The Immunohistochemical Localization of Desmin and Smooth Muscle Actin in the Ovary of the African Giant Rat (Cricetomys gambianus) During the Oestrous Cycle

The aim of this study was to describe the distribution of smooth muscle actin and desmin immunopositive cells in the ovary of the giant rat. In addition, the study describes the morphological changes in the ovary of this species during the oestrous cycle. Healthy secondary and tertiary follicles dominated the ovary during pro-oestrus and oestrus. The theca externa of the tertiary follicles was immunopositive for smooth muscle actin, but immunonegative for desmin. Oestrus was also characterized by the presence of corpora haemorrhagica, which had an outer layer of smooth muscle actin immunopositive cells. Differentiating corpora lutea were observed during metoestrus. A further notable feature of the ovary during metoestrus was the presence of numerous atretic secondary and tertiary follicles. In the later stages of atresia, the follicles were infiltrated by desmin and smooth muscle actin immunopositive cells. Dioestrus was characterized by the presence of non-regressing and regressing corpora lutea. Immunostaining for smooth muscle actin was demonstrated in the enclosing layer of the corpora lutea, as well as in the tunica media of blood vessels within the corpora lutea. The results of this study have shown that morphological changes in the ovary of the giant rat during the oestrus cycle are similar to those of laboratory rodents. Furthermore, the results of the immunohistochemical study indicate that the perifollicular distribution of desmin and smooth muscle actin cells changes during follicular development and atresia.     

Postnatal testicular development and actin appearance in the seminiferous epithelium of the Habu,Trimeresurus flavoviridis

The postnatal testicular development and actin distribution in the seminiferous epithelium were examined by light microscopy, using the testes of the Habu (Trimeresurus flavoviridis; snake) from 0-year-old to 3-year-old. At 0-year-old (about 1 month after birth), the testis was quite small in size, and the seminiferous epithelium was composed of only Sertoli cells and large spermatogonia. Actin immunoreactivity was observed in the peritubular myoid cells, but could not be detected in the seminiferous epithelium. At 1-year-old (about 10 months after birth), the testicular size increased to a great degree. In the seminiferous epithelium, spermatocytes newly appeared. Actin could still not be detected in the seminiferous epithelium. At 2-year-old (about 1 year and 10 months after birth), the testes continued to develop in size. In the seminiferous epithelium, elongate spermatids and round spermatids were frequently seen, in addition to Sertoli cells, spermatogonia and spermatocytes. Thus, active spermatogenesis was clearly recognized at this age. Moreover, the actin distribution in the seminiferous epithelium was observed at the site between Sertoli cells and spermatids, as well as that at adult stage. The immunoreactivity of actin in the peritubular myoid cells gradually increased from 0-year-old to 2-year-old. Conclusively, it seems likely that spermatogenesis in the Habu initiates at 2-year-old, accompanying with the appearance of actin in the seminiferous epithelium.     

Actin in Stratified Squamous Keratinized Epithelium*

Actin filament distribution patterns were revealed in a stratified squamous keratinized epithelium using phalloidin-fluorescent and immunogold labeling techniques applied on bovine luminal pilar as a model tissue. In non-keratinized cell types, actin concentrates on the microfilament-rich cellular cortex as well as on cytoplasmic processes and protrusions. In cornified cells labeling is distributed diffusely over the amorphous cytoplasm. A constant feature in all cell types is plasmalem-mal labeling. Desmosomes exhibit deposition on their plasmalemmal leaflets, the dense central stratum and plaques. Desmosomal as well as cytoplasmic keratinfilament bundles also label for actin, the latter often in a cross-banded manner. These cellular distribution patterns of actin filaments are discussed with respect to the singificance of the microfilaments in the process of cell shape determination, stratification, and cell adhesion.     

Staining patterns for actin and villin distinguish M cells in bovine follicle-associated epithelium

M cells play a central role in the initiation of mucosal immune responses. However, a primary source of difficulty for investigations of this is the lack of an available specific marker for bovine M cells. As M cells possess irregular and short microvilli, we investigated the distribution and localization of the microvillar proteins actin and villin by immunohistochemistry of the gut of calves. In ileum of the calf, actin and villin were clearly and continuously immunostained in the brush border of the villous epithelia, however, discontinuous immunostaining with patches of no staining were observed in follicle-associated epithelium (FAE). Electron microscopy revealed that M cells had irregular microvilli and lacked the typical brush border, and it was inferred that these patches of no staining might be the intercellular crevices of M cells. As the microvilli of M cells were very sparse, there were several areas of weak immunostaining in calf jejunal FAE. These results suggest that M cells in calf FAE are detectable by the absence of staining for actin and villin.     

