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.     

Long hyaline gland discharge and multiple spermatophore formation by the male grasshopper, Melanoplus sanguinipes

ABSTRACT. Discharge from the male accessory reproductive gland (ARG) by the male grasshopper, Melanoplus sanguinipes (Fabr.), has been studied by assay of a characterized product, a glycoprotein LHPI, and the rate of formation of spermatophores. LHPI is an exclusive long hyaline gland (a prominent ARG tubule type) product whose discharge is symmetrical from the bilaterally paired glands. LHPI forms 65% of a viscous secretion that is discharged concomitantly with the spermatophores. Though low ARG reserves in 5-day-old males limit both the number of spermatophores formed and LHPI discharged in copulations ≤6 h, this appears to be the result of shorter copulations. The number of spermarophores formed in 1 h was not impaired by general depletion of ARG protein (by repeated copulations) or by selective depletion of long hyaline gland protein (by unilateral and bilateral long hyaline gland removal), though these manipulations reduced LHPI discharge by 22%, 44% and 100%, respectively. However, 56% of spermatophores formed by males with the long hyaline gland bilaterally ablated failed to uncoil properly. These results indicate LHPI and/or other long hyaline gland proteins may act as lubricants. Unlike spermatophore formation and LHPI discharge, which increased steadily up to 90–120 min then levelled off, transfer of radiolabelled male ejaculate to the spermatheca was very variable. In 90 min copulations, only 1% of the total radioactivity (representing c. 5 μg protein) lost from the ARG complex was transferred to the spermatheca. The importance of male-derived protein in vitellogenesis is discussed.     

Corpus al latum regulation of copulatory behaviour in the male grasshopper, Melanoplus sanguinipes

Abstract The relationship between corpus allatum (CA) regulation of copulatory behaviour and the CA's influence on the development of the accessory reproductive glands was studied in the male grasshopper, Melanoplus sanguinipes (Fabr.). Allatectomy slowed the development of copulatory behaviour; ≤80% of control males 7 days and older mated with females, but allatectomized males did not show comparable levels of copulation until day 19. In a 3 h observation period, the time taken for 50% of males to initiate copulation was c. 60 min for allatectomized males and only c. 10 min for controls. At the age when 50% of males copulated, the accessory glands of allatectomized males were less developed than those of controls; the accessory gland weight was 65%, soluble protein was 72% and reserves of long hyaline protein I were 40% those of controls. Treatment with Juvenile Hormone III on day 2 stimulated accumulation of long hyaline protein I in 9-day-old allatectomized males to the level found in untreated 19-day-old allactec-tomized males, but did not elicit heightened copulation. No positive feedback from the accessory glands appeared necessary to elicit copulation since vasectomy, ablation of the accessory glands or isolation of the terminal abdominal ganglion by ventral nerve cord transection did not inhibit copulatory behaviour in 7-day-old males. Because allatectomy inhibited copulatory behaviour in 7-day-old males in which the accessory glands were removed but not in 19-day-old males without accessory glands, the increased tendency of older allatectomized males to mate with females was not due to release of inhibition caused by continued growth of the accessory reproductive glands.     

Identification,characterization, and developmental profile of a high molecular weight,juvenile hormone-binding protein in the hemolymph of the migratory grasshopper,Melanoplus sanguinipes

In the hemolymph of Melanoplus sanguinipes, a high molecular weight juvenile hormone binding protein (JHBP) was identified by photoaffinity labelling and found to have a M_r of 480,000. The JHBP, purified using native gel electrophoresis followed by electroelution, has an equilibrium dissociation constant for JH III of 2.1 nM and preferentially binds JH III over JH I. Antibody raised against JHBP recognized only the 480,000 band. Under denaturing conditions the native JHBP gave a single band with a M_r 78,000. The antibody against native JHBP recognized only the 78,000 protein in SDS-treated hemolymph samples, indicating that JHBP is a hexamer in this species. The concentration of JHBP fluctuates in both the sexes during nymphal and adult development in parallel with total protein content of hemolymph. © 1995 Wiley-Liss, Inc.     

