Ultrastructural studies of male-incubated ovaries of the gypsy moth,Lymantria dispar

Ovaries from Lymantria dispar females were transplanted into an environment lacking the vitellogenin ligand; i.e., the male milieu. Transmission electron micrographs comparing the terminal oocytes of male-grown ovaries and normal ovaries showed that yolk sphere diameters were reduced in the male-grown oocytes. However, there were larger numbers of these small yolk spheres per unit area of cytoplasm, indicating that the coalescence of endosomes into yolk spheres is reduced in the absence of vitellogenin. Although there are larger numbers of yolk spheres in male-grown oocytes, the smaller diameter of yolk spheres resulted in less area being taken up by yolk spheres per unit area of cytoplasm in male-grown oocytes, yielding lowered yolk production. This lowered yolk production is a result at least in part of the lowered number of coated vesicles per unit area of submembrane space and in part of the reduced interfollicular spaces seen in male-grown ovaries.     

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.     

A correlation between juvenile hormone deficiency and vitellogenic oocyte degeneration inDrosophila melanogaster

Summary It is known from previous work that juvenile hormone (JH) is required to initiate vitellogenin uptake into maturing oocytes ofDrosophila melanogaster, but additional requirements for this hormone during oocyte maturation have not been fully understood. To determine if early vitellogenic oocytes (stages 8 and 9) require JH for continued development, these oocytes were transplanted toDrosophila female and male hosts which were rendered deficient in JH by three methods. Implanted stage 9 and usually stage 8 oocytes were found to degenerate in JH-deficient hosts unless ZR-515, a JH analogue, was applied to the host shortly after implantation.These results were confirmed during in situ ovary development. JH deficiency was produced in gravid females, and ovaries examined at subsequent time intervals were found to be deficient in stage 8–10 oocytes as early as 6 h after treatment. Degenerating oocytes corresponding to these stages were commonly found. ZR-515 prevented oocyte degeneration during at least the first 8 h and continued to support stage 8–10 oocyte development 24 h after application to these females. The results suggest that JH is required not only for initiation but also for continuation of vitellogenin uptake and oocyte development.     

Vitellogenesis and oocyte development in azadirachtin-induced fifth-instar overage nymphs of Locusta migratoria (L.)

Injection of azadirachtin into females of Locusta migratoria at the beginning of the last nymphal instar prevented molting to the adult stage, and many of these locusts survived for long periods as overage fifth-instar nymphs. Overage female nymphs synthesized vitellogenin; maximum vitellogenin content in their hemolymph was 6–7 times higher than that found in normal adult females. The overage female nymphs developed vitellogenic oocytes, but development was retarded to some extent: although vitellogenin did accumulate in the proximal oocytes, their maximum average length was only about 2.8 mm (compared to 6.2 mm in normal adult females) and extensive oocyte resorption was observed. Thus, attainment of adult competence of the organs and processes involved in female reproduction is independent to a considerable extent from the process of overt adult morphogenesis.     

Qualitative and quantitative changes in hemolymph proteins during an ovarian cycle and after insemination in Thermobia domestica (packard) (thysanura,lepismatidae)

Qualitative and quantitative investigations on the hemolymph proteins in the adult firebrat Thermobia domestica were performed during an ovarian cycle in inseminated and noninseminated females. Variations of hemolymph protein concentration were determined by Lowry's method. In addition, the proteins were studied by gradient slab gel electrophoresis using nondenaturing conditions and microdensitometry. Besides five major protein fractions, which are present in both sexes, three female-specific protein bands (vitellogenins) are found in the hemolymph and in maturing oocytes. These vitellogenins have molecular masses of 430, 300 and 240 kiloDalton. In fact, associated with the main 300-kD band, there were two smaller bands (320 and 280 kD) indistinguishable by densitometric measurement. Quantitative changes of vitellogenins are linked to oocyte maturation. These proteins appeared in the hemolymph before ecdysis, at the same time as the first yolk granules in the basal oocytes. They increased after ecdysis during the intense vitellogenic phase and decreased during chorion formation. In noninseminated females, in which all maturing oocytes are resorbed before chorion formation, the level of the 300 kD vitellogenins remained lower than in inseminated females. The quantity of vitellogenins fell only after complete oosorption. Thus insemination caused changes in the relative quantities of the different vitellogenic proteins.     

