Lipids in yolks and neonates of the viviparous lizard Niveoscincus metallicus

Niveoscincus metallicus is a small viviparous skink which provides a substantial amount of yolk to each of its developing embryos although some organic nutrients are also transferred across the placentae. The total amount of lipid present in the yolk of N. metallicus (37% of dry weight) is very much higher than that in the newborns (19% of dry weight), confirming that the yolk is utilised as an energy source during gestation. Triacylglycerols (TAG), which are storage compounds, are the major lipid resource available to the embryos and are present in relatively large amounts in the yolk of N. metallicus. Polar lipids (PL), which form the structural components of membranes, and sterols (ST), which are involved in the synthesis of hormones and vitamins, are also present in the yolk. The proportions of each of these lipid classes differs markedly between yolks and newborns. This may reflect variations in the role played by each lipid class in the provision of nutrients to, and development of, embryos.     

Histochemical studies on the yolk-nucleus in fish oogenesis

Summary A histochemical study of the oogenesis of two species of fresh water fishes, Channa maruleus and Heteropneustes fossilis, was undertaken to reveal the origin, structure, histochemical nature, and function of the so-called yolk-nucleus. The basophilic substance of the yolk-nucleus, which is situated in the juxta-nuclear cytoplasm, gradually accumulates adjacent to the nuclear membrane. It is a homogeneous, spherical mass. In Channa, some basophilic, dense bodies develop in the yolk-nucleus. Histochemical tests show that the yolk-nucleus and dense bodies are rich in RNA and proteins. Mitochondria of lipoprotein composition and lipid inclusions, composed of unsaturated phospholipids, appear in association with the yolk-nucleus. Throughout previtellogenesis, the yolk-nucleus continues to proliferate its basophilic, RNA-containing substance and other inclusions. Finally it disintegrates while lying in the peripheral cytoplasm of the larger oocytes which show the synthesis of yolk bodies. During yolk formation, lipid inclusions and mitochondria start disappearing from view but the RNA-containing substance, originated from the yolk-nucleus of previtellogenesis, continues to persist among the growing yolk bodies. The latter arise de novo from the ground cytoplasm, under the influence of the RNA-containing substance, mitochondria and lipid inclusions of previtellogenesis.This work was carried out in the Department of Zoology, University of Gorakhpur, Gorakhpur, India.Population Council Post-Doctoral Fellow.     

Accumulation of yolk in a caecilian (Gegeneophis ramaswamii) oocyte: A light and transmission electron microscopic study

There is a paucity of information on the female reproductive biology of the caecilian amphibians when compared with the other vertebrate groups. Hence, the accumulation of nutrient reserves in the form of yolk and formation of yolk platelets were studied in Gegeneophis ramaswamii, adopting light microscopic histological and transmission electron microscopy analysis. Previtellogenic as well as vitellogenic follicles were observed in appropriate preparations. On the basis of the source and the routes of entry, we identified four types of yolk precursor materials, precursors 1 to 4. The earliest material appearing in the oocyte consists of abundant lipid vesicles during the previtellogenic phase, i.e., much before the follicular epithelium is fully established. This is a contribution from the oocyte mitochondria, which we identified as yolk precursor material 1, and it is autosynthetic. Once the follicle cell‐oocyte interface is fully established, there is an accumulation of the principal component of the heterosynthetic yolk by sequestration from the blood through the intercellular spaces between follicle cells in a pinocytic process. This we identified as yolk precursor material 2. There was also an indication of a lipidic yolk material synthesis in the follicle cells sequestered from maternal blood through the follicle cells in an endocytic process in which the macrovilli of follicle cells and the microvilli of the oocyte play a role. This we identified as yolk precursor material 3. Contribution to the yolk of peptidic, glycosidic, and/or lipidic material synthesized in the vitellogenic oocyte was also indicated. This we identified as yolk precursor material 4. The sequential development of intercellular associations and indications of synthesis/sequestration of the yolk have been traced. Thus, we report the mechanistic details of synthesis/sequestration of the yolk materials in a caecilian. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

A comparative histochemical study of fish (Channa maruleus) and amphibian (Bufo stomaticus) oogenesis

