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.     

Ultrastructural observations on oogenesis in Drosophila

The ultrastructure of the follicle cells and oocyte periplasm is described during the stages of oogenesis immediately prior to, during, and immediately subsequent to, vitellogenesis. A number of features have not been described previously in Drosophila. Some yolk appears prior to pinocytosis of blood proteins. However, most of the protein yolk forms while the periplasm is filled with micropinocytotic invaginations and tubules derived from the oolemma. These tubules retain the internal layer of material characteristic of coated vesicles and are found to fuse with yolk spheres. No accumulation of electron-dense material in the endoplasmic reticulum or Golgi of the oocyte is found. Both trypan blue and ferritin are accumulated by the oocyte. The follicle cells have an elaborate endoplasmic reticulum during the period of maximum yolk accumulation. Adjacent cells are joined at their base by a zonula adhaerens, forming a band around the cells, and by plaques of gap junctions. Gap junctions are also present between nurse cells and follicle cells. During chorion formation, septate junctions also appear between follicle cells, adjacent to the zonula adhaerens.     

Selective endocytosis,in vitro,by ovarian follicles from Hyalophora cecropia

Ovarian follicles of Hyalophora cecropia, incubated in vitro with isolated and radiolabelled hemolymph and yolk proteins, provided a satisfactory model of in situ vitellogenesis. Uptake of proteins was specific. The follicles accumulated vitellogenin and microvitellin at constant rates for 6 hr, depositing them in the protein yolk spheres of the oocyte. Uptake of these two proteins was saturable by high concentrations of homologous protein and inhibited by p-dinitrophenol. In contrast, two other abundant hemolymph proteins, arylphorin and flavoprotein, were taken up at lower rates, and become concentrated primarily in the basement lamina of the follicle. Their accumulation was not saturable and not inhibited by p-dinitrophenol. The two yolk precursors were accumulated only by follicles at stages known to be vitellogenic, and the rates of uptake were shown to approximate the rates of accumulation of these proteins in situ. The uptake of vitellogenin, but not microvitellin, was enhanced 2- to 3-fold by hemolymph ultrafiltrates. Vitellin from mature eggs was not distinguishable from vitellogenin by the endocytotic apparatus. Finally, endocytotic uptake was not affected by inhibition of protein synthesis. This finding supports the concept of membrane and receptor recycling in yolk formation, and argues against an essential role of the follicle cell product paravitellogenin in the mechanism of hemolymph protein uptake.     

Study of yolk precursor transport in the avian ovary with the use of horseradish peroxidase.

Ovaries of adult Japanese quails were exposed in vivo to the exogenous protein horseradish peroxidase (HRP) for varying lengths of time to investigate ultrastructurally the permeability of the wall of these follicles, the protein uptake capacity of granulosa and oocyte and the kinetics of protein uptake in different stages. There is a sudden increase in permeability of the follicle wall from previtellogenesis to vitellogenesis. This is not due to a loss of sealing (tight) junctions in the granulosa cell layer, but is probably related to a permeability change in the basement membrane. The transition from the slow growth phase to the rapid growth during vitellogenesis is accompanied by a limited widening of the intercellular channels and the concomitant development of a complex endocytotic apparatus in the ooplasm. The slowing down of yolk deposition during the last day before ovulation is accompanied by a narrowing of the intercellular channel width. The granulosa cells show a high intracellular HRP uptake during intermediary yolk formation. Transcytosis through the granulosa cannot be excluded but is probably a minor pathway at certain stages. The light microscopically detectable uptake of HRP by the oocyte coincides with the start of exogenous vitellogenesis. After 90 sec of exposure to HRP (intravenous injection) the tracer can be found in the intercellular channels of the granulosa and in superficially located yolk spheres. On the other hand it takes 10 min for the tracer to traverse the cortex of the oocyte.     

