Accumulation and Spatial Distribution of Poly(A)+RNA in Oocytes and Early Embryos of Drosophila melanogaster

We describe the accumulation and distribution of poly (A)⁺RNA during oogenesis and early embryogenesis as revealed by in situ hybridization with a radio-labeled poly (U) probe. The amount of poly (A)⁺RNA in nurse cell cytoplasm continuously increased untill mid-vitellogenic stage (st. 10), then decreased with the rapid increase of poly (A)⁺RNA in the oocyte (st. 11). The localization of poly (A)⁺RNA at stage 10 was in the anterior region of the oocyte, where it is connected by cytoplasmic bridge to the nurse cells. These observations indicate that most of the poly (A)⁺RNA synthesized in the nurse cells is transferred to the oocyte through the cytoplasmic bridges at stage 10–11. During the remainder of oogenesis (st. 11–14) and during preblastodermal embryogenesis, poly (A)⁺RNA was evenly distributed over the cytoplasm of oocytes and embryos. At blastoderm stage, poly(A)⁺RNA became concentrated in the peripheral region of embryos. Though the somatic nuclei of the blastoderm contained a detectable amount of poly (A)⁺ RNA, the pole cell nuclei did not. The cytoplasmic RNA visualised by acridine orange staining and the poly (A)⁺RNA detected by hybridization with [³H]poly (U) exhibited identical distributions during oogenesis and early embryogenesis. These observations provide a basis to assess the unique distributions of specific RNA sequences involved in early development.     

Scaling of follicular sizes in mammalian ovaries

The scaling of ovarian follicle and oocyte sizes according to body weight ( M , ranging from 0005–500 kg) has been analysed using data obtained from 22 mammalian species in nine orders. The diameters of non-growing (primordial) follicles were correlated significantly with body weight, the relationship being described by the allometric formula y = 0028 M ^0.10. The mean size at which growing follicles began to accumulate extracellular fluid was approximately the same in all species, 0–3 mm diameter. Graafian follicle sizes varied allometrically with body weight as a result of differences in the volumes of follicular fluid rather than those of oocytes, which were relatively similar in eutherian mammals. The statistical significance of the correlation between Graafian and body sizes was increased when the dimensions for an ovulatory quota of follicles were combined because follicles in polyovulating species were disproportionately small. The total Graafian surface areas and volumes were then predicted from body weight by 58–4 M ^0.65 and 18–5 M ^1.06, respectively. Follicular dimensions in the three species of primates were significantly greater than predicted by the allometric relationship. The exponents of these relationships show that the total volume of a set of preovulatory follicles varies approximately isometrically with body weight and, therefore, with the presumptive hormone distribution volume ( M ^1.0). The hypoallometric relationship of follicular surface area demonstrates that, during the course of the evolution of body size, the surface area for secretion has not increased to match the dilution of hormones in the body pool.     

北京油葫芦卵子发生中DNA及RNA动态变化研究

用孚尔根及甲基绿 -派洛宁组织化学染色法了解北京油葫芦Teleogryllusmitratus(Burmeister)卵子发生各时期阶段中卵内DNA及RNA动态变化规律。在卵子发生的最初阶段 ,核中DNA的合成和复制最活跃 ,以后便慢慢减弱 ;而RNA则在第 2阶段合成最旺盛。在卵子发生各个阶段 ,滤泡细胞中DNA ,RNA均为阳性反应 ,并在卵细胞的卵黄形成期活动旺盛 ,为卵母细胞卵黄蛋白形成提供物质基础。卵子发生第 4～ 6阶段 ,滤泡细胞开放时期 ,血淋巴内一些物质可能直接或间接通过滤泡细胞间隙进入卵母细胞内 ,参与卵母细胞的发育和构建。研究表明卵子发生初期卵母细胞的发育和物质构建主要以内源性合成积累为主 ,中后期则有外源性物质的参与。     

