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Fertilization is a complex and multiphasic process, consisting of several steps, where egg-coating envelope's glycoproteins and sperm surface receptors play a critical role. Sperm-associated β-N-acetylglucosaminidases, also known as hexosaminidases, have been identified in a variety of organisms. Previously, two isoforms of hexosaminidases, named here DmHEXA and DmHEXB, were found as intrinsic proteins in the sperm plasma membrane of Drosophila melanogaster. In the present work, we carried out different approaches using solid-phase assays in order to analyze the oligosaccharide recognition ability of D. melanogaster sperm hexosaminidases to interact with well-defined carbohydrate chains that might functionally mimic egg glycoconjugates. Our results showed that Drosophila hexosaminidases prefer glycans carrying terminal β-N-acetylglucosamine, but not core β-N-acetylglucosamine residues. The capacity of sperm β-N-acetylhexosaminidases to bind micropylar chorion and vitelline envelope was examined in vitro assays. Binding was completely blocked when β-N-acetylhexosaminidases were preincubated with the glycoproteins ovalbumin and transferrin, and the monosaccharide β-N-acetylglucosamine. Overall, these data support the hypothesis of the potential role of these glycosidases in sperm-egg interactions in Drosophila., (© 2017 Wiley Periodicals, Inc.)

Sperm-oocyte interaction during fertilization is multiphasic, with multicomponent events, taking place between egg's glycoproteins and sperm surface receptors. Protein-carbohydrate complementarities in gamete recognition have observed in cases throughout the whole evolutionary scale. Sperm-associated α-L-fucosidases have been identified in various organisms. Their wide distribution and known properties reflect the hypothesis that fucose and α-L-fucosidases have fundamental function(s) during gamete interactions. An α-L-fucosidase has been detected as transmembrane protein on the surface of spermatozoa of eleven species across the genus Drosophila. Immunofluorescence labeling showed that the protein is localized in the sperm plasma membrane over the acrosome and the tail, in Drosophila melanogaster. In the present study, efforts were made to analyze with solid phase assays the oligosaccharide recognition ability of fruit fly sperm α-L-fucosidase with defined carbohydrate chains that can functionally mimic egg glycoconjugates. Our results showed that α-L-fucosidase bound to fucose residue and in particular it prefers N-glycans carrying core α1,6-linked fucose and core α1,3-linked fucose in N-glycans carrying only a terminal mannose residue. The ability of sperm α-L-fucosidase to bind to the micropylar chorion and to the vitelline envelope was examined in in vitro assays in presence of α-L-fucosidase, either alone or in combination with molecules containing fucose residues. No binding was detected when α-L-fucosidase was pre-incubated with fucoidan, a polymer of α-L-fucose and the monosaccharide fucose. Furthermore, egg labeling with anti-horseradish peroxidase, that recognized only core α1,3-linked fucose, correlates with α-L-fucosidase micropylar binding. Collectively, these data support the hypothesis of the potential role of this glycosidase in sperm-egg interactions in Drosophila., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The anti-inflammatory, antioxidant, antimicrobial and cytotoxic activities of the hexane (HE), chloroform (CE) and methanol (ME) extracts obtained from the bark of Prunus tucumanensis Lillo were investigated. Both ME and CE extracts displayed a significant in vitro anti-inflammatory activity similar to dexamethasone and to a commercial formulation (Pygeum) used for the treatment of benign prostatic hyperplasia (BPH). ME exhibited powerful antioxidant (67.6% relative to BHT) and free radical scavenging (RC50 = 5 ppm) activities, antimicrobial activities against Staphylococcus aureus and Mycobacterium smegmatis and did not show any cytotoxic effect on human-derived macrophage cells. Chemical analyses showed that (2 R,3 R)-3,5,7,3',5'-pentahydroxyflavan, β-sitosterol and β-sitosterol-3-O-β-D-glucopyranoside (daucosterol) are relevant components of ME.

