bluuebunny/crossref_metadata_2025 huggingface dataset:
145 THE METHYLATION PATTERNS OF POTENTIAL DIFFERENTIALLY METHYLATED REGIONS IN BOVINE Xist, Impact, NDN, AND H19 GENES
Authors: D'Cruz N. T. , Wilson K. J. , Holland M. K.
Published: December 2006

Clinical and laboratory-assisted reproductive techniques such as ICSI have recently been associated with an increased incidence of several syndromes associated with defects in genomic imprinting. Genomically imprinted genes are expressed from only one parental allele and act to regulate growth of the fetus and placenta and brain development/ function. Imprinted genes are controlled by differentially methylated regions (DMRs), whereby one parental allelle (i.e. either maternal or paternal) is epigenetically silenced via methylation. Studies conducted in vitro suggest that culture of embryos and embryo manipulations may perturb the imprinting process. In the current study, the genomic DNA methylation patterns of CpG islands within bovine H19 (27 CpGs analyzed), Impact (36 CpGs), NDN (22 CpGs), and Xist (21 CpGs) were analyzed by bisulfite sequencing. Genomic DNA from a female fibroblast cell line and sperm were chosen for analysis. Potential DMRs for the 4 genes were identified, and semi-nested PCR primers were designed surrounding those regions. Second-round PCR products (2 separate reactions) were mixed, subcloned, and sequenced (n ≥ 10). The fibroblast methylation pattern of the Xist DMR showed consistent methylation in 50% of sequenced clones, with no methylation observed in sperm. The H19 DMR in fibroblast DNA also showed consistent methylation in 25% of sequenced clones, with sperm DNA fully methylated. These results confirm previous studies showing that Xist and H19 are imprinted in cattle. Sequencing of the putative Impact DMR clones indicated no methylation in either cell type, suggesting no imprinting in cattle, tissue-specific imprinting, or that this CpG island (15 bp post ATG) is not the DMR that controls imprinted expression of the Impact gene. The NDN DMR (500 bp post ATG) in sperm was not methylated, whereas the fibroblast cells had a variable methylation pattern. This may be for the same reasons suggested for Impact, but the variability within the CpG island may also be due to in vitro culture conditions resulting in aberrant methylation. This possible culture effect is currently being confirmed through bisulfite sequencing of the gene in an adult tissue. The investigation of methylation patterns in oocytes is also underway. Together, the information gathered will be used to determine the imprinting status of several bovine genes and, in the future, whether any of these imprinted genes are responsible for the increased pregnancy loss and calf abnormalities associated with advanced reproductive technologies.
147 GENE KNOCKDOWN OF MATERNAL- AND EMBRYONIC-EXPRESSED GREEN FLUORESCENT PROTEIN IN MURINE EMBRYOS BY THE INJECTION OF A SHORT INTERFERING RNA (siRNA)
Authors: Iqbal K. , Kues W. A. , Carnwath J. W. , Niemann H.
Published: December 2006

RNA interference (RNAi) is now widely used for gene silencing in various biological systems. Injection of long double-stranded RNAs has been shown to specifically knock down gene expression in mammalian embryos. The utilization of short interfering RNAs (siRNA) to target specific embryonic genes would make this approach flexible and efficient enough for studying physiological functions in development. To demonstrate the feasibility of a single class of siRNA molecules for achieving long-lasting effects after injection into mammalian zygotes, we used siRNAs to knock down expression of the green fluorescent protein (GFP) in transgenic murine embryos of the OG2 transgenic line. Homozygous OG2-animals, carrying the Oct4-GFP transgene, were mated with NMRI animals to produce OG2 hemizygous zygotes, which show a parentally dependent expression pattern of the marker gene. Hemizygous zygotes with a maternally inherited Oct4-GFP gene continuously express the GFP marker; hemizygous zygotes with a paternally inherited Oct4-GFP start transcription of the GFP gene at the 4–8 cell stage, i.e., after onset of embryonic genomic activation. Thus efficacy and duration of gene silencing could be tested under 2 different conditions where (i) GFP mRNA was already present at the time point of injection, and (ii) GFP transcription started 2–3 cell cycles after siRNA injection. Zygotes were microinjected with either a 22-basepair GFP-siRNA or a control siRNA and then cultured in vitro. The siRNAs were conjugated with the fluorochome rhodamine to allow monitoring of injection and subsequent degradation of siRNAs. At the end of the in vitro culture, the developmental stage, the number of nuclei, GFP fluorescence (Table 1), and the GFP mRNA levels were determined by RT-PCR. In conclusion, results demonstrate that injection of a synthetic siRNA is sufficient to knock down a target gene transcript with either maternal or embryonic expression in mammalian embryos. Importantly, maternally derived GFP proteins showed a delayed functional knockdown of GFP for 2 days. The advantages of this approach are that (i) off-target effects of long double-stranded RNAs can be avoided, (ii) siRNAs against any known transcript can be rapidly designed and synthesized, and (iii) developmentally important genes can be silenced in embryos for at least 5 days following injection.

Table 1.siRNA microinjection into zygotes with maternally and
paternally inherited GFP
149 IDENTIFICATION OF DIFFERENTIALLY EXPRESSED PROTEINS IN THE UMBILICAL CORDS OF TERM AND PRE-TERM (35 - 36 WEEKS) INFANTS USING PROTEOMICS
Authors: Lee S. Y. , Cho S. K. , Kwon D. N. , Park J. Y. , Kim J. H.
Published: December 2006

