Study of the synthesis of vitellogenin in intersexual males of Armadillidium vulgare Latreille (oniscoid isopod crustacean): comparison with males and with intact or ovariectomized females

In some natural populations of Armadilidium vulgare, intersex animals are genetic males which are feminized by maternally transmitted symbiotic bacteria. In these intersex males (iM) the fat body synthesizes vitellogenin, although their gonads are testes with hypertrophied--but nonfunctional--androgenic glands. Vitellogenin is present in the hemolymph of males changed experimentally into iM 90 days after inoculation of the feminizing bacteria. During the molting cycle, vitellogenin synthesis in iM varies as in ovariectomized females or in vitellogenic females, with a peak at the stage D1." In A. vulgare, vitellogenin synthesis is a neutral character since it can be observed in a genetic male or in an ovariectomized female; however, it is inhibited by the androgenic hormone. In intersex males, vitellogenin synthesis is the result of their refractoriness to androgenic hormone.     

Ovarian control of oostegite formation in the terrestrial isopod, Armadillidium vulgare (Malacostraca, Crustacea).

The correlation between oostegite formation and ovarian maturation was investigated in the isopod Armadillidium vulgare. Females whose ovaries had been removed and males whose androgenic glands had been removed were used in these experiments. Ovariectomy of puberal females did not stop oostegite formation. Whether oostegites develop in puberal females whose ovaries have been removed depends upon the degree of maturation of the ovaries at the time of removal. On the other hand, ovariectomized juvenile females and andrectomized juvenile males were unable to form oostegites when they attained puberty. An extract of vitellogenic ovaries induced oostegite formation in ovariectomized females and andrectomized males, but not in intact males. The ability of ovarian extracts to induce oostegite formation was dose-dependent. The nature of this ovarian factor that induces oostegite formation in A. vulgare remains to be elucidated.     

Vitellogenin synthesis by fat body and ovary in the terrestrial isopod, Armadillidium vulgare

Vitellogenin synthesis in Armadillidium vulgare was investigated in tissue cultures. The synthesis of vitellogenin was assayed by the incorporation of [35S]methionine into precipitin with anti-vitellin serum. The forms of synthesized vitellogenin were analyzed by polyacrylamide gel electrophoresis and fluorography. The fat body synthesized vitellogenin and its rate was correlated with the molting cycle: a maximum level at stage D and lower levels at A-C and E of the molting cycle. Four forms of vitellogenin (Vg 1-4) were synthesized; the larger forms (Vg 1-2) were prominent. The ovary also synthesized a slight amount of vitellogenin at the end of the molting cycle. The smaller forms (Vg 3-4) were synthesized under cultured condition. Through vitellogenesis in A. vulgare, the fat body must be the principal site of vitellogenin synthesis. Most vitellogenin may be transported from the fat body to the ovary through hemolymph at stage D of the molting cycle.     

The hormonal control of vitellogenin synthesis in the fat body of the female Colorado potato beetle.

Vitellogenin synthesis in the fat body of the female Colorado potato beetle is affected by juvenile hormone (JH) as was demonstrated by its stimulation after topical application of 50 μg JH to allatocardiacectomized prediapause females. The peak of vitellogenin synthesis is reached 5 days after the administration of JH. Allatocardiacectomy in 5-day-old long-day females does not result in decreased rates of vitellogenin synthesis in comparison with the sham-operated control insects. In postdiapausing long-day females which are allatocardiacectomized during diapause, vitellogenin synthesis increases in the absence of JH. It is concluded, therefore, that factors other than JH are involved. β-Ecdysone does not seem to participate in the regulation of vitellogenin synthesis. In virgin long-day females (12 days after adult ecdysis) JH synthesis and protein synthesis are as high as in mated females, although a virgin produces one-sixth the amount of eggs. In the virgin female the rate of vitellogenin synthesis is uncoupled from the rate of egg laying; this suggests a rather autonomous role in the accumulation of proteins by the ovary.     

