Cleavage Formation Research Articles

Autophagy regulates hypoxia‐induced osteoclastogenesis through the HIF‐1α/BNIP3 signaling pathway

Previous studies have implicated that hypoxic stress could enhance osteoclast differentiation; however, the underlying mechanism remains poorly understood. Autophagy is a dynamic lysosomal degradation process that has emerged as an important regulator under hypoxic environment. In the present study, we demonstrate for the first time that autophagy regulates hypoxia-induced osteoclastogenesis in vitro. We found that exposure of RAW264.7 cells to hypoxia (0.2% oxygen) resulted in enhanced osteoclast differentiation, accompanied by the observation of several specific features of autophagy, including appearance of membranous vacuoles, formation of acidic vesicular organelles, cleavage and recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosomes, increase in autophagic flux, as well as up-regulation of autophagy-related gene (Atg) expression. Moreover, suppression of autophagy with DN-Atg5(K130R) or 3-methyladenine (3-MA) significantly attenuated the osteoclast differentiation under hypoxic conditions, indicating the functional significance of autophagy in hypoxia-induced osteoclastogenesis. The data also showed that the activation of autophagy under hypoxic conditions was caused by up-regulated expression of hypoxia-inducible factor-1α (HIF-1α)-dependent Bcl-2 adenovirus E1a 19 kDa interacting protein 3 (BNIP3). Importantly, knockdown of HIF-1α or BNIP3 obviously abrogated hypoxia-induced autophagy activation and osteoclastogenesis enhancement. Collectively, our results highlight the fact that autophagy is a pivotal regulator for hypoxia-induced osteoclast differentiation, which may provide new insight into the pathological processes of osteoclastogenesis under hypoxic stress and help develop new therapeutic strategies for abnormal osteoclastogenesis.

The Kalahari Copperbelt in Central Namibia: Controls on Copper Mineralization

ABSTRACT The Kalahari Copperbelt stretches discontinuously for 800 km from central Namibia to northern Botswana (Figure 1; Borg, 1988; Borg and Maiden, 1989). In central Namibia, copper mineralization, hosted by slate and phyllite, is intermittently developed over more than 60 km of strike of the Kagas Member of the Klein Aub Formation. The strata-bound nature of copper occurrences led early explorers to the conclusion that copper was either syngenetic, i.e., emplaced during deposition of the host strata, or diagenetic, i.e., emplaced into the host strata during burial and compaction. As a result, initial exploration largely focused on “favorable” stratigraphic units as target horizons and for the most part ignored structurally controlled target areas of significant economic potential. Subsequent research at the (presently inactive) Klein Aub Mine and elsewhere in the Namibian part of the belt showed that, although copper concentrations are broadly strata-bound, the structural associations (e.g., the relation to a late reverse fault at Klein Aub) and detailed textural features (e.g., copper in veins, brittle fractures, cleavage-parallel lenticles, and tectonic breccia zones) indicate that copper mineralization was emplaced into structurally controlled sites late in the deformation history of the region. The conclusion of the present review is that economically viable copper accumulations resulted predominantly from one or more regionally extensive but locally structurally controlled hydrothermal events, mostly subsequent to formation of the dominant cleavage. As a result, modern exploration should focus primarily on favorable structures, particularly potential dilatant sites in tectonically complex zones.

Induced temperature-dependent DNA degradation by C60 without photoactivation

This study shows that C60 can degrade pBR322 plasmid DNA at room temperature without photoactivation. The degradation was enhanced by increasing incubation temperature, reaction time or C60 concentration. We also found that superoxide radical anions (O 2 −· ) were formed in the C60 solution. Superoxide dismutase significantly inhibited DNA cleavage and O 2 −· generation induced by C60. These results suggest that DNA cleavage was caused by the formation of reactive oxygen species induced by C60 at room temperature. Furthermore, we demonstrate that the DNA degradation was significantly inhibited by acid amide chemicals such as formamide, and by increased ionic strength of the reaction solution. These results indicate that the DNA conformation stability and the surface properties of C60 are important factors regulating DNA degradation. We propose that C60 can bind DNA, decrease DNA conformation stability, and induce the formation of reactive oxygen species and DNA cleavage at room temperature. Our results provide a possible explanation for the genotoxicity of C60, which should be considered in future use of this particular nano-material.

Formation of Political Cleavage in South Korea: Application of the Lipset-Rokkan Model

Formation of Political Cleavage in South Korea: Application of the Lipset-Rokkan Model

Construction of Ipr1 expression vector and development of cloned embryos in vitro

The purpose of this study was to prepare intracellular pathogen resistance 1 (Ipr1) transgenic donor cells for somatic cell nuclear transfer (SCNT). Based on our current understanding of Ipr1, a macrophage special expression vector pSP-EGFP-Ipr1was constructed. Bovine fetal fibroblasts were transfected with pSP-EGFP-Ipr1. The green fluorescent protein (GFP)-expressing cells were selected and transferred into enucleated bovine oocytes. Then, the rates of oocyte cleavage and blastocyst formation of transgenic cells and non-transgenic cells were observed, respectively. The results showed that reconstructed embryos derived from transgenic cells could successfully develop into blastocysts, most of which were GFP-positive. This study may provide cloned embryos for the production of anti-tuberculosis transgenic animals.

