Identification of a novel Xenopus laevis poly (A) binding protein

Poly (A) binding proteins are intimately implicated in controlling a number of events in mRNA metabolism from nuclear polyadenylation to cytoplasmic translation and stability. The known poly(A) binding proteins can be divided into three distinct structural groups (prototypes PABP1, PABPN1/PABP2 and Nab2p) and two functional families, showing that similar functions can be accomplished by differing structural units. This has prompted us to perform a screen for novel poly(A) binding proteins using Xenopus laevis. A novel poly(A) binding protein of 32 kDa (p32) was identified. Sequence analysis showed that p32 has about 50% identity to the known nuclear poly(A) binding proteins (PABPN1) but is more closely related to a group of mammalian proteins of unknown function. The expression of Xenopus laevis ePABP2 is restricted to early embryos. Accordingly, we propose that p32 is the founder member of a novel class of poly(A) binding proteins named ePABP2.     

乌龟胚胎及仔龟的卵黄代谢

乌龟卵、卵黄、剩余卵黄和仔龟的含水量分别为６８．８７％、６１．４７％、６０．７５％和７６．４３％;干重分别为２．２９１５±０．２１０１ｇ,１．３０７１±０．２９１３ｇ,０．２６８１±０．０２４１ｇ和１．２３０５±０．２９９４ｇ;脂肪含量分别为１８．０３％、３１．７６％、３２．１０％和１９．３０％。卵黄、剩余卵黄和仔龟的热值分别为６．７１ｋｃａｌ／ｇＤＭ,６．７６ｋｃａｌ／ｇＤＭ和４．１０ｋｃａｌ／     

Nitrogen distribution and excretion during embryonic post-yolk sac development inZoarces viviparus

In the viviparous teleostZoarces viviparus (L.) embryonic post-yolk sac development in the ovary is characterized by significant increases in dry weight and nitrogen per embryo, thus indicating an extensive matrotrophic relationship. In the ovarian fluid surrounding the embryos during their intraovarian development, sources of nitrogen were shown to derive from amino acids, urea, ammonia and various cellular components. The level of urea in the ovarian fluid increased significantly from 3.68±0.25 mol urea-N·ml^-1 during early post-yolk sac embryonic development to 6.14±0.44 in late development. The corresponding level of ammonia-N in the ovarian fluid increased from 0.25 to 0.45 mol·ml^-1. An estimation of embryonic nitrogen loss was made by measuring urea and ammonia-N excretion in vitro by post-yolk sac embryos or larvae (i.e. seawater-acclimated embryos). Urea-N constituted an average of 65% of the total nitrogen excreted by the embryos into the ambient medium during a 5-h time-course compared to only 35% in the larvae. Urea-N was excreted at maximum rates during the first hour of the experiment, 0.54±0.09 mol N·g^-1·h^-1 by embryos and 0.35±0.02 by larvae, and then declined to lower levels in both embryos and larvae. A decline after 1 h was also observed for excretion rates of ammonia. In conclusion, the capacity for urea excretion by post-yolk sac embryos ofZ. viviparus may be of adaptive significance for their prolonged stay in ovario. The capacity for excretion of urea seems to decrease after acclimation to sea water.Abbreviations aa amino acid(s) - bm body mass - dw dry weight - NPS ninhydrin-positive substances - sw sea water - ww wet weight     

Effects of acidic water on young-of-the-year smallmouth bass (Micropterus dolomieui)

Synopsis Smallmouth bass (Micropterus dolomieui) populations are declining in areas vulnerable to anthropogenic acidification. To determine if populations may be lost directly from acid effects (i.e., in isolation from synergists), we performed a laboratory study on the effects of acid exposure on growth, survival and histology of young smallmouth bass (3 to 36 d post swimup). We exposed them to declining, then fluctuating acid levels averaging pH 7.4 (control), 5.7 and 5.0. Substantial decreases in survival were observed, as well as damage to tissues. Survival declined with increasing acidity to 43% of that in controls at pH 5.7 and to 4% at pH 5.0. Histological analysis revealed severe changes in the most acidic treatment, with damage to blood, gill, and epithelial tissue. In contrast, growth was less useful as an indicator of response to acidity. Growth in both length and dry weight was greater at the lower pH levels than in controls. This was probably a reflection of lower survival at the lower pH levels, reduced density, and hence less competition for food. Our results suggest that an increase in environmental acidity is sufficient to cause losses in this species, even in the absence of synergists such as heavy metals.School of Forest Resources, University of Georgia, Athens, GA 30602, U.S.A.     

