bluuebunny/crossref_metadata_2025 huggingface dataset:
300 DEVELOPMENT OF TETRACYCLINE-REGULATED GREEN FLUORESCENT PROTEIN EXPRESSING TRANSGENIC RATS BY LENTIVIRAL VECTOR
Authors: Agca C. , Chan A. , Agca Y.
Published: December 2008

Although various methodologies have been available for genetic modification in mouse, such genomic tools are very limited in rats. The aim of the present study was to take advantage of lentiviral gene delivery to develop effective inducible germline transgenic rats. A tetracycline inducible system was designed to control the expression of the green fluorescent protein (GFP) gene. The GFP gene was constructed downstream of tetracycline response element under the control of a mini cytomegalovirus promoter. Additionally, rtTA2s-Ms was constructed at the 3 prime terminus of GFP under the control of an ubiquitin promoter. The resulted construct was named pLV-Tet-GFP. The pLV-Tet-GFP lentiviral vectors were injected into perivitelline space of 115 Sprague-Dawley rat zygotes. Of those 115 zygotes injected, 93 zygotes were transferred into 4 surrogate mothers and 32 live pups were obtained. The presence of transgene in Fo generation was first detected by PCR and subsequently Southern blot analysis in 25 out of 32 pups (78%). A varying level of GFP expression was observed in the 14 Fo pups after feeding them with 2 g L–1 of tetracycline via drinking water for 4 days. Northern blot analysis was performed in the subsequent generations to determine spatial (brain, kidney, liver, and lung tissues) and temporal (day 0, day 4, day 11, and day 30) expression. The results showed expression intensity as being highest in kidney, brain, lung, and liver. There was little expression detected on day 11 and 30. This study suggests that lentiviral vectors can be used to deliver inducible system quite efficiently in rats.

The authors thank Dr. Carlos Lois for providing the lentiviral backbone, Dr. W. Hillen for providing the rtTA2s-Ms fragment, and GuoFu Fang for developing the inducible lentiviral vector.
42 ASSISTED ENUCLEATION IN GOLDEN HAMSTER OOCYTES TREATED WITH COLCHICINE AND DEMECOLCINE
Authors: Li Z. , Wang L. , Li D.
Published: December 2008

The golden hamster is an excellent experimental animal in many research fields. In an effort to establish experimental protocols necessary for cloning golden hamsters, optimized conditions for induced cytoplasmic protrusion and assisted enucleation of golden hamster oocytes with different concentrations of colchicine and demecolcine were examined in this study. The Golden hamsters (female, six weeks of age) were superovulated with eCG (30 IU, ip) followed by hCG (30 IU, ip) at intervals of 72 h. Hamsters were sacrificed at 13.5 h, 15 h and 18 h after hCG injection. The different ages of cumulus–oocyte complexes (COCs) were collected from oviducts and cumulus cells were removed with 0.1% hyaluronidase. (1) denuded oocytes of different ages were treated with 2.5 μg mL–1, 5 μg mL–1, and 10 μg mL–1 of colchicine for 4 h in M199TE, and they were examined each hour; (2) denuded oocytes of different ages were treated with 0.02 μg mL–1, 0.04 μg mL–1, 0.06 μg mL–1, 0.1 μg mL–1, 0.2 μg mL, 0.4 μg mL–1, and 0.6 μg mL–1 of demecolcine for 1 h in M199TE, and they were examined every 15 min; 3) according to the results of processes (1) and (2), cytoplasmic protrusions of oocytes treated with 10 μg mL–1 of colchicine (60 oocytes) or 0.4 μg mL–1 demecolcine (88 oocytes) for 1 h were removed with a micromanipulation pipette. Then the oocytes were examined by being stained with Hoechst 33342, and the percentage of assisted enucleation was compared with that of blind enucleation in which position of the chromosomes was indirectly determined by the location of the first polar body. The results showed that: (1) about 90% of oocytes at 13.5 h and 15 h post hCG injection were induced to form protrusions under the treatment of 10 μg mL–1 of colchicine for 1 h; (2) oocytes of 13.5 h post hCG were very sensive to demecolcine, even the treatment of 0.02 μg mL–1 for 1 h could also induce cytoplasmic protrusions (45.38% of 31 oocytes); (3) when treated with 0.4 μg mL–1 of demecolcine for 1 h, the cytoplasmic protrusion rate of oocytes of 13.5 h–18 h post-hCG could reach to 99–100%; (4) when the cytoplasmic protrusions induced by 10 μg mL–1 of colchicine or 0.4 μg mL–1 of demecolcine for 1 h were removed, assisted enucleation rates were over 80%, significantly higher (P < 0.05) than blind enucleation (31.53% of 60 oocytes). In conclusion, the results of this study demonstrate that both colchicine and demecolcine can induce the hamster oocytes to form cytoplasmic protrusions. The treatments of 10 μg mL–1 of colchicine or 0.4 μg mL–1 of demecolcine for 1 h were the best conditions to induce oocytes (13.5 h–18 h post hCG) to form protrusion. Also, the assisted enucleation rate with colchicine and demecolcine is much higher than that of blind enucleation. These results define conditions for chemical assisted enucleation and should facilitate the development of cloned golden hamsters as an animal model for human diseases.
43 COMPARISON OF THE EFFECTS OF PRE-TREATMENT WITH SODIUM CHLORIDE, SUCROSE AND TREHALOSE ON DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES
Authors: Lin L. , Kragh P. , Purup S. , Du Y. , Zhang X. , Yang H. , Bolund L. , Callesen H. , Vajta G.
Published: December 2008

