Ordered flow of secretion from accessory glands to specific layers of the spermatophore of mealworm beetles: demonstration with a monoclonal antibody

Monoclonal antibodies were produced against the secretory product of the bean-shaped accessory gland (BAG) of male mealworm beetles (Tenebrio molitor). Antibodies from one clone (PL 6.3) recognized a 9,600 dalton protein with a pI of 6.6 which was found in homogenates of the BAG. The PL 6.3 antigen was first detected on Western blots of BAG proteins from 2-day adults, and amounts increased for the next 6 days until reproductive maturation was achieved. The antibody also recognized a polypeptide with a molecular weight (mw) of about 5,000 daltons which we believe to be derived from the larger 9,600 dalton antigen. There are eight types of secretory cells in the BAG. By using light microscopic immunohistochemistry, we localized the antigens recognized by PL 6.3 in cell type 7 (intense staining) and cell type 5 (weak staining). Results from electron microscopic immunocytochemistry showed that antigen PL 6.3 was concentrated in the secretory granules characteristic of each of these two cell types and was absent in all other cell types. PL 6.3 antigens were traced from the BAG into its secretory product and then into the prespermatophoric mass in the ejaculatory duct. The antigen was not randomly mixed with other secretory products of the accessory glands. As it flowed from the BAG and into the ejaculatory duct, it remained in a coherent, precisely localized mass. Within the definitive spermatophore, the PL 6.3 antigen was concentrated in discrete layers of material that line the lumen.     

Molecular dissection of nuptial gifts in divergent strains of Ostrinia moths

Seminal fluid proteins (SFPs) produced in the male accessory glands and ejaculatory duct are subject to strong sexual selection, often evolve rapidly and therefore may play a key role in reproductive isolation and species formation. However, little is known about reproductive proteins for species in which males transfer ejaculate to females using a spermatophore package. By combining RNA sequencing and proteomics, we characterize putative SFPs, identify proteins transferred in the male spermatophore and identify candidate genes contributing to a one‐way gametic incompatibility between Z and E strains of the European corn borer moth Ostrinia nubilalis. We find that the accessory glands and ejaculatory duct secrete over 200 highly expressed gene products, including peptidases, peptidase regulators and odourant‐binding proteins. A comparison between Ostrinia strains reveals that accessory gland and ejaculatory duct sequences with hormone degradation and peptidase activity are among the most extremely differentially expressed. However, most spermatophore peptides lack reproductive tissue bias or canonical secretory signal motifs and aproximately one‐quarter may be produced elsewhere before being sequestered by the male accessory glands during spermatophore production. In addition, most potential gene candidates for postmating reproductive isolation do not meet standard criteria for predicted SFPs and almost three‐quarters are novel, suggesting that both postmating sexual interactions and gametic isolation likely involve molecular products beyond traditionally recognized SFPs.     

Spermatophore of the mealworm beetle: Immunochemical characteristics suggest affinities with male accessory gland

Acid hydrolysis of the spermatophore and of the bean-shaped accessory gland (BAG) of male Tenebrio yields strikingly similar amino acid components; proline, glutamic acid, aspartic acid, and alanine predominate in both. Antisera were prepared against extracts of the spermatophore and BAG. The two antisera shared common cross-reactive male-specific components as shown by both complement fixation tests and immunodiffusion procedures.     

Localization of two phosphorylation sites adjacent to a region important for polymerization on the tail of Dictyostelium myosin

Phosphorylation of the myosin heavy chains of Dictyostelium discoideum is known to be inhibited following chemotactic stimulation of the cells. Effects of dephosphorylation on the assembly of myosin and on its actin-activated ATPase activity raised the question of where the phosphorylated sites are located with respect to sites responsible for polymerization and actin binding. Using seven monoclonal antibodies the binding sites of which were mapped in the electron microscope, two phosphorylation sites, i.e., threonine residues that were phosphorylated by a kinase from D. discoideum, were localized by immunoblotting of chymotryptic fragments. Two of the antibodies bound to the terminal one fifth of the tail and recognized a phosphorylated chymotryptic fragment of 38 kd. The non-phosphorylated form and single and double phosphorylated forms of this fragment were separated by two-dimensional electrophoresis. Antibody labeling of lower mol. wt. polypeptides indicated that both phosphorylation sites were located at least 32 kd from the end of the tail. A non-phosphorylated fragment, that was insoluble at low ionic strength due to polymerization, proved to be an internal cleavage product of the tail. A segment of this fragment necessary for polymerization was mapped adjacent to the phosphorylation sites.     