The influence of juvenile hormone on polyploidy and vitellogenesis in the fat body of Locusta migratoria

Somatic polyploidy is a common developmental feature of endocrine target tissues, including vertebrate liver and insect fat body. Vitellogenin (Vg) synthesis in locust fat body is under strict control of juvenile hormone (JH) and is correlated with DNA synthesis and massive polyploidization of the tissue. We have used this system to concurrently monitor interactions between hormone, specific protein synthesis, and generation of cells with defined ploidy levels. Normal maturation of both males and females were compared, as were animals treated with precocene III and the JH analog ZR-515. Following are the main results and observations: Polyploidization occurs in both males and females but is much more pronounced in the latter. Peak DNA synthesis occurs in females just before onset of Vg synthesis and is coincident with predominant appearance of octoploid (8c) cells. In males DNA synthesis peaks earlier than in females, during transition from 2 c to 4 c cells, and then declines. Suppression of JH production by precocene prevents Vg synthesis completely and also inhibits DNA synthesis and generation of higher ploidy cells in both sexes. Reexposure of withdrawn animals to hormone reversed the foregoing conditions, but the disparity between sexes in timing of DNA synthesis and bulk tissue ploidy level was maintained. The developmental significance of these results is discussed.     

Mechanism of the refractory state of androgen hormone in Armadillidium vulgare Latr. (crustacean, isopod, oniscoid) harboring a feminizing bacteria

In thelygenous lines of Armadillidium vulgare, neo-females and intersex males (iM) with feminizing symbiotic bacteria are not masculinized by an extract from iM androgenic gland, which, however, masculinizes bacterialess genetic females. Injection of iM hemolymph extract masculinizes these genetic females. This indicates that androgenic hormone is present in iM hemolymph. Lack of androgenic hormone activity in thelygenous lines is supposed to result from the action of bacteria on the androgenic hormone receptors. Since a temporary recovery of the male differentiation of iM can be induced by implantation of different parts of central nervous system, bacteria effect is probably indirect, through an action on a neurosecretory system, perhaps one of those controlling the functioning of the androgenic gland.     

Estrone and estradiol participation during exogenous vitellogenesis in the female rainbow trout, Salmo gairdneri

Ovarian steroids were treated for their ability to induce vitellogenin synthesis in the liver of the rainbow trout, Salmo gairdneri. These steroids were chosen because they had been reported to induce vitellogenin in the plasma of teleosts, i.e., estrone, estradiol, and testosterone, or because they were synthesized during vitellogenesis, as was the case with the above steroids and androstenedione and 17α-hydroxyprogesterone. Immature trout were ovariectomized and injected daily for 7 days. The administered doses of steroid were 100, 250, and 500 ng/g body wt. Only estradiol and estrone induced vitellogenin in the plasma; estrone had about 5% of the potency of estradiol. A dose-effect curve was determined for estradiol in the range from 25 to 1000 ng/g body wt. A maximal amount of 7 mg vitellogenin/ml plasma was found following the administration of a dose of 250 ng/g estradiol. Vitellogenin was not present in the plasma of animals treated with saline, nor could it be detected in the liver, not even after the administration of estradiol. Estradiol administration increased the total plasma protein concentration from 35 to maximally 51 mg/ml and decreased the total piver protein concentration from 163 to minimally 118 mg/g. The relative weight of the liver increased from 12.9% to maximally 22.4%. Vitellogenin was not detected in the liver of any of the experimental animals, indicating a low storage and rapid secretion of vitellogenin. The other steroids influenced some of the variables, but never was the total pattern of effect comparable to that of estradiol. Estradiol is found to be the ovarian steroid that physiologically regulates the synthesis of vitellogenin in the liver of the rainbow trout; estrone is less active. Experiments undertaken to determine the effects of the combined presence of estrone and estradiol revealed that estrone was capable of boosting the vitellogenic effect of estradiol when compared to the induced vitellogenin levels following treatment with a combination of testosterone and estradiol. The vitellogenin concentrations induced by a certain dose of one of the combinations of estrone and estradiol approximated the concentrations induced by the same dose of estradiol alone. This effect was independent of the ratio in which estrone and estradiol were administered. These findings could not be explained by conversions of estrone into estradiol or by the vitellogenic activity of estrone alone. The estrone, estradiol, and vitellogenin concentrations in the plasma of the experimental animals were in the same range as determined previously in untreated mature vitellogenic females. Vitellogenin and estradiol levels were found to correlate in experimental animals treated with estradiol (r = 0.627; N = 20). This was not the case in animals treated with combinations of estrone and estradiol. In these animals, however, the sum of estrone and estradiol levels in the plasma correlated with vitellogenin levels (r = 0.724; N = 42). Vitellogenin was not detected in the liver of any of the experimental animals, which indicates a low storage and rapid secretion of vitellogenin. The importance of viewing the sum of estrone and estradiol plasma levels in connection to physiological studies of the regulation of exogenous vitellogenesis in the rainbow trout is discussed.     

