The Origin of Membrane Vesicles in Ram Seminal Plasma

The hypothesis tested in this study was that the membrane vesicles present in ram seminal plasma are of testicular origin, rather than being secreted by the accessory sex glands as has been previously reported for a number of species. Membrane vesicles were present in cellular extracts from reproductive organs and accessory sex glands of six rams, and in the seminal plasma of a further eight rams. When four of the latter rams were subjected to vasectomy, to isolate ejaculate contents to only the secretions of the accessory sex glands, the vesicles were largely eliminated from their ejaculates, while vesicles were still present in the ejaculates of the four control rams. The constituents of the cytoplasmic droplets and membrane vesicles derived from the seminal plasma were compared by transmission electron microscopy (TEM). Vesicles present in the cytoplasmic droplets were similar in morphology but smaller on average than those in the seminal plasma. It was concluded that the membrane vesicles in ram seminal plasma originate from either the cytoplasmic droplets, or a combination of vesicles from the droplets and the epididymis.     

The Endometrium of the Anoestrous Female Pig: Studies on Infiltration by Cells of the Immune System

The aim of this study was to investigate the distribution of immune cells in the endometrium of anoestrous female pigs, five sows in anoestrus by lactation and five pre-pubertal gilts (Swedish Landrace x Swedish Yorkshire). Uterine samples, taken immediately after slaughter, were fixed, embedded in plastic resin and stained with toluidine blue or cryo fixed and stored in a freezer at -70 degrees C until analysed by immunohistochemistry with an avidin-biotin peroxidase method. Immune cells in the surface (luminal) and the glandular epithelium as well as the subepithelial and the glandular connective tissue layers were counted using light microscopy. In the surface (luminal) and the glandular epithelia of gilts and sows, lymphocytes were the predominant immune cells found. There were no significant differences between gilts and sows. Macrophages were detected in the glandular epithelium of sows but not in gilts. In the subepithelial and the glandular connective tissue layers of both gilts and sows, lymphocytes were also the most common immune cells found. The numbers of lymphocytes and macrophages were significantly higher in the sows than in the gilts (p 相似文献   

Licht-und elektronenoptische Untersuchungen an der Membrana limitans gliae superficialis und der Leptomeninx im Rückenmark des Schweines*

Light and electron microscopical examinations on the membrana limitans gliae superficialis and the leptomeninx of the spinal cord of the pig. The membrana limitans gliae superficialis of the pig varies in thickness and consists mainly of processes of fiber-rich astrocytes which, apart from normal mitochondria, contain giant mitochondria up to 7.5 μm in size. On the surface of the membrana limitans is a continuous basement membrane. The pia mater spinalis is fibrous. There are mainly collagenous fibers, but also elastic fibers, microfibrils, fibroblasts, fibrocytes, macrophages, blood vessels and bundles of nerve fibers. The structure of the arachnoid is by and large similar to that of other mammals. The subarachnoid space is lined with a continuous layer of mesothelial cells which in the region of the pia has a discontinuous basement membrane. The mesothelial cells may contain membranous vesicles and have desmosomes and nexus connections. No gaps and pores were observed in this cell layer, and it is thought that this layer prevents direct contact between cerebrospinal fluid and the connective tissue of the leptomeninx. On the basis of structural findings, literature and our own examinations, the problems of cerebrospinal fluid resorption are discussed.     

Cytochemical localization of glycoconjugates in the secretory epithelial cells lining the prostate gland of the miniature pig

The aim of the study was to emphasize several specific functional aspects of the mammalian prostate gland. In this connection, the subcellular localization and characterization of glycoconjugates in the secretory epithelial cells were examined that line the prostate gland of the miniature pig, using cytochemistry, including lectin-gold methods. The results verified a diversified pattern of glycoconjugates in the structures of the secretory cells. The secretory granules and the free surface coat of the plasma membrane contained several sugar moieties such as alpha-D-Man, alpha-D-Glc, alpha-L-Fuc, beta-D-Gal, GalNAc, GlcNAc and NeuAc (sialic acid), whereas the Golgi apparatus exhibited GlcNAc and sialic acid residues. In addition, the cisterns of the endoplasmic reticulum showed alpha-D-Man and alpha-D-Glc moieties. It seems likely that the secretory glycoconjugates demonstrated contribute to favourable conditions for fertilization.     