Morphometric and ultrastructural characterization of Bos indicus preantral follicles

The aim of the present study was to characterize the ultrastructure of zebu cow preantral follicles (PAFs). Ovarian cortex samples were processed for light and transmission electron microscopy. Primordial follicles consisted of an oocyte surrounded by one layer of flattened or flattened-cuboidal granulosa cells. The oocyte contained a large and usually eccentric nucleus. Most organelles were located at the perinuclear ooplasm. Round shaped mitochondria, which contained electron-dense granules, smooth and rough endoplasma reticulum and a Golgi apparatus were also observed. Vesicles and coated pits were often observed in the cortical ooplasm. In primary follicles, the oocyte was surrounded by one layer of cuboidal granulosa cells. Short microvilli were observed on the oolema. Secondary follicles consisted of an oocyte surrounded by a variable number of layers of cuboidal granulosa cells. Small secondary follicles had an ultrastructure very similar to that observed in primary follicles. At this follicular stage, the zona pellucida was beginning to form around the oocyte. In large secondary follicles, the zona pellucida was totally developed around the oocyte. Several granulosa cell projections could be detected that were encroaching into the zona pellucida and protruding towards the oocyte, where gap junctions were observed between oocyte and granulosa cell membranes. Organelles within the oocyte were located at the periphery of the ooplasm, and clusters of cortical granules were observed. Round mitochondria were abundant in all developmental stages. In conclusion, this study described the ultrastructure of zebu cow PAFs, and some unique characteristics could be observed as compared with what has been reported for follicles of Bos taurus cattle.     

Ovarian development, spawning frequency and batch fecundity in Encrasicholina heteroloba (Ruppell, 1858)

A histological and gravimetric analysis of oocyte development in Encrasicholina heteroloba (Ruppell, 1858) indicated that this species spawns serially and has a group-synchronous mode of ovarian development. A six stage maturity scale, based on both external morphology and oocyte composition, was proposed to classify ovarian development in E. heteroloba . The incidence of females with hydrated oocytes and post-ovulatory follicles in samples from two regions; the south Java Sea and Roviana lagoon, in the Solomon Islands were used to estimate spawning frequency. Estimates of mean inter-spawning intervals ranged from around 2 days in fish from Roviana lagoon to up to 16.7 days in fish from the south Java Sea. Batch fecundity was determined from the number of oocytes in the largest oocyte size class in ripe stage ovaries. Batch fecundity was related to size and was significantly greater for a given size in the Roviana lagoon population ( F = 0.081 × L ^4.89, Roviana population; F = 1.682 × L ^2.83, Jepara population).     

Defects in the archenteron of mouse embryos homozygous for the T–mutation

Abstract. The development of the head process was studied in mouse embryos homozygous for the Brachyury (7) mutation at stages between days 7¹/₂ and 8¹/₂ using scanning electron microscopy. Intact T/T embryos removed from deciduae were distinguished from their normal littermates at the presomite stage on the basis of an abnormally short allantois. In the homozygotes so distinguished, the prenotochordal cells were fewer in the archenteron area, and the typical indentation of the archenteron was not observed. These defects are probably early indicators of the abnormality of the notochord previously described for later stages of development.     

Ultrastructure of the follicular epithelium in the ovary of the domestic duck during the period of slow growth of the oocyte]

The work describes ultrastructural changes in the follicular epithelium and ooplasm in the period of the slow oocyte growth. The morphology of follicular cells (well-developed endoplasmic reticulum, numerous free ribosomes) is considered as evidence of their synaptic activity whereas the morphology of ooplasm indicates to its accumulative function. It is supposed that at this stage nutritive substances enter the oocyte through the intercellular spaces and their absorption is brought about by means of microvilli on the oocyte surface. A specific organelle of the avian egg -- so called transosome, is described. Their structure, transposition and possible function are discussed.     