Ovarian development and vitellogenesis in the sawfly,Athalia rosae ruficornis Jakovlev (Hymenoptera,Tenthredinidae)

Summary

Ovarian development in Athalia rosae ruficornis Jakovlev (Hymenoptera: Tenthredinidae) is described. Number of nurse cells per egg chamber is most often around 60 (close to 63 according to the 2ⁿ–1 rule), but in many cases it deviates from this number significantly. Two major yolk proteins [vitellins: large (apparent molecular weight 160–170 kD) and small (48–50 kD] were identified by SDS-PAGE. Western blotting and immunochemical detection using polyclonal antibodies prepared against each of the vitellins revealed that adult female but not male (both haploid and diploid) hemolymph contains vitellogenins corresponding to these vitellins. Vitellogenins become detectable in the hemolymph of late pupae, and vitellins one day later in the oocytes of adults. Transplantation of immature ovaries into the adult male abdomen caused not only significant accumulation of vitellins in the oocyte but also appearance of small amounts of hemolymph vitellogenins in host males. Injection of homogenate of immature ovaries also caused appearance of small amounts of hemolymph vitellogenins in host males.     

Onset of vitellogenin production and vitellogenesis,and their relationship to changes in the midgut epithelium and oocytes in the tickDermacentor variabilis

InDermacentor variabilis (Say), the onset of vitellogenin production and vitellogenesis (up-take of vitellogenin into oocytes) began during the rapid-engorgement feeding period. Mating was required for both vitellogenin production and vitellogenesis to complete the tick's life cycle. Complete immunological identity, as measured by Ouchterlony's double diffusion test, existed between vitellogenin from the fat body, midgut and hemolymph, and vitellin from the ovaries and eggs. Antivitellin antibody did not react with host hemoglobin nor with fat body, midgut, and ovary extracts from feeding females prior to rapid engorgement, feeding unmated females, or unfed or fed males. Some unmated females fed for 13 days and then hand-detached from the host eventually began oviposition after going through a preoviposition period. In these ticks, organ extracts from the midgut, fat body and ovary reacted with antivitellin antibody. The presence or absence of presumed vitellogenic cells in the midgut and yolk bodies in oocytes corresponded with the presence or absence of vitellogenin and vitellogenesis as measured by Ouchterlony's test. Presumed vitellogenic cells increased in size during the preoviposition period. These cells reached their greatest size during the time when the most eggs were being produced, and then declined in size toward the end of oviposition. Vitellogenin was deposited directly into developing yolk bodies in oocytes and was not processed through lysosomes. Feeding was the process that initiated the formation of eggshell cuticle. Detachment from the host was required for the initiation of oviposition.     

Hemolymph juvenile hormone titers in pupal and adult stages of southwestern corn borer [Diatraea grandiosella (pyralidae)] and relationship with egg development

Juvenile hormones I, II and III were monitored in hemolymph of pupal and adult stages of various ages of Diatraea grandiosella females. JH III was the predominant homologue followed by JH II, and JH I was rarely detectable. At day 5 after pupation, no JH was detectable. JH titers increased from 7.5days after pupation to a peak of 24.8ngml(-1) JH II and 26ngml(-1) JH III at adult emergence and then declined to low levels by 24h after emergence. Ovarian development in D. grandiosella parallels changes in hemolymph JH titers, but the role of JH in vitellogenesis is unclear since the time of vitellogenesis initiation has yet to be determined. No apparent vitellogenin deposition was observed in eggs 5days after pupation. Some oocytes were partially vitellogenic by 7.5days after pupation and oocytes continued to grow afterwards, but no oocytes were chorionated during the pupal stage. Chorionated oocytes were observed in 24-h-old female moths. Juvenile hormone is essential for chorion formation in this species, because decapitated pupae treated with 10&mgr;g JH III in corn oil developed chorionated oocytes while decapitated pupae treated with corn oil did not.     

Effect of age,diet, diapause and juvenile hormone on oogenesis and the amount of vitellogenin and vitellin in the twospotted stink bug,Perillus bioculatus (Heteroptera: pentatomidae)

