bluuebunny/crossref_metadata_2025 huggingface dataset:
244 DEVELOPMENT OF SUPEROVULATORY STRATEGIES IN ALPACAS
Authors: Gamarra G. , Gallegos A. , Asparrin M. , Vivanco-Mackie H. W.
Published: December 2006

The objective of the present study was to determine the ovarian response of alpacas to different treatments for follicular development and superovulation. Twenty-nine mature, lactating alpacas, between 31 and 56 days postpartum, managed in the Peruvian highlands (altitude = 4100 m) were randomly distributed into 4 experimental groups. Groups 1 (n = 7) and 3 (n = 8) received a homemade intravaginal sponge containing 60 mg of medroxyprogesterone acetate (MAP; Sigma Chemical Co., St Louis, MO, USA) plus 2 mL of PGF2α (IM; Illiren�; Intervet International, Boxmeer, The Netherlands) on Day 0. Groups 2 (n = 7) and 4 (n = 7) received 2 mL of PGF2α (IM) on Day 0, but did not receive a MAP sponge. All groups received 6 injections (IM) of FSH (Folltropin V�; Bioniche Animal Health, Beltsville, Ontario, Canada) in decreasing dosages of 50, 50, 30, 30, 20, and 20 mg, respectively, every 12 h (at 0700 and 1900 h each day), plus 300 IU of eCG (IM; Folligon�; Intervet International) at the time of the last FSH treatment, with the aim of increasing LH levels. The FSH treatments started on Days 7, 5, 9, and 7 (from Day 0) in groups 1–4, respectively. MAP sponges were removed at the time of the last FSH treatment in groups 1 and 3. All alpacas were naturally mated twice at 12 and 24 h after the last FSH treatment. Alpacas in groups 1 and 2 received 3000 IU of hCG (IM; Corulon�; Intervet International) and alpacas of groups 3 and 4 received 2.5 mL of GnRH (IM; Conceptal�; Intervet International) immediately after the first mating. Seven days after the first mating, ovaries of all alpacas were examined by transvaginal ultrasonography. Ovarian response was estimated by determining the number of CL present on each ovary. The numbers of follicles that were at least 8 mm in diameter were also counted. Data were analyzed as a complete randomized design with 4 treatments. The average number of CL per alpaca was 1.3, 1.00, 1.00, and 0.9 for groups 1 to 4, respectively (P > 0.05). The average number of follicles that were at least 8 mm in diameter per alpaca was 9.4, 20.4, 0.9, and 3.9 for groups 1 to 4, respectively (P ≤ 0.05) with females in group 2 showing the highest response. We conclude that progestin treatment did not affect ovulatory response of lactating alpacas to exogenous gonadotropins. An effective ovarian stimulation strategy for achieving superovulation in alpacas remains to be developed.
248 IN VITRO PRODUCTION OF CATTLE × BUFFALO HYBRID EMBRYOS USING BOVINE OOCYTES AND AFRICAN BUFFALO (SYNCERUS CAFFER CAFFER) EPIDIDYMAL SPERM
Authors: Owiny O. D. , Barry D. M. , Agaba M. , Godke R. A.
Published: December 2006

Interspecies hybridization of bovids occurs between domestic cattle and at least 3 other species: the American bison (Bison bison), yak (Bos grunniens), and banteng (Bos banteng). Birth of a cattle � buffalo hybrid was reported in Russia, but the report was never authenticated. Such hybrids could be important in improving livestock production and managing diseases that impede production in tropical Africa. We investigated hybridization between cattle and their closest African wild relative, the African buffalo (Syncerus caffer caffer). In an attempt to produce pre-implantation cattle � buffalo hybrid embryos in vitro, matured bovine oocytes were subjected to a standard IVF procedure with either homologous (n = 1166 oocytes) or heterologous (n = 1202 oocytes) buffalo epididymal sperm. After IVF, 67.2% of the oocytes inseminated with homologous sperm cleaved. In contrast, insemination with buffalo sperm resulted in a 4.6% cleavage rate. Cleavage was also slower in hybrids than in cattle embryos. Up to 52.2% of cleaved homologous embryos progressed to the morula stage compared with 12.7% for hybrids. No hybrid embryos developed beyond the 16-cell stage, whereas 40.1% of the cleaved bovine embryos developed to the blastocyst stage. Developmental anomalies such as polyspermy, uneven cleavage, vacuolization, and absence of nuclei in some blastomeres were common in the hybrid embryos. We conclude that interspecies fertilization of cattle oocytes with African buffalo sperm occurs in vitro and that the barrier to hybridization is in the early stages of embryonic development. Also, chromosomal disparity is the likely cause of fertilization abnormalities, abnormal development, and subsequent arrest, impairing the formation of pre-implantation hybrid embryos. Investigation into developmental abnormalities, including reciprocal hybridization and genetic studies of the hybrid embryos, are recommended.
256 SWITCH IN THE EXPRESSION OF GENES INVOLVED IN LIPID METABOLISM FOR IN VIVO-MATURED BOVINE OOCYTES AND BLASTOCYSTS
Authors: Algriany O. , Vos P. L. A. M. , Sirard M. A. , Dieleman S. J.
Published: December 2006

