Lysosomes and yolk formation in the oocytes of Asterina gibbosa (Echinodermata,Asteroidea)

Summary

Primary lysosomes appear in the oocytes of A. gibbosa at the end of previtellogenesis. The lysosomes fuse with the cisternae of the endoplasmic reticulum and give rise to yolk globules containing acid phosphatase. The yolk globules then grow by fusion.     

ELECTRON MICROSCOPY OF BASOPHILIC STRUCTURES OF SOME INVERTEBRATE OOCYTES : II. FINE STRUCTURE OF THE YOLK NUCLEI

RNA containing yolk nuclei from the surf clam Spisula solidissima have been studied with the light microscope and with the electron microscope. A variety of structures can be seen with both and a correlation can be made between bodies studied with the electron microscope and those studied with the light microscope. The electron microscope shows many of these structures to be composed of double walled lamellae arranged in space, in various ways. The variety of patterns seen with the electron microscope can be satisfactorily explained on the basis of a hypothetical model. The presence of yolk platelets within the yolk nuclei lends support to classical observations on the synthesis of yolk within yolk nuclei or yolk nuclei derivatives. Small granules (about 100 A) are included within the double walled lamellae and their presence probably accounts for the basophilic nature of the entire body. The presence of small granules attached to electron-dense layers relates the yolk nuclei described here to ergastoplasm discussed by others.     

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.     

A puzzling feature of Colloidal Iron positive and Alcian Blue negative material

Summary A puzzling feature of Colloidal Iron positive and Alcian Blue negative substance is encountered in yolk sac of young larvae of a fish —Tilapia mossambica. This yolk material is PAS positive (proved to be due to neutral mucopolysaccharide) and negative to Toluidine Blue, Azure A (pH 2 to 4.5), Aldehyde Fuchsin and AB pH 1. More work is necessary to establish the exact chemical nature of the CI positive material.     

Influence of Saturated and Polyunsaturated Egg Yolk Phospholipids on Hyperlipidemia in Rats

The supplementation of egg yolk phospholipid (PL) containing phosphatidylcholine (PC) and phosphatidylethanolamine (PE) to a cholesterol-free purified diet causes a reduction in the serum cholesterol level in rats [J. Nutr., 112,1805 (1982)]. The present study was carried out to determine if dietary egg yolk PL also exerts this hypocholesterolemic action in rats given a high cholesterol diet and if this action is influenced by the constituent fatty acids. Egg yolk PL suppressed the elevation of serum cholesterol irrespective of its fatty acid composition, while purified PC had no effect, suggesting that the ethanolamine portion is responsible for this hypocholesterolemic effect. Egg yolk PL and PC containing longer-chain polyunsaturated fatty acids (arachidonic and docosahexaenoic acids) lowered the serum triglyceride level, while their hydrogenated forms did not. The present results, therefore, indicate that the hypolidemic effect of dietary egg yolk PL can be modulated by the combination of the constituent fatty acids as well as the base moieties. This hypolipidemic effect, however, appeared not to be related to the activities of adipocyte lipoprotein lipase and serum lecithin: cholesterol acyltransferase.     

The advantages of LDL (low density lipoproteins) in the cryopreservation of canine semen

