Search

Rocket emissions and debris from spacecraft falling out of orbit are having increasingly detrimental effects on global atmospheric chemistry. Improved monitoring and regulation are urgently needed to create an environmentally sustainable space industry.

The ocean absorbs approximately a quarter of the carbon dioxide currently released to the atmosphere by human activities (Canth). A disproportionately large fraction accumulates in the North Atlantic due to the combined effects of transport by the Atlantic Meridional Overturning Circulation (AMOC) and air–sea exchange. However, discrepancies exist between modelled and observed estimates of the air–sea exchange due to unresolved ocean transport variability. Here we quantify the strength and variability of Canthtransports across 26.5° N in the North Atlantic between 2004 and 2012 using circulation measurements from the RAPID mooring array and hydrographic observations. Over this period, decreasing circulation strength tended to decrease northward Canthtransport, while increasing Canthconcentrations (preferentially in the upper limb of the overturning circulation) tended to increase northward Canthtransport. These two processes compensated each other over the 8.5-year period. While ocean transport and air–sea Canthfluxes are approximately equal in magnitude, the increasing accumulation rate of Canthin the North Atlantic combined with a stable ocean transport supply means we infer a growing contribution from air–sea Canthfluxes over the period. North Atlantic Canthaccumulation is thus sensitive to AMOC strength, but growing atmospheric Canthuptake continues to significantly impact Canthtransports.

The North Atlantic Ocean is a region of intense uptake of atmospheric CO2. To assess how this CO2sink has evolved over recent decades, various approaches have been used to estimate basin‐wide uptake from the irregularly sampled in situ CO2observations. Until now, the lack of robust uncertainties associated with observation‐based gap‐filling methods required to produce these estimates has limited the capacity to validate climate model simulated surface ocean CO2concentrations. After robustly quantifying basin‐wide and annually varying interpolation uncertainties using both observational and model data, we show that the North Atlantic surface ocean fugacity of CO2(fCO2−ocean) increased at a significantly slower rate than that simulated by the latest generation of Earth System Models during the period 1992–2014. We further show, with initialized model simulations, that the inability of these models to capture the observed trend in surface fCO2−oceanis primarily due to biases in the models' ocean biogeochemistry. Our results imply that current projections may underestimate the contribution of the North Atlantic to mitigating increasing future atmospheric CO2concentrations. Robust uncertainties for the recent change in the North Atlantic surface fCO2are determined by using observational‐based and model productsThe increasing North Atlantic surface fCO2is overestimated by ESMs during 1992–2014, and not captured by models' internal variabilitySimulation initialised with biogeochemical observations correct for the models' bias in the trend in surface CO2trends

Future projections indicate the Atlantic Meridional Overturning Circulation (AMOC) will weaken and shoal in response to global warming, but models disagree widely over the amount of weakening. We analyze projected AMOC weakening in 27 CMIP6 climate models, in terms of changes in three return pathways of the AMOC. The branch of the AMOC that returns through diffusive upwelling in the Indo‐Pacific, but does not later upwell in the Southern Ocean (SO), is particularly sensitive to warming, in part, because shallowing of the deep flow prevents it from entering the Indo‐Pacific via the SO. The present‐day strength of this Indo‐Pacific pathway provides a strong constraint on the projected AMOC weakening. However, estimates of this pathway using four observationally based methods imply a wide range of AMOC weakening under the SSP5‐8.5 scenario of 29%–61% by 2100. Our results suggest that improved observational constraints on this pathway would substantially reduce uncertainty in 21st century AMOC decline. The Atlantic Meridional Overturning Circulation (AMOC) is a system of ocean currents that move warm surface waters from the south to the north of the Atlantic Ocean where they cool, sink, and return southward at depth. Changes in the AMOC would have wide‐ranging impacts on our climate. It is predicted to weaken as the climate warms during the 21st century, but the extent of weakening varies among different climate models. We show that AMOC weakening is greatest in models that have a large exchange of water between the AMOC and the Indo‐Pacific Ocean along a specific pathway. The magnitude of this ocean pathway, inferred from four observation‐based estimates of the global overturning circulation, is uncertain. By using these estimates and analyzing the relationship between the aforementioned ocean pathway and AMOC weakening across many climate models, we can predict how the real‐world AMOC will change. Our findings indicate that by 2100, under a high greenhouse gas emission scenario, the AMOC will weaken by 29%–61%. This highlights the importance of reducing differences between observational estimates of the ocean's overturning pathways to reduce uncertainty in future AMOC weakening and to improve the representation of these pathways in climate models. The magnitude of 21st century Atlantic Meridional Overturning Circulation (AMOC) weakening in CMIP6 models is highly correlated with an AMOC pathway into the Indo‐Pacific OceanThe real‐world “Indo‐Pacific diffusive” AMOC pathway inferred from observation‐based estimates is used to constrain future AMOC weakeningUnder high‐end greenhouse gas forcing, AMOC weakening based on this emergent constraint relationship ranges from 29% to 61% by 2100 The magnitude of 21st century Atlantic Meridional Overturning Circulation (AMOC) weakening in CMIP6 models is highly correlated with an AMOC pathway into the Indo‐Pacific Ocean The real‐world “Indo‐Pacific diffusive” AMOC pathway inferred from observation‐based estimates is used to constrain future AMOC weakening Under high‐end greenhouse gas forcing, AMOC weakening based on this emergent constraint relationship ranges from 29% to 61% by 2100

In this work, we use realistic isopycnal velocities with a 3‐D eddy diffusivity to advect and diffuse a tracer in the Antarctic Circumpolar Current, beginning in the Southeast Pacific and progressing through Drake Passage. We prescribe a diapycnal diffusivity which takes one value in the SE Pacific west of 67°W and another value in Drake Passage east of that longitude, and optimize the diffusivities using a cost function to give a best fit to experimental data from the DIMES (Diapycnal and Isopycnal Mixing Experiment in the Southern Ocean) tracer, released near the boundary between the Upper and Lower Circumpolar Deep Water. We find that diapycnal diffusivity is enhanced 20‐fold in Drake Passage compared with the SE Pacific, consistent with previous estimates obtained using a simpler advection‐diffusion model with constant, but different, zonal velocities east and west of 67°W. Our result shows that diapycnal mixing in the ACC plays a significant role in transferring buoyancy within the Meridional Overturning Circulation. Realistic velocity fields used to advect a model tracer in the Southern Ocean produce lateral tracer distributions similar to an observed tracerDiapycnal diffusivities diagnosed from a best fit of the model tracer distribution to observations are 20 times larger in Drake Passage than in the Southeast PacificResults from models using two different velocity field products are consistent with one another, and with the previous result of Watson et al. (2013)

REACHING physicians at the point of care is thought to be the ultimate marketing tool. However, this approach is intrusive and many physicians reject it as invasive, so many pharma [...]