Stimulation of AMP-Activated Protein Kinase Promotes Maturation and Suppresses Spontaneous Activation of Mouse Oocytes.

Our lab has previously demonstrated that AMP-activated protein kinase (AMPK) in mouse oocytes promotes germinal vesicle breakdown (GVB), and meiotic progression to MII. Active AMPK was localized to the germinal vesicle prior to GVB, condensed chromosomes after GVB, spindle poles at metaphase I and II, and the spindle midzone during anaphase. This staining pattern of active AMPK was dependent on microtubule spindle integrity. On the other hand, inhibiting AMPK activity during spontaneous maturation induced spontaneous activation of oocytes. In this study, we further investigated the effects of AMPK activation on the maturation and activation of oocytes. We used the genetically predisposed parthenogenetic activation strains, LT/SvEiJ mice and its related hybrid strain, LT/SvEiJ x C57B/6J (LTBL) to test the effect of AMPK. Oocytes were cultured in either control MEM/BSA or medium supplemented with the AMPK activator, AICAR (0.2 mM), for 17h. There was no sign of PB formation in the control LT/SvEiJ group; however, 29.8% formed PBs in the presence of AICAR. The PB rates for both control and AICAR-treated groups in LTBL oocytes were intermediate between LT/SvEiJ and B6SJL strains. When oocytes from LT/SvEiJ mice were cultured for 30h in either control medium or medium containing AICAR, 22% of the control group oocytes were activated as shown by formation of pronuclei and cleavage of oocytes, and AICAR reduced this number to 1.9%. AICAR had a similar effect on LTBL oocytes,reducing activation from 15.7% to 4%. To determine the critical window of AMPK activity required to convey this suppressive effect on oocyte activation, LT/SvEiJ oocytes were first cultured in either MEM or medium containing AICAR for 17h, then transferred to either MEM or AICAR-supplemented medium for another 13h before of activation was assessed. Oocytes had control levels of activation when they were first cultured in MEM and then transferred to AICAR. In contrast, oocytes showed low levels of activation when they were first cultured in the presence of AICAR even if AICAR was absent during the subsequent 13h. Experiments using B6SJL oocytes showed a similar effect. When superovulated B6SJL oocytes were retrieved from oviducts 16, 20 and 24h post-hCG, 0.5 mM but not 0.2 mM AICAR produced a subtle and transient suppression of activation during 6h of culture. These data indicated an early requirement of AMPK for suppressing premature activation. Immunofluorescent staining of LT/SvEiJ oocytes after 17h of in-vitro culture indicated that oocytes stayed arrested at MI stage. Interestingly, we observed abnormal active AMPK localization in those oocytes. Instead of staining at the both spindle poles, the localization was shifted from the poles in some oocytes and some were missing spindle pole localization. This suggested that the improper functioning of AMPK might also contribute to the defect of LT/SvEiJ oocytes. (poster)

Segregation during cleavage in the mammalian embryo? A critical comparison of whole-mount/CLSM and section immunohistochemistry casts doubts on segregation of axis-relevant leptin domains in the rabbit

Segregation of certain cytoplasmic molecules during cleavage and blastocyst formation that was previously reported to occur in the human and the mouse (Antczak and Van Blerkom Mol Hum Reprod 3:1067-1086, 1997; Antczak and Van Blerkom Hum Reprod 14:429-447, 1999) has been reinvestigated in the rabbit model. Additional methodology was used and two approaches were compared: (1) whole-mount immunohistochemistry followed by confocal laser scanning microscopy (WM-IHC/CLSM) versus (2) IHC performed on histological sections of resin-embedded material (S-IHC). This study concentrates on leptin and cytoskeletal proteins (actin and cytokeratins). With S-IHC, leptin was localized predominantly on the surface of blastomeres which is facing the perivitelline space, and in the extracellular embryonic coats, without any polar asymmetry being detectable along (presumptive) embryonic axes. A polar distribution of leptin with a pattern that could be interpreted as predictive of the prospective embryonic-abembryonic axis was seen only with WM-IHC/CLSM, not with S-IHC, although the latter gave excellent resolution. With both techniques, no differences between blastomeres were detected with respect to actin and cytokeratin patterns, an increased expression of cytokeratin in trophoblast cells occurring no earlier than at blastocyst formation. Artifacts that can occur with the two methodological approaches are critically discussed, as is the possible significance of the findings for theories on the differentiation of trophoblast versus embryoblast and on axis formation in early mammalian development. It is concluded that these data call for cautioning when studying distribution patterns of diffusible molecules with WM-IHC/CLSM technology, whereas patterns obtained with S-IHC are more reliable. Specifically these data cast doubts on previous claims that leptin IHC would allow to monitor cytoplasmic domain segregation occurring during cleavage as an element of early embryonic pattern/axis formation.