Androgenic gland hormone is a sex-reversing factor but cannot be a sex-determining factor in the female crustacean isopods Armadillidium vulgare.

Sex reversal of female isopods, Armadillidium vulgare, has been induced by implantation of the androgenic gland (AG) into individuals after the initiation of morphological sex differentiation. The focus of the present study is to examine whether female gonads are reversed by the androgenic gland hormone (AGH) during the sexually undifferentiated period through postembryonic development in A. vulgare. Instead of injections of AGH, three AGs were implanted into each genetic female at various developmental stages to induce sex reversal. Before implantation fresh AGs were treated with ethanol to stop AGH synthesis, but then still contained AGH. These AGs have been referred to as ethanol-treated AGs (t-AGs). Development of a testis was used as an indicator of gonadal sex reversal. The gonads of genetic females were transformed into testes by implantations of t-AGs during the sex differentiation period. However, when genetic females received implants at sexually undifferentiated stages, development of their gonads was not reversed in the male direction. These results suggest that after the onset of gonadal sex differentiation, AGH is a sex-reversing factor that can turn a female gonad into a male gonad. AGH cannot be a sex-determining factor in female A. vulgare, as undifferentiated gonads of genetic females are not sex reversed by the hormone.     

Social exploitation of vitellogenin

Vitellogenin is a female-specific glucolipoprotein yolk precursor produced by all oviparous animals. Vitellogenin expression is under hormonal control, and the protein is generally synthesized directly before yolk deposition. In the honeybee (Apis mellifera), vitellogenin is not only synthesized by the reproductive queen, but also by the functionally sterile workers. In summer, the worker population consists of a hive bee group performing a multitude of tasks including nursing inside the nest, and a forager group specialized in collecting nectar, pollen, water, and propolis. Vitellogenin is synthesized in large quantities by hive bees. When hive bees develop into foragers, their juvenile hormone titers increase, and this causes cessation of their vitellogenin production. This inverse relationship between vitellogenin synthesis and juvenile hormone is opposite to the norm in insects, and the underlying proximate processes and life-history reasons are still not understood. Here we document an alternative use of vitellogenin by showing that it is a source for the proteinaceous royal jelly that is produced by the hive bees. Hive bees use the jelly to feed larvae, queen, workers, and drones. This finding suggests that the evolution of a brood-rearing worker class and a specialized forager class in an advanced eusocial insect society has been directed by an alternative utilization of yolk protein.     

Effects of androgenic gland ablation on male primary and secondary sexual characteristics in the Malaysian prawn, Macrobrachium rosenbergii (de Man) (Decapoda,Palaemonidae), with first evidence of induced feminization in a nonhermaphroditic decapod

