Structural and ultrastructural features of boar bulbourethral glands

The morphological features of boar bulbourethral glands were examined by light and transmission microscopy. Bulbourethral glands are compound tubuloalveolar glands surrounded by a capsule of dense connective tissue and arranged in multiple lobules formed by endpieces and excretory ducts. Endpieces and excretory ducts are both lined by a single epithelium of mucous cells with a basal nucleus. Epithelial cells accumulate secretory granules containing neutral and carboxylated acid mucosubstances and a small amount of sulphated acid mucosubstances. The ultrastructure of epithelial cells varies according to the secretory cycle. In initial stages, the cells show a columnar shape and secretory granules unevenly distributed in the cytoplasm. As the synthesis of mucosubstances progresses, the amount of the secretory granules increases and the cellular shape becomes pyramidal. Secretory granules can contain inclusions and present differences among them according to their different phases of formation. In pyramidal cells, secretory products are released into the lumen by a merocrine mechanism.     

Purification and partial characterization of the 17 kDa sperm coating protein from boar seminal plasma.

Sperm coating proteins of 16, 17, and 19 kDa have been purified from boar seminal plasma. The 17 kDa protein has been identified as an antigen recognized by monoclonal antibody ACR.3 and is thus identical to low molecular mass zona pellucida binding protein from boar spermatozoa (Moos et al., 1990). The 17 and 19 kDa proteins are glycosylated and tend to form hetero-complexes. The 17 kDa ACR.3 antigen is sequentially released from the sperm cell surface during capacitation and, after induction of the acrosome reaction, the 16 kDa form was also observed. Immunocytochemical studies on boar reproductive tissues have suggested that the seminal vesicle epithelium may be the source of these proteins.     

Origin of a sperm motility inhibitor from boar seminal plasma

We have investigated the origin of the sperm motility inhibitor (SPMI) from boar seminal plasma. SPMI was measured by its capacity to inhibit the motility of demembranated spermatozoa and by an enzyme-linked immunosorbant assay (ELISA). Among the various reproductive and now reproductive tissues and fluids tested, only the seminal vesicle fluid and seminal plasma contained significant amounts of SPMI biological activity and SPMI antigen. Like other seminal vesicle fluid proteins, SPMI is diluted 6- to 8-fold upon ejaculation. By immunohistochemical detection at the light microscope with antibodies obtained from rabbits immunized with SPMI purified from boar seminal plasma, SPMI was found in the cytosol and/or on the plasma membrane bordering the lumen of the seminal vesicles. At the electron microscope level, SPMI appeared to be present only on the surface of the secretory cells. The data indicate that SPMI originates from a single tissue, the seminal vesicle, and suggest that only the mature form present on the luminal surface of the gland can react with the antibody generated from rabbits immunized with the secreted form of SPMI. © 1993 Wiley-Liss, Inc.     

Binding of a major secretory protein from bull seminal vesicles to bovine spermatozoa

Summary The seminal vesicles synthesize in an androgen-dependent manner a neutral protein of 13.5 kDa molecular weight that makes up about 40% of their secretion (major protein). An antiserum against this protein raised in rabbits was used to localize the antigen within the seminal vesicles. In addition to intraluminal secretion of the seminal vesicles and the ampulla of the vas deferens, ejaculated and ampullary spermatozoa revealed an intense immunoreaction, which was restricted to the neck region of the sperm head and the middle piece, while the principal piece of the tail as well as the sperm head were devoid of immunoreactive material. Comparison of spermatozoa taken from the tail of the epididymis with ampullary spermatozoa showed that about 90% of the latter, but only 10–20% of the former presented this distributional pattern of immunoreactive sites. Epididymal epithelium as well as calf seminal vesicle epithelium showed no immunoreactivity with major protein antiserum. Using a pre-embedding staining technique with gold-labeled primary or secondary antibodies, respectively, no immunostaining could be achieved at the ultrastructural level. Incubation experiments of epididymal spermatozoa in EGTA-containing solutions in the absence of calcium resulted in a gradual labilization and eventual loss of the plasma membrane of the sperm middle piece. After removal of (at least part of) the plasma membrane, bound major protein could be visualized immunohistochemically close to the mitochondria of the middle piece using a gold-labeled primary or secondary antibody. The acceptor site for major protein therefore seems to reside inside the plasma membrane of the sperm middle piece. Incubation of epididymal spermatozoa in phospholipase-containing solutions removed the acceptor site from the spermatozoa. Separation by polyacrylamide treatment of proteins from epididymal sperm cells extracted by sodium hydroxide or phospholipase treatment, subsequently transblotted on nitrocellulose sheets and directly labeled with gold-tagged major protein, demonstrated a protein duplet with a molecular weight of 65 and 67 kDa, respectively, which appears to represent the specific binder of major protein underneath the sperm surface. Binding of major protein to this 66 kDa acceptor site is regarded as a physiological event that may be related to the onset of hyperactivated sperm motility.Dedicated to Professor Dr. Th.H. Schiebler on the occasion of his 65^th birthdayThis study was supported by the Deutsche Forschungsgemeinschaft (grant Au 48/7-8)     