Autologous Whole Ram Seminal Plasma and its Vesicle-free Fraction Improve Motility Characteristics and Membrane Status but not In Vivo Fertility of Frozen–Thawed Ram Spermatozoa

Motility characteristics (assessed subjectively and with computer-assisted semen analysis) and membrane status (after staining with chlortetracycline) of washed and non-washed frozen-thawed ram spermatozoa were evaluated after incubation in buffer and buffer containing autologous whole seminal plasma or one of its two fractions: the pellet of membrane vesicles obtained by ultracentrifugation (and used at three times normal protein concentration) or the vesicle-free supernatant fraction. Whole seminal plasma and supernatant, but not membrane vesicles, improved the motility characteristics of spermatozoa after 3 and 6 h of post-thaw incubation compared with the control buffer. Resuspension and incubation with whole seminal plasma, supernatant or membrane vesicles lowered the proportion of acrosome-reacted frozen-thawed spermatozoa compared with the control buffer. Unwashed frozen-thawed semen from three rams, incubated with autologous whole seminal plasma or its fractions and inseminated using cervical or intrauterine artificial insemination, had no effect on pregnancy rates of ewes in synchronized oestrus. However, fertility was higher after laparoscopic than cervical insemination (44.9 vs 12.3%, p < 0.001). In conclusion, resuspension and incubation of frozen-thawed ram spermatozoa in autologous whole seminal plasma or its vesicle-free supernatant fraction improved their motility characteristics and, with membrane vesicles, membrane status, but these benefits were not reflected in improved fertility after cervical or intrauterine insemination.     

Feinstruktur des groβen Netzes von Schaf und Ziege

The ultrastructure of the adipose tissue connective tissue, mesothelium and milky spots forming the greater cmentum of sheep and goats was studied.
The mixture of connective tissue fibers and adipose tissue is arranged in submesothelial, marginal, and central layers. – The mesothelial cells are covered by a surface coat and are characterized by numerous microvilli and vesicles. The number of milky spots varies greatly. They are commonly found on the surface of the omentum and are covered by modified mesothelial cells; their interior contains macrophages and lymphoid cells.     

La vascularisation du feuillet du boeuf)

The blood vascular system of the bovine omasum The blood vessels supplying and draining the omasal wall and laminae are described and are compared with those of other ruminants. A detailed study revealed that the subepithelial vascular networks in the papillae were in part similar to those of intestinal villi. One or more arterioles branch radially and form a capillary network which does not, as in the villus, drain into a central venule but is succeeded by several subepithelial venules. Capillaries and venules differ only in diameter which are 10 μm. and 40 μm. respectively. The walls of both have a complete basal membrane, perforated endothelium and pinocytotic vesicles. This emphasizes their significance in resorption. Inside and between the papillae the capillaries form a dense network which is separated from the basal cells by a thin layer of connective tissue 5 to 6 μm. thick. Hemodynamic problems related to the presence of elastic connective tissue and to the motor activity of the omasum are discussed.     

Elastic Fibre System of the Female Canine Urethra. Histochemical Identification of Elastic, Elaunin and Oxytalan Fibres

The elastic system fibres of the female urethra were investigated in seven dogs of different breeds. The fibres were stained and differentiated with orcein, Verhoeff's iron hematoxylin, and pararosanilin-based aldehyde fuchsin, with and without previous oxidation. Orcein and aldehyde fuchsin revealed some subepithelial longitudinally orientated delicate fibres (elaunin fibres) and numerous coarse longitudinal fibres (elastic fibres) in the deeper subepithelial connective tissue containing the vascular plexus, as well as a network of fibres of different calibers in the periurethral connective tissue. Elastic system fibres were also found in association with the sinusoids of the vascular plexus and the urethral smooth musculature. Verhoeff's iron hematoxylin only reacted distinctly with coarse (elastic) fibres. When applied following oxidation, aldehyde fuchsin disclosed an extensive meshwork of additional delicate fibres (oxytalan fibres) in the subepithelial connective tissue adjacent to the basement membrane. Oxytalan fibres were also discernible in the sinusoidal adventitia of the vascular plexus, as well as between smooth and striated muscle fibres. Due to the predominantly longitudinal orientation of the elastic system fibres and the low urethral resistance to manual expression of urine from the bladder post mortally, when the elasticity of the urethra is still intact and the activity of other continence factors can be excluded, the elastic tissue is not judged to be a major contributory factor to urinary continence.     

