive and -negative cells. To test this hypothesis, we compared expression patterns of the green flourescent protein (GFP) gene introduced by ICSI and PNM into murine and porcine oocytes. For ICSI, 2 to 5 × 105/μL of sperm frozen-thawed in CZB (for mice) or NIM (for pigs) were co-incubated with 2.5 ng/μL of transgene fragments (CAG-EGFP; 3 kb) for 5 min. Murine sperm were microinjected into in vivo-matured oocytes, and porcine sperm into in vitro-matured oocytes. PNM was performed by microinjection of several picoliters of the transgene fragments (10 ng/μL) into pronuclei of in vivo-fertilized oocytes for mice and in vitro-matured and -fertilized oocytes for pigs. ICSI and PNM embryos were cultured in vitro to the morula stage and treated with 0.5% pronase to remove the zona pellucida. These morulae were disassembled into individual blastomeres by pipetting into PBS containing 100 μM EDTA and examined for GFP expression under fluorescence microscopy. As shown in Table 1, the rate of mosaicism in GFP-expressing embryos was significantly lower for ICSI than for PNM (P < 0.01). In addition, GFP-expressing ICSI embryos were likely to contain high percentages, 81 to 100%, of GFP-positive cells, whereas GFP-expressing PNM embryos were significantly less likely to contain such high percentages of GFP-positive cells (P < 0.01). From these results, we conclude that transgenesis by ICSI was less likely to produce mosaic embryos, and that produced transgenic embryos contained higher proportions of transgene-positive cells, although genomic integration remains to be determined.

Table 1.
Transgene expression by ICSI and pronuclear microinjection in murine and porcine embryos

This work was supported by PROBRAIN.
4 GENE EXPRESSION IN CULTURES OF INNER CELL MASSES ISOLATED FROM IN VITRO-PRODUCED AND IN VIVO-DERIVED BOVINE BLASTOCYSTS
Authors: Pant D. , Keefer C.
Published: December 2006

Genetic modification of embryonic stem (ES) cells derived from domestic species could be exploited to produce transgenic animals; however, fully validated ES have not been obtained in domestic species. Recent findings regarding key transcription factors and regulation of pluripotency and self-renewal in murine ES cells may provide keys to enable the derivation of ES in domestic species. The aim of this study was to identify and monitor the expression of candidate genes, which are known to be involved in the maintenance of self-renewal and pluripotency in mouse and human ES cells, during the critical first steps in establishment of primary cultures. Inner cell masses (ICMs) were isolated via manual dissection of 25 to 30 commercial in vitro-produced (IVP) blastocysts (Bomed, Inc., Madison, WI, USA) in each of three separate replicates and from 10 in vivo-derived Day 7-8 bovine blastocysts. On the day of ICM isolation (Day 0), 4-5 ICM clumps were collected for RT-PCR analysis. The remaining isolated ICMs were cultured (4-5 ICM clumps per well) on mitomycin C (Sigma-Aldrich, St. Louis, MO, USA)-inactivated mouse embryonic fibroblasts (STO, ATCC, Manassas, VA, USA). The ICM clumps were cultured in 12-well tissue culture dishes in ES medium consisting of Knockout DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 15% FCS (Hyclone, Logan, UT, USA), 2 mM l-glutamine (Invitrogen), 0.1 mM 2-mercaptoethanol (MP Biomedicals, Irvine, CA, USA), and non-essential amino acids (Sigma). Two to four cultured ICM clumps were collected for RT-PCR analysis on Days 1-4 from IVP embryos and on Days 2, 4, and 6 from in vivo-derived embryos. Total RNA was extracted from the collected samples using the Absolutely RNA Nanoprep Kit (Strategene, La Jolla, CA, USA). First-strand DNAs were synthesized using Superscript III (Invitrogen) and cDNAs were amplified with PfuUltra hotstart PCR mastermix (Stratagene). Primers were designed based on homology between human and mouse sequences and were validated using bovine tissues. In experiments spanning these critical first few days of culture, the pluripotency-related genes (Nanog, Oct-4, Sox-2) and components of the LIF (LIFR, Gp130), BMP (Bmpr1a, Id-1), and Wnt (Beta-catenin, Frizzled) pathways were expressed in the ICM cultures over the 4 days of IVP-ICM cultures and the 6 days of in vivo-derived ICM cultures. These results indicate that the markers of pluripotency and the components of signaling pathways implicated in the maintenance of murine embryonic stem cells are present in ICMs of Day 7-8 bovine blastocysts and continue to be expressed at least during the initial days of culture. Genes (NCAM, Lef1) associated with early differentiation, however, were also expressed. Whether their expression is an indicator of ICM differentiation or of residual contamination with trophectoderm remains to be determined. Further studies will determine whether stimulation of these pathways can facilitate efficient derivation and maintenance ruminant ES cells.
40 EFFECT OF CHEMICAL ACTIVATION ON DEVELOPMENT AND APOPTOSIS OF PREIMPLANTATION PORCINE EMBRYOS DERIVED FROM NUCLEAR TRANSFER
Authors: Im G.-S. , Seo J.-S. , Hwang I.-S. , Kim S.-W. , Park H.-S. , Kim D.-H. , Yang B.-C. , Kong I.-K. , Yang B.-S.
Published: December 2006

