Vitellogenin fluctuations in haemolymph and fat body and dynamics of uptake into oöcytes during the reproductive cycle of Diploptera punctata

Haemolymph and fat body soluble protein titres have been examined during the reproductive cycle of Diploptera punctata, with particular emphasis on the occurrence of vitellogenin and its uptake into the developing oöcytes. Vitellogenin was first detected in the haemolymph of mated females 2 days after adult eclosion at about the same time that vitellin deposition in basal oöcytes began. Peak haemolymph titres of vitellogenin occurred on day 6, correlated with the completion of yolk uptake. Thereafter vitellogenin levels declined and were generally undetectable throughout most of gestation, rising again shortly before parturition in association with the second gonotrophic cycle. Total haemolymph protein levels were not correlated with vitellogenesis.Soluble fat body vitellogenin titres of mated females remained low during the first oöcyte growth period but then rose several-fold at its completion and remained high throughout pregnancy and the second gonotrophic cycle. Total fat body soluble proteins decline after adult eclosion in association with oöcyte growth.Vitellin accumulation in basal oöcytes was related linearly to increase in volume until the onset of chorion formation. Thus no post-vitellogenic growth period was detected.     

Juvenile hormone regulation of structural changes and DNA synthesis in the follicular epithelium of Leucophaea maderae

Juvenile hormone (JH I) stimulates specific morphological and biochemical changes in the follicular epithelium surrounding the terminal oöcytes in Leucophaea maderae. These include extracellular and intracellular structural changes, increased rates of follicle cell DNA synthesis, and elevated follicle cell DNA concentrations.Using females decapitated 24 hr after ecdysis, we have shown that JH I injections stimulate the following structural changes in the follicular epithelium: the appearance of channels between adjacent follicle cells and of spaces between the follicular epithelium and the maturing oöcyte; an increase in follicle cell size; the development of an extensive rough endoplasmic reticulum system; and an enlarged nucleus within each follicle cell. No increase in the number of follicle cells surrounding the developing terminal follicles is found in 7-day JH I-treated females, although the terminal follicles are almost twice as long as those in untreated females.In addition, we have demonstrated that JH stimulates the following biochemical events in the ovary: a 3.5 fold increase in thymidine incorporation into follicle cell DNA, with no subsequent transfer of such DNA to the developing oöcyte, and a 1.4 fold increase in ovarian DNA in 7-day JH-treated females. These data indicated that JH stimulates follicle cell DNA synthesis. The absence of any corresponding division of follicle cells suggests that JH I may induce polyploidy in follicle cells.Extended exposure of decapitated females to JH I does not result in complete ovarian maturation. Although fat bodies in the treated insects continue to display an increasing rate of vitellogenin synthesis, DNA synthesis in the terminal follicles declines rapidly after day 9, and the terminal follicles ultimately degenerate.     

Vitellogenins in the firebrat,Thermobia domestica (Packard): Immunological quantification during reproductive cycles and in relation to insemination (Thysanura: Lepismatidae)

An antiserum raised against the major fractions (240 + 300 Kd) of the vitellins extracted from maturing oöcytes of Thermobia domestica, allowed the immunological relationship between these proteins and their haemolymph precursors (vitellogenins) to be established, as well as the female specificity of these protein fractions. Using rocket immunoelectrophoresis, the vitellogenin concentration in the female haemolymph was measured on each day of a reproductive cycle. The changes in vitellogenin titre appear to be correlated with ovarian maturation: very low levels during the previtellogenic phase and increasing levels during the vitellogenic growth phase of the basal oöcytes. Also, these changes are synchronized with the moulting cycles which persist in the adults of these primitive insects. An insemination at the beginning of the interecdysial period causes a large increase in the vitellogenin titre synchronized with accelerated oöcyte growth.     

