Growth retardation of inner cell mass cells in polyspermic porcine embryos produced in vitro

The in vitro viability of polyspermic pig eggs was investigated. Immature oocytes were matured and fertilized in vitro. Approximately 10 h after insemination, the eggs were centrifuged at 12 000 x g for 10 min and individually classified into two (2PN)- and poly-pronuclear (PPN, 3 or 4 pronuclei) eggs. The classified eggs were cultured in vitro or in vivo. Nuclei numbers of inner cell mass (ICM) and trophectoderm (TE) were compared between 2PN- and PPN-derived blastocysts. The frequency of development in vitro of 2PN and PPN eggs to the blastocyst stage was 53.6% and 40.7%, respectively. The mean number (8.2 +/- 0.7, n = 48) of ICM nuclei of 2PN-derived blastocysts was higher than that (4.2 +/- 0.8, n = 37) of PPN-derived blastocysts (p < 0.001), whereas there was no difference (p > 0.05) in mean numbers of total (46.7 +/- 3.4 vs. 39. 9 +/- 3.9) and TE nuclei (38.5 +/- 2.9 vs. 35.7 +/- 3.3) between the two groups. Development of 2PN and PPN eggs cultured in vivo to the blastocyst stage was 33.3% and 27.4%, respectively. The numbers of ICM and TE nuclei of these embryos cultured in vivo showed a pattern similar to that for the in vitro-produced blastocysts. Additionally, fetuses were obtained on Day 21 from both the 2PN and the PPN groups. This suggests that polyspermic pig embryos develop to the blastocyst stage and beyond, although showing a smaller ICM cell number as compared to normal embryos.     

Daily Oral Oleoyl‐Estrone Gavage Induces a Dose‐Dependent Loss of Fat in Wistar Rats

Objective: To establish whether single daily oral doses of oleoyl‐estrone result in dose‐dependent slimming effects on normal weight rats, and to determine the changes in energy parameters induced by this treatment. Research Methods and Procedures: The effects of a daily oral gavage of oleoyl‐estrone (0, 0.2, 0.5, 1, 2, 5, 10, and 20 μmol/kg per day) in 0.2 ml of sunflower oil given over a 10‐day period were studied in groups, each of which contained six adult female Wistar rats initially weighing 190 to 230 g. A group of intact control rats receiving no gavage was included for comparison. Body weight and food intake were measured daily. Rats were killed on day 10 of treatment, and body composition (protein nitrogen, lipids, and water), liver lipids, and plasma parameters (glucose, triacylglycerols, total cholesterol, free fatty acids, 3‐hydroxybutyrate, urea, aspartate, alanine transaminases, insulin, leptin, and free and acyl‐estrone) were measured. Results: The administration of oleoyl‐estrone resulted in a dose‐dependent loss of body fat, because of a partly maintained energy expenditure combined with decreased food intake. The differences in the energy budget were met by internal fat pools. The changes recorded did not affect the levels of the main plasma energy homeostasis indicators: unaltered glucose, triacylglycerols, free fatty acids, 3hydroxybutyrate, and urea. Protein was accrued even under conditions of severe lipid store drainage. There were no changes in transaminases. No lipid accumulation was recorded in the liver. Plasma insulin and leptin levels decreased with increased oleoyl‐estrone doses, whereas the levels of free and esterified estrone increased with treatment, although not in proportion to the dose received. Discussion: Oral treatment with oleoyl‐estrone resulted in the specific dose‐related loss of fat reserves with little change to other metabolic parameters. These results agree with the postulated role of oleoyl‐estrone as a ponderostat signal.     

Efficient leaf ion partitioning,an overriding condition for abscisic acid-controlled stomatal and leaf growth responses to NaCl salinization in two legumes

Two tree medics contrasting in salt tolerance, Medicago arborea and Medicago citrina, were compared to evaluate the relative importance of abscisic acid on leaf growth and stomatal responses to salt stress. Plants were grown for 30 d in solution culture with 1, 50, 100 or 200 mM NaCl. Salinized plants of M. citrina had lower Na+ and Cl- uptake and maintained better leaf growth than M. arborea. In M. citrina, stomatal conductance was only slightly affected by salt and, in consequence, the salt treatment had no significant influence, neither on the CO2 fixation rate nor the transpiration rate in these plants. Moreover, leaf photosynthetic pigments and soluble protein in M. citrina were increased by the presence of NaCl, while a decrease of both parameters with salt was found in M. arborea. However, leaf and xylem ABA increased only in salt-treated M. citrina, while no differences were found among treatments in M. arborea. The role of ion compartmentation, gas exchange parameters and ABA concentrations in relation to salt tolerance in M. arborea and M. citrina is discussed.     