Low-grade metamorphism around the down-dip limit of seismogenic subduction zones: Example from an ancient accretionary complex in the Shimanto Belt, Japan

Reactions involving clay minerals during low-grade metamorphism at the depth of an ancient accretionary complex in the Shimanto Belt, Kyushu, Japan, were studied by integrated transmission electron microscopy–energy dispersive X-ray spectroscopy and X-ray diffraction analyses of the bulk rock and clay fraction. The analyzed metasediment (the Kitagawa unit) contain an incipient sub-horizontal slaty cleavage. Illite crystallinity data and mica b dimensions indicate that the conditions of metamorphic deformation were anchizone–epizone grade and intermediate pressure. Cleavage formation was linked to two reactions involving clay minerals: (1) the recrystallization of 1 M-dominant matrix mica, inherited from the original sedimentary fabric, into thick, defect-free 2 M 1 packets along cleavage planes; and (2) the formation of chlorite from 7 Å berthierine. Balanced equations among the clay phases, based on compositional data and their relative abundance, suggest that the decomposition of matrix mica resulted in the formation of paragenetic mica and chlorite along the cleavage planes, without significant elemental outflux. Although a modal increase in phyllosilicates is not indicated by the data, the growth of chlorite and mica along cleavage planes may have a large influence on the rheological properties of a décollement and may be related to the occurrence of the seismic–aseismic transition at ~ 350 °C.

Apoptosis-inducing factor contributes to epithelial cell apoptosis induced by enteropathogenic Escherichia coli

The mechanisms by which enteropathogenic Escherichia coli (EPEC) causes intestinal epithelial cell apoptosis remain unclear. We tested the hypothesis that apoptosis-inducing factor (AIF) is involved in apoptosis induced by EPEC. Infection of intestinal epithelial cells in vitro with EPEC led to the mitochondrial and cytosolic accumulation of AIF. This effect was partially dependent on caspase activity. Knockdown of AIF with siRNA blocked cellular apoptosis in response to EPEC infection, as assessed by poly(ADP-ribose) polymerase cleavage and oligonucleosome formation. Taken together, these data suggest that caspase-dependent mobilization of AIF contributes to EPEC-induced epithelial cell apoptosis.

Lithology-dependent reliability of AMS analysis: A case study of the Eocene turbidities in the southern Pyrenees (Aragón, Spain)

Fine-grained sediments are generally better strain-markers in the outer part of orogenic belts and foreland basins because they are more prone to cleavage formation than coarse detrital rocks in diagenetic to very-low metamorphic environments. This also commonly applies to magnetic fabric analysis, thus favoring sampling in fine-grained rocks. In this article, this general assumption is tested sampling for anisotropy of magnetic susceptibility (AMS) analysis along different levels of Bouma sequences in turbidites from the Eocene South-Pyrenean turbiditic basin, above and below the cleavage front. Application of other techniques (LTAMS, AGRM and hard-APARM) indicates that the cleavage imprint in AMS fabrics is carried by paramagnetic particles, in this case, by clay minerals. The results obtained show that: (i) directional results of AMS are similar in the different levels of the Bouma sequence; (ii) the paramagnetic contribution to the susceptibility is independent from the lithology; and (iii) the equivalent paramagnetic contribution to susceptibility (detrital sedimentary particles) does not show differences between a to d (sandstones) and e-level (siltstones and shales).

213 THE EFFECTS OF SERICIN SUPPLEMENTATION IN IN VITRO CULTURE MEDIUM ON THE DEVELOPMENT AND CRYOSURVIVAL OF BOVINE IN VITRO-MATURED - IN VITRO-FERTILIZED EMBRYOS