Uptake of vitellogenin into developing oöcytes of Locusta migratoria

The selective incorporation of vitellogenin into developing locust oöcytes was studied using ¹²⁵I-vitellin. Vitellogenin incorporation does not start before the oöcytes are 1.5 mm in length. It increases rapidly up to a maximum at 4.7 mm oöcyte length and decreases steadily until the eggs are fully developed (6.5 mm). Concentrations of serum proteins and vitellogenin in the haemolymph show parallel changes, vitellogenin titre reaching a maximum of 7.5 mg/ml. Incorporation rates for vitellogenin increase from 1.5 μg/hr/oöcyte (2.2 mm) up to 13.8 μg/hr/oöcyte (4.7 mm). In this range incorporation per unit surface area increases 4-fold. While the vitelline and chorionic membranes are being formed, the incorporation rates as well as the protein concentrations in the haemolymph decrease steadily until the second gonotrophic cycle starts. The hormonal basis for oögenesis and the mechanism for selective uptake of locust vitellogenin are discussed.     

On the relative importance of juvenile hormone and vitellogenin for oöcyte growth in the cockroach Nauphoeta cinerea

The corpora allata of castrated females of Nauphoeta grow only very slightly and do not reach a volume greater than that of the glands of normal females during gestation. These small corpora allata are, however, active and are responsible for the synthesis of vitellogenin (female specific protein) in large amounts. Besides vitellogenin the other haemolymph proteins are also synthesized and accumulated in the haemolymph in much higher concentrations than in normal females. Implanted oöcytes grow in castrated as well as in normal females at about the same rate until the tenth day of the oöcyte maturation period. Thereafter they only grow in castrated females. If castrated and normal females are decapitated, their protein content decreases. At the same time the growth stimulating capacity of their haemolymph decreases at a much faster rate. If oöcytes are implanted in castrated and decapitated females after 4 days they cannot grow any more although the vitellogenin titre of the haemolymph is still much higher than it is at any time in normal females. It can be concluded that vitellogenin alone cannot induce oöcyte growth and that juvenile hormone is necessary as well for vitellogenin synthesis as for its incorporation into the oöcytes. However, in insects rich in vitellogenin juvenile hormone leads to a more rapid oöcyte growth than in insects containing only small amounts of this protein.     

Histological and cytological studies on the fat body of the cockroach Nauphoeta cinerea during the first reproductive cycle

Summary The central fat body of the ovoviviparous cockroach Nauphoeta cinerea was studied during the first reproductive cycle of the female by means of light microscopy, autoradiography and electron microscopy. Comparative studies in larval stages were also undertaken. The fat body of Nauphoeta contains a large amount of lipid droplets and the remaining cytoplasm is very scarce. The cytological cyclicity of the fat body is consistent with the known biochemical rhythms of vitellogenin production. The proteosynthetic apparatus appears about 3 days after imaginal ecdysis, along with vitellogenin. The ribosomal endoplasmic reticulum (RER) shows a tremendous increase by the 7th day of the first cycle. The most active period of vitellogenin production lasts from day 7 to day 12. The proteosynthetic apparatus then returns to an inactive stage and disappears. This inactive condition lasts to the end of the gestation period. The autoradiographic results are consistent with the cytological features.Work carried out under the scientific direction of Prof. M. Lüscher of the University of Bern and supported in part by a grant from the Swiss National Science Foundation (No 3. 633.71).     

Factors responsible for the initiation of a second oöcyte maturation cycle in the ovoviviparous cockroach Nauphoeta cinerea

The factors responsible for the initiation of a second oöcyte maturation cycle were investigated by measuring oöcyte growth, vitellogenin titre, and corpus allatum activity after injection of juvenile hormone and/or removal of the egg-case from pregnant females and by performing ovary and corpus allatum transplant experiments.Egg-case removal in late pregnancy results in immediate oöcyte growth, whereas in early pregnancy oöcyte growth is resumed only after a lapse of time, even after injection of juvenile hormone. This, however, induces an immediate increase in the haemolymph vitellogenin titre. A single injection of 2 or 10 μg of juvenile hormone II first stimulates some oöcyte growth after this lapse of time and later activates the corpora allata, which in turn leads to completion of oöcyte maturation. A repeat injection of 10 μg stimulates continuous oöcyte growth without activating the corpora allata. In the presence of an egg case, activation of the corpora allata is suppressed, even after injection of 2 μg of juvenile hormone III, and the oöcytes do not grow. Injection of higher doses stimulates oöcyte growth and leads to expulsion of the egg case in up to 95% of the females. This, however, is not a direct consequence of the increase in size of the ovaries. Ovary transplant experiment show that in young pregnant females the second generation of oöcyte is not yet competent for growth and that ovaries which are competent can mature in young pregnant females, treated with juvenile hormone, whose egg case has been removed.The results are summarized in a model demonstrating the various factors involved in regulating corpus allatum activity in oöcyte maturation and pregnancy and after application of juvenile hormone. We prepose that the corpus allatum activating effect of exogenous juvenile hormone is mediated by the growing oöcyte and that this activation can be suppressed by the continuous presence of exogenous juvenile hormone.     