The Mediterranean fruit fly Ceratitis capitata (Diptera: Tephritidae) is one of the most destructive agricultural pests, a polyphagus insect of relevant economic importance and is widespread in many regions around the world. It is the best-studied fruit fly pest at genetic and molecular level and much has been learned on its ecology and behaviour. An α-L-fucosidase has been recently hypothesized to be involved in sperm-egg interactions in Drosophila melanogaster and in other Drosophila species. Here, a complete cDNA encoding a putative α-L-fucosidase of the medfly was amplified using the reverse polymerase chain reaction (RT-PCR) with degenerate based on the conserved coding sequence information of several insect α-L-fucosidases, cloned and sequenced (GenBank accession no. FJ177429). The coding region consisted of 1482 bp which encoded a 485-residues protein (named CcFUCA) with a predicted molecular mass of 56.1 kDa. The deduced protein sequence showed 75% amino acid identity to D. melanogaster α-L-fucosidase, and in fact the phylogenetic tree analysis revealed that CcFUCA had closer relationships with the α-L-fucosidases of drosophilid species. The tissue expression analysis indicated that CcFuca was expressed in a single transcript in all tissues, suggesting a ubiquitous localization pattern of the encoded protein. Our findings provide novel insights on a gene encoding a protein potentially involved in primary gamete interactions in C. capitata., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The goal of this study was to identify the genes coding for β-N-acetylhexosaminidases in the Mediterranean fruit fly (medfly) Ceratitis capitata, one of the most destructive agricultural pests, belonging to the Tephritidae family, order Diptera. Two dimeric β-N-acetylhexosaminidases, HEXA and HEXB, have been recently identified on Drosophila sperm. These enzymes are involved in egg binding through interactions with complementary carbohydrates on the surface of the egg shell. Three genes, Hexosaminidase 1 (Hexo1), Hexosaminidase 2 (Hexo2) and fused lobes (fdl), encode for HEXA and HEXB subunits. The availability of C. capitata EST libraries derived from embryos and adult heads allowed us to identify three sequences homologous to the D. melanogaster Hexo1, Hexo2 and fdl genes. Here, we report the expression profile analysis of CcHexo1, CcHexo2 and Ccfdld in several tissues, organs and stages. Ccfdl expression was highest in heads of both sexes and in whole adult females. In the testis and ovary the three genes showed distinct spatial and temporal expression patterns. All the mRNAs were detectable in early stages of spermatogenesis; CcHexo2 and Ccfdl were also expressed in early elongating spermatid cysts. All three genes are expressed in the ovarian nurse cells. CcHexo1 and Ccfdl are stage specific, since they have been observed in stages 12 and 13 during oocyte growth, when programmed cell death occurs in nurse cells. The expression pattern of the three genes in medfly gonads suggests that, as their Drosophila counterparts, they may encode for proteins involved in gametogenesis and fertilization., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Fruit flies in the family Tephritidae are rated among the world's most destructive agricultural pests. The Mediterranean fruit fly Ceratitis capitata is emerging as a model organism to study the fertilization in Insects. Three integral proteins with glycosidase activity are present in the plasma membrane of spermatozoa. The glycosidases have been purified and characterized. We have demonstrated the presence of three enzymes, a β-N-acetylhexosaminidase, an α-mannosidase and an α-l-fucosidase. The molecular mass of the native enzymes estimated by gel filtration was 160 kDa for β-N-acetylhexosaminidase, 310 kDa for α-mannosidase and 140 kDa for α-l-fucosidase. SDS-PAGE showed that β-N-acetylhexosaminidase is a dimer of a single protein of 73 kDa, α-mannosidase consists of six subunits with different molecular weights and α-l-fucosidase is a dimer made up by two different monomers. Characterization of the purified enzymes included glycosylation pattern, pI, optimal pH, substrate preference, kinetic properties and thermal stability. Soluble forms similar to the sperm associated glycosidases are present. Polyclonal antibodies raised against synthetic peptides designed from the predicted products of the Drosophila melanogaster genes encoding β-N-acetylhexosaminidase and α-l-fucosidase were used. Immunofluorescence labelling of spermatozoa showed that the enzymes are present in the sperm plasma membrane overlying the acrosome and the tail. This work represents the first report on the characterization in C. capitata of sperm proteins that are potentially involved in primary gamete recognition., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