A medium containing LDL (Low Density Lipoproteins, the cryoprotective component of chicken egg yolk) was compared with egg yolk for the preservation canine spermatozoa during the freeze–thaw process. Twenty sperm samples taken from 10 dogs were frozen in liquid nitrogen at −196 °C in seven different media: one control medium containing 20% egg yolk, and six test media containing 4%, 5%, 6%, 7%, 8%, and 10% LDL, respectively.Following thawing, sperm motility was assessed using a Hamilton-Thorne Sperm Analyser equipped with the CEROS 12 software. The percentage of motile spermatozoa was 55.3% in the 6% LDL medium (optimal concentration) compared with 27.7% in the egg yolk based medium (p < 0.05).In comparison with the egg-yolk medium, the LDL medium also resulted in an improved preservation of spermatozoa during the freezing process (p < 0.05) in terms of acrosomal integrity (FITC-PSA test), flagellar plasma membrane integrity (HOS test), and DNA integrity (Acridine Orange test).In addition, six Beagle bitches were inseminated twice, via the intra-uterine route, at an interval of 24 h; 200 × 10⁶ spermatozoa that had been previously frozen in the 6% LDL medium were used per insemination. All of the bitches became pregnant (gestation rate of 100%).In conclusion, the 6% LDL medium provides improved protection of the spermatozoa during the freeze–thaw process and a marked improvement in the motility parameters of canine spermatozoa in comparison with the control medium containing egg yolk alone.Finally, the use of LDL as a cryoprotectant for canine semen does not interfere with fertility.     

Comparison of Yolk Production in Seven Pyralid Moth Species

Summary

The yolk proteins of six pyralid moths were analyzed and compared with the yolk proteins of Plodia interpunctella (Hübner). When cross-reacted in an Ouchterlony double immunodiffusion with antiserum raised to either total yolk proteins or purified vitellin from P. tnterpunctella, the yolk proteins of Anagasta kuehniella (Zeller), Cadra cautella (Walker), C. figulilella (Gregson), and Ephestia elutella (Hübner), closely related members of the subfamily Phycitinae, showed strong precipitation lines that consisted of four major yolk polypeptides (YPs). The yolk proteins from Amyelois transitella (Walker) were only weakly reactive, whereas yolk proteins from Galleria mel-lonella (L.) were not precipitated by either antiserum. Abdominal body walls (containing primarily fat body) from late pharate adult females were incubated in vitro and they secreted two major polypeptides that had molecular masses similar to the vitellogenins (YP1 and YP3) from P. interpunctella. In addition, ovarioles from late pharate adult females were incubated in vitro, and they secreted two major polypeptides that had molecular masses similar to YP2 and YP4 from P. interpunctella. When late pharate adult females were injected with ³⁵S-Met, the hemolymph of all species contained vitellogins that were secreted by their respective body walls in vitro. Ovarioles from injected females contained many labeled polypeptides, but there were four major bands that corresponded consistently to the vitellogenins secreted from the fat body and the two major polypeptides secreted from the ovarioles. These data show that the production of the major YPs in these closely related pyralid species is very similar, and that there is considerable conservation of immunological characters of yolk proteins in the subfamily Phycitinae.     

Yolk synthesis in ovarian follicles ofDrosophila

Summary The autonomous synthesis of yolk proteins in ovarian follicles ofDrosophila melanogaster was analyzed. Vitellogenic follicles were labelled with³⁵S-methionine in vitro and the newly synthesized yolk proteins were separated by SDS-polyacrylamide gel electrophoresis. Possible contamination of the follicle preparations caused by adhering fat body cells could be excluded by culturing follicles in males prior to labelling in vitro. When labelled follicles were cut at the nurse cell/oocyte border the three yolk proteins (YP1, YP2, YP3) were found only in posterior fragments containing ooplasm and follicle cells, whereas two radioactive protein bands (A and B) were detected in nurse cells (anterior fragments). The yolk proteins of these five bands were characterized by peptide mapping. Band A protein, migrating a little more slowly than YP2, is closely related to both YP1 and YP2 while band B contains a yolk protein which is very similar to YP3. Hence, the nurse cells have been identified as a site of vitellogenin synthesis within the ovary ofDrosophila.Supported by the Deutsche Forschungsgemeinschaft, SFB 46     

Electronmicroscopical observations on yolk and yolk formation in Ophryotrocha labronica LaGreca and Bacci