The spermatheca of Melanoplus sanguinipes (Fabr.). I. Morphology,histology, and histochemistry

The spermatheca of Melanoplus sanguinipes consists of a preapical and an apical diverticulum, and a long, thin ductus seminalis. Histologically, the three components are identical. The wall of the spermatheca includes a basement membrane, secretory and epithelial cells, and a cuticular intima. Small, discrete bundles of muscle occur outside the basement membrane. In each secretory cell is a large central cavity which connects with a cuticular channel (efferent ductule) running through the epithelial cell to the spermathecal lumen. During sexual maturation, light- and dark-staining vesicles accumulate in the secretory cells and discharge their contents into the central cavity. Simultaneously, glycogen accumulates in the epithelial cells. Allatectomy of newly emerged females renders the secretory cells unable to produce material, an effect which can be reversed by topical application of synthetic juvenile hormone. The secretion contains protein and acidic mucopolysaccharide. After insemination the quantities of secretion in the lumen and of glycogen in the epithelial cells diminish in the preapical diverticulum where almost all sperm are stored. As the number of sperm declines, the secretion and glycogen are replenished.     

Heritability and physiological correlates of migratory tendency in the grasshopper Melanoplus sanguinipes

Abstract. Durations of tethered flights by the North American migratory grasshopper Melanoplus sanguinipes Fabricius are bimodally distributed: most individuals either will not fly, or else will fly for many hours. This observation suggests a simple measure (the ‘one‐hour rule’) for distinguishing migrants from non‐migrants. This measure is repeatable (repeatability = 0.6–0.7). Using laboratory‐reared offspring of grasshoppers from an Arizona population of mixed migratory tendency, a breeding experiment was conducted to determine the heritability of migratory tendency and possible correlated responses to selection on migratory behaviour. When migratory tendency is considered as a threshold trait, the heritability of liability is in the range 0.5–0.6. Most families in the breeding experiment had at least some migrants among their offspring; selection on migratory incidence had a correlated effect on the durations of flights by these individuals. The magnitude of thoracic lipid reserves showed a modest correlated response to selection on migratory behaviour. Thoracic and abdominal lipid reserves in identified migrants are reduced by flight, indicating that lipid is mobilized and consumed during flight in this species.     

优雅蝈螽雄性附腺的超微结构及其腺管提取物对精子束的作用

为阐明优雅蝈螽Gampsocleis gratiosa Brunner von Wattenwyl雄性附腺的结构与功能的关系, 本文利用透射电镜(transmission electron microscope, TEM)技术研究了优雅蝈螽雄性附腺的超微结构, 利用微分干涉相差显微镜(differential interference contrast microscope, DIC)技术并结合雄性附腺匀浆提取物与精子束在体外的短暂培养, 研究了优雅蝈螽雄性附腺对精子束的作用。结果表明： 优雅蝈螽雄性附腺3类腺管组织结构相似, 腺管管壁为单层上皮细胞, 缺少内表皮, 说明其来源于中胚层。上皮细胞富含粗面内质网、 线粒体、 高尔基体, 具有分泌细胞的特点。腺管管腔中分泌物有4种形态, 即电子透明的物质、 电子致密的颗粒物质、 细纤维状物质以及绒球状物质。上皮细胞的分泌方式主要有2种, 即顶质分泌和局部分泌。乳白短腺管的匀浆提取物参与了帽状精子束解聚的过程, 乳白长腺管和透明腺管的匀浆提取物有维持精子束活性的作用。本研究结果为进一步阐明螽斯雄性附腺的生理功能奠定了基础。     

Structure of male accessory glands of Bolivarius siculus (fischer) (Orthoptera,Tettigoniidae) and protein analysis of their secretions

In Tettigoniidae (Orthoptera), male reproductive accessory glands are involved in the construction of a two‐part spermatophore; one part, the spermatophylax, is devoid of sperm and considered a nuptial gift. The morphology, ultrastructure, and secretion protein content of the male reproductive accessory glands from Bolivarius siculus were investigated. Two main groups of gland tubules open into the ejaculatory duct: the “first‐order” glands, a number of large anterior tubules, and the “second‐order” glands, smaller and more numerous tubules positioned posteriorly. Along with a further subdivision of the gland tubules, we here describe for the first time an additional gland group, the intermediate tubules, which open between first and second‐order glands. The mesoderm‐derived epithelium of all glands is a single layer of microvillated cells, which can be either flattened or cylindric in the proximal or distal region of the same gland. Epithelial cells, very rich in RER and Golgi systems, produce secretions of both electron‐dense granules and globules or electron‐transparent material, discharged into the gland lumen by apocrine or merocrine mechanisms, respectively. With one exception, a unique electrophoresis protein profile was displayed by each of the gland types, paralleling their unique morphologies. To assess the contribution of different types of accessory glands to the construction of the spermatophore, the protein patterns of the gland secretions were compared with those of the extracts from the two parts of the spermatophore. All samples showed bands distributed in a wide range of molecular weight, including proteins of very low molecular mass. However, one major high molecular weight protein band (>180 kDa) is seen exclusively in extracts from the first‐order glands, and corresponds to an important protein component of the spermatophylax. J. Morphol., 2009. © 2009 Wiley‐Liss, Inc.     

