Egg Development Neurosecretory Hormone Research Articles

Rapid isolation of a neurohormone from mosquito heads by high-performance liquid chromatography

Methods were developed for the isolation of the egg development neurosecretory hormone, EDNH, from heads of the mosquito Aedes aegypti. This hormone stimulates ecdysone production by ovaries. Methods used for the successful isolation of insulin-like peptides from vertebrate tissues were modified to develop a four-step procedure involving extraction in acidified ethanol, precipitation by neutralization, followed by sequential separation on size-exclusion, ion-exchange and reversed-phase high-performance liquid chromatography columns.

Multiple forms of cerebral peptides with steroidogenic functions in pupal and adult brains of the yellow fever mosquito, Aedes aegypti

In the adult mosquito, Aedes aegypti, the cerebral egg development neurosecretory hormone (EDNH) regulates reproduction by activating the ovaries to synthesize the steroid hormone ecdysone, while during postembryonic development the cerebral prothoracicotropic hormone (PTTH) activates the prothoracic glands to synthesize ecdysone. In each case, ecdysone then drives stage-specific physiological processes. Since EDNH and PTTH share a common steroidogenic function, it is conceivable that they are similar, if not the same, peptide(s). This investigation has begun to address this possibility by determining structural and functional relationships between these two families of neuropeptides. The peptides having EDNH activity in adult and pupal heads of Aedes aegypti were characterized on the basis of their molecular weights and their biological properties in in vitro and in vivo bioassays for EDNH. Gel filtration chromatography of extracts revealed fractions with EDNH activity having molecular weights of ~ 11 kDa and ~ 24 kDa. The similarities in the functional dynamics of these two activities in the different bioassays suggested that comparable moieties existed in pupal and adult brains. The sensitivity of these moieties to proteolytic hydrolysis indicated that they were proteins. Thus at least two EDNH-like peptides appear to be present in pupal and adult heads of Aedes aegypti. The apparent existence of more than one molecular weight form of EDNH has not been reported previously. Since these moieties are present in both pupal and adult heads, it is possible that they have the same steroidogenic function in the two different stages, i.e., to activate ecdysone synthesis, by prothoracic glands in larvae and pupae and by the ovaries in adults.

Inter-species cross-reactivity of the prothoracicotropic hormone of Manduca sexta and egg-development neurosecretory hormone of Aedes aegypti

The cross-reactivities of the big and small forms of prothoracicotropic hormone (PTTH) from pupal brains of Manduca sexta and egg-development neurosecretory hormone (EDNH) from heads of adult Aedes aegypti were examined for PTTH by the in vitro Manduca prothoracic gland assay and for EDNH by the in vitro and in vivo Aedes ovary assays. The synthesis of ecdysone by both larval and pupal prothoracic glands of Manduca was increased in a dose-dependent manner by crude extracts of Aedes aegypti heads, reaching a maximum of approx. 3- and 2-fold, respectively. Gel filtration chromatography of the Aedes head extract revealed a peak of EDNH activity with an apparent mol. wt of 11 kD. This partially purified EDNH did not possess prothoracicotropic activity in the in vitro prothoracic gland assay, nor did any other fractions from the gel filtration column. Similarly, partially purified big and small PTTH did not activate Aedes atropalpus ovaries to synthesize ecdysone in vitro, nor did they cause ovarian maturation in vivo. Thus, it appears that the structural differences between PTTH and EDNH are sufficient enough to prevent functional cross-reactivity. The apparent discrepancy in the results obtained with the crude and partially purified EDNH and PTTHs raises questions about the reliability of bioassays for screening the presence and cross-reactivity of peptide neurohormones in crude extracts.

