Dynamics of vitellogenin uptake in Aedes aegypti as demonstrated by trypan blue

Trypan blue has been shown to be a reliable indicator of the micropinocytotic uptake of vitellogenin by developing oöcytes. Trypan blue was injected into the mosquito Aedes aegypti to determine at what times after the blood meal vitellogenin was taken up. Histological sections examined by light microscopy showed that trypan blue began to be sequestered from 2 to 5 hr after the blood meal. Any association between dye and ovariole ended from 39 to 42 hr after the blood meal, in which period no dye was incorporated into spheres of yolk protein. Of the times investigated in this experiment, the greatest amount of dye was seen in the oöcyte at 24 hr after the blood meal. The onset and conclusion of trypan blue uptake correspond with the related events in the synthesis of vitellogenin by the fat body. The presence of trypan blue in occasional interfollicular spaces suggests that the route of entry of vitellogenin in Aedes aegypti is indeed an interfollicular one.     

凡纳滨对虾卵母细胞卵黄发生的超微结构

利用电镜研究凡纳滨对虾卵母细胞卵黄发生的全过程。结果表明 :凡纳滨对虾卵黄的发生是双源性的。卵黄发生早、中期是内源性卵黄大量合成的阶段 ,卵黄发生中、后期则以外源性卵黄的合成为主。内源性卵黄主要由内质网、线粒体、核糖体、溶酶体、高尔基器等多种胞器活跃参与形成。其中数量众多的囊泡状粗面内质网是形成内源性卵黄粒的最主要的细胞器 ;部分线粒体参与卵黄粒的合成并自身最终演变为卵黄粒 ;丰富的游离核糖体合成了大量致密的蛋白质颗粒并在卵质中直接聚集融合成无膜的卵黄粒 ;溶酶体通过吞噬、消化内含物来形成卵黄粒和脂滴 ,且方式多样 ;高尔基器不直接参与形成卵黄粒。外源性卵黄主要通过卵质膜的微吞饮活动从卵周隙或卵泡细胞中摄取外源物质来形成     

Observations on the penetration and uptake of trypan blue in embryonic membranes of the mouse

Summary Pregnant female Swiss albino mice were injected with 0.4 ml of 1% solution of trypan blue on day 7 1/2, 8 1/2, 10 1/2 and 11 1/2 of gestation. Animals were sacrificed 1–6, 24, 48, 72 and 120 hours after injection and the localization of trypan blue granules was determined in histological sections.Five hours after injection, the dye began to accumulate in the proximal yolk sac epithelium, and within 24 and 48 hours this cell layer was heavily loaded with dye. The dye never became detectable in any other membranes or the embryo proper. This pattern of dye distribution was the same whether injection was initiated at the 7 1/2 8 1/2 day stage of gestation or was delayed until the 10 1/2–11 1/2 day stage.Histochemical tests for acid phosphatase and monoamine oxidase revealed that the cells of the vitelline membrane from embryos of trypan blue-injected mothers stained very much less intensely than the vitelline membrane tissue from saline-injected controls.Implications of these observations for theories of action of trypan blue as a teratogenic agent were discussed.Dedicated to Professor Berta V. Scharrer on her 60th birthday in friendship and gratitude.This study was supported by USPHS Research Grant NB 01716 from the National Institute for Neurological Diseases and Blindness, National Institutes of Health and by Grant NIH ST 1 GM 102This work was carried out in the laboratory of Dr. Max Hamburgh, Department of Anatomy, Albert Einstein College of Medicine, while Dr. Laslo Nebel was a visiting professor on sabbatical leave from the Hebrew University, Hadassah Medical School, Jerusalem, Israel.Acknowledgment is made to Mr. Dan DiDomizio, who collaborated as a technical assistant in this project, in partial fulfillment for research credit as an undergraduate student in the Department of Biology of the City College of New York.     

