Chromosomal radiosensitivity of ataxia telangiectasia cells at different cell cycle stages

Summary X-ray induced chromosomal aberrations in peripheral blood lymphocytes as well as in skin fibroblasts from ataxia telangiectasia patients, and from normal individuals were studied. At all stages of cell cycles—namely G₀, G₁, and G₂, more aberrations were induced in AT cells than in normal cells. In addition, AT cells were sensitive to induction of chromosomal aberrations by tritium beta rays from incorporated radioactive thymidine. Possible reasons for the increased sensitivity of AT cells for induction of chromosomal aberrations by ionizing radiations are discussed.     

Differential sensitivity of muntjac lymphocyte chromosomes to mitomycin C,bromodeoxyuridine and hydroxylamine at different cell-cylce stages

Quantitative and qualitative analyses were made of aberrations induced by 3 hitherto well-known mutagens, mitomycin C (MC), 5-bromodeoxyuridine (BUdR and hydroxylamine hydrocholride (HA), in muntjac chromosomes, during different stages of the cell cycle. The sensitivity ro MC was increased in G₁, reached its maximum in early S and was considerably decreased in late S and G₂ stage treated cells. BUdR induced maximal aberrations when given during the synthetic phase and the cells in G₁ and G₂ were least affected. The sensitivity of the cells to HA in terms of induced chromosomal aberrations increased as they moved through the cell cycle, i.e. more damage was observed in cells treated in late S and G₂ stages than in those treated at G₁ and early S stages. While there were defined patterns of cell-cylce stage-dependent sensitivity for all 3 chemicals, the chromosomal sites being preferentially affected by each were found to be specific and invariant at different stages. Thus, it is presumed that the functional state of such “preferred sites” at one or other stage of the cell cycle is the factor responsible for the stage-dependent sensitivity of a cell towards these chemicals.     

Vasa protein is localized in the germ cells and in the oocyte-associated pyriform follicle cells during early oogenesis in the lizard <Emphasis Type="Italic">Podarcis sicula</Emphasis>

The vasa gene, first identified in Drosophila, is a key determinant for germline formation in eukaryotes. Homologs of vasa have been identified and linked to germline development, in many invertebrates and vertebrates. Here, we analyze the distribution of Vasa in early germ cells (oogonia and oocytes) and previtellogenic ovarian follicles of the lizard Podarcis sicula. During most of its previtellogenic growth, the oocyte in this lizard species is structurally and functionally integrated through intercellular bridges with special follicle cells called pyriform cells. The pyriform cells function similarly to Drosophila nurse cells, but are somatic in origin. In the oogenesis of P. sicula, Vasa is initially highly detected in the oogonia, but its levels decrease in early stage oocytes before the onset of pyriform cell differentiation. In the later stages of oogenesis, the high level of Vasa is related with the nurse function of the pyriform follicle cells. These observations suggest that cells of somatic origin are engaged in the synthesis of Vasa in the oogenesis of this lizard.     

Ultrastructural study of vitellogenesis and oogenesis of Metadena depressa (Stossich, 1883) Linton, 1910 (Digenea,Cryptogonimidae), intestinal parasite of Dentex dentex (Pisces,Teleostei)

The ultrastructural organization of the female reproductive system of Metadena depressa, digenean intestinal parasite of Sparidae (Dentex dentex), was investigated by electron microscopy. The vitellogenesis is divided into four stages: stage I, vitellocytes have a cytoplasm mainly filled with ribosomes and few mitochondria; stage II, beginning of the synthetic activity; stage III, active shell globule clusters synthesis; stage IV, mature vitellocytes are filled with shell globule clusters and generally contain several large lipid droplets. Glycogen granules are grouped at the periphery of the cell. The three stages of the oogenesis process take place in the ovary: stage I, oogonia are undifferentiated small cells located at the periphery of the organ; stage II, primary oocytes possess a higher nucleo-cytoplasmic ratio and a nucleus with a nucleolus and synaptonemal complexes indicating the zygotene-pachytene stage of the first meiotic division; stage III, mature oocytes are located in the proximal region of the organ and possess a cytoplasmic chromatoid body and cortical granules in a monolayer close to the periphery of the cell.     

Identification and genetic localization of mRNAs from ovarian follicle cells of Drosophila melanogaster.