Male Macrobachium rosenbergii (de Man), categorized into three developmental stages according to possession of male gonopore complexes, appendixes masculina, and mature chelipeds, were subjected to bilateral androgenic gland ablation (andrectomy). Andrectomized males initially lacking appendixes masculina and mature chelipeds do not develop them; those initially possessing appendixes masculina and mature chelipeds do not lose them. Growth rate of the appendixes masculina, however, is reduced. Andrectomized males are unable to redifferentiate amputated or accidentally lost appendixes masculina and mature chelipeds. Instead, the appendages regenerate as immature forms. Andrectomized M. rosenbergii possess atrophied testes and vasa deferentia. Histological sections of the testes revealed a reduction in number of spermatogenic lobules. Spermatogonia and primary spermatocytes were commonly found; secondary spermatocytes and spermatids were found only occasionally. Andrectomy appears to inhibit but not prevent meiosis. Feminization occurred in five andrectomized males. Complete feminization, including initiation of oogenesis and development of oviducts and female gonopores, occurred in males andrectomized in the youngest developmental stage. Males andrectomized in later developmental stages were either partially feminized or not feminized. Reimplantation of androgenic glands reverses the effects of andrectomy. Remasculinization, as evidenced by differentiation of appendixes masculina, was noted after the first postimplantation molt. The testes of remasculinized males were normal in gross anatomy and spermatogenic ability. The data indicate a uniform function for the androgenic glands with respect to male primary and secondary sexual characteristics among the Amphipoda, Isopoda, and Decapoda. Past theories on androgenic gland function are modified in light of the present information.     

Ovarian stimulation of juvenile hormone biosynthesis in the viviparous cockroach, Diploptera punctata

A direct radiochemical assay for juvenile hormone (JH) biosynthesis by the corpora allata (CA) showed that the cyclic changes in biosynthesis associated with the gonadotrophic cycle were abolished in the absence of ovaries whether or not the CA were innervated. In ovariectomized animals denervated CA synthesized JH at slightly higher rates than did innervated CA. However, the low rates of JH synthesis in ovariectomized females were sufficient to result in accumulation of vitellogenin in the hemolymph to levels twice those of normal females. Implantation of one ovary 1 week after ovariectomy resulted in a cycle of JH biosynthesis qualitatively similar to that observed during a normal gonadotrophic cycle. Implantation of one ovariole did not result in a detectable cycle of JH biosynthesis whereas normal rates of JH synthesis were observed after implantation of three to six ovarioles. Implantation of four ovaries resulted in a cycle of CA activity more attenuated than that observed in the presence of one ovary. Nonetheless all four ovaries sequestered a normal quantity of vitellin.     

Induction of vitellogenin in primary monolayer cultures of cockerel hepatocytes.

A primary monolayer culture system from cockerel hepatocytes was established. The cultures synthesize and secrete proteins that comigrate with authentic serum proteins on polyacrylamide gels and are found in the same relative abundance. Addition of estradiol increased the synthesis of apoprotein B, found in very low density lipoprotein, under all culture conditions. Vitellogenin synthesis could not be induced directly by estradiol. However, when serum was obtained from cockerels injected with estradiol 4 days before blood collection and included in the culture medium, the cultures secreted a protein identified immunologically as vitellogenin by affinity chromatography. Furthermore, addition of growth hormone or prolactin to cultured cockerel hepatocyte monolayers resulted in the synthesis and secretion of a polypeptide that comigrates with authentic vitellogenin on polyacrylamide gels.     

Evidence for control of vitellogenin synthesis by an ovarian hormone in Orchestia gammarella (Pallas), crustacea; Amphipoda

Females of the amphipod crustacean Orchestia gammarella were bilaterally ovariectomized. Synthesis of vitellogenin completely ceased 5 to 8 days after the operation and was restored by an ovarian transplant. Based on these results, the existence of an ovarian hormone is postulated which controls vitellogenin synthesis. We propose to call this hormone “vitellogenin stimulating ovarian hormone” (VSOH).     

Sex-reversal of male Armadillidium vulgare (Isopoda, Malacostraca, Crustacea) following andrectomy and partial gonadectomy

Evidence is presented for the first time for the complete sex-reversal of genetic males of the terrestrial isopod Armadillidium vulgare into functional females following partial gonadectomy. Fifth-instar males were used. The anterior half of their rudimentary reproductive organs along with the attached androgenic glands were removed. Six months later these animals formed oostegites and laid eggs, and when bred with normal males, produced only male offspring. These results show that in the absence of the androgenic gland, female sex characteristics differentiate and oogenesis occurs in genetic males.     