Summary In Ophryotrocha labronica LaGreca & Bacci mature yolk granules are found only in the ovocyte. Other typical yolk elements are lipid droplets, small vesicular bodies, multivesicular bodies and dense bodies. The two last-mentioned also appear in the accompanying nurse cell and from there obviously pass over unchanged into the ovocyte through a specific intercellular bridge, the fusome.The mature yolk granules are considered as aggregates of mitochondrial, endoplasmic and Golgi material, to which also is added pinocytotically incorporated external material. Mitochondria apparently play a fundamental role in the process, as the multivesicular bodies, most likely the direct precursors to the yolk granules, in all probability represent transformed mitochondria.Labelling with ³H-thymidine during vitellogenesis reveals presence of DNA in the yolk granules. From the labelling pattern, which shows DNA-synthesis both in the ovocyte and the nurse cell nucleus, it is concluded that the labelled material present in the cytoplasm of both cells — most of it in yolk granules and dense bodies — is of nuclear origin. The possible mitochondrial nature of yolk granule DNA is discussed.The author is indebted to Dr. Bertil Åkesson, Zoological Institute, Lund, for kindly supplying the initital material for the Ophryotrocha cultures. The excellent technical assistance of Mrs. Mariann Carleson is gratefully acknowledged. My thanks are also due to Mrs. Siv Nilsson for skilful assistance with the photography. This work has been supported by the Swedish Natural Science Research Council and Kungliga Fysiografiska Sällskapet, Lund.     

Electron microscopy of basophilic structures of some invertebrate oocytes. II. Fine structure of the yolk nuclei

RNA containing yolk nuclei from the surf clam Spisula solidissima have been studied with the light microscope and with the electron microscope. A variety of structures can be seen with both and a correlation can be made between bodies studied with the electron microscope and those studied with the light microscope. The electron microscope shows many of these structures to be composed of double walled lamellae arranged in space, in various ways. The variety of patterns seen with the electron microscope can be satisfactorily explained on the basis of a hypothetical model. The presence of yolk platelets within the yolk nuclei lends support to classical observations on the synthesis of yolk within yolk nuclei or yolk nuclei derivatives. Small granules (about 100 A) are included within the double walled lamellae and their presence probably accounts for the basophilic nature of the entire body. The presence of small granules attached to electron-dense layers relates the yolk nuclei described here to ergastoplasm discussed by others.     

Oogenesis in Centropages typicus (Copepoda, Calanoida)

We carried out a complete study of oogenesis in Centropages typicus using structural, ultrastructural and cytochemical data. The usual stages of oogenesis, i.e. germinative phase, premeiosis, primary and secondary vitellogenesis, were found. The latter two stages were the most typical. Primary vitellogenesis consisted of endogenous yolk accumulations; these substances, probably of lipoprotein or lipoglycoprotein nature, were produced at the granular endoplasmic reticulum level and then stocked in the reticulum cavities. During secondary vitellogenesis, endogenous yolk production continued, but we mainly observed the development of exogenous yolk accumulation (lipid droplets and protein globules) in the ooplasm. These accumulations resulted from the fusion of very numerous pinocytotic vesicles arising from the oolemma and containing substances probably brought to the oocytes by the hemolymph. The effect of various proteases on the vitellus globules caused a more or less marked digestion of their contents, tending to prove their protein nature. The end of vitellogenesis was marked by the appearance of vacuolar formations with dense lamellae which could correspond to cortical granules.     

Ultrastructural studies of differentiation in the oocyte of the polyclad turbellarian,Prostheceraeus floridanus