Implication of gap junction coupling in amphibian vitellogenin uptake

The aim of the present study was to investigate the physiological role and the expression pattern of heterologous gap junctions during Xenopus laevis vitellogenesis. Dye transfer experiments showed that there are functional gap junctions at the oocyte/follicle cell interface during the vitellogenic process and that octanol uncouples this intercellular communication. The incubation of vitellogenic oocytes in the presence of biotinylated bovine serum albumin (b-BSA) or fluorescein dextran (FDX), showed that oocytes develop stratum of newly formed yolk platelets. In octanol-treated follicles no sign of nascent yolk sphere formation was observed. Thus, experiments in which gap junctions were downregulated with octanol showed that coupled gap junctions are required for endocytic activity. RT-PCR analysis showed that the expression of connexin 43 (Cx43) was first evident at stage II of oogenesis and increased during the subsequent vitellogenic stages (III, IV and V), which would indicate that this Cx is related to the process that regulates yolk uptake. No expression changes were detected for Cx31 and Cx38 during vitellogenesis. Based on our results, we propose that direct gap junctional communication is a requirement for endocytic activity, as without the appropriate signal from surrounding epithelial cells X. laevis oocytes were unable to endocytose VTG.     

Extracellular concentrating of proteins in the cecropia moth follicle

Yolk proteins, derived from the blood, are incorporated into the oocytes of insects and certain vertebrates by pinocytosis, but reach the oocyte surface only after penetrating the surrounding follicular epithelium via intercellular channels. In an investigation of the events occurring in these intrafollicular spaces, the dense extracellular material present between the follicle cells and in the oocyte's brush border was extracted from vitellogenic cecropia moth follicles by soaking in physiological saline. Quantitative immunochemical determination of several eluted blood proteins revealed that these components had been more concentrated in the extracellular spaces than in the blood. The average concentration factors were 2.5 for the predominant yolk protein, vitellogenin, and 4.5 for the carotenoid protein. Since injected foreign proteins were also accumulated in the spaces, the concentrating mechanism seemed to act on all available proteins. However, in vitro inhibition of yolk formation with dinitrophenol resulted in a selective increase in the amount of extracellular vitellogenin in follicles which had been previously exposed to a medium low in this protein, suggesting accumulation of a factor with a specific affinity for it. Furthermore under certain conditions vitellogenin was more readily released from the concentrate than was the carotenoid protein. These results indicate that, despite apparent lack of discrimination in the binding of blood proteins in the spaces, extracellular interactions may contribute to the selectivity known to occur during vitellogenesis.     

不同发育时期哲罗鱼卵黄的超微结构

应用透射电镜观察了不同发育时期哲罗鱼(Hucho taimen)卵黄的超微结构.根据哲罗鱼卵黄物质在卵母细胞中的加工合成、积累以及卵母细胞中参与卵黄颗粒形成的细胞器的变化,可将该鱼卵黄发生分为4个特征时期,即卵黄发生前期、卵黄泡期、卵黄积累期和卵黄积累完成期.卵黄发生前期是指卵母细胞发育过程中的卵黄物质开始积累前的时期,此时期核仁不断分裂,出现线粒体云和早期的滤泡细胞层、基层和鞘细胞层;卵黄泡期特点主要是细胞器不断变化产生卵黄泡和皮层泡;卵黄积累期的滤泡膜由内向外依次为放射带、颗粒细胞层、基层和鞘细胞层,此时外源性卵黄前体物质不断经过血液汇集于鞘细胞层,后经微胞饮作用穿过胶原纤维组成的基层,经过多泡体作用转运至颗粒细胞内,在细胞内经过加工和修饰形成小的卵黄蛋白颗粒,卵黄蛋白颗粒经微胞饮穿过放射带进入卵母细胞边缘形成的空泡中,不断积累形成卵黄球;进入卵黄积累完成期,卵黄球体积变大,向细胞中心聚集,填满大部分卵母细胞,卵黄积累完毕.     