Cleavage stage porcine embryos may have differing developmental requirements for karyopherins alpha2 and alpha3

Numerous cellular proteins are able to localize to the nucleus due to the fact that they possess a nuclear localization signal (NLS) in their amino acid sequence. Nuclear localization sequences recognized by the importin alpha/beta heterodimer are found in cellular proteins capable of performing many diverse functions, ranging from chromatin remodeling to cell cycle regulation. Evidence has been presented that suggests individual importin alpha homologues are present at varying levels in different adult tissues. Other data have shown that specific subsets of NLSs found in different cellular proteins are recognized by individual importin alpha homologues with varying affinities. This evidence led us to hypothesize that due to the specific cargoes they carry, the mammalian embryo has different developmental requirements for individual importin alpha homologues. The results of the studies presented here indicate that importin alpha/beta-mediated import occurs throughout early cleavage in the porcine embryo, as determined by a reporter protein microinjection assay, and that multiple importin alpha homologues are present throughout early cleavage, as determined by immunocytochemical analysis. An RNA interference approach was used in an attempt to determine the developmental requirements for specific importin alpha homologues during early cleavage in the porcine embryo. Results from this study showed that fertilized porcine embryos injected with double stranded RNA (dsRNA) corresponding to the importin alpha homologue karyopherin alpha3 had significantly fewer nuclei following four days of culture than did embryos injected with dsRNA for another importin alpha homologue, karyopherin alpha2, or two control groups. This is the first report indicating that mammalian embryos may have differential developmental requirements for specific nuclear trafficking pathways.     

Fellow travellers: a concordance of colonization patterns between mice and men in the North Atlantic region

Background

In the Calvin cycle of eubacteria, the dephosphorylations of both fructose-1, 6-bisphosphate (FBP) and sedoheptulose-1, 7-bisphosphate (SBP) are catalyzed by the same bifunctional enzyme: fructose-1, 6-bisphosphatase/sedoheptulose-1, 7-bisphosphatase (F/SBPase), while in that of eukaryotic chloroplasts by two distinct enzymes: chloroplastic fructose-1, 6-bisphosphatase (FBPase) and sedoheptulose-1, 7-bisphosphatase (SBPase), respectively. It was proposed that these two eukaryotic enzymes arose from the divergence of a common ancestral eubacterial bifunctional F/SBPase of mitochondrial origin. However, no specific affinity between SBPase and eubacterial FBPase or F/SBPase can be observed in the previous phylogenetic analyses, and it is hard to explain why SBPase and/or F/SBPase are/is absent from most extant nonphotosynthetic eukaryotes according to this scenario.

Results

Domain analysis indicated that eubacterial F/SBPase of two different resources contain distinct domains: proteobacterial F/SBPases contain typical FBPase domain, while cyanobacterial F/SBPases possess FBPase_glpX domain. Therefore, like prokaryotic FBPase, eubacterial F/SBPase can also be divided into two evolutionarily distant classes (Class I and II). Phylogenetic analysis based on a much larger taxonomic sampling than previous work revealed that all eukaryotic SBPase cluster together and form a close sister group to the clade of epsilon-proteobacterial Class I FBPase which are gluconeogenesis-specific enzymes, while all eukaryotic chloroplast FBPase group together with eukaryotic cytosolic FBPase and form another distinct clade which then groups with the Class I FBPase of diverse eubacteria. Motif analysis of these enzymes also supports these phylogenetic correlations.

Conclusions

There are two evolutionarily distant classes of eubacterial bifunctional F/SBPase. Eukaryotic FBPase and SBPase do not diverge from either of them but have two independent origins: SBPase share a common ancestor with the gluconeogenesis-specific Class I FBPase of epsilon-proteobacteria (or probably originated from that of the ancestor of epsilon-proteobacteria), while FBPase arise from Class I FBPase of an unknown kind of eubacteria. During the evolution of SBPase from eubacterial Class I FBPase, the SBP-dephosphorylation activity was acquired through the transition ??from specialist to generalist??. The evolutionary substitution of the endosymbiotic-origin cyanobacterial bifunctional F/SBPase by the two light-regulated substrate-specific enzymes made the regulation of the Calvin cycle more delicate, which contributed to the evolution of eukaryotic photosynthesis and even the entire photosynthetic eukaryotes.     