Protein accumulation underlying lifespan extension via ovariectomy in grasshoppers is consistent with the disposable soma hypothesis but is not due to dietary restriction

Reduced reproduction extends lifespan in many experimental animals, but the mechanism by which this occurs is unclear. The disposable soma hypothesis suggests that when reproduction is reduced, more nutrients are allocated to the soma and lifespan is extended. Alternatively, the reproductive tissues or the process of reproduction may have a direct (i.e., non-nutritional) negative effect on lifespan. We used ovariectomized grasshoppers to examine the effects of reduced reproduction throughout the lifespan at the physiological level. We focused on protein, the limiting nutrient for egg production. Ovariectomized females lived significantly longer than sham females. Because both groups ingested similar amounts, the effect was independent of dietary restriction. Despite this, ovariectomized females gained less body mass than sham females. Ovariectomized grasshoppers produced the egg yolk-precursor protein vitellogenin. At the time sham females laid their first clutch, cumulative reproductive protein was similar in ovariectomized and sham females. By advanced ages, however, ovariectomized females had produced about five-fold less cumulative reproductive protein than sham females. In contrast, old ovariectomized females had at least two-fold more hemolymph storage protein. These results are consistent with ovariectomy extending lifespan in part via enhanced protein allocation to storage at the expense of reproduction.     

Tissue-specific sensitivity of chromatin and the vitellogenin gene to micrococcal nuclease after continuous exposure of salmon (Salmo salar) to 17 beta-estradiol.

Smoltified Atlantic salmon (Salmo salar), 2 years old and weighing 217 +/- 13 g, were treated for 2 weeks with 17 beta-estradiol containing silastic capsules implanted intraperitoneally. Control fish received empty capsules. Vitellogenin, present in the blood of both groups of fish, was enhanced by estradiol treatment. Nuclei were isolated from liver, blood cells, and brain and incubated with increasing concentrations of micrococcal nuclease (EC 3.1.31.1). In liver there were more mononucleosomes as a percentage of total chromatin in hormone-treated than in control fish. Using vitellogenin cDNA as a probe the highest hybridization signals were seen when 2 to 4% of the chromatin was digested to mononucleosomes. In blood cell and brain nuclei independent of the extent of the chromatin released the hybridization signals remained low. The expression of the vitellogenin gene in immature females was potentiated by exogenous estradiol to give increased micrococcal nuclease sensitivity of the chromatin without enhancement of the hybridization level. Micrococcal nuclease digestion and hybridization of the vitellogenin gene demonstrated that the hepatic specificity of vitellogenin synthesis is manifested as structural modulations of the chromatin containing the vitellogenin gene.     

Ecdysteroid hormone titer and its relationship to vitellogenesis in the soft tick, Ornithodoros moubata (Acari: Argasidae)

A blood meal is required for reproduction in most argasid female ticks. The blood meal appears to stimulate an organ in the posterior end to produce a fat body stimulating factor (FSF), which is thought to be an ecdysteroid, to induce vitellogenin (Vg) synthesis. In this study, the relationship of vitellogenesis and ecdysteroids was investigated by measuring Vg and ecdysteroid titers while observing oocyte development and oviposition in mated and virgin females. Oviposition occurred from day 10 after engorgement in mated females and continued up to 40-50 days, whereas egg maturation and oviposition did not occur in virgin females. Vg titers in the hemolymph peaked on day 6 after engorgement and subsequently declined in mated females. Interestingly, Vg synthesis occurred and ovarian development progressed to the development of early vitellogenic oocytes in virgin females but oocyte maturation and oviposition did not occur. Topical application of ecdysteroids induced oviposition in fed virgin females indicating that ecdysteroids may induce oviposition. Concentrations of ecdysteroids for 20 days after engorgement revealed several peaks in mated female whole body extracts, but no peaks in virgin female extracts. In the hemolymph of only mated females, ecdysteroid titers showed two peaks that followed the early peak of ecdysteroids in the whole body on day 4 and 6 after engorgement. In addition, ecdysteroids in the reproductive tissues increased with the development of the ovary in mated females and this increase coincided with the latter peaks of the whole body. These observations indicate that physiological elevation of ecdysteroids accelerate Vg synthesis, and may induce egg maturation and stimulate oviposition in fed mated Ornithodoros moubata females.     

