The roles of juvenile hormone and 20-hydroxy-ecdysone during vitellogenesis in isolated abdomens of Drosophila melanogaster

Both juvenile hormone and 20-hydroxy-ecdysone seem to be involved in the regulation of vitellogenesis in Drosophila melanogaster. It is the purpose of this paper to begin to define the functions of these two hormones. Although vitellogenin synthesis does not occur at a high rate in 1-day-old female abdomens isolated from the head and thorax before 0.75 hr after eclosion, both ZR515 (a juvenile hormone analogue) and 20-hydroxy-ecdysone can cause in these preparations vitellogenin synthesis and secretion into the haemolymph. The synthesis and secretion into the haemolymph of all three vitellogenins which are detectable by electrophoresis in sodium dodecyl sulphate-containing gels of polyacrylamide is promoted by both hormones. That result excludes the hypothesis that these two hormones regulate the synthesis of different vitellogenins. A dose-response curve showed that an injection of 0.2 μl of a 10^?6 M 20-hydroxy-ecdysone solution was sufficient to promote vitellogenin synthesis and secretion in isolated abdomens. Ovaries from isolated female abdomens treated with juvenile hormone analogue showed nearly normal amounts of all three vitellogenins and morphologically normal advanced vitellogenic follicles, whereas ovaries from isolated abdomens treated with 20-hydroxy-ecdysone contained little vitellogenin and no vitellogenic follicles. We conclude that under the conditions used, juvenile hormone permits vitellogenin uptake into the oöcyte much more readily than does 20-hydroxy-ecdysone.     

20-Hydroxy-ecdysone and ovarian development in Anopheles stephensi

The characteristics of vitellogenesis and ovarian maturation in the anautogenous Culicid Anopheles stephensi are described. Vitellogenic primary-follicle development from the resting stage is not induced by the injection of 20-hydroxy-ecdysone. However, injection of this hormone does result in premature growth of the penultimate follicle and in the induction of yolk-protein synthesis. Radioimmunoassay of total ecdysteroids supports the hypothesis that 20-hydroxy-ecdysone plays a role in controlling ovarian development. These data, together with published data for Aedes aegypti, are discussed in relation to the control of ovarian development in the Culicidae.     

The allatostatic effect of 20-hydroxyecdysone on the adult viviparous cockroach, Diploptera punctata

The effect of 20-hydroxyecdysone upon the activity of corpora allata (CA) from female Diploptera punctata has been investigated. This ecdysteroid inhibits juvenile hormone (JH) biosynthesis by the CA, whether they have been implanted into a male, or remained in situ within the female. In the female, this inhibition is reflected in reduced oöcyte growth and vitellin content. The allatostatic effect of 20-hydroxyecdysone becomes apparent in vivo within 24 hr. However, no inhibition was observed when the CA were maintained in vitro for 42 hr in medium containing up to 1·10^?5 M 20-hydroxyecdysone. This suggests that the effect of the hormone upon the CA is indirect. These experiments raise the possibility that ecdysteroids play an allatostatic role during the normal gonotrophic cycle in Diploptera.     

Delayed vitellogenesis due to larval food deficiency in the house fly

Oögenesis in normal and small house flies is compared. In small female house flies the onset of vitellogenesis may be delayed by 2–4 days and more. Normal flies fed with sugar only may complete previtellogenic ovarian growth within the first 4–5 days after emergence and may start vitellogenesis immediately when protein is fed after this time. In contrast to this, small flies are not ready to start vitellogenesis when fed proteins after a sugar diet of 5 days. It is suggested that small flies need a protein food for the completion of the previtellogenic period of ovarian growth. Once vitellogenesis has started further ovarian development is normal in small flies.     

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.     

Ultrastructural changes induced by juvenile hormone analogue in oöcyte membranes of apterous4Drosophila melanogaster

Egg chambers from apterous⁴ (ap⁴), a female sterile mutant of Drosophila melanogaster, show none of the microvilli or pinocytotic vesicles which are a prominent feature of the membrane of the wild-type vitellogenic oöcyte. The studies reported here show that a juvenile hormone analogue (ZR515) stimulates formation of microvilli and pinocytotic vesicles in oöcytes of ap⁴ flies. Within 12 hr after topical application of ZR515 to homozygous ap⁴ females the oöcyte membranes exhibit extensive microvilli and pinocytotic activity. The follicle-cell surface adjoining the oöcyte also shows some changes. In vitro studies in which ap⁴ ovaries were incubated in Schneider's Drosophila tissue-culture medium in the presence of ZR515 with or without female haemolymph, or in the absence of ZR515, showed that the analogue acts alone directly on the ovary to cause formation of microvilli and pinocytotic vesicles on the oöcyte membrane.     

Maturation and hatching of eggs from silkworm ovaries preserved in liquid nitrogen

Ovarian imaginal discs prepared from fifth-instar larvae of the silkworm, Bombyx mori were treated with graded concentrations of glycerol, cooled at a rate of 1°C/min to ?35°C and preserved in liquid nitrogen for 2 days or more and then rapidly thawed (500°C/min). The frozen and thawed ovaries were transplanted into fifth-instar female larvae, in which more than 20% of the ovaries developed to produce mature eggs with a chorion according to the state of host development. By parthenogenetic activation, the mature eggs started embryogenesis and hatched to produce larvae. About 50% hatching occurred in the eggs developed in a C 108 × Cambodge host, and about 10% in a C 108 × Aojuku host. The hatched larvae completed post-embryonic development as did the normal larvae.     