Crucial role of the local micro-environment in fate decision of neonatal rat NG2 progenitors

Objectives:  The fate choice of neural progenitor cells could be dictated by local cellular environment of the adult CNS. The aim of our study was to investigate the effect of hippocampal tissue on differentiation and maturation of oligodendrocyte NG2 precursor cells.
Materials and methods:  Hippocampal slice culture was established from the brains of 7-day-old rats. NG2 precursor cells, obtained from a 12-day-old mixed primary culture of neonatal rat cerebral hemispheres, were labelled with chloromethyl-fluorescein-diacetete and seeded on the hippocampal slices. After 7–14 days in co-culture, cells were stained with neural markers.
Results:  NG2 cells differentiated predominantly into oligodendrocytes, presenting various stages of maturation: progenitors (NG2), pre-oligodendrocytes (O4) and finally mature GalC-positive cells. However, except for a few cells with astrocyte-specific S100b staining, a considerable number of these cells differentiated into neurons: TUJ⁺ and even MAP-2⁺ cells were frequently observed. Moreover, a certain population of these cells preserved proliferative properties of primary precursor cells, as revealed by Ki67 expression.
Conclusions:  The neuronal micro-environment provided by the culture of hippocampal slices is potent for induction of neurogenesis from oligodendrocyte NG2⁺/PDGFRα⁺/CNP⁺ progenitor cells and promotes their differentiation not only into macroglia but also into neurons. It also sustains their proliferative capacity. The results indicate the crucial role of the local cellular environment in fate decision of primary NG2⁺ multipotent neural progenitor cells, which may affect their behaviour after transplantation into the central nervous system.     

Morphological adaptation and inhibition of cell division during stationary phase in Caulobacter crescentus

During exponential growth, each cell cycle of the α-purple bacterium Caulobacter crescentus gives rise to two different cell types: a motile swarmer cell and a sessile stalked cell. When cultures of C. crescentus are grown for extended periods in complex (PYE) medium, cells undergo dramatic morphological changes and display increased resistance to stress. After cultures enter stationary phase, most cells are arrested at the predivisional stage. For the first 6–8 days after inoculation, the colony-forming units (cfu) steadily decrease from 10⁹ cfu ml⁻¹ to a minimum of 3 × 10⁷ cfu ml⁻¹ after which cells gradually adopt an elongated helical morphology. For days 9–12, the cfu of the culture increase and stabilize around 2 × 10⁸ cfu ml⁻¹. The viable cells have an elongated helical morphology with no constrictions and an average length of 20 μm, which is 15–20 times longer than exponentially growing cells. The level of the cell division initiation protein FtsZ decreases during the first week in stationary phase and remains at a low constant level consistent with the lack of cell division. When resuspended in fresh medium, the elongated cells return to normal size and morphology within 12 h. Cells that have returned from stationary phase proceed through the same developmental changes when they are again grown for an extended period and have not acquired a heritable growth advantage in stationary phase (GASP) compared with overnight cultures. We conclude that the changes observed in prolonged cultures are the result of entry into a new developmental pathway and are not due to mutation.     

[3H]thymidine labels less than half of the DNA-synthesizing cells in the mouse tumour, carcinoma NT

Abstract. We have studied carcinoma NT, a transplantable mouse adenocarcinoma of spontaneous origin. Cells labelled with [³H]thymidine ([³H]TdR) were restricted to a narrow zone around the periphery of this tumour and were also found in rings up to 50 μ m wide, around isolated blood vessels in the central necrotic area. Labelling with [³H]deoxyuridine ([³H]UdR), another DNA synthesis precursor, produced a very different pattern. The labelled zone around the periphery was much wider than with [³H]TdR, and [³H]UdR labelled cells were found up to 110 μ m from isolated vessels. [³H]iododeoxyuridine ([³H]IUdR) gave the same pattern of labelling as [³H]UdR. In the heavily labelled zone, within 1 mm of the tumour periphery, the labelling index (LI) was 51% after [³H]UdR or [³H]IUdR injection, and only 36% with [³H]TdR.
The data show that at least half of the DNA-synthesizing cells in this tumour did not incorporate [³H]TdR. Previous workers reported cell loss factors for carcinoma NT of 60% calculated from [³H]TdR labelling data and 30% from the rate of loss of [¹²⁵I]UdR. The present work suggests that calculations based on [¹²⁵I]UdR data are more likely to be accurate for carcinoma NT than those using [³H]TdR data.     