The competence of oocytes to develop into healthy offspring is determined by many factors including oocyte quality, embryo culture conditions, and energy requirements. Although glucose metabolism has been studied extensively, little is known about lipid metabolism during oocyte maturation and in the blastocyst. In general, cells can obtain fatty acids (FA) by transport (1) from outside across the cell membrane or by synthesis (2) inside the cell, and loose FA by catabolic processes (3). Therefore, we analyzed mRNA abundance for (1) FA translocase (FAT/CD36) and FA transport protein (FATP1), (2) acetyl-CoA carboxylase (ACC) α and FA synthase (FAS), and (3) AMP-activated protein kinase gamma 1 (AMPK) and carnitine palmitoyltransferase I (CPT-I) in bovine oocytes during resumption of meiosis in vivo vs.in vivo blastocysts. Cyclic Holstein-Friesian cows (n = 16) were treated with oFSH and a controlled LH surge (Knijn et al. 2002 Reproduction 124, 365–375) to collect oocytes at onset of maturation, 2 h before LH, and after the start of germinal vesicle breakdown (6 h after LH). A second group of cows (n = 10) was treated with eCG/anti-eCG (Voset al. 1994 Theriogenology 41, 829–840) to flush blastocysts at Day 7 after ovulation. Following ovariectomy, oocytes from all follicles between 10 and 18 mm were denuded and stored separately at -80�C. Oocytes were selected on the basis of the relative steroid concentration in the follicular fluid, producing 3 replicate pools of 13 competent oocytes each, at both 2 h before and 6 h after LH. Four replicates of 5 expanded blastocysts grade 1 (IETS) each were stored at -80�C. Total RNA was isolated using a microspin column, and DNA was digested (Absolutely RNA Microprep Kit; Stratagene, San Diego, CA, USA) and reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA). Amplification of cDNA was achieved by real-time PCR using specific primers and 2X iQ SYBR Green Supermix (Bio-Rad Laboratories) in duplicate. Relative levels of expression were analyzed using a modified delta-Ct method. Statistical analysis was done using ANOVA and the Tukey post-hoc test. Relative abundance of mRNA for FA transport and catabolism was >10 times higher during oocyte maturation than in blastocysts; CPT-I was not detected in blastocysts. In contrast, transcripts of FA synthesis were sharply increased (>5 times) during the blastocyst stage. Alterations in the expression of these mRNA indicate that they play a key role in mediating cell type-specific differences in energy requirement and that exogenous FA and FA catabolism could be a main source of energy during oocyte maturation. A high level of FA biosynthesis mRNA in blastocysts may reflect high demands of de novo long-chain FA synthesis and elongation that probably are essential for protection against stress.
257 EXPRESSION PROFILING OF GENES CRUCIAL FOR LINEAGE DETERMINATION IN IN VITRO-DERIVED EARLY BOVINE EMBRYOS
Authors: Antonini S. , Lazzari G. , Cillo F. , Galli C. , Colleoni S. , Lagutina I. , Gandolfi F. , Brevini T. A. L.
Published: December 2006

In the early blastocyst, lineage segregation depends on the expression of several key specific transcription factors. In the mouse, commitment to inner cell mass (ICM), lineage is positively regulated by Oct-4, a repressor of trophectoderm (TE) cell fate, and Nanog, which inhibits the formation of extra-embryonic and primitive endoderm. Cdx2, a caudal-type homeodomain protein, is specifically expressed in the nascent TE. The mechanisms that drive Cdx2 segregation to the outside cells are still unclear. However, the expression of Fgf Receptor 2 (FgfR2), restricted to the outside cells, and the role for its ligand, Fgf4, in promoting TE development, suggest that this signalling pathway may act upstream or in parallel with Cdx2. Little information is available on these genes in bovine; therefore the aims of the present study were as follows: (a) to identify and characterize the expression profiles of Cdx2 and FgfR2 variants (IIIc and IIIb) in bovine oocytes and pre-implantation embryos; and (b) to compare their expression patterns in ICM and TE with that of Oct-4 and Nanog. Bovine oocytes and embryos were obtained by in vitro maturation and fertilization; blastocysts at Day 7 post-insemination underwent microsurgery to separate TE from ICM. RNA was isolated from MII oocytes; 2-, 4-, 8-, and 16-cell embryos; morulae; blastocysts; ICMs; and TEs. Semi-quantitative analysis of Cdx2 and FgfR2 expression in oocytes and embryos was performed in the exponential phase of PCR amplification with rabbit globin as exogenous control. In order to exclude false negative results, PCR amplification in isolated TE and ICM was extended to the plateau phase for all genes considered. Fragment identity was confirmed by sequencing. Comparison of bovine Cdx2 cDNA sequence (EMBL AM293662) with databases revealed a 91% and 87% homology with human and mouse, respectively. Cdx2 expression was not detectable in MII oocytes, but increased in 2-cell embryos. Transcript levels decreased at the 4- and 8-cell stages and then increased again in the blastocyst. FgfR2 variants were present as both maternal and embryonic transcripts, because they were detectable throughout pre-implantation development. Cdx2 and FgfR2 IIIc and IIIb expression was restricted to TE cells. Nanog was detected only in ICM, whereas Oct-4 was expressed in both lineages, as previously described in bovine (van Eijk et al. 1999 Bio. Reprod. 60, 1093-1103). In conclusion, the expression profiles of Nanog, Cdx2, and FgfR2 in bovine pre-implantation embryos follow the pattern previously described in the mouse. Their differentially segregated expression is consistent with their role as selector factors of ICM vs. TE fates. The significance of Oct-4 ubiquitous distribution still remains to be elucidated.
This work was supported by FIRB RBNE01HPMX_005, TECLA-MIUR, and EUROSTELLS-ESF.
258 EXPRESSION PATTERN OF THE MATERNAL FACTOR ZYGOTE ARREST 1 (ZAR1) GENE IN OVINE OOCYTES AND PRE-IMPLANTATION EMBRYOS
Authors: Ariu F. , Bebbere D. , Succu S. , Tore S. , Bogliolo L. , Fois S. , Leoni G. G. , Ledda S.
Published: December 2006