Effects of extender, incubation temperature, and added seminal plasma on capacitation of cryopreserved, thawed boar sperm as determined by chlortetracycline staining

The effect on apparent capacitation status of frozen-thawed (FT), washed boar sperm was examined in capacitation-supporting medium at 39 °C without added seminal plasma (SP) or supplemented with either 10 or 20% (v/v) boar SP. The thawed sperm from three boars were washed to remove the egg yolk-based freezing medium (EY) and then incubated for 1–8 h after addition of SP. Capacitation status of the sperm was determined microscopically using chlortetracycline staining patterns. At 1 h after the addition of 10 or 20% (v/v) SP, capacitated sperm decreased from 59.7 to 30.3% and from 59.5 to 26.8%, respectively (P < 0.001). Subsequent studies examined the effect of 10% SP on capacitation status of FT sperm extended in either phosphate buffered saline or commercial thawing extender with or without prior washing of sperm to remove EY and incubated at 17 or 39 °C. No effect of SP resulted from addition to sperm when EY remained or when the temperature was maintained at 17 °C (P > 0.1). These results indicate that SP appears able to reverse capacitation of FT boar sperm, but that this effect is dependent on both temperature and composition of the thawing extender.     

Sexual selection affects the sizes of the mammalian prostate gland and seminal vesicles

The accessory reproductive glands of male mammals contribute the bulk of the secretions in which spermatozoa are transported to the female tract during copulation. Despite their morphological diversity,and the chemical complexity of their products,little is known about the possible effects of sexual selection upon these glands in mammals. Here we consider the seminal vesicles and prostate glands in a sample of 89 species and 60 genera representing 8 Orders of mammals. The sizes of the accessory glands are analysed in relation to body weight and testes weight. Both the seminal vesicles size and prostate size (corrected for body weight) correlate positively with relative testes size in this sample; this finding remains highly significant after application of procedures to correct for possible phylogenetic biases in the data set. The accessory reproductive glands are also significantly larger in those mammals which have large relative testes sizes,and in which the likelihood of sperm competition is greatest. These results support the hypothesis that sexual selection has played an important role in the evolution of the mammalian prostate gland and seminal vesicles.     

Transgelin: An androgen-dependent protein identified in the seminal vesicles of three Saharan rodents

During the breeding season, a major androgen-dependent protein with an apparent molecular weight of 21 kDa was isolated and purified from the seminal vesicles of three Saharan rodents (MLVSP21 from Meriones libycus, MSVSP21 from Meriones shawi, and MCVSP21 from Meriones crassus). The 21-kDa protein was isolated and purified from soluble seminal vesicle proteins of homogenate by one-dimensional polyacrylamide gel electrophoresis (SDS-PAGE). Using polyclonal antibodies directed against POSVP₂₁ (Psammomys obesus seminal vesicles protein of 21 kDa), a major androgen-dependent secretory protein from sand rat seminal vesicles, identified previously as transgelin, we showed an immunological homology with POSVP₂₁ by immunoblotting. These three major androgen-dependent proteins with a same apparent molecular weight of 21 kDa designated as MLVSP₂₁ (Meriones libycus seminal vesicles protein of 21 kDa), MSVSP₂₁ (Meriones shawi seminal vesicles protein of 21 kDa), and MCVSP₂₁ (Meriones crassus seminal vesicles protein of 21 kDa) were localized by immunohistochemistry and identified by applying a proteomic approach. Our results indicated that the isolated proteins MLSVP₂₁, MSSVP₂₁, and MCSVP₂₁ seem to correspond to the same protein: the transgelin. So that transgelin can be used as a specific marker of these rodent physiological reproduction mechanisms.     

