Cleavage Development Research Articles

U-Pb evidence for a 2.15 Ga orogenic event in the Archean Kaapvaal (South Africa) and Pilbara (Western Australia) cratons

Abstract There is geological evidence for widespread deformation in the Kaapvaal craton, South Africa, between 2.2 and 2.0 Ga. In Griqualand West, post-Ongeluk Formation (ca. 2.42 Ga) and pre-Mapedi Formation (>1.91 Ga) folding, faulting, and uplift have been linked to the development of a regional-scale unconformity, weathering horizons, and extensive Fe-oxide mineralization. However, the lack of deformational fabrics and the low metamorphic temperatures (<300 °C) have hampered efforts to date this event. Here we show that metamorphic monazite in Neoarchean shales from four stratigraphic intervals from the Griqualand West region grew at ca. 2.15 Ga, >400 m.y. after deposition. Combined with previous studies, our results show that sedimentary successions across the Kaapvaal craton deposited before ca. 2.26 Ga record evidence for crustal fluid flow at ca. 2.15 Ga, which is locally associated with thrust faulting, folding, and cleavage development. The style of the deformation is similar to that of the Ophthalmian orogeny in the Pilbara craton, Australia, which is interpreted to reflect the northeast-directed movement of a fold-thrust belt between 2.22 and 2.15 Ga. Our results suggest that the Kaapvaal and Pilbara cratons, which some paleogeographic reconstructions place together as the continent Vaalbara, experienced an episode of synchronous folding and thrusting at ca. 2.15 Ga. Deformation was followed by uplift and the development of unconformities that are associated with some of Earth’s oldest oxidative weathering and with the onset of Fe-oxide mineralization.

Timing of Cleavage Development in Relation to Folding and its Implications: An Example from the Deformed Mesoproterozoic Sedimentary Cover of Kaladgi Basin, Southwestern India

ABSTRACT Detailed analysis of cleavage-fold relationships in the Mesoproterozoic cover sediments of Kaladgi basin, south western India, revealed three types of timing relationships between cleavage and folds: (1) cleavage developed earlier than folds. (2) Cleavage developed later at some stage of folding and (3) non-development of cleavages in folds. In the first case cleavage developed earlier than the folding. Evidences that support such an interpretation include high cleavage-bedding angle throughout the fold, asymmetric fanning of cleavage in the folds with large fan angles and rotation of cleavage more in the steep and short limbs of asymmetric fold. In the second case, cleavage initiated later at some stage during folding which is evident from low cleavage fan angles, relatively low cleavage–bedding angles in the limbs of folds and the offset of bedding trace against cleavage trace due to pressure solution. Even though pressure solution is ubiquitous throughout the fold the effects of offset are more pronounced in the limbs compared to that in the hinge due to changing angular relationship of bedding and cleavage throughout the fold. In the third case, cleavage is absent in the folds resulting in its sporadic occurrences and reflects its diachronous nature. The final geometry of cleavage in relation to folds at any location is a combined effect of relative timing of cleavage development and mechanism of folding involving layer parallel shortening, flexural slip, tangential longitudinal strain, syn- to postfold flattening and hinge migration.

Growth and provenance of a Paleozoic subduction complex in the Broken River Province, Mossman Orogen: evidence from detrital zircon ages

A Paleozoic subduction complex dominates the Mossman Orogen developed at the northern extremity of the Tasmanides, eastern Australia. Its southern part, displayed in the Broken River Province, is characterised by dismembered ocean-plate stratigraphy in which turbidite-dominated packages and widespread tectonic mélange development are characteristic. The Broken River complex is characterised by formations with quartzose sandstone alternating with those largely formed of sandstone of more labile character. The two compositional groups are considered to reflect separate, age-significant sedimentary regimes, but their ages have hitherto been poorly constrained. With the use of 1082 concordant detrital zircon ages from 13 samples we provide age control for the complex and track its sedimentary provenance. Of quartzose units, the Tribute Hills Arenite and Pelican Range Formation are late Cambrian–Early Ordovician, and the Wairuna Formation is Middle to Late Ordovician, in age. The more labile units (Greenvale, Perry Creek and Kangaroo Hills formations) are collectively of late Silurian–mid-Devonian age. Development of the complex spanned some 130 Myr. Continent-derived sediment involved in accretion of much the complex, from mid-Ordovician to mid-Devonian, was largely sourced from a nearby magmatic arc of late Cambrian–Devonian age, now represented by granitoid plutons of the Macrossan and Pama igneous associations. An older far-field Pacific-Gondwana sediment source is characteristic of early-phase (late Cambrian–Early Ordovician) accretion, in common with sedimentary units of this age generally developed in the Tasmanides. We consider the complex to have grown largely by underplating that positioned younger components beneath those that are older, with out-of-sequence thrust interleaving of these components occurring late in the accretionary history. A Late Devonian contractional folding and cleavage development (Tabberabberan orogenesis) is uniformly expressed across the entire complex and reflects an abrupt change in plate engagement with imposition of a compressional stress regime.