Vitellogenic oocytes from Perillus bioculatus have two native vitellins, Vt1 and Vt2, with molecular masses of 553 and 228 kDa, respectively. The hemolymph contains a major vitellogenin, Vg, with a molecular mass of 528 kDa that consists of three apoproteins with masses of 177, 84 and 59 kDa, respectively. Antibodies to purified Vt2 reacted with ovary extracts, egg extracts and female hemolymph, but not with male hemolymph in immunodiffusion tests. Western blots showed that anti Vt2 reacted with both Vt1, Vt2 and with Vg. Vitellogenesis starts at an ovarian score of 12 at 2.4 days after emergence. The first cycle of egg development is completed in ovaries with a score of 112 at 7.7 days. During this 5.3 day period, the ovaries of a single female incorporated 1833 &mgr;g of protein to form vitellin. Vitellogenin levels start to increase in females 2.5 days after emergence and reached 17.8 &mgr;g/&mgr;l by 5.5 days. After 5.5 days vitellogenin levels fluctuated between 9.7 and 19.9 &mgr;g/&mgr;l. Most diapausing females contained no ovarian follicles in the vitellarium and their hemolymph contained less than 1 &mgr;g/&mgr;l of vitellogenin. Treating diapausing females with 1 &mgr;g of JH III increased vitellogenin levels over 120-fold. Insects maintained on a liver-based artificial diet had lower vitellogenin levels than the controls at all sample times and did not show an increase in vitellogenin concentration until 11.5 days. Treating insects on the artificial diet with 10 &mgr;g of JH III elevated vitellogenin levels to about a fourth of that found in prey-fed insects of a comparable age. This suggests that females fed the artificial diet have low levels of essential materials needed for vitellogenin production.     

Ovary development and maturation in Nezara viridula (L.) (Hemiptera: Pentatomidae)

Ovary development and maturation of Nezara viridula (L.) were evaluated by examining ovariole morphology and the alterations in the biochemical (protein synthesis related to reproduction) composition of the hemolymph. Quantitative and qualitative protein analyses were performed and ovary structural alterations for the pre-reproductive and reproductive stages were recorded. Total concentration of proteins in female hemolymph gradually increased until the end of the pre-mating stage, remaining unaltered thereafter. Proteins linked to reproduction (vitellogenins) appeared in the hemolymph 10 days after adult emergence and indicated the end of the pre-mating stage. After mating, total protein concentration in the hemolymph was lower compared to virgin females; vitellogenin levels were similar during most of the observation period. Oocyte development and maturation were gradual and age dependent. Ten-day-old females had chorionated oocytes ready for fertilization. Mating did not stimulate oocyte development in N. viridula, but the lack of mating activity appeared to have stimulated oocyte resorption in 17-day-old females.     

Vitellogenesis in Acanthoscelides obtectus (Coleoptera: Bruchidae). I—Oocyte development and vitellogenin in a European strain

Oocyte development was studied in a European strain of the bean weevil, Acanthoscelides obtectus. In the experimental system used, oocyte growth begins after several hours of imaginal life. The lengths of the terminal and penultimate oocytes were measured and correlations between these measurements showed that the latter stopped growing when the former was in the phase of accelerated growth.

Vitellogenin could be immunologically detected in the hemolymph of sexually mature females and was undetectable in males, larvae and in females immediately after imaginal exuviation. The moment of vitellogenin appearance in young females varies from one insect to another.

It appears that no specific external stimulus is required to initiate the synthesis and incorporation of the vitellin protein in this European strain.     

Nonvitellogenic female protein in Musca domestica

During vitellogenesis in Musca domestica, a major hemolymph protein, in addition to vitellogenin, appears preferentially in females. This protein is synthesized by the adult fat bodies, secreted into the hemolymph, and is not taken up by the ovaries during vitellogenesis. We have designated this protein nonvitellogenic female protein (NVFP). It is composed of only one type of polypeptide (M_r=70,000) and occurs in two different forms. Synthesis of NVFP is induced by a protein diet, attaining maximum concentrations in females at the middle of the gonotrophic cycle. In males its maximum concentration never surpasses 10% of the concentration in females. The quantitative variation of the NVFP is cyclic and coincident with the gonotrophic cycles of Musca domestica.     

Regulation of vitellogenesis in the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae)

Studies were undertaken to investigate vitellogenesis and its regulation in female adults of the fall armyworm, Spodoptera frugiperda. A single female-specific protein, likely to be the S. frugiperda vitellogenin (Vg), appeared approximately 5 h after adult eclosion in the hemolymph of virgin females. The concentration of the protein increased with age as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed. A protein with the same relative molecular mass was also present in egg extracts, but absent from hemolymph samples from male moths. The relative molecular mass of the designated S. frugiperda Vg was determined as 164.5+/-2.5 kDa. Vitellogenic oocytes became visible 36-48 h after emergence and egg deposition began on day 3 of adult life. Vg could not be detected in the hemolymph of females decapitated directly after eclosion. When decapitated virgin females were injected with the JH-mimic methoprene (MP), the level of Vg was comparable to that in non-decapitated moths, indicating that vitellogenesis in S. frugiperda depends on juvenile hormone (JH). However, the number of vitellogenic oocytes was somewhat lower than in non-decapitated virgin females. Injection of 20-hydroxyecdysone (20E) promoted Vg production to a similar extent in decapitated female moths, but in contrast to methoprene injection, treatment with 20E never resulted in the production of vitellogenic oocytes. In vitro cultivated ovaries of adult females dissected directly after eclosion produced lower amounts of ecdysteroids than those isolated on day 1 after emergence. Our results suggest a crucial role for 20E in the induction of vitellogenesis in the noctuid S. frugiperda, while JH seems to be essential for the continued uptake of Vg by developing oocytes and may trigger 20E biosynthesis in the ovary.     

