Cytodiffrentiation in the accessory glands of Tenebrio molitor. VI. A congruent map of cells and their secretions in the layered elastic product of the male bean-shaped gland

The morphology of the bean-shaped accessory glands (BAGs) of males of Tenebrio molitor is described. All cells in the secretory epithelium are long and narrow (300–400 mμ × 5 mμ). The seven types of secretory cells are distinguished from one another by the morphology of their secretory granules. Granule substructure varies from simple spheres with homogeneous electrondense contents to complex forms with thickened exterior walls or with crystalline and membranous contents. Individual cell types were mapped by staining whole glands with Oil Red O, and the cell distributions were confirmed by wax histology and ultramicroscopy. The secretions of all seven cell types form a secretory plug composed of seven layers. During mating, the secretory plug from each BAG is forced into the ejaculatory duct by contractions of a sheath of circular muscle. The mirror image plugs from symmetrical BAGs fuse and are transformed into the wall of the spermatophore.     

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%).     

A monoclonal antibody against a structural protein in the spermatophore of Tenebrio molitor (coleoptera)

Monoclonal antibodies were produced against insoluble proteins of spermatophores of Tenebrio molitor. One hybridoma clone produced antibody which recognized two antigens (29.4 and 27.6 kd mol. wt) in the bean-shaped accessory reproductive glands (BAGs) and in the secreted precursor of the insoluble fraction of the spermatophore. At least two molecular weight variants which differed by 1.5–1.7 kd mol. wt daltons are present. Processing accompanies secretion into the BAG lumen, with a reduction of about 4000 daltons in apparent molecular weights.The amount of target antigen during reproductive maturation, its localization and its transport were studied using Western blotting and immunohistochemistry. The monoclonal antibody recognized a protein present in one of the eight secretory cell types (type 3) of the BAG, in the secretory production of this gland, and in discrete layers of the spermatophore ejected from the male. This specific probe, and others currently being produced, will facilitate detailed studies on the process of spermatophore formation.     

Ultrastructure of the ejaculatory duct region producing the male housefly accessory material

The male accessory material of houseflies is produced by what appears to be one type of cell in the anterior part of the ejaculatory duct. The cells of this part of the ejaculatory duct have an extensive rough endoplasmic reticulum, numerous free ribosomes, and many Golgi complexes. Secretion of the accessory material seems to occur as a mixture of release via individual Golgi vesicles, the splitting off of portions of cytoplasm, and the lysis of entire cells. No evidence was found of replacement of lysed cells. Synthesis of the secretory material is most active soon after emergence and after mating. The material accumulates around the duct lumen between the secretory cells and the intima and passes directly through the intima in being discharged from its storage area.     

Ultrastructure of male accessory glands in the scorpionfly Sinopanorpa tincta (Navás, 1931) (Mecoptera: Panorpidae)

The ultrastructure of male reproductive accessory glands was investigated in the scorpionfly Sinopanorpa tincta (Navás, 1931) (Mecoptera: Panorpidae) using light and transmission electron microscopy. The male accessory glands comprise one pair of mesodermal glands (mesadenia) and six pairs of ectodermal glands (ectadenia). The former opens into the vasa deferentia and the latter into the ejaculatory sac. The mesadenia consist of a mono-layered elongated columnar epithelium, the cells of which are highly microvillated and extrude secretory granules by means of merocrine mechanisms. The epithelium of ectadenia consists of two types of cells: the large secretory cells and the thin duct-forming cells. These two types of cells that join with a cuticular duct constitute a functional glandular unit, corresponding to the class III glandular cell type of Noirot and Quennedey. The cuticular duct consists of a receiving canal and a conducting canal. The secretory granules were taken up by the receiving canal and then plunged into the lumen through the conducting canal.     