Pre-term infants born before the end of the 37th week of pregnancy have not been exposed to their mother's womb full-term. Because they are born too soon, many of their biological systems, such as those involving the lungs and liver, are not developed enough to work property on their own. The lungs are not functional in the fetus, and the blood passes directly from the right atrium to the left atrium or from the right ventricle to the aorta. In this study, we analyzed protein profiles in the umbilical cord of term and pre-term infants by 2DE and MALDI-TOF analysis. Protein patterns of the umbilical cord were established using the pH range 4–7, isoelectric focusing (IEF) cell system, and 7.5–17.5% gradient 2-dimensional polyacrylamide gel electrophoresis (2DE). Image analysis was performed following Coomassie brilliant blue G staining to reveal spots, and spot intensities were compared using a PDQuest analysis system. Peptide mass fingerprinting with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and a SWISS-PROT database search were utilized to identify proteins. Around 1000 protein spots were recognized by image analysis for umbilical cords from each treatment. Analysis of umbilical cords from pre-term infants revealed 35 different spots compared with those from full-term infants. Levels of 12 proteins increased, such as serum albumin precursor and tropomyosin, whereas levels of 11 proteins decreased, including 78 kDa glucose-regulated protein and protein disulfide isomerase A3. Another 12 proteins had levels that were different between 35 and 36 weeks of pregnancy. In general, proteins that increased represented precursor and cell mobility or structure-related proteins, whereas proteins that decreased included chaperon-related proteins. Expression levels of this protein were confirmed by Western blot analysis and real-time PCR. The results of this experiment suggest differential protein profiles between umbilical cords of pre-term and full-term infants that may be related to fetal circulation and early birth of infant.
153 IMMUNOCYTOCHEMICAL STUDIES OF OCT-4, SOX-2, AND β-TUBULIN III EXPRESSION IN EARLY PORCINE EMBRYOS
Authors: Schauser K. , Sundkvist T. S. , Vejlsted M. , Maddox-Hyttel P.
Published: December 2006

In the mouse, the transcription factor SOX-2 is known to have at least 2 roles: (1) it acts as a co-factor of the transcription factor OCT-4, the key regulator of pluripotency essential for the development of the inner cell mass/epiblast; and (2) it is involved in the direction of neural development. In this study, we elucidate the localization of SOX-2 in early porcine embryos in relation to that of OCT-4 and the early neuronal marker β-tubulin III. Embryos were flushed from uteri, fixed in 4% paraformaldehyde, and processed for paraffin sectioning. Sections (5 �m) were stained with anti-OCT-3/4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) using the ABC-AEC-method and counterstained with hematoxylin, or processed for double immunofluorescent staining using antibodies against SOX-2 (R&D Systems Europa, Ltd., Abington, Oxon, UK) and β-tubulin III (Sigma-Aldrich Denmark A/S, Copenhagen, Denmark) and counterstaining with Hoechst. The embryos were classified as pre-streak (n = 8), primitive streak (n = 4), neural groove (n = 5), and somite (n = 4) stage (Vejlsted et al. 2006 Mol. Reprod. Dev. 73, 709–718). At the early pre-streak stage, SOX-2 and OCT-4 staining was found in the nuclei and a weak β-tubulin III staining in the cytoplasm of all epiblast cells. At the late pre-streak and the primitive streak stage, SOX-2 staining became polarized to the nuclei in the anterior epiblast region, whereas OCT-4 staining was found in all nuclei of the epiblast and of the forming meso- and endoderm. The β-tubulin III staining was restricted to the epiblast and showed no anterior-posterior polarization. At the primitive streak, when cells were involuting to form the meso- and endoderm, SOX-2 staining of nuclei was absent. At the neural groove stage, the SOX-2 and β-tubulin III staining was localized to nuclei and cytoplasm, respectively, of the same cells and observed in the neural plate and groove. A polarization in SOX-2 staining was observed in an anterior-posterior direction. At the somite stage, the SOX-2 and β-tubulin III staining was again localized to the same cells and observed in the neuropores and neural tube. The SOX-2 staining of the neural tube was polarized in a dorso-ventral direction. At the neural groove and somite stage, the OCT-4 staining gradually disappeared from the epiblast, mesoderm, and endoderm except from scattered cells, presumably primordial germ cells, localized in the endoderm. Our results suggest that also in the porcine embryo SOX-2 plays a dual role, being involved in regulation of both pluripotency and neural development.
157 DYNAMIC CHANGES IN LOCALIZATION OF HP1α DURING BOVINE EMBRYOGENESIS
Authors: Wilson K. J. , Prashadkumar S. , Cooney M. A. , French A. J. , Jans D. A. , Verma P. J. , Holland M. K. , D'Cruz N. T.
Published: December 2006

The Cbx gene family, consisting of Polycomb and Heterochromatin Protein 1 genes, is involved in transcriptional repression, cell cycle regulation, and chromatin remodeling. In Drosophila embryos, the single family member HP1 localizes to the nuclei from the blastoderm stage, nuclear division cycle 10, suggesting that heterochromatin formation occurs around the time of embryonic transcriptional activation (James et al. 1989 Eur. J. Cell Biol. 50, 170–180). In mice, HP1� is absent in the first cell cycle in both parental nuclei following fertilization (van der Heijden et al. 2005 Mech. Dev. 122, 1008–1022). We report the first study of gene expression, mRNA and protein localization, of the Cbx gene HP1� in in vitro-produced bovine embryos. Expression of the HP1� gene was assessed non-quantitatively in amplified cDNA from 3 individual oocytes or embryos for each stage. HP1� (Cbx5) transcripts were detected in all samples except one oocyte, three 4-cell embryos, and one Day 7 blastocyst. The lack of transcripts at the 4-cell stage may reflect an early degradation of maternal transcripts, or indicate that transcript levels were too low to be detected with this assay. Immuno-localization of HP1� was then performed by using an anti-HP1� monoclonal antibody (Upstate, Auspep Pty., Ltd., Parkville, Victoria, Australia), revealing HP1� to be localized evenly in the cytoplasm of oocytes, and then progressing to both cytoplasmic and nuclear staining at the 2-cell stage. This was followed by nuclear staining from the 4-cell stage onward. Higher power investigation of the subnuclear localization of HP1� showed a diffuse type staining pattern within the nuclei of 2- and 4-cell embryos, followed by punctate staining within the euchromatin in the 8-cell embryos, and within the heterochromatin by the 16-cell stage. At the blastocyst stage, staining appeared more diffuse, but localized to the heterochromatin. This suggests that HP1� is localized to the euchromatin early, followed by subsequent localization to the heterochromatin following the MET. HP1� localization to the euchromatin was recently described (Grigoryev et al. 2004 J. Cell Sci. 117, 6153–6162) during a differentiation event in murine lymphocytes. It is evenly distributed throughout the nucleus in quiescent lymphocytes, but localizes to the vicinity of centromeric chromatin during lymphocyte reactivation. HP1-TIF1α interaction was also recently shown to be required during a short period of time within primitive endoderm-like cells for terminal differentiation (Cammas et al. 2004 Genes Dev. 18, 2147–2160). The results thus indicate that HP1� behaves similarly in embryonic differentiation and lymphocyte reactivation, implying a common mechanism of chromatin remodeling in the 2 cellular systems. Collectively, the results suggest that dynamic changes of the nuclear–cytoplasmic and subnuclear distribution of HP1� may play a key role in the bovine MET.
158 LEADING BLASTOMERE OF A 2-CELL-STAGE PORCINE PARTHENOGENETIC EMBRYO CONTRIBUTES TO TROPHECTODERM FIRST
Authors: Won C. , Park S. K. , Choi Y.-J. , Kang H. , Roh S.
Published: December 2006