Egg development in the mosquitoAnopheles albimanus

Summary In the mosquito, Anopheles albimanus, previtellogenic egg development was completed by 48 h after emergence, and vitellogenic growth was completed by 36 h after a blood meal. Ecdysteroid levels reached a peak of 800 pg/female by 18 h, while vitellin levels rose to their maximum 36–48 h after a blood meal. Most of the ecdysteroids present in the female before 36 h behaved as ‘free’ hormone, while after 42 h the ecdysteroids were ‘conjugated’. Injection of 20-hydroxyecdysone into non-blood-fed females induced degeneration of the resting stage oocytes, but vitellogenin synthesis was detectable by autoradiography. Injection of 5 μ of 20-hy-hroxyecdysone into blood-fed decapitated females induced almost precisely normal levels of vitellin. Detailed analysis of the effect of decapitating blood-fed females suggested that the release of factors from the head (e.g., egg development neurosecretory hormone) occurs as an all-or-none phenomenon, and probably occurs twice.

Biosynthesis and distribution of ecdysone and 20‐OH‐ecdysone in Aedes aegypti

AbstractThe distribution and biosynthesis of ecdysone and 20‐hydroxyecdysone (20‐OH‐ecdysone) was followed in sugar‐ and blood‐fed female Aedes aegypti. In both sugar‐ and early blood‐fed animals most of the ecdysteroid determined by radioimmunoassay was found outside the ovary. Twenty‐four to 40 h after blood feeding, however, ecdysteroid was distributed between ovary and carcass in the ratio of 1:1.5. Ecdysteroid titer reached a plateau between 18 to 40 h after the blood meal and decreased thereafter. Analysis of the ecdysteroid titer using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) revealed that both 20‐OH‐ecdysone and ecdysone were synthesized after the blood meal. The ratio of 20‐OH‐ecdysone to ecdysone remained essentially constant and fluctuated in parallel throughout egg development. Chromatography of the early ecdysteroid peak (8 h after feeding) using TLC and HPLC indicated that although it cross‐reacted with ecdysteroid antibodies, it did not have the same elution times as ecdysone and 20‐OH‐ecdysone and is, therefore, probably a precursor of these ecdysteroids. Injections of egg development neurosecretory hormone (EDNH) preparation purified to near homogeneity, into ligated abdomens, induced ecdysteroid synthesis only if the abdomens were first treated with methoprene (12.5 pg). Methoprene at this concentration did not stimulate ecdysteroid synthesis in these abdomens. When blood‐fed females were treated with [4‐14C] cholesterol and analyzed using TLC and HPLC procedures, both [14C]labeled ecdysone and [14C]labeled 20‐OH‐ecdysone were synthesized in the ratio of 1:1.5. This report is the first to show that both ecdysone and 20‐OH‐ecdysone are synthesized in vivo in female A. aegypti.

Egg maturation and ecdysiotropic activity in extracts of mosquito (Aedes aegypti) heads.

Blood-fed, decapitated female Aedes aegypti mosquitoes matured eggs when injected with an extract of heads, but non-blood-fed females did not. The response to head extract was optimal when the head was allowed to remain for 2 hr after feeding. A dose response to injected egg development neurosecretory hormone (EDNH) was observed in vivo that was similar to in vitro dose responses previously reported. Blood-fed decapitated females responded equally well to boiled or unboiled head extract. When blood-fed decapitated females were injected with head extract, ecdysteroid levels increased. Partial purification of head extract using high-pressure liquid chromatography yielded a fraction at 34% acetonitrile that showed egg maturation activity in vivo when injected into blood-fed decapitated females, and ecdysiotropic activity when incubated in vitro with ovaries. In addition, a fraction at 30% acetonitrile was found that showed activity in vivo but not in vitro and may be a precursor. Occasionally, the fraction at 37% acetonitrile showed activity in the in vitro assay but had little activity in vivo and may be a metabolite. These results suggest that the same hormone was being assayed in vivo and in vitro and is EDNH.

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).