Ultrastructure of the ovarian follicles in the placentotrophic Andean lizard of the genus Mabuya (Squamata: Scincidae)

We studied the ultrastructural organization of the ovarian follicles in a placentotrophic Andean lizard of the genus Mabuya. The oocyte of the primary follicle is surrounded by a single layer of follicle cells. During the previtellogenic stages, these cells become stratified and differentiated in three cell types: small, intermediate, and large globoid, non pyriform cells. Fluid‐filled spaces arise among follicular cells in late previtellogenic follicles and provide evidence of cell lysis. In vitellogenic follicles, the follicular cells constitute a monolayered granulosa with large lacunar spaces; the content of their cytoplasm is released to the perivitelline space where the zona pellucida is formed. The oolemma of younger oocytes presents incipient short projections; as the oocyte grows, these projections become organized in a microvillar surface. During vitellogenesis, cannaliculi develop from the base of the microvilli and internalize materials by endocytosis. In the juxtanuclear ooplasm of early previtellogenic follicles, the Balbiani's vitelline body is found as an aggregate of organelles and lipid droplets; this complex of organelles disperses in the ooplasm during oocyte growth. In late previtellogenesis, membranous organelles are especially abundant in the peripheral ooplasm, whereas abundant vesicles and granular material occur in the medullar ooplasm. The ooplasm of vitellogenic follicles shows a peripheral band constituted by abundant membranous organelles and numerous vesicular bodies, some of them with a small lipoprotein core. No organized yolk platelets, like in lecithotrophic reptiles, were observed. Toward the medullary ooplasm, electron‐lucent vesicles become larger in size containing remains of cytoplasmic material in dissolution. The results of this study demonstrate structural similarities between the follicles of this species and other Squamata; however, the ooplasm of the mature oocyte of Mabuya is morphologically similar to the ooplasm of mature oocytes of marsupials, suggesting an interesting evolutionary convergence related to the evolution of placentotrophy and of microlecithal eggs. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Chronological Changes in the Accumulation of Poly(A)+ RNA in Developing Cells of Xenopus laevis, with Special Reference to Primordial Germ Cells

Chronological changes in the accumulation of poly(A)⁺RNA in developing cells of Xenopus laevis were traced by in situ hybridization with ³H-poly(U) as a probe. Almost all the somatic cells acquired ³H-poly(U) binding sites by 3.5 days after fertilization (stage 44). At this stage, the primordial germ cells (PGCs), which had just moved to the genital ridges from the deep endodermal cell mass showed moderate ³H-poly(U) binding activity. The silver grains disappeared from the PGCs after stage 47 (6 days after fertilization), while the somatic cells, which constituted the neural tube, genital ridges, kidney and intestine exhibited strong ³H-poly(U) binding activity. Activity for ³H-poly(U) binding reappeared in the FGCs after stage 52 (21 days after fertilization) when sexual differentiation of gonads became detectable morphologically. The chronological accumulation and disappearance of ³H-poly(U) binding activity in the PGCs in contrast to the constant activity in somatic cells is discussed.     