Ultrastructure of the male accessory glands ofAllacma fusca(Insecta, Collembola)

The accessory glands ofAllacma fusca(L.) (Insecta, Collembola, Sminthuridae) consist of a series of secretory units that are arranged in parallel and open into the ejaculatory duct. Each unit is composed of microvillate cells stacked around a common cavity. Basal cells are involved in ion-control of fluids from the hemocoel to the cavity. The intermediate and apical cells, which have a laminar appearance and contain many microtubules, are involved in the structural integrity of the unit. Supporting cells ensheath the most apical cells. Large openings in the cuticle allow the gland secretion to flow into the ejaculatory duct lumen. These openings are protected by a porous cuticle different from that lining the epithelium of the ejaculatory duct. Conspicuous muscle fibers run along the lateroventral side of the ejaculatory duct beneath the insertion of the accessory glands. The fine structure of the accessory glands indicates that they are type I ectodermic glands as defined by Noirot & Quennedey (1974). Their function could be to control the fluidity of the material for spermatophore formation and to ensure the proper physiological conditions for spermatozoa stored in the ejaculatory duct lumen.     

Ultrastructure of the male reproductive accessory glands in the medfly Ceratitis capitata (Diptera: Tephritidae) and preliminary characterization of their secretions

The morphology and the ultrastructure of the male accessory glands and ejaculatory duct of Ceratitis capitata were investigated. There are two types of glands in the reproductive apparatus. The first is a pair of long, mesoderm-derived tubules with binucleate, microvillate secretory cells, which contain smooth endoplasmic reticulum and, in the sexually mature males, enlarged polymorphic mitochondria. The narrow lumen of the gland is filled with dense or sometimes granulated secretion, containing lipids. The second type consists of short ectoderm-derived glands, finger-like or claviform shaped. Despite the different shape of these glands, after a cycle of maturation, their epithelial cells share a large subcuticular cavity filled with electron-transparent secretion. The ejaculatory duct, lined by cuticle, has epithelial cells with a limited involvement in secretory activity. Electrophoretic analysis of accessory gland secretion reveals different protein profiles for long tubular and short glands with bands of 16 and 10 kDa in both types of glands. We demonstrate that a large amount of accessory gland secretion is depleted from the glands after 30 min of copulation.     

Anatomy and histology of the male reproductive system of the fire ant,Solenopsis invicta Buren (Hymenoptera : Formicidae)

The male reproductive system of the fire ant, Solenopsis invicta Buren (Hymenoptera : Formicidae), consists of the testes, vasa efferentia, vasa deferentia, seminal vesicles, accessory glands, ejaculatory duct, wedge, aedeagal bladder, and external genitalia. The testes in newly eclosed males appear as 4 large white lobes filled with packets of sperm. Each lobe of the testes contains only one follicle. As the testes degenerate, the maturing sperm migrate through the vasa efferentia and vasa deferentia into the seminal vesicles. The seminal vesicles attach to the accessory glands, which are lined with secretory columnar epithelium. The posterior ends of the accessory glands taper and unite to form the ejaculatory duct. A sclerotized wedge is found at the junction of the accessory glands and the ejaculatory duct. An aedeagal bladder, joining the ejaculatory duct posterior to the wedge, is lined with squamous epithelium enveloped by heavy musculature. The ejaculatory duct continues posteriorly to form a distal aedeagus surrounded by 3 pairs of valves, comprising the external genitalia.     

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.     

Identification and characterization of mitochondria autoantigens in progressive systemic sclerosis: identity with the 72,000 dalton autoantigen in primary biliary cirrhosis

Sera from eight out of 62 (14.5%) patients with progressive systemic sclerosis (PSS) reacted by immunoblotting with a 72,000 dalton antigen and one, a patient with concomitant primary biliary cirrhosis (PBC), reacted with the 72,000 dalton and a 47,000 dalton antigen. Reactivity with these antigens was not seen with any of 111 control sera. The antigens with minor variations in m.w. were present in a variety of cultured cells and tissue homogenates from different species. Subcellular fractionation studies localized the antigens to the mitochondria. Of 19 sera from patients with other diseases selected for immunofluorescence staining for anti-mitochondria autoantibody, nine reacted with the 72,000 dalton antigen, seven reacted with both the 72,000 and 47,000 dalton antigens, and three reacted with the 47,000 dalton antigen. These results show that serum reactivity with the 72,000 dalton and 47,000 dalton mitochondria autoantigens is found with some patients with PSS. Because mitochondria autoantibodies that are reactive with the 72,000 dalton and 47,000 dalton polypeptides are also found in patients with PBC, the present finding provides additional support for the association of PSS with PBC. Prior absorption of rat liver homogenate with PBC sera removed PSS serum reactivity with a 63,000 dalton antigen, the equivalent 72,000 dalton antigen in rodents, and vice versa, showing that both PBC and PSS sera recognize the same antigen.     