Embryonic axis formation is important in normal animal development. Some vertebrate and invertebrate eggs display undeniable polarity along a plane known as the animal–vegetal axis. However, axis formation in mammals, which is studied only in mice, is still a point of issue (Hiiragi and Solter 2004 Nature 430, 360–364; Zernicka-Goetz et al. 2005 Nature 434, 391–395). In the present study, we investigated the embryonic–abembryonic axis formation in porcine species. We used porcine parthenogenetic embryos to prevent a topological change of the 2 apposing pronuclei in the egg center caused by polyspermy and to avoid the influence of the fertilization cone, which indicates the sperm entry position. For lineage tracing, DiI, a lipophilic fluorescence dye, was injected into one blastomere per embryo at the 2-cell stage. After this process, as embryos developed into the 3-cell stage, all the embryos were divided into 2 groups named Leading and Lagging, following the location of the DiI oil drop (Leading: the oil drop was positioned in the first-dividing blastomere; Lagging: the drop was positioned in the late-dividing blastomere). The embryos developed into blastocysts after 6 to 7 days of culture in vitro. Only the inner cell mass (ICM) was labeled in 50% (8/16) of the Lagging group. In some Lagging embryos (2/16), ICMs with a small portion of adjacent trophectodermal cells (TE) were labeled and 62.5% (10/16) of Lagging embryos formed the ICM part in total. On the other hand, 60% (6/10) of the Leading embryos formed only distal TE (opposite side of the ICM). Only one of the Leading embryos formed the ICM. The rest of Lagging embryos (37.5%, 6/16) or the Leading group (40.0%, 4/10) showed an even distribution of blastocysts, regardless of the ICM or TE. Oocytes, zygotes, and 2-cell-stage embryos in porcine species tended to show unequally distributed lipid contents and could be distinguished by their different colors and brightness, although not all of them showed significant differences. We injected DiI into a relatively brighter blastomere of the 2-cell-stage embryos and the results were very similar to those obtained from the Leading group (distal TE: 70.6%, 24/34). High lipid contents may be the cause of delayed development of the blastomere of an embryo. Our findings indicated that the Leading blastomere of 2-cell porcine parthenotes formed distal TE first, and that afterward, the Lagging blastomere not only filled the rest of the TE but also contributed to the ICM.
This study was supported by the Research Project on the Production of Bio-organs (No. 200506030601) and Technology Developmental Program (High-Technology Development Project No. 204117-3) for Agriculture and Forestry, Ministry of Agriculture and Forestry, Republic of Korea
160 DETERMINATION OF THE INCIDENCE OF EMBRYONIC MORTALITY IN SYNCHRONIZED EWES DURING THE BREEDING SEASON
Authors: Erdem H. , Saribay M. K. , Tekeli T.
Published: December 2006

The aim of the present study was to evaluate the incidence of embryonic mortality in synchronized Konya Merino ewes (2–4 years of age) during the the breeding season. The flushing procedure started in 126 ewes, beginning 1 week before the breeding period until mating. The ewes were synchronized using progesterone : PMSG; a sponge containing 40 mg florogestone acetate was placed into vagina for 12 days. PMSG (500 IU) was given intramuscularly at the time of sponge removal and ewes were allowed to mate 48-60 hours (Day 0) after PMSG injection. The first pregnancy diagnosis was done transrectally 18 days after mating by using B-mode, linear array, 5 MHz real-time ultrasonography, and it was repeated again on Days 22, 26, 30, and 34. One hundred eleven ewes were pregnant on Day 18, and 191 embryos were detected in 111 pregnant ewes. There were 44 single, 54 twin, and 13 triple pregnancies. Embryonic death was determined in 39 out of 111 ewes (35.1%), and 45 embryos out of the total of 191 were lost (23.6%). Among the ewes which experienced embryonic death, 28 ewes maintained pregnancy while 11 ewes (9 single, 1 twin, and 1 triple pregnancy) did not. Among the embryos that were dead, 9 (20%), 20 (44.4%), and 16 (35.6%) were from single, twin, and triple pregnancies, respectively. Rates of embryonic death in the total number of embryos according to type pregnancy were 20% (9/45), 18.5% (20/108), and 41% (16/39). Incidence of embryonic deaths were 22.2% (10/45), 51.1% (23/45), 22.2% (10/45), and 4.5% (2/45) for Days 22, 26, 30, and 34, respectively. The majority of embryonic deaths were observed by Day 22 in single pregnancies and by Day 26 in multiple pregnancies. Incidence of termination of pregnancy in single pregnancies was significantly higher than for twin pregnancies (P < 0.01). The rate of embryonic mortality between single and twin pregnancies was not significant, whereas it was significant among single and triple and among twin and triple pregnancies (P < 0.05 and P < 0.01, respectively). In conclusion, incidence of embryonic death was similar between single and twin pregnancies but the majority of pregnancy losses were observed in single pregnancies. Although embryonic deaths are observed, maintenance of pregnancy conceals the problem of embryonic mortality in multiple pregnancies. Therefore, applications to improve the survival of embryos during the first month after mating could yield higher numbers of lambs.
162 ESTRUS SYNCHRONIZATION, ARTIFICIAL INSEMINATION, AND PREGNANCY DIAGNOSIS IN WATER BUFFALOES (BUBALUS BUBALIS)
Authors: Hanh N. V. , Huu Q. X. , Uoc N. T. , Nguyen B. X. , Sulon J. , Sousa N. M. , Beckers J. F.
Published: December 2006