Zygote arrest 1 (ZAR1) is an ovary-specific maternal factor that plays essential roles during the oocyte-to-embryo transition. It is evolutionary conserved in vertebrates, and the protein is characterized by the presence of a homeobox zing finger domain, suggesting its role in transcription regulation. Discovered in mice (Wu et al. 2003 Nat. Genet. 33, 187-191), its expression pattern has been analyzed in oocytes and pre-implantation embryos in mice, cattle, and pig (Pennetier et al. 2004 Biol. Reprod. 71, 1359-1366; Brevini et al. 2004 Mol. Reprod. Dev. 69, 375-380; Reprod. Biol. Endocrinol. 21, 4-12). Because no information on Zar1 ortholog in the ovine species is available, the aim of this study was to assess its presence and to analyze its expression pattern in oocytes and pre-implantation embryos. Reverse transcription-polymerase chain reaction (RT-PCR) was performed in germinal vesicle (GV) and IVM metaphase II (MII) oocytes, and, following IVFC, in 2- (2C), 4- (4C), 8- (8C), and 12-16- (12C-16C) cell embryos, morulae, and blastocysts. Primers were designed on the basis of bovine and swine conserved sequences and contained an intron-spanning region. The PCR product was sequenced and the alignment, performed with BLAST (www.ncbi.nlm.nih.gov/BLAST/), confirmed its homology with the Zar1 orthologous genes present in public databases, sharing 98%, 92%, and 89% nucleotide identity with the bovine, porcine, and human sequences, respectively. Real-time PCR revealed that Zar1 is present at its highest level in the GV oocyte, has a major decrease at the MII stage (around 10-fold), remains constant in the embryo first cleavage steps (2C-4C), drops again at the 8C stage (around 30-fold), and disappears in morulae and blastocysts. Our data demonstrate that a Zar1 ortholog is present in the ovine species. Results are also in accordance with the expression patterns characterized in other mammalian species and suggest that Zar1 transcript in the ovine oocyte may be necessary for normal female reproduction in the transition from oocyte to embryonic life.
26 SPERM REPRODUCTIVE POTENTIAL OF FETAL FIBROBLAST CELLS ON A NUCLEAR TRANSFER - CLONED BULL AND ON AN ORIGINAL PATERNAL BULL: A COMPARATIVE ANALYSIS
Authors: Bae S.-H. , Kim D.-H. , Kim S.-W. , Im G.-S. , Lee M.-J. , Ryu J.-W. , Seong H.-H. , Yang B.-S. , Yang B.-C.
Published: December 2006

Recently, cloning technology has produced newborn calves from fetal fibroblast cells and somatic cells. These cloned calves appear to be normal and healthy in terms of reproductive ability. Therefore, we undertook this study to compare the reproductive ability of cloned sperm from the clones themselves and sperm obtained from the frozen semen of the paternal bull (KPN-178). Two cloned bulls from fetal fibroblast cell nuclear transfers (Korean HanWoo Clone-38, 34 months old, and Clone-39, 30 months old) were selected. Sperm from the cloned groups (C-38 and C-39), cloned-38 fresh sperm, and sperm from the paternal bull (KPN-178) were analyzed for the following sperm reproductive characteristics: sperm motility, by computer-assisted sperm analysis (CASA); sperm mitochondrial activity, by flow cytometer after mitotracker staining; and sperm viability, by flow cytometer after staining with live/dead sperm viability kits, both PI and SYBR. Sperm motility was evaluated with Percoll-treated or non-Percoll-treated frozen-thawed spermatozoa. In the Percoll-treated group, total motility of the paternal bull sperm (KPN-178: 75.14%) was significantly lower than that of cloned sperm (C-38: 90.42%; C-39: 87.62%: C-38 fresh: 93.97%; P < 0.05). The non-Percoll-treated group showed different results compared to the Percoll results (P < 0.05), i.e. the total motility of C-38 (92.47%) and C-38 fresh (92.47%) sperm was significantly higher than that of C-39 (79.52%) and KPN-178 (78.41%) sperm. The mitochondrial activity staining rate of KPN-178 (58.22%) sperm was low compared to that of C-38 (88.65%), C-39 (89.45%), C-38 fresh (88.66%) sperm. The sperm viability tests showed similar results (P < 0.05) for all sperm groups: C-38 (PI: 42.53% and SYBR: 46.28%), C-39 (PI: 39.03% and SYBR: 66.18%), C-38 fresh (PI: 34.32% and SYBR: 57.10%), and KPN-178 (PI: 14.43% and SYBR: 47.86%). However, the PI staining of KPN-178 was lower than that of the other sperm groups. Next, in vitro fertilization (IVF) was investigated to compare the reproductive ability of C-38, C-39, KPN-178, and Control (normal bull) sperm. The results, development to the 2-cell stage (157/228, 68.90%; 85/116, 73.28%; 78/114, 68.42%; 61/79, 77.21%, respectively) and blastocyst rates (41/228, 18.0%; 35/116, 30.17%; 26/114, 22.81%; 20/79, 25.32%, respectively) were evaluated (P < 0.05). The IVF rates were not different among cloned bull, paternal bull, and normal bull sperm. A cloned female bovine was successfully impregnated by cloned bull sperm (C-38) using artificial insemination. The calf resulting from this prenancy showed no signs of phenotypical abnormalities. These results suggest that the physiology of cloned bulls and the quality of semen from cloned bulls show no evidence of any deleterious effects.
260 COMPARISON OF HOUSEKEEPING GENE EXPRESSION IN INDIVIDUAL 8-CELL STAGE MOUSE EMBRYOS PRODUCED UNDER DIFFERENT CONDITIONS
Authors: Baji Gal A. , Mamo S. , Bodo S. , Dinnyes A.
Published: December 2006

Real-time PCR has the potential to accurately quantify the mRNA level of selected genes in single cells and individual pre-implantation-stage embryos. The goal of our study was to examine the variations in gene expression within individual embryos of the same stage and between embryos of the same stage but from different sources. In our study, we determined expression level of the 7 most commonly used housekeeping genes in 8-cell-stage mouse embryos produced under different culture conditions. Messenger RNA of 6 embryos each that was derived in vivo, or cultured in vitro from the zygote stage, or derived from oocytes activated parthenogenetically and developed in vitro were extracted individually followed by reverse transcription into cDNA. Optimized real-time PCR was performed for cytoplasmic beta-actin (Actb), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), H2A histone family, member Z (H2afz), hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1), ubiquitin C (Ubc), peptidylprolyl isomerase A (cyclophilin A) (Ppia), and eukaryotic translation elongation factor 1 epsilon 1 (Eef1e1) genes. The results were analyzed, and the percentage standard error of the mean relative expression value was compared for all genes. All 7 genes were presented above the detection limit in all samples. One or two individual embryos showed 2- to 4-fold higher mRNA levels than the average for all genes in the group. The embryos cultured in vitro showed much higher expression levels of H2afz, Ppia, and Eef1e1 genes than those in the in vivo group. The parthenogenetic group was similar to the in vivo group in expression of Actb, H2afz, Hprt, and Eef1e1 genes, but showed significant differences (P < 0.05; Student's t-test) compared to the in vitro group (Table 1). The percent standard error of the mean decreased gradually as the number of samples was increased. The 6 individual embryos in similar groups showed relatively low variability compared to embryos at similar stage but produced in different conditions. Interestingly, the parthenogenetic embryos showed a level of gene expression comparable to that of the in vivo ones, notwithstanding their culture in vitro. In conclusion, morphological observations and similarity in developmental stage alone cannot guarantee the uniformity of embryo samples, and a minimum of 4–6 replicates per treatment is needed. Moreover, we showed that culture condition itself has an effect on housekeeping gene expression, which, if neglected, might result in misinterpretation of data.

