Sugar binding properties of the vitellogenic proteins in the Colorado beetle,demonstrated by hemagglutination and precipitation experiments

Summary The sugar binding properties of 2 important vitellogenic proteins in Colorado beetle hemolymph were demonstrated by hemagglutination and precipitation experiments. The agglutination of human red blood cells by the hemolymph of reproducing females was observed up to a hemolymph dilution of 1/256, irrespective of the blood-group. It increased significantly after trypsinization of the crythrocytes. Vitellogenin 1 was identified as the hemagglutinin. Hemagglutination and hemagglutination inhibition tests showed that this protein has a low affinity for hexosamines and a higher affinity for sulfated polysaccharides. Precipitation tests demonstrated that besides vitellogenin, another major yolk protein, chromoprotein 2, reacts with sulfated polysaccharides. The possibility that there is a specific reaction of the vitellogenic proteins with well defined saccharides on the oocyte surface is discussed. This lectintype reaction may explain the selectivity of yolk precursor endocytosis.     

Yolk sphere formation is initiated in oocytes before development of patency in follicles of the moth,Plodia interpunctella

We describe a provitellogenic stage, a previously unrecognized stage of follicle development in moths, and show that oocytes begin yolk sphere formation prior to the development of patency by the follicular epithelium. The vitellogenic activities of follicles from pharate adult femalePlodia interpunctella (Hübner) were determined by visualizing the subunits of vitellin (YP1 and YP3) and the follicular epithelium yolk protein (YP2 and YP4) using monospecific antisera to each subunit to immunolabel whole-mounted ovaries or ultrathin sections. At 92 h after pupation, yolk spheres that contained only YP2 began to proliferate in the oocytes. The inter-follicular epithelial cell spaces were closed at 92 h making vitellogenin inaccessible to the oocyte, and consequently, the vitellin subunits were not observed in the yolk spheres. YP2 uptake most likely occurred across the brush border from the follicular epithelial cells to the oocyte at this time. At 105 h, the inter-follicular epithelial cell spaces appeared closed yet trace amounts of labeling for vitellin were observed in the spaces and also in the yolk spheres along with YP2. Equivalent labeling for all four YPs in yolk spheres was finally observed at 112 h after pupation when the follicular epithelium had become patent. These data indicate that the provitellogenic stage is an extended transition period between the previtellogenic and vitellogenic stages that lasts for approximately 13 h, and it is marked at the beginning by YP2 yolk sphere formation in the oocyte and at the end by patency in the follicular epithelium.     

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.     

Biochemical and immunocytochemical analysis of vitellogenesis in the olive fruit fly Dacus (bactrocera) oleae (Diptera: Tephritidae)

Synthesis and selective accumulation of the major yolk proteins in the developing oocytes of the species Dacus oleae (Diptera: Tephritidae) was studied biochemically and by immunoelectron microscopy. In the hemolymph of adult females, two yolk proteins precursors (or vitellogenins) have been detected. They each exhibit a similar molecular weight and isoelectric point to their respective mature yolk proteins (or vitellins), while electrophoretic analysis of their synthetic profile shows that their levels in the hemolymph increase rapidly during development. Immunogold electron microscopy of ovarian sections, revealed that the hemolymph vitellogenins reach the oocyte through enlarged inter-follicular spaces and demonstrated vitellogenin synthesis by the follicle cells of the vitellogenic follicles. The newly synthesized vitellogenins follow a distinct secretory pathway into these cells as compared to other components being synthesized at the same time (e.g. the vitelline envelope proteins), since they were found in secretory vesicles that appeared to be differentiated from those destined to participate in the vitelline envelope. The vitellogenin-containing vesicles exocytose their contents directionally into the follicle cell/vitelline envelope boundary, and subsequently the vitellogenins diffuse among the gaps of the forming vitelline envelope and reach the oocyte plasma membrane. Their internalization by the oocyte includes the formation of an endocytic complex consisting of coated pits, coated vesicles, endosomes, transitional yolk bodies, and finally mature yolk bodies, in which the storage of the vitellins and other yolk proteins occur. These results are discussed in relation to data obtained from other Dipteran species.     