Modified environmental stress was reported to improve the developmental competence and cryotolerance of porcine oocytes, such as high hydrostatic pressure (HHP; Du et al. 2008 Cloning Stem Cells, Epub ahead of print) and osmotic stress (Lin et al. 2008 Reprod. Biomed. Online, in press). HHP also improved the cryotolerance of bovine and murine blastocysts (Pribenszky et al. 2005a Reprod. Dom. Anim. 40, 338–344; Pribenszky et al. 2005b Anim. Reprod. Sci. 87, 143–150). In the present study we compared the effects of NaCl with that of concentrated solutions of two non-permeable osmotic agents, sucrose and trehalose on in vitro maturated oocytes. A total of 2050 slaughterhouse-derived porcine cumulus–oocyte complexes (COCs) were matured for 41–42 h, and then put into 800 μL T2 (HEPES-buffered TCM-199 [Earle’s salts] with 2% cattle serum) supplemented with additional NaCl, sucrose or trehalose with the same osmotic level (588 mOsmol) in 4-well dishes and incubated for 1 h at 38.5°C in air. COCs incubated in T2 under the same conditions without supplementation were used as controls. Subsequently COCs were incubated in IVM medium for 1 h at 38.5°C in 5% CO2 with maximum humidity. After this recovery period cumulus cells were removed with 1 mg mL–1 hyaluronidase and pipetting, and oocytes were used as recipients for somatic nuclear transfer with handmade cloning (HMC) method. Porcine fetal fibroblasts were used as nuclear donor cells. Embryo culture was performed in PZM-3 medium (Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) in 5% CO2, 5% O2 and 90% N2 and maximum humidity. Cleavage and blastocyst rates were checked on Day 1 and Day 6, respectively. Cell numbers were counted after fixation in glycerol containing 20 μg mL–1 Hoechst 33342 fluorochrome on Day 6. t-test was performed for statistical calculations with SPSS 11.0 program (SPSS, Chicago, IL, USA). Results are shown in Table 1. Osmotic stress with both permeable and non-permeable agents increased developmental competence of porcine IVM oocytes. NaCl seems to be more appropriate for the purpose, as the other two components resulted in decreased cell number in blastocysts after somatic cell nuclear transfer (SCNT). In conclusion, a simple NaCl pre-treatment of oocytes has improved the in vitro efficiency of porcine SCNT.

Table 1.Developmental competence of porcine HMC embryos derived from oocytes treated with different agents

The authors thank Ruth Kristensen, Anette Pedersen, Janne Adamsen and Klaus Villemoes for their help and excellent technical assistance.
44 ATTEMPTS TO USE SOMATIC CELLS ISOLATED FROM FROZEN BOVINE SEMEN FOR NUCLEAR TRANSFER
Authors: Liu J. , Long C. , Westhusin M. , Kraemer D.
Published: December 2008

Somatic cells in semen are a potential source of nuclei for nuclear transfer to produce genetically identical animals. This is especially important when an animal has died and the only viable genetic material available is frozen semen. In our previous studies, epithelial cells were cultured from fresh ovine and bovine ejaculates and blastocyst stage embryos were produced using these cells (Liu J et al. 2007 Biol. Reprod. special issue, 177; Liu J et al. 2008 Reprod. Fertil. Dev. 20, 102). However, growing cells from frozen semen can be difficult. We hypothesized that nuclei of the somatic cells in frozen semen can be used for nuclear transfer even though the cell membrane and cytoplasm are damaged during the semen freezing process. Electrical fusion or piezo assisted direct injection was applied to introduce nuclei of somatic cells isolated from frozen bovine semen (not cultured) into enucleated bovine oocytes. With electrical fusion, only 5 of the 64 (7.8%) recombined couplets fused, which is lower than our normal fusion rate of approximately 70%. Of the 5 fused embryos, one cleaved and developed to a 4-cell stage embryo. Staining with propidium iodide indicated that less than 10% of somatic cells isolated from frozen bovine semen were viable. These results suggest that it might not be practical to introduce nuclei of the cells in frozen semen into oocytes by fusion due to the high proportions of dead cells. Membranes of the cells in frozen semen were hard and difficult to break by piezo pulses or drawing in and out of the injection pipette. Therefore, whole cell intracytoplasmic injection was applied. After couplet recombination, activation was induced by applying two 0.3 kV cm–1, 55 μs direct-current pulses delivered by an Eppendorf Multiporator (Eppendorf, North America) in activation medium that was composed of 0.28 m Mannitol (Sigma-Aldrich, St. Louis, MO, USA), 0.1 mm CaCl2 (Sigma-Aldrich), and 0.1 mm MgSO4 (Sigma-Aldrich), followed by incubation in 10 μg mL–1 cycloheximide (Sigma-Aldrich) and 5 μg mL–1 cytochalasin B (Sigma-Aldrich) for 5 h in a humidified 5% CO2, 5% O2, and 90% N2 gas mixture at 38.5°C. Of the 299 recombined embryos, 82 (27.4%) either cleaved or were fragmented. Of the 82 embryos, 48 were examined and 42 (87.5%) were determined to be fragmented (contained intact donor cells). Low cleavage rates were observed in embryos produced by direct injection (4.5%), but no further embryonic development occurred. Cells cultured from fresh bovine semen were used as positive controls for whole cell intracytoplasmic injection. Of the 74 recombined embryos, 73 (98.6%) cleaved and 4 developed into blastocysts. These results highlight the difficulty of obtaining viable embryos by employing nuclear transfer and somatic cells obtained from frozen semen samples. Additional research is warranted given the potential value of this approach for recovering lost genetics.
51 DEVELOPMENT OF EQUINE CLONED EMBRYOS DERIVED FROM IN VITRO-MATURED OOCYTES RECEIVING ADULT DERMAL FIBROBLAST CELL NUCLEI
Authors: Skrzyszowska M. , Samiec M. , Mlodawska W. , Kochan J. , Okolski A. , van der Tuin E. , Smorag Z.
Published: December 2008