Activation is one of key factors for improving developmental ability of pre-implantation nuclear transfer (NT) embryos. This study investigated the effect of chemical activation following fusion/activation on the development and apoptosis of pre-implantation porcine embryos derived from NT. Oocytes were aspirated from ovaries collected from a local abattoir, and then matured in TCM-199 for 42 to 44 h. Donor cells were prepared from a 35-day-old porcine fetus. Matured oocytes were enucleated and donor cells were introduced into the perivitelline space. Fusion/activation was conducted with two electric pulse of 1.2 kV/cm for 30 �s. Fused embryos were divided into four groups. The first one was the control without chemical activation; the other three groups were treated with thimerosal (0.2 mM for 10 min; T) and then with dithiothreitol (8 mM for 30 min; DTT), 6-dimethylaminopurine (2 mM for 3 h; 6-DMAP), or cycloheximide (10 �g/mL for 6 h; CH). Treated embryos were cultured in porcine zygote medium-3 (PZM-3) at 38.5�C under 5% CO2 in air for 6 days. Cleavage and blastocyst rate were determined on Days 3 and 6, respectively. Apoptosis was analyzed with a terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay from day 1 to 7. Embryos treated with chemicals following fusion/activation showed significantly higher blastocyst rates compared to control embryos fused/activated by electric pulse alone (12.6% for control vs. 21.1% for DTT, 20.8% for 6-DMAP, 20.6% for CH; P < 0.05). Although total cell number of blastocysts showed no significant difference, the ratio of inner cell mass to trophectoderm was significantly higher (P < 0.05) in embryos with chemical activation than in those without it (11.9 vs. 19.4, 18.1, and 24.1%; P < 0.05). Occurrence of apoptosis was first observed on Day 3, but there was no significant difference among treatments until Day 6. It was significantly increased in embryos with chemical activation on Day 7 compared to control embryos (5.1 vs. 7.1, 7.8, and 7.8%; P < 0.05). These results indicate that chemical activation following fusion/activation could support significantly a higher blastocyst rate for pre-implantation porcine embryos derived from nuclear transfer; however, it can increase occurrence of apoptotic cells at blastocyst stage.
42 DEVELOPMENTAL POTENTIAL AND REPROGRAMMING EFFICIENCY OF BOVINE EMBRYOS CLONED WITH ADULT FIBROBLASTS TREATED BY A DEMETHYLATING AGENT, S-ADENOSYL-HOMOCYSTEINE
Authors: Jeon B.-G. , Perrault S. D. , Rho G.-J. , Betts D. H. , King W. A.
Published: December 2006