Table 1.Relative expression values of the different culture groups (mean ±SE; n =6 embryos)

This work was supported by EU FP6 (MEXT-CT-2003-509582 and 518240), Wellcome Trust (Grant No. 070246), and Hungarian National Science Fund (OTKA) (Grant No. T046171).
264 ANALYSIS OF DNA METHYLATION PATTERN IN PRE-IMPLANTATION-STAGE EMBRYOS DERIVED FROM NUCLEAR TRANSFER USING PORCINE EMBRYONIC GERM CELLS
Authors: Choi D.-H. , Park C.-H. , Lee S.-G. , Kim H.-S. , Son H.-Y. , Lee C.-K.
Published: December 2006

Somatic cell nuclear transfer (SCNT) has been successfully used to produce live cloned offspring in various mammals. However, some studies had reported that cloned embryos by SCNT had many problems in reprogramming or epigenetic modification, such as DNA methylation. DNA methylation is an essential process in epigenetic modification for development, and aberrant methylation in cloned embryos gives rise to abortion, high birth weight, and perinatal death. In this study, embryonic germ (EG) cells were used as donor cells for nuclear transfer. EG cells may have less reprogramming or demethylation than SCNT because these are already in erased status. However, little is known about methylation state or developmental capacity of the EG cell as a donor. The objective of this study was to analyze the methylation pattern of pre-implantation embryos cloned from porcine EG cells. Two regions, PRE-1 and microsatellite (MS), were analyzed for methylation patterns of cloned embryos from porcine EG cells and compared with the pattern of mature oocytes and in vitro-fertilized (IVF) embryos as a control. Cumulus–oocyte complexes were collected from prepubertal gilt ovaries and matured in vitro for 44 h, followed by use for IVF and NT with porcine EG cells. The porcine EG cells were prepared from 28-day-old fetuses after mating; genital ridges were isolated from fetuses, and then transferred into a culture medium on a feeder layer. The number of embryos for analysis was 300 for matured oocytes, 50–80 for 4–8 cell embryos, 30–40 for morulae, and 20–30 for blastocysts. The genomic DNA was isolated from the embryos and treated with bisulfite solution. PCR was performed for the amplification of PRE-1 and MS regions. The PCR products were sequenced by using an automatic DNA sequencer. The methylation rates of the PRE-1 and MS regions in IVF embryos showed that the demethylation process had occurred during the pre-implantation stage, which is a typical phenomenon of in vivo counterparts (Kang et al. 2001 J. Biol. Chem. 276, 39 980). However, compared to IVF embryos, embryos derived from NT using EG cells showed differences at the morula (PRE-1) and blastocyst (MS) stage. These results indicate that porcine EG cells also have problems in reprogramming during NT. For detailed and reliable results, the methylation pattern analysis of the imprinting region, for example, H19 in maternal allele and Igf2 in paternal allele, must be examined.

Table 1.Methylation of PRE-1 and MS regions in embryos derived from IVF and NT using porcine EG cells
268 THE EFFECTS OF METFORMIN ON THE INSULIN SIGNALING PATHWAY ON PORCINE GRANULOSA LUTEIN CELLS IN A PRIMARY CULTURE SYSTEM
Authors: Hossein M. S. , Lee M. S. , Hwang W. S.
Published: December 2006

Metformin (N,N22-dimethylbiguanide) is an oral antihyperglycaemic drug which increases insulin-stimulated glucose uptake as an insulin sensitizing agent (ISA) and has direct effects on ovarian steroidogenesis in human. To investigate the effects of metformin on the insulin signaling pathway on porcine granulosa lutein cells in a primary culture system, we examined mRNA expressions of porcine insulin receptor, insulin-like growth factor-1 receptor, and insulin receptor substrate-1; expressions of downstream targets (Raf, MEK1/2, ERK, PDK1, mTOR, p70, and nuclear factor kB) of the insulin receptor signaling pathway; the luciferase activity of transcription factors activator protein 1; and nuclear factor kB. Granulosa cells were plated in DMEM and 10% fetal bovine serum at a density of 3.5 � 107 in a T-25 tissue culture flask and cultured for 48 h at 37�C in a humidified atmosphere of 5% CO2 and 95% air. Then, porcine granulosa lutein cells (pGLs) were cultured in a serum-free DMEM as a control group or supplemented with 10-5 M metformin, 100 ng mL-1 of insulin, or both for 24 h. The monolayer was collected for RT-PCR and western blot analysis. For transient transfections, porcine granulosa cells were plated in DMEM and 10% FBS at a concentration of 2.5 � 106/well in 24-well culture dishes for 16 h and then transfected with plasmid constructs using FuGene6 Transfection Reagent. Reporter vectors used in these studies were AP-1-luc and NF-κ B-luc. Cells were treated post-transfection with 10-5 M metformin, 100 ng mL-1 insulin, or both for 48 h. The luciferase activity was assayed using the dual luciferase assay kit with a Microlumat LB 96 P luminometer. Metformin with insulin significantly increased mRNA expressions of insulin receptor, insulin-like growth factor-1 receptor, and insulin receptor substrate-1, whereas metformin alone had no significant effect on the expression of genes. Metformin alone and with insulin significantly increased expressions of downstream targets of the insulin receptor signaling pathway except MEK2. Metformin with insulin significantly elicited an induction of luciferase activity in the transfection of activator protein 1 and nuclear factor kB reporter, whereas metformin alone did not. Metformin induced expressions of target molecules in the insulin signaling pathway but had no effect on the luciferase activity of activator protein 1 and nuclear factor kB in porcine granulosa lutein cells. Metformin with insulin increased the luciferase activity of activator protein 1. These results suggest that metformin with insulin could change the function of ovarian granulosa cells induced by activator protein 1 activity.
272 EXPRESSION OF Lef1 TRANSCRIPTION FACTOR AND ITS NOVEL ISOFORMS IN PRE-IMPLANTATION EMBRYOS
Authors: Meece A. , He S. , Keefer C.
Published: December 2006