Gap junctions between the follicle cells and the oocyte during oogenesis in an insect,Tribolium destructor/Coleoptera

Summary Oocyte-follicle cell gap junctions inTribolium occur in all oogenetic stages studied. During early previtellogenesis the junctions are found exclusively between lateral membranes of oocyte microvilli and the membrane of prefollicle cells. In late previtellogenesis and vitellogenesis the junctions are located between the tips of oocyte microvilli and the flat membranes of the follicle cells. During previtellogenesis gap junctions are infrequent, whereas in the phase of yolk accumulation their number increases considerably, exceeding 17 junctions/m² of the follicle cell membrane. It could be shown by microinjection of a fluorescent dye that gap junctions are in a functional state during vitellogenesis. Possible roles of heterologous gap junctions in oogenesis are discussed.     

Cellular and molecular markers of anteroposterior and dorsoventral organisation in the vitellogenic follicles of adult Sarcophaga bullata (Diptera) and dorsoventral orientation of follicles in the ovary

Summary Polar organisation in the follicles of adult Sarcophaga bullata is reflected in the nurse cell-oocyte axis and in the orientation of the two polar cell pairs in the follicular epithelium. The internal organisation of the nurse cell chamber contributes to polarity but not to dorsoventral asymmetry. Dorsoventral asymmetry is correlated with the eccentric position of the germinal vesicle and the orientation of the polar cell pairs; no other follicle cell specialisations are seen. In an ovary, follicles are preferentially orientated with the dorsal side to the centre of the ovary. Cytoskeletal and some haemolymph proteins are molecular markers of polarity. Thus, in pre-vitellogenic stages, tubulin immunoreactivity is higher in the oocyte than in the nurse cells, actin immunoreactivity is the same over the cystocytes and larval serum proteins are restricted to the poles. During vitellogenesis, both actin and tubulin become more concentrated in the nurse cells and larval serum protein 1 accumulated in the polar cells during border cell migration when yolk polypeptides also accumulate in the oocyte. At the end of vitellogenesis a lipophorin is taken up by the oocyte. No molecular marker of dorsoventral asymmetry was identified.     

INTRACELLULAR SYNTHESIS, TRANSPORT, AND PACKAGING OF PROTEINACEOUS YOLK IN OOCYTES OF ORCONECTES IMMUNIS

The incorporation of leucine-³H into either ovarian or oocyte proteins occurs throughout vitellogenesis, but is at a maximum during early phases of this process. The labeling of ovarian and oocyte proteins is inhibited with cycloheximide. Oocytes are permeable to actinomycin D, and this drug does not affect the incorporation of amino acids into oocyte proteins but does block oocyte RNA synthesis. By means of both light microscope and high resolution radioautography, it has been demonstrated that the initial incorporation of leucine-³H under both in vitro and in vivo conditions occurs in elements of the rough-surfaced endoplasmic reticulum in the oocyte. Under pulse-chase conditions, the label subsequently becomes associated with intracisternal (precursor yolk) granules now aggregated within the cisternae of the connected smooth-surfaced endoplasmic reticulum. By 7 days, mature yolk globules are extensively labeled. The results of experiments designed to assess the possible contribution of maternal blood proteins to yolk deposition indicate that such a contribution is minimal. It is concluded that the crayfish oocyte is programmed for and capable of synthesizing the massive store of proteinaceous yolk present in the egg at the end of oogenesis.     

THE DEPENDENCE OF CECROPIA YOLK FORMATION IN VITRO ON SPECIFIC BLOOD PROTEINS

The capacity of cecropia vitellogenic follicles to form yolk during short-term in vitro incubation in female blood was analyzed by labeling with fluorescein-conjugated serum globulin, tritiated cecropia blood proteins, or tritiated amino acid. As judged by fluorescence microscopy or autoradiography, yolk formation during 3–8 hr in vitro was similar in rate and in protein uptake specificity to that observed in vivo. When follicles were incubated in cecropia male blood, 6% gamma globulin, or cecropia saline, the yolk produced was markedly inferior in quality and quantity to that generated in female blood. Purified preparations of vitellogenin, the primary female blood protein deposited in the yolk, were equivalent to whole female blood in supporting yolk formation; this protein seems, therefore, to have a specific stimulatory role. An enhancement of the rate of pinocytosis at the oocyte surface by vitellogenin is postulated.     