Changes in ecdysteroid and juvenile hormone titers in the hemolymph of Galleria mellonella larvae and pupae

The variations in circulating ecdysteroids and juvenile hormones (JH) in Galleria, from the end of the antepenultimate larval stage until emergence of adults, have been determined. The two hormonal families were extracted separately from the same hemolymph sample and quantified by two radioimmunoassays. Juvenile hormone RIA activity was about 35 nM in larvae of the antepenultimate and penultimate stages. It dropped before each molt and increased thereafter. Moreover, it gradually decreased during the last larval instar. In pupae, it was generally low, but it rose drastically during the late pupal development and in young adults. This rise was very much higher in females than in males. Three different RIA-active compounds were found; they were assumed to be JH-I, JH-II, and JH-III according to their retention times in HPLC. The three compounds were present in almost equal concentration in larvae of the penultimate stage: JH-I predominated, however, during the last larval instar. In late pupae, the main hormone was JH-III both in males and in females. There is no clear relationship between ecdysteroid and juvenile hormone changes, except for a female-specific ecdysteroid rise which coincides with the juvenile hormone release in late pupae. This double hormonal stimulation can be involved in the regulation of vitellogenin synthesis and deposition in oocytes.     

Sexual differentiation traits in functional males with female genital apertures (male symbol fga) in the woodlice Armadillidium vulgare Latr. (Isopoda, Crustacea)

This study reports the results of examination of the gonadal morphology and ultrastructural features of the androgenic hormone (AH)-producing androgenic gland cells of laboratory stocks of functional male woodlice, Armadillidium vulgare, with female genital apertures ( male symbol fga), with and without experimentally induced infections of the sex-ratio-distorting endobacterial parasite, Wolbachia. Males ( male symbol fga) have been reported in wild populations containing individuals infected with this maternally transmitted sex-ratio-distorting parasite. We report a reduction of testicular segment (utricle) number, androgenic gland cell hypertrophy, and electron-dense ultrastructural cytological features in male symbol fga males. The presence of the Wolbachia parasite had no effect on the features we examined. These results suggest that male symbol fga males are produced as the result of a delayed expression/action of the male sex-determining AH which causes a "lag-phase" delay in male differentiation in genetic males and is not due to the presence, in genetic females, of a hypothetical, epigenetic "M" gene as suggested by Rigaud and Juchault. Our results favor the interpretation of males as true genetic (ZZ) males in which the delayed AH action appears to involve cellular AH trafficking pathways which may be controlled by an impaired autosomal gene responsible for AH action.     

Effect of androgenic gland ablation on morphotypic differentiation and sexual characteristics of male freshwater prawns, Macrobrachium rosenbergii

Mature males of the freshwater prawn, Macrobrachium rosenbergii (de Man), may change from one to another morphotype, according to a set sequence. Small males may develop into orange-claw males and orange-claw males into dominant blue-claw males. Each of the three morphotypes demonstrates distinctive reproductive behavior and secondary sexual characteristics. The role of the androgenic gland in this morphotypic transformation was examined experimentally by bilateral androgenic gland ablation (andrectomy) of small males and orange-claw males. For andrectomy initiated in the small male morphotype, transformation to the next morphotype was permitted (orange-claw), but subsequent transformation to the blue-claw morphotype was blocked. Andrectomy of orange-claw males did not prevent transformation into the blue-claw. Andrectomy on both small and orange-claw males caused disappearance of the genital papillae and atrophy of the sperm ducts and testes. The growth rates of the andrectomized small and orange-claw males were significantly lower than those of the unoperated and sham-operated controls. We conclude that androgenic gland factors control not only the differentiation of male secondary sexual characteristics but also morphotypic differentiation. Bioassays based on the results of this study will be instrumental in the characterization of such a factor(s).