Lymphoid enhancer-binding factor 1 (Lef1) is a key transcriptional factor in the Wnt signaling pathway. Recently, the Wnt pathway has been shown to function in human and mouse embryonic stem cells; however, its role in ES cell differentiation and self-renewal remains contradictory. Our lab has identified two novel Lef1 isoforms, Lef1Δ2,3,6 and Lef1Δ6, that are transcribed in mouse embryonic stem cells in addition to the full-length Lef1. Both Lef1Δ2,3,6 and Lef1Δ6 contain an N-terminal β-catenin binding domain and a C-terminal HMG DNA binding domain. However, Lef1Δ2,3,6 lacks exons II, III, and VI, and Lef1Δ6 lacks exon VI. Exons II, III, and VI are a part of the context-dependent repression domain, whereas exon VI contains a Groucho binding motif. The absence of these repression domains suggests that Lef1Δ2,3,6 and Lef1Δ6 may be regulated differently than the full-length Lef1. In this study, we determined the expression patterns of Lef1 mRNA and the two isoforms during murine and bovine pre-implantation development using RT-PCR. Total RNA was isolated from pools of 10–20 in vitro-produced bovine embryos at the 2-cell, 4–8 cell, morulae, and blastocyst stages and in vivo-derived mouse embryos at the 8-cell, morula, and blastocyst stages using a Stratagene Nanoprep kit. Reverse transcription was performed using the Invitrogen Supercript III first-strand synthesis system. PCR was then completed using Invitrogen Platinum Taq DNA Polymerase. As determined in preliminary work, full-length Lef1 was expressed in 8-cell-stage mouse embryos but was not detectable in morulae and was only slightly expressed in mouse blastocysts. In contrast, Lef1Δ6 mRNA was not detectable in 8-cell mouse embryos, but became obvious at morula and blastocyst stages. Similarly, in bovine embryos, full-length Lef1 was detected at early cleavage stages (2–8 cells) with undetectable or low levels at the morula and blastocyst stages; however, unlike what was observed in the mouse embryos, Lef1Δ6 was expressed in all stages of bovine embryos tested. Lef1Δ2,3,6 was very low (mouse) or undetected (bovine). Because Lef1 appears to be playing a critical role in response to Wnt differentiation signals in mouse embryonic stem cells (He, unpublished), further exploration of these transcription factors and their regulation in developing mammalian embryos is critical to understanding the process of initial embryonic differentiation.
273 EXPRESSION PATTERN OF CONNEXIN 43 (Cx43) AND POLY-(A) POLYMERASE (PAP) GENES IN BUFFALO (BUBALUS BUBALIS) EMBRYOS PRODUCED IN VITRO
Authors: Mishra D. A. , Chandra V. , Kumar G. S. , Sharma G. T.
Published: December 2006

Polyadenylation of pre-mRNA is carried out by poly-(A) polymerase (PAP), and study of the transcription pattern of this gene is said to indicate the developmental competence of the embryos. Connexin 43 (Cx43) is one of the important gap junction proteins that controls growth, cellular differentiation, and embryonic development. The objective of the present investigation was to study the expression pattern of PAP and Cx43 genes in buffalo (Bubalus bubalis) embryos produced in vitro. Embryos were produced from the slaughterhouse ovaries using standard IVMFC protocol (Rajhans et al. 2006 J. Reprod. Fertil. Dev. 18, 253–254). Briefly, oocytes were aspirated from follicles (2–8 mm in diameter) and matured in vitro in TCM-199 supplemented with 10% FCS and epidermal growth factor (20 ng mL-1) for 24 h. Presumptive zygotes after 18 h of fertilization were cultured in mSOF containing insulin-like growth factor-1 (100 ng mL-1) and β-mercaptoethanol (100 �M) for 9 days or until blastocyst formation, whichever was earlier. Pools of immature (n = 200), in vitro-matured (n = 200), oocytes and embryos (2–4 cell, n = 83; 8–16 cell, n = 80; morula, n = 77), and blastocysts (n = 40) were collected for mRNA isolation. Immature and in vitro-matured oocytes were treated with 1X trypsin-EDTA solution to remove the attached cumulus and then washed with TCM-199 before mRNA isolation to avoid any contamination of these cells during RNA isolation. mRNA from each pool was isolated using a commercially available direct mRNA isolation kit (Oligotex Direct mRNA kit; Qiagen, Valencia, CA, USA). cDNA was prepared using specific reverse primer and M-MLV RT in 20 �L reaction volume following manufacturer's instructions. Polymerase chain reaction was done for 35 cycles with annealing temperatures of 60�C and 58�C for 252 bp of PAP and 425 bp of Cx43, respectively. Amplicons were subjected to restriction endonuclease digestion for further confirmation of expressed genes. RT-PCR amplicon of PAP was digested with HaeIII to obtain characteristic band patterns at 119 bp and 133 bp, and Cx43 RT-PCR amplicon was digested with EcoR1 to obtain characteristic band patterns at 137 bp and 288 bp. While PAP expression could be detected in all stages of developing embryos starting from immature oocytes to blastocyst stage, Cx43 mRNA was detected in immature oocytes to morula stage but not in blastocyst-stage embryos. It could be concluded that the expression patterns of PAP and Cx43 genes in buffalo embryos produced in vitro are similar to those of cattle embryos.
276 PRODUCTION OF TRANSGENIC MINI-PIG CELL LINES EXPRESSING HUMAN CYTOMEGALOVIRUS US2
Authors: Park K. W. , Yoo J. Y. , Choi K. M. , Hong S. P. , Han G. S. , Kim E. J. , Kim S. H. , Kim T. S. , Park S. Y. , Yang B. S. , Im G. S. , Jun K. S. , Heo K. N. , Seol J. G.
Published: December 2006