Hunter syndrome: presence of material cross-reacting with antibodies against iduronate sulfatase

Summary Polyclonal antibodies were obtained from rabbits by injection of iduronate sulfatase purified 35,000-fold from human placenta, after elution of the enzyme from sodium dodecyl sulfate (SDS) polyacrylamide gels. The specificity of these antibodies towards iduronate sulfatase was demonstrated by immunoprecipitation of enzyme activity; the level of other lysosomal hydrolases and sulfatases remained constant. Immunoblot of iduronate sulfatase from various human sources showed that the antibody recognises a polypeptide of mol.wt. 72,000 daltons in placenta and serum, and a form of mol.wt. 60,000 daltons in fibroblasts. No immunoprecipitable peptide was found in urine or in the culture medium of fibroblasts. Polypeptides of the same molecular weight were recognised in serum and in fibroblasts of Hunter patients. The presence of altered proteins in these patients was also shown by competition experiments. The addition of Hunter proteins alters the binding of normal enzyme to the antibody.     

Isolation,partial characterization,and localization of the A and B proteins from the tubular accessory gland of male Tenebrio molitor

In its fully differentiated state, the tubular accessory gland of the male mealworm beetle, Tenebrio molitor, synthesizes five groups of proteins (A, B, C, D₁ and D₂) which are easily distinguished from one another on two-dimensional pI-SDS polyacrylamide gels (Black et al., 1982: Devl Biol. 94, 106–115). In the present work, the A and B proteins have been isolated by preparative gel electrophoresis and Amicon ultrafiltration. The isolation procedure provided two fractions of interest: one contained a mixture of A and B proteins (A/B) and the other consisted of only B proteins. The major proteins in the A class have a molecular weight of 17,900 while those of the B class are 19,000 daltons in size.Antibodies have been produced to the A/B mixture and to the B fraction. Ouchterlony immunodiffusion and straight line immunoelectrophoresis show that the A and B proteins share common immunological characteistics. The proteins from the tubular accessory gland were displayed on one dimensional SDS gels and electrophoretically blotted onto nitrocellulose paper. The antibodies to the A/B mixture recognize A and B bands on these gels. In addition, these antibodies show affinity for C proteins and another band of lower molecular weight.Using the anti-A/B with techniques of immunodiffusion, straight line immunoelectrophoresis, and immunoblotting, we have identified the A and B protein in extracts of soluble proteins in the spermatophore. Furthermore, the A/B proteins have been localized by immunohistochemical techniques within the apical portions of the secretory cells of the tubular gland and also in the lumen of the spermatophore.     

New actin-binding proteins from Dictyostelium discoideum

Dictyostelium discoideum contains a soluble actin-binding protein that caps actin filaments at their fast growing ends. The purified protein consists of two subunits with 34 kd and 32 kd apparent mol. wts. Like similar proteins from Acanthamoeba and bovine brain the capping protein from D. discoideum acts in a Ca²⁺ -independent manner. It lacks severing activity as indicated by its inability to disrupt the stress fibers and the microfilament network in detergent-extracted cells. Two actin-binding proteins from a plasma membrane-enriched fraction were labeled with [¹²⁵I]actin using a gel overlay technique. One of these proteins, with an apparent mol. wt. of 17 kd in SDS-polyacrylamide gels, has been purified from high-salt extracts, the other protein with an apparent mol. wt. of 31 kd has been purified from Triton X-100 extracted membranes. Monoclonal antibodies were raised against D. discoideum severin, α-actinin, the larger subunit of the capping protein, and the 17-kd membrane-associated protein. Immunoblotting of proteins from whole cell lysates showed that all these actin-binding proteins were present in both growth phase and aggregation-competent cells.     

Glycine receptor heterogeneity in rat spinal cord during postnatal development.

Two different isoforms of the inhibitory glycine receptor were identified during postnatal development of rat spinal cord. A neonatal form characterized by low strychnine binding affinity, altered antigenicity, and a ligand binding subunit differing in mol. wt (49 kd) from that of the adult receptor (48 kd) predominates at birth (70% of the total receptor protein). Separation from the adult form could be achieved by either use of a selective antibody or glycine gradient elution of 2-aminostrychnine affinity columns. Both isoforms co-purify with the mol. wt 93 kd peripheral membrane protein of the postsynaptic glycine receptor complex.     