Nuclear trafficking dynamics of Bromodomain-containing protein 7 (BRD7), a switch/sucrose non-fermentable (SWI/SNF) chromatin remodelling complex subunit, in porcine oocytes and cleavage-stage embryos.

In the work presented here, we investigated how bromodomain-containing protein 7 (BRD7), a subunit associated with switch/sucrose non-fermentable (SWI/SNF) chromatin remodelling complexes, is trafficked between cellular compartments during embryo development. SWI/SNF complexes are multi-subunit complexes that contain a core catalytic subunit (SWI/SNF related, Matrix associated, Actin dependent Regulator of Chromatin, subfamily A, member 4, or member 2; SMARCA4 or SMARCA2) and a collection of additional subunits that guide the complexes to their appropriate loci; BRD7 is one of these additional subunits. We hypothesised that BRD7 is exported from the nuclei of porcine oocytes and embryos in a Chromosome Region Maintenance 1 (CRM1)-dependent manner and imported into the nuclei using the karyopherin α/β1 heterodimer. Porcine oocytes and embryos were treated with inhibitors of CRM1-mediated nuclear export and karyopherin α/β1-mediated nuclear import to test this hypothesis. An RNA interference assay and a dominant negative overexpression assay were also performed to determine if karyopherin α7 serves a specific role in BRD7 trafficking. Our findings indicate that BRD7 shuttles between nuclear and cytoplasmic compartments during cleavage development. The shuttling of BRD7 indicates that it serves a unique role in remodelling chromatin during this developmental window.

131 Fatty acid binding protein inhibition as a strategy to reduce the lipid content of in vitro-produced bovine embryos

Lipid accumulation decreases cryopreservation survival of in vitro-produced embryos, reducing pregnancy rate after embryo transfer. Fatty acid binding protein 3 (FABP3) plays a role in lipid transport from cumulus cells to the oocyte during maturation. Blocking this transport could reduce lipid content in the oocyte and embryo and increase cryopreservation survival. This preliminary study aimed to test the effect of α-truxillic acid (FABP-I), a chemical molecule that inhibits FABP3/5 action by receptor competition, on lipid content of matured oocytes and blastocysts after culture. Slaughterhouse-derived cumulus-oocyte complexes were matured with 0 (control), 10, 50, 100 and 500nM FABP-I for 22h. In Experiment 1, 346 oocytes in 3 replicates were fixed following maturation and stained with 1μg mL−1 Nile Red to evaluate total lipid content; maturation was assessed by nuclear staining with 10μg mL−1 Hoechst 33342[ACD1]. In Experiment 2, 876 cumulus-oocyte complexes in 5 replicates were matured for 22h under the same concentrations of FABP-I, then fertilized for 18h and cultured for 7 days. Cleavage and blastocyst development were evaluated on Day 2 and 7, respectively. Blastocysts were fixed at Day 7 and stained with Nile Red. Fluorescence intensity was measured in arbitrary units using ImageJ (NIH), and data was analysed using GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA). In Experiment 1, maturation rate did not differ among treatments (70.2±8.7; P=0.7). There were significant effects of treatment, replicate and interaction of treatment by replicate on fluorescence intensity. Compared with control (23.6±0.6), intensity was lowest in oocytes matured with 500nM FABP-I (21.2±0.6; P<0.01) and highest in the 10nM group (26.5±0.6; P<0.01). Staining intensity tended to decrease in the 100nM group (22.1±0.6; P=0.09) and was not different in the 50nM group (24.0±0.7; P=0.6). In Experiment 2, cleavage rate (75.8±2.9; P=0.3) did not differ and blastocyst development tended to be different among treatments (P=0.06). Compared with the control (33.3±4.8), the 500nM group had lower development (17.0±4.8; P<0.03); 10 and 50nM groups had numerically lower (24.7 and 24.0±4.8) and the 100nM group had the highest development rate (37.3±4.8), although either was significant. Treatment tended to affect fluorescence intensity of blastocysts (P=0.07; n=209), and there were significant effects of replicate and interaction between replicate and treatment. Compared with the control (11.7±1.3), fluorescence intensity was lower in the 50nM group (6.8±1.3; P<0.01), whereas 10nM had a tendency for lower intensity (8.3±1.2; P=0.06). Groups 100 and 500nM were not significantly different from controls (9.4±0.9 and 10.7±1.5, respectively). In conclusion, addition of FABP-I up to 500nM did not affect maturation or embryo cleavage but altered blastocyst development. Exposure to 50nM reduced staining intensity in blastocysts without significant decrease in development, whereas 100nM resulted in numerically lower oocyte staining intensity and higher blastocyst development. Future experiments will evaluate cryopreservation survival of embryos treated with FABP-I, and embryo transfer.