One of the big obstacles in management of reproductive activity in water buffaloes is their silent estrus. This characteristic prevents the determination of time for artificial insemination (AI), resulting in lengthening of the calving interval (Nguyen et al. 1990 Proc. FAO Workshop, Bulgaria, 210–216). Pregnancy-associated glycoprotein (PAG) measurement in maternal blood is an effective method of early pregnancy diagnosis in ruminant species (Zoli et al. 1992 Biol. Reprod. 46, 83–92). The present study describes a preliminary attempt to improve buffalo reproductive performance by association of three different approaches: synchronization of estrus by hormonal treatment, followed by two AI, and finally pregnancy diagnosis based on PAG measurements. A total of 33 buffalo females (4 to 15 years old), with no apparent estrus during a period of 9 to 36 months after last calving, were used. Each female received 2 injections of 5 mL of PGF2α (Intervet, France) 10 days apart (Day −12 and Day −2), one injection of 500 IU of PMSG (SABC, Vietnam) at Day −5, and 1000 IU of hCG (Intervet, France) on the day of AI (Day 0). A second AI was performed 8 h after hCG injection. Blood samples (9 mL) were collected from each female at Days 20, 25, 40, and 45 after AI. Blood samples were withdrawn by jugular venipuncture (heparinized Vacutainer tubes) and kept in a cool box until centrifugation (500g, 10 min). Plasma samples were stored at −20°C until assayed for PAG. Pregnancy diagnosis was considered negative, doubtful, or positive according to the PAG concentrations (<0.6 ng mL−1, 0.6 to 0.8 ng mL−1, and >0.8 ng mL−1, respectively). Clinical observation of females after insemination showed that the total rate of animals that exhibited signs of estrus until Day 45 was 9.1% (3/33). These 3 females were not considered for pregnancy diagnosis. Table 1 shows the results of pregnancy diagnosis in 30 water buffalo females. These results show that fertilization is easily obtained after estrus synchronization in buffaloes and that measurement of PAG concentrations in maternal plasma can be used for pregnancy diagnosis from Day 40 after AI.

Table 1.Number of water buffalo females considered as non-pregnant, doubtful or pregnant on the basis of PAG concentrations

This work was supported by a grant from the Belgian Technical Cooperation.
164 POTENTIAL REGULATION OF PERI-IMPLANTATION OVINE CONCEPTUS GROWTH AND DIFFERENTIATION BY THE WNT SIGNALING SYSTEM
Authors: Hayashi K. , Burghardt R. C. , Bazer F. W. , Spencer T. E.
Published: December 2006

In mice WNT signaling regulates cell fate, differentiation, and growth in the conceptus (embryo and associated extra-embryonic membranes), as well as implantation. We studied various components of the WNT signaling pathway in the ovine uterus during the estrous cycle (C) and pregnancy (P) and in the peri-implantation conceptus. Expression of WNT2, WNT2B, and WNT4 mRNAs was very low in endometria of C and P ewes from Days 10 to 16 and in conceptus trophectoderm (Tr). WNT5A/5B mRNAs were abundant in the stratum compactum stroma, whereas WNT11 mRNA was detected in endometrial epithelia of C and P ewes, but not in conceptus Tr. WNT7A mRNA was localized specifically to luminal (LE) and superficial glandular (sGE) epithelia of Day 10 C and P ewes, was undetectable by Day 12, and then increased up to Day 16 and was maximum on Day 20 only in P ewes. Frizzled receptor (FZD6/8) mRNAs were detected primarily in conceptus Tr and uterine LE and GE, whereas the co-receptor LRP5/6 (low density lipoprotein receptor-related protein) mRNAs were expressed in all uterine cell types and conceptus Tr. Dickkopf (DKK1), a negative regulator of WNT signaling, was detected in stratum compactum stroma after Day 14 in P ewes. CTNNB1 (beta-catenin), a key mediator of canonical WNT signaling, and GSK3B (glycogen synthase kinase-3 beta) and CHD1 (E-cadherin) mRNAs were abundant in endometrial epithelia and in conceptus Tr. Immunoreactive CTNNB1 protein was abundant in LE and GE, and present at lower levels in stroma and myometrium in uteri from C and P ewes. In the conceptus Tr, immunoreactive CTNNB1 protein was abundant in nuclei of the mononuclear and binuclear cells (BNC), as well as in cell adherens junctions. Nuclear CTNNB1 interacts with transcription factors, most notably LEF1/TCF7 (lymphoid enhancer-binding factor 1/transcription factor 7), to regulate gene transcription. LEF1 mRNA was detected in LE and sGE, whereas nuclear TCF7L2 protein was particularly abundant in trophoblast giant BNC. WNT/beta-catenin/TCF7 target genes were also studied. MSX2 mRNA was abundant in conceptus Tr, and MYC mRNA was abundant in BNC of conceptus Tr and endometrial epithelia. Next, ovine Tr (oTr) cells and endometrial stromal (oST) cells were used for mechanistic studies that revealed that transfection of mouse WNT7A stimulated a LEF/TCF-responsive reporter (TOPFLASH), and co-transfection of either dnTCF or SFRP2 (a secreted FZD inhibitor) inhibited WNT7A effects. WNT7A stimulated expression of MSX2 and MYC in oTr cells, and this effect was inhibited by SFRP2. These results implicate the canonical WNT system as a regulator of peri-implantation conceptus growth and differentiation in sheep.
This work was supported by NIH HD38274 and 5 P30 ES09106 funding.
168 ESTABLISHMENT OF TROPHOBLAST STEM CELLS FROM MOUSE ANDROGENETIC AND PARTHENOGENETIC EMBRYOS
Authors: Ogawa H. , Shindo N. , Tanaka S. , Kono T.
Published: December 2006

