Identification and expression analysis of Drosophila melanogaster genes encoding beta-hexosaminidases of the sperm plasma membrane.

Sperm surface beta-N-acetylhexosaminidases are among the molecules mediating early gamete interactions in invertebrates and vertebrates, including man. The plasma membrane of Drosophila spermatozoa contains two beta-N-acetylhexosaminidases, DmHEXA and DmHEXB, which are required for egg fertilization. Here, we demonstrate that three putative Drosophila melanogaster genes predicted to code for beta-N-acetylhexosaminidases, Hexo1, Hexo2, and fdl, are all expressed in the male germ line. fdl codes for a homolog of the alpha-subunit of the mammalian lysosomal beta-N-acetylhexosaminidase Hex A. Hexo1 and Hexo2 encode two homologs of the beta-subunit of all known beta-N-acetylhexosaminidases, which we have named beta(1) and beta(2), respectively. Immunoblot analysis of sperm proteins indicated that the gene products associate in different heterodimeric combinations forming DmHEXA, with an alphabeta(2) structure, and DmHEXB, with a beta(1)beta(2) structure. Immunofluorescence demonstrated that all the gene products localized to the sperm plasma membrane. Although none of the genes was testis-specific, fdl was highly and preferentially expressed in the testis, whereas Hexo1 and Hexo2 showed broader tissue expression. Enzyme assays carried out on testis and on a variety of somatic tissues corroborated the results of gene expression analysis. These findings for the first time show the in vivo expression in insects of genes encoding beta-N-acetylhexosaminidases, the only molecules so far identified as involved in sperm/egg recognition in this class, whereas in mammals, the organisms where these enzymes have been best studied, only two types of polypeptide chains forming dimeric functional beta-N-acetylhexosaminidases are present in Drosophila three different gene products are available that might generate numerous dimeric isoforms.

Purification and characterization of the plasma membrane glycosidases of Drosophila melanogaster spermatozoa.

Previous studies from our laboratory have demonstrated the presence of two integral proteins with glycosidase activity in the plasma membrane of Drosophila melanogaster spermatozoa and we have suggested that these enzymes might have a role in sperm-egg binding. In this study the glycosidases have been purified and characterized. We have evidenced the presence of three distinct enzymes, two beta-N-acetylhexosaminidase isoforms, named HEX 1 and HEX 2, and an alpha-mannosidase. The molecular size of the native enzymes estimated by gel filtration was 158 kDa for beta-hexosaminidases and 317 kDa for alpha-mannosidase. SDS-PAGE showed that HEX 1 and HEX 2 are dimers formed by subunits with different molecular sizes, whereas alpha-mannosidase consists of three subunits with different molecular weights. All the enzymes are terminally glycosylated. Characterization of the purified enzymes included their 4-methylumbelliferyl-substrate preferences, kinetic properties, inhibitor constants and thermal stability. On the basis of substrate specificity, kinetics and the results of inhibition studies, beta-hexosaminidases appear to differ from each other. HEX 1 and HEX 2 are similar to mammalian isoenzyme A and isoenzyme B, respectively. These findings represent the first report on the characterization of sperm proteins that are potentially involved in interactions with the egg in Insects.

Male sterile mutant casanova gives clues to mechanisms of sperm-egg interactions in Drosophila melanogaster.

The plasma membrane of the spermatozoa of Drosophila melanogaster contains two integral proteins with glycosidase activity, beta-N-acetylglucosaminidase and alpha-D-mannosidase. Biochemical analysis and ultrastructural cytochemistry of spermatozoa of the autosomal male sterile mutant casanova reveal that at least one of these enzymes, beta-N-acetylglucosaminidase, is crucial for sperm-egg interactions. casanova sperm are motile, morphologically normal, are transferred to the female at mating, but are unable to fertilize the eggs. The mutation was localised by deficiency mapping to the chromosomal region 95E8-F7. Fluorimetric assays showed that the mutant's sperm have the same level of alpha-D-mannosidase activity as wild-type sperm, whereas beta-N-acetylglucosaminidase activity reaches only 51% of the wild-type level. The biochemical characteristics of alpha-D-mannosidase and of the residual beta-N-acetylglucosaminidase are the same as in wild-type males. Ultrastructural localization of the enzymes indicated that casanova spermatozoa lacks beta-N-acetylglucosaminidase on the plasma membrane covering the acrosome, whereas the location of this glycosidase at the terminal part of the sperm tail is indistinguishable from the wild-type situation. The results strongly suggest that in Drosophila the beta-N-acetylglucosaminidase of the plasma membrane covering the acrosome functions as a receptor for the glycoconjugates on the egg surface. We named the putative egg receptor EROS. This is the first evidence for an egg/sperm recognition system in insects. The mechanism is similar to those known from higher animals.