36 The effects of E-64 on the developmental competence of porcine oocytes vitrified at the germinal vesicle stage

Previous studies reported the activation of the apoptotic cascade by vitrification in mature porcine oocytes (Vallorani et al. 2012 Anim. Reprod. Sci. 135, 68-74) and that the cathepsin B inhibitor E-64 improved developmental competence of bovine oocytes via an antiapoptotic effect (Balboula et al. 2013 Reproduction 146, 407-417). The present study was carried out to test whether E-64 affected the developmental competency of porcine oocytes vitrified at the germinal vesicle stage. Cumulus-enclosed porcine oocytes were vitrified in microdrops and warmed by our method (Somfai et al. 2015 J. Reprod. Dev. 61, 571-579). Then, the oocytes were subjected to in vitro maturation (IVM) for 46h in a chemically defined porcine oocyte medium supplemented with 10ng mL−1 of epidermal growth factor, 10IU mL−1 of eCG, and 10IU mL−1 of hCG and during the first 22h of IVM with 1mM dibutyryl cyclic adenosine monophosphate. Then, cumulus-oocyte complexes were fertilized in vitro and presumptive zygotes were cultured in 50-µL drops of porcine zygote medium-3 for 7 days in 6-well dishes covered by paraffin oil in an atmosphere of 5% CO2, 5% O2, and 90% N2 at 39°C. On Day 5 (Day 0=IVF), the porcine zygote medium-3 was supplemented with 10% (vol/vol) FCS. The effects of 1.0μM of E-64 supplementation during IVM of non-vitrified and vitrified cumulus-oocyte complexes were investigated in a 2×2 factorial design. Survival rates after IVM, cleavage rates on Day 2, blastocyst rates, and total cell numbers in blastocysts on Day 7 were compared among groups. The experiment was replicated 5 times. Results were analysed by ANOVA and Tukey’s multiple comparison test. The percentages of live oocytes were statistically similar when oocytes were matured in the absence or presence of E-64 both in non-vitrified (99.2% v. 99.6%, respectively) and vitrified (94.3% v. 90.8%, respectively) groups. Similarly, IVM without or with E-64 supplementation had no effect on subsequent cleavage and blastocyst development rates in non-vitrified (67.4% v. 71.2% and 38.7% v. 43.2%, respectively) and vitrified (46.8% v. 48.8% and 14.6% v. 22.8%, respectively) oocytes. Irrespective of E-64 treatment, all survival and developmental rates in the vitrified groups were significantly lower (P<0.05) compared with those of their non-vitrified counterparts except for the blastocyst development rate in the E-64-treated vitrified group, which did not differ significantly from those of the non-vitrified groups with or without E-64 treatment. There was no statistical difference in mean blastocyst cell numbers among the groups, ranging between 86.5±15.8 and 118±10.6. In conclusion, E-64 treatment had no effect on embryo production rates, which suggests that in our system, cathepsin-mediated apoptosis during IVM might not be the factor to limit embryo production using either fresh oocytes or those vitrified at the immature stage. This work was supported by JST/JICA SATREPS.

29 Development and survival of bovine vitrified sexed IVF-derived embryos in vitro matured with pituitary or human recombinant follicle-stimulating hormone

Improving in vitro production efficiency involves the development of effective oocyte in vitro maturation conditions. Although >80% of cumulus-oocyte complexes (COC) undergo nuclear maturation, only approximately 30 to 35% of immature bovine COC develop to the blastocyst stage. Also, animal-sourced FSH is typically used in IVF, so an effective alternative using recombinant DNA technology is desirable. The aim of this study was to compare the effect of porcine (p) and recombinant human (rh)FSH concentrations on in vitro performance and post-thaw survival. Ovaries were collected from an abattoir and oocytes were aspirated from 2- to 6-mm follicles. The COC with compact and complete cumulus cell layers were selected and matured in groups of 25 COC in 200µL of M199 medium supplemented with alanyl-glutamine (0.1mM), Na pyruvate (0.2mM), gentamicin (5µg mL−1), epidermal growth factor (50ng mL−1), pLH (5µg mL−1), cysteamine (0.1mM), and 10% FBS for 22 to 24h in humidified air and 5% CO2. Oocytes were divided into the following groups: 1× pFSH (2µg mL−1), 1× rhFSH (0.01 UI mL−1), 1× pFSH+1× rhFSH, 2× pFSH, and 2× rhFSH. After 22 to 24h, fertilization (Day 0) was carried out using female sexed-sorted semen selected with a mini single-continuous 80% layer (PureSperm, Nidacon International AB, Mölndal, Sweden) and diluted to 1×106 sperm mL−1. The SOF-FERT medium was supplemented with fructose (90µg mL−1), penicillamine (3µg mL−1), hypotaurine (11µg mL−1), and heparin (20µg mL−1). After 18h, presumptive zygotes were denuded and cultured under low oxygen tension in groups of 15 to 20 in 50-µL drops of SOF-BSA for 7 days. Also, 2% FBS was added post-fertilization on Day 3.5. Expanded blastocysts were selected based on IETS standards at Day 6.5 to 7 of culture. Only grade 1 expanded blastocysts were vitrified (Cryotop, Kitazato, Tokyo, Japan). Vitrification medium was 15% (vol/vol) ethylene glycol+propylene glycol. Vitrified embryos were thawed in a solution of H199+20% FBS and 0.25M sucrose at 39°C. Thawed embryos were cultured in SOF-BSA+10% FBS under cumulus/granulosa cell monolayer co-culture. Embryo assessment involved post-thaw survival (0h), re-expansion and development progress (24-48h), and hatching of the zona pellucida (72h). A minimum of 4 replicates were performed. Data were analysed using a generalized linear mixed model with logit-link binomial distribution. Media treatment differences were determined using Fisher’s least significant difference test with the Bonferroni correction (α-level=0.05). The FSH origin affected cleavage and embryo development rate but not cryotolerance (Table 1). The results support previous research on low dose versus high dose rhFSH effectiveness and interspecies interaction of FSH on follicular receptors. Table 1.Cleavage, embryo development rate, and cryotolerance of FSH of various origins

