Anomalous neural differentiation induced by 5-bromo-2'-deoxyuridine during organogenesis in the rat

The influence of 5-bromo-2'-deoxyuridine (BrdU) on rat embryo development and neurogenesis was investigated using a rat conceptus culture system during organogenesis (pregnancy days 10-13). The embryos and visceral yolk sacs of conceptuses cultured with BrdU were examined for overall growth, morphological anomalies, incorporation of radiolabeled BrdU into DNA, and neurotransmitter enzyme activities in embryos. In addition, neural tubes from cultured whole embryos were isolated and mechanically dissociated into fragments and cultured again to assess neural cell differentiation into neuron-like cells. BrdU was found to incorporate differentially into embryonic and visceral yolk sac DNA with simultaneous stage-specific retardation and anomalous organogenesis in proportion to the increasing concentrations used. Neural tube differentiation of cultured embryos was markedly altered, and there were morphologically distinct neural anomalies. The neurite outgrowth from neuroblast cells (type 1) of explanted spinal neural tube fragments from BrdU-treated embryos was markedly reduced in length and number compared to those from similar areas of embryos grown without BrdU. In contrast, BrdU at the same doses did not affect differentiation of a number of neural tissue-related enzymes. These results indicate that BrdU incorporation into DNA of primordial embryonic cells significantly affects neurogenesis and differentiation of neurites from neuroblasts, which is a specific neural cytodifferentiation characteristic of neuronal cells.     

Effect of heat stress on conceptus and uterine secretion in the bovine

This study examined the effects of heat stress on conceptus secretion of bovine trophoblastic proteins (bTP) and the uterine secretory environment on Day 17 of pregnancy. After mating to fertile bulls, cows were placed in an environmental chamber at 21 degrees C, 45% RH on Day 7 and were assigned to one of the following treatments on Day 8: Control (21 degrees C, 45% RH), Treatment 1 (37 degrees C, 30% RH) and Treatment 2 (37 degrees C, 40% RH). Cows were slaughtered on Day 17 and each uterine horn was flushed separately with physiological saline to recover the conceptus and uterine contents. The wet weight of the conceptus was recorded and uterine flushings were analyzed for quantitative and qualitative protein changes, prostaglandin F and calcium concentrations. Conceptus tissue was cultured in vitro with 100 muCi of [(3)H]-leucine and the polypeptides released into the medium were analyzed by 2D-PAGE followed by fluorography. Heat stress increased rectal temperature of cows and reduced conceptus wet weight. Uterine content of protein and calcium were increased in the uterine horn ipsilateral to the CL of heat-stressed cows. Although uterine protein increased in heat-stressed cows, no qualitative difference was observed in the polypeptides present in the uterine lumen. Conceptus synthesis and release of bTP were enhanced in treated cows. These responses indicate that heat stress between Days 8 to 17 of pregnancy altered the uterine environment as well as growth and secretory activity of the conceptus.     

Nucleotides in a single mammalian ovum or preimplantation embryo

ATP, ADP, and AMP have been measured jointly on a single mouse ovum or preimplantation embryo using an ultramicrofluorescence technique. The method uses the traditional approach of enzymatic analysis based on changes in the concentrations of nucleotide cofactors, but eliminates the need for enzymatic recycling. It permits the measurement of as little as 10 fmol, and may be adapted for numerous metabolites.     

Whole genome de novo assemblies of three divergent strains of rice,Oryza sativa,document novel gene space of aus and indica

The use of high throughput genome-sequencing technologies has uncovered a large extent of structural variation in eukaryotic genomes that makes important contributions to genomic diversity and phenotypic variation. When the genomes of different strains of a given organism are compared, whole genome resequencing data are typically aligned to an established reference sequence. However, when the reference differs in significant structural ways from the individuals under study, the analysis is often incomplete or inaccurate. Here, we use rice as a model to demonstrate how improvements in sequencing and assembly technology allow rapid and inexpensive de novo assembly of next generation sequence data into high-quality assemblies that can be directly compared using whole genome alignment to provide an unbiased assessment. Using this approach, we are able to accurately assess the ‘pan-genome’ of three divergent rice varieties and document several megabases of each genome absent in the other two. Many of the genome-specific loci are annotated to contain genes, reflecting the potential for new biological properties that would be missed by standard reference-mapping approaches. We further provide a detailed analysis of several loci associated with agriculturally important traits, including the S5 hybrid sterility locus, the Sub1 submergence tolerance locus, the LRK gene cluster associated with improved yield, and the Pup1 cluster associated with phosphorus deficiency, illustrating the utility of our approach for biological discovery. All of the data and software are openly available to support further breeding and functional studies of rice and other species.     

Thermal characterization of Nakagata's mouse sperm freezing protocol

This paper reports the results of an experimental study of the warming and cooling rates achieved using the popular Nakagata Protocol for murine sperm cryopreservation. Problems with the storage and maintenance of the huge number of genetically engineered mouse strains have led to an increased need for murine sperm preservation. Recent studies have begun to focus on optimizing the cryopreservation of murine sperm by carefully studying the effects of cooling and warming rates on sperm survival. In current practice, however, the Nakagata protocol is widely used. The actual cooling and warming rates achieved using the Nakagata protocol have not previously been determined; and the Nakagata protocol has a number of unspecified parameters which we have found can significantly affect cooling rates, warming rates and sperm survival. A detailed study of the thermal response of samples frozen and thawed using the Nakagata protocol reveals that the cooling rates range from 30 to almost 300 °C per minute depending on the exact manner in which the Nakagata protocol is implemented. Warming rates range from 160 °C/min to about 1000 °C/min. Sperm survival depended significantly on the particular cooling rate achieved, and less strongly on the warming rates. Overall, it was found that the particular manner in which the Nakagata protocol was implemented could strongly affect cooling rates and sperm survival; and, consistent with the findings of Mazur and Koshimoto, an optimal cooling rate appears to exist in the range of cooling rates that can be achieved using the Nakagata protocol.