Isolation and characterization of highly purified mosquito oostatic hormone

AbstractOostatic hormone, the hormone that inhibits vitellogenesis in mosquitoes, was purified 7,000‐fold with a recovery of 70% from the ovaries of the mosquito Aedes aegypti. The purification procedure included heat treatment and chromatography on ion exchange and gel filtration columns. The hormone is a small peptidelike molecule of molecular weight 2,200 at pH 4.5, which aggregates into larger molecular species of trimer and octamer at pH 7.0 as determined by gel filtration. The hormone is positively charged at pH 7.8 and has a low Rf at pH 9.4 on disc gel electrophoresis.Injection of purified oostatic hormone (9 ng) into female mosquitoes inhibited yolk deposition and vitellogenin synthesis. Activity of the oostatic hormone in the mosquito ovary increased rapidly following blood feeding and reached a maximum after 48 h. Oostatic hormone of A. aegypti injected into autogenous Aedes taeniorhynchus inhibited egg development. Repeated injections of dilute oostatic hormone at 24 h intervals partially arrested egg development, resulting in 60% reduction in the number of eggs laid. This hormone does not block release of egg development neurosecretory hormone (EDNH) from the mosquito brain but rather appears to act on the ovary.

Juvenile hormone and 20‐hydroxyecdysone as primary and secondary stimuli of vitellogenesis in Aedes aegypti

AbstractFemale Aedes aegypti that were fed blood and immediately abdominally ligated did not deposit yolk. Injection of 20‐hydroxyecdysone (1.5–5.0 ng) or topical application of juvenile hormone (JH) analogue methoprene (25 pg) did not induce vitellogenesis in these abdomens. When blood‐gorged ligated abdomens were treated with both hormones, however, vitellogenesis was stimulated in 60% of treated animals. Rocket immunoelectrophoresis indicated that vitellin concentration per follicle in treated animals was similar to that in intact controls. When ligated abdomens were first treated with methoprene and immediately injected with a crude head extract of egg development neurosecretory hormone, vitellogenin synthesis was induced at a rate similar to that in blood‐fed controls. Methoprene at this concentration (25 pg), did not cause an increase in whole‐body ecdysteroid titers. Larger amounts of methoprene (1.65 ng) were needed to stimulate egg development and ecdysteroid production.Implantation of ecdysone‐secreting ovaries into ligated abdomens did not stimulate vitellogenesis in the recipients. However, in recipients that were first treated with methoprene (25 pg), implantation of ecdysone‐secreting ovaries resulted in normal egg development.These experiments indicate that the appearance of JH precedes 20‐hydroxyecdysone in stimulating vitellogenesis following blood feeding in Ae. aegypti.

Purification and partial characterization of mosquito egg development neurosecretory hormone: Evidence for gonadotropic and steroidogenic effects

AbstractEgg development neurosecretory hormone (EDNH), a hormone that stimulates vitellogenesis in mosquitoes, was purified 10,000‐fold from the mosquito Aedes aegypti. The purification procedure included chromatography on hydrophobic, ion exchange, and gel filtration columns, and preparative electrophoresis to give an almost homogeneous preparation. The hormone is a polypeptide monomer of molecular weight of 18,700 ± 500 as determined from SDS electrophoresis and gel filtration chromatography. Using lowpressure chromatography throughout the purification procedure, the hormone was recovered at a high yield (39%). The amount of EDNH in a mosquito is about 0.6‐1.6 ng, corresponding to 32–85 fmol.Injection of purified EDNH into female mosquitoes resulted in the conversion of [14C]cholesterol into labeled ecdysone and 20‐hydroxyecdysone which were separated and identified using thin‐layer chromatography and high‐performance liquid chromatography separation procedures.Egg development and vitellogenin synthesis were also induced when EDNH was injected into several mosquito species, indicating that the hormone is not species‐specific.This report is the first to show that a purified preparation of EDNH has both steroidogenic and a gonadotropic effects on female mosquitoes.