Animal cloning by somatic cell nuclear transfer (SCNT) has been successfully applied to several species although with low efficiency and often associated with severe abnormalities. These poor outcomes are thought to be a consequence of aberrant DNA methylation patterns that result from incomplete epigenetic reprogramming of the transplanted nucleus into recipient oocytes. Telomerase, an enzyme not expressed in most somatic cells, should be expressed in cloned embryos. Therefore its activity has been used as an index of reprogramming in SCNT embryos. The objective of this study was to investigate the DNA methylation status of donor fibroblasts treated with a non-cytotoxic transmethylation inhibitor, S-adenosyl homocysteine (SAH), and to assess the relative telomerase activity (RTA) and developmental potential of SCNT embryos derived from such cells. Adult ear skin fibroblasts were cultured in DMEM supplemented with 0, 0.5, 1.0, or 2.0 mM SAH for 144 h by daily media change prior to nuclear transfer. The SAH-treated fibroblasts were immunostained with a fluorescein isothiocyanate (FITC) conjugated 5-methylcytosine antibody and the relative fluorescence intensity (RFI) was analyzed using a fluorescence microscope equipped with an Openlab" program (Improvision, Coventry, UK). RTA was measured in Day 8 SCNT blastocysts using the real-time quantitative telomeric repeat amplification protocol (RQ-TRAP). Fibroblasts treated with 0.5, 1.0, and 2.0 mM SAH showed lower levels of DNA methylation compared to nontreated controls, and the values did not differ among the treatment groups. Cleavage rates did not differ between the SCNT embryos derived from 0.5 mM SAH-treated cells and nontreated control cells (92.3% vs. 91.3%, respectively). However, the rates of blastocyst development and hatching were significantly (P < 0.05) higher in SCNT embryos derived from 0.5 mM SAH treated donor cells compared to controls (60.0 and 40.0% vs. 34.3 and 26.4%, respectively). Moreover, RTA of the 0.5 mM SAH SCNT embryos was significantly (P < 0.05) increased (1.5-fold) in relation to controls. S-adenosyl homocysteine treatment induces global DNA demethylation in donor fibroblasts and enhances the blastocyst frequencies for bovine SCNT embryos that also exhibit greater telomerase activity levels. These results suggest that use of hypomethylated donor somatic cells increases the developmental potential for SCNT embryos by enhancing the nuclear reprogramming efficiency.
This work was funded by NSERC, OMAF, OCAG, and CRC.
43 PRODUCTION OF PORCINE EMBRYOS BY NUCLEAR TRANSFER OF BONE MARROW MESENCHYMAL STEM CELLS
Authors: Jin H.-F. , Mohana Kumar B. , Kim J.-G. , Song H.-J. , Balasubramanian S. , Choe S.-Y. , Rho G.-J.
Published: December 2006

Recent experimental evidence indicates that adult stem cells are more desirable than somatic cells for nuclear transfer (NT) because of their easy reprogrammability to resemble the genome of the zygote (Zhu et al. 2004 Biol. Reprod. 71, 1890-1897). Mesenchymal stem cells (MSCs) are a heterogeneous population of uncommitted and lineage-committed cells and may have a more flexible potential as donor cells for NT. The aim of this study was to characterize an isolated population of porcine MSCs from bone marrow and to compare the developmental potential of cloned (IVF) embryos with MSCs and porcine fetal fibroblasts (pFFs) by assessing the cleavage and blastocyst rate, total cell numbers, inner cell mass (ICM) ratio and apoptosis. MSCs were obtained from the aspirated bone marrow of 6-8-month-old pigs. Cells were centrifuged, resuspended, and plated in advanced-DMEM (ADMEM) supplemented with 5% fetal bovine serum (FBS). The differentiation potential was demonstrated by culture of MSCs (passage 3) under conditions that were favorable for adipogenic, osteogenic, and chondrogenic development (Pittenger et al. 1999 Science 284, 143-147). Oil red O staining revealed that MSCs produced lipid droplets after incubation in adipogenic media. Following osteoinduction, MSCs exhibited robust alkaline phosphatase activity and cells later transformed into mineralized nodules as demonstrated by von Kossa staining. Histological staining of proteoglycan indicated chondrogenic differentiation. Cumulus-oocyte complexes were matured, fertilized, and cultured by the following method (Abeydeera et al. 2000 Theriogenology 54, 787-797). NT embryos were produced as described by Kim et al. (2005 Mol. Rep. Dev. 70, 308-313). Cleavage rate was significantly (P < 0.05) higher in IVF embryos than in NT embryos derived from MSCs and pFFs (84.5% � 4.6 vs. 52.2% � 5.4 and 50.8% � 5.2, respectively). However, blastocyst rates in IVF embryos and NT embryos derived from MSCs (20.6% � 2.5 and 18.5% � 3.0) did not differ but these rates were significantly (P < 0.05) higher than that for NT embryos derived from pFFs (9.5% � 2.1). Total cell numbers and the ratio of ICM to total cells among embryos developed in NT from MSCs (29.4 � 5.2 and 0.38 � 0.08, respectively) were significantly (P < 0.05) higher than for those from pFFs (22.6 � 5.5 and 0.18 � 0.12, respectively). Proportions of TUNEL-positive cells in NT embryos from pFFs (12.8 � 2.5) were significantly (P < 0.05) higher than in those from MSCs (8.6 � 1.8) and in IVF embryos (4.6 � 1.5). The results clearly demonstrate that multipotent bone marrow MSCs can make a suitable alternative to fibroblasts as donor cells and have a greater potential for producing viable cloned porcine embryos.
This work was supported by Grant No. R05-2004-000-10702-0 from KOSEF, Republic of Korea.
44 IN VITRO AND IN VIVO SURVIVABILITY ON VITRIFIED EMBRYOS OBTAINED BY BOVINE SOMATIC CELL NUCLEAR TRANSFER
Authors: Kaneyama K. , Kobayashi S. , Matoba S. , Hashiyada Y. , Imai K. , Yonai M. , Saito N.
Published: December 2006

