The Regulation of yolk polypeptide synthesis in Drosophila ovaries and fat body by 20-hydroxyecdysone and a juvenile hormone analog

In adult female Drosophila melanogaster an increase in the synthesis and secretion of three yolk polypeptides (YPs) occurs during the first 24 hr after eclosion. During organ culture, these same polypeptides are synthesized and secreted into the medium by both fat body and ovaries. Two hormones, 20-hydroxyecdysone (20-HE) and a juvenile hormone analog (ZR-515) stimulate synthesis and secretion of YPs into the hemolymph of isolated female abdomens. The present experiments were undertaken to compare synthesis of YPs in normal females with YP synthesis in preparations deprived of anterior endocrine glands, and to find which hormone stimulates synthesis in the different organs. Separation of hemolymph proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that at eclosion incorporation of [³⁵S]methionine into YP1 and YP2 was low and was barely detectable in YP3. Over the next 24 hr the rate of label incorporation increased for all the YPs. Isolation of female abdomens at eclosion prevented this increase in label incorporation but did not entirely abolish YP synthesis. Application of either ZR-515 or 20-HE to isolated abdomens stimulated up to ninefold label incorporation into three polypeptides which comigrated with YPs from normal vitellogenic females. The response of isolated abdomens to ZR-515 or 20-HE was first detectable between 90 and 135 min after hormone application. The stimulated bands were confirmed to be YPs by a comparison of peptide digests of each of the three labeled polypeptides with those of the yolk polypeptides from intact vitellogenic females. The hypothesis that the two hormones might act on different organs was tested by treating isolated female abdomens with various concentrations of either ZR-515 or 20-HE and then culturing the stimulated organ in vitro with [³⁵S]methionine. The fat body responded to both hormones by synthesizing and secreting into the culture medium polypeptides which comigrated with the YPs found in hemolymph, whereas the ovary produced similar polypeptides only after ZR-515. These secreted polypeptides were confirmed to be YPs by repeating the experiment using organs from heterozygotes for both YP2 and YP3 electrophoretic variants. Such organs synthesized five polypeptides which comigrated with the corresponding yolk polypeptides. These findings are discussed in relation to a hypothesis for the action of the two hormones.     

Sex determining genes and vitellogenin synthesis in Drosophila melanogaster

Summary This study investigates the relationship between sexual phenotype and ability to synthesize vitellogenin (yolk proteins, YPs) in Drosophila. Various mutations were used to transform XX and XY animals into intersexes or pseudomales (Table 1). The presence or absence of YPs in the haemolymph and in the fat body was determined by SDS gel electrophoresis, fluorography, and precipitation of YPs with anit-YP antibody (see Fig. 1). YPs were synthesized whenever the flies displayed at least some female morphological characteristics, regardless of their sex chromosome constitution (Table 1; Fig. 2). Pseudomales (definition see p. 1) did not produce detectable amounts of YPs despite their female XX-karyotype. Immature ovaries, transplanted into adult males or pseudomales, developed normally and synthesized YPs, but the fat bodies of the host males or pseudomales were not induced to synthesize YPs. Vitellogenesis was, however, induced in the fat bodies of males and pseudomales by injection of 20-hydroxyecdysone (ecdysterone) (Fig. 3). The results are interpreted to mean that the sexual pathways are controlled by a small number of key genes that regulate the synthetic activities of many sex-specific genes. However, the female-specific YP genes can be activated with ecdysterone although the genetic signals are set for male differentiation.     

Ovarian yolk-protein synthesis in Drosophila melanogaster

The ovaries and fat bodies of Drosophila melanogaster adult females both synthesize yolk polypeptides. In a series of experiments it has been shown that the ovaries become competent to mature in an adult male host, where only ovarian synthesis occurs, very early in metamorphosis and synthesis of yolk polypeptides begins in a time-dependent sequence related to the age of the ovary when transplanted. Maturation of ovaries occurs prior to eclosion when they are transplanted to an earlier developmental stage showing that neither the event of eclosion not the adult environment is essential in triggering yolk-polypeptide synthesis by the ovary. When metamorphosing ovaries are transplanted to a female host they take up host yolk polypeptides from the haemolymph, but this does not lead to the implanted ovary developing substantially better than in a male host where only synthesis by the ovary can occur. The regulation of ovarian yolk-polypeptide synthesis therefore appears to be autonomous to the ovary itself. There may be a trigger early in metamorphosis which induces competence in the ovary so that it subsequently initiates yolk-polypeptide gene expression at eclosion.     

