Morphological and Gene Expression Changes in Cattle Embryos from Hatched Blastocyst to Early Gastrulation Stages after Transfer of In Vitro Produced Embryos

A detailed morphological staging system for cattle embryos at stages following blastocyst hatching and preceding gastrulation is presented here together with spatiotemporal mapping of gene expression for BMP4, BRACHYURY, CERBERUS1 (CER1), CRIPTO, EOMESODERMIN, FURIN and NODAL. Five stages are defined based on distinct developmental events. The first of these is the differentiation of the visceral hypoblast underlying the epiblast, from the parietal hypoblast underlying the mural trophoblast. The second concerns the formation of an asymmetrically positioned, morphologically recognisable region within the visceral hypoblast that is marked by the presence of CER1 and absence of BMP4 expression. We have termed this the anterior visceral hypoblast or AVH. Intra-epiblast cavity formation and the disappearance of the polar trophoblast overlying the epiblast (Rauber’s layer) have been mapped in relation to AVH formation. The third chronological event involves the transition of the epiblast into the embryonic ectoderm with concomitant onset of posterior NODAL, EOMES and BRACHYURY expression. Lastly, gastrulation commences as the posterior medial embryonic ectoderm layer thickens to form the primitive streak and cells ingress between the embryonic ectoderm and hypoblast. At this stage a novel domain of CER1 expression is seen whereas the AVH disappears. Comparison with the mouse reveals that while gene expression patterns at the onset of gastrulation are well conserved, asymmetry establishment, which relies on extraembryonic tissues such as the hypoblast and trophoblast, has diverged in terms of both gene expression and morphology.     

Standardizing Immunohistochemistry: A New Reference Control for Detecting Staining Problems

A new standardized immunohistochemistry (IHC) control for breast cancer testing comprises formalin-fixed human epidermal growth factor receptor 2, estrogen receptor, or progesterone receptor peptide antigens covalently attached to 8-µm glass beads. The antigen-coated beads are suspended in a liquid matrix that hardens upon pipetting onto a glass microscope slide. The antigen-coated beads remain in place through deparaffinization, antigen retrieval, and immunostaining. The intensity of the beads’ stain provides feedback regarding the efficacy of both antigen retrieval and immunostaining. As a first report, we tested the sensitivity and specificity of the new IHC controls (“IHControls”). To evaluate sensitivity, various staining problems were simulated. IHControls detected primary and secondary reagent degradation similarly to tissue controls. This first group of IHControls behaved similarly to tissue controls expressing high concentrations of the antigen. The IHControls were also able to detect aberrations in antigen retrieval, as simulated by sub-optimal times or temperatures. Specificity testing revealed that each antigen-coated bead was specific for its cognate IHC test antibody. The data support the conclusion that, like tissue controls, IHControls are capable of verifying the analytic components of an immunohistochemical stain. Unlike tissue controls, IHControls are prepared in large bulk lots, fostering day-to-day reproducibility that can be standardized across laboratories.     

Glycogen phosphorylase ‘b’ in Dictyostelium: stability and endogenous phosphorylation

Summary The slime mold Dictyostelium discoideum has two forms of the enzyme glycogen phosphorylase. The inactive phosphorylase b form requires 5 AMP for activity and is present in early development. The active phosphorylase a form is 5 AMP independent and occurs during later development. We here show that the 92 kd b enzyme subunit exists either as a singlet or a doublet upon SDS-PAGE, depending on the method of sample extraction. In the presence of exogenously added Mn²⁺ and ATP, the phosphorylase b shows apparent conversion into a 5 AMP independent form as measured by enzyme activity. In addition, Mn²⁺ and ATP also support an in vitro phosphorylation of the 92 kd phosphorylase b subunit. We also demonstrate phosphorylation of the b enzyme subunit in vivo by ³²-P incorporation into the enzyme protein. A protein kinase responsible for the observed in vitro phosphorylation of the phosphorylase b subunit is characterized.     

Effects of feeding enrichment on behavior of three species of captive bears

Bears are extremely popular among the zoo-going public, yet while many zoo exhibits have undergone dramatic design changes in recent years, most bears continue to be housed in moated grottos constructed largely of gunite. In these traditional exhibits they frequently demonstrate stereotypic locomotor patterns and are often encouraged by the public to beg. Thus, the manner in which most captive bears are exhibited does not facilitate conservation education. It is possible, however, to provide bears with opportunities to demonstrate species-typical feeding and foraging behaviors, even in standard exhibits. Subjects were four individuals of three bear species. Feeding enrichment was provided to one bear per week during three mornings during the summers of 1989 and 1990. Overall, animals were more active, less passive and less often engaged in abnormal behaviors during sessions with enrichment. Effects showed individual variation and were more profound during the second year of the study, when a greater variety of enrichment items was presented. These results suggest that simple and inexpensive methods of enrichment may have a significant, positive influence on the behavior of captive bears. © 1992 Wiley-Liss, Inc.     