东方扁虾卵子发生的超微结构

根据卵细胞的形态、内部结构特征及卵母细胞与滤泡细胞之间的关系,东方扁虾的卵子发生可划分为卵原细胞、卵黄发生前卵母细胞、卵黄发生卵母细胞和成熟卵母细胞等四个时期。卵原细胞胞质稀少,胞器以滑面内质网为主。卵黄发生前卵母细胞核明显膨大,特称为生发泡;在靠近核外膜的胞质中可观察到核仁外排物。卵黄发生卵母细胞逐渐为滤泡细胞所包围;卵黄合成旺盛,胞质中因而形成并积累了越来越多的卵黄粒。东方扁虾卵母细胞的卵黄发生是二源的。游离型核糖体率先参与内源性卵黄合成形成无膜卵黄粒。粗面内质网是内源性卵黄形成的主要胞器。滑面内质网、线粒体和溶酶体以多种方式活跃地参与卵黄粒形成。卵周隙内的外源性物质有两个来源:滤泡细胞的合成产物和血淋巴携带、转运的卵黄蛋白前体物。这些外源性物质主要通过质膜的微吞饮作用和微绒毛的吸收作用这两种方式进入卵母细胞,进而形成外源性卵黄。内源性和外源性的卵黄物质共同参与成熟卵母细胞中富含髓样小体的卵黄粒的形成。卵壳的形成和微绒毛的回缩被认为是东方扁虾卵母细胞成熟的形态学标志。       

Immunocytochemical localization of ribonuclease in yolk granules of adult Rana cttesbeiana oocytes

To determine the localization of the pyrimidine-guanine sequence-specific ribonuclease in Rana catesbeiana (bullfrog) oocytes, the RNase was first isolated and used to prepare a specific rabbit antiserum. Only one protein of similar molecular size to the RNase was immunoprecipitated from ovary homogenate by the antiserum, but two bands were observed by Western blotting analysis. These two proteins were shown by further purification of antibody and Western blotting analysis to have similar antigenicity. Immunoprecipitation and Western blotting of tissue homogenates showed that the RNase was found predominantly in the ovary, but not in other tissues. The specific localization of the RNase was determined by immuno-electron microscopy of oocyte sections incubated with the specific antiserum; the yolk granules, but not other organelles, were found to contain the RNase. Most of the RNase was evenly distributed in the lateral amorphous area of the yolk granule but not in the central yolk crystal area which contains stored vitellogenin proteins. Our results indicate that the RNase is compartmentalized in the yolk granules of oocytes, which might prevent damage to cellular RNAs.     

Fluorescence of Trypan blue in frozen-dried embryos of the rat

Freeze-drying and fluorescence microscopy techniques were combined to create a sensitive method for the visualization of the teratogenic dye, Trypan blue, in both protein-bound and free forms. In the development and initial application of this method, visceral yolk sacs of several gestational ages as well as normal appearing, 12-day embryos obtained from dye-injected rats were utilized. Observations on paraffinized sections of the yolk sac placentae demonstrated that only the protein-bound form of the dye exists in the yolk sac cavity whereas both forms of the dye exist in supranuclear regions of cells of the visceral endoderm. Paraffin sections of the normal appearing, 12-day embryos displayed the protein-bound form of dye within lumina of mid- and hind-gut, and both forms of dye in the primitive mucosa of mid- and hind-gut. The advantages of the method are derived not only from the use of fluorescence microscopy but also from the avoidance of solvents that are employed in more routine microtechniques.     

Cytotoxicity as measured by trypan blue as a potentially confounding variable in the in vitro alkaline elution/rat hepatocyte assay