The purpose of our study was to determine the in vitro developmental competences of equine NT embryos reconstructed with adult dermal fibroblast cells. Frozen/thawed fibroblast cells, whose mitotic cycle had been synchronized at G1/G0 stages through a contact inhibition of their migration and proliferative activity under total confluency, were used as a source of nuclear donor cells in the somatic cell cloning procedure. In vitro-matured oocytes were used as recipient cells for fibroblast cell nuclei. The compact cumulus–oocyte complexes (cpCOCs) were collected from abattoir-derived mare ovaries and selected for in vitro maturation. The cpCOCs were cultured in TC-199 medium supplemented with 5 mU mL–1 follicle-stimulating hormone (FSH), 10% fetal bovine serum (FBS) and 75 μg mL–1 kanamycin monosulfate (kanamycin A) for 30 h at 38.2°C in a 100% water-saturated atmosphere of 5% CO2 and 95% air. Cumulus-denuded in vitro-matured oocytes were incubated in the maturation medium supplemented with 0.4 μg mL–1 demecolcine for 40 min. The treated oocytes were subsequently transferred into TC-199 medium containing 4 mg mL–1 BSA-V and 5 μg mL–1 cytochalasin B. Metaphase chromosomes, which had been allocated into the chemically-induced protrusion of the plasma membrane, were removed microsurgically. The chemically-assisted enucleation was accomplished by gently aspirating the ooplasmic cone, which contained the condensed chromosome mass, with the aid of a beveled micropipette. The single nuclear donor cells were inserted into perivitelline space of previously enucleated oocytes. Fibroblast cell-ooplast couplets were fused with two consecutive DC pulses of 2.4 kV cm–1 for 30 μs. After a 1.5-h delay, nuclear transfer-derived oocytes were chemically activated by exposure to 5 μm L–1 calcium ionomycin for 5 to 7 min, followed by their incubation in B2 medium with addition of 2 mm L–1 6-dimethylaminopurine (6-DMAP) for 4 h. Reconstructed embryos were in vitro cultured in B2 medium for 2 days. Afterwards, cleaved embryos were co-cultured with Vero cells in B2 medium supplemented with 10% FBS for 5 to 6 days up to morula/blastocyst stages. From among 88 in vitro cultured cpCOCs, 55 (62.5%) acquired meiotic nuclear and cytoplasmic maturity state after reaching the Metaphase II stage. A total of 55 enucleated oocytes underwent reconstruction and 44/55 (80.0%) were successfully fused with nuclear donor cells. Out of 44 cultured NT embryos, 21 (47.7%) were cleaved. The frequencies of cloned embryos that reached the morula and blastocyst stages were 6/44 (13.6%) and 3/44 (6.8%), respectively. In conclusion, the cell nuclei of in vitro cultured adult dermal fibroblast cells, which had undergone the contact inhibition, were able to direct the preimplantation development of equine cloned embryos to morula and blastocyst stages.

This work was supported by the Scientific Net of Animal Reproduction Biotechnology.
53 THE EFFECT OF ELECTRICAL PULSE ON DEVELOPMENT OF NT BOVINE EMBRYOS FROM CARTILAGE CELLS
Authors: Tas A. , Arat S. , Cetinkaya G. , Akkoc T. , Bagis H.
Published: December 2008

In some previous NT studies, it was reported that oocyte activation starts with electrical stimulation applied before chemical activation (Rzucidlo SJ et al. 2004 Reprod. Fertil. Dev. 16(1,2), 157; Arat S et al. 2006 Reprod. Fertil. Dev. 18(1,2), 119). Therefore; one of the several parameters affecting reprogramming of somatic cells is the fusion parameter and time. Electrical stimulation has also an effect on embryo quality (Milazzotto MP et al. 2008 Reprod. Dom. Anim. 43, 319–322). The objective of this study was to examine the effect of fusion parameter and time on blastocysts development rates on somatic cell nucleus transfer(SCNT). Bovine oocytes isolated from slaughterhouse ovaries were matured in TCM199 supplemented with 10% fetal bovine serum (FBS), sodiumpyruvate, penicillin/streptomycin, epidermal growth factor (EGF), bFSH, and bLH in a humidified atmosphere of 5% CO2 in air for 18 h. Single cells derived from cartilage tissue of Anatolian Black Cow were inserted into the perivitelline space of the enucleated oocytes. In the first experiment, NT couples were fused by 2.66 kV cm–1, 30 μs, 1 pulse (Group 1) in sorbitol fusion buffer (0.25 m sorbitol, 0.1 mm Calcium acetate, 0.5 mm Mg Acetate). After one hour of the first fusion nonfused NT couples were refused by 1.40 kV cm–1, 40 μs, 1 pulse (Group 2). After fusion, all fused NT couples in group 1 (1.5 h after the first fusion) and 2 (1.5 h after the second fusion) were activated using a combination of cytochalasin D (2.5 μg mL–1) and cycloheximide (CHX, 10 μg mL–1) for 1 h and CHX alone for 4 h. Following activation, reconstructed oocytes were cultured in Sage cleavage medium supplemented with 8 mg mL–1 BSA for 72 h and then developing embryos were cultured in Sage blastocyst media (Tang R et al. 2006 Human Reproduction 21(5), 1179–1183) supplemented with 4 mg mL–1 BSA + 5% FCS for an additional 4 days. Differences among groups were analyzed by one-way ANOVA after arcsin square transformation(P = 0.05). There was no significant difference on fusion rates observed between Group 1(49.4%) and Group 2(47.5%). However, the result showed that the blastocyst development was seriously decreased after second fusion(Group 1: 30.8% and group 2: 2.5%). This was considered as a negative effect of the electrical stimulation which was applied twice on NT couples. In the second experiment; development rates of embryos were compared from NT couples fused 24 or 28 hours after maturation. In this experiment, fusion was applied one time. NT couples fused approximately 24 and 28 h postmaturation were considered as early and late, respectively. The results showed that there was no significant differences between the two groups on the blastocysts development rates (31.6% early and 28.4% late). According to the these results, 4 h difference at oocyte ages was not an effective parameter on the blastocyst development rates of NT embryos.