Late embryo cleavage as an indicator of chromosome aneuploidy by pre-implantation genetic screening

During in vitro fertilization (IVF), not all patients receive good quality oocytes or embryos. This study investigated the frequency of aneuploidy and mosaicism in embryos obtained from parthenogenetic oocytes and poor morphological embryos that also exhibited late cleavage development by preimplantation genetic screening (PGS). Twenty late cleavage embryos under IVF treatment were cultured until day 5, and one of the blastomeres was biopsied. The embryos were divided into three groups (parthenogenetic oocytes and poor-quality embryos with dark cytoplasm, several vacuoles or fragments in the cytoplasm; arrested embryos; and good-quality embryos). All samples were tested for abnormalities using array-comparative genomic hybridization (a-CGH). Twenty late cleavage-stage embryos were biopsied and analyzed for aneuploidy. A total 17 of 20 (85%) embryos were aneuploid and three (15%) were euploid. The incidence of aneuploidy in good-quality embryos was 75%, while poor-quality embryos displayed a higher frequency (86.7%) of aneuploidy. The parthenogenetic oocytes were invariably aneuploid (100%). Based on the embryo-grading system by Veeck, embryo grades 1 and 2 exhibited a lower incidence of aneuploidy (75%), while grades 3 and 4 showed 1.33-times higher aneuploidy rates (RR = 1.33; p < 0.001). The numerical defects of the chromosomes increased with the percent of fragmentation, with fragmented embryos showing 1.32-times greater aneuploidy rates than the non-fragmented ones. Embryos with uneven blastomeres showed 1.07-times higher aneuploidy rate when compared with embryos showing even blastomeres (p < 0.0064). Late embryo cleavage results in a higher incidence of aneuploidy and mosaicism upon comparison with early embryo cleavage.

New insights into asymmetric folding by means of the anisotropy of magnetic susceptibility, Variscan and Pyrenean folds (SW Pyrenees)

Magnetic fabric allows to unravel the petrofabrics of sedimentary rocks and to assess their deformational history. The use of this technique, in addition to classical structural field observations in the limbs of seven asymmetric folds in the Pyrenees, helps to determine the differences of internal deformation as well as the folding kinematics. Three folds developed during the Variscan Orogeny in Ordovician and Devonian rocks, and four folds developed during the Pyrenean Orogeny in Eocene rocks, are studied. Folds show a variety of structural locations, in different thrust sheets of the Southern Central Pyrenees, different cleavage development, age, geometry and lithology. Sampling follows an equivalent lithological layer in the two limbs, except for one case, of the selected folds. Results show a modified tectonic magnetic fabric in most sites with the magnetic lineation on the tectonic foliation plane. A larger scattering of the magnetic lineation (maximum magnetic anisotropy axis) and a higher intensity of the preferred orientation of minerals (eccentricity of the anisotropy of magnetic susceptibility - AMS ellipsoid) is better observed in the overturned (short) limb of the asymmetric Variscan folds than in the normal (long) limb. On the other hand, the shape parameter in Alpine folds is generally larger in the overturned (short) limb then in the normal (long) one. A good clustering of the minimum magnetic anisotropy axes is observed in all limbs. The combination of the AMS data with the structural data helps to understand and better constrain the deformation degree in these asymmetric folds and to unravel the deformational history.

Chromatin remodeling in mammalian embryos.