New insights in the male anatomy,spermatophore formation,and sperm structure in Atyidae: The red cherry shrimp Neocaridina davidi

This study aims to analyze the functional anatomy of the male reproductive system in Neocaridina davidi, a very popular ornamental species of caridean shrimp. Mature males were cold‐anaesthetized and their reproductive systems were dissected for histological and histochemical analysis, while the spermatozoa and spermatophore wall ultrastructure were analyzed under transmission electron microscopy. The male reproductive system consisted of two coiled testes, which were continuous with the vasa deferentia. Testes were positioned on the dorsal side of the cephalothorax above the hepatopancreas, and comprised seminiferous tubules where spermatogenesis occurred. Each vas deferens (VD) was a long tube dorsolaterally positioned with respect to the hepatopancreas, and increased in diameter at the distal end. Three regions could be recognized in the VD: proximal, middle, and distal. The proximal region had a cylindrical epithelium with secretory cells. The middle region (or typhlosole) had a dorsal fold (or typhlosole) with a thick columnar epithelium and high secretory activity. The spermatophore was a continuous cord with three acellular layers, which were mainly characterized by the presence of neutral glycoconjugates and proteins. The sperm morphology was distinct from the inverted cup‐shaped spermatozoa observed in the majority of caridean shrimps. The spermatozoa in specimens of N. davidi were spherical in shape, with a cross‐striated, single, short spike, and arranged in clusters of three or four sperm cells. The composition of the spermatophore, and the arrangement and form of the spermatozoa, seem to be unique in comparison to other species of Caridea.     

Conjugation of [125I]ubiquitin to cellular proteins in permeabilized mammalian cells: comparison of mitotic and interphase cells.

[125I]Ubiquitin introduced into permeabilized hepatoma tissue culture (HTC) cells rapidly forms conjugates with endogenous proteins. A characteristic pattern of low mol. wt conjugates is obtained which includes the ubiquitinated histone, uH2A, and unknown molecular species with MrS of 14, 23, 26 (two bands) and 29 kd. A broad spectrum of higher mol. wt conjugates is also produced. The formation of all conjugates is absolutely dependent on ATP, and upon depletion of ATP they are rapidly broken down. The 14, 23 and 29 kd species are found in all subcellular fractions examined. uH2A is located exclusively in the nuclear fraction. The pair of 26 kd bands is specifically associated with the ribosome fraction. A considerable percentage of the higher mol. wt conjugates sediments with the small particle (100,000 g) fraction in the ultracentrifuge but is solubilized with deoxycholate, indicating that there are many membrane-associated conjugates. The pattern of ubiquitin conjugation in interphase and metaphase cells was compared. The incorporation of ubiquitin into uH2A was markedly reduced in metaphase cells whereas its incorporation into other low mol. wt conjugates and into high mol. wt conjugates was affected slightly, if at all. This shows that the known decrease of uH2A levels in metaphase is due to a specific effect on histone ubiquitination and not to a general decrease in ubiquitination activity or increase of isopeptidase activity. Changes in the levels of uH2A during mitosis measured by immunoblotting were similar to those estimated in permeabilized cells. These experiments indicate that permeabilized cells provide a useful approach to the study of rapidly turning over ubiquitin conjugates in mammalian cells.     

A cellular protein phosphorylated by the avian sarcoma virus transforming gene product is associated with ribonucleoprotein particles

In chick embryo fibroblasts transformed by Rous sarcoma virus (RSV) the tyrosine phosphorylation of a cellular protein of 34,000 daltons mol. wt. (34 kd) is greatly enhanced; this was shown to be catalyzed by the phosphotransferase activity of RSV transforming protein pp60src. We report here that in cytoplasmic extracts of both normal and transformed cells, in the presence of magnesium ions, the majority of the 34-kd protein is associated with large structures and that a fraction of 34 kd appears to be associated with ribonucleoprotein particles (RNPs). In addition, upon u.v. light cross-linking of RNA to protein in normal or transformed cells, an anti-34 kd serum immunoprecipitates RNA fragments of apparent low sequence complexity as detected by T1 fingerprint analysis. Our results indicate that the 34-kd protein may play a role in the cell at the level of RNPs.     

