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1.
It has been reported that Pasteurella haemolytica leukotoxin (LKT) induces morphologic changes in bovine leukocytes consistent with apoptosis in vitro, but DNA fragmentation was not observed. We investigated whether bovine lymphocytes undergo DNA fragmentation during LKT-induced apoptosis. Bovine peripheral blood lymphocytes were isolated by density gradient centrifugation and exposed to LKT or inactive pro-LKT protein from a lktC- mutant strain. After exposure, DNA fragmentation in lymphocytes was quantified colorimetrically by diphenylamine assay and visualized by agarose gel electrophoresis. At high LKT concentrations, bovine lymphocytes were lysed, but at low concentrations, LKT caused DNA fragmentation characteristic of apoptosis. Maximal DNA fragmentation in bovine lymphocytes was induced by 0.1 TU ml(-1) LKT following 3 h exposure, but only background level of DNA fragmentation was observed with the inactive pro-LKT. Equine lymphocytes that are resistant to LKT intoxication did not show DNA fragmentation following exposure to LKT. Preincubation of LKT with a neutralizing anti-LKT monoclonal antibody inhibited LKT-induced DNA fragmentation. Electrophoresis of DNA from bovine lymphocytes treated with 0.1 TU ml(-1) LKT demonstrated the typical 'ladder' pattern of internucleosomal DNA cleavage, the hallmark of apoptosis associated with activation of endonucleases. LKT-induced DNA fragmentation was inhibited by 0.5 mM ZnCl2, an endonuclease inhibitor. The results indicated that LKT at low concentrations induced apoptotic cell death of bovine lymphocytes, which may play a role in initiation and persistence of P. haemolytica infection.  相似文献   

2.
Programmed cell death (PCD), or apoptosis, is initiated in response to various stimuli, including virus infection. A number of studies have shown that deregulation of apoptosis is an important feature of virus-induced immunosuppression for various viral diseases. In the present study, CapHV-1 was found to cause apoptosis in mitogen-stimulated as well as nonstimulated caprine peripheral blood mononuclear cells (PBMC). Apoptotic index, as quantified by fluorescent dyes, revealed a significant increase in the percentage of apoptotic cells at 24 and 48 h postinfection as compared to their respective noninfected controls. Apoptosis specific internucleosomal laddering in DNA from CapHV-1 infected PBMC was seen in agarose gel electrophoresis. No DNA fragmentation was observed in control noninfected PBMC. Virus-induced apoptosis was reduced by Z-VAD-FMK, an aspecific caspase inhibitor, by AC-DEVD-CHO (caspase-3-specific) and AC-VEID-CHO (caspase-6-specific) treatment. PCD in CapHV-1 infected peripheral blood mononuclear cells occurs at the G0/G1 phase of the cell cycle. However, penetration of virus particles and infection was not required for PCD, as UV-inactivated CapHV-1 induced apoptosis of mitogen-stimulated bovine peripheral blood mononuclear cells in vitro.  相似文献   

3.
The present study was conducted to investigate the effects of storage time and temperature of porcine ovaries on the quality and nuclear maturation in vitro of oocytes obtained from stored ovaries and their subsequent development after in vitro fertilization. The ovaries were stored in physiological saline for 0, 3, 6, 9 and 12 h at various temperatures (4, 15, 25 and 35 C). The pH of follicular fluid obtained from the ovaries, DNA fragmentation of the oocyte nucleus and meiotic competence of oocytes were examined. Some oocytes from ovaries stored at 15, 25 and 35 C for 6 h were fertilized in vitro, and then cultured for 7 days to examine the ability of embryos to develop to the blastocyst stage. When the ovaries were stored at 35 C, the pH of follicular fluid decreased and the proportions of oocytes with DNA fragmented nuclei increased as the storage time was prolonged, and the storage of ovaries for 6, 9 and 12 h resulted in lower maturation rates of oocytes. When the ovaries were stored at 4, 15, 25 and 35 C for 6 h, the storage at higher temperatures (> or =15 C) decreased the pH of follicular fluid and induced nucleic DNA fragmentation in higher proportions of oocytes. None of the oocytes from ovaries stored at 4 C reached metaphase II. The storage of ovaries at 15 C reduced the rates of in vitro fertilized oocytes and subsequent embryo development, but there were no significant differences in the rates of fertilization and blastocyst formation between oocytes from ovaries stored at 25 C and 35 C. Our findings indicate that the storage of ovaries at 25-35 C for 6 h is effective for maintaining the developmental competence of porcine oocytes even though the development rates were lower than those of ovaries stored at 35 C for 3 h.  相似文献   

