Nuclear binding sites for juvenile hormone III in the male accessory reproductive glands of Melanoplus sanguinipes

Summary

We have identified a potential nuclear juvenile hormone (JH) receptor in the long hyaline tubules (LHT), part of the male accessory reproductive gland (MARG) of M. sanguinipes. The MARG was incubated in vitro with [³H]JH III, and the distribution of the [³H]JH III among the cellular fractions of the LHT was determined. Some 37±4% of the radioactivity was associated with the crude nuclear pellet, while the cytosolic, microsomal and mitochondrial fractions contained 30±3%, 23±2% and 10±1%, respectively. The bound JH III was measured in nuclear extracts of LHT from males up to 15 days post-eclosion. These results revealed that JH binding increased in an age-dependent manner up to day 7, then levelled off to day 12, to increase again on day 14. The nuclear-binding component in the LHT had a very strong affinity for JH III, with a K_D value of 0.8 nM. Our observations are considered in relation to the potential site and mode of action of JH.     

Growth of the male accessory gland in adult locusts: Roles of juvenile hormone,JH esterase,and JH binding proteins

The participation of juvenile hormone (JH) in the regulation of growth and protein synthesis in the accessory reproductive gland of male Locusta migratoria has been investigated. After elimination of endogenous JH with ethoxyprecocene, the accessory gland failed to grow, but growth was restored by a single application of the JH analog, pyriproxyfen. Pyriproxyfen appeared to stimulate total protein synthesis by 3 h, with a significant effect by 12 h, in contrast to 24 h observed in fat body. The dose curve for stimulation of protein synthesis 12 h after applying pyriproxyfen gave an ED₅₀ of 0.1 μg; the dose curve for gland growth at 72 h was biphasic, with steps at about 0.01 μg and 10 μg, suggesting two phases in JH action. SDS-PAGE analysis showed several components that were stimulated by pyriproxyfen, the effect being strongest in an 11 kDa band. A 5 kDa component was enhanced in the soluble and reduced in the particulate fraction after precocene treatment. The accessory gland contained JH esterase activity at levels about 100 times those in fat body or hemolymph, and was higher in precocene treated locusts. Binding activity for [³H]10R -JH III was high in cytosolic and nuclear fractions, and was identified immunologically as due to the previously described hemolymph JH binding protein. The results indicate that the mode of action of JH in the accessory gland may differ from that in the fat body. The presence of intracellular JH binding protein suggests a direct action of JH within the gland, that may be modulated by JH esterase. © 1995 Wiley-Liss, Inc.     

Protein production in components of the accessory gland complex of male Melanoplus sanguinipes (Insecta: Orthoptera)

Protein production during sexual maturation or after allatectomy (followed by juvenile hormone replacement therapy) has been examined in the long hyaline glands, short hyaline glands, white glands, and seminal vesicles, which make up the accessory gland complex of male Melanoplus sanguinipes. During maturation, the amount of protein in the long hyaline glands increases about 14-fold, and in each of the other components between 5- and 6-fold. Most protein accumulates between days 3 and 5, and this is reflected in high levels of incorporation of radiolabelled leucine in this period.

The components show differential sensitivity to the effects of allatectomy. After this operation, the protein content of, and incorporation of radiolabel into, the long hyaline glands remain near the day 0 level. In the white glands and short hyaline glands, allatectomy also has a marked, though less severe, effect on protein synthesis and accumulation. The seminal vesicles are least affected by allatectomy and continue to accumulate protein (though more slowly) to about 60% of the normal level by day 10. Juvenile hormone compounds applied topically to allatectomized insects on day 2 restored the ability of the gland components to accumulate proteins, though to differing degrees. JHI is the most effective compound, stimulating synthesis and accumulation of protein to near normal levels by day 10, whereas application of JHIII or Stauffer's synthetic JH led to only partial restoration of protein synthesis in the glands.     

Juvenile hormone stimulation of sperm activator production in male monarch butterflies

A sperm activating substance is demonstrable in the male Monarch butterfly ejaculatory duct, but only during reproductively active stages. Experiments using neck-ligatures and allatectomies, with or without injections of juvenile hormone isomers, show that the post-eclosion production of sperm activator is stimulated by juvenile hormone.     