Hormonal regulation of ovarian ecdysteroid production in the autogenous mosquito, Aedes atropalpus

The effects of juvenile hormone (JH) and egg development neurosecretory hormone (EDNH) on ovarian ecdysteroid production during vitellogenesis in the autogenous mosquito, Aedes atropalpus, were investigated using in vitro techniques coupled with radioimmunoassay (RIA) and high-pressure liquid chromatography (HPLC). Normal females were characterized by quantitative, qualitative, and temporal patterns of in vitro ovarian ecdysteroid production. Females decapitated at emergence showed little ovarian ecdysteroid production and did not undergo vitellogenesis. A 500-ng dose of JH-I applied topically to decapitated females restored normal patterns of ecdysteroid production. In both normal and experimental females, ecdysone constituted the major portion of the ecdysteroids secreted by the ovaries in vitro. However, significant amounts of other RIA-active materials were detected, one of which was probably 20-OH-ecdysone. Fat body incubations indicated that these tissues produce little RIA-active material during the peak of vitellogenesis. During that period, the ovaries were the major source of ecdysteroid. Various doses of JH-I, applied to adbomens isolated at emergence, enhanced ovarian responsiveness to subsequent applications of head extracts containing EDNH both in vivo and in vitro. A 500-ng dose of JH-I, applied topically to blood-fed, decapitated Aedes aegypti, stimulated a significant increase in in vitro ovarian ecdysteroid production. Similarities between these data and those demonstrating prothoracicotropic effects of JH in the Lepidoptera are discussed.

Effects of house fly oostatic hormone on egg development neurosecretory hormone action in Aedes atropalpus

AbstractOostatic hormone, prepared from mature female house flies, Musca domestica, was examined for its effect on processes mediated by egg development neurosecretory hormone (EDNH). When injected into newly emerged adult female mosquitoes, Aedes atropalpus, oostatic extracts inhibited vitellogenic growth of the ovaries. Comparison of oostatic activities in heads, thoraces, and abdomens of house flies showed that the major portion was present in the abdomens, apparently associated with the ovaries. In contrast to untreated vitellogenic females, no ecdysone or 20‐hydroxyecdysone could be detected in female mosquitoes injected with the oostatic extract. Vitellogenic growth and ecdysteroid levels were suppressed in decapitated females injected with a mixture of extracts containing EDNH and oostatic activities. Since the normal vitellogenic and steroidogenic responses to EDNH can be suppressed by simultaneous injection of EDNH and oostatic material, it is suggested that oostatic hormone acts at a level subsequent to EDNH release. However, we cannot rule out at this time that EDNH release, itself, is also affected.

Partial purification of egg development neurosecretory hormone with reverse-phase liquid chromatographic techniques

We report the use of reverse-phase liquid chromatographic techniques for the isolation of a steroidogenic neuropeptide (EDNH) from mosquito heads. Activity of fractions was assayed by measuring the ability of ovaries to produce ecdysteroid in vitro . Dose response profiles using crude head extracts or partially purified EDNH were nearly identical, indicating that the methods of preparation did not alter biological activity. EDNH activity eluted from a reverse-phase HPLC (RP-HPLC) column primarily near 35 percent acetonitrile using a linear gradient. Methods developed with an analytical RP-HPLC column were successfully adapted for preparative work. Active fractions from the preparative RP-HPLC were further purified on a second analytical column under isocratic conditions at 30% acetonitrile. Two adjacent UV absorbing peaks were found, each with EDNH activity. Activity was sensitive to proteolysis.

Release of egg development neurosecretory hormone in Aedes aegypti and Aedes taeniorhynchus induced by an ovarian factor