Summary Comparative histochemical studies on the fish (Channa maruleus) and amphibian (Bufo stomaticus) oogenesis demonstrate a great similarity in the growth and differentiation of their egg follicle. The ooplasm, germinal vesicle and egg-membranes show distinct morphological and cytochemical changes during previtellogenesis and vitellogenesis.During previtellogenesis the various components of the follicle are engaged in the synthesis of protoplasm as shown by the proliferation of yolk nucleus substance, mitochondria and some lipid bodies in the ooplasm and of nucleoli in the germinal vesicle. The substance of the yolk nucleus consisting of proteins, lipoproteins and RNA first appears adjacent to the nuclear membrane. Numerous mitochondria of lipoprotein composition, and some lipid bodies consisting of unsaturated phospholipids lie in association with the yolk nucleus which forms substratum for the former. The lipid bodies, present inside the germinal vesicle, follicular epithelium, and adjacent to the plasma membrane in association with some pinocytotic vacuoles, have been considered to play a significant role in the active transport of some substances from the environment into the ooplasm and from the latter into the germinal vesicle. The follicular epithelium itself is very poorly developed, negating its appreciable role in the contribution of specific substances into the oocyte, which seem to be contributed by the germinal vesicle showing a considerable development of nuclear sap, basophilic granules and nucleoli consisting of RNA and proteins; many large nucleoli bodily pass into the cytoplasm during the previtellogenesis of Channa, where their substance is gradually dissolved. The intense, diffuse, basophilic substance of the cytoplasm is believed due to free ribosomes described in many previous ultrastructural studies.During vitellogenesis, the various deutoplasmic inclusions, namely carbohydrate yolk, proteid yolk and fatty yolk, are deposited in the ooplasm. The carbohydrate yolk bodies rich in carbohydrates originate in association with the plasma membrane and correspond to vesicles and cortical granules of previous studies. The proteid yolk consisting of proteins and some lipoproteins, and fatty yolk containing first phospholipids and some triglycerides and then triglycerides only are deposited under the influence of yolk nucleus substance, mitochondria and cytoplasm. The mitochondria and yolk nucleus substance foreshadow in some way the pattern of these two deutoplasmic inclusions and persist at the animal pole of mature egg while the other inclusions of previtellogenesis disappear from view. The pigment granules, which also show a gradient from the animal to vegetal pole in Bufo, are also formed in association with yolk nucleus substance and mitochondria. Some glycogen also appears in both the species. The nuclear membrane becomes irregular due to the formation of lobes. The lipid bodies of the germinal vesicle come to lie outside the nuclear membrane, suggesting active transport of some substances into the ooplasm; many nucleoli bodily pass into the ooplasm of Bufo, where they are gradually absorbed. The amount of basophilic granules is considerably increased in the germinal vesicle during vitellogenesis. Various egg-membranes such as outer epithelium, thin theca, single-layered follicular epithelium, zona pellucida or vitelline membrane surround the vitellogenic oocytes. The zona pellucida formed between the oocyte and follicle cells consists of a carbohydrate-protein complex. The follicle cells show lipid droplets, mitochondria and basophilic substance in their cytoplasm. The various changes that occur in the components of the follicle during vitellogenesis seem to be initiated by gonadrotrophins formed under the influence of specific environmental conditions.The author wishes to express sincere appreciation and gratitude to Dr. Gilbert S. Greenwald, who has made the completion of this investigation possible.Ph. D. Population Council Post-doctoral Fellow.     

Cytochemistry of yolk elements in the egg of the tunicate Molgula

Summary Cytochemical techniques demonstrate two types of yolk elements (compound yolk and lipid yolk) in the egg of the tunicate (Molgula manhattensis). The compound yolk elements consisting of proteins, carbohydrates, lipoproteins and triglycerides arise under the influence of cell organelles. A few lipid yolk elements staining moderately for phospholipids are also formed. The distribution and cytochemistry of cell organelles have also been described briefly in growing oocytes, test cells and follicular epithelial cells.     

Cytochemistry of yolk elements in the Amphioxus egg

Summary The Amphioxus egg develops compound yolk and lipid yolk, besides the cortical vacuoles described previously. The compound yolk elements consisting of carbohydrates, proteins, lipoproteins, and triglycerides originate within the ooplasmic masses that are constituted by the yolk nucleus substance and mitochondria. The lipid yolk elements, which are poorly developed, stain for phospholipids; the exact mode of their formation could not be determined. The behaviour and cytochemistry of organelles (yolk nucleus and mitochondria) have also been described.     