Dynamics of vitellogenin uptake in Aedes aegypti as demonstrated by trypan blue

Trypan blue has been shown to be a reliable indicator of the micropinocytotic uptake of vitellogenin by developing oöcytes. Trypan blue was injected into the mosquito Aedes aegypti to determine at what times after the blood meal vitellogenin was taken up. Histological sections examined by light microscopy showed that trypan blue began to be sequestered from 2 to 5 hr after the blood meal. Any association between dye and ovariole ended from 39 to 42 hr after the blood meal, in which period no dye was incorporated into spheres of yolk protein. Of the times investigated in this experiment, the greatest amount of dye was seen in the oöcyte at 24 hr after the blood meal. The onset and conclusion of trypan blue uptake correspond with the related events in the synthesis of vitellogenin by the fat body. The presence of trypan blue in occasional interfollicular spaces suggests that the route of entry of vitellogenin in Aedes aegypti is indeed an interfollicular one.     

Phase polymorphism in Schistocerca gregaria: Assessment of juvenile hormone synthesis in relation to vitellogenesis

Juvenile hormone (JH) synthesis by the corpora allata of gregarious and solitarious phase females of Schistocerca gregaria was determined in vitro during the penultimate and last stadia as well as during the first gonotrophic period of adults. Generally, the corpora allata of solitarious females showed higher rates of JH synthetic activity. In addition, in adult females there was a temporal difference between the corpora allata activities of gregarious and solitarious locusts, the latter exhibiting relatively higher rates of JH synthesis early in the first gonotrophic period. The corpus allatum volumes of solitarious females were also generally larger than those of their gregarious counterparts; there was no synchrony between fluctuations in JH synthetic activity and changes in corpus allatum volume in either phase.The early onset of relatively high JH synthetic rates in solitarious females was correlated with the early detection, by rocket immunoelectrophoresis, of vitellogenin in the haemolymph and vitellin in the oöcytes. Vitellogenin appeared in the haemolymph on day 4 in solitarious females and on day 6 in gregarious females and vitellin appeared in the oöcytes on days 6 and 8 respectively. Oöcyte length at which vitellogenesis was first detected was 1.8 mm for gregarious and 1.3 mm for solitarious females. However, despite the accelerated onset of both vitellogenin synthesis and uptake, oöcyte maturation time of solitarious females was longer. In both gregarious and solitarious females, vitellogenin titres increased until oöcytes reached a length of about 4 mm and declined thereafter. Vitellin content of ovaries increased proportionately to oöcyte growth until they attained a length of 5.0 mm. The subsequent increase in length of oöcytes to maturity is attributed to postvitellogenic growth, possibly by hydration.     

Vitellogenin accumulation in the fat body and haemolymph of Locusta migratoria in relation to egg maturation

Vitellogenins first appear in the fat body of Locusta migratoria during subphase I of vitellogenesis and increase to a constant level during subphase II. A second increase occurs shortly before the oöcyte attains maximal size. Vitellogenin content of fat body subsequently returns to that of subphase I, appropriate to the size of the subterminal oöcyte. The absolute amount of vitellogenin in the fat body is low compared to that found in the haemolymph. Fat body and haemolymph vitellogenins have immunological properties similar to oöcyte yolk proteins—when challenged with oöcyte protein antiserum. They exhibit similar electrophoretic mobility in polyacrylamide gel electrophoresis and are complex glyco-lipoproteins.     