4.
The purpose of the present study was to investigate the development of follicles and incidence of apoptosis in vitrified neonatal mouse ovaries cultured in vitro in the presence of leukemia inhibitory factor (LIF). The vitrified and non-vitrified ovaries of 1-week-old mouse were cultured in the presence or absence of LIF for 7 days. At the beginning and at the end of culture period in each ovary of all groups of study the mean area and the development of ovarian follicles were analyzed; moreover, the incidence of apoptosis was assessed by transmission electron microscopy, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) method, DNA laddering and caspase-3/7 activity technique. The hormonal assay was done on the conditioned media collected during culture period. The proportion of preantral follicles and the levels of hormones increased in all cultured groups and it was significantly higher in LIF treated groups than in their control (P < 0.001). The ultrastructural characteristics of cell death, DNA fragmentation and TUNEL positive signals were prominent in vitrified cultured ovaries. The level of caspase-3/7 activity was higher in vitrified cultured ovaries.  相似文献   

5.
This study describes the morphology of salivary glands of Rhipicephalus (Boophilus) microplus female ticks at beginning of feeding (24-48 h of attachment) and semi-engorged (4-5 days of attachment) to verify the degenerative characteristics of these organs and the secretory phase in which the process begins. At the beginning of feeding, secretion granules had been observed only in the cytoplasm of cells b, c1, c2, c4 (type II acinus) and d (type III acinus), as well as large nuclei with regular and preserved morphology. In the semi-engorged females the acini presented few normal cells, few partially preserved ones, and the remaining ones in several stages of degeneration, that is, with retraction and cytoplasmic vacuolization, and nuclei with chromatin in several stages of condensation, picnotic and/or in fragmentation. In type I acinus and in the excretory ducts of the studied glands, at both feeding stages, no degenerative characteristic was observed. In females of R. (B.) microplus, the salivary glands degenerate asynchronically and precociously when compared with those of others tick's species.  相似文献   

6.
The present study examined the salivary glands of Rhipicephalus sanguineus males at days 0, 3, and 7 post-detachment from the host. Degeneration of this organ occurred in the three stages and it advanced as time away from the host progressed. Thus, characteristics of degeneration were more prominent in males at day 7 post-detachment than in males at day 0 post-detachment. In males at day 0 post-detachment, type I acini were intact; while in other stages these acini exhibited signs of degeneration. In type II acini of individuals at day 0 post-detachment, cells a, c1-c5, c8, and indeterminate were identified. Only c1 and c8 were intact. The remaining cell types were undergoing degeneration, as well as all cells d-f in type III acini, and all g in type IV acini. In males at day 3 post-detachment from the host, all cells (a, c1-c5, c8 and indeterminate) of type II acini, cells d and e in type III acini, and g in type IV were undergoing degeneration. In some Indeterminate acini, the boundaries of cells still could be distinguished, while in others, only a cytoplasmic mass was observed. At day 3 post-detachment, apoptotic bodies were present. In males at day 7 post-detachment from the host, the degeneration process progressed. All cells a, c1, c3-c5, c8 and indeterminate in type II, and d and e in type III acini were undergoing degeneration. Type IV acini still contained remnants of secretion and in Indeterminate acini, only a cytoplasmic mass could be observed. At this stage, apoptotic bodies were also present. The present study still revealed that cells of salivary glands of R. sanguineus males when degenerating undergo the following changes: (a) decrease in secretion production with or without granule breakage, (b) changes in nuclear morphology, (c) cytoplasm shrinkage, (d) loss of cell shape, (e) loss of cell boundaries, and (e) cytoplasmic vacuolation. Together, these changes result in cell fragmentation with release of apoptotic bodies.  相似文献   