Cellular Commitment for Post-Gastrular Increase in Alkaline Phosphatase Activity in Xenopus laevis Development

Xenopus embryos were dissociated into cells and cultured in Ca²⁺-free medium to study the relationship between the cell-to-cell interaction and macromolecular synthesis. Under the conditions, cells did not aggregate at all, and remained isolated even while they were dividing actively. Synthesis of DNA and protein as studied by the incorporation of (³H)thymidine and (³H)leucine proceeded as in the aggregating cells. Also, the activity to synthesize rRNA, 5S RNA, and heterogeneous RNA as determined by the incorporation of (³H)uridine was not impaired. Such an increase in the activity of alkaline phosphatase, as occurs in embryos after the gastrula stage, was found to be inhibited greatly when early-blastula cells were cultured in the non-aggregating conditions. However, we found here that the inhibition was not observed with cells isolated from late-blastulae. Therefore, it appears that the increase in the activity of alkaline phosphatase during post-gastrular stages is dependent on some cellular commitment which may be established by cell-to-cell contact during the blastula stage.     

Synthesis of Collagen-Like Proteins in Embryonic Organs of the Sea Urchin, Hemicentrotus pulcherrimus

In sea urchin embryos exposed to ¹⁴C-proline at 20°C for 3 hr at the gastrula, prism or pluteus stage, ¹⁴C-radioactivity was found in hot acid-extractable proteins, in which more than 4% of the radioactivity was detectable in hydroxyproline residues. In these embryos, ¹⁴C-radioactivity in collagen-like proteins was found in the archenteron, spicule and embryo-wall cells. The rate of synthesis of collagen-like proteins was highest in the archenteron in the mid-gastrula stage, in the embryo-wall cells in the prism stage and in the spicule in the pluteus stage. The rate of synthesis decreased in the archenteron and increased in embryo-wall cells in the period between the mid- and late-gastrula stages, when the rate of synthesis in the spicule was quite low. Thereafter, the rate decreased slightly in the embryo-wall cells, was maintained in archenteron and increased markedly in the spicule. The rates of synthesis of collagen-like proteins are high in these embryonic organs at stages at which development and growth respectively, occur in embryos. Therefore, synthesis of collagen-like proteins probably supports morphogenesis in these embryonic organs.     

BIOCHEMICAL AND CYTOLOGICAL EXAMINATION ON THE INITIATION OF RIBOSOMAL RNA SYNTHESIS DURING GASTRULATION OF XENOPUS LAEVIS

Embryos of Xenopus laevis at stage 6 were labeled with ¹⁴CO₂ for 4 hr and then allowed to develop under nonradioactive conditions until they reached stage 9, 10, 11 or 12. RNA was extracted and electrophoresed on a polyacrylamide-agarose gel. From the pattern of newly synthesized RNAs, the incorporation into 18S and 28S ribosomal RNAs was measured. At the same time, the specific radioactivity of nucleoside triphosphates in the acid-soluble fraction was determined. On the basis of the results obtained, the absolute amounts of the RNAs synthesized were calculated. The results show that the synthesis of the ribosomal RNAs begins, or is at least markedly activated, around stage 10. Moreover, cytological examination has shown that cells with nucleolated nuclei appeared between stages 9 and 10 and increased thereafter.
Thus, from the results of these studies along two different lines, it can safely be concluded that the initiation of 18S and 28S RNA synthesis takes place around stage 10.     

Exogut Formation by the Treatment of Sea Urchin Embryos with Ascorbate and α-Ketoglutarate

In sea urchin embryos at the stages from hatch out to the pluteus stage, [¹⁴C]proline incorporation into hot trichloroacetic acid TCA-extractable proteins occurred during an exposure to [¹⁴C]proline for 3 hrs at 20°C. The rate of [¹⁴C]proline incorporation into hot TCA-extractable proteins was higher in gastrulae and plutei than in blastulae. Percentage of [¹⁴C]hydroxyproline residue to whole radioactivity of the hot TCA-extractable proteins was quite low at the blastula stage and increased exponentially during futher development. Production of [¹⁴C]hydroxyproline residue at the blastula stage, as well as at the later stages, was stimulated by ascorbate and α-ketoglutarate, activators of protocollagen proline hydroxylase, and inhibited by α, α'-dipyridyl, an inhibitor of this enzyme. It is also probable that the enzyme in the embryos is not fully activated because of low amounts of activating substances. These suggest that blastulae,…, also have a potency of protocollagen hydroxylation. Blastula kept in sea water containing ascorbateand α-ketoglutarate became undeveloped embryo with large exogut. Gastrula developed normally to pluteus even in the presence of these compounds. The embryos, kept in sea water containing these compounds from fertilization to hatch out, also developed normally. Exogut formation in the embryos treated by these compounds, as well as normal archenteron formation, was inhibited by α, α'-dipyridyl.     