Although many studies have been conducted on somatic cell nuclear transfer, there are only a few reports on cryopreservation of reconstructed embryos after nuclear transplantation. The objective of this study was to examine in vitro or in vivo development of vitrified blastocysts obtained by nuclear transfer. Nuclear transfer was carried out according to the procedure of Goto et al. (1999 Anim. Sci. J. 70, 243–245), and conducted using abattoir-derived oocytes and cumulus cells derived by ovum pickup from Holstein and Japanese Black cows. Embryos were vitrified as described by Saito et al. (1998 Cryobiol. Cryotech. 43, 34–39). The vitrification solution (GESX solution) was based on Dulbecco's PBS containing 20% glycerol (GL), 20% ethylene glycol (EG), 0.3 M sucrose (Suc), 0.3 M xylose (Xyl), and 3% polyethylene glycol (PEG). The blastocysts were equilibrated in three steps, with 10% GL, 0.1 M Suc, 0.1 M Xyl, and 1% PEG for 5 min (1); with 10% GL, 10% EG, 0.2 M Suc, 0.2 M Xyl, and 2% PEG for 5 min (2) and GESX solution (3). After transfer to GESX, equilibrated embryos were loaded to 0.25-mL straws and plunged into liquid nitrogen for 1 min. The vitrified blastocysts were warmed in water (20°C) and diluted in 0.5 M and 0.25 M sucrose for 5 min each. Equilibration and dilution procedures were conducted at room temperature (25–26°C). After dilution, the vitrified blastocysts were cultured in TCM-199 supplemented with 20% fetal calf serum and 0.1 mM β-mercaptoethanol at 38.5°C under gas phase of 5% CO2 in air. In Experiment 1, survival rates after vitrification were compared between the nuclear transfer and the IVF blastocysts. Survival rates of vitrified nuclear transfer blastocysts (n = 60, Day 8) at 24 and 48 h were 70.0% and 56.7%, respectively, and those of vitrified IVF blastocysts (n = 41) were 82.9% and 82.9%, respectively. There were no significant differences in survival rates at 24 and 48 h between the two groups. In Experiment 2, one (VIT-single) or two (VIT-double) vitrified and one (nonVIT-single) or two (nonVIT-double) nonvitrified reconstructed blastocysts per animal were transferred into Holstein dry cows. The result of Experiment 2 is shown in Table 1. This experiment demonstrated that the vitrification method in this study can be used for cloned embryo cryopreservation but the production rate should be improved.