Determination and distribution of a female-specific protein in the brown planthopper, Nilaparvata lugens Stal (Homoptera: Delphacidae)

Electrophoretic analyses of hemolymph and body or ovary homogenates from reproducing females, males, and 5th instar nymphs of the brown planthopper, Nilaparvata lugens Stal, revealed a protein band of 175 kDa in females. An immunoblot test using antibody against this protein showed a positive reaction with a 175 kDa protein from female body or ovary homogenates. It is likely that this protein in hemolymph is vitellogenin (Vg). Distribution of Vg was determined by immunofluorescence and immunogold labeling techniques. The results showed that the positive immunofluorescence reactions were present in yolk particles, the intercellular space of follicle cells, hemolymph, and the epithelial plug of ovarioles. In addition, the yeast-like symbiotes (YLS) in mycetocytes of adults and various nymphal instars as well as those free in hemolymph or entering oocytes also exhibited a positive reaction. Electron micrographs showed that immunogold particles were found most in yolk mass and YLS over other tissues. Especially the YLS in various developmental stages all contained immunogold particles, implying that the symbiote is somewhat related with production of the female-specific protein.     

Differential sorting of constitutively co-secreted proteins in the ovarian follicle cells of Drosophila

Conventional and freeze-fracture electron microscopy, immuno-electron microscopy of ovarian cryosections and confocal immunofluorescence were used to analyze the ovarian distribution of the major protein classes being secreted by the follicle cells during the vitellogenic and choriogenic stages of Drosophila oogenesis. Our results clearly demonstrated that at vitellogenic stages the follicle cells co-secrete constitutively vitelline membrane and yolk proteins that are either sorted into distinct secretory vesicles or they are segregated in different parts of bipartite vesicles by differential condensation. Following their exocytosis only the vitelline membrane proteins are incorporated into the forming vitelline membrane. The yolk proteins (along with their hemolymph circulating counterparts) diffuse through gaps amongst the incomplete vitelline membrane and are internalized through endocytosis by the oocyte where they are finally stored into modified lysosomes referred to as alpha-yolk granules. The unexpected immunolocalization of vitelline membrane antigens in the associated body of the alpha-yolk granules may indicate that this structure is a transient repository for the proteins being internalized into the oocyte along with the yolk proteins. In the early choriogenic follicle cells the vitelline membrane and early chorion proteins were found to be co-secreted and to be evenly intermixed into the same secretory vesicles. These findings illuminate new details concerning the follicle cells secretory and oocyte endocytic pathways and provide for the first time evidence for condensation-mediated sorting of constitutively secreted proteins in Drosophila.     

Vitellogenesis in Drosophila: sequestration of a yolk polypeptide/invertase fusion protein into developing oocytes

The mechanism of yolk deposition into developing oocytes of Drosophila was investigated by following the fate of a reporter protein fused to a vitellogenin, or yolk polypeptide (YP). Embryos were transformed with a hybrid gene consisting of the promotor and amino terminal 430 codons of the Yp2 gene fused to the cytoplasmic form of the invertase gene from the yeast Saccharomyces cerevisiae. RNA hybridization experiments with established lines of transformed flies showed that the hybrid gene was expressed in female fat bodies and ovaries but not in any male cells. Immunoblotting and endoglycosidase digestion showed that the hybrid protein was secreted from fat body cells via the secretory pathway, transported in hemolymph, and sequestered into developing oocytes. Transfusion experiments with hemolymph and pure invertase showed that sequestration of invertase depended on its attachment to YP. Immunocytochemistry demonstrated that the hybrid protein became localized in yolk granules as oocytes developed. Females homozygous for the fusion gene are generally sterile; their eggs containing the hybrid protein often collapse and their embryos fail to develop, suggesting that the structure of the yolk polypeptides is important for embryonic development. These experiments show that YP2 carries structural information sufficient to direct a reporter protein from fat body cells, through the hemolymph, and into the yolk granules of developing oocytes. This work provides a means of identifying the features of yolk polypeptides that are responsible for their deposition into yolk during oogenesis.     