The mammalian embryo undergoes a dramatic amount of epigenetic remodeling during the first week of development. In this review, we discuss several epigenetic changes that happen over the course of cleavage development, focusing on covalent marks (e.g., histone methylation and acetylation) and non-covalent remodeling (chromatin remodeling via remodeling complexes; e.g., SWI/SNF-mediated chromatin remodeling). Comparisons are also drawn between remodeling events that occur in embryos from a variety of mammalian species.

Developmental biology and potential use of Alboglossiphonia lata (Annelida: Hirudinea) as an \u201cEvo-Devo\u201d model organism

BackgroundThe need for the adaptation of species of annelids as “Evo-Devo” model organisms of the superphylum Lophotrochozoa to refine the understanding of the phylogenetic relationships between bilaterian organisms, has promoted an increase in the studies dealing with embryonic development among related species such as leeches from the Glossiphoniidae family. The present study aims to describe the embryogenesis of Alboglossiphonia lata (Oka, 1910), a freshwater glossiphoniid leech, chiefly distributed in East Asia, and validate standard molecular biology techniques to support the use of this species as an additional model for “Evo-Devo” studies.ResultsA. lata undergoes direct development, and follows the highly conserved clitellate annelid mode of spiral cleavage development; the duration from the egg laying to the juvenile stage is ~7.5 days, and it is iteroparous, indicating that it feeds and deposits eggs again after the first round of brooding, as described in several other glossiphoniid leech species studied to date. The embryos hatch only after complete organ development and proboscis retraction, which has not yet been observed in other glossiphoniid genera. The phylogenetic position of A. lata within the Glossiphoniidae family has been confirmed using cytochrome c oxidase subunit 1 (CO1) sequencing. Lineage tracer injections confirmed the fates of the presumptive meso- and ectodermal precursors, and immunostaining showed the formation of the ventral nerve system during later stages of development. Further, the spatiotemporal expression of an EF-hand calcium-binding protein Calsensin ortholog was characterized, which showed a specific pattern in both the ventral and peripheral nervous systems during the later stages.ConclusionsOur description of the embryonic development of A. lata under laboratory conditions provides new data for further comparative studies with other leech and lophotrochozoa model organisms. Moreover, it offers a basis for the establishment of this species as a model for future “Evo-Devo” studies.

ARID1A, a component of SWI/SNF chromatin remodeling complexes, is required for porcine embryo development.

Mammalian embryos undergo dramatic epigenetic remodeling that can have a profound impact on both gene transcription and overall embryo developmental competence. Members of the SWI/SNF (Switch/Sucrose non-fermentable) family of chromatin-remodeling complexes reposition nucleosomes and alter transcription factor accessibility. These large, multi-protein complexes possess an SNF2-type ATPase (either SMARCA4 or SMARCA2) as their core catalytic subunit, and are directed to specific loci by associated subunits. Little is known about the identity of specific SWI/SNF complexes that serve regulatory roles during cleavage development. ARID1A, one of the SWI/SNF complex subunits, can affect histone methylation in somatic cells; here, we determined the developmental requirements of ARID1A in porcine oocytes and embryos. We found ARID1A transcript levels were significantly reduced in 4-cell porcine embryos as compared to germinal vesicle-stage oocytes, suggesting that ARID1A would be required for porcine cleavage-stage development. Indeed, injecting in vitro-matured and fertilized porcine oocytes with double-stranded interfering RNAs that target ARID1A, and evaluating their phenotype after seven days, revealed that the depletion of ARID1A results in significantly fewer cells than their respective control groups (p < 0.001).

A 3D Voronoi and subdivision model for calibration of rock properties

Rock cleavages consist of the cracks between blocks and the internal faults in blocks. The built-in modeling module in the Three-dimension Distinct Element Code cannot accurately describe the internal structure and the cleavage development characteristics of rock. This study combined 3D Voronoi block elements and scanning electron microscope images, to construct a numerical model equivalent to the rock meso structures. Three strategies are proposed to refine the initial model by block subdivision to achieve a function that allows cleavages in the model to cross through the blocks. A physical uniaxial compression test was performed in the laboratory to calibrate the mechanical parameters of the siltstone and the calibrated parameters then were validated by a Brazilian disc test. Sensitivity analysis was conducted to investigate the relationship between the meso-parameters and the model’s macro-response, and a calibration procedure was established for accurate replication of rock mechanical behavior. The statistics and analyses of failure characteristics for joints in the model showed that when the compressive and tensile strength values peaked, the percentages of tensile failures were approximately 37% and 10%, respectively. Comparing the cleavages after sample failure in lab tests with the cleavage development paths predicted by the models, the compressive and tensile characteristics were analyzed and the results confirmed the reliability of applying the 3D Voronoi block and subdivision modeling method in the numerical simulation study of the mechanical properties of the rock.