Mobilization of lipid and carbohydrate reserves in the migratory grasshopper Melanoplus sanguinipes

Abstract. The North American migratory grasshopper Melanoplus sanguinipes Fabricius (Orthoptera: Acrididae) exhibits heritable variation in predisposition to make long-duration flights, and performance of long-duration flight enhances reproductive output. As a first step in understanding the physiological basis of these phenomena, we examined the mobilization of lipid and carbohydrate reserves during flight and in response to injection of extracts of the corpora cardiaca. Extract of conspecific corpora cardiaca elevates the concentration of haemolymph lipid. Both synthetic locust adipokinetic hormone I (AKH I) and synthetic Locusta migratoria AKH II raise the concentration of lipid in the haemolymph. However, although AKH I is more active than AKH II in locusts, dose-response curves for the two peptides are similar in M.sanguinipes. Neither extract of conspecific corpora cardiaca nor locust AKH I affects haemolymph carbohydrate in this species. Haemolymph carbohydrate and total glycogen reserves are Diminished by tethered flight; in contrast, haemolymph lipid is elevated by flight. Grasshoppers identified as presumptive migrants or non-migrants do not differ significantly in body composition. Total lipid reserves did not decrease measurably after extended flight, even though total reserves of carbohydrate do not appear to be sufficient to maintain the durations of flight performed.     

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.     

Histochemical studies on the male accessory gland of Acrida turrita (Insecta: Orthoptera)

The male accessory reproductive glands of Acrida turrita includes fifteen pairs of tubules opening into the ejaculatory duct. Four of them are white and eleven hyaline glands. Histochemically the hyaline glands are further differentiated into seven types (HG1-HG7). The secretion of all the glands taken together includes polysaccharide (neutral mucopolysaccharide, chondroitin sulphate and sialomucin), protein (-NH2 and -SH groups, and tyrosine, histidine, tryptophan and arginine amino acids) and neutral lipid. The possible role of the accessory glands based on their histochemical analysis, has been speculated.     

Histochemical studies on the male accessory gland of Oxya velox (Insecta: Orthoptera)

The male accessory gland of Oxya velox includes fourteen pair of tubules opening into the ejaculatory duct. Histochemically these tubules can be distinguished into six types (AG1--AG6). Their secretion is the mixture of polysaccharides (Glycogen, sialomucin and neutral mucopolysaccharides), proteins (-NH2 and SH groups, tyrosine, tryptophan, histidine and arginine) and lipids (neutral and phospholipids). The possible role of the accessory glands based on their histochemical analysis has been speculated.     

Male insect accessory glands: Functions and control of secretory activity

Summary

Paralleling the diversity of the class Insecta, the male accessory glands (MAG) exhibit a wide range of form, and their secretion serves a variety of functions, including spermatophore and mating plug formation, sperm activation, provision of nutrients to females, and, through production of fecundity-enhancing and/or receptivity-inhibiting substances, modification of female reproductive behavior. In most insects, juvenile hormone (JH) is important in the regulation of MAG secretory activity; specifically, JH controls the production of particular proteins in the secretion. However, the production of some proteins appears not to be influenced by JH; rather, their synthesis is regulated by ecdysteroids. During sexual maturation, JH and ecdysteroids seem to interact to bring about a specified temporal sequence of protein synthesis in the MAG.     