Distribution of lipids from the yolk to the tissues during development of the water python (<Emphasis Type="Italic">Liasis fuscus</Emphasis>)

Energy metabolism during embryonic development of snakes differs in several respects from the patterns displayed by other reptiles. There are, however, no previous reports describing the main energy source for development, the yolk lipids, in snake eggs. There is also no information on the distribution of yolk fatty acids to the tissues during snake development. In eggs of the water python (Liasis fuscus), we report that triacylglycerol, phospholipid, cholesteryl ester and free cholesterol, respectively, form 70.3%, 14.1%, 5.7% and 2.1% of the total lipid. The main polyunsaturate of the yolk lipid classes is 18:2n-6. The yolk phospholipid contains 20:4n-6 and 22:6n-3 at 13.0% and 3.6% (w/w), respectively. Approximately 10% and 30% of the initial egg lipids are respectively recovered in the residual yolk and the fat body of the hatchling. A major function of yolk lipid is, therefore, to provision the neonate with large energy reserves. The proportion of 22:6n-3 in brain phospholipid of the hatchling is 11.1% (w/w): this represents only 0.24% of the amount of 22:6n-3 originally present in the egg. This also contrasts with values for free-living avian species where the proportion of DHA in neonatal brain phospholipid is 16–19%. In the liver of the newly hatched python, triacylglycerol, phospholipid and cholesteryl ester, respectively, form 68.2%, 7.7% and 14.3% of total lipid. This contrasts with embryos of birds where cholesteryl ester forms up to 80% of total liver lipid and suggests that the mechanism of lipid transfer in the water python embryo differs in some respects from the avian situation.Abbreviations ARA arachidonic acid - DHA docosahexaenoic acidCommunicated by G. Heldmaier     

Fatty acid esterification in the yolk sac membrane of the avian embryo

The transfer of lipid from the yolk to the avian embryo is mediated by the yolk sac membrane (YSM). Some, but not all, of the published morphological evidence supports the view that the lipid undergoes a cycle of hydrolysis and re-esterification during translocation across the YSM. The present study aims to test this view by investigating the capacity of the YSM to perform esterification of free fatty acids to form acyl-lipids. YSM pieces (area vasculosa), obtained from the chicken embryo at day 10 of development, were incubated in vitro in medium containing [¹⁴C]-palmitic acid. Radioactivity was rapidly incorporated into the tissue lipid indicating a high capacity for esterification. The incorporation was linear with time during the 1-h incubation. Approximately 84% of the incorporated label was recovered in triacylglycerol, 12% was incorporated into phospholipid and less than 1% was detected in cholesteryl ester. [¹⁴C]-palmitic acid was incorporated primarily at the sn-1/3 positions in the triacylglycerol molecule and at the sn-1 position of phospholipid. The incorporation of label into tissue pieces obtained from the non-vascularized peripheral region of the YSM (area vitellina) was much more limited than that observed for the area vasculosa. The results support the hypothesis that yolk lipid is hydrolyzed and re-esterified during transfer across the YSM.Abbreviations YSM yolk sac membrane - VLDL very-low density lipoprotein Communicated by G. Heldmaier     

The ultrastructure of the yolk nucleus during early cleavage of Nassarius reticulatus L. (Gastropoda,Prosobranchia)

Summary In all blastomeres of Nassarius from 4- to 16-cell stage yolk nuclei occur. Most of them are spherical bodies, lying juxtanuclearly between the nucleus and the apical plasmalemma. Strangely they are not ultrastructurally uniform but fall into two categories (Fig. 5): Type I is a massive spherical accumulation of mitochondria embedded in a dense intermitochondrial substance, which appears to contain both granules and filaments. Type II is a ball of radially arranged small Golgi stacks clustered around a centre of Golgi vesicles and other organelles embedded in a ground cytoplasm structurally similar to the intermitochondrial substance of type I. The function of both types of yolk nuclei is unknown. These segmentation yolk nuclei have nothing to do with yolk synthesis any more. On the other hand there are no indications that yolk nuclei occurrence is correlated with the break-down of yolk because neither lipid droplets nor protein yolk granules are observed in or beside the yolk nuclei.We wish to thank Mrs. Chr. Mehlis for her valuable technical assistance as well as Professor J. Bergérard for the excellent working conditions on the Station biologique at Roscoff (France).     