烟夜蛾雄蛾性附腺因子对雌蛾性信 息素合成的抑制作用

烟夜蛾Helicoverpa assulta处女蛾在交配后1 h,其性信息素滴度即显著降低,72 h内未见恢复。生测结果表明,烟夜蛾性信息素合成抑制因子主要来源于雄蛾性附腺。不同日龄雄蛾性附腺提取物的抑制活性无显著差异。光暗期对其活性具显著影响,暗期中雄蛾的性附腺物质对雌蛾性信息素合成具有较强抑制作用,而光期中雄蛾的性附腺物质不具抑制活性。在暗期的不同时间处理,对处女蛾性信息素合成的抑制作用无显著差异。雄蛾性附腺提取物对雌蛾性信息素合成的抑制作用与注射剂量有明显的相关性,0.2 ME（雄蛾当量）是产生显著抑制作用的最小剂量。对交配雌蛾注射性信息素生物合成激活神经肽（PBAN）提取物后,其性信息素合成又可恢复,这说明雌蛾交配后,性信息素滴度降低的原因是由于缺少了PBAN的调控。     

Juvenile hormone synthesis by ring glands of the blowfly Lucilia cuprina

The major radiolabelled product released from ring gland and brain-ring gland complexes of third instar larval and pre-pupal stages of the sheep blowfly Lucilia cuprina upon incubation with L-[methyl-³H]methionine corresponded to one diastereomer of juvenile hormone III bisepoxide (JHB₃). Endocrine glands incubated with the juvenile hormone precursor 2E,6E-farnesoic acid released increased quantities of JHB₃, together with significant amounts of juvenile hormone III but not the isomeric methyl 2E-6,7-epoxyfarnesoate. Synthesis of JHB₃ was developmentally and neurally regulated. Ring glands and brain-ring gland complexes from third instar larvae released more JHB₃ than comparable preparations from pre-pupae, while isolated corpus allatum segments of the gland were more active than intact brain-gland complexes. These results reinforce the emerging status of JHB₃ as the characteristic juvenile hormone of dipteran insects. Arch. Insect Biochem. Physiol. 34:239–253, 1997. © 1997 Wiley-Liss, Inc.     

Mating in Heliothis virescens: Transfer of juvenile hormone during copulation by male to female and stimulation of biosynthesis of endogenous juvenile hormone

Studies were undertaken to determine whether adult males of Heliothis virescens transfer juvenile hormone (JH) to females during copulation, and an in vitro radiochemical assay was used to determine whether mating causes an allatotropic effect, i.e., stimulation of JH biosynthesis by corpora allata (CA). In vitro, CA from 3-day-old mated females synthesized and released approximately 2.5 times total JH as that of CA from comparably aged virgin females. Of the homologues, JH II exhibited significant increase in mated females; JH I also increased but not significantly. JH III remained similar to that of virgin females. This is the first demonstration of an allatotropic effect of mating in moths. In contrast to the female, CA of virgin males did not produce any JH, but accessory sex glands (ASG) in 3-day-old males synthesized small amounts of JH. Immediately after adult emergence, male ASG contained approximately 1.5 ng JH I and II, which increased by 12 h after emergence and remained at this high level up to 54 h after emergence. JH III was barely detected in ASG. JH in ASG of mated male immediately after uncoupling was depleted almost completely, and 24 h later recovered to levels comparable to that of 54-h-old virgin male. Virgin female bursa copulatrix did not contain any JH, but mated female bursa, immediately after uncoupling, had JH at levels comparable to that observed in virgin male ASG. By 6 h after uncoupling, JH levels decreased dramatically in mated female bursa. These data suggest the transfer of JH to females by the male. Arch. Insect Biochem. Physiol. 38:100–107, 1998. © 1998 Wiley-Liss, Inc.     