Mouse androgenetic and parthenogenetic embryos, which have two sets of paternal and maternal genomes, respectively, are lethal until Day 9.5 of pregnancy, because the growth of the extraembryonic tissues including placenta is inadequate. These results suggest that both parental genomes are involved in placental development. However, the reason for placental deficiency in androgenetic and parthenogenetic embryos has been unclear. Trophoblast stem (TS) cells, which have the ability to differentiate into trophoblast lineage, have been used to elucidate the mechanism of differentiation and gene function in the development of the placenta. In this study, to characterize trophoblast lineage in androgenetic and parthenogenetic embryos, we examined TS cells from these embryos, and assessed their ability to differentiate into trophoblast lineage. The ovulated MII oocytes from B6D2F1 (C57BL/6 � DBA2) female mice were used for the uniparental embryo production. In order to produce parthenogenetic embryos, the oocytes were activated in SrCl2 and cytochalasin B. The androgenetic embryos were produced by in vitro fertilization using enucleated oocytes. These uniparental embryos were cultured for 3.5–4.5 days to develop into blastocysts. TS-like cell lines were established from these blastocysts, as described previously (Tanaka et al. 1998 Science 282, 2072–2075). TS-like cells were cultured without feeder cells in conditioned medium containing FGF4 and heparin to maintain the stem cell conditions. The expression of 5 TS cell maker genes (Eomes, CdX2, Fgfr2, Ap2a, and Errb) in TS-like cells was analyzed by RT-PCR and northern blotting. In addition, the localization of CDX2 was detected using immunostaining. To cause their differentiation into giant cells, TS-like cells were cultured for 6 days in TS medium without FGF4 and heparin. The giant cells were detected by the expression of 2 giant cell marker genes, Hand1 and Pl-1. TS cells from fertilized embryos between B6D2F1 male and female mice were also established as controls. Five TS cell marker genes were expressed both in androgenetic TS-like cells (ATS) and in parthenogenetic ones (PTS). The CDX2 gene was localized in the nucleus in both ATS and PTS; however, the number of CDX2-positive cells decreased in PTS. After 6 days of differentiation, Hand1 and Pl-1 were expressed and giant cells were detected in differentiated derivatives in ATS. On the other hand, giant cells were not detected in differentiated derivatives in PTS. These results suggest that parental genomes may regulate the gene expression independently to differentiate into trophoblast lineage. In conclusion, TS-like cells from uniparental embryos are useful tools to aid the understanding of the function of the parental genomes during placental development.
171 CELLULAR EXPRESSION, LOCALIZATION, AND SECRETION OF OVINE Mx2 (oMx2) IN OVINE GLANDULAR EPITHELIAL CELLS (oGE)
Authors: Toyokawa K. , Assiri A. M. , Ott T. L.
Published: December 2006

Mx proteins are antiviral proteins that belong to the dynamin super-family of large GTPases. In a number of species Mx proteins were shown to be important components of the innate response to viral infection. Work in our laboratory showed that during early pregnancy Mx proteins are up-regulated in the ruminant uterus by conceptus-derived IFN tau. Transient oMx1 knockdown in ovine glandular epithelial cells (oGE) reduced oMx1 secretion and secretion of other unconventionally secreted proteins without affecting secretion via the classical secretory pathway. We further showed that oMx1 was present in uterine flushes from pregnant ewes. We recently characterized another Mx protein, oMx2, in sheep. The newly characterized oMx2 has higher homology with bovine Mx2 (93%) than with oMx1 (60%). The purpose of this study was to characterize cellular expression and localization of oMx2 in oGE cells and in cell lines derived from the luminal epithelium (oLE) and stroma (oSC). In addition we determined if oMx2 was secreted by oGE cells in vitro. Expressions of oMx1 and oMx2 were low to undetectable in all three lines in the absence of IFN tau and were increased by IFN tau (P < 0.01). Induction of oMx1 was greatest in oGE cells (8.4-fold increase; P < 0.01), whereas expression of oMx2 was greatest in oSC cells (19.9-fold increase; P < 0.01). Immunofluorescence analysis confirmed the high level of expression of oMx1 and oMx2 in response to IFN tau in oGE cells. Immunofluorescence analysis revealed that oMx2 co-localized with lamin A/C and nucleoporins, whereas oMx1 was distributed throughout the cytoplasm in oGE cells, suggesting that oMx2 is a nuclear membrane-associated protein. SignalP analysis indicated that oMx2 lacked a traditional leader sequence. Using an inhibitor of the classical secretory pathway, Monensin, we showed that oMx2 levels were not reduced in response to Monensin treatment. The oMx1, oMx2, and ISG15 levels in oGE-conditioned culture medium actually increased in secretions in response to Monensin (2.7-fold, 4.6-fold, and 2.9-fold increases, respectively; P < 0.1), whereas there was no effect of Monensin on �2MG levels (P > 0.1). Results show that oMx2 expression, like that of oMx1, is regulated by IFN tau. The functional significance of the different cellular localization between oMx1 and oMx2 is not clear, but may suggest that these related proteins possess distinct cellular functions. Finally, this is the first report that oMx2 is secreted, perhaps via poorly described unconventional secretory pathways. The potential intracellular and extracellular functions of oMx2 are currently being investigated.
This work was supported by USDA grant 2002-02398 and NIH NCRR grant P20-RR15587-01 to T.L.O.
177 ISOLATION AND CULTURE OF MOUSE ENDOMETRIAL CELLS IN A THREE-DIMENSIONAL CULTURE SYSTEM
Authors: Fu T.-Y. , Tang P.-C. , Ju J.-C.
Published: December 2006