Distribution of actin filaments in the seminiferous epithelium of the Habu,Trimeresurus flavoviridis

The distribution of actin filaments was examined in the seminiferous epithelium of the Habu (Trimeresurus flavoviridis; snake), by transmission electron microscopy and fluorescence histochemistry. By transmission electron microscopy, actin filaments were clearly found only at the site between Sertoli cell and spermatid without a lattice‐like structure. Fluorescence histochemistry showed a weak labelling of actin filaments in the seminiferous epithelium, whereas these findings seem to be common among reptiles, they are different from those in mammals. Additionally, the bundles of actin filaments adjacent to the plasma membrane of Sertoli cells, appeared in other reptiles, were not observed in the Habu.     

Role of actin microfilaments in canine distemper virus replication in vero cells

Several studies have indicated that viruses require a specific cytoskeletal structure for replication in host cells. In this study, we examined the role of actin fiber in the replication of canine distemper virus (CDV), belonging to the Morbillivirus genus of the family Paramyxoviridae. For this purpose, we used two actin depolymerizing agents, cytochalasin-D (C-D) and mycalolide-B (ML-B). In Vero cells, C-D disrupted actin fibers distributed in the cytosol, but peripheral actin fibers remained intact. On the other hand, ML-B completely disrupted the actin fibers distributed in both areas. Treatment of Vero cells with C-D or ML-B inhibited the replication of CDV. Double staining of CDV-infected Vero cells with antibody to N-protein and rhodamine-phalloidin revealed the presence of N-protein in mid-cytoplasm. However, the N-protein was specifically localized at the submembrane region in the presence of C-D, whereas it was clustered in the presence of ML-B. Viral mRNA levels of N- and H-proteins were rather increased by treatment with C-D or ML-B. The treatment with ML-B strongly inhibited N-protein expression, whereas C-D only slightly inhibited N-protein expression. These results suggest that actin microfilaments distributed in the cytoplasm and on the membrane region in host cells may have a different role in the process of CDV replication.     

Immunohistochemical demonstration of myoid cells in the testis and its excurrent ducts in the domestic fowl

(1) Immunohistochemical methods and three antibodies (against actin, desmin and smooth muscle actin) were used to demonstrate the myoid cells in the domestic fowl testis and its excurrent ducts. (2) A positive reaction to actin, smooth muscle actin and desmin was found in the myoid cells of peritubular tissue of the testis and in rete testis, ductuli efferentes and ductus epididymidis. (3) In the testis myoid-reactive cells form a single layer. In the rete testis, ductuli efferentes and the ductus epididymidis reactive myoid cells form a main component of the stroma. (4) Positive reaction to actin, smooth muscle actin and desmin was also observed in the myoid cells of the tunica albuginea and in the wall of blood vessels in the testis and epididymis, indicating a contractile function for the testicular capsule.     

Actin filament alterations in rat hepatocytes induced in vivo and in vitro by microcystin-LR, a hepatotoxin from the blue-green alga, Microcystis aeruginosa