Change of specific T cells in an emerging neonatal infectious disease induced by a bacterial superantigen

A new epidemic, NTED, has recently occurred in Japan. The cause of NTED is a bacterial superantigen, TSST-1. The aim of the present study was to analyze the change in Vβ2⁺ T cells reactive to TSST-1 in NTED in order to establish T-cell-targeted diagnostic criteria for NTED. Blood samples from 75 patients with clinically diagnosed NTED were collected from 13 neonatal intensive care units throughout Japan. We investigated the percentages of Vβ2⁺, Vβ3⁺ and Vβ12⁺ T cells and their CD45RO expressions in the samples using flow cytometry. In 18 of the 75 patients, we conducted multiple examinations of the T cells and monitored serial changes. The Vβ2⁺ T-cell population rapidly changed over three phases of the disease. Whereas the percentage of Vβ2⁺ T cells was widely distributed over the entire control range, CD45RO expression on Vβ2⁺ T cells in CD4⁺ in all 75 patients was consistently higher than the control range. Patients cannot necessarily be diagnosed as having NTED based on expansion of Vβ2⁺ T cells alone in the early acute phase. Instead, CD45RO expression on specific Vβ2⁺ cells is a potential diagnostic marker for a rapid diagnosis of NTED. We present three diagnostic categories of NTED. Fifty patients (66.7%) were included in the category 'definitive NTED'. It is important to demonstrate an increase of Vβ2⁺ T cells in the following phase in cases of 'probable NTED' or 'possible NTED'.     

Effect of Verticillium lecanii on biological characteristics and life table of Serangium japonicum (Coleoptera: Coccinellidae), a predator of whiteflies under laboratory conditions

Effects of entomopathogenic fungus Verticillium lecanii on biological characteristics and life table of Serangium japonicum , a predator of whiteflies against five different conidial concentrations (1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, and 1×10⁸ conidia/mL) were studied under laboratory conditions. The developmental periods for all immature stages (from eggs, 1st, 2nd, 3rd, 4th instar nymph and pupae up to emergence) among the treatments were significantly different when compared to that of control, and the longest development period was observed as treated with 1×10⁸ spore/mL. However, no significant difference on the percent survival of all immature stages was observed among the treatments and control. Also, there were no significantly different effects of V. lecanii on mean generation time, intrinsic rate, the finite rate of increase and longevity of S. japonicum among the treatments and control.     

Denitrifying population dynamic and evolution of nitrogen gas during a composting process

Abstract Little information exists about nitrogen losses through microbial activity during treatment of solid urban waste (SUW) by processes such as composting. In the present study, in addition to evaluating the pattern of nitrogen losses by denitrification at different stages of the process, a comparison between the method of Pochon and Tardieux, and an improved gas chromatographic method for estimating denitrifying populations was undertaken, Though the MPN (Most Probable Number) enumerations were higher using the colorimetric method than the gas chromatographic one, the patterns of the two graphs showing numbers of denitrifiers during composing were the same. The highest numbers were revealed immediately after loading the reactor (10⁷–10⁸/g d.w.), lower numbers of denitrifiers were found in the second sampling corresponding to the thermophilic phase (10³–10⁴/g d.w.). These numbers increased gradually as the waste material stabilized (10th to 123rd day of composting) to again reach values of 10⁷–10⁸/g d.w.