RNA synthesis in ovarian follicles of Drosophila melanogaster was studied by methods which eliminate experimentally induced alterations in gene expression. Gel electrophoresis of follicular RNA, labeled after injection of precursors into females, revealed qualitative and quantitative differences in synthesis during the course of oogenesis. A highly heterogeneous group of poly(A)-containing RNAs is produced during much of the course of follicular development. However, post-vitellogenic stages synthesize a small number of stage-specific poly(A)-containing RNAs. During this period, RNA synthesis is known to take place primarily in the follicle cells, which are engaged in the production of the endochorion and exochorion. Two intense bands of nonmitochondrial poly(A)⁺ RNA are labeled between stage 11 and early stage 13. The synthesis of a more heterogeneous group of very small poly(A)-containing RNAs characterizes the last part of oogenesis, stages 13 and 14. Evidence is presented to show that these RNAs are specifically localized in the follicle cells of the egg chamber. We propose that they represent mRNAs for chorion proteins. In situ hybridization of preparations of late stage poly(A)-containing RNA to salivary gland chromosomes revealed two major sites of complementarity, 7E11 and 12E, as well as several minor sites. Experiments in which RNAs were separated on gels prior to hybridization in situ suggested that both the major stage 12-specific RNA bands contained molecules which were complementary to DNA in the 7E11 region. It is particularly interesting that this site is within a small chromosomal interval known to contain the gene ocelliless. Females homozygous for ocelliless have been shown to produce structurally abnormal chorions (Johnson and King, 1974).     

Identification and cytogenetic localization of vitelline membrane messenger RNAs in Drosophila

During stages 9 and 10 of oogenesis in Drosophila the major proteins involved in vitelline membrane (VM) formation are synthesized and secreted by the somatic follicle cells surrounding the oocyte. To identify potential mRNAs involved in VM protein synthesis, newly synthesized poly(A)-containing RNA from egg chambers of different developmental stages was studied. Urea-agarose gel electrophoresis revealed two RNA bands in stage 10 egg chambers in the size range expected for those which encode the smaller VM proteins. These RNA bands, T₁ and T₂, are specifically enriched in stage 10 follicle cell preparations. In vitro translations in reticulocyte lysates in the absence and presence of microsomal membranes showed both RNA bands code for products that are synthesized in precursor forms which are processed to species that comigrate with VM proteins. T₂ directed the synthesis of processed species that comigrated with the 23- to 24-kDa and 17.5-kDa VM proteins (J. Fargnoli and G. L. Waring, 1982, Dev. Biol. 92, 306–314) while the T₁ translation product comigrated with the 14-kDa protein. To determine the cytogenetic location of the genes encoding T₁ and T₂ RNAs, radiolabeled T₁ and T₂ RNAs were hybridized in situ to salivary gland chromosomes. The results suggest that the structural genes coding for the small vitelline membrane proteins are localized at two sites on the second chromosome: 39DE and 42A.     

Oogenesis and the ovarian cycle in Salamandra salamandra infraimmaculata Mertens (Amphibia; Urodela; Salamandridae) in fringe areas of the taxon's distribution

Reproductive cycle and oogenesis were studied in specimens of Salamandra salamandra infraimmaculata Mertens that inhabit fringe areas of the taxon's distribution in the Mediterranean region. Both ovarian mass and length are correlated significantly with body mass and length. Ovarian length is also correlated with the number of oocytes. During the oogenetic cycle six stages in oocyte development were recognized. Three occur during previtellogenesis: stage 1, in which oogonia divide and form cell nests; stage 2 in which oogonia differentiate into oocytes; and stage 3, in which the oocyte cytoplasm increases in volume. In the vitellogenic phase two additional stages, 4 and 5, were recognized: stage 4, in which lipid accumulates in vacuoles in the periphery followed by the appearance of yolk platelets near the cytoplasmic margin; and stage 5, in which oocyte volume increases rapidly due to increased number of yolk platelets until it reaches its maximal size. During postvitellogenesis one stage was recognized: stage 6, in which the beginning of maturation is characterized by movement of the nucleus toward the animal pole. Oogenesis continues year-round. The first four stages were seen in all ovaries examined. The ovarian cycle is independent of season and reproductive stage apart from the number of mature, postvitellogenic oocytes that increases following gestation toward the beginning of spring (March-April). J. Morphol 231:149–160, 1997. © 1997 Wiley-Liss, Inc.     