This study was supported by a grant from TUBITAK TOVAG 104O360 and KAMAG, Turkey (106G005).
59 THE INFLUENCE OF DILUENT EGG YOLK PARTICLES ON THE ACCURACY OF FLOW CYTOMETRIC ANALYSES OF SPERM FUNCTIONAL CHARACTERISTICS
Authors: Alhaider A. K. , Watson P. F.
Published: December 2008

Flow cytometry is an important tool to assess semen quality and sperm function, especially after cryopreservation, but the presence of egg yolk particles in many semen diluents can distort the results. The aims of the present study were to investigate the effect of the presence of egg yolk in the semen diluent on the outcome of flow cytometric analysis of dog spermatozoa and to explore the use of Laser Dye Styryl-751 (LDS-751), a permeant DNA stain with an emission wavelength of 712 nm, to exclude egg yolk particles from the analysis of plasma membrane fluidity (Merocyanine 540 and Yo-Pro-1) and intracellular Ca2+ (Fluo-3 and PI). Flow cytometric analyses were carried out on a Coulter Epics XL flow cytometer (Beckman-Coulter Inc., Fullerton, CA). Data were analyzed using EXPO32TM ADC software (Beckman-Coulter). A paired t-test was used to test whether gating out egg yolk particles using LDS-751 affected the analysis. Method agreement analysis was undertaken to assess the agreement between the 2 staining protocols (i.e. with or without LDS-751). The proportion of spermatozoa with high plasma membrane fluidity was significantly (P < 0.05) greater after gating out egg yolk particles; the measurements of high membrane fluidity spermatozoa were on average 7.7 percentage points greater than without gating out egg yolk as shown by the method agreement analysis (SD = 9.35). Similarly, the proportion of spermatozoa with high intercellular Ca2+ was significantly (P < 0.01) greater after gating out egg yolk particles; the proportions of spermatozoa with high intracellular Ca2+ were 5.95 percentage points greater when egg yolk particles were gated out compared with measurements in the presence of egg yolk particles (SD = 5.43). The method agreement shows that both methods show close agreement with and without egg yolk particles (i.e. results mostly fall within the mean of differences between pairs of repeated measurements ±2 SD). Therefore, the 2 methods are measuring the same phenomena. However, the change in measurement values with exclusion of egg yolk particles is significant. Therefore, accuracy is increased; that is, closer to the true value. In conclusion, using MC540/Yo-Pro-1 and Fluo-3/PI staining alone in the presence of egg yolk particles resulted in underestimation of the proportions of high plasma membrane fluidity and high intracellular Ca2+ spermatozoa, respectively. LDS-751 could be used with other fluorescent stains that target organelles such as mitochondria and acrosomes with multicolor flow cytometric analysis to exclude egg yolk particles.
63 VITRIFICATION OF BOVINE IN VITRO-PRODUCED AND IN VIVO EMBRYOS
Authors: Frers L. , Hepburn J. , Mandriaza Munoz J. , Forsyth J. , Strongman K.
Published: December 2008

There is increasing interest in vitrification as a method of cryopreserving bovine embryos, especially previously problematic embryos such as IVP and Jersey cattle embryos, which, it has been suggested, have lower tolerance to cryopreservation because of the high lipid content of the embryo interfering with water movement out of the cells. Jersey embryos were demonstrated to have lower pregnancy rates following cryopreservation compared with Holstein embryos [Steel R et al. 2004 Reprod. Fertil. Dev. 16(Suppl. 2), 120 abst]. The first trial aimed to compare the subsequent pregnancy rate of vitrified and fresh in vitro-produced embryos. Embryos were produced by 9 Friesian cows through transvaginal recovery and in vitro production (IVP) over a 10-week period. The embryos produced during the first 6 weeks were all vitrified and warmed during the last 4-week period concurrent with the transfer of fresh embryos produced from the same donor cows at that time. Vitrification and warming were performed using a technique previously reported (Peachey B et al. 2005 Reprod. Fertil. Dev. 17, 199 abst) using the CVM method (Lindemans W et al. 2004 Reprod. Fertil. Dev. 16, 174 abst). All embryos were transferred by the same experienced technician into randomly assigned synchronized recipients of the same herd. All recipients were scanned for pregnancy 40 days after transfer. Data (Table 1) were compared by Fisher exact test and showedno significant difference (P = 0.761) between pregnancy rates of vitrified-warmed embryos and fresh embryos. This result demonstrates that vitrification is a valuable technique in cryopreservation of IVP embryos. In a second trial, pregnancy rates of in vivo-produced Jersey embryos after slow freezing and vitrification were compared. Embryos were flushed from 6 Jersey cows and randomly divided into 2 groups to be cryopreserved. The first group of 12 embryos (DT) were cryopreserved by slow freezing (0.3°C min–1 to –35°C) in 1.5 m ethylene glycol + 0.1 m sucrose. The second group of 12 embryos (VIT) were vitrified and warmed, using the same technique as described above. All embryos were transferred on the same day, by the same experienced technician, to randomly assigned recipient cows in the same herd. The pregnancy results for VIT were 7/12 (58%) and for DT were 4/12 (33%). The data were compared by logistic regression (Genstat v. 10, VSN International, Hemel Hempstead, UK) to account for donor effect, and no significant difference (P = 0.462) was found. It is proposed that the lack of significant difference may be due to the small numbers in the trial but that the results are promising enough to warrant further use and evaluation of this technique in the cryopreservation of Jersey cattle embryos.