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.     

Development and physiology of follicular atresia during ovarian growth in the house fly, Musca domestica

Atretic follicles regularly occur in the ovary of the house fly, Musca domestica. The frequency of ovarian follicular atresia and the proportion of atretic follicles per ovary are related to the stage of oögenesis and to the age of the females. Only vitellogenic follicles may become atretic. The atresia may occur at any stage of vitellogenesis, though most follicles become atretic in mid-vitellogenesis. Atretic follicles are completely resorbed within 24–36 hr. The follicle cells may play a synthesizing role during growth and disintegrating one during follicle resorption. The induction of glycogen synthesis by the cessation of RNA and protein synthesis and by vitellogenesis in normal follicles is discussed. The same processes occur prematurely in the atretic follicle which can be thus distinguished by a high content of glycogen.     

Some aspects of oögenesis in the stable fly Stomoxys calcitrans (Diptera: Muscidae)

Two distinct patterns of blood ingestion in the female stable fly, Stomoxys calcitrans L. were observed, based on whether or not the female was mated. Mating is not required for oögenesis, but is necessary for oviposition. Virgin females develop eggs but, in the absence of mating, they retain eggs until death. In stable flies, oögenesis appears to be atypical among other dipterans as the penultimate follicles develop and deposit up to 50% yolk before the terminal follicles are matured. Oösorption was seen in the penultimate follicles of virgin females retaining eggs. Accessory-gland implants initiated oviposition in virgin females. However, the total number of eggs laid by such females was 50% less than the number of eggs laid by mated flies.     

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.     

Vitellin and vitellogenin concentrations during oögenesis in the first gonotrophic cycle of the house fly,Musca domestica

The house fly, Musca domestica, contains at least two native vitellin and two vitellogenin proteins. Both vitellins appear to have an identical vitellogenin partner. The major native vitellin has a mol. wt of 281 K Daltons, and the major native vitellogenin has a mol. wt of 283 K Daltons. These proteins are composed of three subunits with mol. wt of 48, 45 and 40 K Daltons. The relationship of the subunits to the native proteins is not known.Haemolymph vitellogenin levels are cyclical during oögenesis with no detectable amounts in previtellogenic flies and low levels in postvitellogenic flies. The highest level of vitellogenin, 10.5 μg/μl, occurred in flies with stage-7 ovaries. The vitellogenin levels during oögenesis fit a parabolic curve and the fat body vitellogenin content during oögenesis showed this same pattern.Uptake of vitellogenin into the ovary during each stage of oögenesis also fit a parabolic curve and produced a high linear correlation with haemolymph vitellogenin levels. The greatest uptake was 37 μg/stage and occurred during stage 6.     

What determines the number of ovarioles in a fly ovary?

The relation between the size of a fly and the number of ovarioles in its ovary was investigated in Phormia regina and Sarcophaga bullata. Small flies of varying size were produced by taking larvae prematurely off the food. The smallest flies thus obtained were derived from larvae only $\text{1}\text{8}$ of the weight of a normal larva. The number of ovarioles in an ovary is directly proportional to the size of the fly and, in the extreme case, is about $\text{1}\text{5}$ the normal number in Sarcophaga and about $\text{1}\text{3}$ in Phormia. Larvae prematurely taken off the food, but fed again after starving for several days, grow to normal or almost normal size and develop ovaries with about the normal number of ovarioles. Small or re-fed Sarcophaga do not show any changes in the anatomy of individual ovarioles but in Phormia disorders in ovariole development and a consequent reduction of fertility are frequent. The number of ovarioles remains identical from the early pupal stage all through the development of the pharate fly and then through ovarian development in the adult fly: it is determined by the size of the larva when it was taken off the food. This shows that it is not lack of space in a small adult fly abdomen which determines the number nor the occurrence of degenerative processes during ovarian development.     

Endocrine influence upon the development of vitellogenic competency in Oncopeltus fasciatus

Immunoelectrophoretic analysis of the blood of precocious adult females of Oncopeltus demonstrated the presence of the A form of vitellogenin but no detectable AB form, the form present in mature adult-female haemolymph. Although the appearance of the AB form could be induced by topical treatment of precocious adult females with juvenile hormone, no yolk deposition was induced in these females. Histological examination revealed that the ovaries of precocious adult females reached only a previtellogenic stage of development with or without treatment with juvenile hormone. Topical treatment of normal larvae and adults with the hormone demonstrated that vitellogenin synthesis could not be induced with juvenile hormone treatment alone until after adult emergence. Since the precocious adult females are chronologically younger than normal last-stage larval instars, we suggest that a transition period during which juvenile hormone is absent (i.e. the precocious moult in treated animals; the final moult in control animals) must occur before some tissue of the precocious or normal adult females, presumably the fat body, becomes competent to respond to further exposure to the hormone by making the AB form of vitellogenin. The ovary requires a similar transition period prior to becoming capable of depositing vitellogenin; however, the times when the fat body and the ovary become competent to respond to the transition period differ.