We have studied the presence of four sperm glycosidases, alpha-mannosidase, alpha-L- fucosidase and two beta-hexosaminidase isoforms, in 11 species of the genus Drosophila spanning approximately an evolutionary 60 MY period, and in Scaptodrosophila lebanonensis, belonging to the ancestor genus Scaptodrosophila. These enzymes had been previously identified in Drosophila melanogaster as putative receptors for glycoconjugates of the egg surface. Alpha-mannosidase and beta-hexosaminidases are intrinsic proteins of the sperm plasma membrane in species closely related to D. melanogaster as well as in the divergent species D. willistoni, D. hydei, D. virilis, and S. lebanonensis. Alpha-L-fucosidase is restricted to the species of the genus Drosophila. Alpha-mannosidase and beta-hexosaminidases have been purified and characterized in all species. Their molecular masses and optimal pHs are similar in all the species, whereas interspecific differences in enzyme activities were detected. Cross-species comparison of kinetic parameters indicated a relationship between enzyme efficiency and phylogenetic relatedness. Beta-hexosaminidases were the most efficient enzymes. Lectin cytochemistry suggested the presence of carbohydrate residues complementary to the glycosidases on the eggshell at the site of sperm entry in all species. Bioinformatic analysis of the coding sequences of beta-hexosaminases and alpha-L-fucosidase and of their predicted products showed no evidence of positive selection of the genes coding for these enzymes and a high degree of sequence identity of the predicted polypeptides among the species of the genus Drosophila. Collectively, our findings indicate that the Drosophila sperm glycosidases are structurally and functionally conserved and strengthen the hypothesis of their involvement in the interactions with the egg surface.

Fucosylated glycoconjugates play a role in a wide variety of biological processes, including immune responses, signal transduction, ontogenic events and pathogenesis of several human diseases. Alpha-L-fucosidases, which are responsible for their processing, have been demonstrated to be involved in lysosomal storage disease, inflammation, cystic fibrosis, cancer development and in the interactions between gametes in vertebrates as well as invertebrates. The sequence and comparative genomic analysis of these glycosyl hydrolases and the study of their evolutionary relationships appear therefore to be of considerable interest. In this work we carried out extensive similarity searches and comparative analyses to identify sequences encoding alpha-L-fucosidases. We have identified novel alpha-L-fucosidase coding sequences in worms, insects, sea urchin, ascidians, fish, chicken, amphibians, mammals and various bacteria resulting in a total of 39 alpha-L-fucosidase sequences. Two alpha-L-fucosidases that are present in all vertebrates likely reflect a distinct biological role for paralogous genes. Comparative sequence analysis of all metazoan alpha-L-fucosidases reveals a broad conservation of features, including the aspartate residue that constitutes the catalytic nucleophile. However, a cysteine which is thought to be part of the active site is also conserved in metazoa but not in arthropods, where it is replaced by an alanine. Phylogenetic analysis suggests a gene duplication event very early in metazoan evolution with the subsequent differential loss of isoforms in various metazoan lineages.

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.

Drosophila is emerging as a model organism to investigate egg fertilization in insects and the possible conservation of molecular mechanisms of gamete interactions demonstrated in higher organisms. This study shows that the spermatozoa of several species of Drosophila belonging to the melanogaster group have a plasma membrane associated alpha-L-fucosidase with features in common with alpha-L-fucosidases from sperm of other animals, including mammals. The enzyme has been purified and completely characterized in D. ananassae, because of its stability in this species. The sperm alpha-L-fucosidase is an integral protein terminally mannosylated, with the catalytic site oriented toward the extracellular space. It has a M(r) of 256 kDa and a multimeric structure made up by subunits of 48 and 55 kDa. Enzyme characterization included kinetic properties, pI, optimal pH, and thermal stability. A soluble form of the enzyme similar to the sperm associated alpha-L-fucosidase is secreted by the seminal vesicles. Synthetic peptides designed from the deduced product of the D. melanogaster gene encoding an alpha-L-fucosidase were used to raise a specific polyclonal antibody. Immunofluorescence labeling of spermatozoa showed that the enzyme is present in the sperm plasma membrane overlying the acrosome and the tail. Lectin cytochemistry analysis of the egg surface indicated that alpha-L-fucose terminal residues are present on the chorion with a strongly polarized localization on the micropyle. The alpha-L-fucosidase of Drosophila sperm plasma membrane appears to be potentially involved in gamete recognition by interacting with its glycoside ligands present on the egg surface at the site of sperm entry.