Strain histories from the eastern Central Range of Taiwan: A record of advection through a collisional orogen

In the eastern Central Range of Taiwan there is a regional variation in the orientation of maximum finite stretch across the slate belt, with down-dip maximum stretch found in the western Central Range and along-strike maximum stretch in the eastern Central Range. Incremental strain histories from syntectonic fibers in pyrite pressure shadows indicate a progressive change in extension direction from down dip to along strike during deformation, there is a corresponding temporal variation in stretching direction shown in samples from the eastern edge of the Central Range, a pattern that mimics the regional west-to-east spatial variation. These observed temporal and spatial strain distributions are used to evaluate the kinematics associated with slaty cleavage development during advection through the Taiwan orogenic system. The subduction zone beneath the island of Taiwan is influenced by two types of obliquity that have the potential to generate the observed along-strike stretching. First, the plate motion vector of the Philippine Sea plate relative to the Eurasian plate is slightly oblique to the regional strike of the mountain range, which could result in partitioning of strike slip shearing into the interior of the collision. Second, the north-south Luzon volcanic arc on the Philippine Sea Plate is obliquely oriented relative to the northeast-southwest edge of the Eurasian continental margin, which could result in lateral extrusion of the ductile core of the range. Incremental strain histories in cleavage-parallel samples represent a time-for-space equivalence where the stretching direction is fixed relative to the position within the mountain belt architecture (e.g., the topographic divide), and temporal variations in the eastern central Range reflect lateral advection through the strain field in response to accretionary and erosional fluxes. Incremental strain histories in cleavage perpendicular samples show both clockwise and counter-clockwise rotation of strain, indicating the possibility of bulk coaxial strain and lateral extrusion.

138 THE RELATIONSHIP BETWEEN THE NORMALITY OF FIRST CLEAVAGE, THE GENE EXPRESSION IN BLASTOMERES, AND THE ABILITY TO DEVELOP TO THE BLASTOCYST STAGE IN IVF-DERIVED BOVINE 2-CELL STAGE EMBRYOS

Early first and second cleaved embryos after IVF associated with even blastomeres without fragments or protrusions were found to be a potent criterion for the selection of embryos with high developmental competence (Sugimura et al. 2012 PLOS ONE 7, e36627). The aim of this study was to examine the relationship between an early normal first cleavage pattern, the transcript abundance, and their development to the blastocyst stage in each blastomere in 2-cell stage bovine embryos. The IVF-derived bovine embryos were cultured individually in well-of-the-well culture dishes in CR1aa medium supplemented with 5% calf serum and 0.25 mg mL−1 linoleic acid albumin at 38.5°C in 5% CO2, 5% O2, and 90% N2. The first embryonic cleavage was categorized as being either normal (occurring within 28 h after IVF with 2 even blastomeres without fragment or protrusion) or abnormal (2 uneven blastomeres, with/without fragment/protrusion and/or later than 28 h after IVF). Then, cleaved embryos were placed in 0.5% actinase-E in Ca- and Mg-free PBS and blastomeres were separated by pipetting (n = 85; 4 replicates). In each embryo, one blastomere was subjected to quantitative RT-PCR to analyse the expression of developmentally important genes. The remaining blastomere was subsequently cultured in an individually identifiable manner to verify their ability to develop to the blastocyst stage. Primers were designed for 12 target genes related to pluripotency, cell cycle, metabolism, pregnancy reorganization, placentation, and fetal growth (OCT4, ATP1A1, CCNB1, CDH1, COX1, CTNNB1, GLUT8, MNSOD-3, SOX2, DYNLL1, IGFBP3, and PMSB1) and a reference gene (PPIA). Transcript abundance of target genes in individual blastomeres was compared between embryos showing normal and abnormal cleavage. Values were normalized to the average values of the reference genes and all the means were compared by the Student t-test. Blastomeres resulted from normal cleavage developed to the blastocyst stage on Day 7 to 8 (Day 0 = IVF) at significantly higher rates than those resulted from abnormal cleavage (65.7% v. 37.5%, respectively, P &lt; 0.05). Transcript abundance of OCT4 was significantly higher in blastomeres associated with all abnormal cleavage than in those associated with normal cleavage (P &lt; 0.05). The expression of CCNB1, COX1, ATP1A1, GLUT8, and PMSB1 in blastomeres associated with normal cleavage and blastocyst development was higher than that in those of abnormal cleavage (P &lt; 0.05). However, the level of OCT4, CCNB1, COX1, ATP1A1, and PMSB1 was lower in blastomeres associated with normal cleavage but failure of blastocyst development than those in blastomeres showing abnormal cleavage (P &lt; 0.05). Our results reveal that significantly higher expression of CCNB1, COX1, ATP1A1, and PMSB1 in blastomeres at the 2-cell stage in bovine embryos with superior developmental competence compared with those showing abnormal cleavage and low competence. Research was supported by JSPS KAKENHI (26450388).