桑天牛雄性附腺内容物组分分析

测定桑天牛Apriona germaric(Hope)雄性附腺内容物中各组分含量及变化情况。结果表明,内容物中可溶性蛋白占总腺管鲜重的8.39%;总糖占总鲜重的4.21%;海藻糖和游离氨基酸分别占总鲜重的0.50%、0.25%。随虫龄的增大,内容物中各组分含量不断降低。交配过程中,雄性附腺内容物部分转移到雌虫,交配后附腺内容物中总糖、海藻糖、游离氨基酸等组分含量均降低,蛋白质组分含量先升高再降低,48h即恢复可到交配前水平。大、小附腺内各组分含量变化有差异。     

Development of secretory activity in the long hyaline gland of the male migratory grasshopper,Melanoplus sanguinipes (Fabr.) (Orthoptera : Acrididae)

Changes in the ultrastructure of epithelial cells from long hyaline glands of male Melanoplus sanguinipes (Fabr.) (Orthoptera : Acrididae) have been examined during sexual maturation and after allatectomy. In newly emerged males, the long hyaline gland epithelium is composed of 1–3 cell layers. The cells contain almost no rough endoplasmic reticulum, inconspicuous Golgi complexes, and large numbers of free ribosomes and polysomes. Within 24 hr, the cells undergo considerable reorganization to form a 1-cell-thick layer. Changes in cytostructure include proliferation of the rough endoplasmic reticulum and the development of several elaborate Golgi complexes. The developing lumen contains a coarse fibrous material. By 3 days postemergence, columnar epithelial cells are clearly capable of considerable synthesis and export of secretory protein. Rough endoplasmic reticulum, and large, elaborate Golgi complexes are the major structural features of the cytoplasm. From day 3 to sexual maturity (day 7), no major ultrastructural changes occur, although massive accumulation of secretion in the lumen causes the epithelium to become cuboidal or flattened. Isoelectric focusing of soluble proteins from long hyaline gland secretions shows that maturing glands contain increasing numbers and quantities of secretory proteins.Allatectomy has minor effects on long hyaline gland ultrastructure. A reduction in the density of rough endoplasmic reticulum and ribosomes suggests that glands from operated males are metabolically less active. This is confirmed by qualitative and quantitative changes in the amount of secretion as revealed by isoelectric focusing. The observations are discussed in terms of the juvenile hormone control of long hyaline gland maturation.     

Juvenile hormone III produced in male accessory glands of the longhorned beetle,Apriona germari,is transferred to female ovaries during copulation

We report on juvenile hormone (JH) biosynthesis in vitro by male accessory glands (MAGs) in the longhorned beetle, Aprionona germari, accompanied by the transfer of JH from males to females during copulation. JH was extracted from the MAGs and separated by reversed‐phase high‐performance liquid chromatography. JH III was identified as the major JH by gas chromatography–mass spectrometry. A radiochemical assay and a non‐radioactive method were used to measure the in vitro rate of JH biosynthesis by the MAGs. After 4 h of incubation with ³H‐methionine in the medium, the radioactivity in the MAGs substantially increased. In a separate assay, incubation of the MAGs with non‐radioactive methionine for 4 h resulted in a 39% increase in JH III. Seven‐day‐old males were injected with medium 199 containing ³H–methionine and 24 h later they were mated with virgin females. Hemolymph and the MAGs were collected from the mated males and hemolymph, ovaries and eggs were collected from the mated females for assaying radioactive JH. The radioactivity incorporated into JH in the MAGs was transferred to the females during copulation and later transferred into their eggs. Assayed 1 h after copulation, JH III level in the MAGs decreased 42% and the content of JH III in the male hemolymph did not change, whereas the content of JH III in the female hemolymph and ovaries both increased. © 2010 Wiley Periodicals, Inc.     

昆虫雄性附腺功能蛋白研究进展

昆虫雄性附腺蛋白是精液蛋白的主要来源,对雌雄虫生殖过程具有重要生理功能,按功能可分为精包结构蛋白和功能蛋白两类。精包结构蛋白参与精包的形成;功能蛋白在交配过程中随精子一起转移到雌虫体内,导致雌虫行为和生理的深刻变化,如降低雌虫再交配率、提高产卵量、促进精子转移、储存和竞争等。随着对昆虫雄性附腺功能蛋白研究的深入,特别对果蝇附腺功能蛋白的详细研究,从分子水平上阐述蛋白质序列与功能的关系,明确其作用机制,可为进一步阐明昆虫生殖和进化机制等提供新依据。     

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.