Rat hepatocytes treated in vitro with A2RA, an angiotensin II receptor antagonist, displayed increased level of DNA-strand breaks as determined by alkaline elution, without an appreciable increase in cytotoxicity as determined by a trypan blue dye exclusion assay at harvest. The alkaline elution profile appeared to have two components: a rapidly eluting component detected in the first fraction collected (often associated with DNA from dead or dying cells), followed by a more slowly eluting component detected in the subsequent fractions. Further analysis of hepatocytes treated with A2RA by pulsed-field gel electrophoresis and neutral elution revealed significant levels of DNA double-strand breaks. Electron microscopy (EM) showed pronounced damage to mitochondria; although cell blebbing was seen using both EM and light microscopy, the plasma and nuclear membranes appeared intact when examined by EM. Cellular ATP levels decreased precipitously with increasing doses of A2RA, falling to less than 10% of control values at a dose of 0.213 mM A2RA, a concentration showing 100% relative viability by trypan blue at harvest. Thus, whereas in our experience trypan blue dye exclusion accurately reflects cytotoxicity induced by the majority of test agents, in this rather unusual case, trypan blue did not accurately reflect compound-induced cytotoxicity at harvest since there was no concurrent loss of membrane integrity. However, when hepatocytes treated with A2RA were incubated for either 3 h or 20 h in the absence of compound, a sharp, dose-dependent decline in viability was observed using trypan blue dye exclusion. Together with the initial, dose-dependent drop in the alkaline elution curve, these data suggest that the observed DNA double-strand breaks arose as a consequences of endonucleolytic DNA degradation associated with cytotoxicity, rather than by a direct compound-DNA interaction. Since DNA double-strand breaks behave under alkaline denaturing conditions as two single-strand breaks and can therefore produce increases in the alkaline-elution slope values, a necessary criteria for a valid positive result in this assay is that cytotoxicity by trypan blue dye exclusion will not be greater than 30%. Our data, however, indicate that interpretation of the elution assay as a test for genotoxicity can still be confounded by the failure of the trypan blue dye exclusion assay to reflect cytotoxicity in the unusual instance when there is no concurrent, immediate loss of membrane integrity.     

Dissimilar Staining Properties of Purified and Certified Toluidine Blue

Certified toluidine blue (National Aniline Co.). applied to sections of frog blastulae, stained the nuclei light blue and left the yolk platelets either unstained or light blue. Purified toluidine blue (also National Aniline Co.) stained the nuclei a deep blue and the yolk platelets a brilliant pink with deep blue borders. Some of the observations suggest that this difference in staining behavior is due to the presence of an inhibitor in the certified dye, which suppresses the metachromatic staining of the platelets and reduces the intensity of the nuclear staining. Unsuccessful attempts were made to remove the inhibitor by salting out the certified dye and washing it with alcohol or by extracting it with chloroform. Details of these attempts, and of other experiments designed to identify the stainable substrates in the yolk platelets are given in the text.     

Oocyte-follicle cell differentiation in two species of amphineurans (Mollusca), Mopalia mucosa and Chaetopleura apiculata

Differentiating oocytes and associated follicle cells of two species of amphineurans (Mollusca) Mopalia muscosa and Chaetopleura apiculata have been studied by techniques of light and electron microscopy. In addition to the regularly occurring organelles, the ooplasm of young oocytes contains large, randomly situated, basophilic regions. These regions are not demonstrable in mature eggs. As oocytes differentiate, lipid, pigment and protein-carbohydrate yolk bodies accumulate within the ooplasm. Concomitant with the appearance of pigment and the protein carbohydrate containing yolk bodies, the saccules of the Golgi complex become filled with a dense material. Associated with the Golgi complex are cisternae of the rough endoplasmic reticulum which are filled with an electron opaque substance which is thought to be composed of protein synthesized by this organelle. That portion of the cisternae of the endoplasmic reticulum facing the Golgi complex shows evaginations. These evaginations are thought to finalize into protein containing vesicles that subsequently fuse with the Golgi complex. Thus, the Golgi complex in these oocytes might serve as a center for packaging and concentrating the protein used in the construction of the protein containing pigment or protein-carbohydrate yolk bodies. The suggestion is made that the Golgi complex may also synthesize the carbohydrate portion of the formentioned yolk bodies. In an adnuclear position in young oocytes are some acid mucopolysaccharide containing vacuolar bodies. In mature eggs, these structures are found within the peripheral ooplasm and we have referred to them as cortical granules. There is no alteration of these cortical granules during sperm activation.     