156 OVIDUCTAL FLUID SUPPLEMENTATION DURING MATURATION AND FERTILIZATION IMPROVES IN VITRO EMBRYONIC DEVELOPMENT IN PIGS

Oviducal fluid has a major role in the maturation of gametes and the process of fertilization. The objective of this study was to determine the effects of oviducal fluid supplementation in vitro, during oocyte maturation and IVF on fertilization characteristics and early embryonic development rates. Oocytes from aspired aspirated mature follicles (3–6 mm diameter) were obtained from a local abattoir. During the last 24 h of maturation, oocytes (n = 1303) were placed into maturation media supplemented either 1% (vol/vol) or 5% (vol/vol) thawed snap-frozen oviducal fluid. Fertilization was performed using pooled frozen-thawed semen from 3 different boars. During IVF, the fertilization medium was supplemented with 1% (vol/vol) or 5% (vol/vol) oviducal fluid. Fertilization characteristics were evaluated 12 h after IVF and rates of embryonic cleavage and blastocyst development were observed at 48 and 144 h after IVF, respectively. Data were analysed using ANOVA with the main effects including treatment, well, and replicate. Chi-square analysis was used to determine percentages of embryos reaching the different developmental stages for each treatment. There were no significant differences in the percentages of oocytes that reached metaphase II by the end of maturation or in sperm penetration rates after IVF. However, oocytes treated with 1% (vol/vol) oviducal fluid during the end of maturation and IVF (33.33 ± 2.61) and 5% (vol/vol) oviducal fluid during maturation (33.33 ± 2.66) or IVF (39.53 ± 3.78) had significantly less (P &lt; 0.05) incidence of polyspermic penetrations and a significantly higher (P &lt; 0.05) incidence of male pronuclear formation (87.50 ± 4.01; 86.67 ± 4.83; 86.05 ± 3.19, respectively) compared with no oviducal fluid supplementation. Oocytes supplemented with 5% (vol/vol) oviducal fluid during maturation and IVF had significantly lower (P &lt; 0.05) incidences of polyspermic penetration (27.91 ± 2.50) and significantly higher (P &lt; 0.05) percentages of embryos reaching the 2-cell stage (81.76 ± 3.72) and blastocyst stage of development (37.74 ± 1.09) by 48 and 144 h, respectively, compared with all other groups. The results of this study suggest that supplementing 5% (vol/vol) oviducal fluid during maturation and IVF improves the success rates of in vitro embryo development in pigs.

182 MATURATION OF BOVINE CUMULUS-OOCYTE COMPLEXES WITH FOLLICLE FLUID VARYING IN ESTRADIOL CONTENT AFFECTS CUMULUS CELL EXPANSION WITHOUT AFFECTING SUBSEQUENT EMBRYO DEVELOPMENT IN VITRO

The objective of this work was to determine how characteristics of bovine follicle fluid (FF; especially oestradiol content) affect cumulus cell expansion and oocyte competence. In the first study, FF was collected from abattoir-derived ovaries and pooled separately for large follicles (≥10 mm) and small follicles (≤3 mm). A portion of the FF from each category was charcoal stripped. These 4 types of FF were then used as the primary ingredient (75% vol/vol) in oocyte maturation media. A separate control group lacking FF but containing BSA was included to monitor potential impacts of protein on outcomes (control; without FF). Some of the cumulus-oocyte complexes (COC; n = 250) were matured in individual drops for analysis of cumulus expansion (photographed and measured at 0 and 21 h of maturation). Other COC (n = 770) were matured in groups of 12 to 25 in the previously described media, and then subjected to IVF procedures. Cleavage rates were recorded on Day 3, and blastocyst rates were recorded on Day 8 post-fertilization. Cumulus cell expansion was greatest when COC were matured in medium containing FF from large follicles, wherein it even exceeded the controls (P &lt; 0.02). Maturation in FF from small follicles resulted in cumulus expansion that was intermediate between large and control. Maturation in charcoal-stripped FF severely restricted cumulus cell expansion (P &lt; 0.05) compared with those matured in untreated FF. Despite the observed improvement in cumulus cell expansion, COC that had been matured in media containing FF were less likely to cleave (P &lt; 0.05) and also less likely to develop to the blastocyst stage (P &lt; 0.01) than those matured in control medium. Cleavage and blastocyst rates did not differ among any of the maturation media containing FF. In the second study, oestrous cycles of 9 crossbred cows were synchronized and FF samples were collected 36 to 42 h after prostaglandin F2α injection. Samples from individual cows were categorized as having high oestradiol (&gt;800,000 pg mL−1; H) or low oestradiol concentrations (&lt;800,000 pg mL−1; L). The FF was retained for use in in vitro experiments, where it was added to maturation media (20% vol/vol). Cumulus-oocyte complexes (n = 1,775) were randomly distributed into treatments across 12 in vitro maturation/fertilization replicates (H and L, balanced within replicate; 4 replicates/cow). Each replicate included the following 3 control groups: maturation medium containing BSA without FF, maturation medium without BSA with abattoir-derived FF, and maturation medium without BSA and without FF. The COC were matured in their assigned medium for 21 h, and then all COC were subjected to IVF procedures. Cleavage rates were recorded on Day 3, and blastocyst rates were recorded on Day 7 and 8 post-fertilization. Oestradiol content of the FF (H v. L) did not affect oocyte cleavage nor blastocyst rates on Day 7 or 8. The results of these studies indicate that although FF improves cumulus cell expansion during maturation in vitro, it does not result in higher rates of cleavage or blastocyst development regardless of oestradiol content.