Table 1.
Comparison of survival rates of vitrified or nonvitrified cloned embryos after transfer
46 ANTI-APOPTOTIC EFFECT OF INSULIN-LIKE GROWTH FACTOR-1 AND ITS RECEPTOR ON DEVELOPMENT OF PORCINE PRE-IMPLANTATION EMBRYOS PRODUCED BY IN VITRO FERTILIZATION AND SOMATIC CELL NUCLEAR TRANSFER
Authors: Kim S. , Lee S. H. , Kim J. H. , Jeong Y. W. , Koo O. J. , Mi Park S. , Lee E. G. , Hashem A. , Hossein M. S. , Lee B. C. , Kang S. K. , Hwang W. S.
Published: December 2006

Insulin-like growth factor (IGF)-I is a receptor-mediated autocrine and/or paracrine growth and/or survival factor for mammalian embryo development. It is known to promote the growth and development of mouse pre-implantation embryos. The present study was designed to investigate the effects of IGF-I (50 ng/mL), anti-IGF-I receptor (IGR-IR) antibody (0.05 �g/mL), and their combination on porcine pre-implantation embryo development. Furthermore, the mechanism underlying the embryotropic effects of IGF-I was evaluated by monitoring the incidence of apoptosis and expression of apoptosis-related genes. In both IVF and SCNT embryos, culturing with IGF-I increased the rate of blastocyst formation and this embryotropic effect was neutralized by culturing with IGF-I and anti-IGF-I receptor antibody. Significant effects on the development of blastocysts (P < 0.05) were found in IVF (16.9, 22.6, 9.3, and 13.5% for control, IGF-I, anti-IGF-IR antibody, and their combination, respectively) and SCNT (13.2, 21.0, 5.4, and 15.7%) embryos. Culturing IVF and SCNT embryos with IGF-I significantly increased the total number of cells in IVF blastocysts (58.3, 72.4, 41.1, and 55.2; P < 0.05), and SCNT blastocysts (49.2, 60.1, 35.2, and 43.1; P < 0.05), and it decreased the number of apoptotic nuclei in IVF blastocysts (3.9, 2.8, 5.5, and 3.9; P < 0.05) and SCNT blastocysts (4.6, 3.0, 6.1, and 4.9; P < 0.05). These effects of IGF-I were also neutralized by culturing with IGF-I along with anti-IGF-IR antibody. Expression of the anti-apoptotic Bcl-2 gene was increased, whereas expression of the pro-apoptotic Bax gene was decreased in both IVF and SCNT embryos cultured with IGF-I. In both IVF and SCNT embryos, anti-IGF-IR antibody along with IGF-I neutralized the effect of IGF-I on expression of Bcl-2 and Bax genes. In conclusion, the present study demonstrated that IGF-I through its specific receptors improved the developmental competence of IVF and SCNT embryos by decreasing the incidence of apoptosis and regulating apoptosis-related genes in porcine pre-implantation embryos.
This study was supported by grants from the Korean MOST (Top Scientist Fellowship) and MAF (Biogreen 21 #20050301-034-443-026-01-00).
53 EVALUATION OF NUCLEAR REPROGRAMMING IN CLONED MINIATURE PIG EMBRYOS USING GENES OF Oct-4 FAMILY
Authors: Lee E. , Lee S. H. , Kim S. , Jeong Y. W. , Kim J. H. , Koo O. J. , Hashem A. , Park S. M. , Hossein M. S. H. , Son H. Y. S. , Lee Ch.-K. , Kang S. K. , Lee B. C. , Hwang W. S.
Published: December 2006

Xenotransplantation as a source of organs is a rapidly expanding field which can save thousands of human lives each year. Cloned miniature pigs have been considered as a model system for xenotransplantation. However, the efficiency of somatic cell nuclear transfer (SCNT) is extremely low, with most clones resulting in early lethality and several aberrancies. Possible explanation of the developmental failure of SCNT embryos is related to insufficient reprogramming of the somatic cell nucleus. In order to test this, we analyzed the reprogramming capacity of differentiated fibroblast cell nuclei and undifferentiated germ cell nuclei with Oct-4 and four Oct-4-related genes (Ndp5211, Dppa2, Dppa3, and Dppa5) as molecular markers using quantitative reverse transcription-polymerase chain reaction (RT-PCR). Oct-4 expression patterns were similar among IVF-derived embryos and cloned embryos derived from fibroblasts or germ cells during pre-implantation embryo development. However, the expression level was significantly lower (P < 0.05) in hatched blastocysts of fibroblast clones compared to other hatched blastocysts. Also, 9 of 13 cloned morulae and 12 of 40 cloned blastocysts failed to reactivate at least one of the five tested genes, whereas all of the germ cell clones and control embryos correctly expressed these genes. Analysis with miniature pig fetuses collected at Day 30 of gestation revealed that normal and cloned fetuses successfully expressed these genes. In conclusion, our results suggest that analysis of expression of Oct-4 and related genes could be a reliable marker for evaluating the reprogramming status of transplanted donor nuclei in cloned embryos. The reprogramming of fibroblast cloned embryos is highly error-prone. This may contribute to their embryonic lethality because cloned embryos that fail to reactivate the marker genes may fail to be successfully implanted.
This study was supported by grants from the Ministry of Science and Technology (Top Scientist Fellowship), and the Biogreen 21-1000520030100000.
55 INFLUENCE OF DONOR CELL TYPE ON THE DEVELOPMENT OF PORCINE NUCLEAR TRANSFER EMBRYOS
Authors: Lee K. , Fodor W. L. , Machaty Z.
Published: December 2006