The ultrastructure of Strongylus vulgaris-mediated equine chronic mesenteric arteritis

Cells found in the intima and media of the cranial mesenteric artery of a mature mare with chronic arteritis were identified as smooth muscle cells and occurred in association with collagen and elastin fibres. As no fibroblasts were demonstrable within these regions, the smooth muscle cells were the likely source of the extracellular matrix. In contrast, the abnormal adventitis from the same artery contained abundant fibroblasts which are considered to be the source of the adventitial collagen.     

Characterization and Localization of Membrane Vesicles in Ejaculate Fractions from the Ram, Boar and Stallion

Membrane vesicles, separated by differential centrifugation from the seminal plasma, were detected in the sperm-rich ejaculate fractions of four boars and three stallions, and in the whole ejaculates of seven rams. The volume and percentage of vesicles, determined by a stereological technique, were higher in the sperm-rich than in the post-sperm-rich fractions of the boar and stallion ejaculates, and no vesicles were detected in the pre sperm-rich fractions. Vesicles were examined by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). For boar, stallion and ram semen, the mean (+/- s.e.m.) vesicle diameters were 130.9 +/- 3.22 (range 18-577), 164.1 +/- 4.42 (range 15-671) and 159.7 +/- 2.92 nm (range 22-986), respectively, although they were not significantly different (p = 0.709). The vesicles had approximately round (TEM) or spherical shape (SEM), were surrounded by single, double or multi-laminar membranes, and were trapped within ample amorphous material, sometimes containing short, flattened membranous elements. The majority of the vesicles had a clear interior but some contained granule-dense material. Ram membrane vesicles, purified from ultracentrifuged plasma by size exclusion chromatography, kept their round shape and the amorphous material was less evident compared with the sections taken before purification. This is the first report to identify seminal plasma membrane vesicles in the different fractions of ejaculated semen in the boar and stallion, and confirms their presence in ram seminal plasma. The origin and function of these vesicles are yet to be elucidated.     

Chicken seminal fluid lacks CD9- and CD44-bearing extracellular vesicles

The avian seminal fluid (SF) is a protein-rich fluid, derived from the testis, the rudimentary epididymis and, finally, from the cloacal gland. The SF interacts with spermatozoa and the inner cell lining of the female genital tract, to modulate sperm functions and female immune responsiveness. Its complex proteome might either be free or linked to extracellular vesicles (EVs) as it is the case in mammals, where EVs depict the tetraspanin CD9; and where those EVs derived from the epididymis (epididymosomes) also present the receptor CD44. In the present study, sperm-free SF from Red Jungle Fowl, White Leghorn and an advanced intercross (AIL, 12th generation) were studied using flow cytometry of the membrane marker tetraspanin CD9, Western blotting of the membrane receptor CD44 and electron microscopy in non-enriched (whole SF) or enriched fractions obtained by precipitation using a commercial kit (Total Exosome Precipitation Solution). Neither CD9- nor CD44 could be detected, and the ultrastructure confirmed the relative absence of EVs, raising the possibility that avian SF interacts differently with the female genitalia as compared to the seminal plasma of mammals.     

Light and electron microscopic study of the thoracic respiratory air sacs of the fowl

There are conflicting reports in the existing literature on the nature of the epithelial lining and the content of the supporting connective tissue of the respiratory air sacs of birds. The present study describes the light and electron microscopic structure of the thoracic air sacs of the fowl. A simple squamous epithelium lined the greater part of the thoracic air sacs. The squamous cells characteristically contained vesicles filled with lamellar or myelinoid material. Localized areas of cuboidal to columnar ciliated epithelium were randomly distributed and often associated with underlying blood vessels. Isolated ciliated cells first appeared on squamous or low cuboidal cells and increased in frequency as the cells became taller. Occasional basal and goblet cells were seen between the ciliated columnar cells. A fibrous connective tissue stroma supported the epithelium. Fine elastic fibres were particularly prevalent immediately below the epithelium. Isolated smooth muscle myocytes were present in the connective tissue stroma. A sheet of smooth muscle extended some distance into the membrane from the attachment of the latter to the body wall. Numerous small blood vessels, lymphatics and occasional nerve bundles were observed in the stroma.     