Ovariectomized Aedes aegypti do not synthesize vitellogenin after a blood meal, unless an ovary from a blood-fed donor is implanted. Decapitation, however, prior to implantation inhibits vitellogenin synthesis. A female ovariectomized and decapitated 6 hr after a blood meal, synthesizes vitellogenin if an ovary from a blood-fed donor is implanted. On the other hand, females that are fed on blood and immediately decapitated can not be stimulated to synthesize vitellogenin with implanted ovaries removed from blood-fed donors. These experiments led to the hypothesis that the blood meal stimulates the ovary to secrete a corpus cardiacum stimulating factor, that in turn promotes release of egg development neurosecretory hormone stored in the corpus cardiacum. Injection of 20-hydroxy-ecdysone or ovarian extract prepared from ovaries removed from unfed females does not release egg development neurosecretory hormone. Thus corpus cardiacum stimulating factor is not 20-hydroxy-ecdysone, and ovaries removed from unfed females do not store it. The rate of inactivation of egg development neurosecretory hormone released from the corpus cardiacum after a blood meal was investigated by implanting an ovary into females that were blood fed for various intervals than decapitated and ovariectomized. Seventy per cent of implants grow when the operation is done 18 hr after feeding, and 30% when the operation is done between 18 and 24 hr after feeding, indicating that egg development neurosecretory hormone is stable for the first 18 hr after a blood meal. Aedes taeniorhynchus females ovariectomized 24 hr after adult emergence do not synthesize vitellogenin. When such a female is implanted with an ovary removed from a sugar-fed or blood-fed Aedes aegypti donor vitellogenin synthesis is initiated, and the implant grows. Decapitation prior to implantation inhibit vitellogenin synthesis and implants do not grow. These results indicate that corpus cardiacum stimulating factor is not species specific.

A neurosecretory hormone-releasing factor from ovaries of mosquitoes fed blood

In mosquitoes, a hormone (egg development neurosecretory hormone or EDNH), produced by the medial neurosecretory cells and stored in the corpus cardiacum soon after eclosion, is released after a blood meal, and vitellogenesis begins a few hours later. When either the ovaries or the neurosecretory cells and corpus cardiacum are removed before the blood meal, vitellogenin is not synthesized. Therefore, we tested the hypothesis that the release of EDNH from the corpus cardiacum is dependent on the secretion of a releasing factor from the ovaries. Using a bioassay for EDNH in the corpus cardiacum, we found that the gland of an ovariectomized female remained active after blood feeding, and therefore, has not released EDNH. When an ovary was implanted before the blood meal, the corpus cardiacum was inactive, and therefore, had released EDNH. We concluded that the ovaries secrete an EDNH-releasing factor, and that this factor and EDNH must both be in circulation before vitellogenesis can begin. Although releasing factor alone did not stimulate vitellogenesis, it was the rate limiting process that controlled the onset of vitellogenesis. Using a bioassay for the EDNH-releasing factor from the ovaries and using rocket-immuno-electrophoresis, we showed that a Culex ovary, but not an Anopheles ovary, could replaces Aedes ovaries as a source of the releasing factor. In Ae. aegypti, EDNH-releasing factor was required again after oviposition in order to reinitiate the vitellogenic process in females that took a second blood meal. Thus, the releasing factor is part of the mechanism regulating cyclic egg maturation in mosquitoes.

Ovarian ecdysone secretion is controlled by a brain hormone in an adult mosquito

In the mosquito Aedes aegypti, a blood meal triggers egg development. Early workers used the simple technique of decapitation, or neck ligation, to show that a factor from the head was required for normal egg development1–4. Lea subsequently demonstrated that the source of this factor was the medial neurosecretory cells of the brain5. The hormone from these cells, which he termed the egg development neurosecretory hormone (EDNH), is stored in the corpus cardiacum (CC) and released shortly after a blood meal6. Previous work had demonstrated that the ovary begins to produce ecdysone after a blood meal8. Considerable evidence suggests that the ecdysone, after being converted to 20-hydroxyecdysone, stimulates the synthesis and secretion of vitellogenin by the fat body8–12. The vitellogenin is taken up by the growing oocytes and becomes part of the yolk. Analysis of the amount of 20-hydroxyecdysone present in females after a blood meal showed that significant quantities were first seen 4–6 h after a blood meal8. Vitellogenin synthesis begins at about the same time13. The early decapitation experiments of Gillett demonstrated that the head was necessary for 4–8 h after a blood meal for egg development to proceed normally3. A similar critical time was found for the control of vitellogenin synthesis by the brain hormone7. The coincidence of this timing suggested to us that the ovary was the target of the brain hormone. The experiments reported here were designed to test the hypothesis that the brain hormone released after a blood meal acted on the ovary, causing the synthesis of ecdysone. Our results confirm this.