Sexual phenotype and vitellogenin synthesis in Drosophila melanogaster

An ovary transplanted from a Drosophila melanogaster female into a male will mature and form morphologically normal yolk-filled oocytes. Since it has been supposed that the yolk polypeptides come only from the female fat body, it was hypothesized that the implanted ovary induces the fat body of the male host to synthesize and secrete yolk polypeptides (YPs). To test this hypothesis, fat body preparations from females, untreated males, and males containing transplanted ovaries were cultured in vitro with ³⁵S-methionine and the medium was examined for the presence of newly labeled YPs. Female fat body secreted newly labeled YPs, but no freshly synthesized YPs were secreted by fat bodies from untreated males or from males containing transplanted ovaries. In vitro cultured ovaries, however, both from females and from male hosts did secrete newly synthesized YPs. Therefore, the YPs in an ovary that matured in a male come mainly from endogenous synthesis by the implanted ovary. To find whether males were responsive to the hormones that stimulate YP production in isolated female abdomens, we treated males with the juvenile hormone analogue ZR-515 and with 20-hydroxyecdysone. The latter, but not the former, was able to cause synthesis and secretion of three bands migrating precisely as YPs in SDS gels. Partial peptide digests of the 20-hydroxyecdysone-stimulated polypeptides in males showed them to be identical with those stimulated by 20-hydroxyecdysone or ZR-515 in isolated female abdomens and with the three YPs found in normal female hemolymph. Finally, YP synthesis was assayed in mutants that affect the phenotypic sex of a fly. It was found that flies bearing two X chromosomes and the mutations dsx, dsx^D, ix or three sets of autosomes continued to make YPs, but tra-3-pseudomales did not. These results suggest that the process of sex determination involves steps leading to synthesis of an ecdysteroid in females, which then activates synthesis of the YPs by the fat body. A hypothesis is suggested to explain the fact that two different hormones can stimulate YP synthesis and two different organs can synthesize YPs.     

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.     

Rhipicephalus sanguinius: localization of vitellogenin synthesis by immunological methods and electron microscopy

In ovipositing Rhipicephalus sanguinius (Latrelle), complete immunological identity existed between vitellogenin from the midgut, fat body, and hemolymph and vitellin from eggs. This supported the hypothesis that the same vitellogenin was synthesized by both the midgut and fat body, then released into the hemolymph and transported to the ovary. Vitellogenin was taken up unaltered by the oocytes during vitellogenesis to become vitellin. Antivitellogenin did not react with host (dog) hemoglobin. Transmission electron microscopy showed specialized cells with large amounts of rough endoplasmic reticulum, Golgi complexes, and secretory granules in the midgut and fat body of ovipositing females that were absent in the midgut and fat body of fed males. It is suggested that these cells synthesize vitellogenin.     

Immunochemical identification and partial characterization of female-specific serum proteins in white-edged rockfish,Sebastes taczanowskii

Synopsis Female-specific serum proteins (FSSPs) in white-edged rockfish,Sebastes taczanowskii, were identified and partially characterized by immunochemical procedures. Two FSSPs, which clearly reacted with antiserum against egg proteins, were confirmed in the serum of mature females, and estrogen treatment induced similar FSSPs in the serum of mature males. Hence, the FSSPs were considered to be vitellogenin. The vitellogenin concentration in female fish was high during the vitellogenic period and low during gestation, parturition and the recovery period, indicating that vitellogenin is used only for yolk formation in the oocytes and not as a direct nutritional source for developing embryos during gestation. On the other hand, an FSSP (FS3), which was considered not to be vitellogenin, was also identified in the sera of mature females and males after estradiol-17β administration by using an antiserum (a-FS3) that removed the components of the male serum and egg extracts from the anti-mature female serum antiserum. Moreover, immunohistochemical observation with a-FS3 illustrated that FS3 was a major constituent of the ovarian fluid but not of vitellogenic oocytes. The cross-reactivities of these FSSPs among seven viviparous rockfishes demonstrated that vitellogenin existed in the sera of all rockfishes studied belonging to the generaSebastes andSebastiscus, whereas FS3 was not present in several species ofSebastes.     