ORGANIZATION AND REPLICATION ACTIVITY OF THE MITOCHONDRIAL MASS OF OOGONIA AND PREVITELLOGENIC OOCYTES IN XENOPUS LAEVIS

The origin of the mitochondrial mass, previously well characterized in Xenupus diplotene oocytes, has been traced up to oogonia by means of electron microscopy. A polarized organization of the oogonia and of the oocytes of the succeeding stages was observed. The mitochondrial cloud was found to be built up in the centriolar region near the site where the chromosomes will be implanted along the nuclear envelope at the "bouquet" stage. Autoradiographic studies of thymidine incorporation into mitochondrial DNA suggest that mitochondrial DNA synthesis is active throughout this early period of oogenesis.     

Comparative induction of unscheduled DNA synthesis by physical and chemical agents in non-proliferating primary cultures of rat hepatocytes

The incorporation of [³H]thymidine into DNA due to unscheduled DNA synthesis (UDS) induced by N-OH-2-acetylaminofluorene (N-OH-AAF), aflatoxin B₁ (AFB₁), ethyl methanesulfonate (EMS) and ultra-violet light was quantitated by autoradiography and by scintillation spectrometry on acid precipitable macromolecules or DNA insolated by isopycnic banding in cesium chloride (CsCl). Dose-dependent increases in UDS due to N-OH-AAF and AFB₁ treatment were found. Only 2-fold increases at the highest dose levels were found, however, when incorporated [³H]thymidine was quantitated by scintillation spectrometry. Seven, 11, and 25-fold increases in UDS induced by AFB₁, N-OH-AAF and ultra-violet light, respectively, were found when incorporated [³H]thymidine was quantitated by autoradiography, indicating a high sensitivity for detecting ‘long patch’ repair by this technique. Scintillation spectrometry was completely ineffective in detecting EMS-induced UDS, whereas autoradiography demonstrated a small, but significant induction in [³H]thymidine incorporation at high dose levels. The non-proliferative nature of the primary hepatocyte prohibits the uniform radioactive prelabeling of DNA, necessary in other techniques, for the detection of ‘short patch’ repair induced by compounds such as EMS. Therefore, the sensitivity of the primary cultured rat hepatocyte in conjunction with UDS for detecting DNA damage caused by mutagens and carcinogens which induce ‘short patch’ repair may be limited to the autoradiographic analysis of the unscheduled incorporation of [³H]thymidine.     

Site and timing of synthesis of tubulin and other proteins during oogenesis in Drosophila melanogaster

Protein synthetic patterns during oogenesis in Drosophila melanogaster were examined; in particular the site, time, and rate of tubulin synthesis and accumulation during oogenesis were determined. Ovarian proteins were labeled with [³⁵S]methionine in vivo or in organ culure in vitro, and the proteins synthesized in egg chambers of specific developmental stages displayed by two-dimensional gel electrophoresis. A dissection technique was devised to examine proteins synthesized in each of the three cell types present in stage 10B egg chambers. The majority of proteins which were resolved by two-dimensional gel electrophoresis, including tubulin and actin, were synthesized throughout oogenesis and, at least to some extent, in each of the stage 10B cell types. Protein synthesis specific to developmental stage and/or cell type was also observed; for example, two nonchorion proteins were synthesized only in follicle cells and primarily at stage 10. A sensitive and specific radioimmune assay was developed in order to quantitate tubulin accumulation. Synthesis of several α-tubulin subunits and one β-tubulin subunit was observed. The tubulin content per egg chamber increased from 3 ng in stage 9 to 17 ng in stage 14, a period of about 13 hr. An accumulation rate of 1 ng/hr suggests that tubulin mRNA can account for about 4% of the total, nonmitochondrial, poly(A)⁺ RNA of the egg. Analysis of separated cell types at stage 10B revealed that both the follicle and nurse cells synthesize and accumulate appreciable amounts of tubulin. The stage 10B oocyte contains relatively little tubulin but actively synthesizes it. These two complementary analyses demonstrate that the tubulin present in the egg is synthesized within the oocyte-nurse cell syncytium, first in the nurse cells and later in the oocyte.     