Implantation of mammalian embryos occurs only during a restricted narrow window. The endometrium becomes highly receptive for the embryos during this period of time. The objective of this study was to establish an in vitro culture system for pre- and post-implantation mouse embryos. In Experiment 1, mouse uterine horns were excised at Day 3.5 post-coitus. After being washed with Dulbecco's phosphate-buffered solution (DPBS), the uterine horns were cut open and incubated with 0.05% trypsin in DPBS at 4°C for 2 h. After trypsinization, the tissues were incubated at 37°C for an additional 30 min. The solution containing epithelial cell suspension was recovered after 30 s of vortexing. The trypsinized uterine horns were then cut into 1-mm3 pieces and digested with collagenase (type I, 1 mg mL-1 in DPBS) at 37°C for 3 h with vigorous shaking. At the end of digestion, the solution was filtered through 40-µm nylon mesh and the flowthrough containing stromal cells was collected. The isolated epithelial and stromal cells were characterized by their morphology and immunocytochemistry. Both types of cells showed positive immunocytochemical reaction with desmin antibody. The cultured epithelial cells formed polyhedral shapes, and more than 95% expressed epithelium-specific protein, cytokeratin-18. On the other hand, most of the spindle-like stromal cells had no signal for cytokeratin-18 expression, although a few scattered cells were positively labeled. In Experiment 2, for construction of the 3-dimensional culture system, epithelial cells obtained by the method described in Experiment 1 were seeded on an artificial basal membrane (ECMatrixTM; Millipore/Upstate/Chemicon, Temecula, CA, USA) with underlying stromal cells embedded in the type I collagen matrix. The whole system was settled in a Millicell® (Millipore) hanging in a 24-well culture plate, and immersed in DMEM medium supplemented with 10% fetal bovine serum, 20 ng mL-1 epidermal growth factor, 63.5 nmol progesterone, and 7.14 nmol 17β-estradiol. The morphology of epithelial cells on the matrix became cuboidal after 48 h of culture. Additionally, the columnar cells with a basal nucleus were observed on the paraffin wax sections. In Experiment 3, mouse E3.5 embryos were recovered and cultured in this established culture system. Normal hatching and/or attachment of the blastocysts were observed after 2 days of culture. In conclusion, our results showed that epithelial cells formed morphologically columnar monolayers and apparently interacted with blastocyst embryos. Successful construction of this model system would facilitate the study of early embryo development through the implantation stage.
189 EFFECT OF PROGESTERONE AND EPIDERMAL GROWTH FACTOR ON IN VITRO-PRODUCED EIGHT-CELL BOVINE EMBRYOS IN A SERUM-FREE CULTURE MEDIUM
Authors: Merlo B. , Iacono E. , Mari G.
Published: December 2006

The role of progesterone (P4) and epidermal growth factor (EGF) in early bovine embryo development is still not clear. P4 has been administered at different times of embryo development, and a direct effect on IVF-derived bovine 8-cell embryos has been noted even if there was an interference due to the P4 vehicle (Ferguson et al. 2005 Reprod. Fertil. Dev. 17, 219 abst). EGF has been added to the culture medium from the presumptive zygote stage at different concentrations, resulting in improved blastocyst rates compared to that in control medium (Mtango et al. 2003 Theriogenology 59, 1393–1402; Sirisathien et al. 2003 Anim. Reprod. Sci. 77, 21–32), and gave results similar to those with 5% or 10% FCS (Palasz et al. 2000 Anim. Reprod. Sci. 58, 229–240). The objective if this experiment was to determine the effect of P4 and EGF on development of in vitro-produced bovine embryos when administered alone or in combination at the 8-cell stage in the absence of serum. In vitro-produced bovine 8-cell embryos were randomly allotted to treatments: (1) control, SOFaaBSA medium (BSA, 16 mg mL−1; n = 198); (2) P4, SOFaaBSA + P4 (15 ng mL−1 in ethanol; n = 198); (3) EGF, SOFaaBSA + EGF (25 ng mL−1; n = 200); (4) P4 + EGF, SOFaaBSA + P4 (15 ng mL−1 in ethanol) + EGF (25 ng mL−1; n = 201); and (5) FBS, SOFaaBSA + FBS (5%; n = 197). In order to minimize the toxic effect of ethanol, it was allowed to evaporate from the culture dish and then medium was added. All in vitro procedures were carried out at 38.5°C in a humidified atmosphere of 5% CO2 in air; presumptive zygotes were cultured in SOFaaBSA until 8-cell stage. Embryo development was evaluated on Day 6 and on Day 8 after IVF (Day 0), and rates calculated from 8-cell embryos. The study was done in 4 replicates and chi-square test was used for statistical analysis (Statistica for Windows; Stat Soft Inc., Tulsa, OK, USA); significance was assessed at P < 0.05. Results are reported in Table 1. No differences were found in the number of morulae between P4 and control, between P4 + EGF and FBS, and between P4 + EGF and EGF (P > 0.05), whereas the combination P4 + EGF was better than P4 alone (P < 0.05). Blastocyst rate was not different (P > 0.05) among EGF, P4 + EGF, and FBS groups. P4 achieved an higher (P < 0.05) blastocyst rate than control but it was lower (P < 0.05) than that of P4 + EGF or FBS. In conclusion, P4 alone improves embryo development from the 8-cell embryo to the blastocyst stage in a serum-free culture system, and EGF alone achieves a blastocyst rate not significantly different from that of FBS; furthermore, the combination of P4 and EGF can be considered the most suitable as an alternative to FBS because similar results were obtained in terms of both morulae and blastocysts.