The morphologic effects of microcystin-LR (MCLR) were examined in vitro and in vivo to identify the specific cell type(s) affected and to characterize the actin filament changes occurring in hepatocytes. Male Sprague Dawley rats were used for all studies. For in vitro studies, hepatic cells were isolated by collagenase perfusion of liver, while parenchymal cells (hepatocytes) and nonparenchymal cells were prepared by pronase digestion and metrimazide gradient centrifugation. Cell suspensions and and primary hepatocyte monolayer cultures were treated with MCLR at doses up to 10 micrograms/ml; cultured hepatocytes were also treated with phalloidin or cytochalasin B at a dose of 10 micrograms/ml; and rats were treated intraperitoneally with MCLR at 180 mg/kg. Cultured hepatocyte preparations and frozen liver sections were stained with rhodamine-labeled phalloidin for filamentous actin. In cell suspensions, MCLR did not affect nonparenchymal cells but caused rapid, progressive, blebbing of the plasma membrane in hepatocytes. In cultured hepatocytes, MCLR caused plasma membrane blebbing as well as marked reorganization of actin microfilaments. These alterations were dose and time dependent. Cultured hepatocytes treated with phalloidin or cytochalasin B also showed extensive plasma membrane blebbing and actin filament alterations; however, actin filament changes were morphologically distinct from those induced by MCLR. In vivo, MCLR-induced hepatocyte actin alterations occurred at the same time as, or slightly preceded, histologic changes that began 30 minutes after dosing. These studies suggest that early MCLR-induced morphologic changes occurring both in vivo and in vitro are due to alterations in hepatocyte actin filaments.     

Localization of smooth muscle actin in the iridocorneal angle of normal and spontaneous glaucomatous beagle dogs

Purpose To date, our knowledge of the canine trabecular meshwork (TM) with regard to contractility is incomplete. It is important to understand the potential contractile capability within the TM and possible changes associated with spontaneous hypertensive glaucoma. To that end we have examined the presence of actin, including smooth muscle (SM) actin, in the normal and glaucomatous canine iridocorneal angle (ICA) morphologically and immunohistochemically. Methods Sections from the ICAs of 12 Beagles with inherited glaucoma (3 months to 6 years old) and age‐matched normal Beagles were treated with target retrieval, protein and power blocked and sequentially incubated with the primary antibody (rat anticanine SM actin) and the secondary antibody (rabbit antirat immunoglobulin), followed by peroxidase labeled streptavidin and incubation with substrate‐chromogen solution (AEC). Smooth muscle fibers that lined an artery within canine heart tissue were used as positive controls. Separate specimens were prepared for ultrastructual observation. Results Ultrastructurally, cells within the inner, posterior region of the corneoscleral TM and outer, posterior region of the uveal TM contained many microfilaments, 6 nm in diameter (i.e. actin). Immunohistochemistry demonstrated that cells within these regions possessed SM actin, having been greatest posteriorly, but extended anteriorly to a lesser extent. In the preglaucomatous affected dog the localization pattern for SM actin was identical to that seen in the normal dogs. With the progression of the disease the pattern disappeared. Conclusions The interior presence of myofibroblastic cells within the canine ICA suggests that these cells and the smooth muscle cells of the ciliary body along the same plane of orientation function to facilitate the removal of aqueous humor and are likely to be influenced by vascular mediators. The contractile apparatus for the ICA in the dog with inherited glaucoma appeared identical to that of the normal dog prior to expression of the disease, but weakened as the disease progressed.     

Temporary disturbance of actin stress fibers in swine kidney cells during pseudorabies virus infection

Rounding and loosening of cells is a consequence of infection with pseudorabies virus (PrV), both in vitro and in vivo. These changes in the normal structure of the cell may be the result of cytoskeletal changes. Immunofluorescence staining of actin filaments and microtubule bundles was performed to examine whether PrV induces a reorganization of these cytoskeletal components in infected swine kidney (SK) cells. Every 2h until 12h post-inoculation (p.i.), cells were washed in cytoskeleton stabilizing buffer (CSB), fixed with paraformaldehyde and washed again with CSB. Cells were permeabilized with a 1/1000 dilution of Triton X-100 and actin filaments were stained by incubating cells with phalloidin-Texas Red. Staining of microtubules was done by incubating the cells subsequently with mouse monoclonal anti-alpha-tubulin and goat anti-mouse IgG-FITC. During the course of infection, actin fibers of SK cells were rearranged in the following sequence: (1) disappearance of thick actin stress fibers between 4 and 6h p.i., (2) complete loss of stress fibers between 6 and 8h p.i., and (3) reappearance of thin stress fibers starting from 10h p.i. In contrast to herpes simplex virus 1 (HSV1) or equine herpesvirus 1 (EHV1), PrV infection did not induce changes in the cellular microtubule network. PrV infection induces a temporary disassembly of actin stress fibers.     