Xenotransplantation has the potential to resolve the chronic shortage of donor organs if immunological barriers can be overcome. In particular, the initial type of rejection following xenotransplantation is acute cellular rejection by host CD8+ cytotoxic T lymphocyte (CTL) cells that react to the donor class I major histocompatibility complex (MHC). The human cytomegalovirus (HCMV) glycoprotein US2 specifically targets class I MHC heavy chains for dislocation from the endoplasmic reticulum (ER) membrane to the cytosol, where they are degraded by the proteasome. In this study, the recombinant expression vector pCX-US2 was stably transfected into mini-pig fetal fibroblasts by lipofection. The integration of US2 into the host genome was confirmed by PCR and Southern blot assay. The reduction of swine leukocyte antigen class I (SLA-I, MHC protein class I) by US2 was detected by flow cytometry analysis (FACS). FACS analysis of US2 clonal cell lines demonstrated substantial reductions in SLA surface expression. The decrease in the level of class I MHC expression for US2 clonal cell lines ranged from 22 to 34% relative to the non-transfected control. US2 clonal cell lines were also tested to determine if the resulting reduction in cell surface SLA would reduce in vitro cytotoxicity by CTL. The US2 clonal cell line demonstrated 5- to 6-fold reduction of specific lysis by primed CD8+ CTL. In conclusion, US2 can directly protect pig clonal cell lines from human CTL cells. These results indicate that the expression of US2 in pig cells may provide a new approach toward overcoming CTL-mediated immunity to xenotransplantation.
This work was supported by the National Livestock Research Institute (6132-211-303-1).
281 EXPRESSION PROFILE AND KNOCKDOWN ANALYSIS OF A FUNCTIONALLY UNKNOWN DD2-2 GENE IN MOUSE PRE-IMPLANTATION EMBRYOS
Authors: Shin S. , Matsumoto K. , Amano T. , Saeki K. , Hosoi Y. , Iritani A.
Published: December 2006

Zygotic gene activation (ZGA) starts at the G2 phase at the 1-cell stage in the mouse. However, the molecular mechanism of ZGA has not been completely elucidated. We have investigated the molecular functions of many gene clusters, DD clones obtained by differential display assays for ovulated eggs at the M II stage and 1-cell stage embryos at the G2 phase. As a result, we have identified a functionally unknown gene, whose sequence did not match a known transcript in the gene bank DD2-2 gene. Here, we report the expression profile and knockdown analysis of the DD2-2 gene in mouse pre-implantation embryos. Nucleotide sequence analysis of the DD2-2 cDNA revealed that the open reading frame of 1056 bp encodes a protein of 351 amino acids with a predicted molecular mass of 41.5 kDa. The deduced amino acid sequence indicated that DD2-2 protein might be a soluble protein without a signal peptide. We first investigated the expression profiles of DD2-2 in pre-implantation embryos by quantitative real-time PCR using an ABI PRISM 7300 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). To investigate the effect of knockdown of the DD2-2 gene on the development of pre-implantation embryos, we injected pβ-act/antisenseDD2-2/IRES/EGFP into male pronuclei of embryos at 7 to 9 h after insemination (hpi) and observed the development of embryos that showed EGFP expression at 24 hpi. Real-time PCR analysis of pre-implantation embryos showed that maternal DD2-2 mRNA at a low level significantly increased up to the early 2-cell stage, and significantly decreased by the 4-cell stage and later, suggesting that DD2-2 gene specifically expresses at major ZGA. In the knockdown analysis, EGFP-positive embryos with pβ-act/antisenseDD2-2/IRES/EGFP showed a lower rate of development to the 4-cell stage and later, compared with that of EGFP-positive embryos with pβ-act/luc+/IRES/EGFP [72% (94/130) vs. 54% (71/131); P < 0.05], indicating that the knockdown of DD2-2 by antisense RNA resulted in a inhibition of pre-implantation development. In conclusion, the DD2-2 gene, a functionally unknown gene, may play an important role in pre-implantation development.
This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Mext and by a grant for the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technology Excellence of the JST.
282 GENE EXPRESSION PATTERNS AND QUALITY OF BOVINE EMBRYOS PRODUCED UNDER DIFFERENT OXYGEN CONCENTRATIONS
Authors: Son W. J. , M. K. B.  , Jeong Y. J. , Balasubramanian S. , Choe S. Y. , Rho G. J.
Published: December 2006