Identification of Importin α 7 Specific Transport Cargoes Using a Proteomic Screening Approach

The importin α:β complex is responsible for the nuclear import of proteins bearing classical nuclear localization signals. In mammals, several importin α subtypes are known to exist that are suggested to have individual functions. Importin α 7 was shown to play a crucial role in early embryonic development in mice. Embryos from importin α 7–depleted females stop at the two-cell stage and show disturbed zygotic genome activation. As there is evidence that individual importin α subtypes possess cargo specificities, we hypothesized that importin α 7 binds a unique set of intracellular proteins. With the use of a collection of in vitro and in vivo binding assays, importin α 7 interaction partners were identified that differed from proteins found to bind to importin α 2 and 3. One of the proteins preferentially binding importin α 7 was the maternal effect protein Brg1. However, Brg1 was localized in oocyte nuclei in importin α 7–deficient embryos, albeit in reduced amounts, suggesting additional modes of nuclear translocation of this factor. An additional SILAC-based screening approach identified Ash2l, Chd3, Mcm3, and Smarcc1, whose nuclear import seems to be disturbed in importin α 7–deficient fibroblasts.The nuclear compartment is spatially separated from the cytoplasm by the nuclear envelope. The nuclear pores, which are embedded in the nuclear membrane, are the gateway for intracellular molecules that must traverse the nuclear envelope to enter or exit the nucleus. Small molecules can pass through the nuclear pores via passive diffusion; molecules weighing more than 40 kDa must be transported actively through the nuclear pore (1). According to the transport direction, carrier proteins that mediate these nuclear trafficking events are called importins or exportins, known collectively as karyopherins. Nuclear trafficking mediated by the importin α:importin β heterodimer is perhaps the best characterized nuclear import pathway. Here, importin α (or karyopherin α) serves as an adaptor molecule that binds cargoes containing classical nuclear localization signals (NLSs)¹ in their primary amino acid sequence. Upon cargo binding, importin α binds to importin β (karyopherin β 1), forming a trimeric transport complex that moves through the nuclear pore into the nucleus. In the nucleoplasm, RanGTP binds to importin β, leading to a conformational change in importin β and to the dissociation of the transport complex. The cargo is released to the nucleoplasm and can fulfill its function, whereas importins α and β are recycled back to the cytoplasm, where they can perform the next round of import (for reviews, see Refs. 2–4).There is only one importin α and one importin β protein present in yeast. However, multiple importin α isoforms, each transcribed from a different gene, are found in higher eukaryotes. Three importin α subtypes have been identified in Caenorhabditis elegans and Drosophila melanogaster, and up to seven importin α isoforms have been identified in mammals (5–7). These importin α isoforms can be grouped into three subfamilies based on sequence similarity (8). Little is known as to why multiple importin α isoforms exist in higher eukaryotes, but there is evidence that each importin α subtype has a tissue-specific expression pattern and distinct cargoes containing classical NLSs (9–12).We have recently shown that importin α 7 is required for embryonic development in mice (13). Oocytes from importin α 7 null females ovulate but produce embryos that fail to develop beyond the two-cell stage. To elucidate the molecular mechanisms behind this phenotype, we were especially interested in the identification of importin α 7 binding partners. Therefore, the aim of this study was to combine in vivo and in vitro screens to identify an importin α 7 subtype-specific cargo set. Through GST pull-down and co-immunoprecipitation experiments, we were able to identify a unique set of importin α 7 interaction partners that are involved in RNA processing, chromosome organization, and chromatin modification. Among them we found Brahma-related gene 1 (Brg1), also known as smarca4 or Baf190a, a known maternal effect protein required for early development in the mouse (14). An additional approach utilizing stable isotope labeling by amino acids in cell culture (SILAC) was used to further narrow down the list of potential importin α 7 specific cargoes. Hereby, we identified Ash2l, Chd3, Mcm3, Mcm5, and Smarcc1, whose nuclear levels were clearly decreased in importin α 7–deficient fibroblasts.     

The chemokine receptors ACKR2 and CCR2 reciprocally regulate lymphatic vessel density

Macrophages regulate lymphatic vasculature development; however, the molecular mechanisms regulating their recruitment to developing, and adult, lymphatic vascular sites are not known. Here, we report that resting mice deficient for the inflammatory chemokine‐scavenging receptor, ACKR2, display increased lymphatic vessel density in a range of tissues under resting and regenerating conditions. This appears not to alter dendritic cell migration to draining lymph nodes but is associated with enhanced fluid drainage from peripheral tissues and thus with a hypotensive phenotype. Examination of embryonic skin revealed that this lymphatic vessel density phenotype is developmentally established. Further studies indicated that macrophages and the inflammatory CC‐chemokine CCL2, which is scavenged by ACKR2, are associated with this phenotype. Accordingly, mice deficient for the CCL2 signalling receptor, CCR2, displayed a reciprocal phenotype of reduced lymphatic vessel density. Further examination revealed that proximity of pro‐lymphangiogenic macrophages to developing lymphatic vessel surfaces is increased in ACKR2‐deficient mice and reduced in CCR2‐deficient mice. Therefore, these receptors regulate vessel density by reciprocally modulating pro‐lymphangiogenic macrophage recruitment, and proximity, to developing, resting and regenerating lymphatic vessels.