Yolk polypeptide synthesis in the fat body of Sarcophaga bullata: Localization,hormonal induction and cell-free translation

The fat body of adult Sarcophaga bullata consists of different cell-types. The yolk polypeptides (YPs) are localized in secretory granules in the cytoplasm of female trophocyte fat body cells while the oenocytes and larval fat body cells are immunonegative. An antiserum against the larval serum protein 1 of Drosophila crossreacts on immunoblotting with several polypeptide bands in the haemolymph with mol. wt ∼80 kD. This antiserum specifically reacts with some storage granules of the persisting larval fat body cells and not with the other fat body cell types. The trophocyte fat body cells of male Sarcophaga treated with 20-OH-ecdysone, display a similar granular type of immunoreaction with an anti-YP antiserum as in vitellogenic females. Moreover, 20-OH-ecdysone induced in the fat body of males, in contrast to methoprene, synthesis of mRNA coding for YPs to a level as high as that in vitellogenic females, as shown in the reticulocyte lysate cell-free system.     

Biosynthesis of Drosophila yolk polypeptides

The three yolk polypeptides (YPs) of Drosophila are synthesized and secreted by female fat body and ovarian follicle cells, sequestered by pinocytosis into oocytes, and finally deposited into yolk granules. The biosynthesis of the YPs was studied using two-dimensional gels. Labeling the YPs with [³⁵S]-cysteine, an amino acid found only near the amino terminus of YP1 and YP2, showed that an amino terminal peptide is removed from YP1 and YP2 shortly after or during translation. Intermediates in YP biosynthesis corresponding in electrophoretic mobility to pancreatic membrane-processed primary translation products were also detected in a 5-min pulse label with [³⁵S]-methionine. Genetic variants that alter YP structure were used to identify which YP precursor comes from which Yp gene. Pulse labeling with [³⁵S]-methionine revealed that all three YPs becomes more negatively charged, that YP1 and YP2 become heterogeneously charged, and that YP1 gains in apparent molecular weight within 15 min after translation. Injecting female flies with radioiabeled sugars or orthophosphate revealed that the YPs are glycosylated and phosphorylated. Treating hemolymph proteins with phosphatase showed that phosphorylation is responsible for much of the change in charge and increase in molecular weight of the maturing YPs. These experiments with wild-type flies provide a basis for the analysis of mutations at the Yp genes which alter the structure of individual YPs.     

Identification, synthesis, and characterization of the yolk polypeptides of Plodia interpunctella

The mature eggs of Plodia interpunctella were found to contain four major polypeptides. These yolk polypeptides (YPs) were found to have approximate molecular weights of 153,000 daltons (YP1), 69,000 daltons (YP2), 43,000 daltons (YP3), and 33,000 daltons (YP4) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In addition, we found YP1 was resolved by a 5% polyacrylamide gel into two separate polypeptides of 153,000 and 147,000 daltons. All of the YPs could be labeled in vivo or in vitro with [35S]-methionine. Yolk peptide 1 and YP3 were synthesized by fat body of pharate adult and adult females and secreted into the hemolymph. Yolk peptide 2 and YP4 were synthesized and secreted into incubation medium by ovaries that contained vitellogenic oocytes, but these polypeptides were not found in the hemolymph. Fat bodies of males synthesized and secreted an immunoprecipitable polypeptide similar to YP3 as well as immunoprecipitable polypeptides larger than 200,000 daltons that had no counterparts in the oocytes. Peptide mapping by protease digestion showed each YP to be cleaved into unique fragments, suggesting that no precursor-product relationship exists between the YPs. Ion exchange chromatography and gel permeation chromatography separated that yolk proteins into two groups with approximate molecular weights of 462,000 and 264,000 daltons. By resolving these peaks on SDS-PAGE, it was found that YP1 and YP3 formed the 462,000-dalton yolk protein and YP2 and YP4 formed the 264,000-dalton yolk protein.     

Ovarian defects in hybrids of the species pair Drosophila virilis and Drosophila texana

In interspecific matings between Drosophila virilis and Drosophila texana female sterility is observed in F₂ hybrid females. A previous study has shown that no vitellogenin synthesis occurs in the fat body of sterile hybrid females. The results presented in this paper show that hybrid ovaries of sterile females transplanted into the abdomens of females of the parental species are not able to develop upon maturity. With few exceptions, the hybrid ovaries remained alive in the host environment, but their oocytes failed to develop to vitellogenic stages. Thus, in hybrid females between Drosophila virilis and Drosophila texana sterility is the result of defects in both the two main developmental processes of egg maturation, the synthesis of vitellogenins in the fat body and the uptake of vitellogenins by the ovary. Dev Genet 20:47–52, 1997. © 1997 Wiley-Liss, Inc.     