Table 1.Pregnancy rates of fresh v. vitrified/warmed IVP embryos
EVALUATION OF DEVELOPMENT AFTER CRYOPRESERVATION OF IN VITRO-PRODUCED BOVINE EMBRYOS
Authors: Nicacio A. C. , Simões R. , Peres M. A. , Gonçalves J. S. A. , Paula-Lopes F. F. , Meirelles F. V. , Assumpção M. E. O. A. , Visintin J. A.
Published: December 2008

The inefficiency of embryo cryopreservation protocols limits the broad use of in vitro production (IVP) of bovine embryos. The aim of this work was to identify the damage caused by cryopreservation and embryo culture of IVP bovine embryos after thawing by in vitro development before and after cryopreservation. Cumulus–oocyte complexes were in vitro-matured, fertilized, and cocultured on granulosa cells in SOF with amino acids (SOFaa) supplemented with FCS. Expanded blastocysts (n = 600) harvested on Days 7 to 9 were submitted to controlled freezing [controlled group: 10% ethylene glycol (EG) for 10 min and 1.2°C min–1 cryopreservation], quick-freezing [quick group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s], or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. The embryos of the control group were not exposed to cryoprotectant or cryopreservation method, and the hatching rate was evaluated on Day 12 post-insemination. The straws (quick and vitrification groups) were first placed on nitrogen vapor (0.8 cm over the liquid nitrogen) for 2 min and then immersed in liquid nitrogen. Embryos were thawed in air for 10 s followed by a 25°C water bath for 20 s. Embryos were rehydrated in PBS + 0.2% BSA + 0.3 m sucrose and PBS + 0.2% BSA for 3 min each. To evaluate development of frozen–thawed embryos, they were cocultured on granulosa cells in TCM-199 or SOFaa both supplemented with FCS for 4 days. Hatching rate of the control group was 46.1%. Data were analyzed by PROC MIXED model of SAS System for Windows®. For TCM-199, the controlled group hatching rate was 44.65 ± 5.94%, quick group did not hatch, and vitrification group showed hatching rates of 9.4 ± 6.8%. For SOFaa, the controlled group hatching rate was 11.6 ± 3.4%, embryos submitted to the quick group did not hatch, and the vitrification group showed hatching rates of 8.7 ± 4.5%. Values were significant at P < 0.05. The controlled group showed a difference compared with the other groups of cryopreservation in both media (TCM-199 and SOFaa). However, TCM 199 showed high rates of re-expansion and hatching. In conclusion, the culture medium influences embryo development after cryopreservation, and TCM-199 is more appropriate than SOFaa.

Financial support by FAPESP (04/05335-1).
75 GLUCOSE CONCENTRATION OF FREEZING EXTENDER MODULATES THE TYROSINE PHOSPHORYLATION PATTERN OF FROZEN-THAWED BOAR SPERMATOZOA
Authors: Rodríguez-Gil J. E. , Hernández M. , Rivera M. M. , Ramió-Lluch L. , Ballester J. , Fernández-Novell J. M. , Vazquez J. M. , Martinez E. A. , Roca J.
Published: December 2008

The optimization of freezing extenders is an essential issue for enhancing boar sperm cryosurvival. The aim of the present study was to disclose the role of glucose concentration of freezing extender on the metabolic activity of frozen–thawed spermatozoa. To achieve it, pooled sperm-rich ejaculate fractions from 5 mature and fertile boars (3 ejaculates per boar) were collected using the gloved-hand method. After centrifugation (2400g for 3 min), the sperm pellet was split into 7 aliquots. The aliquots were diluted to a final concentration of 1 × 109 sperm mL–1, in a Tris-citric extender supplemented with 20% egg-yolk, 3% glycerol, and 0, 0.05, 2, 4, 10, 55, or 185 mm glucose. All the extenders were adjusted to a pH of 6.8 and 310 mOsm kg–1 to avoid osmolarity effects. Extended semen samples were dispensed into 0.5-mL straws, and frozen in a programmable cell freezer at 20°C min–1. Thawing was carried out in a water bath at 37°C for 20 s. Afterward, an analysis of protein phosphorylation in tyrosine residues was carried out through bi-dimensional electrophoresis followed by a Western blot analysis. This analysis indicated that sperm samples frozen in extenders without glucose showed specific changes in the tyrosine phosphorylation pattern compared with fresh sperm. Furthermore, the addition of glucose in increasing concentrations to the freezing extender was accompanied by a concentration-dependent decrease in the overall tyrosine phosphorylation pattern, especially in proteins with a molecular weight ranging from 150 to 200 kDa and an acidic isoelectric point (pI). The maximal decrease was observed in spermatozoa frozen in the extender containing 185 mm glucose, in which an additional decrease in the tyrosine phosphorylation of proteins ranging from 60 to 80 kDa, and a basic pI was also observed. These results suggest that glucose is a modulator in the resistance of boar sperm to support freezing and thawing process, because the precise protein phosphorylation pattern of spermatozoa is directly linked to their functional status. In this way, a precise control of the glucose concentration of the freezing extender would be required to improve boar sperm cryoresistance.