Oocyte differentiation in the polyclad turbellarian Prostheceraeus floridanus has been examined to determine the nature of oogenesis in a primitive spiralian. The process has been divided into five stages. (1) The early oocyte: This stage is characterized by a large germinal vesicle surrounded by dense granular material associated with the nuclear pores and with mitochondria. (2) The vesicle stage: The endoplasmic reticulum is organized into sheets which often contain dense particles. Vesicles are found in clusters in the cytoplasm, some of which are revealed to be lysosomes by treatment with the Gomori acid phosphatase medium. (3) Cortical granule formation: Cortical granules are formed by the fusion of filled Golgi vasuoles which have been released from the Golgi saccules. The association between the endoplasmic reticulum and Golgi suggests that protein is synthesized in the ER and transferred to the Golgi where polysaccharides are added to form nascent cortical granules. (4) Yolk synthesis: After a large number of cortical granules are synthesized, yolk bodies appear. They originate as small membrane-bound vesicles containing flocculent material which subsequently increase in size and become more compact. Connections between the forming yolk bodies and the endoplasmic reticulum indicate that yolk synthesis occurs in the ER. (5) Mature egg: In the final stage, the cortical granules move to the periphery and yolk platelets and glycogen fill the egg. At no time is there any evidence of uptake of macromolecules at the oocyte surface. Except for occasional desmosomes between early oocytes, no membrane specialization or cell associations are seen throughout oogenesis. Each oocyte develops as an independent entity, a conclusion supported by the lack of an organized ovary.     

Further morphological and histochemical studies on the yolk nucleus and associated cell components in the developing oocyte of the Indian wall lizard

In March through April when the oocyte growth in the ovaries of the wall lizard (Hemidactylus) is very rapid, the yolk nucleus continues to persist through various stages of previtellogenesis. This persisting yolk nucleus and associated cell components have been studied with histochemical techniques. The spherical and dense yolk nucleus stains for protein, lipoprotein and RNA. It does not form any close morphological association with the other cell components such as the mitochondria, lipid bodies (L₂), spaces or canals, diffuse sudanophilic substance and dense bodies, which are arranged into three zones round the yolk nucleus proper. The mitochondria stain for lipoprotein; the L₂ bodies consist of phospholipid; the spaces do not contain any material demonstrable with histochemical techniques; and the ooplasm containing the diffuse sudanophilic substance and dense bodies shows lipoprotein, protein and RNA. Eventually, the yolk nucleus disintegrates, and its substance as well as the other cell components are distributed in the cortical ooplasm of oocytes which are ready to form the yolk bodies. Concepts of the origin, morphology, cytochemistry and function of the yolk nucleus in the oocytes of invertebrates and vertebrates, which have come about recently through the application of cytochemical and submicroscopical techniques, are discussed.     

Trans-order yolk protein can stimulate endocytic activity in Drosophila oocytes

Vitellogenins (Vgs) and mature yolk from some non-Dipteran insects can be recognized by Drosophila melanogaster oocyte Vg receptors and incorporated via receptor-mediated endocytosis into nascent yolk spheres (NYS). It had previously been assumed that only Vgs of Drosophila or other Dipterans could be so endocytosed. Drosophila ovarian follicles from 4-day old females were incubated in the presence of physiological salt solution (PSS) containing some fluorescent TexasRed-Dextran (Dex-red) or PSS-Dex-red in which either female hemolymph, or vitellin (mature yolk) from lysed oocytes was present from any of the following: (1) Drosophila (Diptera); (2) Oncopeltus (milkweed bug, Hemiptera); (3) Acteaus (luna moth, Saturniidae Lepidoptera); (4) Papilio (swallowtail butterfly, Papilionidae Lepidoptera); or (5) Xylocopa (carpenter bee, Hymenoptera). Under incubation conditions, any NYS would become fluorescent due to non-specific fluid-phase uptake. Ovarian follicles incubated in PSS-DexRed alone or in PSS with hemolymph from males did not carry out endocytosis detectable by this technique, but all other treatments listed above did.     