Fluorescence of trypan blue in frozen-dried embryos of the rat

Summary Freeze-drying and fluorescence microscopy techniques were combined to create a sensitive method for the visualization of the teratogenic dye, Trypan blue, in both protein-bound and free forms. In the development and initial application of this method, visceral yolk sacs of several gestational ages as well as normal appearing, 12-day embryos obtained from dye-injected rats were utilized. Observations on paraffinized sections of the yolk sac placentae demonstrated that only the protein-bound form of the dye exists in the yolk sac cavity whereas both forms of the dye exist in supranuclear regions of cells of the visceral endoderm. Paraffin sections of the normal appearing, 12-day embryos displayed the protein-bound form of dye within lumina of mid- and hind-gut, and both forms of dye in the primitive mucosa of mid- and hind-gut. The advantages of the method are derived not only from the use of fluorescence microscopy but also from the avoidance of solvents that are employed in more routine microtechniques.Supported in part by the Oregon Heart Association and by the Medical Research Foundation of Oregon, grant 7513     

The dynamics of yolk deposition in the desert locust Schistocerca gregaria

Injection of the protein dye Fast Green or the fluid-phase probe fluorescein dextran into the haemolymph of vitellogenic female desert locusts (Schistocerca gregaria) resulted in their incorporation into oocytes. We used Fast Green to study the physical dynamics of yolk deposition during vitellogenesis. Timed maternal injections of Fast Green reveal that yolk deposition and oocyte growth are inextricably linked during vitellogenesis, and that little or no yolk movement occurs within oocytes prior to embryogenesis. The yolk granules laid down early during vitellogenesis lie at the centre of the egg, with yolk granules deposited later packed around these, such that they lie progressively closer to the eventual egg surface. In contrast, during early embryogenesis yolk granules migrate in a manner that closely resembles the movement of early cleavage nuclei. We find fluorescein dextran to be a clear, robust and developmentally inert marker for the timing of maternal injections relative to vitellogenesis in S. gregaria, and we propose its use in parental RNAi or morpholino knockdown experiments. With such experiments in mind, we show that fluorescein-labelled DNA oligonucleotides are internalized within oocytes during vitellogenesis. However, neither Fast Green, fluorescein dextran nor fluorescein-labelled DNA oligonucleotides are detectably transferred from yolk granules to embryonic cells during embryogenesis, and our initial attempts at parental RNAi using maternal injections of dsRNA targeted to late vitellogenesis have proved unsuccessful.     

The ultrastructural patterns of germinal and yolk granules in oocytes and embryonic cells of the holothurian Apostichopus japonicus

The yolk germinal granules in oocytes and embryonic cells of Apostichopus japonicus were studied by transmission electron microscopy. Analysis of the features of synthesis and utilization of yolk granules made it possible to reveal ultrastructural criteria to distinguish between granules of the forming and utilized yolk, and germinal granules. Based on these findings, the authors suppose that identification of germ plasm elements in oocytes and embryonic cells of A. japonicus is quite possible with ultrastructural analysis only, and does not require utilizing molecular markers.     

The ultrastructural patterns of germinal and yolk granules in oocytes and embryonic cells of the holothurian Apostichopus japonicus

The yolk germinal granules in oocytes and embryonic cells of Apostichopus japonicus were studied by transmission electron microscopy. Analysis of the features of synthesis and utilization of yolk granules made it possible to reveal ultrastructural criteria to distinguish between granules of the forming and utilized yolk, and germinal granules. Based on these findings, the authors suppose that identification of germ plasm elements in oocytes and embryonic cells of A. japonicus is quite possible with ultrastructural analysis only, and does not require utilizing molecular markers.     

Uptake of the yolk protein, lipovitellin, by developing crustacean oocytes

A variety of cytochemical techniques were used to demonstrate how crustacean lipovitellin accumulates within the egg. It was found that a protein serologically identical to the lipovitellin of yolk spheres was present in the hemolymph of vitellogenic crustaceans, but was absent from the hemolymph of males and immature females.In the three crustacean species studied (Uca pugilator, Cambarus clarkii, and Libinia emarginata), pinocytosis of fluorescein-conjugated lipovitellin and trypan blue occurred only during those periods when oocytes were accumulating yolk.It may be concluded from the present studies that yolk spheres develop in crustacean eggs primarily through micropinocytotic uptake of lipovitellin from the hemolymph, although other oocyte proteins appear to be made in the oocyte.     