Differentiation of cytoplasmic organelles and storage of yolk during vitellogenesis in Hemidiaptomus ingens and Mixodiaptomus kupelwieseri (Copepoda,Calanoida)

These investigations concern two freshwater calanoid copepods Hemidiaptomus ingens and Mixodiaptomus kupelwieseri. The first aspect of the research relates to the processes involved in the formation and the differentiation of the ooplasmic organelles at the time of primary vitellogenesis. During this phase, a number of complex associations develop in the ooplasm. They consist chiefly of nuage-like structures, corresponding to extruded nuclear material, and vesicular formations, some arising from the nuclear envelope and the others neoformed in the ooplasm. These associations represent centers of maturation for ribosomes and synthesis for reticulum membranes. Annulate lamellae may be observed near these associations. Biogenesis of the reticulum always precedes the differentiation of the Golgi apparatus. Indeed, the dictyo-somes develop in characteristic complexes including endoplasmic reticulum cisternae and numerous vesicles resulting from intensive blebbing from cisternae. The second aspect of this research concerns yolk synthesis and accumulation of hyaloplasmic inclusions. A preliminary synthesis of yolk occurs early in these complexes and becomes more important after achievement of Golgi apparatus biogenesis. However, the most important yolk storage results from exogenous molecules and consists of complex globules, which develop into the ooplasm during secondary vitellogenesis. Formation of these globules is associated with the accumulation of two categories of inclusions in the hyaloplasm, i.e., lipid droplets and clusters of glycogen particles. At the end of vitellogenesis, a new type of endogenous material develops into small cisternae localized in the cortical ooplasm. © 1993 Wiley-Liss, Inc.     

Verteilungsmuster und Ultrastruktur des Glykogens in der Oocyte vonMusca domestica

Summary Fully grown oocytes ofMusca domestica contain large amounts of glycogen distributed in a characteristic pattern. Three cytoplasmic layers can be distinguished: 1. The periplasm which is free of carbohydrates and merely contains some lipid and protein yolk. 2. A zone of large glycogen clods. 3. Adjacent to this the central ooplasm where numerous lipid droplets and protein yolk spheres are found beside medium size glycogen clods. The glycogen areas are not surrounded by membranes, in contrast to the other yolk inclusions. Some possible interpretations of this ooplasmic pattern, which is already established during oogenesis, are discussed.     

Incorporation de la vitellogénine tritiée dans les ovocytes du Crustacé Amphipode Orchestia gammarellus (Pallas) / Incorporation of tritiated vitellogenin into the oocytes of the crustacean amphipod Orchestia gammarellus (Pallus)

Summary

During the secondary vitellogenesis the oocytes of Orchestia gammarellus accumulate yolk spheres and lipid droplets. We studied the uptake of tritiated vitellogenin by the oocyte and its accumulation in the yolk spheres.     

Composition, accumulation and utilization of yolk lipids in teleost fish

Lipid reserves in teleost eggs are stored in lipoprotein yolk and, in some species, a discrete oil globule. Lipoprotein yolk lipids are primarily polar lipids, especially phosphatidylcholine and phosphatidylethanolamine (PE), and are rich in (n–3) polyunsaturated fatty acids, especially 22:6(n–3) (docosahexaenoic acid, DHA). Oil consists of neutral lipids and is rich in monounsaturated fatty acids (MUFA). Egg lipids are derived from dietary fatty acid, fatty acid mobilized from reserves and possibly fatty acid synthesized de novo. There is selective incorporation of essential fatty acids, particularly DHA, into yolk lipids and discrimination against incorporation of 22:1(n–11). Lipid is delivered to the oocyte by vitellogenin, which is rich in polar lipids, and likely also by other lipoproteins, especially very low density lipoprotein, which is rich in triacylglycerol (TAG). All classes of lipid may be used as fuel during embryonic and larval development and MUFA are preferred fatty acids for catabolism by embryos. Catabolism of oil globules is frequently delayed until latter stages of development. In some species, DHA derived from hydrolysis of phospholipid may be conserved by transfer to the neutral lipid. Recent work has expanded knowledge of the role of DHA in membrane structure, especially in neural tissue, and molecular species analysis has indicated that PE containing sn-1 oleic acid is a prime contributor to membrane fluidity. The results of this type of study provide an explanation for the selection pressures that influence yolk lipid composition. Future work ought to expand knowledge of specific roles of individual fatty acids in embryos along with knowledge of the ecological physiology of ovarian recrudescence, environmental influences on vitellogenin and yolk lipid composition, and the control of yolk lipid accumulation and utilization.     