Supported by CICYT (AGL2005-00760 and AGL2004-04756-C02-02/GAN), Madrid and GERM (04543/07), Murcia, Spain.
84 DEVELOPMENTAL EFFECTS OF CHOLESTEROL-ENRICHED HYPERLIPIDIC DIETS IN RABBITS
Authors: Chavatte-Palmer P. , Laigre P. , Dahirel M. , Picone O. , Lamothe L.
Published: December 2008

The long-term effects of low birth weight on the onset of adult metabolic diseases have been described in several species. Low birth weight is usually due to maternal food restriction or placental insufficiency during pregnancy. In contrast, the effects of maternal high planes of nutrition or dietary unbalance have been less studied. In previous work, we have shown that the administration of a hypercholesterolemic (0.2%) hyperlipidic (8%) diet to rabbit does throughout puberty and subsequent pregnancy significantly affected fetal and postnatal development of their offspring. The aim of this study was to determine the relative role of cholesterol and of the n3/n6 fatty acid ratio on fetal development using the same model. Twenty-five does were fed from 10 weeks of age with 1 of 5 diets: S or SC (soy oil, 9% lipids, n3/n6 = 0.15 without or with 0.1% cholesterol), R or RC (rapeseed, 8% lipids, n3/n6 = 0.38 without or with 0.1% cholesterol), or Co (2% lipids, no cholesterol). Does were bred at 18 weeks of age. The growth of 4 fetuses per dam was monitored by weekly ultrasound scanning (Chavatte-Palmer P et al. 2008 Theriogenology). All pups were weighed at birth. Additionally, fetuses and placentas from a further 2 to 4 does per treatment group were collected at 15 days of pregnancy. Data were analyzed by ANOVA using group, number of fetuses, and dam as variables. Because different measurements were taken at different stages, the analysis of repeated measures could not be performed. Although maternal lipid intake was significantly increased in all treated groups, does adjusted their intake so that caloric intake and doe body weight were not statistically different between groups. Mean litter size was not different between groups. Group and, to a lesser extent, dam, but not litter size, significantly affected fetal development. Conceptus length was reduced on Day 9 of pregnancy in SC (–14%) and RC (–5%) compared with Co (P < 0.05). Fetal length was reduced on Day 15 in groups RC and S (both –9%, P < 0.05) but not in SC. Fetal abdominal perimeter measurements were smaller in SC, RC, and R at Day 22 and in SC at Day 28 (P < 0.05). Birth weight was decreased in all groups (–10, –18, –21, and –7% in SC, S, RC, and R, respectively compared with Co). Postmortem analyses did not show significant differences in fetal weight at Day 15, but placentas were significantly heavier in S compared with all other groups (P < 0.05). In conclusion, long-term cholesterol- and lipid-enriched diets induced intrauterine growth retardation in does with variable effects according to the n3/n6 fatty acid ratio and cholesterol dietary content, with more significant effects observed in the cholesterol-enriched diets. These effects are observed very shortly after implantation, indicating that oocyte and/or embryonic development may be affected. Further work is in progress to evaluate these early effects, using micro-array analysis of gene expression in the embryos.
90 ENDOGENOUS MODIFICATIONS OF AURORA B AND AURORA C KINASE EXPRESSION IN MOUSE OOCYTES AND EARLY EMBRYOS
Authors: Lima C. A. , Huntress V. , Overström E. W.
Published: December 2008

The Aurora (Aur) proteins are a family of serine/threonine kinases that play fundamental roles in controlling M-phase progression. Previous reports have shown that AurB is vital for proper completion of karyokinesis and cytokinesis in somatic cells. The role of AurC in somatic cells has been found to be much less significant, whereas it appears to play an important role in spermatogenesis. The role of these Aur proteins is not well characterized in mouse oocytes and early embryos. The objective of this study was to assess changes of AurB and AurC mRNA and protein expression in mouse oocytes and early embryos as development progresses through the activation of the zygotic genome. Oocytes and embryos were collected from the oviducts of hormone-stimulated CF-1 mice. After culturing for varying amounts of time, cumulus-denuded samples were either fixed for immunofluorescence microscopy studies or lysed for analysis of mRNA levels through the use of reverse transcription-PCR (RT-PCR). Samples were processed for immunofluorescence using markers of spindle morphology (tubulin) and AurB. Analysis of relative levels of AurB and AurC mRNA were assessed by RT-PCR methods. Marked differences were observed in the localization of AurB when unfertilized oocytes or prezygotic genome activation (ZGA) embryos were compared with post-ZGA samples. There was no evidence of AurB protein localized to the mitotic spindle or resultant midbody in oocyte and early embryo samples. Embryos fixed post-ZGA demonstrated AurB localization, as is conventionally found in somatic cells. The AurB protein was found co-localized with DNA in metaphase stage blastomeres and associated with the midbody in blastomeres near completion of cytokinesis. Relative levels of AurB mRNA were not found to be significantly different when pre- and post-ZGA samples were compared. A significant decrease in relative levels of AurC mRNA was observed in fertilized pre-ZGA samples when compared with unfertilized oocyte counterparts. These observations demonstrate significant differences in the status of AurB and AurC mRNA levels and protein localization in mouse oocytes and early embryos when compared with somatic cells. Given earlier reports showing the vital role of AurC in spermatogenesis, the elevated levels of AurC mRNA observed in prefertilization oocytes may be indicative of a similar role of AurC during oogenesis. Elucidating temporal and localization details of Aur expression is vital to gaining further understanding of cell-cycle regulation in oogenesis and early embryogenesis.
A mouse model for
EML4-ALK
-positive lung cancer
Authors: Soda Manabu , Takada Shuji , Takeuchi Kengo , Choi Young Lim , Enomoto Munehiro , Ueno Toshihide , Haruta Hidenori , Hamada Toru , Yamashita Yoshihiro , Ishikawa Yuichi , Sugiyama Yukihiko , Mano Hiroyuki
Published: December 2008