Plasma membrane association and preliminary characterization of Drosophila sperm surface glycosidases.

Previous studies have identified beta-N-acetylglucosaminidase (GlcNAc'ase) and (alpha-mannosidase activities on the Drosophila melanogaster sperm surface which may have a role in fertilization. The aim of this study was to investigate their linkage to the sperm plasma membrane. We verified that glycosidases are not peripherally adsorbed to the cell surface by evaluating their resistance to release by KI, by buffered salt solutions of high ionic strength or alkaline buffers. Glycosidases were released from the sperm surface by detergents and, only to a minor extent, by mild proteolysis. Differential detergent solubilization pointed out that Triton X-114 was the most effective releasing agent for GlcNAc'ase and CHAPS for mannosidase. No activity was released from the membrane by a phosphatidylinositol-specific phospholipase C (PI-PLC). The released forms were quite hydrophilic in phase separation experiments with Triton X-114. This finding indicates the presence of a hydrophobic domain limited to a single transmembrane helix or/and the presence of an extensive glycosylation. The use of a Con-A binding assay demonstrated that both the enzymes are glycosylated. The molecular weight of the released glycosidases estimated by gel filtration was 158 kDa for GlcNAc'ase and 317 kDa for mannosidase. These results suggest that Drosophila melanogaster GlcNAc'ase and mannosidase are mannosylated integral membrane proteins that would function as exoenzymes with their active sites accessible in the extracellular space.

Mechanism of monocyte-macrophage-mediated transmission of HIV.

Observations from our laboratory support the theory that HIV-infected monocyte-macrophages present in genital tract secretions have an important role in sexual transmission of HIV. Light and electron microscopy were used to study the behavior of HIV-infected, primary human monocytes. These cells progress on surfaces, putting forward a leading pseudopod from which they secrete HIV. When added to cultures of CD4-, cervix-derived epithelial cells, monocytes advanced between epithelial cells while secreting virus anteriorly. Epithelial cells subsequently become productively infected. Infection of epithelia could be blocked by sera from HIV-seropositive individuals. These findings support the supposition that transmission of HIV may occur via cell-mediated infection of intact epithelia. The observations also hint at the possibility that HIV-infected monocyte-macrophages in semen or cervical-vaginal secretions could cross intact epithelia by passing between epithelial cells. To test this hypothesis supravital-stained mouse macrophages were inoculated into the vaginas of mice. Four hours later numerous stained cells were observed in the connective tissue beneath the vaginal epithelium and in the iliac lymph nodes. We speculate that direct infection of epithelial cells and/or cell trafficking across epithelia may be involved in sexual transmission of HIV.

Glycosidases are present on the surface of Drosophila melanogaster spermatozoa.

We investigated the presence of enzymes on the surface of Drosophila melanogaster spermatozoa that might bind the carbohydrate residues of the egg shell. Spectrophotometric and fluorimetric studies were used on whole spermatozoa to assay galactosyltransferase and glycosidase activities. No galactosyltransferase is present on the sperm surface, whereas two glycosidases, beta-N-acetylglucosaminidase (GlcNAc'ase) and alpha-mannosidase (Man'ase), have been evidenced. They have an optimal pH of 6-6.5 and 4, respectively. The same glycosidases were detected as soluble forms probably secreted by the seminal vesicle epithelium. We suggest that these enzymes might be involved in the recognition of alpha-mannose and beta-N-acetylglucosamine residues present on the egg shell at the site of sperm entry.

A role for cell migration in the sexual transmission of HIV-1?