The host-seeking inhibitory peptide, Aea-HP-1, is made in the male accessory gland and transferred to the female during copulation

Male accessory glands (MAGs) of insects are responsible for the production of many of the seminal fluid proteins and peptides that elicit physiological and behavioral responses in the post-mated female. In the yellow fever mosquito, Aedes aegypti, seminal fluid components are responsible for stimulating egg production, changing female behavior away from host-seeking toward egg-laying and mating refractoriness, but hitherto no behavior-modifying molecule from the MAGs has been structurally characterized. We now show using mass spectrometry and HPLC/ELISA that the MAG is a major site of synthesis of the biologically active decapeptide, Aea-HP-1 (pERPhPSLKTRFamide) that was first characterized by Matsumoto and colleagues in 1989 from mosquito head extracts and shown to have host-seeking inhibitory properties. The peptide is localized to the anterior portion of the MAG, occurs at high concentrations in the gland and is transferred to the female reproductive tract on copulation. Aea-HP-1 has a pyroglutamic acid at the N-terminus, an amidated carboxyl at the C-terminus and an unusual 4-hydroxyproline in position 4 of the peptide. The structure of the peptide with its blocked N- and C-termini confers resistance to metabolic inactivation by MAG peptidases; however the peptide persists for less than 2h in the female reproductive tract after copulation. Aea-HP-1 is not a ligand for the mosquito sex peptide/myoinhibitory peptide receptor. A. aegypti often mate close to the host and therefore it is possible that male-derived Aea-HP-1 induces short-term changes to female host-seeking behavior to reduce potentially lethal encounters with hosts soon after insemination.     

Juvenile hormone regulation of male accessory gland activity in the red flour beetle, Tribolium castaneum

Male accessory gland proteins (Acps) act as key modulators of reproductive success in insects by influencing the female reproductive physiology and behavior. We used custom microarrays and identified 112 genes that were highly expressed in male accessory glands (MAG) in the red flour beetle, Tribolium castaneum. Out of these 112 identified genes, 59 of them contained sequences coding for signal peptide and cleavage site and the remaining 53 contained transmembrane domains. The expression of 14 of these genes in the MAG but not in other tissues of male or female was confirmed by quantitative real-time PCR. In virgin males, juvenile hormone (JH) levels increased from second day post adult emergence (PAE), remained high on third day PAE and declined on fourth day PAE. The ecdysteroid titers were high soon after adult emergence but declined to minimal levels from 1 to 5 days PAE. Feeding of juvenile hormone analog, hydroprene, but not the ecdysteroid analog, RH-2485, showed an increase in size of MAGs, as well as an increase in total RNA and protein content of MAG. Hydroprene treatment also increased the expression of Acp genes in the MAG. RNAi-mediated knock-down in the expression of JHAMT gene decreased the size of MAGs and expression of Acps. JH deficiency influenced male reproductive fitness as evidenced by a less vigor in mating behavior, poor sperm transfer, low egg and the progeny production by females mated with the JH deficient males. These data suggest a critical role for JH in the regulation of male reproduction especially through MAG secretions.     

蜱雄性附腺分泌物及其功能的研究进展

交配是蜱类繁殖过程的关键环节,诱发雌蜱发生一系列的生理变化,并最终产卵。蜱雄性附腺分泌物在交配过程中发挥着重要作用,具有保护、活化精子,促进受精、卵巢发育和卵黄发生的功能,并对雌蜱的生殖生理行为等产生影响,如诱导雌蜱快速吸血和加速产卵。本文在简要分析蜱雄性附腺结构和分泌物生化特性基础上,系统阐述了蜱雄性附腺分泌物中各种功能因子的研究现状,着重论述其在精子获能、诱导雌蜱吸血、促进雌蜱卵巢发育和卵黄发生等方面的进展,并对未来研究提出了展望,以期为此领域的研究拓展思路。     

Male accessory gland substances and the control of sexual receptivity in female Culex tarsalis