Hepatopancreas is the extraovarian site of vitellogenin synthesis in black tiger shrimp, Penaeus monodon

The site of yolk protein synthesis in crustaceans has long been a subject of controversy. The vitellogenin gene structure was partially reported only very recently in Macrobrachium rosenbergii, after which the hepatopancreas was confirmed as the extraovarian site of vitellogenin synthesis in that species. Ovaries are the most frequently reported as the site of yolk protein synthesis in penaeid shrimp. Using cDNA reversed-transcribed from mRNA isolated from the hepatopancreas of vitellogenic female shrimp, Penaeus monodon, we found that its deduced amino acid sequence had high identity of 48% with that from M. rosenbergii vitellogenin. A similar location of the intron in the sequenced region of genomic DNA was also found between these two species. We therefore concluded that the hepatopancreas the extraovarian site of vitellogenin synthesis in P. monodon in vivo. The partial structure of vitellogenin gene is presented in this study.     

Hepatopancreas and ovary are sites of vitellogenin synthesis as determined from partial cDNA encoding of vitellogenin in the marine shrimp,Penaeus vannamei

Summary

The site of yolk protein synthesis in crustaceans has long been a subject of controversy. A portion of the vitellogenin gene structure was reported recently in a freshwater giant prawn (Macrobrachium rosenbergii) and black tiger shrimp (Penaeus monodori), in which the hepatopancreas was confirmed to be the extraovarian site of vitellogenin synthesis. The ovary is also frequently reported to be the site of yolk protein synthesis in penaeid shrimp. The same PCR product was obtained using cDNA from the hepatopancreas or the ovary as a template. The deduced amino acid sequence of Vg in P. vannamei showed high identities of 57% and 78% with those from M. rosenbergii and P. monodon, respectively. The same location of the intron in the sequenced region of genomic DNA was also found between these three species. We therefore concluded that the hepatopancreas and ovary are sites of vitellogenin synthesis in P. vannamei. The partial structure of the vitellogenin gene is further presented.     

Metabolism and distribution of yolk DNA in embryos ofOphryotrocha labronica La Greca and Bacci

Summary Exposure of the polychaeteOphryotrocha labronica to³H-thymidine during vitellogenesis leads to substantial incorporation of label in the ooplasm, especially in yolk granule DNA. In embryos from oocytes labelled in this way it was possible to follow the amount and localization of the labelled material (DNA) throughout early development by means of light microscopical and electron microscopical autoradiography; liquid scintillation measurements also were carried out.Within the embryonic cells the bulk of the labelled DNA was localized in the yolk granules and noticeable amounts were associated with minor structural elements, whereas mitochondria and lipid droplets were only slightly labelled. Nuclear labelling was weak. Early development was found to be characterized by rapid loss of labelled DNA, so that larvae, ready to leave the egg packs, retained only about 30% of the amount originally present.It was concluded that yolk granule DNA cannot be a store of precursor material for nuclear DNA synthesis, as has been suggested sometimes, but most likely represents an informative DNA which upon release from the yolk granules is rapidly metabolized. Possible roles for yolk granule DNA is discussed.The excellent technical assistance of Mrs. Siv Nilsson and Mrs. Annagreta Petersen is gratefully acknowledged. This work has been supported by the Swedish Natural Science Research Council and Kungliga Fysiografiska Sällskapet, Lund.     