Regulation of the vitellogenin receptor during Drosophila melanogaster oogenesis

In many insects, development of the oocyte arrests temporarily just before vitellogenesis, the period when vitellogenins (yolk proteins) accumulate in the oocyte. Following hormonal and environmental cues, development of the oocyte resumes, and endocytosis of vitellogenins begins. An essential component of yolk uptake is the vitellogenin receptor. In this report, we describe the ovarian expression pattern and subcellular localization of the mRNA and protein encoded by the Drosophila melanogaster vitellogenin receptor gene yolkless (yl). yl RNA and protein are both expressed very early during the development of the oocyte, long before vitellogenesis begins. RNA in situ hybridization and lacZ reporter analyses show that yl RNA is synthesized by the germ line nurse cells and then transported to the oocyte. Yl protein is evenly distributed throughout the oocyte during the previtellogenic stages of oogenesis, demonstrating that the failure to take up yolk in these early stage oocyte is not due to the absence of the receptor. The transition to the vitellogenic stages is marked by the accumulation of yolk via clathrin-coated vesicles. After this transition, yolk protein receptor levels increase markedly at the cortex of the egg. Consistent with its role in yolk uptake, immunogold labeling of the receptor reveals Yl in endocytic structures at the cortex of wild-type vitellogenic oocytes. In addition, shortly after the inception of yolk uptake, we find multivesicular bodies where the yolk and receptor are distinctly partitioned. By the end of vitellogenesis, the receptor localizes predominantly to the cortex of the oocyte. However, during oogenesis in yl mutants that express full-length protein yet fail to incorporate yolk proteins, the receptor remains evenly distributed throughout the oocyte.     

An autoradiographic and cytophotometric study of oogenesis in a pulmonate snail,Planorbarius corneus

Summary The spatial and temporal patterns of macromolecular syntheses in oocytes and somatic auxiliary cells of the snail Planorbarius corneus have been investigated by autoradiography and cytophotometry. Oogenesis has been divided into three stages, comprising early meiosis up to diplotene (stage I), previtellogenetic growth phase (stage II), and vitellogenesis (stage III). No DNA synthesis was found in any oocyte stage. In stage-I oocytes, only nucleoli were found labelled with ³H-uridine. Oocyte nuclei of stage II and III actively synthesize RNA in nucleoli and chromosomes. The most intense incorporation of uridine in chromatin probably occurs during the previtellogenesis — vitellogenesis transition period during which cytological findings suggest well developed lampbrush chromosomes. RNA synthesis in amphinucleoli of stage-III oocytes is restricted to basophilic nucleolar parts, whereas acidophilic parts (protein bodies) neither synthesize nor store RNA. During vitellogenesis oocytes incorporate amino acids into yolk platelet proteins. Radioactive proteins are found in yolk platelet precursors 5 h after injection of the tracer and in yolk platelets 3 h thereafter. The labelling pattern suggests that oocytes synthesize certain hitherto unidentified yolk components. No evidence for the participation of follicle cells in synthesis and transport of vitellogenic proteins has been obtained from autoradiography. Cytological findings suggest an important role for these cells in oogenesis. They are highly active in RNA and protein synthesis. Cellular differentiation is accompanied by polyploidization of the nuclei which attain a highest DNA content of 256 c. Polyploidization probably occurs in incremental steps as indicated by complete endomitotic chromosomal cycles. Autoradiographs show that, during vitellogenesis, oocytes do not incorporate significant amounts of glucose, and only certain follicle cells were labelled with glucose, probably indicating the synthesis of glycogen.     