Table 1.Eight-cell bovine embryo development in SOFaaBSA medium in presence of P4, EGF, P4+EGF, or FBS
190 INFLUENCE OF IN VITRO CULTURE OF BOVINE EMBRYOS ON THE STRUCTURE OF THE ZONA PELLUCIDA
Authors: Mertens E. , Besenfelder U. , Gilles M. , Hölker M. , Rings F. , Havlicek V. , Schellander K. , Herrler A.
Published: December 2006

The zona pellucida is an extracellular structure at the direct interface between the maternal and embryonic sides, through which all signals of the embryo–maternal dialogue as well as nutritional factors have to pass. Up to now there has been no investigation as to whether in vitro culture influences the structure of the zona pellucida compared to that of in vivo embryos. Therefore, in vitro (oocyte, zygote, 2-, 4-, 8-, 16-cell, morula, and Day 7 blastocyst, using the protocol published by Nganvongpanit et al. 2006 Reproduction 131, 861–874) and in vivo (zygote, 4-cell, morula, and blastocyst) embryos were prepared for microscopical investigation. In total, 191 in vitro and 99 in vivo embryos of quality 1 and 2 from at least 2 replicates were used. Araldit-embedded embryos were semi-thin-sectioned and stained with hematoxilin. A morphometrical evaluation was performed to determine the percentage of the more reticular outer part compared to the total thickness of the zona. Furthermore, the total thickness of the zona pellucida was compared between in vitro and in vivo embryos. In parallel, embryos were analyzed by scanning electron microscopy. Up to the 16-cell stage, the zona of in vivo and in vitro embryos is similar, but in vivo morulae and blastocysts show significantly thicker zonae than the in vitro stages. The reticular part of the zona is thicker in in vivo embryos than in in vitro embryos (30.2 � 2.1% vs. 12.4 � 1.8%, respectively). Investigating the pores of the zona pellucida, in vivo morulae/blastocysts show smaller-sized pores than in those derived in vitro. Most of the in vivo morulae/blastocysts are totally covered by secreted granules, and therefore the pores could not be investigated. Furthermore, 30-50% of the in vitro embryos show partly degenerated outer layers of the zona pellucida. This investigation demonstrates that in vitro and in vivo zonae pellucidae are significantly different, reflecting a negative influence of the IVM and IVC.
207 INFLUENCE OF THE INTERVAL BETWEEN THAWING TO TRANSFER ON PREGNANCY RATES OF FROZEN - THAWED DIRECT-TRANSFER EMBRYOS
Authors: Bo G. A. , Peres L. C. , Pincinato D. , de la Rey M. , Tribulo R.
Published: December 2006

An experiment was designed to evaluate the effect of the interval between thawing to deposition of the embryo into the uterine horn on pregnancy rates of in vivo-produced frozen–thawed embryos in 1.5 M ethylene glycol (direct transfer). Data were collected from 1122 embryo transfers performed in the same farm (Estancia El Mangrullo, Lavalle, Santiago del Estero, Argentina) during the spring and summer of 2004/05 and 2005/06 (6 replicates, ambient temperature between 20 and 40�C). Recipients used in all replicates were non-lactating, cycling, multiparous Bos taurus � Bos indicus crossbred cows with body condition score between 3 and 4 (1 to 5 scale) that were synchronized using fixed-time embryo transfer protocols. Briefly, the synchronization treatments consisted of the insertion of a Crestar ear implant (Intervet, Sao Paulo, Brazil) or a progesterone-releasing device (DIB; Syntex SA, Buenos Aires, Argentina), plus 2 mg of estradiol benzoate (EB; Syntex) intramuscularly (IM) on Day 0, and 400 IU of eCG (Folligon 5000; Intervet, or Novormon 5000; Syntex) IM plus 150 �g d-cloprostenol IM (Preloban; Intervet, or Ciclase; Syntex) on Day 5. Progestin devices were removed on Day 8 and all cows received 1 mg of EB IM on Day 9. All cows were examined by ultrasonography on Day 16 and those with a luteal area >76 mm2 (by calculating the area of the CL minus the area of the cavity) received, on Day 17, frozen–thawed embryos by nonsurgical transfer. All embryos were Grade 1, and all were frozen in 1.5 M ethylene glycol at the Embryo Plus Laboratory (Brits, South Africa). After being stored in liquid nitrogen, the embryos were plunged directly (no air thawing) in a 30�C water bath for 30 s, and then transferred to the recipient cows by either one of two technicians. Based on the interval between thawing and transfer, the transfers were classified as being in one of 3 groups: Group 1: <3 min; Group 2: 3 to 6 min; and Group 3: 6 to 16 min. The main reason for delayed transfers beyond 6 min was the replacement of one recipient for another because of difficulty in threading the cervix (1% of the total transfers) or a recipient falling down into the chute or with very bad disposition and behavior. Pregnancy was determined by ultrasonography 28 to 35 days after fixed-time embryo transfer, and data were analyzed by logistic regression. There were no effects of replicate, technician, CL area, recipient body condition score, embryo stage, and time from thawing to transfer on pregnancy rates. Pregnancy rates in the 3 thawing to transfer intervals were: Group 1: 215/385, 55.8%; Group 2: 372/655, 56.8%; Group 3: 42/82, 51.2%; P > 0.6. These results may be interpreted to suggest that there is no significant effect of time from thawing to transfer (up to 16 min) in direct transfer embryos using Bos taurus � Bos indicus recipients transferred at a fixed time.
209 VIABILITY OF PORCINE EMBRYOS VITRIFIED BY THE METAL MESH VITRIFICATION METHOD AFTER SURGICAL OR NONSURGICAL TRANSFER
Authors: Fujino Y. , Nakamura Y. , Kobayashi H. , Nakano S. , Suzuki C. , Yoshioka K.
Published: December 2006

