Ecdysone during ovarian development in Locusta migratoria

Considerable amounts of ecdysteroids are produced during each ovarian cycle in adult females of Locusta when vitellogenesis is almost completed. The hormonal molecules are synthesized at the end of the maturation of the terminal oöcytes during each cycle, at the time when vitellogenesis is almost completed. No synthesis takes place in the absence of ovarian development (allatectomy, ovariectomy), whereas extirpation of the prothoracic glands at the beginning of adult life does not affect ecdysteroid production. More than 95% of the total ecdysteroid content of female adults can be recovered from the ovaries. In vitro studies show that the ovaries produce ecdysteroids and convert labelled cholesterol into ecdysone. Microsurgical experiments indicate that this synthesis takes place in the follicle cells surrounding the oöcyte. The newly synthesized ecdysteroids do not enter massively into the blood, but pass into the oöplasm where they are progressively converted to polar compounds; as a result, at the end of each ovarian cycle, egg-laying corresponds to the disappearance of ecdysteroids from the female insects, the hormonal molecules can easily be recovered from the eggs. A gas chromatographic analysis coupled to mass spectrometry shows that the principal ecdysteroid synthesized by the adult females of Locusta is by far ecdysone. Ecdysterone, the paramount ecdysteroid of the larvae of Locusta, is not present in noticeable amounts in the female adult of this species.     

Experimental evidence for a neuroendocrine control of ecdysone biosynthesis in adult females of Locusta migratoria

The considerable increase in ecdysteroid concentration which occurs in normal Locusta ovaries at the end of each cycle of oöcyte maturation is prevented if the median neurosecretory cells of the pars intercerebralis are cauterized, or if the corpora cardiaca are excised 24 hr before the onset of ecdysone synthesis in normal females. Implantation of additional brain-corpora cardiaca complexes into young vitellogenic females advances the time of ecdysone synthesis by some 12 hr. Oöcyte growth itself is not affected in these different types of experiments.It is inferred from the data of the present study that ecdysone synthesis in the follicle cell epithelium of maturing Locusta ovaries is stimulated by a neurohormone produced in the median neurosecretory cells of the pars intercerebralis and secreted into the blood via the corpora cardiaca.     

Oöcyte resorption during ovarian development in the blowfly Lucilia cuprina

Nulliparous females of a normal anautogenous strain of Lucilia cuprina mature all of their primary oöcytes after feeding ad lib on sheep's liver. Females fed measured inadequate amounts of protein-rich materia either fail to mature any oöcytes or mature less than their full complement. These mature oöcytes are smaller than in ad lib fed females. In females maturing no oöcytes, ovarian development ceases with all oöcytes in a pre-vitellogenic or early vitellogenic stage. When females mature only some of their oöcytes, the remainder are resorbed in early vitellogenesis. Few females mature less than 100 oöcytes, if they mature any at all.     

Ecdysteroid levels during adult reproductive and embryonic developmental stages of Sarcophaga bullata (Sarcophagidae: Diptera)

Ecdysteroid levels were determined during the period of the adult reproductive cycle in the ovovivparous fleshfly Sarcophaga bullata. Low levels were found in males during the 10 days following eclosion while the entire adult female showed a significant peak of ecdysteroid activity at 190 h post eclosion (i.e. during embryogenesis). When the female reproductive tract was analyzed it was observed that the ovaries became vitellogenic a short time after a protein meal was offered at 96 h post eclosion: at 140 h, ecdysteroid activity was recorded in the developing oöcytes. The major peak of ecdysteroids during the reproductive cycle was found in developing embryos at 190 h. The significance of these releases of ecdysteroids is discussed in relation to major embryonic developmental events.     

Precocene I and II inhibition of vitellogenic oöcyte development in Drosophila melanogaster

Adult female Drosophila melanogaster were exposed to precocene I and II, antiallatropin compounds which result in juvenile hormone deficiency in many insects. The presence of juvenile hormone in Drosophila adults was evaluated by examining vitellogenic oöcyte development, a process regulated by juvenile hormone in these flies. Both precocenes reduced the number of vitellogenic oöcytes present 43 hr after exposure in a dose-dependent manner. Precocene I was effective when applied to either newly eclosed females prior to vitellogenic oöcyte development or to gravid females. Precocene I was also effective in decapitated females, indicating that the action of the compound is not mediated by the brain. Corpus allatum volume, presumably a reflection of secretory activity, increased between 0 and 24 hr after eclosion in control females but not in precocene-treated females even after 48 hr. However, when females were removed from precocene medium, gland volumes increased within 48 hr to approximately those of control flies. This result is consistent with the reversibility of the precocene effect on Drosophila adults. These results suggest that precocene acts on the corpus allatum of Drosophila adult females to produce juvenile hormone deficiency.     