Endosomes transfer yolk proteins to lysosomes in the vitellogenic oocyte of the trout

The internalization of the yolk proteins has been investigated by electron microscopy and cytochemistry in the oocyte of the trout which stores up large quantities of yolk. The oocyte evolution has been followed for 18 months in a homogeneous group of animals. Anionic ferritin has been injected during vitellogenesis. The results indicate that as in other oocytes the yolk proteins are absorbed by coated vesicles during vitellogenesis. But unlike most other oocytes the yolk proteins are then transferred via typical endosomes to a conspicuous lysosomal compartment built up very early at the onset of the cytoplasmic differentiation of the oocyte e.i. 10 months earlier. During vitellogenesis yolk progressively accumulates in this lysosomal compartment. Injected anionic ferritin follows the same pathway of internalization. These findings indicate that in this oocyte, the whole yolk cycle presumably represents an adaptation of a general cellular activity, the receptor-mediated endocytosis, largely amplified, sequenced and spread over several months.     

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     

Major protein changes during vitellogenesis and maturation of Fundulus oocytes

The protein content of various size follicles was measured in Fundulus heteroclitus and indicated four phases of increase relative to follicle volume: Phases I (previtellogenic; estimated to be less than 0.01 mg/mm3), II (vitellogenic; 0.20 mg/mm3), III (early maturation; 0.03 mg/mm3), and IV (late maturation; 0 mg/mm3). A pronounced and rapid size increase occurs during maturation due to hydration, but protein uptake, which was also documented cytologically, contributes to about 16% of the volume increment during early maturation. Protein incorporation appears to stop abruptly at the time of germinal vesicle breakdown, most likely reflecting an altered physiological state of the oocyte. SDS-polacrylamide gel electrophoretic patterns of various size follicles indicated that five major protein bands (molecular weights = 122, 103, 45, 26, and 20 k) accumulate during vitellogenesis and presumably are proteolytically derived from a 200-kDa vitellogenin precursor. During maturation, the 122- and 45-kDa proteins disappear and several new, lower molecular weight bands appear. Proteolysis of specific yolk proteins may thus help generate part of the osmotic pressure gradient required for water uptake during oocyte maturation.     

OVARIAN NUTRITIONAL RESOURCES DURING THE REPRODUCTIVE CYCLE OF THE HEMATOPHAGOUS DIPETALOGASTER MAXIMA (HEMIPTERA: REDUVIIDAE): FOCUS ON LIPID METABOLISM

In this study, we have analyzed the changes of the ovarian nutritional resources in Dipetalogaster maxima at representative days of the reproductive cycle: previtellogenesis, vitellogenesis, as well as fasting‐induced early and late atresia. As expected, the amounts of ovarian lipids, proteins, and glycogen increased significantly from previtellogenesis to vitellogenesis and then, diminished during atresia. However, lipids and protein stores found at the atretic stages were higher in comparison to those registered at previtellogenesis. Specific lipid staining of ovarian tissue sections evidenced remarkable changes in the shape, size, and distribution of lipid droplets throughout the reproductive cycle. The role of lipophorin (Lp) as a yolk protein precursor was analyzed by co‐injecting Lp‐OG (where OG is Oregon Green) and Lp‐DiI (where DiI is 1,10‐dioctadecyl‐3,3,30,30‐tetramethylindocarbocyanine) to follow the entire particle, demonstrating that both probes colocalized mainly in the yolk bodies of vitellogenic oocytes. Immunofluorescence assays also showed that Lp was associated to yolk bodies, supporting its endocytic pathway during vitellogenesis. The involvement of Lp in lipid delivery to oocytes was investigated in vivo by co‐injecting fluorescent probes to follow the fate of the entire particle (Lp‐DiI) and its lipid cargo (Lp‐Bodipy‐FA). Lp‐DiI was readily incorporated by vitellogenic oocytes and no lipoprotein uptake was observed in terminal follicles of ovaries at atretic stages. Bodipy‐FA was promptly transferred to vitellogenic oocytes and, to a much lesser extent, to previtellogenic follicles and to oocytes of ovarian tissue at atretic stages. Colocalization of Lp‐DiI and Lp‐Bodipy‐FA inside yolk bodies indicated the relevance of Lp in the buildup of lipid and protein oocyte stores during vitellogenesis.     