Preparation ofN-acetylneuraminic acid from delipidated egg yolk

Egg yolk, a large proportion of the egg, was studied for the preparation ofN-acetylneuraminic acid (Neu5Ac). The delipidated hen egg yolk (DEY; 500 kg containing 0.2% w/w, Neu5Ac) was hydrolysed with HCl (pH 1.4) at 80 °C and neutralized with NaOH (pH 6.0). The mixture was filtered and electrodialysed until the conductivity was 240 µS cm^–1. The filtrate was applied on a column of Dowex HCR-W2 (20–50 mesh), followed by a column of Dowex 1-X8 (200–400 mesh). The latter column was washed with water, and then eluted with a linear gradient of HCO₂H (0–2m). The eluates containing Neu5Ac were concentrated using a reverse osmosis membrane and, finally, rotary evaporated at 40 °C. The residue was then lyophilized to yield 500 g Neu5Ac. The purity of Neu5Ac was >98% (TBA method). HPLC, NMR spectroscopy and TLC chromatography of the product obtained from the DEY showed that Neu5Ac was the sole derivative present in egg yolk. The DEY, a byproduct from egg processing plants, was found to be an excellent source for the large-scale preparation of Neu5Ac.Abbreviations Neu5Ac N-acetylneuraminic acid - Neu5Gc N-glycolylneuraminic acid - DEY delipidated egg yolk - HPLC high performance liquid chromatography - TLC thin layer chromatography - NMR nuclear magnetic resonance - IR infrared spectroscopy Presented at the 11th International Symposium on Glycoconjugates, Toronto, Canada.     

Vitellogenesis in Varroa jacobsoni,a parasite of honey bees

Reproduction in Varroa jacobsoni occurs only in cells of the capped honey bee brood. Female mites were sampled at different times after cell sealing and ovaries containing a vitellogenic oocyte of the first gonocycle were examined under an electron microscope. It was found that the cytoplasmic connection between the lyrate organ and the oocyte persists far into the vitellogenic growth phase. In addition, a large amount of yolk material is taken up from the haemolymph. All ultrastructural features characteristic of vitellogenesis, such as microvilli, coated pits, vesicles and growing yolk platelets, are present. If more than four Varroa females live in an overcrowded brood cell, they appear to be in stress conditions and their vitellogenic oocytes may become atretic. Alterations typical for oocyte degradation and oosorption were observed in such situations.     

Transesterification of phosphatidylcholine in <Emphasis Type="Italic">sn</Emphasis>-1 position through direct use of lipase-producing <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> cells as whole-cell biocatalyst

The enzymatic process presents an advantage of producing specified phospholipids that rarely exist in nature. In this study, we investigated the regiospecific modification of phosphatidylcholine (PC) in the sn-1 position using immobilized Rhizopus oryzae. In a reaction mixture containing egg yolk PC and exogenous lauric acid (LA) in n-hexane, lipase-producing R. oryzae cells immobilized within biomass support particles (BSPs) showed a much higher transesterification activity than lipase powders. To improve the product yield, several parameters including substrate ratio and reaction time were investigated, resulting in the incorporation of 44.2% LA into the product PC after a 48-h reaction. The analysis of the molecular structure showed that a large proportion of exogenous LA (>90%) was incorporated in the sn-1 position of the enzymatically modified PC. Moreover, the BSP-immobilized R. oryzae maintained its activity for more than 12 batch cycles. The presented results, therefore, suggest the applicability of BSP-immobilized R. oryzae as a whole-cell biocatalyst for the regiospecific modification of phospholipids.     

Development of yolk sac and chorioallantoic membranes in the Lord Howe Island skink,Oligosoma lichenigerum

