Induction of DNA synthesis by 2,4-dichlorophenoxyacetic acid during callus induction in Jerusalem artichoke tuber tissues

During the induction of DNA synthesis in Jerusalem artichoke (Helianthus tuberosus L.) tuber by 2,4-D, the 2-¹⁴C-2, 4-D from the agar medium rapidly incorporated into the ethanol soluble and insoluble fractions. Although the 2,4-D level in the ethanol soluble fraction decreased on transplantation of the tissue from the 2-¹⁴C-2,4-D medium to medium without the auxin, its level in the buffer-soluble and -insoluble macromolecular fractions increased. The purified, buffer-insoluble macromolecules were chromatin. The 2,4-D binding to chromatin particularly increased during DNA synthesis. The histone contents of chromatin decreased as DNA synthesis progressed. The polyacrylamide gel electrophoretic patterns of the histones showed a decrease in the moderately lysine-rich histone fraction as compared to other fractions. Thus, the decrease in the histone level caused by 2,4-D and the presence of the 2,4-D moderately lysine-rich histone complex may be closely related to the induction of DNA synthesis by 2,4-D in cells.     

VITELLOGENIN IN THE EGGS OF THE COCKROACH, BLATTELLA GERMANICA: PURIFICATION AND CHARACTERIZATION

Vitellogenin in the eggs of Blattella germanica was solubilized with solutions at high salt concentrations and high pH. This protein was purified by ammonium sulfate precipitation, acetic acid precipitation, DEAE-cellulose chromatography, and hydroxylapatite chromatography, into a chromatographically homogeneous state. By sucrose density gradient centrifugation, the purified vitellogenin was resolved into two components. The relative amounts of the two components varied according to the pH of the solution. An equilibrium seemed to exist in the interconversion between them when the conditions of the solution were fixed. It is suggested that aggregation and disaggregation of the vitellogenin molecules may account for the apparent heterogeneity.     

Localization of Acrosomal Proteinase after Vesculation of the Acrosome

The freshly ovulated egg is covered by the zona pellucida and follicular-cell layer. The former is reported to dissolved by acrosomal proteinase and the latter may be dispersed by hyaluronidase. If these two enzymes have different targets, two possibilities can be taken into consideration concerning the timing of release of the two enzymes from the acrosome.
Since hyaluronidase is considered to be released immediately after the acrosomal reaction at the follicular-cell layer, this work was undertaken to examine the timing of release of acrosomal proteinase after vesiculation of the acrosome, by demonstrating the site of the proteinase histochemically. The results indicated that the acrosomal proteinase disappeared immediately after vesiculation of the acrosome. This result suggests that spermatozoa that show vesiculation of the acrosome in the follicular-cell layer probably lose acrosomal proteinase in this layer. Thus the spermatozoon probably does not attack the follicular-cell layer and zona pellucida, one by one by release sequential of hyaluronidase and acrosomal proteinase.     

Can Once Neuronally Differentiated Cells of Neural Retina Be Lentoidogenic in Cell Culture?*

Cells dissociated from neural retina of 3.5-day-old chick embryos transdifferentiated extensively into lens cells under the conditions of a cell culture for 3 to 4 weeks. In early satges of cell culture by about 10 days, cultures consisted of small round cells often with cytoplasmic processes(N-cells) and flattened epithelial cells (E-cells). Only N-cells were stained with a fluorescent dye Merocyanine 540. When cells harvested from early cultures were separated into two fractions by centrifugation in Percoll gradient, the specific activity of choline acetyltransferase was much higher in the fraction consisting mainly of N-cells than in other fraction mainly of E-cells. Continuous daily observations as well as cinematographic observations of living cultures indicate that lentoid bodies were often formed in the locations where clusters of N-cells had been found in early stages of culturing. The possibility of transdifferentiation of N-cell clusters into lentoid bodies is discussed.     

Effect of Auxin and Cytokinin on Newly Synthesized Proteins of Cultured Douglas Fir Cytoledons

Newly synthetized cyloplasmic proteins of Douglas fir cotyledon culture capable of initiating adventitious bud formation (bud culture) were compared with those of morphologically distinct types of cultures, bud-callus culture (some adventitious buds with callus), and callus culture (no adventitious buds; only callus). SDS polyacrylamide gel electrophoretic profiles of double labeled (¹⁴C-and ³H-) proteins showed that a persistent increase of low molecular weight proteins (16,000 to 20,000 daltons) was associated with bud culture. Similar electrophoretic profiles of proteins extracted in the absence and in the presence of protease inhibitor (4-mcthylbenzene sulfonyl fluoride) or phenolic compounds absorbents (Dowex I-X8 and polyvinylpolypyrolidine) were obtained indicating that the detected low molecular weights of these proteins represented the real monomeric state of these molecules.     