Female qualities in males: Vitellogenin synthesis induced by ovary transplants into the male silkworm, Bombyx mori

Female qualities in males are common in vertebrates but have not been extensively reported in insects. Vitellogenin (Vg) is highly expressed in the female fat body and is generally required for the formation of yolk proteins in the insect egg. Vg upregulation is generally regarded as a female quality in female oviparous animals. In this study, we found that Bombyx mori Vg (BmVg) is especially highly expressed in the female pupa. Downregulation of the BmVg gene in the female pupa by RNA interference (RNAi) interfered with egg formation and embryonic development, showing the importance of BmVg in these processes. So, we used BmVg as a biomarker for female qualities in the silkworm. Hematoxylin-eosin staining and immunofluorescence histochemistry showed that ovary transplants induced BmVg synthesis in the male pupa fat body. Ovaries transplanted into male silkworms produced only a few eggs with deformed yolk granules. These results suggested that the amount of BmVg in the male silkworm was insufficient for eggs to undergo complete embryonic development. After 17-beta-estradiol was used to treat male pupae and male pupal fat bodies, BmVg was upregulated in vivo and in vitro. These findings indicated that the male silkworm has innate female qualities that were induced by a transplanted ovary and 17β-estradiol. However, in silkworms, female qualities in males are not as complete as in females.     

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.     

Genetic analysis of Drosophila vitellogenesis: A molecular weight variant of yolk polypeptide-2 in Drosophila simulans

Summary To assess the likelihood of finding genetic variants for the three major yolk polypeptides (YPs) within the species Drosophila melanogaster, YPs from the five species most closely related to D. melanogaster were investigated. The relative positions of the three YPs were characteristic for each species, and in all cases the mobilities of the YP in the ovary corresponded to that of the YP in the hemolymph of the same species.Different stocks of Drosophila simulans were found to have either of two forms of yolk polypeptide-2 (YP2). The YP2^S polypeptide migrated more slowly than YP2^F by an apparent molecular weight difference of about 700 daltons. The genetic factor responsible for this difference mapped to locus 35 on the X chromosome. The Yp2 allele present specified the mobility of the YP2 polypeptide in both the hemolymph and the ovary. YP1 and YP3 were the same in both Yp2 ^S and Yp2 ^F stocks indicating that they are not affected by the Yp2 gene. Densitometric scans of gels showed that there was more than twice as much YP2^F as YP2^S in the ovaries and hemolymph of homozygous animals. Yp2 ^S/Yp2 ^F heterozygotes contained both fast and slowly migrating YP2. The amount of each YP2 in Yp2 ^F/Yp2 ^S heterozygotes was about half that found in each homozygote. These dosage results suggest that this locus is the structural gene. Peptide mapping showed that the structural element contributing to retarded mobility of YP2^S is unlikely to reside at either end of the molecule. These experiments suggest a cytogenetic location in which to concentrate further investigations on the genetic regulation of YP2 synthesis.     

Vitellogenesis in the stable fly,stomoxys calcitrans

• 1.1. No female specific proteins were found in the stable fly hemolymph by polyacrylamide gel electrophoresis.
• 2.2. Six major yolk polypeptides (YP1, YP2, YP3, YP4, YP5 and YP6) have been identified in the stable fly. Their mol. wt as determined by SDS-polyacrylamide gel electrophoresis are 41,100, 42,600, 44,100, 46,600, 48,900 and 50,600, respectively.
• 3.3. YP3 was purified and antibody made against it. By using the antibody and in vitro organ culture the stable fly yolk polypeptides were shown to be synthesized exclusively by the ovaries and not the fat body.
• 4.4. The stable fly yolk polypeptides are immunologically similar to yolk proteins of other related flies.

     

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.     

Ecdysteroids in adult males and females of Drosophila melanogaster

Ecdysteroid titres in whole flies and different tissues of adult male and female Drosophila were determined at various times after eclosion using a radioimmunoassay. The ecdysteroid titre decreased as the flies matured after eclosion. The differences in titre between males and females can be accounted for by their difference in body weight. The ecdysteroids were found to be distributed throughout several tissues. At eclosion not all of the ecdysteroid complement present could be accounted for by that found localised in tissues. After maturation of the flies the ecdysteroids in various tissues can account for the majority of that detected in whole-fly extracts. Ecdysteroids were produced during in vitro culture of various tissues, but the quantities detected were low by comparison with ring glands of wandering 3rd-instar larvae. Neither the ovaries nor the abdominal body walls (fat body) seem to be a major source of hormone, and they are only able to convert minute quantities of ecdysone to the biologically active form, 20-hydroxyecdysone, in vitro. The amounts of 20-hydroxyecdysone present were measured using high performance liquid chromatography and radioimmunoassay. We tentatively suggest that the differential experession of the yolk-protein-genes in the fat bodies of males and females does not result from differences in hormone titres between them.     