Embryonic development after nuclear transfer is very low; the majority of cloned embryos do not survive the pre-implantation stage. Recent reports indicate that the characteristics of nuclear transfer embryos depend on the type of nuclear donor cells. It has been suggested that development after nuclear transfer improves if less differentiated cells are used as nuclear donors. The aim of the present study was to investigate the developmental potential of nuclear transfer embryos reconstructed using differentiated and non-differentiated cells. Two types of non-differentiated cells, skin stem cells and olfactory bulb progenitor cells, were used; fetal fibroblasts were used as differentiated control. Prior to nuclear transfer, the differentiated state of the cells was characterized by Oct-4 immunocytochemistry (Chemicon International, Inc., Temecula, CA, USA); Oct-4 is known to be expressed by pluripotent cells only. During nuclear transfer, the cells were transferred into the perivitelline space of in vitro-matured enucleated oocytes. After fusion, reconstructed oocytes were activated by an electrical pulse followed by incubation in 10 �g/mL cycloheximide and 5 �g/mL cytochalasin B for 5 h. The embryos were subsequently cultured in NCSU-23 medium for 6 days; their developmental data were recorded and compared by ANOVA. Non-differentiated cell types showed strong Oct-4 expression, whereas the marker protein was completely absent in fetal fibroblast cells. A total of 161 embryos were reconstructed using skin stem cells, 171 embryos from olfactory bulb progenitor cells, and 189 embryos from fibroblasts. Of the skin stem cell-derived embryos, 32.9% cleaved, and during subsequent culture, 5.6% developed to the morula/blastocyst stage. In the olfactory bulb progenitor cell group, 19.8% cleaved, and the percentage of embryos that developed to the morula/blastocyst stage was 4.7%. In the control group, 22.7% cleaved; the morula/blastocyst formation was 2.6%. Embryos reconstructed from skin stem cells showed superior cleavage rate compared to embryos from the other cell types (P < 0.05). Also, morula/blastocyst formation from skin stem cells was significantly higher than that from fetal fibroblasts (P < 0.05), and morula/blastocyst formation from olfactory bulb progenitor cell-derived embryos also tended to be higher compared to control embryos (P = 0.08). Furthermore, the formation of morulae/blastocysts per cleaved embryos was the highest in embryos reconstructed with olfactory bulb progenitor cells (23.5% vs. 17.0% using skin stem cells and 11.6% using fibroblasts) implying that embryos from olfactory bulb progenitor cells may have higher developmental potential in later stages of development. The results demonstrate that nuclei of different donor cells support development to various degrees and confirm previous reports that using non-differentiated cells as nuclear donors increases the efficiency of nuclear transfer in the pig.
57 DEFECTIVE PLACENTAL VASCULARIZATION AND TROPHISMS IN FULL TERM SOMATIC CELL SHEEP CLONES
Authors: Loi P. , Matsukawa K. , Palmieri C. , Della Salda L. , Ptak G.
Published: December 2006