Atmospheric CO2concentrations over glacial–interglacial cycles closely correspond to Antarctic temperature patterns. These are distinct from temperature variations in the mid to northern latitudes, so this suggests that the Southern Ocean is pivotal in controlling natural CO2concentrations. Here we assess the sensitivity of atmospheric CO2concentrations to glacial–interglacial changes in the ocean’s meridional overturning circulation using a circulation model for upwelling and eddy transport in the Southern Ocean coupled with a simple biogeochemical description. Under glacial conditions, a broader region of surface buoyancy loss results in upwelling farther to the north, relative to interglacials. The northern location of upwelling results in reduced CO2outgassing and stronger carbon sequestration in the deep ocean: we calculate that the shift to this glacial-style circulation can draw down 30 to 60 ppm of atmospheric CO2. We therefore suggest that the direct effect of temperatures on Southern Ocean buoyancy forcing, and hence the residual overturning circulation, explains much of the strong correlation between Antarctic temperature variations and atmospheric CO2concentrations over glacial–interglacial cycles.

Before implantation into the uterine wall, the mammalian embryo undergoes a period of cell division, cell shape change, and cell differentiation leading to the formation of an outer epithelium, the trophectoderm. The trophectoderm is the part of the embryo that initiates uterine contact and, after transformation to become the trophoblast, uterine invasion. Similar to the kidney nephron, the trophectoderm is a transporting epithelium with distinct apical and basolateral membrane domains; its function is to facilitate transepithelial Na+and fluid transport for blastocoel formation. That transport is driven by Na,K-adenosine triphosphatase (ATPase) localized in basolateral membranes of the trophectoderm. Preimplantation embryos express multiple a and ß subunit isoforms of Na,K-ATPase, potentially constituting multiple isozymes, but the basolaterally located a1ß1 isozyme appears to function uniquely to drive fluid transport. Embryos unable to express a1 subunits because of targeted deletion of the gene are able to form a blastocoel, but they fail to maintain their integrity and expire during the peri-implantation period. Preimplantation embryos also express the ? subunit, a modulator of Na,K-ATPase activity, but targeted deletion of that gene did not reveal an essential developmental role. The preimplantation embryo offers a unique model for understanding the roles of Na,K-ATPase subunit isoforms in epithelial development and transepithelial transport.

New insights into the early development of large mammals are becoming available through the measurement of differential mRNA levels in oocytes and preimplantation embryos. These advances in knowledge are rapidly picking up in pace, mainly owing to the advantages brought by new molecular biology approaches being developed. The possibility of amplifying the starting material and therefore making measurements in single embryo units is now feasible. With these tools, the evaluation of variations in gene expression patterns during the preimplantation period or the impact of culture on mRNA levels is now possible. However, it is important to keep in mind that these methods still have limitations associated with sample preparation or the use of the appropriate controls. Even proper methods of analysis are very important to achieve the full benefit of the application of these tools. The present paper describes some of the potential, as well as limitations, of mRNA level analysis in early embryos, especially for microarray analysis. We have generated a bovine cDNA array (>2000 clones) that contains expressed sequence tags (ESTs) collected from various preimplantation development stages. Using this chip, we have initiated the characterisation of global mRNA level patterns of several key developmental stages from the immature oocyte to the blastocyst stage. As expected, the hybridisation results indicate very different expression profiles involving hundreds of genes when comparing oocyte and blastocyst samples to a reference mRNA sample made from a pool of ESTs from pooled somatic tissues. Although this array is still in its preliminary stage and the EST bank has not been processed to contain only unigenes, it is already a very useful tool for discovering candidate genes that may play important roles during early embryonic life.

We utilized SIVmne infection of Macaca fascicularis to assess the efficacy of DNA vaccination alone, and as a priming agent in combination with subunit protein boosts. All SIVmne structural and regulatory genes were expressed using the human cytomegalovirus Immediate Early-1 promoter in plasmids that directed the formation of virus-like particles in vitro. Macaques (n = 4) were immunized intradermally and intramuscularly four times over 36 weeks with 3 mg plasmid DNA. A second group (n = 4) received two DNA priming inoculations followed by two intramuscular boosts consisting of 250 μg recombinant Env gp160 and 250 μg recombinant Gag-Pol particles in MF-59 adjuvant. These regimens elicited modest cellular immunity prior to challenge. Humoral immune responses to Env gp160 were elicited and sustained by both vaccine protocols, and as expected antibody titers were higher in the protein subunit-boosted animals. Neutralizing antibodies prior to challenge were measurable in two of four subunit-boosted macaques. The two vaccine regimens elicited comparable helper T cell responses at the time of challenge. Vaccinees and mock-immunized controls (n = 4) were challenged intrarectally at week 38 with uncloned SIVmne. Following challenge all macaques became infected, but both vaccine regimens resulted in reduced peak virus loads (p = 0.07) and significantly improved maintenance of peripheral CD4+ T cell counts postchallenge (p = 0.007, DNA alone and p = 0.01, all vaccinees). There was no significant difference between the two vaccine groups in levels of plasma viremia or maintenance of CD4+ T cell counts postchallenge.

New insights into the early development of large mammals are becoming available through the measurement of differential mRNA levels in oocytes and preimplantation embryos. These advances in knowledge are rapidly picking up in pace, mainly owing to the advantages brought by new molecular biology approaches being developed. The possibility of amplifying the starting material and therefore making measurements in single embryo units is now feasible. With these tools, the evaluation of variations in gene expression patterns during the preimplantation period or the impact of culture on mRNA levels is now possible. However, it is important to keep in mind that these methods still have limitations associated with sample preparation or the use of the appropriate controls. Even proper methods of analysis are very important to achieve the full benefit of the application of these tools. The present paper describes some of the potential, as well as limitations, of mRNA level analysis in early embryos, especially for microarray analysis. We have generated a bovine cDNA array (>2000 clones) that contains expressed sequence tags (ESTs) collected from various preimplantation development stages. Using this chip, we have initiated the characterisation of global mRNA level patterns of several key developmental stages from the immature oocyte to the blastocyst stage. As expected, the hybridisation results indicate very different expression profiles involving hundreds of genes when comparing oocyte and blastocyst samples to a reference mRNA sample made from a pool of ESTs from pooled somatic tissues. Although this array is still in its preliminary stage and the EST bank has not been processed to contain only unigenes, it is already a very useful tool for discovering candidate genes that may play important roles during early embryonic life.