Development of the yolk sac of squamate reptiles (lizards and snakes) differs from other amniote lineages in the pattern of growth of extraembryonic mesoderm, which produces a cavity, the yolk cleft, within the yolk. The structure of the yolk cleft and the accompanying isolated yolk mass influence development of the allantois and chorioallantoic membrane. The yolk cleft of viviparous species of the Eugongylus group of scincid lizards is the foundation for an elaborate yolk sac placenta; development of the yolk cleft of oviparous species has not been studied. We used light microscopy to describe the yolk sac and chorioallantoic membrane in a developmental series of an oviparous member of this species group, Oligosoma lichenigerum. Topology of the extraembryonic membranes of late stage embryos differs from viviparous species as a result of differences in development of the yolk sac. The chorioallantoic membrane encircles the egg of O. lichenigerum but is confined to the embryonic hemisphere of the egg in viviparous species. Early development of the yolk cleft is similar for both modes of parity, but in contrast to viviparous species, the yolk cleft of O. lichenigerum is transformed into a tube‐like structure, which fills with cells. The yolk cleft originates as extraembryonic mesoderm is diverted from the periphery of the egg into the yolk sac cavity. As a result, a bilaminar omphalopleure persists over the abembryonic surface of the yolk. The bilaminar omphalopleure is ultimately displaced by intrusion of allantoic mesoderm between ectodermal and endodermal layers. The resulting chorioallantoic membrane has a similar structure but different developmental history to the chorioallantoic membrane of the embryonic hemisphere of the egg. J. Morphol. 2012. © 2012 Wiley Periodicals, Inc.     

The formation of the mesoderm in urodelean amphibians VI. The self-organizing capacity of the induced meso-endoderm

Summary During the years 1973 and 1974, non-dorsal/ventral (non-d/v)-polarized blastulae were made of animal (upper) ectodermal caps and vegetal (lower) endodermal yolk masses by means of the destruction of the d/v polarity of the yolk mass endoderm by disaggregation, stirring and subsequent reaggregation. A simultaneous dis- and reaggregation of the upper ectodermal cap, as performed in the 1970 and 1971 experiments, led to the same results. The destruction of d/v polarity in the reconstituted blastulae, under maintenance of their animal-vegetal polarity, did not therefore prevent dorsal axis formation. The formation of single, double, triple or multiple dorsal axes in these reconstituted embryos must be based upon the self-organizing capacity of the induced meso-endoderm, since the alternative assumption of the re-establishment of mesoderm-inducing centre(s) in the reaggregated yolk mass endoderm seems very unlikely. The formation of single, double, triple or multiple axes must be the result of a competition between mesodermal aggregation centres arising in an initially evenly-induced zone of meso-endoderm. These initial centres which compete for like cells, attract each other, resulting, e.g. in their fusion into a single dorsal axis system, or in the survival of two, three or more centres in an initially balanced spatial configuration. Intensification of the meso-endoderm-inducing capacity, by using dorsal instead of entire yolk endoderm mass and by using two instead of one dorsal yolk mass, led to a more complete type of dorsal axis formation and to a higher number of axes formed (a higher percentage of triple and multiple axes than double and single ones) in one and the same reaggregate. The possible nature of the self-organizing capacity of the induced meso-endoderm has briefly been discussed, emphasizing the fundamental significance of the self-organizing capacity of embryonic anlagen in developmental processes. Offprint requests to: P.D. Nieuwkoop     

Liquid culture of the entomopathogenic nematode,Steinernema colombiense: evolution of the nematode concentration,particles volume fraction,rheological properties and surface tension

ABSTRACT

The rheological properties and surface tension (σ) of culture broths with suspended particles are factors that affect the performance of fermentation processes. This article presents the first report concerning the evolution of suspended particles, rheological properties and σ of culture broths during the liquid culture of Steinernema colombiense and its symbiont bacterium, in a medium containing 1.25% (w/v) egg yolk. Cultures started with 1087?infective-juveniles/mL and finished with 53,583?individuals/mL after 10 days. Culture broths were moderately concentrated suspensions with total particles volume fraction in the range 0.08–0.14. The suspended particles were nematodes of different sizes depending on their developmental stage, and egg yolk particles. Rheological properties corresponded to pseudoplastic fluids with flow behaviour index values ?0.3 (dimensionless), greatly determined by the symbiotic bacterium activity. After nematode inoculation, the apparent viscosity values (η_a; Pa s) for the whole culture broth and its supernatants were in the ranges 0.010–0.018 and 0.008–0.015, respectively. Concerning σ, their values were in the range 31.22–37.74?mN/m, probably determined by the contents of egg yolk.