We report on cloning experiments designed to explore the causes of peri- and post-natal mortality of cloned lambs. A total of 93 blastocysts obtained by nuclear transfer of somatic cells (granulosa cells) were transferred into 41 recipient ewes, and pregnancies were monitored by ultrasound scanning. In vitro-derived, fertilized embryos (IVF; n = 123) were also transferred in order to assess oocyte competence; naturally mated ewes (n = 120) were analyzed as well. Cloned embryos developed to the blastocyst stage and implanted at the same rate as IVF embryos. After Day 30 of gestation, however, dramatic losses occurred, and only 12 out of 93 (13%) clones reached full-term development, compared to 51 out of 123 (41.6%) lambs born from the IVF control embryos. Three full-term lamb clones were delivered stillborn as a result of placental degeneration. A further five clone recipients developed hydroallantois. Their lambs died within 24 h following delivery by Caesarean section; their carcasses displayed degenerative lesions in liver and kidney resulting from the severe hydroallantois. One set of twins was delivered by assisted parturition at Day 150, but died 24 h later due to respiratory distress syndrome. The remaining two clone recipients underwent Caesarean section; the corresponding two lambs displayed signs of respiratory dysfunction and died at approximately one month of age due to a bacterial complication. Blood samples collected from the cloned lambs after birth revealed a wide range of abnormalities indicative of kidney and liver dysfunction. Macroscopical and histopathological examination of the placentae revealed a marked reduction in vascularization, particularly at the apex of the villous processes, as well as a loss of differentiation of the trophoblastic epithelium. Our results strongly suggest that post-mortality in cloned lambs is mainly caused by placental abnormalities.
61 GENERATING A HERD OF TRANSGENIC DAIRY GOATS BY SOMATIC CELL NUCLEAR TRANSFER
Authors: Melican D. , Hawkins N. , Gavin W.
Published: December 2006

Transgenic dairy goats expressing recombinant therapeutic molecules in the milk may offer an alternative manufacturing method compared to traditional cell culture. Typically, a herd of transgenic progeny can be efficiently generated by naturally mating a transgenic buck with multiple does. However, the ability to rapidly generate a herd of transgenic progeny from a female transgenic founder by natural mating represents a challenge. While a herd of transgenic progeny can be generated from a female transgenic founder by superovulation and either in vitro fertilization or embryo flushing, the time period required is dependent on the number and gender of progeny needed. In this work we determined the ability of using skin cells from two transgenic founder female dairy goats as karyoplasts for nuclear transfer to produce a herd of transgenic female dairy goats. Two transgenic founder female dairy goats expressing a recombinant therapeutic human protein in the milk were generated by nuclear transfer using a transfected fetal cell line. Primary cultures of transgenic skin cells were established from these two founder lines using tissue samples obtained by biopsy. The cells were used as nuclear donors following three or four days of low-serum culture [0.5% fetal bovine serum (FBS)]. Donor cells were simultaneously fused and activated with in vivo produced oocytes from superovulated does. Nuclear transfer embryos were transferred to synchronized surrogate recipients at 24 to 48 h post-fusion and activation. Pregnancies were determined by ultrasonography starting at approximately Day 28 post-fusion and activation and then monitored weekly. The results of work conducted during two defined breeding periods are summarized in Table 1. While there were no significant differences in the number of Day 50 pregnancies (17 vs. 27), there were significantly more term pregnancies (14 vs. 12) and offspring produced (20 vs. 15) during the traditional (September through December) compared with the nontraditional (January through May) breeding seasons, respectively (P < 0.05). In this work, 35 female NT offspring were produced, requiring 13 months from the start until the last offspring was born. These results demonstrate that somatic cell nuclear transfer using transgenic skin cells as karyoplast donors is an alternative method to produce a herd of transgenic female dairy goats.

Table 1.
Caprine transgenic herd development by nuclear transfer
62 REPROGRAMMING EVENTS AND DEVELOPMENTAL COMPETENCE OF RHESUS MONKEY EMBRYOS PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER
Authors: Mitalipov S. , Zhou Q. , Byrne J. , Ji W.-Z. , Wolf D.
Published: December 2006