The objective of this study was to investigate the feasibility of the silk protein sericin as an alternative protein supplement for bovine embryo cultures. The effects of sericin supplementation in in vitro culture (IVC) medium on the development and cryosurvival of bovine IVM-IVF embryos were investigated. Cumulus–oocyte complexes were collected from 2- to 8-mm follicles of ovaries obtained from a local abattoir. They were matured for 20 to 22 h in TCM-199 supplemented with 5% fetal bovine serum (FBS), 0.002 AU mL–1 of FSH, and 1 μg mL–1 of oestradiol-17β at 38.5°C under an atmosphere of 5% CO2 in air. After IVF (Day 0), presumptive zygotes were cultured in SOF medium (IVC medium) containing amino acids and supplemented with either 5% FBS (FBS group: n = 400) or sericin at 3 different concentrations (wt/vol; 0.05%: n = 493; 0.1%: n = 419; or 0.15%: n = 520; sericin groups) at 38.5°C under an atmosphere of 5% CO2, 5% O2, and 90% N2 for 5 days. They were then transferred into each IVC medium supplemented with 0.1 mM β-mercaptoethanol and cultured for an additional 4 days (9 days in total). Cleavage rates were recorded on Day 2 of IVC. The excellent expanded blastocysts harvested on Days 7 and 8 were used for freezing (FBS group: n = 51, 0.05% sericin group: n = 56, 0.1% sericin group: n = 44, 0.15% sericin group: n = 44). They were frozen in m-PBS supplemented with 1.5 M ethylene glycol, 0.1 M sucrose, 20% calf serum, and 4 mg mL–1 of BSA. After thawing, they were cultured in TCM-199 supplemented with 20% FBS and 0.1 mM β-mercaptoethanol under the same atmosphere used for IVC for 72 h. Rates of the embryos that reexpanded and developed to the hatching and hatched blastocyst stages were determined at, respectively, 24, 48, and 72 h after thawing. Rates of cleavage and blastocyst formation were expressed as mean ± SD and were analysed by ANOVA. The post-thaw survival rates of frozen embryos were analysed by Fisher’s exact test and chi-square test. Four replications were performed. Cleavage and blastocyst formation rates did not differ among the groups (FBS: 61.6 ± 15.1 and 22.1 ± 3.5%; 0.05% sericin: 71.6 ± 12.0 and 19.4 ± 6.7%; 0.1% sericin: 70.3 ± 4.7 and 18.4 ± 5.6%; 0.15% sericin: 66.9 ± 10.3 and 17.9 ± 6.9%, respectively). There were no significant differences in post-thaw survival rates after 24 h among the FBS (88.2%), 0.05% sericin (92.9%), 0.1% sericin (84.1%), and 0.15% sericin (93.2%) groups. However, post-thaw survival rates after 72 h in the 0.05% sericin (83.9%) and FBS (82.4%) groups were significantly higher than those in the 0.1% sericin (56.8%) and 0.15% sericin (61.4%) groups (P < 0.05). These results indicate the feasibility of sericin as an alternative protein supplement for bovine embryo culture. Additionally, in this study, 0.05% sericin was shown to be the best concentration for survival of the resultant embryos after freezing and thawing.

294 PRODUCTION AND CHARACTERIZATION OF PUTATIVE NUCLEAR TRANSFER EMBRYONIC STEM CELLS DERIVED FROM HANDMADE CLONED EMBRYOS USING EMBRYONIC STEM CELLS, LYMPHOCYTES, AND ADULT FIBROBLAST CELLS AS DONOR CELLS IN GOAT

The handmade cloning technique has been a relatively recent addition in the field of nuclear transfer. In the present study, attempts were made to efficiently derive stem cells from handmade cloned (HMC) embryos in goat using adult fibroblast cells, embryonic stem (ES) cells, and lymphocytes as donor cells, and to characterise the derived putative nuclear transfer ES (ntES) cells for their stemness. Efficiency of the donor cells for nuclear transfer was also compared, and an overall cleavage and morula formation rates of 62.44 ± 3.9% and 35.30 ± 3.86%, 75.45 ± 3.92% and 45.84 ± 3.86%, and 56.38 ± 3.92% and 29.09 ± 3.86% were obtained from adult fibroblasts, ES cells, and lymphocytes, respectively. A significant difference was found between ES cells and the other 2 donor cells in terms of cleavage and morula formation. However, no such difference existed between fibroblasts and lymphocyte donor cells. Stem cell colonies were successfully derived from HMC embryos obtained from all 3 different donor cells. The rate of primary colony formation was 61.66 ± 4.62% for fibroblast-donor-cell-derived embryos. This rate was 59.91 ± 4.62% for ES-donor-cell-derived embryos and 62.49 ± 4.62% for lymphocyte-donor-cell-derived embryos. The putative ntES colonies were positively characterised for TRA-1-60, TRA-1-81, SSEA-1, SSEA-4, OCT-4, SOX-2, and Nanog by immunocytochemistry and RT-PCR. Results indicated that ES cells had better efficiency as donor cells in cloned embryo production than did adult fibroblasts and lymphocytes. The finding also suggested that terminally differentiated cell-like lymphocytes can also be reprogrammed. Moreover, there was no difference between the different donor-cell-derived HMC embryos in terms of ntES cell derivation. The study has established an efficient protocol for putative ntES cell derivation from HMC embryos. This could be of substantial significance because patient-specific ntES cells have proven therapeutic significance. The authors acknowledge N.D.R.I for the financial and infrastructural assistance.

129 DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES AFTER IN VITRO MATURATION AND IN VITRO CULTURE UNDER COMPARATIVE OXYGEN TENSION