ABSTRACT. Homogenates of accessory glands from male Culex tarsalis Coquillet (Diptera; Culicidae) were fractionated using gel filtration column chromatography. A component of low molecular weight capable of suppressing female sexual receptivity was obtained and partially characterized. Proteins present in homogenates of isolated accessory glands formed aggregates with other proteins in extracts of whole mosquitoes. Consequently, previous reports of the molecular weight of biologically active components in homogenates from accessory glands from other mosquito species may be inaccurate.
When female Cx. tarsalis were mated with ³H-leucine labelled males. radioactivity could be found throughout the body of the female 24 h after mating. Autoradiography showed that the radioactivity was incorporated into the cells of various organs, and was not specifically localized in a particular target tissue. Approximately 30% of the total radioactivity transferred to the female was found in the head and thoracic regions, and proteins from the head had a specific activity twice that of the thoracic proteins. Bioassay of isolated body parts from mated and virgin females showed that injections of head tissues from mated females into virgin females suppressed sexual receptivity. These results suggest that the head is involved in the physiological mechanisms resulting in refractory mating behaviour.     

Juvenile hormone esterases in diapause and nondiapause larvae of the European corn borer, Ostrinia nubilalis

Haemolymph levels of juvenile hormone esterase, 1-naphthyl acetate esterase, and juvenile hormone were measured in synchronously staged diapause and nondiapause larvae of the European corn borer, Ostrinia nubilalis. Juvenile hormone esterase levels were monitored using juvenile hormone I as a substrate while juvenile hormone titres were measured with the Galleria bioassay. Haemolymph of nondiapause larvae showed two peaks of juvenile hormone hydrolytic activity: one near the end of the feeding phase and a smaller one just prior to pupal ecdysis. These peaks of enzyme activity correlated well with the low levels of haemolymph juvenile hormone. Juvenile hormone titres were high early in the stadium then showed a second peak during the prepupal stage coinciding with low esterase activity. Diapause haemolymph had peak juvenile hormone esterase activity nearly 4 times the nondiapause level, reaching a peak near the end of the feeding phase. Diapause-destined larvae retained high juvenile hormone titres even during the rise of the high esterase levels. 1-naphthyl acetate esterase levels did not correlate with the juvenile hormone esterase levels in either the diapause or nondiapause haemolymph. High levels of 1-naphthyl acetate esterase activity were associated with moulting periods.     

Ecdysterone antagonism,mimicry, and synergism of juvenile hormone action on the Monarch butterfly reproductive tract

Previous research on Monarch butterflies has shown that juvenile hormone (JH) stimulates the development of the ovary and certain reproductive glands of both sexes. Ecdysterone injections into intact Monarchs demonstrate that low doses of this hormone inhibit ovarian development, and higher doses stimulate the male and female reproductive glands. In addition, experiments using neckligatured adults show that ecdysterone stimulates the reproductive glands of both sexes, in the apparent absence of JH, with the most pronounced effect being observed on the female colleterial gland. Other studies with neck-ligatured animals demonstrate that ecdysterone also synergizes with JH on the female gland and all three male glands. The feasibility of using Monarch reproductive glands for studies on the mode of action and interaction of JH and ecdysterone, and the possibility of a rôle of ecdysterone in the normal regulation of Monarch oögenesis, are discussed.     

The nature and titre of juvenile hormone in the Colorado potato beetle, Leptinotarsa decemlineata

This paper describes the nature and titre of juvenile hormone at different developmental stages of the Colorado potato beetle, Leptinotarsa decemlineata Say, determined by a selective mass-spectroscopic detection technique, High levels of juvenile hormone III were observed in long-day beetles, whereas low titres occurred in pre-diapause and diapause adults. The level of juvenile hormone III in larvae was low compared with reproductive adults, whereas hardly any juvenile hormone could be detected in pupae. We were not able to detect juvenile hormones I or II. The results agree well with previously reported data using the Galleria bioassay.     