Expression of cyclooxygenase-2 (COX-2) and prostaglandin E2(PGE2) receptor 2 (EP2) are necessary for rodent cumulus expansion in vivo. Prostaglandin E2receptor 3 (EP3) has been detected in bovine preovulatory follicles and corpora lutea. The current experiments examined the effect of PGE2on bovine cumulus expansion in vitro and expression of COX-2, EP1, EP2, EP3, and EP4 mRNAs in bovine cumulus-oocyte complexes (COCs) at 0, 6, 12, 18, and 24 h time points during maturation in vitro. Concentrations of PGE2above 50 ng/ml resulted in moderate cumulus expansion of bovine COCs, but expansion did not occur in the absence of serum. COX-2 mRNA expression increased in bovine COCs at 6 h and 12 h of maturation, then decreased. EP2 mRNA was detectable by reverse transcription-polymerase chain reaction at all time points. EP3 mRNA expression increased in COCs from 0 to 6 h and remained at this higher level through the culture period. Very low levels of EP4 mRNA expression were detectable, but EP1 was not detected in bovine COCs. Because EP receptor mRNAs and COX-2 mRNA are expressed in bovine COCs, there exists the potential for a prostaglandin autocrine/paracrine regulatory pathway during oocyte maturation. Differential expression of the EP3 mRNA among varying COC classes indicates that this gene product may be a useful marker of oocyte competence. Although the PGE2pathway is involved in cumulus expansion, serum factors are required to mediate PGE2-induced expansion.

Na+,K+-ATPase plays an essential role in mammalian blastocoel formation (cavitation) by driving trans-epithelial sodium transport. Previously, the α1 and β1 subunit isoforms of this enzyme were identified in preimplantation mouse embryos and were assumed to be responsible for this function. Here we show that mRNAs encoding an additional α subunit isoform (α3) and the remaining two β subunit isoforms are also present in preimplantation embryos. Whereas α3 mRNA accumulates between the four-cell and the blastocyst stages and thus results from embryonic transcription, the same could not be demonstrated for β2 and β3 mRNAs. Immunoblot analyses confirmed that these subunits are present in cavitating embryos. Using confocal immunofluorescence microscopy we found that α1 and β1 subunits are concentrated in the basolateral membranes of the trophectoderm while being equally distributed in plasma membranes of the inner cell mass. In contrast, α3, β2, and β3 subunits were not detected in plasma membranes. Our current assessment, therefore, is that as many as six isozymes of Na+,K+-ATPase could be involved in preimplantation development although it is primarily the α1β1 isozyme that is responsible for blastocoel formation. Our findings imply that the regulation of sodium transport within the preimplantation mouse embryo is more complex than had been appreciated.

The objectives were 1) to investigate the effects of oocyte maturation in serum-free and amino acid-supplemented defined media on oocyte transcript levels, blastocyst cell number, and apoptosis; 2) to investigate the influence of oocyte maturation culture atmosphere on blastocyst development, total cell number, and apoptosis; and 3) to examine the influence of epidermal growth factor (EGF) during oocyte maturation on blastocyst cell number and apoptosis. The results demonstrate that blastocysts derived from in vitro maturation, fertilization, and embryo culture protocols undergo apoptosis but that apoptotic levels are not greatly influenced by the oocyte maturation environment. Amino acid supplementation of oocyte maturation media was associated with enhanced developmental frequencies, increased blastocyst cell number, and elevated oocyte maternal mRNA levels. Oocyte maturation with supplemented synthetic oviduct fluid medium (cSOFMaa) resulted in blastocyst cell numbers comparable to those observed with Tissue Culture Medium 199 + newborn calf serum. Blastocyst development was reduced following oocyte maturation under a 5% CO2, 7% O2, 88% N2culture atmosphere. EGF supplementation of oocyte maturation medium resulted in a concentration-dependent increase in blastocyst development but did not influence blastocyst total cell number or apoptosis. Our findings indicate that cSOFMaa medium is an effective base medium for bovine oocyte maturation.

Recent landmark achievements in animal cloning have demonstrated that the events of cell differentiation can, in principle, be reversed. This reversal necessarily requires large-scale genetic reprogramming, of which little is known. In the present study we characterized the extent to which blastocyst stage-specific mRNA expression would be conserved in bovine embryos produced by nuclear transfer (NT) using fetal fibroblasts as nuclei donors (FF NT). The mRNA pool of FF NT embryos was compared with that of NT embryos reconstructed from embryonic blastomeres (Emb NT), with embryos produced under in vivo or in vitro conditions, and finally with fibroblast cells. Embryo/cell-specific mRNA pools were contrasted using differential display methodology. Random oligonucleotide primer pair combinations were used to subfractionate mRNA populations and represent individual mRNAs as copy DNA (cDNA) bands ranging in size from 100 to 800 base pairs. Regardless of whether bovine blastocysts developed in vivo or in vitro, or were derived after nuclear transplantation with embryonic blastomeres or fetal fibroblasts, their mRNA profile was highly conserved and distinct from that of fetal fibroblast cells. There was approximately 95% conservation in cDNA banding patterns between FF NT, Emb NT, and in vivo derived blastocysts, when compared with in vitro derived blastocysts. In contrast, the cDNA banding in fibroblasts was only 67% conserved with in vitro derived blastocysts (p < 0.0001), indicating that dramatic changes in gene transcription are induced by nuclear transplantation. After nuclear transplantation, gene expression in fetal fibroblasts is reprogrammed so to mimic that of preimplantation embryo development. Future characterization of these changes will be invaluable for the identification of suitable cell types to serve as nuclear donors for embryo reconstruction and provide information that can be used to improve the efficiency of cloning animals by nuclear transplantation.

There have been several recent advances in our understanding of the geochemistry of iron and its effect on the marine biota. In this contribution, we highlight two such advances, namely results of the Ironex experiments in the equatorial Pacific and the recent publication of the first global data set for iron concentrations in the oceans. These have profound consequences for our understanding of the factors that set the pre-anthropogenic concentration of carbon dioxide in the atmosphere, and how these may have changed between glacial and interglacial time. Some of these consequences we are able to quantify and explore, but others open new questions for which we have as yet no answers.