The in vitro developmental abilities of porcine oocytes are generally increasing steadily at a similar ratio to those of in vivo embryos. However, it has been suggested that the in vitro culture system for the development of porcine embryos is not optimal. In this study, we investigated the effect of 2 oxygen concentrations (5 and 20%) on porcine embryo development during in vitro maturation and in vitro culture and analyzed differences in gene expression of resulting blastocysts. Oocytes were recovered by aspiration of slaughterhouse ovaries and then matured in tissue culture medium (TCM) 199 supplemented with 10% porcine follicular fluid (pFF), epidermal growth factor (EGF), insulin, pyruvate, cystine, and gonadotropin. Matured oocytes were then activated parthenogenetically, cultured in PZM-3 media for 7 days. In vitro maturation (M group) of oocytes was carried out under two oxygen concentration (5 and 20%) in terms of nuclear maturation (polar body extrusion; Exp. 1). The developmental differences between 5% oxygen culture group and 20% oxygen culture group during in vitro culture (C group) of embryos after parthenogenetic activation was investigated in terms of first cleavage and blastocyst formation (Exp. 2). Relative mRNA abundance of multiple genes in blastocysts was analyzed for transcript abundance of genes related with metabolism (GLUT1, LDHA), oxidative response (MnSOD, GPX1), apoptosis (BAX, Bcl2), and developmental competence (CCNB1, IGF2R; Exp. 3). The results show there were no significant differences in maturation rate between 2 oxygen concentrations during in vitro maturation (83 v. 86%). It was thought that cumulus cells surrounding oocytes might have attenuated oxidative stress, but number of resulting blastocysts were (P < 0.05) increased in 5% IVC group when compared with 20% IVC group (18.67 v. 14.09%, respectively). Moreover, the M20C5 group (23.01%) had a beneficial effect on in vitro culture compared with M5C5 (14.32%), M5C20 (10.30%), and M20C20 (17.88%) groups. Total cell numbers were not significantly different among groups. According to mRNA abundance data of multiple genes, each group altered the expression of genes in various patterns. Therefore, it could be concluded that high oxygen tension during in vitro maturation and low oxygen tension during in vitro culture might alter the expression of multiple genes related to oocyte competence and improve (P < 0.05) embryo development, but not blastocyst quality. This study was supported by MKE (#2009-67-10033839, #2009-67-10033805), NRF (#M10625030005-508-10N25), BK21 for Veterinary Science, IPET (#109023-05-1-CG000), and Hanhwa L&C.

84 COMPARISON BETWEEN IN VIVO AND IN VITRO PRODUCED EMBRYOS WITH FORSKOLIN AFTER VITRIFICATION

The high concentration of lipids on embryos reduces their viability after cryopreservation. The use of drugs to modify their metabolism has been used to produce embryos with greater resistance to cryopreservation. The present experiment aimed to induce cytoplasmic lipolysis in in vitro produced (IVP) bovine embryos using forskolin (Sigma-Aldrich, St. Louis), which raises the levels of intracellular cAMP. Nelore cow cumulus–oocyte complexes (COC) were matured in TCM 199 and fertilized with frozen Nelore bull semen. Presumptive zygotes were cultured in SOFaa supplemented with BSA in the presence of 2.5% FCS. Embryos were kept in a humidified atmosphere with 5% CO2, 5% O2, and 90% N2 at 38.5°C. On Day 6, embryos were divided into 2 groups: 2.5% FCS (2.5% FCS without forskolin) and 2.5% FCS + F (2.5% FCS plus 10 μM forskolin). Embryo cleavage was recorded on Day 3 (IVF: D0), and blastocyst production on Day 7. Embryo viability was estimated by the index of total number of cells per embryos observed after the staining with propidium iodite and Hoechst 33342. In vitro produced embryos were compared with embryos obtained in vivo from Nelore cows. Embryos were vitrified using the protocol developed by Campos-Chillòn et al. (2006). After thawing, the re-expansion rate and cell number were again estimated (after 12 h). For statistical analysis, percentage cleaved and percentage blastocyst, percentage re-expansion, and total number of cells were transformed using the arcsine transformation (√y/100) and analysed using ANOVA followed by Tukey’s test. The level of significance adopted was 5%. No statistical differences were observed between IVP embryos concerning cleavage (2.5% FCS: 87.48 ± 9.52 and 2.5% FCS + F: 85.13 ± 7.57) and blastocyst formation rates (2.5% FCS: 46.8 ± 13.28 and 2.5% FCS + F: 46.31 ± 11.39). Also, no differences were detected in total number of cells per embryos (2.5% FCS: 162.4 ± 43.3; 2.5% FCS + F: 147.6 ± 35.3 and in vivo: 143.5 ± 11.5) when IVP and in vivo produced embryos were compared. After vitrification the re-expansion rate was similar between IVP and in vivo produced embryos (2.5% FCS: 75.07 ± 9.81; 2.5% FCS + F: 81.09 ± 10.90 and in vivo: 86.40 ± 18.62). But the total cell number of IVP embryos was significantly lower than the in vivo produced embryos [2.5% FCS: 42.6 ± 17.2a (P < 0.001); 2.5% FCS + F: 59.9 ± 46.2a (P < 0.05) and in vivo: 124.2 ± 12.9b]. If one side the viability cell between IVP and in vivo produced embryos is the same, after vitrification in vivo produced embryos showed greater resistance after the vitrification. The addition of forskolin did not interfere in embryo production or quality, but this drug was not efficient in increasing embryo tolerance after vitrification. FAPESP 07/53505-1.