Cross-validation of techniques for measuring lipid content of bovine oocytes and blastocysts

The main objective was to test and validate a fluorescence approach to quantify lipid content of individual bovine oocytes and blastocysts. For Experiment 1, denuded oocytes were evaluated, as well as in vitro-produced blastocysts in a factorial design: cows versus feedlot heifers; three additives during Days 2.5-7.5 of culture (Control; 10% FCS; 0.3 μM phenazine ethosulfate (PES), an electron acceptor that oxidizes NADPH); and two blastocyst stages (early versus expanded). All blastocysts were graded subjectively for darkness (1 = clear … 4 = dark). In Experiment 2, denuded oocytes were used to measure lipid content in a factorial design of: cows versus heifers and four subjective darkness grades (1 = clear … 4 = dark). To quantify lipids, oocytes and 7.5 d blastocysts were fixed and then stained with 1 μg/mL Nile Red dye in mPBS overnight. A digital photograph of the equatorial part of the oocyte and embryo was taken at 200×, and fluorescence intensity (Arbitrary Fluorescence Units, AFU) was measured with Image Pro software. Reverse images of the same photographs were used to count numbers of cytoplasmic lipid droplets of various sizes (LC). The linear regression equation of LC with AFU in oocytes had an r² = 0.84, and for blastocysts r² = 0.91. The LC and AFU also had similar coefficients of variation from the ANOVA for blastocysts (38 vs 44%, respectively). Treatment differences were of similar magnitude with both procedures: lipid content in oocytes and blastocysts from heifers and cows was similar (P > 0.1); PES reduced lipid accumulation, and FCS increased it relative to the Control for AFU (18.6 vs 46.6 vs 36.9 units, respectively), and LC (1763 vs 4081 vs 3310, respectively; all, P < 0.01). Early blastocysts resulted in more lipid accumulation per unit area than expanded ones based on AFU (41.5 vs 26.6) and LC (3519 vs 2583; both P <0.01). There was a strong relationship (P < 0.01) between subjective oocyte and blastocyst darkness and lipid content. The less labor intensive fluorescence staining was a reliable technique for quantifying lipid droplets in oocytes and blastocysts.     

Capsule walls as barriers to oxygen availability: Implications for the development of brooded embryos by the estuarine gastropod Crepipatella dilatata (Calyptraeidae)

Encapsulation of developing embryos imposes potential restrictions, because the capsule wall must allow for adequate inward diffusion of oxygen and for increased diffusion of oxygen as metabolic demand increases with continued development. Samples of egg capsules from the gastropod Crepipatella dilatata were used to document surface characteristics, composition of the different capsule wall layers, and alterations in wall thickness during development. The diffusion coefficient and capsule wall permeability were determined experimentally for capsules containing embryos at different developmental stages. We also determined oxygen consumption rates for various embryonic stages and for nurse eggs, which provide food for embryos during development. The capsule wall of C. dilatata possesses 2 differentiated layers: the external capsular wall (ECW) and the internal capsular wall (ICW). The ECW is compact and fibrous, features that remain invariable during development, and lacks surface features that might make some portions of the capsule wall more permeable to oxygen than others. On the other hand, the ICW is initially spongy and thick, but significantly decreases in thickness over time, particularly before the embryos begin feeding on nurse eggs. Although the capsule wall is a serious barrier to diffusion, permeability to oxygen increases over time by 112% due to the dramatic thinning of the inner capsule wall layer. Nurse eggs consume oxygen but at very low rates, supporting the idea that they correspond to living embryonic cells that have stopped their development. Respiration measurements indicated that embryos are initially supplied with enough oxygen within the egg capsules to carry out the activities characteristic of embryogenesis, even though the capsular walls show their maximum thickness and lowest permeability at this time. However, as the embryo develops its velum and becomes more active, capsule wall thickness decreases and capsule permeability to oxygen increases. Correspondingly, the oxygen demands of metamorphosed but still encapsulated specimens are approximately 135% higher than those of pre-metamorphosed sibling embryos.     