Seasonal Changes in the Immunolocalization of Cytoskeletal Proteins and Laminin in the Testis of the Black‐Backed Jackal (Canis mesomelas)

Manipulation of the reproductive activity of jackals is dependent on a thorough understanding of the reproductive biology of this species. This study describes seasonal morphological changes in the adult testis of the black‐backed jackal in relation to the immunoexpression of the basement membrane marker, laminin and the cytoskeletal proteins, cytokeratin, smooth muscle actin and vimentin. Laminin was immunolocalized in basement membranes surrounding seminiferous tubules, as well as in basement membranes associated with Leydig, peritubular myoid and vascular smooth muscle cells. Scalloped basement membranes enclosed seminiferous tubules in regressing testes. The seminiferous epithelium and interstitial tissue in all animals studied were cytokeratin immunonegative. Smooth muscle actin was demonstrated in vascular smooth muscle cells, as well as in peritubular myoid cells encircling seminiferous tubules. Vimentin immunoreactivity was exhibited in the cytoplasm of Sertoli cells, Leydig cells, vascular endothelial cells, vascular smooth muscle cells and fibrocytes. Vimentin immunostaining in Sertoli, Leydig and peritubular myoid cells varied depending on the functional state of the testis. The results of the study have shown that dramatic seasonal histological changes occur in the testes of the jackal. In addition, the use of immunohistochemistry accentuates these morphological changes.     

枯草芽孢杆菌拮抗病原菌对细胞微丝的影响

本研究旨在探讨枯草芽孢杆菌拮抗2种病原菌(鼠伤寒沙门氏菌和产肠毒素大肠杆菌)对人结肠癌细胞Caco-2细胞中微丝的影响。将枯草芽孢杆菌及上述2种病原菌加到Caco-2细胞上,用异硫氰酸荧光素-鬼笔环肽标记细胞微丝,在倒置荧光显微镜下观察细胞微丝的变化;利用Western blotting检测细胞微丝调节蛋白Rac1蛋白的表达变化。结果显示正常Caco-2细胞内微丝呈散在的平行排列;加鼠伤寒沙门氏菌后微丝呈小区域聚集;加产肠毒素大肠杆菌后细胞微丝重排聚集在细胞中央放射状分布或分布紊乱;单独加枯草芽孢杆菌后细胞微丝排列方式变化不大;同时分别加2种病原菌和枯草芽孢杆菌后,大部分的细胞内微丝排列整齐,只是在少部分细胞内存在微丝的聚集。产肠毒素大肠杆菌、鼠伤寒沙门氏菌可使细胞微丝调节蛋白Rac1的表达显著上调,而枯草芽孢杆菌则可抑制病原菌对Rac1的影响。本试验证明产肠毒素大肠杆菌、鼠伤寒沙门氏菌可影响细胞微丝的表达和分布;而枯草芽孢杆菌及其培养上清液可抑制病原菌的这种破坏作用。     

Actin polymerization enhances Pasteurella haemolytica leukotoxicity

Pasteurella haemolytica leukotoxin is cytotoxic to bovine leukocytes, causing increased cell membrane permeability, osmotic swelling, release of cytosolic proteins and cell lysis. These studies were designed to test if leukotoxin causes release of the cytoskeletal protein, actin, from bovine leukemia cells and if purified actin-influenced bacterial growth or leukotoxin production. Culture supernatants caused a 7-fold decrease in viability of bovine leukemia cells and increased cell permeability that was accompanied by release of beta-actin into the cell culture supernatant. Exposing P. haemolytica to purified actin solutions induced the conversion of monomeric G-actin to polymerized F-actin. This conversion was partially inhibited by bovine P. haemolytica immune, but not pre-immune, serum. Loss of streptomycin resistance following treatment of the organism with acridine orange ablated the polymerizing activity. Incubation of P. haemolytica in the presence of purified F-actin did not affect growth but resulted in culture supernatant that had 3.0-3.9-fold greater leukotoxicity compared to medium alone or medium containing G-actin, heat-denatured actin or albumin. The effect of actin on leukotoxicity was concentration-dependent and directly associated with increases in secreted leukotoxin. The interaction between P. haemolytica and actin is potentially detrimental to the host by inducing polymerization of actin into insoluble filaments and by enhancing leukotoxicity.     