Dating slate belts using 40Ar/39Ar geochronology and zircon ages from crosscutting plutons: A case study from east-central Maine, USA

We report the ages of cleavage development in a normally intractable lower greenschist facies slate belt, the Central Maine-Aroostook-Matapedia belt in east-central Maine. We have attacked this problem by identifying the minimum ages of muscovite in a regional Acadian cleavage (S1) and in a local ductile fault zone cleavage (S2) using 40Ar/39Ar geochronology and the ages of crosscutting plutons. Our success stems from the regional low-grade metamorphism of the rocks in which each crystallization event preserves a40Ar/39Ar crystallization age and not a cooling age. Evidence for recrystallization via a pressure solution mechanism comes from truncations of detrital, authigenic, and in some rocks S1 muscovite and chlorite grains by new cleavage-forming muscovite and chlorite grains. Low-blank furnace age spectra from meta-arkosic and slaty rocks climb from moderate temperature Devonian age-steps dominated by cleavage-forming muscovite to Ordovician age-steps dominated by a detrital muscovite component. S1- and S2-cleaved rocks were hornfelsed by granitoids of ∼407 and 377 Ma, respectively. The combination of these minimum ages with the maximum metamorphic crystallization ages establishes narrow constraints on the timing of these two cleavage-forming events, ∼410 Ma (S1) and ∼380 Ma (S2). These two events coincide in time with a change in the plate convergence kinematics from the arrival of the Avalon terrane (Acadian orogeny), to a right-lateral transpression arrival of the Meguma terrane in the Neoacadian orogeny.

History of faulting in the Northern Hastings Block, southern New England Orogen

ABSTRACTBrittle failure is common in the Devonian to Permian rocks in the Northern Hastings Block (NHB) and is manifested by faults of different orientation and kinematic histories, but the timing of fault movement is not well defined. In this study, faults in the NHB were analysed with the map pattern of cross-cutting faults used to estimate the relative time of movement and relationship to other faults. We defined five episodes of faulting or fault reactivation that affected the NHB. The Yarras Fault System on the southwestern side of the NHB and the Parrabel Fault and related faults on the eastern side of the NHB are the two major fault systems responsible for transporting and rotating the NHB in the late Carboniferous. Faults on the eastern, northeastern and northern part of Parrabel Dome started and stopped moving after emplacement of the Hastings Block and before the intrusion of the Werrikimbe Triassic granitoids. We suggested that the movement on the major bounding faults is related to the accommodation of the NHB to the folding and cleavage development in the adjoining Nambucca Block, and is associated with the earliest part of the Hunter–Bowen Orogeny. Limited dextral movement on the extensions of the Taylors Arm Fault System caused minor displacements in the northeastern part of the NHB during the Late Triassic. Some small faults cut the Triassic granitoids or Triassic Lorne Basin sediments indicating tectonic activity continued post-Triassic.

RAPID COMMUNICATION: Nerve growth factor influences cleavage rate and embryo development in sheep.

Recent information about Nerve growth factor (NGF), a protein traditionally associated to the nervous system that regulates survival and maturation of developing neurons, suggests that it may exert action also on different levels in the reproductive system. The aim of this study was to evaluate the effect of NGF added during in vitro oocyte maturation, fertilization or in vitro embryo development in sheep. Nerve growth factor was supplemented to the culture medium at 0, 100, or 1,000 ng/mL, during either in vitro maturation (Exp. 1), in vitro fertilization (Exp. 2), or in vitro culture (Exp. 3). In addition, NGF mRNA expression was determined in cumulus cells and oocytes. Nerve growth factor induced early cleavage when added during oocyte maturation or fertilization, improved embryo development when added during fertilization, and had no significant effect when added during embryo culture. In general, the effect was more evident with 100 rather than 1,000 ng/mL (P < 0.05). Expression of endogenous NGF was not detected in oocytes, and increased in cumulus cells when 1,000 ng/mL of NGF was added during fertilization, but not during maturation and embryo culture. In conclusion, the addition of NGF during oocyte maturation and fertilization affects in vitro cleavage and embryo development in sheep. We suggest a possible effect of this growth factor on oocyte maturation and mainly on the fertilization process.