Seasonal variations in the morphology of the seminal vesicles of the African straw-coloured fruit bat (Eidolon helvum)

The study investigated seasonal changes in the morphology of the seminal vesicles of Eidolon helvum in a typical African tropical environment. Seminal vesicles of forty-eight bats were examined during the early rainy, late rainy and peak dry seasons, using gross anatomical, histological and ultrastructural techniques. The interlobular connective tissue septa from the external capsule demarcated the glandular parenchyma into numerous lobules of tubulo-alveolar glands. A simple cuboidal epithelium of mono- or bi-nucleated cuboidal cells lined the glandular mucosa during the rainy season. This was replaced by a bistratified cuboidal epithelium, whose upper layer of cells showed evidence of degeneration, during the dry season. The PAS-positive secretory products appeared to be numerous during the rainy season, but few and clumped together during the dry season. The mean gross weight, interlobular septal thickness, acini diameter and epithelial height of the glands varied significantly with season. Ultrastructural features of the secretory cells during the rainy season showed well-developed cytoplasmic organelles, numerous electron lucid secretory vesicles and electron-dense granules. Secretory epithelial cells of the seminal vesicles are very active during the rainy season, while regeneration of degenerated epithelium in preparation for the next reproductive cycle occurs during the dry season.     

Ultracytochemical demonstration of glycoproteins in the eccrine glands of the digital pads of the North American raccoon (Procyon lotor)

In the eccrine glands of the digital pads of the North American raccoon (Procyon lotor), ultrastructural localization of glycoproteins with various saccharide residues was studied using preferably lectin cytochemical methods. Secretory granules observed in the dark cells exhibited glycoproteins with different terminal sugars (alpha-D-mannose, beta-D-galactose, beta-N-acetyl-D-glucosamine and sialic acid). The cytoplasm of the clear cells contained numerous glycogen particles. Several sugars were also detectable in the surface coat of the plasma membrane of the secretory cells. The results obtained could be helpful in understanding secretion production and cell related secretion functions of the eccrine glands of the raccoon digital foot pads.     

Metastasizing seminal vesicle adenocarcinoma in a Wistar rat

An adenocarcinoma in the seminal vesicles of a 15-month-old male Wistar rat from a 30-month inhalation study is described. The rat was killed because of cachexia, apathy and a large palpable mass in the abdominal cavity. Macroscopic examination of the abdominal cavity revealed a 3.8 cm x 3.2 cm yellow-grey to pink mass, firm to soft in consistency. The cut section revealed cystic spaces. Histologically, the mass consisted of epithelial cells arranged in glandular and solid patterns with abundant amounts of connective tissue. Epithelial tumour cells were round-to-cylindrical with round-to-oval basophilic nuclei and one or two prominent nucleoli and a distinct eosinophilic cytoplasm. The glandular structure contained clusters of macrophages in their lumen with eosinophilic cytoplasm and indented nuclei. Extensive necrosis and reactive inflammation were present. The histological features of the small nodules in the pancreas and on the surface of the liver, rectum and urinary bladder resembled those of the primary tumour in the seminal vesicles. Based on these criteria, the neoplasm (mass) was diagnosed as an adenocarcinoma of the seminal vesicles. The immunohistological examination confirmed the diagnosis, i.e. immunostaining was positive for cytokeratins (4, 7, 14, 15, 18, and 19), vimentin, PCNA, and ED(1).     

Epithelial-stromal tumor of the seminal vesicles in the transgenic adenocarcinoma mouse prostate model