Determinants of sperm transfer in the scorpionfly Panorpa cognate: male variation, female condition and copulation duration

Recent studies suggest that sperm production and transfer may have significant costs to males. Male sperm investment into a current copulation may therefore influence resources available for future matings, which selects for male strategic mating investment. In addition, females may also benefit from actively or passively altering the number of sperm transferred by males. In the scorpionfly Panorpa cognata, the number of sperm transferred during copulation depended on copulation duration and males in good condition (residual weight) copulated longer and also transferred more sperm. Moreover, sperm transferred and stored per unit time was higher in copulations with females in good condition than in copulations with females in poor condition. Males varied greatly and consistently in their sperm transfer rate, indicative of costs associated with this trait. The duration of the pairing prelude also varied between males and correlated negatively with the male's sperm transfer rate, but no other male character correlated significantly with male sperm transfer rate. The results are consistent with strategic mating effort but sperm transfer could also be facilitated by the physical size of females and/or females in good condition may be more cooperative during sperm transfer.     

Evidences of the extraglandular origin of two secretory proteins in the male accessory reproductive glands of Chilo partellus (Pyralidae,Lepidoptera, Insecta)

Summary

The male accessory reproductive glands (MARG) play an important role in contributing proteins for the formation of the spermatophore, a structure that transports sperm from the male to the female. Several studies have shown that these proteins could be either of intraglandular or extraglandular origin. SDS-PAGE analysis revealed proteins in the adult MARG of Chilo partellus, which comigrate with the larval hemolymph proteins (LHP). Ouchterlony's immunodiffusion showed that the antiserum raised against MARG extract cross-reacts with the proteins present in the adult hemolymph and fat body. Immunoprecipitates of labelled hemolymph proteins with anti-LHP or anti-MARG proteins reveal two polypeptides that comigrate with LHPs. Thus, there appear to be some proteins of extraglandular origin in the MARG of C. partellus, and the LHP could belong to this category.     

Juvenile hormone titres and metabolism during starvation-induced supernumerary larval moulting of the tobacco hornworm, Manduca sexta L.

When tobacco hornworm (manduca sexta) larvae are starved for 5 days immediately after ecdysis to the 5th instar, then fed normal diet, they undergo a supernumerary moult instead of metamorphosis. During starvation the titre of juvenile hormone in the haemolymph increased to a maximum of 3 ng juvenile hormone I equivalents/ml (determined by the black Manduca larval bioassay) on the fourth day of starvation, then began a decline which continued through the subsequent feeding period. The changes in juvenile hormone titre were not attributable to changes in haemolymph volume during starvation (only a 5% decrease) and subsequent feeding. During starvation the esterase activity of the haemolymph declined 4-fold with a 2-fold larger decrease in the DFP-insensitive, presumably juvenile hormone specific, esterase activity. Both the total and the juvenile hormone-specific esterase activity then increased as a function of larval weight during the subsequent feeding period. As growth was slow in the prolongedly starved larvae, sufficient juvenile hormone was present at the time of prothoracicotropic hormone (PTTH) and ecdysteroid release at the beginning of the fourth day of feeding to prevent metamorphosis.     

Juvenile hormone acid methyl transferase is a key regulatory enzyme for juvenile hormone synthesis in the Eri silkworm, Samia cynthica ricini

During the period of adult emergence in the Eri silkworm, Samia cynthia ricini, the corpora allata (CA) are apparently reactivated in females, but not males. This creates a significant sexual dimorphism in juvenile hormone (JH) synthesis by CA. To determine the underlying molecular mechanisms in this process, we cloned cDNAs of two enzymes involved in the JH synthesis pathway: 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) and juvenile hormone acid methyl transferase (JHAMT). Both Samcri-HMGR and -JHAMT mRNAs were detected in CA almost exclusively. However, their expression patterns were different from each other. During the period of adult emergence, Samcri-HMGR was expressed in CA at a constantly high level suggesting it plays little role for the regulation of JH synthesis. In contrast, the patterns of both Samcri-JHAMT mRNA level and enzyme activity were closely correlated with the patterns of JH synthesis, CA reactivation, and sexual dimorphism of JH synthesis. In addition, JHAMT mRNA levels were paralleled JH synthesis in the fifth-instar larvae of S. cynthia ricini and the pharate adults of the silkworm Bombyx mori. We infer from these results that JHAMT is a key regulatory enzyme for JH synthesis in the Eri silkworm.