Simultaneous and successive localization of two antigens in the same tissue section using 3,3'-diaminobenzidine and 1-naphthol basic dye

We describe two procedures for the simultaneous and successive localization of two antigens in the same tissue section. In the simultaneous staining procedure, the first antigen was localized using 3,3'-diaminobenzidine (DAB), while the second antigen was stained using the 1-naphthol basic dye (1-NBD) method. The colour of the second antigen depended on the basic dye used, and no mixing of colours was observed when the two antigens were localized in different cells or structures. However, sequential double staining proved to be more convenient for the demonstration of two antigens in the same cell. In this procedure, the first antigen was stained using 1-NBD, and the interesting microscopic fields were photographed. The basic dye was then completely removed, and the second antigen was stained using DAB.     

An immunohistochemical study of the distribution of ABH antigens in human submandibular glands at the light and electron microscopic levels.

The distribution of blood group antigens ABH in submandibular glands was studied at light and electron microscopy levels by applying ImmunoGold Silver Staining (IGSS) and post-embedding ImmunoGold (IGS) methods, respectively. In IGSS treated samples, a cytoplasmic and a surface form of antigen localization were discernible in the glandular parenchyma. The former was restricted to most mucous cells and to scattered serous cells: A and B antigens were demonstrated in mucous cells of A and B type glands, while H antigen appeared in most mucous and occasional serous elements regardless of the blood type of donors. The latter appeared as a strong H reactivity on cell surfaces of serous acini and ducts regardless of the patient blood type. The IGS method was applied both on non-osmicated samples embedded in LR White resin and on osmicated, Epon embedded samples. In non-osmicated tissues, antigen labelling was revealed in secretory granules and cell surfaces. Positive secretory granules were found in most mucous cells and occasional serous, intercalated, and striated duct cells. A and B antigens weakly reacted in mucous cells of A and B type glands, respectively, while strong H reactivity was seen in mucous, serous, intercalated and striated duct cells of glands of all types. Surfaces labelled with H antigen were found on both lumenal and basolateral membranes of striated ducts in glands of all types. IGS method applied on osmicated, Epon embedded samples, selectively revealed blood group antigens in secretory granules of serous cells but not in the apical vesicles of striated ductal cells. Cell surfaces were completely unreactive.     

Immunocytochemical evidence for vasopressin receptors.

An electron microscopic study was made of mouse pituitaries immunocytochemically stained with anti-lysine vasopressin (LVP) as the primary antiserum in the unlabeled antibody peroxidase-anti-peroxidase procedure. Vasopressin (VP) was identified in the neurosecretory granules of the neural lobe which stained with peroxidase anti-peroxidase molecules. Electron density was induced in secretory granules of the pars intermedia (PI), both in the melanocyte stimulated hormone and ACTH cell types, probably indicating VP molecules attached to binding (receptor) sites. Omission of anti-LVP abolished staining both in the neural lobe and the PL Anti-LVP absorbed with antigen, by admixing with LVP, abolished staining in the neural lobe but not in the PI; according to optical density measurements the PI showed a +/- 22% staining increase over controls. Staining intensity in the PI probably reflects occupancy of binding (receptor) sites for VP. Exposure of PI granules to LVP before the usual staining sequence resulted in +/- 48% increased staining. In water-deprived mice with high endogenous VP titers, staining was +/- 33% and +/- 40% more intense than in normal mice. Solid phase absorbed and eluted antibodies to LVP provided additional proof that staining in both neural lobe and PI could be attributed to anti-LVP. Results indicate that binding or receptor sites for VP are located on secretory granules in the PL Possible physiological significance is discussed.     