The issue of how human immunodeficiency virus-1 (HIV-1) enters the body following sexual contact has been the subject of considerable controversy. Several possible routes for the initial infection have been suggested [1-6], including the possibility that the transmission is mediated by HIV-1-infected lymphocytes or macrophages in serum and female genital tract secretions, rather than by free virus. We recently reported that HIV-1-infected, activated primary monocytes can migrate between epithelial cells grown in confluent monolayer cultures in vitro [7]. We report here on experiments carried out in mice to test the hypothesis that mononuclear blood cells are capable of migrating through intact epithelia, and thus of carrying a virus into an animal. We placed double-stained, activated mononuclear blood cells into the vaginas of mice; four hours later, numerous double-stained cells were observed in the connective tissue beneath the vaginal epithelium and the iliac lymph nodes of the experimental mice. We speculate that such migration may be involved in the sexual transmission of HIV-1.

Directional budding of human immunodeficiency virus from monocytes.

Time-lapse cinematography revealed that activated human immunodeficiency virus (HIV)-infected monocytes crawl along surfaces, putting forward a leading pseudopod. Scanning electron micrographs showed monocyte pseudopods associated with spherical structures the size of HIV virions, and transmission electron micrographs revealed HIV virions budding from pseudopods. Filamentous actin (F-actin) was localized by electron microscopy in the pseudopod by heavy meromyosin decoration. Colocalization of F-actin and p24 viral antigen by light microscopy immunofluorescence indicated that F-actin and virus were present on the same pseudopod. These observations indicate that monocytes produce virus from a leading pseudopod. We suggest that HIV secretion at the leading edges of donor monocytes/macrophages may be an efficient way for HIV to infect target cells.

Ultrastructural and cytochemical analysis of sperm dimorphism in Drosophila subobscura.

In Drosophila subobscura the male produces two classes of motile spermatozoa that differ in total length and nucleus length. The significance of this within-ejaculate polymegaly is obscure. We have carried out an ultrastructural and cytochemical analysis of both sperm morphs to understand their possible role at fertilization. Computer-aided analysis was used to clarify the complex three-dimensional structure of the spermatozoa. Short and long spermatozoa have a similar architecture. The axoneme is of the classic insect type and, together with the major mitochondrial derivative, runs for almost the whole sperm length. The axoneme ends just below the sperm apex with a centriole adjacent to the acrosome. Minor differences between the two types of sperm are related to acrosome size, nucleus morphology and relationship between nucleus and minor mitochondrial derivative. Cytophotometry of Feulgen stained samples indicated that long and short spermatozoa contain a similar amount of DNA. Both short and long spermatozoa are transferred and stored in the female upon mating. As they have similar ultrastructural and cytochemical characteristics, both sperm are potentially functional in egg penetration and karyogamy.

Glycoconjugates of the surface of the spermatozoa of Drosophila melanogaster: a qualitative and quantitative study.

The important roles played by glycoconjugates in the recognition of gametes and in fertilization are well documented. In the present study, the nature and distribution of carbohydrate residues of the plasma membrane of spermatozoa of Drosophila melanogaster were characterized by use of FITC-conjugated lectins as probes. The plasma membrane bound agglutinins from Concanavalia ensiformis (Con A) and Pisum sativum (PSA), native and succinylated agglutinins from wheat germ (WGA and s-WGA), the A4 isoform of agglutinin-I from Griffonia simplicifolia (GSA-I A4), and, to a lesser extent, the lectins from Dolichus biflorus (DBA), lotus tetragonolobus (LTA), and Glycine maximus (SBA). Each lectin gave a specific pattern of binding. The extent of binding of Con A, WGA, s-WGA, and GSA-I A4 over the acrosomal region was greater than over nonacrosomal regions, indicating the concentration of alpha-mannose/alpha-glucose, beta-N-acetylglucosamine, and alpha-N-acetylgalactosamine residues in this area of the plasma membrane. The surface of the sperm failed to react with lectins from Ricinus communis (RCA-I), Limulus polyphemus (LPA), and Limax flavus (LFA) and with the B4 isoform of agglutinin-I from Griffonia simplicifolia-I (GSA-I B4). The plasma membrane over the nucleus did not react with any of the lectins tested. Quantitative analysis of binding of Con A, s-WGA, and GSA-I A4 to spermatozoa showed that only Con A bound consistently to the sperm surface, showing high affinity for the acrosomal area of the plasma membrane. The other lectins tested bound only to limited and variably sized fractions of the total population of sperm.(ABSTRACT TRUNCATED AT 250 WORDS)

Lymphocyte-facilitated infection of epithelia by human T-cell lymphotropic virus type I.