Ovarian maturation in Leucophaea maderae: Juvenile hormone regulation of thymidine uptake into follicle cell DNA

Our research demonstrates that juvenile hormone (JH I) stimulates thymidine incorporation into ovarian follicle cell DNA in the ovoviviparous cockroach, Leucophaea maderae.A rapid, quantitative method for monitoring ³H-thymidine incorporation into ovarian DNA, in vitro, is described. Cultured ovarian tissue from L. maderae incorporates ³H-thymidine into DNA at a linear rate between 16 and 120 min; analysis of the incorporated label revealed at least 98% of it to be in DNA.Using L. maderae females that had been mated 7 days after adult emergence, we monitored the following biochemical phenomena during the 18–22 day period of terminal oöcyte growth: (1) ³H-thymidine incorporation into ovarian DNA: (2) general protein synthesis in fat body; and (3) specific fat body vitellogenin synthesis.Decapitation of mated females with maturing oöcytes arrested both ovarian DNA synthesis and fat body vitellogenin synthesis. Substantial restoration of both types of synthesis was induced by injection of JH I. The resumption of thymidine incorporation into DNA was localized in the follicular epithelium of the terminal oöcyte.In decapitated virgin females, injection of JH I stimulated oöcyte growth and ³H-thymidine incorporation into ovarian DNA. Dose and time response curves indicate that peak stimulation of ovarian DNA synthesis occurred between 72 and 96 hr after administration of a single optimal dose of 25 μg JH I. The concurrent manifestation of ³H-thymidine uptake into ovarian DNA and activity within the fat body indicates that a similar hormonal mode of action may be operative with respect to both tissue types in virgin females.     

Rate of follicle growth,change in follicle volume and stages of macromolecular synthesis during ovarian development in Musca domestica

At 21°C the first egg generation takes 6 days to develop. During this period the oöcyte volume increases by a factor of ×5000, and compared with an oögonium the growth factor amounts to ×100,000. The growth rate of the oöcyte increases with each stage of oögenesis, until at stage 5 it reaches 2.5 × 10⁶ μm³/hr. About 35% of the substance stored in the oöcyte originates from the nurse chamber. 30% of the volume is formed in the stage where the oöcyte synthesizes almost exclusively glycogen. Accordingly, about 30% of the volume is provided by the euplasmatic protein synthesis and by the yolk uptake.     

Haemolymph vitellogenin,juvenile hormone,and oöcyte growth in the adult cockroach Nauphoeta cinerea during first pre-oviposition period

In the cockroach Nauphoeta cinerea the incorporation of a protein of low solubility into the oöcytes begins at day 5 of its adult life. An immunologically identical protein appears in the haemolymph two days earlier. The concentration of this protein, i.e. ‘vitellogenin’ in the haemolymph increases up to the onset of yolk incorporation into the oöcytes. During ovarian development no correlation could be detected between vitellogenin titre and several other parameters (ovary dry weight, length of the basal oöcytes, haemolymph protein concentration, body weight and age when ovulation occurred). In young females vitellogenin titre depends on the age, i.e. the volume of the corpora allata and hence on the presence and the titre of JH. During the period of egg maturation the total haemolymph protein concentration generally tends to drop while materials not precipitable by trichloracetic acid circulate at higher concentration after ecdysis and before ovulation.Early decapitation prevents vitellogenin synthesis and oöcyte growth, but when JH is applied to decapitated females, the normal vitellogenin titre is re-established, ovarian development, however, cannot be fully resumed. A dose-response curve shows that serial application of the hormone is much more effective than single large doses. Farnesylmethylester, a JH mimic, is about a hundred times less active, but more persistent than JH. Copulation seems to enhance the synthesis and release of endogenous JH, while food and water uptake are necessary to guarantee and optimal ovarian development. JH and high vitellogenin titre never restore ovarian development in females deprived of food and/or water or in those decapitated shortly after ecdysis.     