Distribution and accumulation of storage protein-1 in ovary of Hyphantria cunea Drury

Storage protein-1 (SP-1) is a major storage protein found in the hemolymph and fat body of Hyphantria cunea. In this study, the uptake and accumulation of SP-1 into the ovary of H. cunea was investigated using biochemical and immunocytochemical methods. SP-1 in H. cunea has a high methionine content (4.6%) but is not female-specific, like other high methionine storage proteins. In the 6-day-old pupal ovary, SP-1 was detectable in trace amounts but accumulated to significant levels toward the end of the pupal stage. After adult emergence, SP-1 rapidly decreased in the ovarian follicles and remained low in the egg. This suggest that SP-1 is either extensively modified or degraded, causing a loss of its antigenic property in the ovary after adult emergence. During vitellogenesis, SP-1 is present in the hemolymph and penetrates through the tunica propria to reach the perioocytic space. From there, SP-1 is incorporated into yolk bodies. These results clearly show that SP-1 is taken up by the developing oocyte. Its disappearance suggests that SP-1 might be an amino acid reservoir for providing precursors for egg formation, in contrast to yolk proteins, which are utilized during postembryonic development. Arch. Insect Biochem. Physiol. 37:115–128, 1998. © 1998 Wiley-Liss, Inc.     

Yolk protein endocytosis by oocytes in Drosophila melanogaster: immunofluorescent localization of clathrin, adaptin and the yolk protein receptor

The process of yolk protein (YP) uptake by developing oocytes in Drosophila melanogaster has been investigated by immunofluorescent localization of the endocytosis proteins, clathrin, alpha-adaptin and the putative yolk protein receptor (YP receptor). Data suggests that YPs from the follicle cells are trafficked into the oocyte during early stages of vitellogenesis, and that hemolymph YPs are sequestered by nurse cells adjacent to the developing oocyte during late stages of vitellogenesis. Yolk proteins were immunolocalized to both follicle cells and nurse cells during these processes. Diapausing female Drosophila melanogaster undergo a pre-vitellogenic arrest of ovarian development associated with the absence of ovarian alpha-adaptin, clathrin and putative YP receptor. Diapause termination by transfer of whole animals from 11 degrees C to 25 degrees C, or by 20-hydroxyecdysone injection, results in the appearance of immunopositive material in the nurse cells for all three proteins between 12 h and 16 h post upshift and within four days of injection. Immunopositive material was not noted in the follicle cells during diapause termination. In vitro warming of diapausing ovaries, or incubation in the presence of 1 &mgr;M 20-hydroxyecdysone failed to initiate early vitellogenic development suggesting that diapause termination requires factor(s) external to the ovary. Western blotting analysis of extracts of 24 h post-eclosion wild type and ap(56f) females identified putative yolk protein receptor with a molecular weight of 208 kDa and clathrin with a molecular weight of 178 kDa.     

Ovarian follicular atresia is mediated by heterophagy, autophagy, and apoptosis in Prochilodus argenteus and Leporinus taeniatus (Teleostei: Characiformes)