In the present study the abundance of mRNAs for eukaryotic translation initiation factors eIF-1A (formerly known as eIF-4C), -2α, -4A, -4E, and -5 was examined in in vivo-derived mouse embryos throughout preimplantation development using a semiquantitative reverse transcription-polymerase chain reaction assay. Although the mRNA profile for each gene is unique, only mRNA for eIF-1A transiently increases during embryonic gene activation (EGA) at the 2-cell stage, and this was confirmed by an independent hybridization-based assay. In in vitro-developed bovine embryos, mRNA for eIF-1A was transiently detected at the 8-cell stage, when the major activation of the genome occurs in this species. As in the mouse, detection in 8-cell bovine embryos was sensitive to the transcriptional inhibitor α-amanitin. It was also observed at the same time relative to cleavage in embryos cultured in defined medium under a reduced oxygen environment, and in medium supplemented with serum and somatic cells in 5% CO2in air. Neither the chronology of early cleavage divisions nor the yield of bovine blastocysts differed in these culture media. Our results suggest that transient expression of eIF-1A in the mouse and cow is a conserved pattern of gene expression associated with EGA in mammals.

The reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the patterns of expression for several growth factor ligand and receptor genes during ovine preimplantation development. Transcripts for insulin-like growth factor (IGF)-I, IGF-II, and the receptors for insulin and IGF-I were detected throughout ovine preimplantation development from the 1-cell to the blastocyst stage. Transforming growth factor α (TGFα) transcripts were also detected throughout ovine preimplantation development. The mRNAs encoding basic fibroblast growth factor (bFGF) were detected in all stages of the ovine preimplantation embryo, although the relative abundance of this transcript consistently decreased from the 1-cell to the blastocyst stage, suggesting that it may represent a maternal transcript in early sheep embryos. Transcripts encoding ovine trophoblast protein (oTP) were detected only within blastocyst-stage embryos. Primary ovine oviduct cell cultures express the transcripts for IGF-II, IGF-I, TGFα, bFGF, TGFβ1, and the receptors for insulin and IGF-I, suggesting that paracrine growth factor circuits may exist between the oviduct epithelium and the early ovine embryo. Transcripts for insulin, epidermal growth factor (EGF), and nerve growth factor (NGF) were not detected in any stage of the ovine preimplantation embryo or within the oviduct cell preparations. The expression of growth factor transcripts very early in mammalian development would predict that these molecules fulfil a necessary role(s) in supporting the progression of early embryos through the preimplantation interval. Our future efforts will be directed to understanding the nature of these putative regulatory pathways.

The roles of medium composition, serum source, embryo coculture, and culture under low O2conditions on the development of in vitro-matured and in vitro-fertilized (IVMF) ovine zygotes were investigated in three separate experiments. In the first experiment, the proportion of cocultured IVMF zygotes developing to the blastocyst stage was significantly higher (38.0% vs. 3.5%; p< 0.05) than that of non-cocultured zygotes treated within three embryo culture media (TCM-199 + 10% fetal bovine serum [FBS]; bicarbonate-buffered, glucose-free synthetic oviduct fluid medium [mod-SOFM] + 10% FBS; and bicarbonate-buffered BSA-free Tyrode’s salt solution [mod-TALP] + 10% FBS) under a 5% CO2atmosphere in air. In a second experiment, a significantly higher (p< 0.05) proportion of cocultured zygotes placed in TCM-199 medium survived to the blastocyst stage (37.4% blastocysts vs. 23.4% in mod-SOFM). No significant effect of serum (FBS vs. human serum [HS]) was observed on embryonic development, but coculture was confirmed to exert a significant influence on development to the blastocyst stage. In the final experiment, survival of the embryo under a reduced oxygen (5% CO2:5% O2:90% N2) atmosphere was investigated. In contrast to results in the initial experiments, embryonic survival was significantly higher (p< 0.05) in the non-cocultured treatment groups (21.9% blastocysts vs. 0.4% for cocultured zygotes). Serum source also had a significant (p< 0.05) influence upon the development of non-cocultured zygotes: 32.3% of zygotes cultured with HS progressed to the blastocyst stage vs. 11.5% of zygotes cultured in FBS-supplemented medium. These results have characterized two distinct culture environments, each capable of supporting the development of high frequencies of unselected IVMF zygotes to the blastocyst stage in vitro.

A number of growth factor ligand and receptor genes are expressed by early embryos of the mouse, rat, cow, and sheep. Several growth factors have stimulatory effects on early embryos when added exogenously to the medium for culture of preimplantation mammalian embryos. Growth factor supplementation of simple defined medium for culture of early bovine embryos leads to a higher percentage of embryos that can cleave and develop beyond the eightcell stage compared to those in un-supplemented medium. Cultured preimplantation bovine embryos do not express as broad a range of antioxidant enzymes as do mouse embryos. Bovine primary oviduct coculture cells constitutively express the same set of antioxidant enzymes as the mouse embryo and may contribute to protection of the embryo against damage from free oxygen radicals during in vitro culture. Preliminary experiments to analyze growth factor gene expression in cloned bovine embryos indicate that nuclear transplantation alters the normal pattern of expression during early embryonic development.

Expression of the trophoblast interferon, bovine trophoblast protein-1 (bTP-1), has been studied in embryos produced by in vitro maturation-in vitro fertilization (IVM-IVF). No bTP-1 production was noted until after embryos had reached the expanded blastocyst stage and had begun to hatch (Days 8–9 post-fertilization). Single blastocysts comprising 115 ± 22 cells released 1.0 ± 0.1 units of interferon activity/24 h. Amplification of conceptus mRNA by reverse transcription-polymerase chain reaction procedure with bTP-1-specific oligonucleotides confirmed that bTP-1 transcripts were present in blastocysts but were not detectable at earlier stages. Although cultured blastocysts produced by IVM-IVF procedures continued to secrete bTP-1 for a few days, they failed to attach to the substratum and form outgrowths, and soon lost structural integrity. However, when Day 8 blastocysts/morulae were transferred to the uteri of synchronized cows, recovered 4 days later, and placed into individual cultures, they attached and formed outgrowths that produced large amounts of bTP-1 (> 2000 units/culture/24 h after 14 days). Embryos thus first expressed bTP-1 when a functional trophectoderm was first formed, and induction did not require a period of in vivo development. However, continued viability of the blastocyst and bTP-1 production were not sustained in vitro and may require some exposure to the uterine environment.