First evidence of DAAM1 localization in mouse seminal vesicles and its possible involvement during regulated exocytosis

Dishevelled-associated activator of morphogenesis 1 (DAAM1) is a protein belonging to the formin family, which regulates, together with the small GTPase RhoA, the nucleation and the assembly of actin fibres through Wnt-Dishevelled PCP pathway. Its role has been investigated in essential biological processes, such as cell polarity, movement and adhesion during morphogenesis and organogenesis. In this work, we studied the expression of DAAM1 mRNA and protein by PCR and Western blot analyses and its co-localization with actin in adult mouse seminal vesicles by immunofluorescence. We show that both proteins are cytoplasmic: actin is evident at cell–cell junctions and at cell cortex; DAAM1 had a more diffused localization, but is also prominent at the apical plasmatic membrane of epithelial cells. These findings support our hypothesis of a role of DAAM1 in cytoskeletal rearrangement that occurs during the exocytosis of secretory vesicles, and in particular concerning actin filaments. We were also able to detect DAAM1 and actin association in the smooth muscle cells that surround the epithelium too. In this case, we could only speculate the possible involvement of this formin in muscular cells in the maintenance and the regulation of the contractile structures. The present results strongly suggest that DAAM1 could have a pivotal role in vesicle exocytosis and in the physiology of mouse seminal vesicles.     

Investigation of the uterine structural changes in the experimental model with polycystic ovary syndrome and effects of vitamin D treatment: An ultrastructural and immunohistochemical study

In this study, we aimed to investigate the structural changes seen in the endometrium in experimental PCOS rat model and the effects of vitamin D treatment on these changes at immunohistochemical and electron microscopic levels. 24 prepubertal female rats were divided into three groups. Two groups were injected with dehydroepiandrosterone and one of them was treated with 1,25(OH)2 D3 at the same time. The control group was injected with sesame oil. At the end of the 28th day, the blood samples were collected. Uterus tissues were prepared for light and electron microscopic examinations. Epithelial, stromal and endometrial thickness measurements were investigated. Immunohistochemical staining was applied against caspase-3 and Ki-67. Serum AMH and estradiol levels were higher in PCOS group compared to the control group. Serum progesterone levels were similar in all groups. Endometrial, epithelial and stromal thickness measurements were increased in PCOS group compared to the control group, and decreased in the vitamin D treatment group compared to the PCOS group. Light and electron microscopic results of PCOS group showed an increase in apoptosis and proliferation. In the PCOS group, immunohistochemical staining of caspase-3 and Ki-67 were found to be higher than in the control group, but stainings were decreased with vitamin D treatment compared to PCOS group. Structural changes observed in endometrium may be related to implantation problems seen in patients with PCOS. Our studies suggest that vitamin D therapy may be beneficial in these patients.     

Phosphorylcholine-binding proteins from the seminal fluids of different species share antigenic determinants with the major proteins of bovine seminal plasma

The major proteins of bovine seminal plasma, BSP-A1, BSP-A2, BSP-A3, and BSP-30kDa (collectively named BSP proteins) bind to phospholipids containing the phosphorylcholine moiety. An affinity purification method using a p-aminophenyl phosphorylcholine-Agarose (PPC-Agarose) affinity matrix was developed for their purification. In this study, we investigated the distribution of BSP-like analogues in seminal fluid of the human, porcine, hamster, mouse, and rat using this affinity matrix. Alcohol precipitates of the seminal plasma/seminal vesicle secretions (SP/SVS) were further delipidated using isopropyl ether:n-butanol (60:40). The protein preparations obtained were solubilized in a minimal volume of buffer A (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.02% NaN₃), dialyzed against the same buffer, and applied to a PPC-Agarose column connected to a FPLC system. The unbound material was washed out and the adsorbed proteins eluted with buffer A containing 10 mM phosphorylcholine (PrC) and 10 M urea. The fractions were separated by SDS-PAGE, stained or transferred onto a nitrocellulose membrane, and probed with rabbit polyclonal anti-BSP antibodies. Anti-BSP cross-reacting proteins were detected in the seminal fluids of all the species investigated. Moreover, many of these proteins bound to the affinity matrix. The BSP proteins and their immunoreacting analogues appear to be ubiquitous in mammals and may possibly be involved in a common function such as in the modification of the lipid content of the sperm plasma membrane. © 1993 Wiley-Liss, Inc.     