Ultrastructure et cycle annuel des glandes annexes de l'appareil genital male de Campodea remyi Denis (Apterygota : Diplura : Campodeidea)

The accessory glands of the male reproductive system of Campodea remyi (Apterygota : Diplura : Campodeidea) consist of similar glandular units, each of which is made of a long secretory cell with a sieve at the tip of its extracellular cavity and a cell forming the excretory canalicule. During the annual cycle of sexual activity, they show morphological differences in terms of their size and the number of secretory granules. When ecdysis occurs, the cuticle of the ejaculatory duct and canalicules, the sieves, and the apical part of the glandular cells disappear. The gland's secretory products make up the spermatophore stalks.     

Immunocytochemical localization of renin in the submandibular gland of the mouse.

Renin was localized in the submandibular gland of the adult mouse at light and electron microscopic levels by the unlabeled antibody enzyme method of Sternberger. At the light microscopic level, renin was confined to the granular convoluted tubule (GCT) segment of the gland with considerable variation among GCT cells in intensity of staining. Some GCT cells failed to stain for renin. The pattern of staining was the same in the gland of male and female mice, but in the glands of females GCT segments were smaller and less numerous. At the electron microscopic level, staining for renin was also confined to the GCT cells, and was localized exclusively to the secretory granules. The intensity of staining of the secretory granules within a given GCT cell varied; some cells contained only minimally reactive or negative secretory granules. All other organelles within the GCT cell, except condensing vacuoles, failed to stain.     

Ia-specific mixed leukocyte reactive T cell hybridomas: analysis of their specificity by using purified class II MHC molecules in synthetic membrane system

This study was undertaken to determine the nature of the antigens recognized in allogeneic and syngeneic mixed leukocyte reactions (MLR). Specifically, we wished to determine whether Ia antigens alone were recognized by MLR-reactive T cells, or whether the specificity was determined by the corecognition of non-MHC antigens together with syngeneic or allogeneic Ia. To do this we used 11 T cell hybrids that were characterized as being specific for Iad and were tested their capacity to respond to isolated I-Ad or I-Ed that had been incorporated into liposomes and had bound to the surface of glass beads. Of nine alloreactive T cell hybrids (five I-Ad-and four I-Ed-specific), seven were shown to be responsive to the relevant isolated Ia antigen on glass beads. Also, two of two syngeneic I-Ad-specific T cell hybrids responded to I-Ad on the glass beads. One of the two alloreactive T cell hybrids that failed to respond to the relevant Ia antigen on glass beads was shown to be specific for an antigen in fetal calf serum (FCS) that was recognized in the context of the allo-Ia antigen (I-Ed), because when intact accessory cells were used, a response by this hybrid was only observed when FCS was present in the assay culture medium or when the accessory cells were pre-pulsed with FCS. The possible involvement of FCS antigens and non-Ia accessory cell antigens in the stimulation of the nine T cell hybrids that responded to isolated Ia on glass beads was evaluated. T cell hybrids that were grown and were tested in serum free medium were still capable of reacting to Ia on beads. The isolated Ia preparations used were greater than 90% pure, and their capacity to stimulate the T cell hybrids did not correlate with the degree of contamination with non-Ia proteins. We conclude from these studies that the majority of T cells that respond to allogeneic or syngeneic Ia bearing stimulator cells are specific for the Ia antigens themselves, and do not require the co-recognition of other non-Ia antigens; nor is there any requirement for Ia antigen processing for this recognition.     

Expression of blood group A and B antigens in nuclear heterochromatin in mucous cells of human cervical glands.