This study evaluated Na/K-ATPase α1- and α3-subunit isoform polypeptide expression and localization during bovine preattachment development. Na/K-ATPase cation transport activity from the one-cell to blastocyst stage was also determined by measuring ouabain-sensitive86Rb+uptake. Both α1- and α3-subunit polypeptides were detected by immunofluorescence to encircle the entire cell margins of each blastomere of inseminated zygotes, cleavage stage embryos, and morulae. Immunofluorescent localization of α1-subunit polypeptide in bovine blastocysts revealed an α1 immunofluorescence signal confined to the basolateral membrane margins of the trophectoderm and encircling the cell periphery of each inner cell mass (ICM) cell. In contrast, α3-subunit polypeptide immunofluorescence was localized primarily to the apical cell surfaces of the trophectoderm with a reduced signal present in basolateral trophectoderm regions. There was no apparent α3-subunit signal in the ICM. Analysis of86Rb+transportin vitrodemonstrated ouabain-sensitive activity throughout development from the one-cell to the six- to eight-cell stage of bovine development.86Rb+uptake by morulae (day 6 postinsemination) did not vary significantly from uptake detected in cleavage stage embryos; however, a significant increase was measured at the blastocyst stage (P< 0.05). Treatment of embryos with cytochalasin D (5 μg/ml) did not influence86Rb+uptake in cleavage stage embryos. Cytochalasin D treatment however was associated with a significant rise in ion transport in morulae and blastocysts (13.49 and 61.57 fmol/embryo/min, respectively) compared to untreated controls (2.65 and 22.83 fmol/embryo/min, respectively). Our results, for the first time, demonstrate that multiple Na/K-ATPase α-subunit isoforms are distributed throughout the first week of mammalian development and raise the possibility that multiple isozymes of the Na/K-ATPase contribute to blastocyst formation.

Bovine oviductal monolayer and vesicle primary cultures express insulin-like growth factor (IGF)-I and -II mRNAs and polypeptides. Early bovine embryos also express IGF-I, IGF-II, IGF-I receptor, IGF-II receptor, and insulin receptor mRNAs. This study reports the expression of IGF binding protein (IGFBP) mRNAs and polypeptides in bovine oviduct primary cultures and IGFBP mRNAs in preattachment embryos. Release of immunoreactive IGF-I and IGF-II by oviduct cultures and bovine blastocysts was also determined. IGFBP-2, -3, -4, and -5 transcripts were observed in oviduct primary cultures throughout an 8-day interval. IGFBP-1 and -6 mRNAs were consistently not detected in the oviduct. Messenger RNAs encoding IGFBPs -2, -3, and -4 were detected throughout bovine preattachment development, while transcripts encoding IGFBP-5 were detected only in blastocysts. IGFBP-1 and -6 transcripts were not detected in early embryos. Ligand blot analysis with 125I-labeled IGF-II revealed the presence of four prominent polypeptide bands of approximate molecular masses 24, 31, and 36 kDa, and a broad band extending from 46 to 53 kDa, in conditioned media samples prepared from oviduct primary cultures. Western immunoblot analysis confirmed the identity of the 24-kDa, 31-kDa, and 36-kDa species as IGFBP-4, -5, and -2, respectively. Levels of the release of IGF-II from oviductal vesicle cultures were significantly greater than levels observed for monolayer cultures (p< 0.005). No significant difference in the levels of IGF-I release between monolayer and vesicle cultures was observed. Pools of 10 blastocysts released on average 36.2 ± 3.9 pg of IGF-II per embryo, while the release of embryonic IGF-I was below the levels of detection for our assay. The results suggest that maternally derived IGF may be regulated by IGFBPs to support bovine preattachment development.

Genetic, molecular and cellular analyses of the HLA-D region of the major histocompatability complex (MHC) in man have led to the definition of three different products. Two of these, DR and MB (the latter also known as DC (ref. 1) and LB-E (ref. 2)) are defined with serological reagents; the third, known as SB (ref. 3) and PL-3 (ref. 4) is defined with primed lymphocyte typing (PLT) cells5. The classical features attributed to HLA-D region encoded (class II) molecules are that they are cell-surface dimers consisting of a structurally conserved α-chain noncovalently associated with a polymorphic β-chain and that they are found primarily on B lymphocytes, some monocyte populations, endothelial and certain other cells6. Using these criteria a monoclonal antibody,. B7/21, was described as reactive with HLA-DR (ref. 7). We have now re-evaluated B7/21 antibody reactivity using mutant lymphoblastoid cell lines. It appears that this antibody does not react with the molecularly defined D region products described to date but instead, recognizes a class II antigen with distinctive molecular characteristics. We provisionally refer to this antigen as FA.

We review aspects of the influence of the marine biota on climate, focusing particularly on their role in mediating surface temperatures via their influence on atmospheric carbon dioxide (CO2) and dimethyl sulphide (DMS) concentrations. Variation in natural CO2concentrations occurring over 103to 105years are set by oceanic processes, and in particular by conditions in the Southern Ocean, so it is to this region that we must look to understand the glacial–interglacial changes in CO2concentrations. It seems likely that marine productivity in the Southern Ocean is limited by a combination of restricted iron supply to the region and insufficient light.Plankton–produced DMS is thought to influence climate by changing the numbers of cloud condensation nuclei available in remote regions; the efficiency of this mechanism is still unknown, but calculations suggest it may be a powerful influence on climate. It has a much shorter time–scale than the CO2effect, and as a consequence may well be a player on the ‘global change’ timescale. The direction of both the CO2and the DMS mechanisms is such that more marine productivity would lead to lower global temperatures, and we speculate that the overall effect of the marine biota today is to cool the planet by ca. 6°C as a result of these two mechanisms, with one–third of this figure being due to CO2effects and two–thirds due to DMS.While the marine biota influence climate, climate also influences the marine biota, chiefly via changing atmospheric circulation. This in turn alters ocean circulation patterns, responsible for mixing up sub–surface nutrients, and also influences the transport of nutrients, such as iron, in atmospheric dust. A more vigorous atmospheric circulation would be expected to increase the productivity of the marine biota on both counts. Thus during glacial time, the colder and drier climate might be expected to stimulate greater marine productivity than occurs today. Since more production leads to greater cooling by reduction in CO2and increase in DMS, the marine biota–climate system appears to have been in positive feedback in the glacial–interglacial transition, with the changes in the climate system being reinforced by changes in the marine biota. In the context of anthropogenic change, we cannot at present say what sign the feedback on climate will have, because we have no clear idea whether circulation will become more or less vigorous in the future.

To study the impact of follitropin delta for ovarian stimulation on embryo development and quality compared with that of follitropin alfa or beta in in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles.

The mixing of heat and chemical constituents across density strata in the ocean plays a major role in ocean circulation and in the response of the climate to thermal and chemical perturbations. We are developing a tracer technique which, in conjunction with fluid dynamical measurements, promises to improve our understanding and our estimates of such diapycnal mixing. In a prototype tracer experiment in Santa Monica Basin, 50km west of Los Angeles, two tracers, sulphur hexafluoride (SF6) and per-fluorodecalin (PFD), have been injected on an isopycnal surface near the centre of the basin. After 50 days, the tracers had mixed along the isopycnal surface to nearly every part of the basin, although relatively little had penetrated to the basin walls. The diapycnal spreading of the tracer distributions during this first stage of the experiment yielded an apparent eddy diffusivity of 0.33±0.08 cm2s−1at the ambient density gradient of 4.0±0.5×10−9g cm−4.