Studies on the mechanism of trypan blue teratogenicity in the rat developing in vivo and in vitro

Trypan blue is a potent teratogen in vivo and in vitro in the rat. Many of the abnormalities produced by trypan blue--including swollen neural tube and pericardium, subectodermal blisters, hematomas, and generalized edema--may result from altered fluid balance in and around the embryo. The present study demonstrates relationships between changes in the fluid environment around the embryo and appearance of anomalies. Rat embryos were exposed in utero or in vitro to trypan blue during the early period of organogenesis. Both exposures resulted in defects that are typical of trypan blue treatment. Osmolality of exocoelomic fluid (ECF) was measured on gestation day 10 in vivo and day 12 in vitro, both after 48 hr of exposure to trypan blue. In both cases ECF osmolality was significantly lower than controls. This was correlated with the presence of edema-related anomalies in the embryo. On gestation day 11 in vivo, three days after maternal injection of trypan blue, ECF osmolalities were significantly higher than controls; however, there was tremendous variability in this parameter in day 11 treated embryos, and some had ECF osmolalities below the control range. Increased frequency of abnormalities was correlated with abnormal ECF osmolality, below and above the control range. Trypan blue probably exerts its teratogenic effects by disturbing the function of the visceral yolk sac. The movements of an amino acid and a monosaccharide across the visceral yolk sac were measured on gestation day 12 embryos in vitro. This aspect of yolk sac function was not altered by trypan blue exposure. Ultrastructure of the visceral yolk sac was observed after trypan blue exposure in vivo and in vitro. Endodermal cells in trypan blue-treated yolk sacs contained fewer large, electron dense lysosomes than controls. These were replaced by numerous small vacuoles, which may contain trypan blue. Trypan blue causes osmotic changes in the rat embryo in vivo and in vitro. These changes are correlated with embryonic malformations. Alterations in yolk sac ultrastructure indicate that trypan blue affects the function of this membrane.     

Ultrastructural observations of the early and late stages of gorgonian coral (Junceella juncea) oocytes

The developmental oogenesis of gorgonian coral was investigated at the histological level. The objective of this study was to examine and improve the understanding of Junceella juncea oogenesis using ultrastructural methods, such as histological sectioning and transmission electron microscopy. At least three types of yolk materials were observed in this study: yolk body, lipid granules and cortical alveoli. Some of the complex yolk materials were encompassed by concentric or arched layers of smooth and rough endoplasmic reticulum and the Golgi complex in early stage oocytes. Different types of vesicles were found in both early and late stage oocytes and some granules could be seen inside the empty vesicles. This may be a possible method for elaborating complex yolk materials. Homogeneous yolks from different types of inclusions were abundant and the autosynthesis of yolk may be a major mechanism in J. juncea oocytes. This is the first report of the ultrastructural observation of oogenesis in gorgonian coral species using transmission electron microscopy. Our study obtained relatively detailed information at the ultrastructural level, and it provides an overview of the oocyte ultrastucture of the gorgonian coral J. juncea.     

The ovarian cycle histochemistry and its relationship with hepatopancreas weight in the blue crab Callinectes danae (Crustacea: Portunidae)

Zara, F.J., Gaeta, H.H., Costa, T.M., Toyama, M.H. and Caetano, F.H. 2011. The ovarian cycle histochemistry and its relationship with hepatopancreas weight in the blue crab Callinectes danae (Crustacea: Portunidae). —Acta Zoologica (Stockholm) 00 :1–13. Several studies use macroscopic patterns of the ovarian development in crustaceans. Here, we examined the relationship between ovary histochemistry, changes in gonadosomatic and hepatosomatic indices against the macroscopic pattern of the ovarian development in Callinectes danae. Animals were collected in the south coast of São Paulo State, Brazil. Ovaries were macroscopically classified as juvenile, rudimentary, developing, intermediary, mature, and rudimentary ovigerous. Samples were fixed in 4% paraformaldehyde, processed for historesin, and stained with HE, protein, and neutral and acid polysaccharides detection. The juvenile oocytes are not enclosed by follicular cells and have fewer yolk nuclei being less intense in PAS reactivity than rudimentary oocytes. Developing oocytes show yolk granules and thick follicular cells. Yolk granules were positive for proteins and neutral polysaccharides. The intermediary stage is marked by a qualitative increase in yolk granules and the onset of chorion formation. In mature oocytes, cytoplasm is completely filled by yolk granules and the chorion is completely formed. Ovigerous ovaries have several atretic follicles and large quantities of hemocytes in the process of tissue reorganization. In C. danae, the changes in cell, goandosomatic and hepatosomatic indices coinciding with macroscopic observations and any combination of different macroscopic stages in a single pattern should be avoided.     