After the addition of human T-cell lymphotropic virus type I (HTLV-I)-infected lymphocytes to enterocyte monolayers, the lymphocytes adhered via microvilli from both cell types and shed virus onto the enterocyte surface. Virus fused with the epithelial membrane and infected these cells as confirmed by electron microscopic immunocytochemistry, in situ hybridization, and amplification by polymerase chain reaction.

Expression of D-amino acid oxidase in Rhodotorula gracilis under induction conditions: a biochemical and cytochemical study.

D-amino acid oxidase is expressed to a high level in the yeast Rhodotorula gracilis (0.3% of total cell protein) through induction by D-alanine in a defined growth medium. Monospecific polyclonal antibodies against pure enzyme were obtained. Western blot analysis showed that the enzyme is synthesized as the mature polypeptide. The localization of the enzyme was investigated by immunoelectron microscopy using the postembedding immunogold technique and by submicroscopic enzyme cytochemistry. D-Amino acid oxidase was detected in peroxisomes, and quantitation of immunoelectron microscopic data indicated that the enzyme is exclusively confined to these organelles. Immunoelectron microscopic observations are in complete agreement with biochemical data showing that the enzyme is not expressed in the absence of D-alanine. Morphometric analysis demonstrated that induction of D-amino acid oxidase synthesis is associated with a 241% increase of peroxisome volume density and with a 31% increase of peroxisome size as compared to cells grown on non-inducing medium.

Structure of the cumulus oophorus at the time of fertilization.

Ultrastructural and morphometric techniques were employed to examine the ovulated cumulus oophorus of hamsters and rats. Observations on cumuli prepared in a variety of ways including different chemical fixation techniques and cryofixation freeze substitution were compared. It was concluded that the cumulus mucus is not arranged in lamellae or granules as has previously been suggested but is composed of molecules which form very fine filaments when properly fixed. Morphometric analysis of cumuli fixed either in situ or after being explanted into medium revealed that the distance between neighboring cumulus cells was greater with increasing distance from the oocyte. Morphometry revealed that, when placed into medium, the cumulus expands possibly due to hydration. Thus physiological experiments carried out on cumuli should be performed very shortly after cumuli are isolated. From their ultrastructure cumulus cells appear to be actively involved in protein synthesis and secretion as well as steroid production.

Concanavalin A binding sites on the surface of Drosophila melanogaster sperm: a fluorescence and ultrastructural study.

The distribution of alpha-D-mannose/alpha-D-glucose terminal residues in the plasma membrane of Drosophila melanogaster spermatozoon has been investigated by fluorescence and electron microscopy using concanavalin A (Con A) labeling. The results indicate the presence of distinct domains on the sperm surface. Intense binding of Con A to the plasma membrane is highly restricted to the acrosomal region and to the endpiece of the tail. In the former, Con A receptors are not homogeneously distributed, suggesting the presence of microdomains in the acrosomal area. The main part of the tail contains very few Con A binding sites, which are confined to specific areas of the membrane. The sperm surface overlying the nucleus is completely negative. The labeling pattern is unchanged after storage in the female before fertilization. A preliminary analysis of the surface of mature oocytes using fluorochrome-conjugated horseradish peroxidase indicates that D-mannose binding molecules are specifically associated with the chorion of the micropyle anterior part, which might therefore be the site of a preliminary interaction between egg and spermatozoon.

Immunoelectron microscopic localization of D-amino acid oxidase in rat kidney and liver.

The intracellular localization of D-amino acid oxidase in rat kidney and liver has been investigated using the indirect immunogold postembedding technique. Different fixation and embedding conditions for optimal preservation of antigenicity and fine structure have been tested. Immunolabelling was possible only in tissues embedded in polar resins (glycol methacrylate and Lowicryl K4M). In kidney the enzyme was demonstrable only in the peroxisomes of the proximal tubule, where it was associated with the peroxisome core. The enzyme was present in all the peroxisomes of the proximal tubule and appeared to be codistributed with catalase. Control experiments and quantitative analysis confirmed the specificity of the D-amino acid oxidase immunolocalization. All the other cells in kidney failed to demonstrate any labelling. In liver, the immunolabelling was present in the matrix of the hepatocyte peroxisomes, whereas no traces of the enzyme were found in the nucleoid. The intensity of the immunolabelling in liver peroxisomes was lower than in kidney. No specific labelling was observed in cells other than hepatocytes.