Various factors are known to influence the survival and development of in vitro-produced embryos, including co-culture with somatic cells, antioxidants, and O2 tension. Studies in several species report that embryo development and quality were enhanced at low O2 concentrations. This study compared the effects of 2 O2 concentrations on IVP embryo development, embryo quality, and gene expression to those of in vivo counterparts. Cumulus–oocyte complexes were matured in vitro in TCM-199 with hormones and 10% FCS, and inseminated in TALP medium. Presumptive zygotes were cultured in SOF medium under either 5% or 20% O2 in air. In triplicate, sets of 5 embryos at the 2-cell, 4-cell, 8-cell, 16-cell, morula, and Day 7 blastocyst stages were used for analyzing the expression patterns of apoptotic (Bax and Bcl2), metabolism (Glut-1 and Glut-5), stress (Sox, Hsp70, and G6PDH), compaction (Cx43), oxidation (PRDX5, NADH, and MnSOD), and implantation (VEGF and IFN-tau) genes using real-time quantitative PCR. The expression of each gene was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Statistical analysis was performed with Bonferroni and Duncan tests by ANOVA (P < 0.05). Cleavage rates did not differ among groups. Blastocyst and hatched blastocyst development in 5% O2 was significantly (P < 0.05) higher than in 20% O2. Total cell number of in vivo blastocysts was significantly (P < 0.05) higher than that of IVP blastocysts. ICM ratio and apoptosis of in vivo blastocysts were significantly (P < 0.05) lower than for IVP blastocysts. The relative abundances (RAs) of Glut-1, Glut-5, MnSOD, NADH, PRDX5, Cx43, Bcl2, and IFN-τ were significantly (P < 0.05) higher in in vivo embryos, whereas the RAs of Sox, G6PDH, Hsp70, Bax, and VEGF were significantly (P < 0.05) lower than for IVP counterparts. In conclusion, culture at 5% O2 concentration resulted in higher rates of development to the blastocyst stage, higher total cell numbers, and decreased apoptosis. Furthermore, differences in expression of genes including Glut-1, Glut-5, Sox, G6PDH, Hsp70, Bax, Bcl2, Cx43, PRDX5, NADH, MnSOD, VEGF, and IFN-τ may prove useful in determining optimal culture conditions.
This work was supported by ARPC (204119-03-SB010), Republic of Korea.
283 TRANSCRIPTIONAL ANALYSIS OF BIOPSIES DERIVED FROM IN VITRO-PRODUCED BOVINE BLASTOCYSTS IN RELATION TO PREGNANCY SUCCESS AFTER TRANSFER TO RECIPIENTS
Authors: Tesfaye D. , El-Sayed A. , Hoelker M. , Rings F. , Jennen D. , Tholen E. , Sirard M. A. , Schellander K.
Published: December 2006

Early embryonic mortality is a recognized cause of reproductive failure in cattle leading to the loss of a large number of potential calves, retarded genetic progress, and significant loss of money and time in rebreeding cows. The purpose of this work was to address the relationship between the transcriptional profile of embryos, using the microarray technique, and pregnancy success based on blastocyst biopsies taken prior to transfer to recipients. Biopsies (3 to 40% of the intact embryo) were taken from IVP Day 7 blastocysts (n = 98) and 60 to 70% was transferred to recipients (2-year-old Simmental heifers) after re-expansion. Based on the success of pregnancy, biopsies were pooled in 3 groups: those resulting in no pregnancy (G1), resorption (G2), and calf delivery (G3). Gene expression analysis of these groups of biopsies was performed using a home-made bovine pre-implantation-specific array (with 219 clones) and bovine cDNA array (BlueChip) (with 2000 clones) (Sirard et al. 2005 Reprod. Fertil. Dev. 17, 47–57). Approximately 3 �g of amplified RNA from pooled biopsies (10 biopsies each) was used as a template in reverse transcription reactions incorporating amino-modified dUTPs into labeled cDNA using the CyScribeTM post-labelling kit (Amersham Bioscience, Freiburg, Germany). The synthesized cDNAs from all 3 groups were differentially labeled using Cy3 and Cy5 dyes. Slides were scanned using a GenePix 4000B scanner and images were analyzed using GenePix Pro Version 4.0 software (Axon Instruments, Union City, CA, USA). Data were analyzed using Significant Analysis for Microarray (SAM) software (http//www.stst.stanford.edu/tips/SAM). Quantitative real-time PCR was used to confirm the differentially expressed genes revealed by microarray experiments. Results revealed that 52 genes were differentially regulated between G1 and G3, and 58 genes were differentially regulated between G2 and G3. Biopsies resulting in calf delivery were enriched with genes necessary for implantation (COX-2 and CDX2), signal transduction (PLAU), polyamine biosynthesis (ODC1), response to oxidative stress (TXN), growth factor (BMP15), and placenta-specific 8 (PLAC8). Biopsies that ended up with resorption were enriched with transcripts involving protein phosphorylation (KRT8) plasma membrane (OCLN), and glucose metabolism (PGK1, AKR1B1). Biopsies resulting in no pregnancy were enriched with transcripts involving inflammatory cytokines (TNF), protein amino acid binding (EEF1A1), transcription factors (MSX1, PTTG1), glucose metabolism (PGK1, AKR1B1), and CD9, which is an inhibitor of implantation. In conclusion, we generated direct candidates of blastocyst-specific genes that determine the fate of the embryo after transfer.
286 EFFECTS OF PORCINE FOLLICULAR FLUID DURING IN VITRO MATURATION OF OOCYTES ON IN VITRO FERTILIZATION AND EMBRYONIC DEVELOPMENT
Authors: Agung B. , Otoi T. , Fuchimoto D. , Senbon S. , Onishi A. , Nagai T.
Published: December 2006

When used as a solo maturation medium for oocytes, porcine follicular fluid (pFF) promoted male pronucleus formation (MPF) of oocytes after in vitro maturation (IVM), using a static system, and in vitro fertilization (IVF) in pigs (Naito et al. 1988 Gamete Res. 21, 289–295). However, the developmental competence of oocytes matured in pFF after IVM/IVF has not been reported. This study was conducted to assess the ability of pFF as a maturation medium to support IVM/IVF of porcine oocytes and their subsequent in vitro development. pFF, including cumulus–oocyte complexes (COCs), was aspirated from follicles (2–5 mm in diameter) of prepubertal crossbred gilt ovaries, and large clusters of follicular cells (FC) were removed from pFF by filtration through 212 �m of mesh. All of the COCs in filtered pFF were collected, and COCs with compact cumulus cells were selected for IVM. Also, small clusters of FC were collected by centrifugation of the filtered pFF, and pFF without any cells was prepared by centrifugation and used as a maturation medium (MpFF) after supplementation with FSH and antibiotics. COCs were transferred to 3.5 mL (in a 15-mL test tube) of MpFF with FC (5.2 � 106 cells mL-1) and cultured for 44–48 h at 38.5�C in 5% O2 and 5% CO2 using the rotating culture system. As a control group, COCs were cultured in 2 mL of MpFF without FC in a 35-mm Petri dish by the standard static culture system. After maturation, culture oocytes were co-incubated (IVF) for 5 h with frozen–thawed sperm in vitro, as reported previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041), and then some of them were fixed 10 h after IVF to assess the fertilization status; the rest of them were cultured in PZM (Yoshioka et al. 2002 Biol. Reprod. 60, 112–119) for 7 days to assess their early embryonic development. All of the data were analyzed by ANOVA. Oocytes cultured with FC in the rotating system (R group) showed significantly higher sperm penetration (71.0%), MPF formation (70.5%), and normal fertilization (monospermic fertilization with female and male pronuclei; 31.5%) rates than those in the control group (56.0%, 56.9%, and 17.6%, respectively; P < 0.05). Also, the R group showed significantly higher rates of 8-cell embryos at 2 days after IVF and blastocyst formation at 7 days after IVF than those of the control group (17.2% vs. 8.3% and 10.9% vs. 4.5%, respectively; P < 0.05). These results indicate that porcine oocytes matured in pFF supplemented with FC using the rotating system have the ability to be penetrated by sperm and form MPF, and to develop to the blastocyst stage at higher rates, than oocytes cultured in the standard static maturation culture system. In conclusion, the pFF can be a sole and simple maturation culture medium useful for the in vitro production of blastocysts in pigs.
288 KINETICS OF FERTILIZATION, DEVELOPMENT, AND SEX RATIO OF IN VITRO-PRODUCED BOVINE EMBRYOS OBTAINED WITH FOUR DIFFERENT BULLS
Authors: Alomar M. , Tasiaux H. , Remacle S. , George F. , Paul D. , Donnay I.
Published: December 2006