Relationship between feeding,mating, vitellogenin production and vitellogenesis in the tickDermacentor variabilis

Anti-vitellin IgG directed againstDermacentor variabilis egg vitellin was used in sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gradient gel immunoblots to detect the presence of vitellin and its precursor, vitellogenin, in the organs of feeding adults and in the immature stages of this tick. Vitellin polypeptides were found in the egg, larvae, nymph, and in the unfed adult stages of both sexes. Vitellin polypeptides were first detected in the ovary of mated females during the rapid-engorgement feeding, period. These polypeptides were also present in the ovaries of ovipositing females, unmated females fed for extended periods, and fed unmated females that were detached from the host and held for 12 h before dissection. The same anti-vitellin antibody was used in immunoblots to monitor the appearance of vitellogenin in the organs and hemolymph of female ticks. Immunoreactive peptides of vitellogenin were found in the fat body, midgut, and hemolymph of pre-rapid-engorging mated and unmated females. These polypeptides were not found in fed males nor in Malpighian tubes of feeding or ovipositing females Our data supported the following conclusions: 1) presence of immunoreactive vitellogenin in the adult female fat body, hemolymph, and midgut was, dependent upon feeding; 2) in mated feeding females, we could not detect the uptake of vitellogenin by the ovary until rapid engorgement; 3) in unmated females, vitellogenesis did not, begin unless prolonged feeding occurred; and 4) during the early developmental stages of this tick, vitellin served as an embryonic nutrient reserve and as a reserve against starvation between feedings.     

Changes in biosynthesis and degradation of juvenile hormone during breeding by burying beetles: a reproductive or social role?

Burying beetles, Nicrophorus orbicollis, depend on the location of an unpredictable resource, a small vertebrate carcass, for reproduction. When they discover a carcass, they undergo a correlated rapid rise in titers of juvenile hormone (JH) in the hemolymph and ovarian development. This study investigates the regulation of the changes in JH during breeding in both male and female burying beetles and the role of JH in ovarian development. JH biosynthesis by the corpora allata (CA), measured in vitro, increased in females within an hour of their discovery of a carcass and increased later in males. After returning to low rates as oviposition began, JH biosynthesis rose again 3 days later in females but not in males. Neither the ovaries nor testes synthesized JH. There was a concomitant fall in JH esterase activity within 12 h of discovery of the carcass in both males and females. Although the rise in JH titers and biosynthesis and the fall in JH esterase is correlated with ovarian development, application of methoprene or JH III in the absence of a carcass did not result in vitellogenin uptake by the oocytes. Therefore, we conclude that, in spite of the rapid rise in JH before oviposition, it is not sufficient to regulate vitellogenin synthesis and/or its uptake by the ovaries. We suggest that its role has been preempted to organize social behavior and coordinate parental behavior between mates.     

Autonomous yolk protein synthesis in ovaries ofDrosophila cultured in vivo

Summary Immature ovaries ofDrosophila mercatorum were injected into young larvae and into adult males ofD. mercatorum, D. melanogaster, D. hydei, D. virilis, andZaprionius vittiger. These homo- and heteroplastic transplantations allow normal vitellogenesis to occur in the donor ovary. By SDS gel electrophoresis, we identified the major species-specific yolk proteins of mature eggs (stage 14) which were exclusively of donor-specific origin. Other experiments withD. hydei andZ. vittiger showed that, when females were used as hosts, the host-specific yolk proteins became incorporated into the donor eggs. When two immature ovaries, one ofD. mercatorum and one ofD. hydei, were co-cultured in males, again only the donor-specific yolk proteins were found in the mature eggs implying that these yolk proteins were not released into the host hemolymph.A parthenogenetic strain ofD. mercatorum was used to demonstrate the ability of transplanted immature ovaries to produce viable eggs which can give rise to fertile adults.The role of the species-specific yolk proteins is discussed with respect to the dual origin of these proteins during normal vitellogenesis, i.e., an autonomous synthesis within the ovary itself in addition to the well-known production by the fat body. Further experiments with pupae as hosts indicate that even in the absence of juvenile hormone and in the presence of high doses of ecdysone, vitellogenesis can proceed within the donor ovary.Based on these experiments, a new hyopthesis on the hormonal control of vitellogenesis inDrosophila is presented. We propose that yolk proteins derived from the fat body are controlled by juvenile hormone, whereas the independent and autonomous vitellogenesis within the ovary itself is controlled by endogenously synthesized ecdysone.