We investigated apoptosis, cell proliferation antigen (PCNA), and heat shock protein (HSP70) during ovarian follicular atresia in two freshwater teleost species from the São Francisco River basin, Brazil: curimatã-pacu, Prochilodus argenteus and piau-jejo, Leporinus taeniatus. Fishes were maintained in captivity after the reproductive period and ovarian regression was assessed by gonadosomatic index for three stages: early, advanced, and late regression. Follicular atresia was analysed by light and transmission electron microscopy, as well as by TUNEL and immunohistochemistry for HSP70 and PCNA. During early regression, atretic follicles exhibited zona pellucida breakdown, yolk degeneration, and hypertrophied follicular cells (e.g. granulosa in mammals). Intense heterophagy to engulf the yolk, and autophagy were detected in the follicular cells during advanced and late atresia. The TUNEL assay detected DNA fragmentation, mainly in late follicular atresia. The apoptosis rate of the follicular cells increased up to 10% during follicular atresia in both species and was negatively correlated with follicular area. Immunohistochemistry reaction for HSP70 stained the follicular cells strongly during advanced atresia, when they are intensively involved in yolk engulfment, whereas the reaction for PCNA labelled theca cells. We inferred that heterophagy, autophagy, and apoptosis contributed to follicular atresia in teleost ovaries, thereby achieving a more efficient removal of the degenerating oocyte and dying follicular cells. Additionally, HSP70 may protect the follicular cells before apoptosis when they are involved in yolk engulfment, and cell proliferation in the theca contributed to ovarian remodelling.     

Ovarian follicular atresia is mediated by heterophagy,autophagy, and apoptosis in Prochilodus argenteus and Leporinus taeniatus (Teleostei: Characiformes)

We investigated apoptosis, cell proliferation antigen (PCNA), and heat shock protein (HSP70) during ovarian follicular atresia in two freshwater teleost species from the São Francisco River basin, Brazil: curimatã-pacu, Prochilodus argenteus and piau-jejo, Leporinus taeniatus. Fishes were maintained in captivity after the reproductive period and ovarian regression was assessed by gonadosomatic index for three stages: early, advanced, and late regression. Follicular atresia was analysed by light and transmission electron microscopy, as well as by TUNEL and immunohistochemistry for HSP70 and PCNA. During early regression, atretic follicles exhibited zona pellucida breakdown, yolk degeneration, and hypertrophied follicular cells (e.g. granulosa in mammals). Intense heterophagy to engulf the yolk, and autophagy were detected in the follicular cells during advanced and late atresia. The TUNEL assay detected DNA fragmentation, mainly in late follicular atresia. The apoptosis rate of the follicular cells increased up to 10% during follicular atresia in both species and was negatively correlated with follicular area. Immunohistochemistry reaction for HSP70 stained the follicular cells strongly during advanced atresia, when they are intensively involved in yolk engulfment, whereas the reaction for PCNA labelled theca cells. We inferred that heterophagy, autophagy, and apoptosis contributed to follicular atresia in teleost ovaries, thereby achieving a more efficient removal of the degenerating oocyte and dying follicular cells. Additionally, HSP70 may protect the follicular cells before apoptosis when they are involved in yolk engulfment, and cell proliferation in the theca contributed to ovarian remodelling.     

莫桑比克非鲫卵黄形成的电镜观察

运用透射电镜观察了莫桑比克非鲫卵母细胞的生长.根据卵母细胞的大小和内部结构特征,将其分为四个时期:卵母细胞生长早期:卵黄泡形成期:卵黄积累期:卵黄积累完成期.本文着重研究了主要卵黄成分--卵黄球的形成过程.卵黄球属外源性卵黄,由卵母细胞通过微胞饮作用吸收肝脏合成的卵黄蛋白原后形成的.在卵黄大量积累前,卵母细胞内的线粒体和多泡体聚集成团,构成卵黄核,继而线粒体大量增殖,线粒体形状发生改变,形成同心多层膜结构,为大量的卵黄物质积累提供场所.最终形成的卵黄球由被膜、卵黄结晶体和两者之间的非结晶区三部分组成.       