123 AGGREGATION OF CLONED EQUINE EMBRYOS: IMPROVEMENT OF IN VITRO AND IN VIVO DEVELOPMENT

Development of cloned equine embryo is still inefficient. The aim of our study was to assess the aggregation of zona-free genetically identical cloned embryos as a strategy to improve in vitro and in vivo development. Oocyte collection, maturation, cloning, and activation procedures were performed as described by (Lagutina et al. 2007 Theriogenology 67, 90–98). After activation, reconstructed embryos (RE) were cultured in DMEM/F12 with 5% of FBS in the well of well system in 3 different groups: I, only one RE per well; II, two RE per well; and III, three RE per well. Cleavage and blastocyst formation (7 to 8 days) of all experimental groups was assessed. At day 8, some embryos of each group were either fixed to determine Oct-4 expression by immunocytochemistry or transferred transcervically to a synchronized mare. Pregnancies were assessed by ultrasound from 7 days after embryo transfer until day 45 to 50 of pregnancy every 7 to 10 days, and sizes of vesicles and embryos were measured. In advanced pregnant mares, combined thickness of the uterus and the placenta (CTUP) and serum progesterone levels were also determined. The remaining embryos obtained from each group were maintained in culture from day 7 until day 15. Blastocysts growth was determined every 24 h. In vitro development, on a per-well and RE basis, was compared using the chi-square test. Statistical differences were observed in cleavage among groups I and II (P = 0.0088) and groups I and III (P = 0.0004): (I: 91/111, 82%; II: 74/78, 95%; III: 62/62, 100%). Blastocyst rates differed between groups I and III (I: 10/111, 9%; III: 23/62, 37%); no difference was observed with group II (11/78, 14%). There was no difference on blastocyst rates based on the number of aggregated RE (I: 10/111, 9%; II: 11/156, 7%; III: 23/184, 12.5%). The highest pregnancy rate was obtained in group III (I: 1/3, 33%; II: 2/5, 40%; III: 3/4, 75%). Sizes of vesicles and embryos did not differ statistically in such groups. The CTUP and serum progesterone levels were considered normal (<1.2 cm; >8 ng mL–1, respectively) in ongoing pregnancies. We did not observe any differences in Oct-4 expression patterns among groups. Even though statistical differences were found, surprisingly all embryos grew in vitro until day 15 with good rates and the biggest embryo reached 4.25 mm. Embryo aggregation improved in vitro development of equine cloned embryos until day 7, and pregnancies rates were higher. The in vivo sizes of vesicles and embryos were normal for all groups, and in vitro development beyond day 7 showed the high viability of embryos. To conclude, aggregation of cloned equine embryo does not imply extra oocytes because there is no statistical difference in the number of blastocysts obtained per oocytes used to achieve RE. It is also a good strategy to improve in vitro embryo development without alterations on in vivo progress. This is the first report of pregnancies from aggregated equine cloned embryos, and the first healthy cloned foal from South America, confirmed by STR analysis, was born recently derived from group II. Stumpo, Ignacio, Paola Barboza, and Don Antonio staff.

61 EFFECT OF ROOM TEMPERATURE HOLDING PROCEDURE ON ABILITY OF OOCYTES TO MATURE AND DEVELOP IN VITRO AFTER EQUINE SOMATIC CELL NUCLEAR TRANSFER

In Korea, it takes time to transport the ovaries of mares to the laboratory because horses are slaughtered only on Jeju island. Also, initiation of in vitro maturation (IVM) may be a little more delayed because of the oocyte collection by scraping of the follicular wall. It was reported that holding procedure of equine oocytes before IVM did not affect the developmental competence after intracytoplasmic sperm injection (Choi et al. 2006 Theriogenology 66, 955–963). The aims of present study were 1) to investigate the meiotic competence of equine oocytes held before IVM according to the type of oocytes, and 2) to examine the in vitro development after somatic cell nuclear transfer (SCNT). Cumulus–oocyte complexes (COCs) were recovered by scraping and washing of the follicular wall with Dulbecco’s modified Eagle medium (D-MEM) supplemented with 0.05% PVA, and classified as compact (Cp) or expended (Ex) depending on the expansion of cumulus or granulosa cells. 2 types of IVM procedures were compared: 1) COCs were matured immediately in IVM medium (TCM-199 supplemented with 5 mU mL–1 FSH, 50 ng mL–1 EGF, and 10% FBS) at 38.5°C under 5% CO2 in air for 24 to 27 h, and then held in holding medium (40% TCM-199 with Earle’s salts, 40% TCM-199 with Hanks’ salts, and 20% FBS) at room temperature for 6 to 7 h (control); or 2) COCs were initially held in holding medium for 6 to 7 h, and then matured in IVM medium for 24 to 27 h (holding). For SCNT, matured oocytes (pooled) were enucleated and electrically fused with equine skin fibroblasts (2.25 kV cm–1, 20 μs, 2 pulses). Fused couplets were activated with 5 μM ionomycin for 4 min followed by 5 h culture in 2 mM 6-DMAP, and cultured in D-MEM supplemented with 10% FBS and 50 ng mL–1 EGF at 38.5°C under 5% CO2, 5% O2, and 90% N2 for 7 to 9 days. Cleavage and blastocyst formation were evaluated on Days 2 and 8, respectively. All analyses were performed using SAS (version 9.1; SAS Institute Inc., Cary, NC, USA). 4 replicates were conducted from May to June 2010. In Ex oocytes, the maturation rate of the holding group (71.4%; 10/16) was not different from that of the control (65.6%; 44/73), and the rate of degenerated oocytes (4.8%; 1/16) in the holding group was not different from that in the control (5.6%; 5/73). However, in Cp oocytes, the degeneration rate of the holding group (65.0%; 31/49) was higher (P < 0.001) than that of the control (28.4%; 23/83), and the maturation rate of the holding group (20.6%; 12/49) was slightly lower (P = 0.07) than that of the control (46.0%; 38/83). After SCNT, the cleavage rate of the holding group (66.7%; 8/9) was not different from that of the control (60.8%; 14/25), and the rates of blastocyst formation of the control and the holding group were 8.1% (2/25) and 16.7% (2/9), respectively. Although the holding procedure may influence to the degeneration of Cp oocytes, it is considered that the developmental competence of equine oocytes held before IVM is not affected after SCNT.