Development of captive bongos (Tragelaphus eurycerus): Activity budgets and developmental milestones

The development of changes in visual orientation, posture, activity budgets, and behavioral milestones was examined in five bongo calves which were observed from birth to 4 or 6 months of age. The only significant change in the calves' visual orientation was an increase in orientation to food. Reclining declined significantly over weeks, while standing increased and moving did not change. Overall, stationary behaviors dominated the activity budget, but decreased significantly over the study. The category that included locomotion, object exploration, and auto-play did not change, while forage and rumination increased significantly. Contrary to expectation, the decline in suckling was not significant. Affiliative interactions with the dam decreased significantly during the study, but increases in affiliation with other bongos were not significant. Levels of agonistic behavior between the calves and their dams and other herd members were low throughout the study, and did not change significantly. One female calf achieved several developmental milestones later than the others. It is suggested that these differences may have been influenced by the dominance status of her dam. The results conform to the general developmental pattern of “hider” bovids. Bongos, however, show somewhat different patterns of social development than eland, which are reported to be more precocial in expression of aggressive and sexual behaviors. © 1992 Wiley-Liss, Inc.     

An interspecific linkage map of mouse chromosome 15 positioned with respect to the centromere

We have used an interspecific backcross between C57BL/6J and Mus spretus to derive a molecular genetic linkage map of chromosome 15 that includes 25 molecular markers and spans 93% of the estimated length of chromosome 15. Using a second interspecific backcross that was analyzed with a centromere-specific marker, we were also able to position our map with respect to the chromosome 15 centromere. This map provides molecular access to many discrete regions on chromosome 15, thus providing a framework for establishing relationships between cloned DNA markers and known mouse mutations and for identifying homologous genes in mice and humans that may be involved in disease.     

Expression of human salivary protein genes

Human proline-rich proteins (PRPs) are polymorphic, homologous in sequence, and linked in a cluster called the human salivary protein complex (SPC). Recently this complex was localized to human chromosome band 12p13.2 (Mamulaet al., Cytogenet. Cell Genet. 39:279, 1985). We have isolated a PRP cDNA, EO27, from a human parotid gland library, identified it by DNA sequencing, and used it to study the molecular and cellular biology of PRP production. Cell-free translation and mRNA characterization with EO27 indicate that the numerous PRPs seen in saliva are produced from relatively few, large precursors, probably by posttranslational cleavage. This supports an hypothesis originally proposed by Friedman and Karn in 1977 (Am. J. Hum. Genet. 29:44A;Biochem. Genet. 15:549) and later supported by biochemical studies (Karnet al., Biochem Genet. 17:1061, 1979) and molecular studies (Mamulaet al., Fed. Proc. 43:1522, 1984; Maedaet al., J. Biol. Chem. 260:1123, 1985). EO27 was also used in this study to localize PRP mRNA production to the acinar cells of the parotid gland byin situ hybridization.     

Behavioral treatment of irritable bowel syndrome: A 1-year follow-up study

Sixteen clients afflicted with irritable bowel syndrome (IBS) were reassessed 1 year following completion of a multicomponent treatment package incorporating progressive muscle relaxation, thermal biofeedback, cognitive therapy, and IBS education. For the 14 patients who kept a 2-week symptom diary, significant reductions in ratings of abdominal pain and tenderness, diarrhea, and flatulence were obtained comparing pretreatment and follow-up symptom-diary ratings. Eleven of 14 clients were improved over pretreatment levels, 57% met the criteria for clinical improvement of at least a 50% reduction in major symptom scores, and all but 1 of 16 rated themselves as subjectively improved.     

Myelogenesis and estimation of the number of axons in the anterior commissure of the chick (Gallus gallus)

Summary By use of light- and electron microscopy the anterior commissure of the chick was studied at different times during development. Between the 19th day of incubation and the 35th day after hatching the cross-sectional area of the anterior commissure, as determined from mid-sagittal sections, undergoes a 6-fold increase in size. Thereafter the area remains fairly constant. The total number of fibres in the anterior commissure was estimated to be 89000. The full complement of fibres is already present by the 19th day of incubation. Myelogenesis occurs mainly between the 19th day of incubation and the 35th day after hatching, concomitant with the increase in cross-sectional area. From the 35th day after hatching, myelinated fibres comprise approximately 40% of the total number of fibres. The median diameter of unmyelinated fibres is about 0.35–0.40 m. The median diameters of myelinated axons and fibres are 0.8–1.0 m and 1.1–1.3 m, respectively.