The aim of the present study was to evaluate viability of porcine embryos vitrified by the metal mesh vitrification (MMV) method after surgical or nonsurgical transfer. Prepubertal gilts were treated with eCG and hCG (= Day 0), and then inseminated artificially. Expanding blastocysts that were about 200 �m in diameter were collected on Day 7. The embryos were equilibrated in 7.5% ethylene glycol (EG) + 7.5% DMSO + 20% fetal bovine serum (FBS) in PBS at 37�C for 5 min, and then transferred into 15% EG + 15% DMSO + 0.6 M trehalose + 20% FBS in PBS for 1 min. Embryos in groups of 5 were transferred in a minimum volume of the vitrification solution (less than 1 �L) onto stainless steel mesh (75 �m screen size, 1.5 mm in width by 10 mm in length, with a 3-mm right-angle bend), and then plunged into liquid nitrogen. The mesh was stored in a 1.8-mL cryotube submerged in liquid nitrogen. Warming and dilution were performed by moving the mesh from liquid nitrogen into 0.5 M trehalose + 20% FBS in PBS at 37�C for 5 min. Embryos were rinsed twice in NCSU37 + 10% FBS (mNCSU37) for 5 min. After being vitrified, embryos in groups of 20 per recipient were suspended in modified NCSU37 medium and then transferred into gilts either by surgical transfer (5 gilts) or by nonsurgical transfer (6 sows). For surgical transfer, embryos suspended in 0.1 mL of medium were transferred into the uterine horn at 15 cm above the uterine body, which was about 35 cm from the external uterine orifice. For nonsurgical transfer, an intrauterine catheter made from polyethylene tubing (1.2 m long, 3.0 mm outer diameter, 0.5 mm inner diameter) was used. A spiral guide inserted through the vagina into the cervix was used to guide the catheter into one uterine horn. The catheter was moved through the cervix and along the uterine horn. Then, embryos suspended in 1 mL of medium were transferred. Pregnancy was assessed by ultrasonography at 30 days post-estrus. With surgical transfer, 4 of 5 recipients became pregnant, and 3 gilts farrowed a total of 21 (10, 8, 3) live piglets; the fourth gilt aborted one fetus on Day 34. With nonsurgical transfer, 3 of 6 sows became pregnant. The present study demonstrates that vitrified porcine embryos can develop after both surgical and nonsurgical transfer to recipients. As a practical measure, nonsurgical transfer of vitrified porcine blastocysts may be used instead of surgical transfer.
21 RELATIONSHIP BETWEEN DURATION OF ESTRUS BEHAVIOR DETERMINED BY RADIOTELEMETORY SYSTEM AND THREE FACTORS (MILK PRODUCTION, STEROID HORMONE, AND OVULATION TIME) IN DAIRY COWS
Authors: Yoshioka H. , Matsuda H. , Sasaguri K. , Yonai M.
Published: December 2006

Many studies have reported on the low fertility of dairy herds during the past several years. The object of this study was to investigate the relationships between duration of estrus and various factors (milk production, peripheral steroid hormones, and period from estrus to ovulation). All primiparous and multiparous Holstein cows (n = 62) were fitted pedometers (Gyuho; Comtec, Miyazaki, Japan) that measure total counts of walking activity per hour and send the individual cow
210 FACTORS AFFECTING CONCEPTION RATE IN LACTATING HOLSTEIN RECIPIENTS
Authors: Jardina D. T. G. , Rodrigues C. A. , Vasconcelos J. L. M.
Published: December 2006

This study evaluated embryo transfers from Holstein donors in lactating Holstein recipients. It was performed at the Santa Rita Farm, Descalvado, SP, Brazil, between January 2005 and January 2006. Holstein recipients were milked 3 times a day and fed with total mixed ration. Embryos were obtained by a superstimulation protocol with two 3-mg norgestomet ear implants (Crestar�; Intervet, Sao Paulo, Brazil) and an IM injection of 3 mg of estradiol benzoate (EB) on random days of the estrous cycle. Four days later, 500 IU FSH (Pluset�; Laboratorios Calier de Argentina) was divided into 8 decreasing doses given IM 12 h apart. With the seventh FSH injection, 0.530 mg of sodium cloprostenol (PGF2�, Ciosin�; Coopers Brazil, Sao Paulo, Brazil) was given IM; implants were removed with the eighth FSH injection. Twelve hours later, 250 �g gonadorelin (Fertagyl�: Intervet, Brazil) was given IM, followed by two AI 12 and 24 h later. Ova/embryos were recovered 6.5 days after the first insemination. Recipients (n = 2543) received an injection of PGF2� and were observed in estrus (n = 1322). Presence of CL was evaluated by rectal palpation of cows that presented heat, and embryo transfers (n = 873) were performed with a nonsurgical method 6 to 8 days after heat. Pregnancy diagnoses were performed by ultrasonography (Aloka 500, probe 5.0 MHz; Aloka) on Days 25 and 39. Embryonic mortality was considered when any recipient pregnant on Day 25 was open on Day 39. Data were analyzed by logistic regression and variables included in the model were: donor status (lactating or non-lactating), recipient CL external size classification (1 > 2 > 3), donor–recipient synchrony (-1, 0, +1), fresh or frozen embryos, embryonic development (Mo, Bi, Bl, Bx – IETS), and embryo quality (degree 1, 2, 3 – IETS). The conception rate (CR) on Day 25 was influenced by donor–recipient synchrony (-1: 37.7%; 0: 46.9%; +1: 45.6%; P = 0.053). Embryonic development (Mo: 45.6%; Bi: 41.8%; Bl: 46.9%; Bx: 43.8%), embryo quality (1: 44.2%; 2: 47.4%; 3: 42.3%), CL external size classification (CL 1: 47.9%; CL 2: 43.5%; CL 3: 30.0%), embryo condition (fresh: 45.8%; frozen: 43.1%), and donor status (lactating: 45.5%; non-lactating: 47.08%) did not affect conception rate. The CR on Day 39 was also affected by donor–recipient synchrony (-1: 29.9%; 0: 34.9%; +1: 38.2%; P = 0.021). Embryonic mortality was affected by donor status (lactating, 16.6% vs. non-lactating, 24.0%; P < 0.10). These data suggest that donor–recipient synchrony is a major factor affecting conception rate in lactating Holstein recipients.