Considerable evidence suggests that human immunodeficiency virus (HIV)-infected macrophages and/or lymphocytes may mediate sexual transmission of HIV. We and others have previously demonstrated that when vitally stained donor mouse lymphocytes or macrophages are placed in the vaginas of mice, some of the stained cells can later be found in the iliac lymph nodes. The aim of this study was to assess the extent of mononuclear cell trafficking from the vagina and to test the possibility that carrageenan formulation, a sulfated polysaccharide formulation containing 3% PDR98-15 carrageenan (PC-515; FMC Biopolymer, Rockland, ME), a vaginal microbicide, would prevent vaginal transmigration of macrophages. When supravitally stained mouse macrophages and T cells were inoculated into the vagina of recipient mice, discrete numbers of donor cells migrated to the recipient iliac and inguinal lymph nodes and spleen. When recipient mice were preinoculated with the carrageenan formulation, the number of macrophages in lymph nodes and spleen was reduced by >90%. In contrast, a methylcellulose formulation, which is believed to be inactive, did not significantly reduce migration to the lymphoid organs. Our findings suggest that the carrageenan formulation blocks cell trafficking of macrophages from vagina and that blocking does not result from cytotoxicity. Blocking cell trafficking may help to prevent sexual transmission of HIV.

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.

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.

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.

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.

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.

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.

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.

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)

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.

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.

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.

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.

Ten testicular biopsies from adult males with agenesis of the vas deferens have been investigated with light and electron microscopy. The ultrastructural study of SERTOLI cells has demonstrated that: (a) the junctions between SERTOLI cells and between SERTOLI and germinal cells retain their normal ultrastructural features. (b) Lanthanum permeates SERTOLI cells tight junctions, but its diffusion towards the adluminal compartment is prevented. (c) SERTOLI cells present unusual amounts of cytoplasmic inclusions, especially lipofuscin granules. A relationship between these inclusions and phagocytosis has not been demonstrated. (d) "Dark" SERTOLI cells which show alterations of their typical junctional complex have been frequently observed, although their frequency varies among the different patients. The results are discussed in relation to diagnostic significance of the ultrastructural study of testicular biopsies.

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.

Cytochrome oxidase activity via cytochrome c, as demonstrated by the diaminobenzidine procedure, has been employed in this electron microscope cytochemical study to determine the respiratory potency, integrity and fate of the Arbacia sperm mitochondrion at fertilization and during early embryogenesis. The sperm mitochondrion remained intact and was intensely positive for cytochrome oxidase activity both during and after penetration into the egg. The mitochondrion remained highly reactive throughout zygote formation, up to the eight-cell stage. The sperm mitochondrion formed many projections and buds in the cytoplasm of immature oocytes, monospermic and polyspermic eggs, and in blastomeres. At all stages of early embryogenesis, close juxtaposition and structural contact were observed between the highly reactive sperm mitochondrion and the less reactive egg mitochondria. The results suggest that following fertilization the mitochondrion of the sea urchin spermatozoon retains some degree of metabolic autonomy within the ooplasm. The structural integrity of the paternal mitochondrion is maintained along with a functional respiratory enzyme system (cytochrome c-a3). The hypothesis that the fertilizing sperm mitochondrion may have some relevance to sea urchin development is discussed.

Events following the binding of Concanavalin A and cationized ferritin to the surface of normal human peripheral blood lymphocytes were followed using ultracytochemical methods. In this paper, we described a technique involving microdensitometry, which was employed in order to make a comparative cytochemical quantitation of the dense horseradish peroxidase-diaminobenzidine reaction products representing the states of Concanavalin A receptors under various conditions of distribution and redistribution. The technique presents a direct approach to the analysis of various probes that are currently being employed for studying the nature and distribution of various cell surface components. An attempt was also made to analyze the distribution of anionic sites, and their lateral redistribution is compared with the mobility of Concanavalin A receptors. The data presented suggests an independent mobility of receptors for Concanavalin A and cationized ferritin.

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.

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.

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.

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.