DIFFERENTIATION AND TRANSDIFFERENTIATION IN VITRO OF NEURAL RETINA FROM MUTANT CHICKENS WITH HYPERPLASIC LENS EPITHELIUM1)

Mutant chickens, Hy-1 and Hy-2, show abnormalities in growth and differentiation of the lens epithelium. In this study, neural retinal cells (NR cells) from 3.5-day-old embryos of these mutants were cultured, and the differentiation in vitro was compared with the cells of the normal strain. Hy-1 cells in vitro were characterized by a delay in the first appearance of neuronal cells (N-cells) and by excessive production of this cell type at later stages. By contrast, the Hy-2 cells were indistinguishable from the normal cells in the early phase of culturing. In spite of the marked difference of Hy-1 NR cells in neuronal differentiation up to about 7 days in culture, the transdifferentiation of lens and pigmented cells occurred to a similar extent and with the same time schedule as cultures of normal cells. A number of lentoid bodies were formed by about 10 days. The relative composition of the three major classes of crystallins in transdifferentiated lens cells was almost identical between normal and Hy-1 strains. The results were discussed in comparison with the previous results of cell culture of NR of 8-day embryonic mutant chickens, and it was concluded that the process of transdifferentiation in cell culture is different between NR from 3.5-day-old and 8-day-old embryos.     

DEVELOPMENT OF THE EMBRYONIC CHICKEN THYMUS III. UNEQUAL DIVISION OF LYMPHOID CELLS

Cell division of thymus lymphoid cells from 11- to 17-day old embryonic chickens, as well as chickens just after hatch was investigated on cell smears stained with Giemsa. Unequally dividing cells were observed in the developmental stage of thymocytes. At the telophase of such cells, the cytoplasm of one of two future daughter cells was apparently larger in amount and was sometimes stained deeper than the cytoplasm of its counterpart. Unequal division was also observed in pro-, meta- and anaphase; sometimes a dividing cell had a large cytoplasmic process belonging to one hemisphere, suggesting that only one of the two daughter cells would receive the cytoplasmic process through cell division.
The incidence of unequal division calculated by a rough estimation was around 10% of the total cell division between 11 and 13 days of embryonic development, and decreased progressively thereafter.     

TRANSPLACENTAL EFFECT OF ETHINYL ESTRADIOL ON MOUSE VAGINAL EPITHELIUM *

The effect of prenatal administration of ethinyl estradiol (EE) on the vaginal epithelium of adult mice was examined histologically. The mice were the offspring of JCL/ICR strain mice given orally 0.02 mg/kg body weight/day or 0.01 mg/kg/day of EE dissolved in olive oil from day 11 to day 17 of gestation at a stage when the urogenital sinus has just appeared in the embryos. The control mice were offspring of those fed with the vehicle alone. Autopsies were performed at 10 to 14 weeks of age. Another group of mice exposed to 0.02 mg/kg/day of EE or vehicle alone in utero, were spayed at 16 weeks of age and killed at 32 weeks of age. In the experimental nonspayed mice, hyperplasia of the vaginal epithelium with intense cornification was seen. The epithelium was significantly thicker than in the controls and showed an EE dose-response relation. One of the 16 mice exposed to 0.01 mg/kg/day of EE in utero had cystic or gland-like structures in the stroma and mucus-secreting cells in the surface epithelium consisting of columnar cells. In some experimental spayed mice, vaginal hyperplasia with cornified epithelium and hypertrophy of the ovarian interstitial tissue without corpus luteum were seen. These results indicate that EE can cross fetal membranes and affect undifferentiated cells in the urogenital sinus and/or Müllerian epithelium.     

IMMUNOHISTOCHEMICAL DETECTION OF VITELLOGENIN IN THE OVARY AND FAT BODY DURING A REPRODUCTIVE CYCLE OF THE COCKROACH, BLATTELLA GERMANICA

Vitellogenin was immunohistochemically demonstrated in the ovary and fat body during a reproductive cycle. In terminal oocytes of 4-day adults, vitellogenin began to appear in yolk spherules at the cell periphery. The vitellogenin-containing spherules increased in size and number to occupy the whole cell 2 days later. In the female fat body, vitellogenin began to appear in 3-day adults. It was distributed diffusely in the cytoplasm, at a much lower concentration than in the oocytes, during the vitellogenic period. In 11-day adults whose vitellogenesis had terminated, a higher concentration of vitellogenin was found in the cytoplasmic inclusions of the fat body. Vitellogenin was not detected in the male fat body.