Transnational networks and kin states: the Turkish minority in Bulgaria, 1878–1940

This paper focuses on the development of national identity and the formation of political organizations in the Turkish minority in the early years of the formation of the Bulgarian state from 1878 to the 1940s. It studies transnational aspects of nationalism, which is usually considered territorially bounded, by studying the impact of connections between the Turkish minority and the Ottoman Empire and later with Turkey. In addition to studying inter-state relations and their reflection in the legal and political sphere, this paper studies the flow of ideas across borders, transnational networks among political activists, and the resulting cleavage formation. Findings show that transnational connections and actors played dual roles. The circulation of political activists, contributed to the formation of national organizations which played a crucial role in (re)formulating national identity. Transnational connections increased political activity in the name of the Turkish minority. Through these organizations the community was able to make collective demands from the Bulgarian state. However, transnational connections carried debates and divisions in the Ottoman Empire and Turkey to the Turkish minority in Bulgaria. By dividing the community, they decreased the capacity for collective action.

Ordovician deformations in the Pyrenees: new insights into the significance of pre-Variscan (‘sardic’) tectonics

AbstractTwo deformational events which developed prior to the Variscan structures can be characterized in the Palaeozoic rocks of the Pyrenees: a Middle (?) Ordovician folding event and a Late Ordovician fracture episode. The Middle (?) Ordovician folding event gives rise to NW–SE- to N–S-oriented, metric- to hectometric-sized folds, without cleavage formation or related metamorphism. These folds can account for the deformation and uplift of the pre-Upper Ordovician (Cambro-Ordovician) sequence and for the formation of the Upper Ordovician unconformity. Ordovician folds control the orientation of the Variscan main-folding-phase minor structures, fold axes and intersection lineation in the Cambro-Ordovician sediments. The Late Ordovician fracture episode gave rise to normal faults affecting the lower part of the Upper Ordovician series, the basal unconformity and the underlying Cambro-Ordovician metasediments. Displacement of some of these faults diminishes progressively upwards of the series and tapers off in the upper part of the Upper Ordovician rocks, indicating that the faults became inactive during Late Ordovician times before deposition of the Ashgillian metasediments. Normal faults can be linked to the Upper Ordovician volcanic activity, which has been extensively described in the Pyrenees. The aforementioned deformation episodes took place after the Early Ordovician magmatic event, which gave rise to a large volume of plutonic rocks in the Pyrenees as in other segments of the European Variscides. This Middle Ordovician contractional event separated two extensional events in the Pyrenees from Early Ordovician to Silurian times. This event prevents us from assuming the existence of a continuous extensional regime through Ordovician and Silurian times, and suggests a more complex evolution of this segment of the northern Gondwana margin during the Ordovician.