The transgenic adenocarcinoma mouse prostate (TRAMP) model, designed for researching human prostatic cancer, was genetically engineered to harbor a transgene composed of the simian virus 40 Large-T/small-t antigen promoted by the rat probasin gene. In addition to prostatic neoplasms, the TRAMP mouse develops tumors in the seminal vesicles. This study was conducted to evaluate the pathology and histogenesis of TRAMP seminal vesicle neoplasms. Tissues of accessory sex organs harvested from 72 TRAMP mice of various ages (11-40 weeks of age) were fixed in neutral buffered formalin and stained with hematoxylin and eosin, desmin, 5-bromo-2'-deoxyuridine (BrdU, treated animals only), and SV40 Large-T antigen (SV40-Tag). In the seminal vesicles, we found neoplastic stromal cells that emerged multicentrically just beneath the epithelium, densely packed between the epithelium and the smooth muscle layer. These stromal cells frequently exhibited mitotic figures and showed BrdU incorporation and SV40-Tag protein expression in the nuclei and immunopositivity for desmin. The proliferative mesenchymal cells were lined by cuboidal to columnar epithelium. Some of the larger papillary, polypoid lesions exhibited a phyllodes pattern resembling that seen in mixed epithelial-stromal tumors of the breast, prostate, and seminal vesicles of humans. Although the epithelium was negative for SV40-Tag and showed only occasional incorporation of BrdU, it clearly participated in the biphasic proliferation, forming papillary, cystic, and tubuloglandular structures. No conclusive evidence of malignancy (invasion or metastasis) was identified. Our recommended diagnosis of this lesion in the seminal vesicles is epithelial-stromal tumor.     

Effect of male accessory gland secretions on sensitivity of porcine sperm acrosomes to cold shock, initiation of motility and loss of cytoplasmic droplets

Hyaluronidase release was used as an index of acrosomal membrane damage during cold shock of epididymal boar sperm and ejaculated sperm from intact and vesiculectomized boars. Sperm were also incubated with seminal plasma from intact and vasectomized boars to examine the contributions of male accessory gland secretions. Acrosomal membranes of epididymal sperm were more resistant to cold shock than those of ejaculated sperm. Only 36% of the hyaluronidase released by ejaculated sperm was released by the epididymal sperm in spite of similar hyaluronidase content of the sperm. Preincubation of epididymal sperm in seminal plasma from both intact and vasectomized boars increased resistance to cold shock by 60 to 80%. Initial dilution of epididymal sperm with seminal plasma, rather than Ringer-fructose buffer, was associated with low progressive motility and with retention of cytoplasmic droplets. In contrast, acrosomal membranes of ejaculated sperm from intact and vesiculectomized boars exhibited similar sensitivity to cold shock, releasing hyaluronidase capable of forming .20 and .19 mumol N-acetylglucosamine from hyaluronic acid/10(8) sperm in 8 min. Moreover, seminal plasma from vasectomized boars had no effect on acrosomal sensitivity to cold shock of ejaculated sperm from vesiculectomized boars.     

外泌体的生物学功能及其在动物遗传育种中的应用

外泌体是一种广泛存在于多种细胞间质中的囊泡,多种细胞在正常情况下或在细胞外刺激等应激条件下都能产生外泌体,来自不同细胞类型的外泌体作用机制及功能有很大区别。外泌体可通过膜蛋白与靶细胞膜蛋白结合、膜蛋白碎片与细胞膜上受体结合、膜与靶细胞膜直接融合等方式传递生物活性分子,从而参与机体免疫、细胞分化等过程。由于外泌体具有来源天然、穿越屏障能力好、生物相容性强等特点,在许多研究领域均受到广泛的关注。作者介绍了外泌体的形态特点、形成过程、作用机制、分离纯化、生物学特性、功能及应用;总结了近年来外泌体在畜禽研究中的应用。外泌体不仅可应用于动物遗传育种中,还可作为工具或载体来研究动物体内各种生化途径。该综述可为外泌体的相关研究及应用提供参考。     

The risk of using monoclonal or polyclonal commercial antibodies: controversial results on porcine sperm CD44 receptor identification

Presence of the hyaluronan (hyaluronic acid, HA) receptor CD44 on spermatozoa has been difficult to pursue, mostly obeying to the use of different commercial mono‐ and/or polyclonal antibodies, often lacking proper controls. Here, we describe how the presence (Western blotting) and specific location (immunocytochemistry) of the CD44 receptor differs in ejaculated pig spermatozoa depending on the type of antibody and protocol used. While we were able to detect binding to spermatozoa and mark its presence in the sperm membrane, the use of blocking peptides clearly indicated that only the monoclonal antibody could confirm the specific presence and location of the CD44 receptor, whereas the polyclonal antibody was detecting multiple presumed CD44 isoforms or degraded proteins thus proving unspecific. These results call for strict protocols when attempting immunological determination of sperm membrane receptors.