The scheduling of spawning with the moult cycle in Northern krill (Crustacea: Euphausiacea): a strategy for allocating lipids to reproduction

Summary

Euphausiids moult and grow throughout their life, which implies sharing of resources between growth and reproduction for adult krill. In the Northern krill, Meganyctiphanes norvegica (M. Sars), female krill produce eggs cyclically. Spawning moult cycles alternate with vitellogenic moult cycles for lipid yolk accumulation. Histology shows that lipids are associated with the R cells of the digestive gland in both sexes, with the yolk platelets of mature oocytes and with the fat body cell membranes and blood lacunae in reproducing females. Mature female krill can have a total lipid content twice as high as males, mostly due to accumulation in the ovary, the fat body and the haemolymph. In contrast, in males, as well as in non-reproducing females, the highest percentage of lipids is found in the digestive gland and the haemolymph. In Meganyctiphanes norvegica, the most abundant lipid fractions are polar lipids and triglycerides, the latter being relatively low in reproducing female gonad and fat body. Triglycerides are believed to be a pure energy source and polar lipids are essential for membrane development in embryos. The fatty acid content and composition of the triglyceride and polar lipid fractions in females are different from males, related to both reproductive and dietary processes. Higher levels of polyunsaturated fatty acids (PUFA) in the polar lipid fraction were found in reproductive females. During the non-reproductive season, the converse was found, indicating the specific role PUFA and other fatty acids play in growth and egg production. Adaptive processes linked to reproduction were studied comparatively in three populations of the Northern krill—Clyde Sea (W, Scotland), Kattegat (E, Denmark), Ligurian Sea (Mediterranean)—all differing considerably in climatic and trophic conditions. Such adjustments in lipid synthesis and storage are viewed as reproductive strategies developed by the Northern krill in response to different environmental conditions.     

Evidence for synthesis in vitro of neutral lipids by isolated oocytes of Perinereis cultrifera (Annelida,Polychaeta). A biochemical and autoradiographic study

Summary

Isolated oocytes of Perinereis cultrifera have been incubated in culture media with added [³H]glycerol, [¹⁴C]butyric acid or [¹⁴C]oleic acid. The principal neutral lipid synthesized was triacylglycerol, although incorporation of radioactivity into other lipid categories (sterol, fatty acid, wax ester) was also observed. A more significant percentage of triacylglycerol was labelled after incubation with [³H]glycerol and [¹⁴C]oleic acid than with [¹⁴C]butyric acid. With this precursor, monoacylglycerol appears to be the class of lipid compartment which initially show the most radioactivity. Electron microscopic autoradiography has revealed that labelling after incorporation of glycerol was mainly localized on the lipid droplets but not on the yolk granules. A second metabolic pathway is represented by phospholipid membrane synthesis.     

Lipid composition,fatty acid profiles,and lipid‐soluble antioxidants of eggs of the Hermann’s tortoise (Testudo hermanni boettgeri)

The major lipid classes, their fatty acid profiles, and the amounts of the lipid‐soluble components, vitamin E, vitamin A, and carotenoids, were determined for egg yolks of the Hermann’s tortoise (Testudo hermanni boettgeri) with the aim of identifying any features that may potentially impair the adaptation of this endangered species to deteriorations in habitat. Total lipid formed 16% (wt/wt) of the fresh yolk and consisted of (wt/wt) 74.4% triacylglycerol, 18.1% phospholipid, 3.0% cholesteryl ester, and 3.4% free cholesterol. Despite a diet based on green plants, contributing α‐linolenic acid as the main polyunsaturate, this fatty acid formed only 3.8% of the total mass of fatty acid of the total lipid. The main acyl component of the yolk lipids was the monounsaturated fatty acid, oleic acid, which formed 45.6% of the total. The most striking feature of the yolk composition was the almost complete lack of two nutrients, docosahexaenoic acid and vitamin A, which are essential for the developing embryo. Although it is feasible that the embryo synthesizes docosahexaenoic acid from yolk‐derived α‐linolenic acid and also converts yolk‐derived β‐carotene to vitamin A, the yolk is poorly endowed with both these precursors. The stringencies displayed by the yolk composition in this species may limit the flexibility to adapt to changes in the availability of food items when the habitat is threatened. Zoo Biol 20:75–87, 2001. © 2001 Wiley‐Liss, Inc.