Testicular biopsy of azoospermic men with vas deferens malformation using two different techniques.

A morphological analysis was carried out on testicular biopsies of men with obstructive azoospermia as consequence of vas deferens malformations as compared with biopsies from fertile men. For each case the biopsies were processed with two different techniques: routine histological procedure, and semithin sections of specimens processed for electron microscopy. Four parameters were considered: tubular morphology, testicular biopsy score count (TBSC), tubular diameter, and germinal cell density. The data were quantified and analyzed by statistical tests. The biopsies of azoospermic men present a higher frequency of tubular sections with "tubular blockage," "sloughing" and cellular degeneration, lower values of TBSC, and lower germinal cell density. The results appear to be of relevant interest in relation to the existence of long-term testicular alteration following vasectomy.

Similarities and differences in the ultrastructure of two hormone-dependent and one independent human breast carcinoma grown in athymic nude mice: comparison with the rat DMBA-induced tumor and normal secretory mammocytes.

The human breast cancer cell lines MCF-7 (ER positive), ZR 75-1 (ER positive) and MDA-MB 231 (ER negative) form solid tumors within one week following inoculation into athymic nude mice. Tumor formation by MCF-7 and ZR 75-1 cells was dependent upon estrogen, whereas MDA-MB 231 cells formed tumors in ovariectomized mice with or without supplemental estrogen. Ultrastructural comparison of tumors formed by the three human breast carcinoma lines in athymic nude mice indicated that lactoperoxidase activity, milk protein and fat globule formation were virtually absent from all three tumors. The estrogen-dependent tumors (MCF-7, ZR 75-1), however, had more desmosomes, intermediate-sized microfilaments and collagen than the estrogen-independent tumor (MDA-MB 231). When the ultrastructure of the three human tumors was compared to the hormone-dependent, DMBA-induced rat mammary carcinoma and to the normal lactating rat mammocytes, the following observations were evident: a) the estrogen-dependent human tumors closely resembled the normal rat tissue in the distribution of desmosomes and collagen, b) the rat mammary carcinoma differed from both the estrogen-dependent and -independent human tumors, in having milk protein, milk fat globules and intense lactoperoxidase activity. The results indicate that these hormone-dependent and -independent human mammary tumors maintained in athymic nude mice differ markedly in their ultrastructure from the lactating rat mammocytes and the rat DMBA-induced mammary carcinoma.

Quantitative cytochemistry of the diaminobenzidine cytochrome oxidase reaction product in mitochondria of cardiac muscle and pancreas.

The rate constant (k) of the cytochrome oxidase reaction under optimal conditions for cytochemical staining (i.e., 15 min fixation, incubation for 180 min for heart, 120 min for pancreas) can be used as a measure of the enzyme concentration within mitochondria. The rate constant derived from microdensitometric measurements of the mass thickness of the 3,3'-diaminobenzidine (DAB) cytochrome oxidase reaction in cristae times correlated data derived from morphometry on the surface density of cristae (SVcristae/Vmit micron-1) and the volume density of mitochondria per cell (Vmit/Vcell) has been used to determine the respiratory index (RI) of these tissues according to the following equation: RI = k(SVcristae/Vcell). Using this formula, the RI of cardiac muscle tissue was computed to be 33 times the RI of pancreas under the conditions of our experiments. The greater cristae surface density and the large mitochondrial volume density in cardiac muscle and high k value accounted for the higher RI of cardiac muscle.

Ultrastructural study of the decapitated sperm defect in an infertile man.

An infertile man presented a spermiogram in which 100% of the spermatozoa displayed separation of head from tail at the level of the proximal centriole. Most tails were normally structured and ended anteriorly with the proximal centriole covered by a continuous plasma membrane. In a small percentage of tails a rudimentary connecting piece was surrounded by a minute cytoplasmic mass and the middle piece was missing, whereas the chromatoid body and the spindle-shaped body were still present. Finally, a few tails had a large cytoplasmic mass surrounding either regular connecting and middle pieces or a rudimentary connecting piece continuous with the main piece. Tails of the first type had good forward motility, although the pattern of movement appeared altered. The other types were immotile or motile but without forward progression. In the loose heads the implantation fossa had failed to differentiate. The separation of heads from tails appeared to be the result of a specific morphogenetic defect and took place at different stages of spermatid differentiation, giving rise to the structurally different types of tails.