The between-bulls variation in in vitro fertility and the shift of sex ratio toward male embryos are two problems affecting the in vitro production (IVP) of bovine embryos. Our objective was to evaluate the possible correlation between the kinetics of fertilization, embryo development, and the sex ratio of the resulting embryos. In a first experiment, and using frozen-thawed semen of 4 different AI bulls, the kinetics of pronucleus (PN) formation was evaluated at 8, 12, and 18 h post-in vitro insemination (hpi) after fixation and staining with Hoechst 33342. Fertilized oocytes were classified in 3 PN stages: PN1: showing the first signs of sperm head decondensation; PN2: with two pronuclei of different sizes, the two being far from each other; and PN3: showing two symmetric pronuclei of equal size, close to each other. Differences between bulls were observed at each time point, but were greater at 12 hpi than at 8 or 18 hpi. At 8 hpi and 12 hpi, bull C showed a significantly faster PN formation by comparison with the 3 other bulls (chi-square test: P < 0.05), whereas at 18 hpi, the proportion at each of the PN stages was similar to that of bulls A and D, with bull B showing delayed PN development. In a second experiment, a standard IVP procedure was conducted with the 4 bulls to determine cleavage and blastocyst rates. The timing of first cleavage was measured using time-lapse cinematography. Compared with those of bull B, the embryos generated with bull C led to significantly higher Day 7 blastocyst yields (31.3 � 9.5% vs. 21.9 � 6.7%; ANOVA: P < 0.05). Moreover, the embryos from bull C reaching the blastocyst stage cleaved faster (first cleavage at 23.1 � 2.1 hpi vs. 25.4 � 2.7 hpi for bull B; ANOVA: P < 0.05). In a third experiment, 65 to 76 Day 8 blastocysts were sexed per bull. Embryo sexing was performed by PCR using the co-amplification of a Y-specific bovine SRY sequence and an autosomal btRep-137 sequence. Only blastocysts obtained with bull C showed a shift in sex ratio toward male embryos (76.0% male embryos vs. 53.8% for bull B; chi-square test: P < 0.05), whatever the size of the blastocyst. The shift in sex ratio was already present at the 2-cell stage (64.2% male embryos; n = 53; chi-square test: P < 0.05). In conclusion, for 2 out of 4 bulls, a correlation was observed between the kinetics of PN formation, the timing of first cleavage, and the sex ratio of the resulting embryos.
291 EFFECTS OF ANTIOXIDANT AND CULTURE MEDIUM ON THE DEVELOPMENT OF PORCINE IN VITRO-MATURED - IN VITRO-FERTILIZED EMBRYOS
Authors: Choe C. , Son D.-S. , Choi S.-H. , Cho S.-R. , Kim H.-J. , Yeon S.-H. , Han M.-H.
Published: December 2006

Most cells cultured in vitro are exposed to the risk of injury by free oxygen radicals (FOR). However, some of FOR-induced injury could be reduced by the antioxidants and culture medium used for in vitro embryos. This study was undertaken to examine the effects of the antioxidant and culture medium on the development of porcine in vitro-matured–in vitro-fertilized embryos. In Experiment 1, we treated the porcine oocytes in NCSU23 medium with various concentrations of β-mercaptoethanol (β-ME) to determine the effective concentration of antioxidants during IVM of porcine oocytes. In Experiment 2, we tested different culture media to find the proper culture conditions for in vitro porcine embryos. The porcine oocytes that were matured in NCSU23 medium and then fertilized in mTBM medium were cultured in NCSU23 or porcine zygote medium-5. All steps (maturation, fertilization, and development) were carried out in vitro. Differences were analyzed among treatments using the general linear model (GLM) procedure in the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA). The results were summarized as follows. Various concentrations of β-ME showed different developmental rates in porcine embryos. The rates of blastocyst formation at Day 7 after IVF were 9.2 � 1.8 (n = 65), 10.0 � 4.2 (n = 80), 17.5 � 1.1 (n = 63), 20.7 � 1.7 (n = 82), and 14.6 � 1.4 (n = 82) in oocytes treated with β-ME at 0, 10, 25, 50, and 100 �M during IVM, respectively. Of the concentrations of β-ME tested, 50 �M β-ME markedly increased the rates of blastocyst formation at Day 7 (P < 0.05). The rates of blastocyst formation at Day 7 in the NCSU23 and PZM-5 culture media of porcine IVF-derived embryos were 18.8 � 2.6 (n = 96) and 15.6 � 7.1 (n = 77), respectively. The developmental rates were slightly increased in NCSU23, compared with those in PZM-5, but there were no significant differences (P < 0.05) between the NCSU23 and PZM-5 media. In conclusion, these results suggest that the addition of 50 �M β-ME in the IVM medium can improve developmental the rates of porcine embryos in vitro.