EML4-ALK is a fusion-type protein tyrosine kinase that is generated in human non-small-cell lung cancer (NSCLC) as a result of a recurrent chromosome inversion, inv (
2
)(p21p23). Although mouse 3T3 fibroblasts expressing human EML4-ALK form transformed foci in culture and s.c. tumors in nude mice, it has remained unclear whether this fusion protein plays an essential role in the carcinogenesis of NSCLC. To address this issue, we have now established transgenic mouse lines that express EML4-ALK specifically in lung alveolar epithelial cells. All of the transgenic mice examined developed hundreds of adenocarcinoma nodules in both lungs within a few weeks after birth, confirming the potent oncogenic activity of the fusion kinase. Although such tumors underwent progressive enlargement in control animals, oral administration of a small-molecule inhibitor of the kinase activity of ALK resulted in their rapid disappearance. Similarly, whereas i.v. injection of 3T3 cells expressing EML4-ALK induced lethal respiratory failure in recipient nude mice, administration of the ALK inhibitor effectively cleared the tumor burden and improved the survival of such animals. These data together reinforce the pivotal role of EML4-ALK in the pathogenesis of NSCLC in humans, and they provide experimental support for the treatment of this intractable cancer with ALK inhibitors.
Genome-wide expression profiling reveals distinct clusters of transcriptional regulation during bovine preimplantation development in vivo
Authors: Kues W. A. , Sudheer S. , Herrmann D. , Carnwath J. W. , Havlicek V. , Besenfelder U. , Lehrach H. , Adjaye J. , Niemann H.
Published: December 2008

Bovine embryos can be generated by in vitro fertilization or somatic nuclear transfer; however, these differ from their in vivo counterparts in many aspects and exhibit a higher proportion of developmental abnormalities. Here, we determined for the first time the transcriptomes of bovine metaphase II oocytes and all stages of preimplantation embryos developing in vivo up to the blastocyst using the Affymetrix GeneChip Bovine Genome Array which examines approximately 23,000 transcripts. The data show that bovine oocytes and embryos transcribed a significantly higher number of genes than somatic cells. Several hundred genes were transcribed well before the 8-cell stage, at which the major activation of the bovine genome expression occurs. Importantly, stage-specific expression patterns in 2-cell, 4-cell, and 8-cell stages, and in morulae and blastocysts, were detected, indicating dynamic changes in the embryonic transcriptome and in groups of transiently active genes. Pathway analysis revealed >120 biochemical pathways that are operative in early preimplantation bovine development. Significant differences were observed between the mRNA expression profiles of in vivo and in vitro matured oocytes, highlighting the need to include in vivo derived oocytes/embryos in studies evaluating assisted reproductive techniques. This study provides the first comprehensive analysis of gene expression and transcriptome dynamics of in vivo developing bovine embryos and will serve as a basis for improving assisted reproductive technology.
Cytokinin signaling regulates cambial development in poplar
Authors: Nieminen Kaisa , Immanen Juha , Laxell Marjukka , Kauppinen Leila , Tarkowski Petr , Dolezal Karel , Tähtiharju Sari , Elo Annakaisa , Decourteix Mélanie , Ljung Karin , Bhalerao Rishikesh , Keinonen Kaija , Albert Victor A. , Helariutta Ykä
Published: December 2008

Although a substantial proportion of plant biomass originates from the activity of vascular cambium, the molecular basis of radial plant growth is still largely unknown. To address whether cytokinins are required for cambial activity, we studied cytokinin signaling across the cambial zones of 2 tree species, poplar (
Populus trichocarpa
) and birch (
Betula pendula
). We observed an expression peak for genes encoding cytokinin receptors in the dividing cambial cells. We reduced cytokinin levels endogenously by engineering transgenic poplar trees (
P. tremula
×
tremuloides
) to express a cytokinin catabolic gene,
Arabidopsis CYTOKININ OXIDASE 2
, under the promoter of a birch
CYTOKININ RECEPTOR 1
gene. Transgenic trees showed reduced concentration of a biologically active cytokinin, correlating with impaired cytokinin responsiveness. In these trees, both apical and radial growth was compromised. However, radial growth was more affected, as illustrated by a thinner stem diameter than in WT at same height. To dissect radial from apical growth inhibition, we performed a reciprocal grafting experiment. WT scion outgrew the diameter of transgenic stock, implicating cytokinin activity as a direct determinant of radial growth. The reduced radial growth correlated with a reduced number of cambial cell layers. Moreover, expression of a cytokinin primary response gene was dramatically reduced in the thin-stemmed transgenic trees. Thus, a reduced level of cytokinin signaling is the primary basis for the impaired cambial growth observed. Together, our results show that cytokinins are major hormonal regulators required for cambial development.
How social status shapes race
Authors: Penner Andrew M. , Saperstein Aliya
Published: December 2008

We show that racial perceptions are fluid; how individuals perceive their own race and how they are perceived by others depends in part on their social position. Using longitudinal data from a representative sample of Americans, we find that individuals who are unemployed, incarcerated, or impoverished are more likely to be seen and identify as black and less likely to be seen and identify as white, regardless of how they were classified or identified previously. This is consistent with the view that race is not a fixed individual attribute, but rather a changeable marker of status.
Superfamily phenomena and motifs of networks induced from time series
Authors: Xu Xiaoke , Zhang Jie , Small Michael
Published: December 2008

We introduce a transformation from time series to complex networks and then study the relative frequency of different subgraphs within that network. The distribution of subgraphs can be used to distinguish between and to characterize different types of continuous dynamics: periodic, chaotic, and periodic with noise. Moreover, although the general types of dynamics generate networks belonging to the same superfamily of networks, specific dynamical systems generate characteristic dynamics. When applied to discrete (map-like) data this technique distinguishes chaotic maps, hyperchaotic maps, and noise data.
ERK-mediated regulation of leukotriene biosynthesis by androgens: A molecular basis for gender differences in inflammation and asthma
Authors: Pergola Carlo , Dodt Gabriele , Rossi Antonietta , Neunhoeffer Eva , Lawrenz Barbara , Northoff Hinnak , Samuelsson Bengt , Rådmark Olof , Sautebin Lidia , Werz Oliver
Published: December 2008