Effect of semen quality in transgenic boars on the developmental competence of preimplantation embryos

The aim of this study was to compare the fertilising capacity of sperm from 6 transgenic (TG) and 6 non-transgenic (NTG) boars based on analyses of embryos resulting from insemination with sperm from these particular boars. Expanded blastocysts were collected from five groups of synchronised gilts (six gilts per group) inseminated by TG boars bearing a gene construct containing the human α1,2-fucosyltransferase gene and by NTG boars. The ejaculates used for insemination were analysed to detect apoptotic changes using two fluorescence methods: an assay to assess early changes in the membrane integrity of the sperm using the YO-PRO-1 fluorophore and an assay for phosphatidylserine (PS) translocation across the plasma membranes using fluorescein-labelled Annexin-V. Our results, using a combination of YO-PRO-1 and PI fluorophores, revealed no significant differences in the percentage of sperm subpopulations between non-transgenic and transgenic boars (P < 0.01). Moreover, the second fluorescent probe also revealed no significant differences between the average values of live (Ann-V⁻/PI⁻), early apoptotic (Ann-V⁺/PI⁻), and late apoptotic/early necrotic sperm (Ann-V⁺/PI⁺) as calculated for TG and NTG boars. Only the percentage of necrotic sperm (Ann-V⁻/PI⁺) was significantly different (P < 0.05) between transgenic and non-transgenic boars (3.4% ± 2.7; 7.2% ± 2.1, respectively). The quality of the preimplantation embryos at the blastocyst stage was determined by counting the number of cells, observing a TUNEL-positive reaction and by caspase-3 labelling. We found that expanded blastocysts that were derived from gilts inseminated with TG and NTG boar semen showed almost no DNA fragmentation (80%) and 70% caspase-3 activity. The expanded blastocysts that were derived from gilts inseminated with TG and NTG boar semen did not differ significantly in their DNA fragmentation, and there were no differences in caspase-3 activity. These results revealed a positive correlation between the percentage of blastocysts with TUNEL-positive nuclei and the percentage of blastocysts with caspase-3 activity (r = 0.9787; P < 0.0001).     

Cryopreservation of Human Embryos

Cryopreservation of human embryos from the 2-cell stage up to the morula stage is a safe procedure which has been carried out for the last 25 years. Experience with blastocyst cryopreservation is still limited and pregnancy rates after the use of frozen, thawed blastocysts vary extremely. Vitrification has improved the success of embryo cryopreservation. However, this technique cannot yet be considered as a routine procedure. Despite all of the advantages for infertile couples, cryopreservation of human embryos creates severe ethical problems, because of surplus frozen embryos which either have to be destroyed or perhaps used for research. Embryo adoption may provide a solution to solve imminent medical, ethical and social problems.     

Expression analysis of the NDRG2 gene in mouse embryonic and adult tissues

N-myc downstream-regulated gene 2 (NDRG2) is believed to be involved in cell growth events. However, its exact function is still unknown. To elucidate the role of this gene, we used an anti-Ndrg2 monoclonal antibody in immunohistochemistry and immunofluorescence assays to analyze the expression pattern of Ndrg2 protein in mouse embryos at various gestational ages and in a variety of adult mouse tissues. Ndrg2 immunoreactivity was generally localized to the cytoplasm. During mouse development, Ndrg2 expression was observed in many developing tissues and organs including the heart, brain, lung, gut, liver, kidney, skeletal muscle, cartilage, chorion, epidermis, and whisker follicles. Ndrg2 expression was developmentally dynamic, being generally lower in the early stages of development and markedly increasing during later stages. Ndrg2 expression was also observed in a variety of adult mouse tissues, particularly in the heart and brain. This is the first demonstration of Ndrg2 protein expression in both embryonic and adult mouse tissues. Our results suggest that NDRG2 plays important roles in histogenesis and organogenesis.This study was supported by grants from the National Key Basic Research and Development Program (no. 2002CB513007), the National Natural Science Foundation of China (nos. 30370315 and 30171044) and PCSIRT04-59.