The influence of the brain hormone on retention of blood in the mid-gut of the mosquito Aedes aegypti (L.). III. The involvement of the ovaries and ecdysone.

Most female mosquitoes require a blood-meal in order to produce mature oöcytes. An egg development neurosecretory hormone (EDNH), which is produced in the medial neurosecretory cells (m.n.c.) of the brain and stored in the corpus cardiacum, is released into the haemolymph following the ingestion of blood and is essential for the promotion of ovarian development to maturity. It has been shown that a factor from the m.n.c., presumably EDNH, is necessary if the blood-meal is to be retained in the mid-gut until the oöcytes approach maturity. The present paper shows that retention is not a direct result of the action of EDNH, but is dependent on the ovaries and may well involve ecdysone. Removal of the ovaries before a blood-meal leads to early haem-defaecation, but delay can be restored by injection of ecdysterone. Sub-threshold feeders and mosquitoes decapitated immediately after the intake of blood, each of which would be expected to eliminate the blood-meal early, also show a delay in the onset of haem-defaecation when injected with ecdysterone. Further, both in ovariectomized insects and sub-threshold feeders the time of onset of haem-defaecation is associated with the dose of ecdysterone given.

The influence of the brain hormone on retention of blood in the mid-gut of the mosquito Aedes aegypti (L). II. Early elimination following removal of the medial neurosecretory cells of the brain.

Development of the oöcytes beyond the resting stage to maturity in mosquitoes is triggered by an egg development neurosecretory hormone from the brain. This hormone is produced by the medial neurosecretory cells of the pars intercerebralis, and in anautogenous species is stored in the corpus cardiacum until a blood-meal is taken. Removal of the head of the mosquito Aedes aegypti (L.), shortly after completion of a blood-meal, has previously been shown not only to prevent oöcyte maturation, but also to result in early elimination of the blood-meal. The work described in this paper shows that it is a factor originating in the medial neuro­-secretory cells, presumably egg development neurosecretory hormone, which is essential if the blood-meal is to be retained in the mid-gut until the oöcytes are nearing maturity.

The role of isoleucine in differential egg production by the mosquito Aedes aegypti linnaeus (Diptera: Culicidae) following feeding on human or guinea pig blood

1. 1. Egg production by the Newbam strain of Aedes aegypti was significantly greater following feeding on guinea pig blood than on an equal volume of human blood. 2. 2. One prominent difference between the bloods is the lower isoleucine content of human blood. When fed with either whole human blood or red blood cells suspended in saline, both supplemented with isoleucine, egg production by these females increased to the level of those fed guinea pig blood. 3. 3. The addition of isoleucine to guinea pig blood did not increase egg production. 4. 4. A nutritional role of isoleucine in ovarian development, rather than a trigger for the release of egg development neurosecretory hormone (EDNH), was proved by the following: (a) Isoleucine-poor human blood was as effective in causing EDNH release within 2 hr following feeding as was the isoleucine-richer guinea pig blood. (b) A positive correlation exists between the isoleucine content of ingested media and the degree of follicular maturation. (c) In the presence of isoleucine, fat body organ cultures incorporated greater amount of labelled amino acid into soluble protein (vitellogenins) than in its absence. 5. 5. Sufficiency of isoleucine content in the feeding media resulted in less follicular resorption, and hence a higher level of follicular maturation.

Peptides as stimulators of egg development neurosecretory hormone release in the mosquito Aedes aegypti

1. 1. The supernatant (peptides) from heat coagulated trypsin-digested blood, stimulated egg development neurohormone release in female Aedes aegypti. 2. 2. An amino acid mixture, or proteins in the presence of trypsin inhibitor, were ineffective in causing EDNH release.