Using a post-embedding immunogold labeling procedure, we found that monoclonal antibody against A (MAb-A) or B antigen (MAb-B) reacted with nuclear heterochromatin regions, as well as secretory granules, in mucous cells of human cervical glands. Systematic and critical observation of specimens from 24 individuals of different blood groups revealed that the labeling pattern with MAb with strictly dependent on the blood group (A,B, or O) of the donors, i.e., MAb-A reacted with the heterochromatin from blood group A and AB but not with B and O individuals. Labeling with MAb-B was also specific for the heterochromatin from blood group B donors. On the other hand, MAb against H antigen did not react with the heterochromatin from any individuals examined, despite the fact that H antigens were detected by the MAb in secretory granules. Such specific reactions provide evidence that certain types of blood group-related antigens exist in the nuclear heterochromatin in mucous cells of human cervical glands. In contrast to the secretory granules in which ABH antigens were recognized by blood group-specific lectin, heterochromatin regions had little or no affinity for these lectins. Furthermore, the secretory status of individuals affected the staining intensity with MAb in secretory granules but not in the heterochromatin. These results suggest that the blood group substances found in the heterochromatin may have different molecular properties from those in the secretory granules, although both have the same determinant structures of ABH antigens.     

Ducts of the labral glands of Leptestheria dahalacensis (Crustacea: Branchiopoda: Spinicaudata)

Inside the labrum of Leptestheria dahalacensis are situated three types of large epidermal gland cells, whose ducts open onto the outer dorsal surface of the labrum. SEM revealed that the thin ducts of the A-type gland cells open out behind the epipharynx at the end of small, conically shaped protuberances, the two paired ducts of the B-type gland cells lead into the distal portion of the labrum, and the external opening of the single duct of the C-type gland cells lies on the dorsal lobe of the labrum. The ducts of the three different gland cell types have the same fundamental constitution, but vary in diameter. Each secretory unit consists of a pair of gland cells (A, B, or C) and a secretory duct. The duct is formed by ring-shaped folding of one anteroposteriorly elongated epidermal cell (duct cell), whose ends adhere closely to one another. A further ring-folded epidermal cell (accessory cell), but flattened in shape, is interposed, like a sleeve-connection, between the gland cells and the duct cell. The reservoirs of gland cells open into the lumen of the duct. Discontinuous deposits of highly electron-dense matter are present on the plasma membrane of the accessory cell delimiting the initial part of the duct lumen, while the plasma membrane of the duct cell facing the lumen is cuticularized. The cytoplasm of the accessory cell, on examination by TEM, appears quite similar to that of the duct cell, except for the different distribution and greater abundance of microtubules. Similarly organized tricellular tegumental glands also commonly occur in other Crustacea, both Malacostraca and non-Malacostraca. Possible functions of secretions from the three different types of gland cells present in the labrum of L. dahalacensis are discussed.     

Comparative study of the structure of the reproductive system of dwarfish males of Glyphina betulae (Linnaeus, 1758) and Anoecia (Anoecia) corni (Fabricius, 1775) (Hemiptera, Aphididae)

The morphology and ultrastructure of the male reproductive system of dwarfish males of the monoecious aphid species Glyphina betulae (subfamily Thelaxinae) and the heteroecious species Anoecia (Anoecia) corni (subfamily Anoeciinae) are described. The testicular follicle of these species has the form of a single sac, the proximal parts of the vasa deferentia are slightly (G. betulae) or strongly (A. (A.) corni) expanded, the accessory glands are sack-shaped, and in G. betulae asymmetric and strongly elongated, whereas the ejaculatory duct is short.In both species only mature spermatozoa have been found within the testicular follicles, i.e. the consecutive stages of spermatogenesis have not been observed in adult males. Our studies also show that the testicular follicle, vasa deferentia, accessory glands and ejaculatory duct are histologically very simple. They are composed of more-or-less flattened epithelium of a secretory type, and thin muscle fibres. The epithelial cells are rich in rough endoplasmic reticulum, mitochondria and small vacuoles. The vasa deferentia, especially in G. betulae, are filled with an electron-dense secretion which, as was shown by histochemical staining, contains proteins and polysaccharides. We suggest that the maximum secretory activity of these epithelial cells occurs, as does spermatogenesis, during larval stages, so that the short living adult males are immediately ready for copulation as in other aphids with normal-sized males.