Male-grown eggs in Hyalophora: Deficient follicle cell secretion as well as protein and lipid yolk deposition

Ovaries transplanted to male Lepidoptera during late larva or pupal stages produce smaller and fewer chorionated eggs than those remaining in place or transplanted to other females. Small size is shown here in Hyalophora cecropia to result not only from a lack of vitellogenic hemolymph proteins but also from dysfunction of the follicular epithelium. Several aspects of egg formation can proceed normally in the male environment, including RNA deposition by the nurse cells, the conversion of lipophorin to a very high density form as the oocyte endocytoses it, and the customary period of osmotic swelling between the end of yolk deposition and the beginning of chorion formation. But as would be expected, male-grown eggs lack vitellogenin and contain very little microvitellogenin. They also contain lower than normal amounts of lipophorin, which is related to the male's poor ability to replace this protein as the oocyte removes it from the hemolymph. A low phospholipid content can be attributed to the absence of vitellogenin and a low triglyceride droplet content to the shortage of lipophorin. Two other deficiencies, however, could not be directly explained by the low levels of vitellogenic hemolymph proteins: paravitellogenin and chorion, both secretions of the follicle cells, are deposited in significantly reduced amounts. Males of this species, in addition to lacking sufficient vitellogenic proteins and lipids in their hemolymph, are thus unable to fully support the secretory activities of the follicle cells.     

Morphological and histochemical studies on oogenesis in Callosobruchus analis Fabr. (Bruchidae-Coleoptera)

The ovary in Callosobruchus analis consists of telotrophic ovarioles with the so called nurse cells confined to one chamber at the anterior end of the ovariole. There are three types of lipids in the ovary: (1) L₁ bodies that are present in the early oocytes, in the posterior prefollicular tissue and in the follicular epithelium and contain unsaturated phospholipids; (2) L₂ bodies that have a complete or incomplete sheath of phospholipids and a triglyceride core; (3) L₃ bodies that are formed of highly saturated triglycerides. Lipids are absent from the trophic tissue. In a mature oocyte the L₁ and L₂ bodies are cortical in distribution while the L₃ bodies are centrally located. The mitochondria contain lipoproteins with RNA. The yolk spheres are acid mucopolysaccharides and protein in nature. The precursors of the yolk spheres appear first in the cortical coplasm and are absent from the follicular epithelium or the trophic tissue. The nucleolus of the oocyte shows evidence of extrusions that are believed to pass into the ooplasm. There are no nutritive cords connecting the trophic tissue to the oocytes; nor is there any evidence of any histochemically demonstrable nutritive material being contributed to the oocyte by the trophic tissue. The circumstantial evidence points towards a contribution of the raw materials to the oocyte by the haemolymph either through or in between the follicular epithelium in some soluble form or as submicroscopic particles.     

Adsorptive endocytosis of vitellogenin,lipophorin, and microvitellogenin during yolk formation in Hyalophora

Yolk in Hyalophora cecropia is a mixture of proteins that are derived from the extracellular medium. We have measured for five of these proteins the number of moles deposited in each egg, the molarity of their precursors in the hemolymph at a midpoint in vitellogenesis (day 18 of adult development), and the degree to which they are concentrated by the oocyte, relative to inulin. The proteins were isolated by gel permeation and ion exchange chromatography and used to generate antibodies in rabbits. Preliminary studies established that yolk proteins are essentially quantitatively extractable in media suitable for measuring antigen concentrations by precipitation with antibodies and that yolk and hemolymph forms of the five proteins have, effectively, the same antibody-binding specificities as the isolated standards. Content per egg was about 900 pmol for vitellogenin, 600 pmol for microvitellogenin, and 300 pmol for lipophorin. By contrast, two hemolymph storage hexamers, arylphorin and a flavoprotein, occurred at less than 3 pmol per egg. In principle, yolk precursors are taken in both as solutes in the fluid phase of the endocytotic vesicles and as ligands adsorbed to vesicle membranes. Measurements of inulin uptake indicated that fluid phase endocytosis could account for only 4% of vitellogenin, 1% of microvitellogenin, and 15% of lipophorin in the yolk, when hemolymph precursors are at their day 18 concentrations. By the same comparison, arylphorin and flavoprotein appear to be excluded from the yolk, relative to inulin.     