Cyclization of o‐aminobenzamide (I) with various benzylic and aliphatic alcohols proceeds smoothly under Ru‐catalyzed hydrogen transfer conditions to afford quinazolines (III).

A wide range of secondary and tertiary amines as well as amides and sulfonamides is available.

AMPK (AMP-regulated kinase) is a kinase involved in nutrient- and stress-sensing. In general, its role is to conserve ATP by shutting down anabolic pathways and increasing activity of transport and catabolic pathways. AMPK is known to be dysregulated in diabetes and obesity and is a target of some diabetic drugs such as metformin. There is a role for AMPK in mammalian oocyte maturation, however, little is known of its role in later embryo development. Two-cell mouse embryos were cultured in KSOM under 5% oxygen conditions in the presence of increasing concentrations of AICAR, an AMPK activator, or Compound C, an AMPK inhibitor. Blastocyst development was evaluated at 48h of culture. Embryo diameters were measured, and embryos fixed for immunofluorescent staining. Concentrations of AICAR above 250 micromolar, decreased blastocyst development and embryo diameter (250: 10.9 ± 3.3% and 76.29 ± 1.86 microns and 1000: 1.0 ± 1.0% and 71.27 ± 0.65 microns vs. 0: 44.6 ± 10.8% and 108.18 ± 2.69 microns, P < 0.05). Diameter was also decreased at with the 100 micromolar dose of AICAR, 84.54 ± 2.45 microns, P < 0.05. Most embryos seemed to arrest at the morula stage. Similarly, Compound C at 100 micromolar decreased blastocyst development to 9.2 ± 5.5% and 85.82 ± 1.56 microns compared to the DMSO control at 39.5 ± 14.9% and 114.50 ± 2.34 microns, P < 0.05. Using confocal microscopy, embryos that were arrested by AICAR treatment appeared to have lower immunofluorescence for ZO-1, a tight junction protein, in the membrane and possibly lower expression of actin. Interestingly, the active phosphorylated AMPK protein appeared to be expressed mostly in the cytoplasm in the inner cell mass, and in the nucleus, perinuclear area and in the membrane of trophectoderm cells. Membrane staining has only been observed in a few cell types. AMPK activation may need to be regulated within a narrow window to ensure proper embryo development. Research supported by NSERC to A.J.W.(poster)

The catalysis comprises oxidation by hydrogen transfer of the alcohols to the corresponding ketones and a CH activation/alkene insertion step.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

RNA binding proteins (RBP) are expressed in mammalian cells and they affect storage, splicing, localization, adenylation, half-life and translation of mRNAs. Previously, we detected expression of ELAVL1, KHSRP and ZFP36 RBPs in mouse embryos by real-time PCR and immunofluorescence and showed abundance of two RBPs and one target mRNA were affected by culture. Generally, ELAVL1 increases expression of target mRNAs while KHSRP and ZFP36 decrease mRNA expression. Furthermore, all are known to be regulated by MAPK, especially p38. The roles of environmental stress and MAPKs in affecting RBP and target mRNA expression were investigated. Three potential ARE-containing targets were chosen based on the likelihood that their expression would be culture-responsive, GCLC (glutathione synthesis), SLC2A1 (GLUT-1, glucose transport) and CAT-1 (amino acid transport). We investigated two methods of inducing environmental stress: 1) hyperoxia (H, 20% O2) versus low oxygen (L, 5% O2); and 2) culturing embryos in a good culture medium, KSOMaa (K) versus a poor medium without amino acids, Whitten's (W), ± the p38 MAPK inhibitor, SB220025 (10 ìM) or 3) culture in K or W ± the ERK inhibitor U0126 (10 ìM). In expt 1, embryos were cultured from the 2 cell stage in KSOMaa under a 5% or 20% O2 atmosphere to approximately 96h post hCG, and only blastocysts were collected. In expts 2 and 3, embryos were cultured from 2-cell to blastocyst in KSOMaa in 5% O2, then embryos were washed twice and cultured in a) K; b) K + inhibitor, c) W; d) W + inhibitor for approximately 18h. Embryos were frozen, reverse transcribed with oligodT and used for real-time PCR. GCLC (P < 0.001) and CAT-1 mRNAs (P< 0.01) were expressed at higher levels in H vs. L medium, and there was a tendency towards higher expression of SLC2A1 in H than L (P < 0.065). In expt. 2, there were no significant differences due to media, but p38 MAPK inhibition caused expression of CAT-1 mRNA to be markedly reduced (P < .001). In experiment 3, GCLC mRNA was more highly expressed in W than K (P < 0.003), and there was a tendency towards higher expression of CAT-1 mRNA in W than K (P < 0.06) but no differences in the presence of the ERK inhibitor. Further experiments will examine the role of the JUNK/SAPK pathway and examine whether stress affects mRNA half-life. We have therefore identified a culture sensitive stress response pathway that mediates affects on transcript levels likely by affecting mRNA stability rather than transcription. Culture stress acting through the p38 MAPK pathway may influence embryonic mRNA transcript levels via RBP mediated mechanisms controlling target mRNA abundance. Variations in mRNAs levels observed in cultured embryos may therefore occur due to effects to mRNA stability and half-life in addition to transcriptional control mechanisms. Research support by the Natural Science and Engineering Council of Canada (NSERC).(poster)