昆虫卵子发生调控因素的研究进展

动物卵原细胞形成成熟卵细胞的过程称为卵子发生。昆虫卵子发生在卵巢里进行,经历了卵原细胞的增殖、卵母细胞的生长和卵母细胞的成熟三个阶段。在此过程中,昆虫卵子的发生受到很多内外因素的影响,如基因、细胞因子和环境等。卵子的发生影响着成熟卵细胞的质量以及卵细胞与精子的结合。本文就影响昆虫卵子发生的因素进行综述。     

Morpho-physiological variations in response to NaCl stress during vegetative and reproductive development of rice

The complex nature of plant resistance to adverse environmental conditions, such as salinity and drought requires a better understanding of the stress-induced changes that may be involved in tolerance mechanisms. Here we investigate stress-related morpho-physiological effects during vegetative and reproductive growth in two Japonica rice cultivars (Bomba and Bahia) exposed to a range of NaCl concentrations from the seedling stage. The stress-related detrimental effects were observed either earlier or to a higher extent in cv. Bomba than in Bahia. Damages to the photosynthetic apparatus were related to loss of chlorophyll (Chl) and to a decrease of the maximum potential efficiency of PSII (F _v /F _m), affecting negatively net CO₂ assimilation rate (P _N). Stress-related leaf anatomical alterations were analysed during the vegetative and reproductive stages. The size of bulliform cells as well as dimensions related to the vascular system increased under mild stress but decreased in the longer term or under higher stress level. The pattern of the anatomical alterations observed at the reproductive stage under 20 mM NaCl was reflected in poor panicle development and yield loss, with effects more pronounced in cv. Bomba than in Bahia. In summary, our results show that some physiological and, particularly, leaf anatomical responses induced by NaCl stress are distinctive indicators of sensitivity to salt stress in rice cultivars.     

In vitro biosynthesis of liver cytochrome P450 mature peptide sub-unit by translation of isolated poly(A)+ mRNA from normal and phenobarbital induced rats

The biosynthesis of a cytochrome P₄₅₀ peptide sub-unit by the in vitro translation of total hepatic poly (A)⁺ mRNA in an heterologous cell-free-system is described. The ability of the liver poly (A)⁺ RNA preparations from normal and phenobarbital induced rats to promote protein synthesis and the identification of in vitro synthesized proteins revealed the presence of a cytochrome P₄₅₀ peptide sub-unit presenting the same apparent molecular weight of the native peptide. This fact demonstrates that rat liver poly (A)⁺ mRNA fraction contains an important amount of cytochrome P₄₅₀ peptide messages. Total poly (A)⁺ RNA from rats in an early phenobarbital induction stage exhibits a higher cytochrome P₄₅₀ template activity in good agreement with the enhancement of this hemeprotein concomitantly observed in vivo, in the liver microsomes, it is also concluded that cytochrome P₄₅₀, peptide sub-unit, induced in rat liver by phenobarbital, is translated in its mature form.     

Viscosity regulates apolipoprotein A-1 gene expression in experimental models of secondary hyperlipidemia and in cultured hepatocytes

This study analyzes the relationship of plasmatic colloid osmotic pressure (P_CO) and viscosity with the different hyperlipidemic stages observed in rats with acute liver damage induced by carbon tetrachloride (CCl₄) and in rats with nephrotic syndrome induced by puromycin amino nucleoside (PAN). In both animal models viscosity increases were associated with the induction of the hyperlipidemic stage characterized by an increase of high density lipoproteins (HDL) and steady-state levels (SSL) of apo A-1 mRNA. In both animal models P_CO decreased at early stages of the disease when hyperlipidemia was characterized principally by an increase of total cholesterol and triacylglycerols, but was not associated with the induction of HDL and apo A-1 mRNA. To confirm the in vivo findings, we studied the effect of viscosity on apo A-1 gene expression in an in vitro model using cultured hepatocytes. When medium viscosity was maintained below physiological values, an induction of the SSL of apo A-1 mRNA was observed. By contrast, when medium viscosity was raised to values similar or higher than the physiological range, the SSL of apo A-1 mRNA decreased steadily and after 24 h incubation an almost total inhibition was observed. These results suggest that in both experimental animal models of secondary hyperlipidemia, small viscosity changes below the physiological range, most probably in the interstitial fluid, can induce apo A-1 gene expression at the mRNA level, and that when viscosity reaches physiological values, apo A-1 gene expression is inhibited. Both effects were shown in cultured hepatocytes.     