Effects of polyanions and polycations on the trehalose phosphate synthetase of Mycobacterium smegmatis

When tested as activators on the trehalose phosphate synthetase [UDP-d-glucose:d-glucose 6-phosphate α-d-glucosyltransferase, EC 2.4.1.15 (46)] from Mycobacterium smegmatis, heparin was the best, various other sulfated polysaccharides (especially chondroitin 4- and 6-sulfates, dermatan sulfate, heparan sulfate, and γ-carrageenan) and polynucleotides were good, but hyaluronic acid, d-galacturonan, dextran sulfate, and keratan sulfate, were poor. Digestion of chondroitin sulfate with hyaluronidase destroyed the activating ability, but separation of the digestion products on Sephadex G-100 resin gave large-molecular-weight componentns that still showed activating ability. A sulfated tetra- or octa-saccharide isolated from chondroitin sulfate did not activate the enzyme, nor did they prevent the activation by chondroitin sulfate, suggesting that these small polyanions do not bind to the enzyme. Among polycations, poly-dl-ornithine (mol. wt. 15,600 daltons) was the best inhibitor of the enzyme followed by poly-l-lysine (mol. wt. 4,000 daltons), poly-d-lysine (mol. wt. 70,000 daltons), poly-d,l-lysine (mol. wt. 35,000 daltons), and then poly-l-ornithine (mol. wt. 120,000 daltons); polyglycine, polyleucine, and polyhistidine showed no effect. In all cases, more polycation was required to inhibit the enzyme when heparin was used as the activator than when chondroitin sulfate was used. The order of mixing of various reaction components was important for the extent of inhibition, the greates inhibition being observed when polyanion and polycation were mixed before the addition of enzyme, and the smallest when polyanion and enzyme were mixed before the addition of polycation.     

Purification and characterization of a proline-rich secretory protein that is a precursor to a structural protein of an insect spermatophore

The spermatophore or sperm sac of Tenebrio molitor (yellow mealworm beetle) is an acellular structure composed mostly of structural proteins, termed spermatophorins. The proteins are derived from the bean-shaped accessory reproductive glands of the male and are assembled into the multilayered structure within the ejaculatory duct. Homogenates of the secretory plug from this gland were used as immunogens for the production of monoclonal antibodies, including one identified as PL 21.1 which recognizes an antigen in the gland and the spermatophore. With the aid of gel filtration and immunoaffinity chromatography with a PL 21.1, we isolated a glandular secretory protein that is a precursor to a spermatophorin with similar electrophoretic mobility. On native polyacrylamide gels, the antigen from gland homogenates has an apparent molecular mass of 370 kDa. On sodium dodecyl sulfate gels, the antigen from the gland and that from the spermatophore have apparent molecular masses of 23 kDa. According to immunoblots of sodium dodecyl sulfate gels, the 23-kDa glandular antigen is organ-specific and adult-specific. By immunocytochemistry with PL 21.1, we found the antigens to be restricted to secretory vesicles of only one cell type in the gland and to a discrete layer in the outer wall of the spermatophore. The 23-kDa secretory antigen is distinguished by being high in glutamic acid/glutamine (15.4%) and in proline (25.2%).     

Biochemical analysis of an antigen produced by both human sex chromosomes.

It has been argued on both evolutionary and functional grounds that genes must be shared by the mammalian sex chromosomes. The only direct evidence for such genes is our previous finding that loci on the human X (MIC2X) and Y (MIC2Y) chromosomes encode a species-specific cell surface antigen recognised by the monoclonal antibody 12E7. These loci map to the regions of the sex chromosomes which pair at meiosis, and MIC2X has been shown to escape X-inactivation. We have used immunoprecipitation and Western blot analysis combined with one- and two-dimensional polyacrylamide gel electrophoresis to compare the products of MIC2X and MIC2Y. The human specific molecule recognised by the 12E7 antibody is a membrane-associated polypeptide of mol. wt. 32.5 kd and pI = 5.0. No difference in size or charge has been detected between X and Y encoded forms of this molecule confirming that MIC2Y is a functional homologue of MIC2X. An intracellular polypeptide of mol. wt. 29 kd and pI = 7.0 present in the cytoplasm of both human and mouse cells is also recognised by the 12E7 antibody.     