Mouse preimplantation development is characterized by two distinct morphological events: Compaction and Blastocyst Formation. Compaction is initiated at the 8-cell stage where the assembly of adherens (AJ) and tight junction proteins at the surface of the outer blastomeres begin the process of polarization of the outer cells and eventually provides a permeability seal in the outer cells of the early embryo. In concert with the formation of this seal, the Na/K ATPase establishes an ionic gradient across the trophectoderm (TE), which promotes the movement of fluid through aquaporins to form the blastocyst cavity. The blastocyst consists of two cell types: the TE, which will form the embryonic portion of the placenta, and the inner cell mass (ICM), which consists of the precursor cells that will become the embryo proper. Although we understand the role of many of the proteins involved in blastocyst formation, we have not elucidated how those proteins are regulated throughout preimplantation development. Of these, CDH1, an AJ associated protein, and the ATP1B1, a Na/K ATPase associated protein, are regulated by the transcription factors SNAI1 and SNAI2 in many cell types. SNAI1 and SNAI2 normally function to initiate epithelial-to-mesenchymal transition throughout mouse development. However, SNAI1 and SNAI2 are also oncogenes and aberrant expression of these proteins will promote cancer metastasis. My hypothesis is that Cdh1 and Atp1b1 are both regulated by SNAI1 and SNAI2 throughout mouse preimplantation development. Real-time RT-PCR was applied to determine the steady state mRNA levels of Snai1 and Snai2 throughout preimplantation development. Snai1 steady state mRNA expression increases at the 2-cell stage and then decreases until the 8-cell stage. The steady state mRNA levels returns to the 1-cell level in the blastocyst. Snai2 steady state mRNA levels decrease at the 2-cell stage during the maternal-to-zygotic transition. Expression begins to increase after the 4-cell stage and reaches its peak at the morula stage. Steady state mRNA then decreases at the blastocyst stage. Immunofluorescence was applied to detect SNAI1 and SNAI2 throughout preimplantation development and revealed that SNAI1 and SNAI2 are asymmetrically localized in each blastomere at the 2-cell and the 4-cell stage. SNAI1 and SNAI2 become exclusively distributed to only the outside cells of the developing embryo from the 8-cell stage onward. In the blastocyst, SNAI1 and SNAI2 are only detectable in the TE. SNAI1 and SNAI2 have a unique localization pattern throughout preimplantation development indicative of a role in lineage specification and TE differentiation. Loss-of-Function and Gain-of-Function studies will be applied to determine whether SNAI1 and SNAI2 play a role in TE differentiation and Cdh1 and Atp1b1 regulation. Together, these results provide further insight into the biochemical pathways that regulate blastocyst formation and provide new insight into the normal function of two oncogenes, SNAI1 and SNAI2 during preimplantation development. Research supported by the Canadian Institutes of Health Research.(poster)

The endoplasmic reticulum (ER) is the cellular organelle responsible for the production and maturation of secretory and transmembrane proteins. Nascent, unfolded polypeptide chains enter the ER lumen where they are folded and undergo post-translational modification by ER chaperone proteins. ER stress is defined as an accumulation of unfolded or malfolded proteins in the ER lumen, occurring as a result of physiological or environmental changes to the cell. In response, cells activate the unfolded protein response (UPR), an evolutionary conserved mechanism that alleviates this stress. Due to the large protein load that is continually being secreted throughout preimplantation development, we hypothesize that the ER stress pathway plays an active role in the adaptive ability of the preimplantation embryo to its environment. To characterize the expression of mRNAs encoding UPR constituents during mouse preimplantation development, embryos were collected from superovulated MF1 females to obtain a developmental series (1-cell to blastocyst stage). Total RNA isolated from embryo pools (20 per pool) representing each stage was reversed transcribed using oligo-dT priming and Sensiscript reverse transcriptase, prior to application of standard polymerase chain reaction methods. To investigate whether the ER stress pathway can be induced during preimplantation development, morulae and blastocysts were cultured in 0.1 µg/mL and 0.5 µg/mL tunicamycin for 4 and 12 hours. Total RNA was extracted, and gene expression levels of ER chaperones were quantified using real-time PCR. RT-PCR results confirm that BIP, ATF4, CHOP, IRE1alpha, XBP1-U, ASK1, AFT6 and CSP12 mRNAs are present at the blastocyst stage. Additionally, qPCR results demonstrated that BIP, GRP94 and ATF4 mRNAs are initially high at the 1-cell stage, undergo a significant depletion between 1 and 2-cell stages and then increase markedly with each cell division, reaching high levels once again at the blastocyst stage. However p58IPK mRNA levels remained low from the two-cell stage onwards. Both morulae and blastocysts cultured in either 0.1 µg/mL and 0.5 µg/mL tunicamycin displayed an elevation in GRP94 and p58IPK mRNAs, indicating UPR activation. Additionally, RT-PCR results demonstrate that XBP1 splicing is favoured in tunicamycin treated embryos. Our results demonstrate that ER stress response pathways are present and functional during preimplantation development. Future studies will examine the role of the UPR as an adaptive mechanism and will investigate if culture conditions induce UPR activation, by comparison to in vivo derived embryos. Research supported by operating funds from NSERC.(poster)

The investigation of the mechanisms controlling preimplantation development is necessary to improve our assessment of embryonic development and to apply assisted reproductive technologies efficiently. The Na+/K+-ATPase plays a pivotal role during preimplantation development; the period between oocyte-sperm union and uterine wall invasion. Activity of the Na+/K+-ATPase establishes a trans-epithelial ionic gradient that facilitates the movement of water to form the fluid-filled blastocyst cavity. Reaching the blastocyst stage is necessary for implantation, and consequently, successful pregnancy. The Na+/K+-ATPase is implicated in regulating tight junction assembly and function and most recently in extracellular protein interactions. Signal transduction properties of the Na+/K+-ATPase have been discovered following binding of cardiotonic steroids (CTS), such as ouabain, to its α subunit leading to SRC activation. We hypothesize that pump activation induced by ouabain leads to SRC activation, resulting in the regulation of tight junction formation and function during mouse preimplantation development. To investigate this hypothesis, embryos were collected following application of superovulation methods to MF1 female mice. Preimplantaion embryos of the desired stage were subjected to CTS treatment or Src Family Kinase (SFK) inhibition followed by immmuofluorescence. Morula stage embryos were treated with 10-3 M, 10-5 M, 10-7 M, 10-9 M ouabain or bufalin to characterize affects of CTS treatment on blastocyst formation. Treatment with 10-5 M, 10-7 M, and 10-9 M CTS for 30 h did not significantly affect the proportion of morulae that progressed to the blastocyst stage. However, embryos treated with 10-3 M CTS did not progress to the blastocyst stage. Next experiments investigated the effects of treating blastocysts with 10-3 M, 10-4 M, and 10-5 M ouabain for 2 or 10 min to characterize influences of ouabain treatment on SRC phosphorylation by whole-mount immunofluorescence. Reduced Tyr418 phosphorylation after treatment with 10-3 M ouabain for 10 min was apparent when compared to control embryos. Next experiments treated morulae in PP2 (SFK blocker) for 18 h. Embryos treated with PP2 displayed a reversible blockade of development and did not cavitate using 20 μM, 30 μM, or 50 μM PP2. Morulae treated with SU6656, a more potent and specific SFK inhibitor, for 18hr also displayed an inability to cavitate using 1, 5, 10 μM doses. RT-PCR analysis was used to detect mRNAs encoding all SFKs during preimplantation development. mRNAs encoding SRC and YES were detectable at the blastocyst stage while mRNAs encoding FGR, LCK, LYN, FYN, HCK, and BLK were not present in blastocysts. These studies contribute to our understanding of the mechanisms controlling preimplantation development. Activation of SRC through phosphorylation of tyrosine 418 is necessary for mouse blastocyst formation. The presence of a ouabain-induced Na+/K+-ATPase signaling transduction pathway via SRC was revealed in the mouse blastocyst. Future experiments will investigate the effect of SFK inhibition on trophectoderm tight junction function. Research supported by operating grant from the Canadian Institutes of Health Research.(poster)