Immunohistochemical evaluation of the muscularis mucosae in the ruminant forestomach

The muscularis mucosae and condensed fibrous layer of the ruminant forestomach were studied by immunohistochemistry using specific antibodies against alpha-smooth muscle actin (alphaSMA) and gamma-smooth muscle actin (gammaSMA). The specimens were collected from the rumen, reticulum and omasum of cattle, water buffalo, sheep, goat, Barbary sheep, Japanese serow, sika deer and mouse deer. The muscularis mucosae showed immunoreactivity for both alphaSMA and gammaSMA. On the other hand, the condensed fibrous layer appearing between the propria mucosa and tela submucosa was immunoreactive only for alphaSMA except for that in the goat and Barbary sheep reticulum which is intermingled with gammaSMA immunoreactivity. The distribution of muscularis mucosae and/or condensed fibrous layer varied among the compartments of forestomach and ruminant species. In the rumen, only the condensed fibrous layer was detected. On the other hand, the omasum contained only the muscularis mucosae. In the reticulum, both were detected. The amount of the condensed fibrous layer in the reticulum varied among different species in the following order of abundance: goat > Barbary sheep > sika deer> sheep > water buffalo > cattle and Japanese serow. Smooth muscle cells of external muscle layer were immunoreactive for alphaSMA and gammaSMA whereas those of blood vessels and pericytes were immunoreactive only for alphaSMA. The present findings on the actin immunoreactivity and distribution profile of muscularis mucosae and the condensed fibrous layer provide additional knowledge to further understand the histophysiological specialization of the different compartments of the ruminant forestomach.     

Primary ureteral giant cell sarcoma in a Pomeranian

An 8-year-old male neutered Pomeranian dog was presented to the Veterinary Teaching Hospital at Oregon State University for surgical treatment of hydronephrosis of the left kidney and a left cranial abdominal mass. A primary ureteral mass was found during exploratory surgery, and the mass was resected and ureteral anastomosis was performed. Cytologic evaluation of the mass revealed 3 distinct cell populations, including a large number of multinucleated giant cells, a moderate number of thin spindle-shaped cells, and cohesive clusters of transitional epithelial cells. The cytologic diagnosis was giant cell sarcoma. The diagnosis was confirmed by histologic examination, and immunohistochemical staining was performed. The spindle-shaped cells and multinucleated giant cells were both immunoreactive for vimentin and spindle-shaped cells for S-100. Tumor cells did not express wide-spectrum cytokeratin, broad-spectrum muscle actin, smooth muscle actin, sarcomeric actin, desmin, BLA36, Mac 387, synaptophysin, neuron-specific enolase, glial fibrillary acid protein, or von Willebrand factor. These findings are most consistent with an anaplastic sarcoma with giant cells. This is the first case report of a primary ureteral giant cell sarcoma in a dog.     

Development of the glandular epithelium of the bovine parotid gland during ontogenesis

The development of the parotid gland was examined in 36 bovine embryos and foetuses with a crown-rump-length (CRL) from 28 up to 1000 mm by light, transmission electron microscopical and actin-immunohistochemical methods. The anlage of the parotid gland in an embryo with 28 mm CRL can be found at the lateral angle of the primitive oral cavity as a local thickening of the epithelium. During the second month, the differentiation of primary ducts and endbuds starts and a lumen develops in the primary ducts. At the end of the second month a lumen appears in the terminal endbuds. In the immature endpiece cells first secretory granules can be seen from a CRL of 240 mm. In the third month differentiation between intra- and inter-lobular ducts is possible. Immature myoepithelial cells present as a basal layer of flattened cells between the epithelial cells and the basement membrane at the end of the second month. During further development they increase in number, become more flattened and form long cellular processes. At the end of the fourth month isolated actin filament bundles are formed, which were also detected by an antibody against smooth muscle actin. The actin filaments condense continuously until they fill the cell processes completely at the end of foetal development.     