High cyclic AMP levels in young chick embryonic hearts.

Acid phosphatase (Acph) activities and protein content were measured in developing ovaries of adult flies. Acph and protein increased approximately logarithmically for the first 2 days of adult life and then plateaued at about 80 and 35 times, respectively, the levels present at eclosion. The specific activity of Acph was constant for the first 15 hr and then increased by a factor of three over the next 2 days. Analysis of staged follicles showed that the specific activity of Acph starts to increase at stage 10. Ovaries from homozygotes for Acph-1ⁿ⁴, a null activity mutant, showed constant low specific activity, indicating that this gene codes for the major ovarian Acph. Ovarian transplantations between Acph-1ⁿ⁴ and wild type showed that Acph is made by the ovary. Ovaries from isolated abdomens failed to increase in Acph activity or protein, but treating isolated abdomens with ZR-515, a juvenile hormone analog, caused nearly normal levels to be attained. Ovaries of the female sterile mutant ap⁴ failed to develop Acph activity unless they were implanted into a normal host or treated with ZR-515. Ovaries from the female sterile mutant fs(3)L3 developed no increase in Acph activity even when treated with ZR-515. The results demonstrate that the activity of a genetically localized enzyme is controlled by a chemically defined hormone in a genetically favorable higher eucaryote.     

Hormonal control of ovarian development in the silkworm,Bombyx mori

Hormonal control of ovarian development was examined in Bombyx mori. The weight of the ovary increased suddenly by 3 days after pupal ecdysis, and vitellogenin could be immunologically detected in the ovary at that time. The ecdysteroid titers during pupal-adult development, quantified by radioimmunoassay, increased from day 0 to day 2. Ovarian development was arrested for a long period in brainless pupae and isolated pupal abdomens. Injection of 20-hydroxyecdysone into such preparations stimulated development of the ovaries, and vitellogenin could be detected in ovaries 2 days after injection. The results suggest that 20-hydroxyecdysone acts by stimulating the growth of ovary.     

Mutant yolk proteins lead to female sterility in Drosophila.

Specific mutations in the yolk protein genes, yp1 and yp2, of Drosophila melanogaster cause the yolk proteins (YPs) they encode to precipitate, ultimately resulting in female sterility. YPs of the yp1 mutant fs(1)1163 are secreted normally but then precipitate as globules and occasionally as crystalline fibers in the subbasement membrane space of the fat body (Butterworth et al., 1991, J. Cell Biol. 112, 727-737). The present ultrastructural and immunological studies of the fat body of the yp2 mutant fs(1)K313 show that YP also precipitates as globules in the same tissue compartment. The globules are also incapable of passing into the hemolymph but they are morphologically distinct from those of fs(1)1163. Similar analyses were performed on developing oocytes in wild type and both mutant strains. YP-containing aggregates, ultrastructurally similar to those in the fat body of each respective mutant, were found in the space between the plasmalemma and the vitelline membrane and embedded within the membrane itself. The evidence suggests that the precipitates interfere with the correct assembly of the eggshell membranes, leading to the sterile phenotype. Immunogold studies demonstrate that newly synthesized YPs in the normal and mutant strains share secretory vesicles with putative, vitelline membrane proteins and that the translocation of follicle cell YP is not through the membrane along the interfollicular spaces but directly through the plasmalemma facing the oocyte. Further the YP precipitates in the mutants permit visualization of the polarity of exocytosis of YP from the follicle cells.     

Genetic and Hormonal Regulation of Vitellogenesis in Drosophila

In Drosophila the female-specific yolk protein, or vitellogenin,is synthesized in the fat body. In D. melanogaster, vitellogenin,is first detected in the female hemolymph at the time of adulteclosion and in the ovaries 20 hours later, suggesting differentregulatory mechanisms for the processes of synthesis and uptake.Transplantations of pupal or immature adult ovaries into D.melanogaster adult males induce vitellogenin synthesis, implicatingan ovarian agent in the control of synthesis. Larval ovariesfail to stimulate synthesis. Female-sterile mutants with rudimentaryor previtellogenic ovaries synthesize and accumulate large quantitiesof vitellogenin in the hemolymph, but not in the ovaries. Transplantationof these rudimentary ovaries into males induces vitellogeninsynthesis, suggesting that the ovarian inducing agent does notoriginate from the germ cells. Treatment of the homozygous female-sterilemutants with juvenile hormone stimulates the uptake of vitellogeninby the ovary in some strains. This shows that juvenile hormoneplays a role in vitellogenin uptake. The potential importanceof Drosophila vitellogenesis for studies of gene regulationis discussed.