5-Lipoxygenase initiates the biosynthesis of leukotrienes, lipid mediators involved in normal host defense and in inflammatory and allergic disorders. Despite an obvious gender bias in leukotriene-related diseases (e.g., asthma), gender aspects have been neglected in studies on leukotrienes and 5-lipoxygenase. Here, we show that leukotriene formation in stimulated whole blood or neutrophils from males is substantially lower compared with females, accompanied by changed 5-lipoxygenase trafficking. This is due to gender-specific differential activation of extracellular signal-regulated kinases (ERKs). The differences are directly related to variant male/female testosterone plus 5α-dihydrotestosterone levels, and addition of 5α-dihydrotestosterone to female blood or neutrophils reduced the high (female) LT biosynthesis capacity to low (male) levels. In conclusion, regulation of ERKs and leukotriene formation by androgens constitutes a molecular basis for gender differences in the inflammatory response, and in inflammatory diseases such as asthma.
Efficient gene targeting in
Drosophila
by direct embryo injection with zinc-finger nucleases
Authors: Beumer Kelly J. , Trautman Jonathan K. , Bozas Ana , Liu Ji-Long , Rutter Jared , Gall Joseph G. , Carroll Dana
Published: December 2008

We report very high gene targeting frequencies in
Drosophila
by direct embryo injection of mRNAs encoding specific zinc-finger nucleases (ZFNs). Both local mutagenesis via nonhomologous end joining (NHEJ) and targeted gene replacement via homologous recombination (HR) have been achieved in up to 10% of all targets at a given locus. In embryos that are wild type for DNA repair, the products are dominated by NHEJ mutations. In recipients deficient in the NHEJ component, DNA ligase IV, the majority of products arise by HR with a coinjected donor DNA, with no loss of overall efficiency in target modification. We describe the application of the ZFN injection procedure to mutagenesis by NHEJ of 2 new genes in
Drosophila melanogaster
:
coil
and
pask
. Pairs of novel ZFNs designed for targets within those genes led to the production of null mutations at each locus. The injection procedure is much more rapid than earlier approaches and makes possible the generation and recovery of targeted gene alterations at essentially any locus within 2 fly generations.
Lung epithelial apoptosis in influenza virus pneumonia: the role of macrophage-expressed TNF-related apoptosis-inducing ligand
Authors: Herold Susanne , Steinmueller Mirko , von Wulffen Werner , Cakarova Lidija , Pinto Ruth , Pleschka Stephan , Mack Matthias , Kuziel William A. , Corazza Nadia , Brunner Thomas , Seeger Werner , Lohmeyer Juergen
Published: December 2008

Mononuclear phagocytes have been attributed a crucial role in the host defense toward influenza virus (IV), but their contribution to influenza-induced lung failure is incompletely understood. We demonstrate for the first time that lung-recruited “exudate” macrophages significantly contribute to alveolar epithelial cell (AEC) apoptosis by the release of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) in a murine model of influenza-induced pneumonia. Using CC-chemokine receptor 2–deficient (CCR2−/−) mice characterized by defective inflammatory macrophage recruitment, and blocking anti-CCR2 antibodies, we show that exudate macrophage accumulation in the lungs of influenza-infected mice is associated with pronounced AEC apoptosis and increased lung leakage and mortality. Among several proapoptotic mediators analyzed, TRAIL messenger RNA was found to be markedly up-regulated in alveolar exudate macrophages as compared with peripheral blood monocytes. Moreover, among the different alveolar-recruited leukocyte subsets, TRAIL protein was predominantly expressed on macrophages. Finally, abrogation of TRAIL signaling in exudate macrophages resulted in significantly reduced AEC apoptosis, attenuated lung leakage, and increased survival upon IV infection. Collectively, these findings demonstrate a key role for exudate macrophages in the induction of alveolar leakage and mortality in IV pneumonia. Epithelial cell apoptosis induced by TRAIL-expressing macrophages is identified as a major underlying mechanism.
Quasiconformally homogeneous planar domains
Authors: Bonfert-Taylor Petra , Taylor Edward
Published: December 2008

In this paper we explore the ambient quasiconformal homogeneity of planar domains and their boundaries. We show that the quasiconformal homogeneity of a domain

D
D

and its boundary

E
E

implies that the pair

(
D
,
E
)

(D,E)

is in fact quasiconformally bi-homogeneous. We also give a geometric and topological characterization of the quasiconformal homogeneity of

D
D

or

E
E

under the assumption that

E
E

is a Cantor set captured by a quasicircle. A collection of examples is provided to demonstrate that certain assumptions are the weakest possible.
Repression of Nascent Strand Elongation by Deregulated Cdt1 during DNA Replication inXenopusEgg Extracts
Authors: Tsuyama Takashi , Watanabe Saori , Aoki Ayako , Cho Yunje , Seki Masayuki , Enomoto Takemi , Tada Shusuke
Published: December 2008

Excess Cdt1 reportedly induces rereplication of chromatin in cultured cells and Xenopus egg extracts, suggesting that the regulation of Cdt1 activity by cell cycle-dependent proteolysis and expression of the Cdt1 inhibitor geminin is crucial for the inhibition of chromosomal overreplication between S phase and metaphase. We analyzed the consequences of excess Cdt1 for DNA replication and found that increased Cdt1 activity inhibited the elongation of nascent strands in Xenopus egg extracts. In Cdt1-supplemented extracts, overreplication was remarkably induced by the further addition of the Cdt1-binding domain of geminin (Gem79-130), which lacks licensing inhibitor activity. Further analyses indicated that fully active geminin, as well as Gem79-130, restored nascent strand elongation in Cdt1-supplemented extracts even after the Cdt1-induced stalling of replication fork elongation had been established. Our results demonstrate an unforeseen, negative role for Cdt1 in elongation and suggest that its function in the control of replication should be redefined. We propose a novel surveillance mechanism in which Cdt1 blocks nascent chain elongation after detecting illegitimate activation of the licensing system.