The role of factors from the head in the regulation of egg development in the mosquito Aedes aegypti

The relationships between the release of factors from the head after blood-feeding, subsequent levels of ecdysteroids and vitellin, and the ultimate maturation of eggs in Aedes aegypti were investigated. Females were decapitated at various times after a blood meal, at 20 or 48 h after feeding the animals were dissected and divided into two groups, those with arrested oöcytes (yolk length < 100 μm) and those with maturing oöcytes (yolk length > 100 μm). These yolk lengths correspond with the levels of oöcyte growth believed to accompany the proposed initiation and promotion phases of egg development. Animals dissected at 20 h were assayed for ecdysteroid by radioimmunoassay; those dissected at 48 h were assayed for vitellin by rocket immunoelectrophoresis.Non-blood-fed unoperated females contained 8% as much ecdysteroid as blood-fed controls and no measurable vitellin. Females with arrested oöcytes (< 100 μm) were obtained only if decapitations were performed before 8 h; these females had about 20% of the ecdysteroids and 8% of the vitellogenin normally found in blood-fed animals. Females decapitated between 2 and 8 h with maturing oöcytes contained 50–60% as much ecdysteroid and vitellin as blood-fed unoperated controls. Normal ecdysteroid and vitellin levels were reached only when decapitations were delayed for 12 and 24 h, respectively. The number of developing oöcytes was also decreased by early decapitation and was closely correlated with vitellin levels.We conclude that the egg development neurosecretory hormone is released twice, once before 8 h and once after 8 h, to control ecdysteroid levels. We also suggest the presence of other factors from the head that control vitellin levels, the number of developing oöcytes, and the early growth of the oöcyte (initiation).     

Synthesis and deposition of yolk protein in adult Drosophila melanogaster

Newly eclosed Drosophila melanogaster females contain only previtellogenic stage oöcytes and no immunologically detectable female specific haemolymph protein. During the subsequent 48 hr the concentration of female specific protein in the haemolymph rises to a plateau value of 21 μg/μl; at this time yolk protein represents about one third of the total haemolymph protein in adult females. The first mature (stage 14) oöcytes are observed at 48 hr post eclosion. The female specific haemolymph protein and the major protein from mature oöcytes are electophoretically and immunologically the same or very similar. Injection of alpha amanitin into newly eclosed females inhibits the development of mature oöcytes and the degree of inhibition depends on the age of the female at the time of injection. Phenocopies of non-vitellogenic mutants result when alpha amanitin is injected into newly eclosed females; after 36 hr post eclosion no visible inhibition of vitellogenesis (as observed morphologically at 72 hr post eclosion) can be produced by alpha amanitin.     

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.     

Determinants of oöcyte degeneration in Drosophila melanogaster

During normal oögenesis in many insects some of the oöcytes fail to mature; instead they degenerate and are resorbed. In this work oöctte degeneration was investigated in Drosophila melanogaster females and found to be limited to early vitellogenic stages (stages 8–10). Even when retained for up to 18 days by females, mature (stage 14) oöcytes showed unaltered protein patterns after separation by SDS polyacrylamide electrophoresis, indicating that protein breakdown, which is characteristic of degeneration, does not occur in chorionated oöcytes.A number of environmental parameters were shown to influence the percentage of degenerating oöcytes in females. Strong responses as reflected by increased stage-8 and 9 oöcyte degeneration were found in females subjected to suboptimal (but not starvation) medium, virgin females, females mechanically unable to oviposit, and females unable to locate suitable oviposition sites. Little or no response was seen in females subjected to crowding, however, since all of these environmental parameters except adult crowding have been shown to decrease fecundity, and therefore the rate of oöcyte production, it is suggested that oöcyte degeneration is a strategy for decreasing the rate of oöcyte production in Drosophila.     

Influence of a juvenile hormone analogue on vitellogenin synthesis and oögenesis in larvae of Nauphoeta cinerea

In experiments on the synthesis of the vitellogenic protein, farnesylmethylester, a juvenile hormone (JH) analogue, was injected into female Nauphoeta cinerea larvae at various stages during their development. Two and 4 days after injection, 2 μl of haemolymph were assayed in a vitellogenin immunodiffusion test. In second last and last instar larvae less than 6 days before adult ecdysis, high doses (100 μg) of farnesylmethylester are necessary to induce vitellogenin synthesis, whereas older last stage larvae and decapitated adults respond to small doses (1 μg) with the synthesis of vitellogenin. It seems that the competence to synthesize the vitellogenic protein changes at the time of induction of the moulting process. If farnesylmethylester is injected into last instar larvae with a supposedly high titre of ecdysone, the vitellogenic protein can be detected in the haemolymph of a small percentage of animals only.Oöcyte maturation can be observed in last instar larvae injected after the fifth to ninth day with farnesylmethylester. The observed volume changes of the corpora allata suggest that an absence of JH for a short time is necessary for the oöcytes to become competent to grow. Last instar larvae treated with farnesylmethylester become larval-adult intermediates with partly developed oöcytes, demonstrating a simultaneous juvenilizing and gonadotropic influence of the JH analogue. In last instar larvae injected with farnesylmethylester a partial degeneration of already maturing oöcytes is induced at the time when the ecdysone titre is supposedly high and the possible reasons for this are discussed.     