Early stages in the yeast secretory pathway are required for transport of carboxypeptidase Y to the vacuole

Temperature-sensitive secretory mutants (sec) of S. cerevisiae have been used to evaluate the organelles and cellular functions involved in transport of the vacuolar glycoprotein, carboxypeptidase Y (CPY). Others have shown that CPY (61 kd) is synthesized as an inactive proenzyme (69 kd) that is matured by cleavage of an 8 kd amino-terminal propeptide. sec mutants that are blocked in either of two early stages in the secretory process and accumulate endoplasmic reticulum or Golgi bodies also accumulate precursor forms of CPY when cells are incubated at the nonpermissive temperature (37°C). These forms are converted to a proper size when cells are returned to a permissive temperature (25°C). Vacuoles isolated from sec mutant cells do not contain the proCPY produced at 37°C. These results suggest that vacuolar and secretory glycoproteins require the same cellular functions for transport from the endoplasmic reticulum and from the Golgi body. The Golgi body represents a branch point in the pathway: from this organelle, vacuolar proenzymes are transported to the vacuole for proteolytic processing and secretory proteins are packaged into vesicles.     

Sexual interactions in red-spotted newt populations

Two alternative reproductive tactics (amplexus and spermatophore transfer interference) and three sexual interference tactics (spermatophore transfer interference, pseudofemale behaviour, amplexus interference) are described for male red-spotted newts, Notophthalmus viridescens. The two reproductive tactics yield equal insemination rates for males displaying these behaviours. The three sexual interference tactics decrease the potential fitness of amplectic males either directly, by displacing them from receptive females and/or duping them into fruitless spermatophore depositions, or indirectly, by increasing the time spent in amplexus with no concomitant increase in probability of successful insemination.     

Infection increases the value of nuptial gifts, and hence male reproductive success, in the Hymenolepis diminuta-Tenebrio molitor association

During copulation, male insects pass accessory gland components to the female with the spermatophore. These gifts can affect female reproductive behaviour, ovulation and oviposition. Here, we show that female mealworm beetles, Tenebrio molitor, mated with males infected with metacestodes of the rat tapeworm, Hymenolepis diminuta, produced significantly more offspring than those mated with uninfected males. There is a significant positive relationship between parasite intensity in the male and reproductive output in the female. Infection results in a significant increase in bean-shaped accessory gland (BAG) size. We suggest that infected males pass superior nuptial gifts to females and discuss the confounding effects of infection in male and female beetles upon overall fitness costs of infection for the host and the likelihood that the parasite is manipulating host investment in reproduction.     

Homologies between paraflagellar rod proteins from trypanosomes and euglenoids revealed by a monoclonal antibody

A Nonidet P 40 insoluble fraction was isolated from Trypanosoma brucei and was used to raise a monoclonal antibody (5E9). The antigen was localized by indirect immunofluorescence in the flagellum of T. brucei and of two species of euglenoids, Euglena gracilis and Distigma proteus. In immunoblot analysis, 5E9 appeared to bind to paraflagellar rod proteins PFR1 and PFR2 of T. brucei (72000 and 75000 mol. wt.) and of E. gracilis (67000 and 76000 mol. wt.). The presence of a common epitope in paraflagellar rod proteins from species of trypanosomes and euglenoids shows that despite distinct structures of the rods some identical domain exists in the proteins that could be involved in their supramolecular assembly into a similar organelle. The antigenic determinant defined by 5E9 was also shown to be present in a 87000 molecular weight polypeptide located in the proximal part of the flagellum of Crithidia oncopelti in which a paraflagellar rod is not detectable at the ultrastructural level.     

Single subunit structure of the human thyrotropin receptor.

We have produced rabbit antibody against a synthetic peptide corresponding to N-terminal region of the extracellular domain of human thyrotropin receptor (hTSH-R) (N peptide, aminoacid residues 29-57). Western blot analysis revealed that N-peptide antibody recognized recombinant hTSH-R stably expressing in CHO-K1 cells as a mol. wt. about 104 kDa regardless in the presence or absence of disulfide-reducing agent. The band was not detected in untransfected CHO-K1 cells and no band was also stained by the antibody absorbed with N-peptide. In a reducing condition, the antibody also bound the rat receptor from FRTL5 cells as the same molecular size (104 kDa). These results clearly indicate that TSH-R is composed of a single subunit and that two subunit model for the TSH-R may reflect artifactual proteolytic cleavage of the receptor during membrane preparation.