Changes in follicle cell morphology,ovarian protein synthesis and ovarian DNA synthesis during oöcyte maturation in Leucophaea maderae: Role of juvenile hormone

Changes in follicle cell morphology were correlated with changes in rates of protein synthesis and DNA synthesis by the ovary during ovarian maturation in Leucophaea maderae. During the vitellogenic period of oöcyte development, which lasts approx, 15 days, morphological changes in the follicle cells are accompanied by moderate rates of ovarian protein synthesis and rapid rates of ovarian DNA synthesis. At approx. 15 days after mating, the shape of the follicle cells changes from cuboidal to squamous, ovarian DNA synthesis is arrested, and ovarian protein synthesis increases slightly. During the final period of oöcyte development, which lasts approx, two days, the interfollicular channels between the follicle cells have disappeared and the squamous follicle cells, which contain an extensive rough endoplasmic reticulum, deposit a chorion around the mature oöcyte. These morphological changes are accompanied by a radical increase in ovarian protein synthesis, while ovarian DNA synthesis remains arrested. Immediately before ovulation, ovarian protein synthesis starts to decline, reaching a minimal level 24 hr post-ovulation.Ovarian maturation is dependent on the presence of juvenile hormone (JH) only during the vitellogenic stage of oöcyte development. Decapitation of insects at any point during the first 10 days after mating arrests the synthesis of DNA and retards the synthesis of protein by the ovary, resulting in degeneration of the oöcyte. Subsequent injection of JH restores both events to normal levels within 72 hr. Decapitation on or after the tenth day following mating does not alter normal oöcyte development, chorion deposition, ovulation or egg case formation.Primary induction of protein synthesis in ovaries from virgin females can be achieved by either an in vivo or in vitro exposure of the tissue to JH, thus confirming a site of action for JH to be ovarian tissue. Electrophoretic analysis of the soluble proteins from JH-exposed ovaries in vivo reveals that JH stimulates general protein synthesis, rather than the synthesis of a specific major protein such as vitellogenin.     

Protein synthesis in the fat body of Leucophaea maderae during vitellogenesis

During the early stages of vitellogenesis in Leucophaea, vitellogenin accounted for most if not all of the secreted protein synthesized by the fat body. Synthesis began about 5 days after mating and continued until 24 hr or so before the formation of the oötheca. Ligation resulted in the degeneration of the oöcytes, the first evidence of which was seen within 24 hr. Ligation also curtailed the synthesis of vitellogenin at about the same time. Isolated abdomens treated with an analog of juvenile hormone commenced vitellogenesis within 12 to 24 hr and measureable oöcyte growth occurred after 5 days. Despite continued synthesis of vitellogenin, the oöcytes in isolated abdomens always degenerated.     

Evidence for the involvement of transglutaminase in the uptake of vitellogenin by Xenopuslaevis oöcytes

The yolk protein, vitellogenin, is sequestered by the developing oöcyte by receptor-mediated endocytosis, the process by which cells bind and internalize extracellular proteins. Endocytosis of a variety of proteins follows a similar pathway, whereby internalization of receptor-bound ligand takes place over clathrin-coated regions of the cell membrane. The protein crosslinking enzyme, transglutaminase, has been reported to be essential for the receptor-mediated endocytosis of insulin and α₂-macroglobulin. In this study, the presence of transglutaminase activity was demonstrated in the $\text{Xenopus}\text{laevis}$ ovary and was effectively inhibited by poly L-lysine, an inhibitor of vitellogenin uptake, and dansylcadaverine, a known inhibitor of transglutaminase activity. Two other less poteint inhibitors of transglutaminase, methylamine and bacitracin produced partial inhibition of the ovarian enzyme. Furthermore, dansylcadaverine and methylamine were found to inhibit the appearance of vitellogenin in the yolk platelets of the oöcyte.     

Vitellogenin synthesis in blood-fed Aedes aegypti in the absence of the head,thorax and ovaries

A blood meal initiates oöcyte maturation in Aedes aegypti, and we have used rocket immunoelectrophoresis to investigate the function of midgut, ovaries, and head in the onset of vitellogenin synthesis. Non-blood-fed females and those fed blood (by enema) containing soybean trypsin inhibitor never contained vitellogenin. This demonstrates that the pressure of an undigested blood meal on stretch receptors of the midgut plays no role in the induction of vitellogenin synthesis, rather the stimulus is a digestion product of blood.When females were ovariectomized or decapitated and then fed blood, the haemolymph contained newly synthesized vitellogenin 24 h later. This was also demonstrated in isolated ovariectomized abdomens. Apparently, induction of vitellogenin synthesis does not require factors from either the head, thorax, or ovaries. When ovariectomy or decapitation was postponed after a blood meal, the level of vitellogenin in the haemolymph rose. Therefore, interaction of factors from the head and ovaries maintain the synthesis needed for oöcyte maturation.     