Cytoskeletal changes in <Emphasis Type="Italic">Eimeria bovis</Emphasis>-infected host endothelial cells during first merogony

The first merogony of Eimeria bovis takes place in lymphatic endothelial cells of the ileum, resulting in the formation of macromeronts up to 250 mum. In this study, we investigated the host cell cytoskeleton (actin filaments, microtubules, spectrin, vimentin intermediate filaments) associated with parasitic development in vitro by confocal laser scanning microscopy (CLSM) using primary bovine umbilical vein endothelial cells (BUVEC) and bovine spleen lymphatic endothelial cells (BSLEC) as host cells. No prominent changes in the host cell cytoskeleton were detected 1-3 days after E. bovis sporozoite invasion. With ongoing meront maturation a significant increase in microtubules and actin filaments close to the parasitophorous vacuole (PV) was found. Mature macromeronts within the PV were completely enclosed by these cytoskeletal elements. Our findings suggest, that in order to guarantee the survival of the host cell on the enlargement of macromeronts, E. bovis needs not only to augment but also to rearrange its cytoskeletal system.     

MSTN基因对牛肌动蛋白细胞骨架调节通路的影响

为探究肌肉生长抑制素（myostatin,MSTN）对牛骨骼肌生长发育的作用机制,本研究前期利用定量蛋白质组学与磷酸化蛋白质组学分析野生型蒙古牛（MG.WT）和MSTN^+/-蒙古牛（MG.MSTN^+/-）腿臀肌肌肉组织中蛋白质水平和磷酸化修饰水平的差异变化,使用已建立的牛骨骼肌卫星细胞体外诱导成肌分化模型,检测设计合成的MSTN siRNA （si-MSTN）干扰效果;采用实时荧光定量PCR和Western blotting方法检测转染si-MSTN的增殖期（GM）和分化第3天（DM3）牛骨骼肌卫星细胞中肌动蛋白细胞骨架调节通路相关基因的mRNA和蛋白水平的表达变化,研究敲低MSTN表达对肌动蛋白细胞骨架调节通路的影响。结果显示,在MSTN^+/-蒙古牛肌肉组织中共鉴定到16个肌动蛋白细胞骨架调节通路相关基因表达丰度上调;转染si-MSTN细胞中的MSTN表达水平极显著降低（P<0.01）;在转染si-MSTN的GM期牛骨骼肌卫星细胞中,肌动蛋白细胞骨架调节通路相关基因ENAH、ACTN4和Cdc42的mRNA水平均显著升高（P<0.05）,PFN1、RhoA和ACTN4的蛋白水平均显著或极显著升高（P<0.05;P<0.01）;在转染si-MSTN的DM3牛骨骼肌卫星细胞中,ENAH、CFL1、SCIN和Cdc42基因mRNA水平均显著升高（P<0.05）,RhoA基因mRNA水平极显著升高（P<0.01）,PFN1和ACTN4的蛋白水平均显著升高（P<0.05）。结果表明,干扰MSTN可以促进肌动蛋白细胞骨架调节通路相关基因的表达,探明了MSTN可能通过介导肌动蛋白细胞骨架调节通路影响牛骨骼肌卫星细胞增殖和成肌分化的分子机制,为进一步研究MSTN对牛成肌分化的调控机制提供参考。     

A peripheral primitive neuroectodermal tumor with generalized bone metastases in a puppy

A peripheral primitive neuroectodermal tumor (pPNET), most consistent with a human Ewing's sarcoma, is described in a 5-month-old male Australian Shepherd puppy. The first tumor site detected was in the left frontal bone of the skull with apparent subsequent rapid metastases to multiple sites in the axial and appendicular skeleton and bone marrow, kidneys, and perihyphophyseal meninges. Radiographically, all bone lesions were lytic and there was also a humeral bone fracture. Histologically, the tumor was diagnosed as a small round blue cell tumor. At this stage, the differential diagnosis included a lymphoma, rhabdomyosarcoma, and a PNET of the peripheral nervous system. However, the cells had positive expression of triple neurofilament antigens as detected immunocytochemically. The cells were negative for a broad panel of canine-specific leucocyte cell marker antigens for desmin, smooth muscle actin, synaptophysin, and CD99. Ultrastructurally, the cells contained occasional dense core neurosecretory granules and intermediate filaments with intercellular desmosomal-like junctions and abundant glycogen clusters. Based on the age of the dog, the clinical history, the distribution of gross lesions, histologic characteristics of a small round blue cell tumor, and immunocytochemical and ultrastructural evidence of neuroectodermal differentiation, a diagnosis of a pPNET similar to a human Ewing's sarcoma was made.