Proteinase inhibitors in aggregated forms of boar seminal plasma proteins

Boar seminal plasma proteins were separated by gel chromatography on Sephadex G-75 into five fractions (I–V). Serine proteinase inhibitors were found mainly in the protein fraction with relative molecular weight 5–25 kDa. Small amounts of these inhibitors were also found in the high molecular weight protein fraction (M_r>100 kDa). The protein fraction containing most of the proteinase inhibitory activity was further separated by RP HPLC. Isolated proteins were characterized by SDS electrophoresis and immunoblotting, N-terminal amino acid sequencing and by determination of the proteinase inhibitory activity. In the fraction containing proteinase inhibitors, also β-microseminoprotein (β-MSP), AQN 1 and lactoferrin were identified. The possible existence of complexes of protein components in the fraction with relative molecular weight 5–25 kDa was studied in detail using gel chromatographic separation on Sephadex G-50. A part of proteinase inhibitors with M_r 8 kDa was eluted together with AQN 1 spermadhesin. An interaction of isolated spermadhesin AQN 1 and proteinase inhibitor was shown.     

Early meiosis in Rhynchospora pubera L. (Cyperaceae) is marked by uncommon ultrastructural features

The family Cyperaceae has an unusual microsporogenesis in which tetrad formation does not occur. In addition, other cytological features are important, such as the occurrence of holokinetic chromosomes and post-reductional meiosis. We have examined the ultrastructural features of the pollen mother cell (PMC) of Rhynchospora pubera. Anthers of several sizes were analyzed using light and transmission electron microscopy. The PMC before meiosis presented a central nucleus and a regular profile of the nuclear envelope. During prophase I, the nucleus was in the abaxial region of the cell. This cellular polarization was accompanied by other marked ultrastructural features in the nuclear envelope. Morphological changes involved dilations of perinuclear cisterns and polarization of the nuclear pore complexes. The results show that polarization occurs in the initial phases of microsporogenesis in R. pubera, unlike other plant species.     

p-Chlorophenylalanine-induced proliferation of the seminal vesicle epithelium

Summary Intraperitoneal injection of p-chlorophenylalanine (pCPA) methylester (100 mg/kg body weight) results in an activation of the lysosomal system of the secretory cells in the rat seminal vesicle and an elevation of the activities of lysosomal enzymes within 15 min following the injection. Large autophagic vacuoles are formed, sequestering rough endoplasmic reticulum and part of the Golgi apparatus within 2 h. Shortly after the activation of the lysosomal system an elevation of both DNA and protein synthesis is measured biochemically. 6 h subsequent to the injection a wave of mitoses of the secretory cells begins, reaching a maximum 6 h later and then declining within 3 h. About 12 h following the injection a second rise in lysosomal activity begins, declining within 24 h. The entire sequence of lysosomal and proliferative activities is inhibited in antiandrogen-pretreated rats. Deduced from these findings the following hypothesis of growth regulation of the accessory sex glands is advanced: enhanced loss of intracellular material during autophagocytosis diminishes the intracellular concentration of a substance curtailing cell division below its effective threshold resulting in division of the secretory cells. The prerequisites of this mechanism are (i) a sufficient distributive capacity of the stroma for hormones (androgens) and metabolic precursors, and (ii) sufficient capacity of the basal cells for transporting the precursors to the secretory cells. Sloughing of the secretory cells separates them from these auxiliary structures (stroma and basal cells) and enables the basal cells to divide.The financial support of the Deutsche Forschungsgemeinschaft is gratefully acknowledged     

Statistical modeling of ultrastructural features of murine dermal collagen under chronic low-dose whole body X-irradiation

The present study investigated the changes in ultrastructural features of dermal collagen fibrils of mice following exposure to different cumulative chronic low-dose X-irradiation through digital image analysis-based statistical modeling. Pubertal mice were X-irradiated and dorsal skin biopsies were collected and processed for transmission electron microscopic (TEM) analysis. TEM features of collagen fibrils showed alteration in the cross-sectional area, population density and in the axial periodic pattern of light and dark bands. The mathematical analysis of histogram data from TEM images revealed some adaptive behavior in collagen structures of the X-irradiated group. This finding indicated that exposure to chronic low-dose X-radiation induced an altered steady state with adaptive variation in dermal collagen fibrils in irradiated mice.     