Vitellogenic and embryogenic activity of the microtubule disruptor vinblastine following ingestion by the wasp Bracon hebetor

The reproductive performance of Bracon hebetor females was adversely affected by the consumption of sub-lethal doses of vinblastine. While all oögenic cell types present in the gonads at the time of treatment displayed various degrees of fecundity and/or fertility depression, the transitional cells proved to be the least susceptible to vinblastine damage. Vitellogenically active oöcytes were most sensitive to vinblastine. In these oöcytes development was blocked at or prior to the terminal growth phase. Oviposited eggs which did arise from the exposed vitellogenic oöcytes were few in number and characterized by aberrant morphology. Embryogenic effects were predominantlydue to pre-blastoderm formation damage and most pronounced in oöcytes exposed during the most advanced stages of gonadal development, late vitellogenesis through meiotic metaphase I. Reduced egg hatchability also occurred in exposed undifferentiated oögonial cells but the effect was less severe than that seen in the more mature oögenic cells. All observed effects could be accounted for by vinblastine's selective interference with microtubule-dependent processes. Fecundity effects were most closely associated with oöcyte cortex and possibly follicular cell damage which prevented vitellogenic growth beyond the terminal growth phase. Fertility effects were caused by the inhibition of early embryonic karyokinesis with the most plausible target being mitotic spindle formation.     

The ultrastructure and functions of the ovariole sheath and tunica propria in the flour moth

The ultrastructure of the ovariole sheath, tunica propria, and associated cells is described. The sheath contains circular and longitudinal muscles and completely covers each ovariole except at the clefts between the oöcyte chambers where pores occur. Muscular movements cause haemolymph components to be pumped through these pores to the oöcyte at a rate faster than can be explained by diffusion. The tunica propria consists of two intercellular layers of fine granules and is not considered to have elastic properties. It may act simply as a valve for incoming haemolymph proteins. Directly beneath the sheath pores are found macrophages containing bacteria in various states of digestion. These may protect the oöcytes from bacterial infections. The interfollicular cells found between the chambers also contain bacteria and are considered to be mycetocytes.     

The effects of starvation and subsequent feeding on juvenile hormone synthesis and oöcyte growth in Schistocerca americana gregaria

The effect of starvation on the synthesis of C₁₆ juvenile hormone (JH) and the growth of terminal oöcytes was assessed in Schistocerca americana gregaria at two times during adult life: before activation of the corpora allata and during the first gonotrophic cycle. In both groups, starvation resulted in a decline in JH synthesis within 2–3 days and rates of synthesis remained low throughout the experimental period. The growth rate of oöcytes which were not vitellogenic at the time of starvation was depressed whereas the percentage of resorption of vitellogenic oöcytes increased dramatically with starvation. Although the percentage of resorption increased in animals with vitellogenic oöcytes, some mature oöcytes were produced, particularly in animals in which the oöcytes were greater than 5 mm in length at the time of starvation. This suggests that oöcyte maturation can be divided into two distinct phases—an early phase of vitellogenesis associated with high rates of JH synthesis and a late phase, in oöcytes greater than 5 mm, associated with much lower rates of JH synthesis.Stimulation of JH synthesis by farnesenic acid in 5-day starved animals resulted in high rates of JH synthesis, indicating that starvation did not appreciably alter the enzymic activities of the final two stages in JH synthesis. Thus rate limitation did not occur at these stages.Feeding of 5-day starved animals resulted in a transient increase in the rate of JH synthesis. However, rates of JH synthesis and oöcyte growth remained subnormal throughout the observation period, suggesting that the effects of starvation cannot be entirely reversed by feeding. Thus starvation may decrease the reproductive potential of the females.