Cloning and expression of the yolk protein of the tsetse fly Glossina morsitans morsitans

Two major families of nutritional proteins exist in insects, namely the vitellogenins and the yolk proteins. While in other insects only vitellogenins are found, cyclorraphan flies only contain yolk proteins. Possible sites of yolk protein synthesis are the fat body and the follicle cells surrounding the oocyte. We report the cloning of the yolk protein of the tsetse fly Glossina morsitans morsitans, a species with adenotrophic viviparity. The tsetse fly yolk protein could be aligned with other dipteran yolk proteins and with some vertebrate lipases. In contrast to the situation in most fly species, only a single yolk protein gene was found in the tsetse fly. Northern blot analysis showed that only the ovarian follicle cells, and not the fat body represents the site of yolk protein synthesis.     

Oocyte growth in the sheepshead minnow: Uptake of exogenous proteins by vitellogenic oocytes

The structure of the vitellogenic follicle of the sheepshead minnow, Cyprinodon variegatus, is described. Follicles enlarge primarily by protein yolk accumulation (vitellogenesis) and subsequently increase in size by hydration. This study uses the electron-dense tracer, horseradish peroxidase, and a larger heterologous protein,Xenopus laevis [³H]vitellogenin, to follow the fate of exogenous proteins from the maternal circulation to yolk spheres of the growing oocyte. Materials appear to leave the perifollicular capillaries via an interendothelial route, traverse the theca and the patent intercellular channels of the follicular epithelium and the pore canals of the vitelline envelope. At the oocyte surface they are incorporated via micropinocytosis and translocated to growing yolk spheres in the peripheral ooplasm. In contrast to other studies on oocyte growth in teleosts which suggest that yolk is an autosynthetic product, this study substantiates the importance of heterosynthetic processes during oocyte growth in C. Variegatus.     

Production and fates of unique organelles (transosomes) in ovarian follicles of Gallus domesticus under various conditions. II

Production and fates of transosomes (sacs of ribosomes made in the follicular cells of an ovarian follicle and subsequently passed to the cytoplasms of the oocyte) have been studied by electron microscopy in ovaries of young chicks, a testosterone-treated hen, aged hens which had ceased laying eggs and a "non-layer" mutant. Study was also made of "primitive yolk" (vacuoles present in both follicular cells and ooplasms of small follicles of normally laying hens). It was found that both transosomes and vacuoles of primitive yolk were present in small oocytes of young chicks, and "non-layer" mutants. However, the transosomes deep within the ooplasms were present within lysosomal vesicles in both of these instances and the vacuoles containing primitive yolk were patently abnormal in the "non-layer" mutant. Very few transosomes or primitive yolk vacuoles were present within the ooplasms of follicles from a testosterone-treated hen or from those of aged hens which were no longer laying. In both of these latter cases such bodies were present in the follicular cells. However, many transosomes were seen to be in the process of being lysed within the cytoplasms of these follicular cells.     

PROTEIN UPTAKE IN THE OOCYTES OF THE CECROPIA MOTH

The formation of yolk spheres in the oocyte of the cecropia moth, Hyalophora cecropia (L.), is known immunologically to result largely from uptake of a sex-limited blood protein. Recent electron microscope analyses of insect and other animal oocytes have demonstrated fine structural configurations consistent with uptake of proteins by pinocytosis. An electron microscope analysis of the cecropia ovary confirms the presence of similar structural modifications. With the exception of two apparently amorphous layers, the basement lamella on the outer surface of the follicular epithelium and the vitelline membrane on the inner, there is free access of blood to the oocyte surface between follicle cells. Dense material is found in the interfollicular cell space and adsorbed to the outer surface of the much folded oocyte membrane. Pits in the oocyte membrane and vesicles immediately under it are lined with the same dense material not unlike the yolk spheres in appearance. Introduction of ferritin into the blood of a developing cecropia moth and its localization adsorbed to the surface of the oocyte, and within the vesicles and yolk spheres of the oocyte cortex, is experimental evidence that the structural modifications of the oocyte cortex represent stages in the pinocytosis of blood proteins which arrive at the oocyte surface largely by an intercellular route. Small tubules attached to the yolk spheres are provisionally interpreted as a manifestation of oocyte-synthesized protein being contributed to the yolk spheres.