59 PRODUCTION OF HANDMADE CLONED GOAT EMBRYOS WITH TWO TYPES OF DONOR CELLS AND CULTURED IN THREE MEDIA

The study was carried out to see the developmental efficiency of handmade cloned goat embryos with 3 different media: RVCL (Research Vitro Cleave, Cook, Brisbane, Australia), EDM (Embryo Development Media) and modified SOF (mSOF) and 2 types of donor cells: fetal fibroblast and adult fibroblast. Oocytes were isolated from abattoir goat ovaries, matured in maturation medium, and incubated in 5% CO2 in air at 38.5°C for 24 h. Then, the oocytes were made cumulus free by treatment with hyaluronidase (0.5 mg mL-1) and zona free by pronase (2 mg mL-1). Protrusion cone formation in oocytes was found 95 to 100% in T20 (TCM-199 + 20% FBS). The zona-free oocytes were bisected with an ultra-sharp micro blade on the basis of visible protrusion cones on the surface of oocytes using T20 medium containing 2.5 μg mL-1 cytochalasin-B. Fetal and adult fibroblast cells were used from confluent monolayer at passage 5 after trypsinizing in 0.25% trypsin-EDTA. One somatic cell was attached with one enucleated demioocyte by phytohemagglutinin and further fused with another enucleated demioocyte through electric pulse with a combination of alternating current (4 V) and direct current (2.10 kV cm-1 for 5 μs with a single pulse) in fusion medium (0.3 M mannitol, 0.1 mM MgCl2, 0.05 mM CaCl2, and 3 mg mL-1 BSA). Then, triplets were chemically activated with 5 μM Ca ionophore for 5 min and 2 mM 6-DMAP for 4 h and cultured in the 3 media. Cleavage and morulae formation were observed at Day 7 from 183 triplets with fetal fibroblasts as donor cells in media RVCL (78.60 ± 2.23, 38.97 ± 2.1), mSOF (72.62 ± 1.89, 33.81 ± 1.9), and EDM (73.96 ± 1.66, 26.20 ± 2.04), respectively. Simultaneously, cleavage and morulae formation were observed at Day 7 from 203 triplets with adult fibroblasts as donor cells in media RVCL (73.97 ± 3.57, 33.14 ± 2.68), mSOF (76.22 ± 4.36, 26.15 ± 0.99), and EDM (65.97 ± 3.11, 20.78 ± 2.77), respectively. Among the 3 media, morulae formation was significantly higher in RVCL. Hence, in the subsequent experiment, RVCL medium was used exclusively in culture for 172 triplets. Cleavage and morulae formation at Day 7 was not significantly different (P < 0.05) in 2 types of donor cells; fetal fibroblasts (77.46 ± 3.65, 38.70 ± 2.66) and adult fibroblasts (75.74 ± 3.04, 33.77 ± 1.43), respectively. The data were analyzed using SYSTAT 7.0 (Systat, SPSS Inc., Chicago, IL, USA) after arcsine transformation, one-way ANOVA followed by Fisher’s LSD test. PCR analysis was performed with highly polymorphic 286-bp fragment of MHC-II DRB gene of cloned embryo and its donor cell. Similar bands were observed in both the cloned embryos and fibroblast cells in agar gel electrophoresis. In conclusion, development of handmade cloned embryos was higher in RVCL medium compared with the other two media tested, and efficiency of morulae formation was similar in both types of donor cells. Further study is required to optimize blastocyst production.

118 ARE LIPID CONTENTS AND APOPTOSIS DETERMINANTS OF IN VITRO-PRODUCED BOVINE EMBRYO SUSCEPTIBILITY TO VITRIFICATION?

Supplementation of fetal calf serum (FCS) during culture of bovine in vitro-produced embryos (IVPE) has been correlated with lipid accumulation, representing a limiting factor on embryo cryotolerance. In the present experiment we studied lipid content and apoptosis in Nelore IVPE cultured in different concentrations of FCS. The experimental design was a 4 × 2 × 2 factorial, in which embryos, exposed or not to the metabolic regulator, phenazine ethosulfate (PES), were cultured in 4 concentrations of FCS. In 16 replicates, 560 IVPE were vitrified after cultured in SOFaa supplemented with 0, 2.5, 5 or 10% FCS. On Day 2.5 of culture, one-half of the embryos in each group were treated with 0.3 μM of PES and then returned to standard culture conditions (5% O2, 5% CO2 and 90% N2 atmosphere at 38.5°C). All embryos were vitrified on Day 7 (Campos-Chillòn LF et al. 2006 Theriogenology 65, 1200-1214). After warming, embryos were placed into culture for 12 h under standard conditions. A sample of fresh embryos was stained with Sudan Black B for quantification of small (<2 μm), medium (2-6 μm), and large (>6 μm) cytoplasmic lipid droplets. Apoptosis was analyzed, before and after vitrification, using TUNEL. As a positive control (CV), in vivo-produced embryos were collected from a superovulated Nelore cow. Data were analyzed using ANOVA followed by Tukey’s test; and Pearson’s linear correlation (P < 0.05). The results indicate that FCS concentration and PES treatment had no influence (P > 0.05) on cleavage and blastocyst formation rates (mean of 85.7 ± 2 and 36.6 ± 5, respectively). The total number of cells in fresh embryos was also similar (P > 0.05) among groups (mean of 134.1 ± 28). Additional results are shown in Table 1. Elevated concentrations of FCS increased lipid accumulation (r = 0.9) and the percentage of apoptotic cells in fresh and vitrified embryos, reducing the re-expansion rate after warming. Moreover, the number of apoptotic cells observed in fresh embryos was strongly correlated with the apoptosis observed after vitrification (r = 0.95). Although PES was able to reduce lipid accumulation, it was not efficient at preventing apoptosis in vitrified embryos. In summary, the results of this experiment indicate that the concentration of FCS during culture did not influence cleavage and blastocyst formation in bovine IVPE. We conclude that the apoptosis rate in fresh embryos is a key factor affecting survival after vitrification. Table 1.Lipid droplets, re-expansion, apoptosis of in vivo and IVPE Acknowledgements: FAPESP (07/57766-4).