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.     

Hormonal aspects of oögenesis in the flies Phormia regina and Sarcophaga bullata

The corpora allata (CA) and median neurosecretory cells (MNC) of Phormia regina and Sarcophaga bullata become active with increasing age of the fly, on a diet of sugar alone. To prevent or retard oögenesis the CA or MNCs must be removed shortly after emergence, with subsequent protein meals. Topical JH application partially compensates for CA or MNC removal. This shows that the MNC activate the CA, and not vice versa. The trauma of either operation slightly depresses egg development.Injection of ecdysone into both species in the stage of initial yolk deposition causes the primary oöcytes to degenerate. This leads to development of the penultimate oöcytes. Older and younger egg stages are not sensitive to ecdysone. In P. regina the application of JH to females with developing primary oöcytes stimulates yolk deposition in the penultimate oöcytes.     

Yolk deposition in Douglas-fir beetle oöcytes: Possible rôle of RNA synthesis in the follicular epithelium

RNA synthesis and morphological changes in the follicular epithelial cells of oöcytes of Douglas-fir beetle, Dendroctonus pseudotsugae were studied during the reproductive phase. Inhibition of synthesis of DNA dependent RNA by actinomycin D injections blocked yolk deposition in the oöcytes as well as oviposition within the normal period. A mixture of radioactively labelled haemolymph and ovarial proteins was deposited as yolk proteins in the oöcytes of normal beetles. Such proteins were not deposited in the oöcytes of females injected with actinomycin D; the blockage of yolk deposition persisted even when such females were treated extraneously with juvenile hormone.     

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.     

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.     

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.     

Haemolymph ecdysteroid levels in female tsetse fly Glossina fuscipes (Diptera) during the first reproductive cycle: A comparison between virgin and mated females

The fluctuations in haemolymph ecdysteroid levels were recorded by radioimmunoassay during the first two ovarian cycles of the tsetse fly, Glossina fuscipes. During the first ovarian cycle, the patterns of ecdysteroid levels are similar in virgin and mated females. In virgins, the first oöcyte is not ovulated and ecdysteroid levels during the second vitellogenesis remain low. In mated females, vitellogenesis of the second oöcyte is concomitant with growth of the first larva in the mother's uterus and ecdysteroid levels are higher than during the first cycle. Two ecdysteroid increases were recorded in mated females and not in virgins. One coincides with vitellogenesis of the second oöcyte and the other occurs at the end of pregnancy and finishes after larviposition. The roles of ecdysteroids in the regulation of vitellogenesis, ovulation, pregnancy and larviposition are discussed.     

Ecdysteroids during oögenesis in the ovoviviparous cockroach Nauphoeta cinerea

By using thin-layer chromatography and high-pressure liquid chromatography combined with radioimmunoassay as well as gas chromatography-mass spectrometry we have identified and quantified ecdysteroids in ovaries and haemolymph of adult female Nauphoeta cinerea. Our analyses demonstrate the presence of ecdysone and 20-hydroxyecdysone, the latter being clearly predominant in all stages investigated. Titre determinations of free ecdysteroids in ovaries show that the 20-hydroxyecdysone concentration is highest (approximately 400 ng/g) at the beginning of chorion formation, suggesting an involvement in this process. Towards ovulation, the titre of free ecdysteroid drops and is low in the newly ovulated egg case. Measurement of immunoreactive highly polar products demonstrates that their concentration remains on a low level throughout the oöcyte maturation period; hydrolysis experiments with Helix pomatia enzymes reveal that, compared to the free ecdysteroids in the ovary, only small quantities of ecdysteroids are present as Helix hydrolysable conjugates. If one compares the quantities of free ecdysteroids in the ovary with those in the haemolymph it becomes apparent that the concentration in the haemolymph is about 10 times lower than that in the ovary.In vitro incubation of follicle cells from oöcytes at stages around chorion formation reveals that these cells are able to produce ecdysone and 20-hydroxyecdysone, and incubation with [³H]-ecdysone demonstrates that ecdysone is efficiently converted to 20-hydroxyecdysone in a stage-dependent manner. These observations strongly suggest that the follicle cells are the site of ecdysteroid biosynthesis and of C-20-ecdysone hydroxylation.A comparison of these findings with observations made of other insects such as locusts and mosquitoes demonstrates significant differences in quality, composition, titre fluctuation and distribution of ecdysteroids in adult females from different species and suggests that these ecdysteroids might fulfil multiple and various biological functions.     

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.     

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.