Ultrastructural features of the adult hermaphrodite gonad of Caenorhabditis elegans: relations between the germ line and soma.

Genetic and embryological experiments have established the Caenorhabditis elegans adult hermaphrodite gonad as a paradigm for studying the control of germline development and the role of soma-germline interactions. We describe ultrastructural features relating to essential germline events and the soma-germline interactions upon which they depend, as revealed by electron and fluorescence microscopy. Gap junctions were observed between oocytes and proximal gonadal sheath cells that contract to ovulate the oocyte. These gap junctions must be evanescent since individual oocytes lose contact with sheath cells when they are ovulated. In addition, proximal sheath cells are coupled to each other by gap junctions. Within proximal sheath cells, actin/myosin bundles are anchored to the plasma membrane at plaque-like structures we have termed hemi-adherens junctions, which in turn are closely associated with the gonadal basal lamina. Gap junctions and hemi-adherens junctions are likely to function in the coordinated series of contractions required to ovulate the mature oocyte. Proximal sheath cells are fenestrated with multiple small pores forming conduits from the gonadal basal lamina to the surface of the oocyte, passing through the sheath cell. In most instances where pores occur, extracellular yolk particles penetrate the gonadal basal lamina to directly touch the underlying oocytes. Membrane-bounded yolk granules were generally not found in the sheath cytoplasm by either electron microscopy or fluorescence microscopy. Electron microscopic immunocytochemistry was used to confirm and characterize the appearance of yolk protein in cytoplasmic organelles within the oocyte and in free particles in the pseudocoelom. The primary route of yolk transport apparently proceeds from the intestine into the pseudocoelom, then through sheath pores to the surface of the oocyte, where endocytosis occurs. Scanning electron microscopy was used to directly visualize the distal tip cell which extends tentacle-like processes that directly contact distal germ cells. These distal tip cell processes are likely to play a critical role in promoting germline mitosis. Scanning electron microscopy also revealed thin filopodia extending from the distal sheath cells. Distal sheath filopodia were also visualized using a green fluorescent protein reporter gene fusion and confocal microscopy. Distal sheath filopodia may function to stretch the sheath over the distal arm.     

Ultrastructural and cytochemical effects of trypan blue on TSH stimulation of thyroid follicular cells

Summary Ultrastructural and cytochemical techniques were used to study the effects of trypan blue on the response of mouse-thyroid cells to exogenous stimulation by thyroid stimulating hormone (TSH). The dye delayed the response to TSH resulting in decreased colloid-droplet formation in the apical region of the cells. The dye did not stop the shift of trimetaphosphatase activity from lysosomes to phagolysosomes. The duration of the TSH-induced response was shorter in the dye treated thyroids. Small vesicles, with trimetaphosphatase reaction product, were found near Golgi elements, phagolysosomes, and the plasma membrane facing the intercellular space of adjacent follicle cells. Their enzyme activity was not affected by exposure to the dye. These data indicate that the primary effect of trypan blue on the response of thyroid follicle cells to TSH stimulation was reduced endocytosis in the apical region resulting in fewer colloid droplets.     

Oogenesls in the terrestrial hermit crab,coenobita clypeatus (decapoda,anomura): II. Vitellogenesis

Oocytes from the land hermit crab, Coenobita clypeatus, in various stages of vitellogenesis were examined by light and electron microscopy. Early vitellogenic oocytes are characterized by accumulations of discrete vesicles of endoplasmic reticulum in the perinuclear cytoplasm. As oocytes develop, the endoplasmic reticulum becomes abundant, and numerous Golgi complexes are seen. There is a well developed Golgi-endoplasmic reticulum interaction. Within the confines of the reticulum are discrete intracisternal granules, which can be seen coalescing into electron-dense yolk bodies. Lipid accumulation is seen throughout the cytoplasm. Coincident with the burst of intra-oocytic metabolism are oolemma modifications and micropinocytosis, which provide ultrastructural evidence for extra-oocytic yolk production. The mature oocyte contains numerous yolk and lipid vesicles of varying electron density that comprise both intra- and extra-oocytic substrates.