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

The investigation of the mechanisms controlling preimplantation development is necessary to optimize embryonic culture conditions, to improve our ability to assess embryonic developmental capacity prior to embryo transfer and to apply assisted reproductive technologies in the most efficient ways possible. The Na-K ATPase plays a pivotal role in preimplantation development, the period from oocyte-sperm union to uterine wall invasion. Activity of the Na-K ATPase pump, after assuming its basolateral position in the trophectoderm, establishes a trans-epithelial ionic gradient that facilitates the movement of water across the epithelium to create the fluid-filled blastocyst cavity. Reaching the blastocyst stage is crucial for implantation, and consequently, successful pregnancy. The Na-K ATPase is also implicated in regulating tight junction assembly and function and most recently in extracellular protein interactions. Signal transduction properties of the Na-K ATPase have been discovered upon binding of cardiotonic steroids (CTS), such as ouabain, to its α subunit leading to SRC activation. We hypothesize that pump activation induced by ouabain leads to SRC and subsequent MAPK activation, resulting in the regulation of trophectoderm tight junction formation and function during preimplantation development. To investigate this hypothesis, embryos were collected following application of superovulation methods applied to MF1 female mice. Morula stage embryos were subjected to treatment with 10-3M, 10-5M, 10-7M, 10-9M ouabain or bufalin to characterize affects of CTS treatment on blastocyst formation. Treatment with 10-5M, 10-7M, and 10-9M concentrations for up to 30 hr did not significantly affect the proportion of morulae that progressed to the blastocyst stage. However, embryos treated with 10-3M ouabain and bufalin did not progress to the blastocyst stage. Embryos treated with 10-5M, 10-7M, and 10-9M concentrations displayed a trend for increased blastocyst diameter and volume compared to untreated controls. Next experiments investigated the effects of treating blastocysts with ouabain at 10-3M, 10-3.5M, 10-4M, 10-4.5M and 10-5M in KSOMaa medium for 1, 5, and 10 minutes to characterize influences of ouabain treatment on SRC phosphorylation by detecting variations in SRC phosphorylation using whole-mount immunofluorescence methods. The results from these experiments demonstrated an increase in phosphorylated SRC between KSOM controls and 10-3.5M ouabain-treated embryos. Those treated with 10-4M ouabain for 1 minute exhibited a marked increase in Src phosphorylation with a steady decrease at 10-4.5M and 10-5M concentrations. In addition RT-PCR methods were used to detect mRNAs encoding all SRC family members during preimplantation development. Preliminary results indicate that only mRNAs encoding SRC are detectable during preimplantation development while mRNAs encoding Lyn, Lck, Fyn and Yes are not. Our results demonstrate the likelihood of a CTS Na-K ATPase mediated mechanism for activating SRC during preimplantation development. Our future experiments will investigate the consequences of SRC blockade on preimplantation development and the role of SRC signaling in regulating tight junction assembly and function during preimplantation development. These studies contribute to our understanding of the mechanisms controlling preimplantation development. Research supported by operating grant from the Canadian Institutes of Health Research.

Oogenesis is characterized by the accumulation of large stores of maternal mRNAs that drive development at least until the maternal-zygotic transition, when the embryo begins to transcribe and translate its own mRNAs. Over 200 RNA binding proteins are expressed in the Drosophila embryo, and they affect storage, localization, adenylation, half-life and translation of maternal mRNAs. It is therefore very likely that RBPs will have an important influence on mRNA stability and turnover during mammalian oogenesis and early embryogenesis. We may discover that RBP expression and function is affected by culture environment and embryonic gene expression may be affected via changes in RBP target mRNA half-life, polyadenylation or translation. We have recently reported the expression of several RBPs in the mouse oocyte and early embryo. Here we report that both K homology splicing regulatory protein (KHSRP) and zinc finger protein 36 (TTP, tristetraprolin) are expressed at the mRNA and protein levels in mouse oocyte to blastocyst stage embryos. Pools of mouse oocytes-blastocysts were collected, cultured from the 2 cell stage in KSOMaa medium, frozen and prepared for PCR (regular and real-time) or fixed in 2% paraformaldeyde and used for confocal immunofluorescent localization. KHSRP protein was expressed from the GV oocyte to blastocyst stage in both the nucleus and cytoplasm. ZFP36 protein was expressed from the GV oocyte to the blastocyst stage in the nucleus, nuclear envelope and the cytoplasm. Next we investigated the influences of culture medium on RBP expression. Recent reports have indicated that the p38 MAPK pathway is a potent mediator of mRNA half-life and/or translation and we sought to investigate whether RBPs, including ELAVL1 (HuR), KHSRP, ZFP36 and target mRNAs gamma glutamyl-cysteine ligase (GCLC) and glucose transporter 1 (SLC2a1) abundances are altered during embryo culture. Variations in embryonic transcript abundance following exposure to culture are well reported in the literature and it is widely presumed that variations in mRNA levels reflect alterations in mRNA transcription. However, it is equally possible that culture influences are directed via changes in RBP mediated mRNA polyadenylation and stability. Embryos were collected at the 2 cell stage and cultured in either KSOMaa (low stress) or Whitten's media (high glucose, higher stress) in 5% O2 atmosphere. Embryos were collected at the 4 cell, 8 cell, morula and blastocyst stages, frozen, reverse transcribed with oligodT and used for real-time PCR. ELAVL1 mRNA increased in both media from the 4 cell to the blastocyst stage and KHSRP mRNA also increased with stage. In contrast, ZFP36 was most abundant at the 4 cell stage. GCLC was higher at the morula and blastocyst stages in Whitten's than KSOMaa media while SLC2a1 was higher in blastocysts in KSOMaa compared to Whitten's. These studies demonstrate that expression of RBP changes during preimplantation development. In addition, alterations in RBP levels are accompanied with changes in the expression of their known targets. Thus culture may influence embryonic mRNA transcript levels via RBP mediated mechanisms controlling target mRNA stability. Research supported by the Natural Sciences and Engineering Research Council of Canada.