Successful reprogramming of somatic cell nuclei after nuclear transfer requires active remodeling by factors present in the nonactivated cytoplast. High levels of maturation promoting factor (MPF) activity are associated with this remodeling process which includes nuclear envelope breakdown (NEBD), premature chromosome condensation (PCC), and spindle formation. In this study, we examined the extent of nuclear remodeling in monkey somatic cell nuclear transfer (SCNT) embryos by monitoring the dynamics of lamin A/C appearance, as detected immunocytochemically, following fusion of donor cells with recipient cytoplasts. In the control, intracytoplasmic sperm injection (ICSI) fertilized embryos, lamin A/C was readily detected at the pronuclear stage but disappeared in early cleaving embryos only to reappear by the morula stage in association with the activation of the embryonic genome. We initially documented lack or incomplete NEBD and PCC in SCNT embryos in the form of retention of lamin A/C signal emanating from the donor nucleus. This observation was consistent with premature cytoplast activation due to the manipulation procedures. SCNT embryos produced by this approach typically arrested at the morula stage. Significant modifications in nuclear transfer protocols were then employed. Optimization of procedures resulted in robust NEBD and PCC, as indicated by loss of lamin A/C signal from the donor cell. Also, significant improvement of SCNT embryo development in vitro was observed, with a markedly improved blastocyst formation rate (21%). Several different fetal and adult somatic cell types screened as nuclear donors supported blastocyst development. SCNT blastocysts displayed a pattern of Oct-4 expression similar to that of sperm fertilized counterparts, indicative of efficient nuclear reprogramming. However, no pregnancies were established following a preliminary trial of 8 embryo transfers with 48 cloned embryos. Nevertheless, our results represent a breakthrough in efforts to produce cloned monkeys and should provide the resources required for the derivation of embryonic stem cells from SCNT blastocysts.
63 IMPROVEMENT OF SEMEN-DERIVED EPITHELIAL CELL PROLIFERATION BY FIBROBLAST CO-CULTURE
Authors: Nel-Themaat L. , Gómez M. C. , Wirtu G. , Cole A. , Bondioli K. R. , Dresser B. L. , Pope C. E. , Godke R. A.
Published: December 2006

We previously isolated epithelial-like cells (ELC) from sheep (Ovis aries) and eland (Taurotragus oryx) semen (Nel-Themaat et al. 2004 Reprod. Fertil. Dev. 16, 152) and subsequently developed a system to separate ELC before plating (Nel-Themaat et al. 2005 Reprod. Fertil. Dev. 17, 314). Cells attached and proliferated in only 50% and 31% of the attempts, respectively. Therefore, the purposes of the present study were to improve ELC proliferation by co-culture with inactivated 3T3 mouse fibroblasts and to characterize the obtained cells. Semen fractions from two mature Gulf Coast Native rams (n = 20 ejaculates) and one common eland bull (n = 2 ejaculates) were plated on feeder layers (A), on collagen with feeder cell inserts (B), or on collagen alone (C). For B and C, cell attachment and division were assessed; proliferation and passage 1 (P1) confluence were evaluated for A, B, and C. No difference in attachment rates between B (80%) and C (70%) were found for ovine cells, but (P < 0.05) cells divided more times in B (80%) than in C (35%). All colonies in A (60%) and B (70%) reached P1 confluence and no difference was detected between A and B, but less proliferation (10%) and P1 confluence (5%) were observed in C. Therefore, contact between epithelial cells and feeders was not necessary for growth stimulation by the feeder cells. No difference among A, B, and C was detected for eland ELC proliferation (100%), but no P1 confluence was observed in C. Ram cells were subsequently characterized by immunohistochemical detection of keratin and vimentin, as well as morphology. The 3T3 cells cross-reacted with keratin, and characterization was thus performed mainly on morphology and vimentin expression. Distribution of vimentin microfilaments differed between different epithelial morphologies and fibroblasts. Expression in epithelial cells was faint and patchy in confluent colonies and located around cytoplasmic extremities in semi-confluent colonies. In 3T3 cells, expression was very prominent throughout the cytoplasm and around the nucleus. For treatments A and B, 63 and 57%, respectively, were characterized as only epithelial cells; 25 and 36%, respectively, appeared to contain a mixture of epithelial and fibroblast cells; and 13 and 7%, respectively, contained only fibroblast cells. Only one sample was evaluated from treatment C and only keratin was detected in the epithelial-like colony. We conclude that culture of semen-derived ELC is markedly improved by 3T3 fibroblast co-culture. Further research on conditioned media may simplify the system and reduce chances of 3T3 cell contamination.