Germ line dependence of the deep orange maternal effect in Drosophila

Pole cell transplantations were used to determine the tissue specificity of maternal effects in Drosophila. The deep orange maternal effect is shown to be germ line autonomous. A cytoplasmic injection assay was used to determine when the dor⁺ substance could be detected in the developing oocyte. The dor⁺ substance is present during the early stages of vitellogenesis but could not be detected in the yolk of the embryo after blastoderm cellularization.     

Aspects of the reproductive biology of the bass, Dicentrarchus labrax L. I. An histological and histochemical study of oocyte development

Histological and histochemical studies of oocyte development in the bass, Dicentrarchus labrax L., showed that three types of inclusions are formed during vitellogenesis. Lipid yolk accumulates first as lipid droplets, followed by protein yolk in the form of discrete protein yolk granules. The third type of inclusion are the small cortical alveoli (intravesicular yolk/yolk vesicles, i.e.'carbohydrate yolk') which form in the peripheral cytoplasm after both the lipid and protein yolk have started to accumulate. While the protein yolk granules maintain their structural integrity through to maturation, forming a densely packed zone in the mid-outer cortex, the lipid yolk droplets continually coalesce and migrate centripetally, forming a prominent zone of large lipid droplets in the inner-mid cortex. From the histological study of oocyte development, a number of distinct developmental stages are delineated, while gross examination of the paired ovary revealed that, depending on its stage of development, it can be placed into one of seven maturity stages.     

Oogenesis and ovarian histology in two populations of the viviparous lizard Sceloporus grammicus (Squamata: Phrynosomatidae) from the central Mexican Plateau

The annual histological changes in ovarian morphology (oogenesis, follicular atresia, and corpus luteum) are described for the Mexican lizard Sceloporus grammicus, in two populations that inhabit contrasting environments (vegetation categories, climate, precipitation, and temperature) from Hidalgo State, Mexico. Two germinal beds were situated on the dorsal surface of each ovary of this species. In both the populations, oogenesis involves two major processes: previtellogenesis and vitellogenesis. The histological changes during previtellogenesis are similar to those for other reptilian sauropsids, whereas vitellogenesis differs and the features of this last process are described for the first time. In early previtellogenesis, primary oocytes have fibrillar chromosomes and the ooplasm stains slightly. The primordial follicles are surrounded by a granulosa composed of cuboidal follicular cells. During late previtellogenesis, the oocyte had an eccentric nucleus with lamp‐brush chromosomes and multiple nucleoli. The granulosa becomes multilayered and polymorphic, containing three cell types: small, intermediate, and pyriform. The zona pellucida was homogeneous and clearly observed. In early vitellogenesis, the oocyte showed several small acidophilic granules distributed in the center and the periphery of the oocyte. As vitellogenesis progresses, the yolk platelets move toward the central area of the oocyte and they fuse to form acidophilic and homogeneous yolk. Lipid droplets were distributed irregularly in the ooplasm of the oocyte. In Zacualtipán, the results revealed a strong seasonal reproductive activity. Females had vitellogenic follicles from July to September, and pregnant females were founded from September to March. In Tizayuca, the results showed an unusual pattern of reproductive activity. Females with vitellogenic follicles and pregnant females were found throughout the year, indicating continuous reproduction. We suggest that the observed differences in reproductive activity from these populations indicate adaptative fine tuning in response to local environmental conditions. These results contribute to the knowledge of variation in vitellogenesis and reproductive strategies of this species and among spiny lizards overall. J. Morphol. 275:949–960, 2014. © 2014 Wiley Periodicals, Inc.