Accumulation of mitochondrial DNA during oogenesis in Xenopus laevis

The mitochondrial DNA (mtDNA) content of Xenopus laevis oocytes at various stages of oogenesis has been determined by molecular hybridization with ³H-labeled complementary RNA (cRNA). The previtellogenic oocyte less than 250 μm in diameter (stage 1) contains 0.95 ± 0.47 ng of mtDNA. Accumulation of mtDNA proceeds until stage 4 (500–750 μm diameter oocyte), by which time a steady-state level of 4.28 ± 0.40 ng/oocyte is attained. Using the hybridization assay, the stage 6 (full-grown) Xenopus oocyte contains 4.51 ± 0.69 ng of mtDNA, compared to the previously reported value of 3.8 ng determined by direct measurement on the unfertilized egg. There appears to be a reasonable correlation, therefore, between the termination of mtDNA accumulation and the dispersal of the juxtanuclear, mitochondrial aggregate (Balbiani body) at the onset of vitellogenesis in Xenopus. It is concluded that the enormous complement of oocyte mitochondria is accumulated well before the end of oocyte growth and is maintained at a constant level during the remainder of oogenesis, through maturation, fertilization, and on into early development.     

Studies of the induction of mitotic gene conversion by ultraviolet irradiation: II. Action spectra

Action spectra for the induction of intragenic mitotic recombination (gene conversion) at the trp5 locus by UV are presented for three cell stages (T₀, T₉ and T₁₆) taken from synchronously growing cultures of Saccharomyces cerevisiae. The spectra over the range from 230 to 300 nm were taken mostly in 5-nm steps. The peak of action spectra was significantly shifted, regardless of the stage, toward the longer wavelengths as compared with that of the absorption spectrum of DNA (258 nm) or even that of thymine (265 nm). In one extreme case (T₁₆), the peak was shifted 17 nm from the absorption peak of DNA. Further, the spectrum changed its shape at the cell stage advanced from non-dividing (unbudded) (T₀) to a dividing phase (T₁₆). Furthermore, the induction cross section decreased by a large factor (about 40), regardless of the wavelength, in going from T₀ to T₁₆. From observations of the high photoreversibility of induced conversions, the major primary damage was thought to be pyrimidine dimers in the DNA.One plausible explanation, though not quite satisfactory from the quantitative viewpoint for these findings was that the increasing RNA during growth would screen the incident UV differentially with respect to the stage. If this explanation is correct, thymine dimers may still be considered, in spite of the shifts and deformations in the action spectra, as the major primary damage that triggers the long series of processes leading to gene conversion. Conventional methods for obtaining action spectra are discussed in comparison with the present method, which was based on sensitivity parameter a in the proposed dose (t)-frequency (f) relation, f = (at)^α (α is the multiplicity parameter).     

拟目乌贼卵子发生与卵巢发育

为了丰富拟目乌贼(Sepia lycidas)生物学资料, 为人工育苗与养殖提供理论依据, 采用解剖学和组织学的方法, 对水泥池养殖条件下拟目乌贼卵子发生和卵巢发育进行了研究。结果表明: 经过6个月水泥池养殖, 平均体重为256.34 g, 最大体重达到457.08 g, 个别发育成熟, 绝大部分未达性成熟。卵子发生不同步, 根据细胞形态、细胞大小、滤泡细胞形态和卵黄形成情况可分为卵原细胞阶段(卵原细胞期)、原生质生长阶段(无滤泡期、单层滤泡期和双层滤泡期)、间质生长阶段(滤泡内折早期、滤泡内折中期和滤泡内折晚期)和营养质生长阶段(卵黄发生早期、卵黄发生晚期和成熟期), 共4个阶段10个时期。卵巢发育根据外观形态、性腺指数变化和切面上各期细胞所占的比例, 可分为形成前期、形成期、小生长期、大生长期、成熟前期和成熟期6个时期。拟目乌贼繁殖周期为一年。