The ultrastructural architecture of the adult Schistosoma japonicum tegument

The tegument of the adult blood fluke Schistosoma japonicum is in direct contact with the host blood and immune systems. A comprehensive understanding of the ultrastructure of the tegument is crucial to the understanding of how the parasite maintains itself within the mammalian host. Important functions such as nutritional uptake and immune evasion are suspected functions of the tegument and this review discusses these aspects and presents some insights into some of these crucial functions. Transmission electron microscopy has allowed the identification of ultrastructural features of the adult S. japonicum, some of which differ from the reported features of other schistosome species. Morphological differences within the tegument of the adult S. japonicum are noted between sexes, among different regions of the worms and between aspects along the length of the parasite. Differences included variations in the ultrastructure, size and number of tegumental bodies and mitochondria within the matrix, and differences in the relative area of the apical surface of the tegument. Functions of the various components of the tegument matrix and specialised functions of different regions of the male and female parasites are discussed based on ultrastructural findings and previously reported biochemical and molecular data.     

Purification and characterization of a sperm motility-dynein ATPase inhibitor from boar seminal plasma.

A sperm motility inhibitor from boar seminal plasma was purified. The purification procedure included dialysis against 0.1 M Tris-HCl containing 0.1 mM DTT and chromatographies on SP-Sephadex C-25 and Phenyl-Sepharose CL-4B. With this procedure, the seminal plasma motility inhibitor (SPMI) preparation was highly purified with a 18% recovery of inhibitory activity. The molecular weight of SPMI in native conditions has been estimated at 50,000 by molecular sieving, but 3 polypeptides with molecular weights of 14,000, 16,000 and 18,000 were observed following polyacrylamide gel electrophoresis in denaturing conditions. SPMI is a thermolabile basic protein that is stable between pH 6 and pH 11. The observations that SPMI effects on motility of demembranated spermatozoa are reversed by Mg.ATP and that SPMI inhibited bull dynein ATPase in a concentration-dependent manner suggest that this protein blocks the motility of demembranated spermatozoa by interfering with dynein arm function.     

Observations on the marginal ruffles of an established fibroblast-like cell line

Summary LW13K2 cells, a clone of a spontaneously in vitro transformed derivative of embryonic Lewis rat fibroblastic cells, were studied by phase contrast cine-light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The ruffles found at the advancing edge of cells grown on glass substrates in vitro form and recede in a period of less than one min if they do not make an attachment of the substrate. If they fail to make an attachment they may form pinocytotic channels near the leading edge as described by Price (1972) and/or collapse, generally backwards, towards the cell body. The spines which appear to reinforce the membranous ruffles are the last structures to disappear, and accumulate in an irregular array behind the ruffling edge; this area is behind that in which pinocytosis occurs. In comparison with the sparse numbers of ribosomes found in the trailing edge, they are present in notable concentrations near the leading, ruffling edge of the cell. No membrane vesicles have been found in or near the ruffling edges at the ruffle-spine concentration zone.     

Characterization of low Mr zona pellucida binding proteins from boar spermatozoa and seminal plasma.

A group of low Mr (16 kDa-23 kDa) glycoproteins on ejaculated boar spermatozoa have been shown to have high affinity for homologous zona pellucida glycoproteins (ZPGPs). These ZPGP binding proteins are derived from seminal plasma as shown by their absence from epididymal spermatozoa and their presence in seminal plasma as identified by N-terminal amino acid sequence analysis. They bind to ZPGPs by a polysulphate recognition mechanism similar to that found for proacrosin-ZPGP interactions. The haemagglutination activity of boar seminal plasma is also associated with these low Mr glycoproteins. It is suggested that they play a role in regulating the rate of sperm capacitation and survival in the female reproductive tract.     

Structural characterization of the hydrocarbon degrading bacteria–oil interface: implications for bioremediation

Hydrocarbon degrading bacteria, enriched from an in situ bioremediation site in Long Valley, AZ emulsified and colonized the surface of waste engine oil. The application of a partial dehydration conventional embedding protocol for ultrathin-section transmission electron microscopy preserved the hydrocarbon degrading bacteria–surfactant–oil interface. Bacterial adsorption to oil occurred in association with a highly charged, amphipathic bacterial surfactant interface (25–50 nm thick). This biosurfactant completely encapsulated the emulsified oil droplets demonstrating that less than 1% surfactant (by volume) is required to emulsify waste hydrocarbon during or to promote biodegradation. Growth on oil appeared to occur by the uptake of tens of nm-sized droplets of emulsified oil.