7.
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8.
Canine coronavirus induces apoptosis in cultured cells   总被引：1，自引：0，他引：1  
Canine coronavirus (CCoV) is widespread in dogs in several countries and causes mild enteric illness evolving to severe enteritis in young pups. In in vitro cultures canine coronaviruses generally induce extensive cell death, however nature of the events leading to cell death remains largely unknown. We analysed the induction of cytopathic effect by CCoV in a canine fibrosarcoma cell line (A-72) in order to characterize the apoptotic effect in homologous cell system. Following CCoV infection A-72 cell line, which is permissive to CCoV, showed reduced growth rate, as detected by MTT assay, a standard colorimetric assay for measuring cellular proliferation, and underwent to apoptotic death. Starting from 24h after CCoV infection, cells morphology appeared dramatically changed, with cells rounding and detachment from culture surface. Morphologic and biochemical features of apoptosis, such as blebbing of the plasma membrane, translocation of phosphatidilserine to cell surface and annexin V positive staining, nuclear fragmentation, apoptotic bodies formation and DNA laddering, were detected in CCoV-infected cells. Propidium iodide staining of infected culture indicated the appearance of hypodiploid DNA peak corresponding to apoptotic cell population. Commonly to other animal coronavirus infection caspase-3 is likely to contribute to the execution phase of apoptosis induced by CCoV in A-72 cells since we found activation of enzymatic activity as well as procaspase-3 activating cleavage. Apoptotic death of infected cells is detrimental as it causes cell and tissue destruction as well as inflammatory responses. Therefore in the case of CCoV associated gastroenteritis, apoptosis of epithelial mucosa cells may be responsible for pathology induced by CCoV infection.  相似文献   

9.
In this study, we performed experiments to evaluate the extend of the process of apoptotic cell death by foot-and-mouth disease virus (FMDV). Apoptosis can also occur in some virus-infected cells, and ability of viruses to either inhibit or promote apoptosis may influence the pathologic outcome of infection. In this study, to determine if apoptosis plays a role in the outcome of FMDV infection in swine, we evaluated apoptosis in diseased tissues collected from pigs inoculated with two different stains of FMDV (O1 Campos and O Taiwan). And host cell DNA fragmentation in diseased tissue from animals which were infected with either virus was evaluated by occurrence of a laddering pattern characteristic of apoptosis. Infection of cultured keratinocytes from swine tongue failed to demonstrate apoptosis in the first few hours of infection, suggesting that cell-to-cell correlation between viral antigen and apoptotic changes, e.g. cytokine secretions by immune system cells, could be critical to initiating apoptosis. Consistent with this finding, we were able to detect the pro-inflammatory cytokine TNF-alpha in diseased tissues. A clear difference in the pathogenicity of the two different FMDV isolates to pigs was not demonstrated in our study.  相似文献   

10.
The objective of this study was to determine whether neutrophil apoptosis and their consequent elimination by macrophages from the mammary gland is modulated by an infection caused by Staphylococcus aureus (S. aureus). The study was performed on twenty mammary glands of 5 virgin heifers. A buffered physiological solution (PBS) was administered as a means of control into the mammary glands of the heifers and after 168 h, the glands were inoculated with S. aureus. The samples of cell populations were obtained by lavages of the mammary glands in 4 intervals (24, 48, 72 and 168 h) after the experimental infection. Flow cytometry was used for determination of Annexin-V positivity and propidium iodide (PI) negativity of neutrophils. Light microscopy was used for determination of neutrophil karyopyknosis. Cytochemistry was used for the detection of myeloperoxidase-positive (MPO+) macrophages. Instillation of S. aureus resulted in an intramammary infection which persisted during the following experimental period. The total number of both Annexin-V-positive and PI negative neutrophils and karyopyknotic neutrophils peaked at 24 h after both of PBS and S. aureus administration. The highest percentages of Annexin-V-positive and PI negative neutrophils and karyopyknotic neutrophils were detected 48 and 168 h after PBS and S. aureus administration, respectively. The total number of MPO+ macrophages was the highest 24 h and 48 h after PBS and S. aureus administration, respectively; the percentage of MPO+ macrophages was the highest at 72 h in both cases. The dynamics of resolution of mastitis caused by S. aureus was very similar to the resolution of inflammatory response of the mammary gland after PBS administration. Mechanisms of cell pathogen elimination as well as inflammation resolution were very intensively involved; nevertheless, the mammary gland infection persisted. An early inclusion of the mechanisms of an acute inflammatory resolution thus paradoxically led to chronic infection.  相似文献   

11.
Salivary glands from males of 3 Dermacentor species (D andersoni, D variabilis and D occidentalis) that were infected with either the Virginia or Idaho isolate of Anaplasma marginale as nymphs or adults were examined for colonies of A marginale by use of light and electron microscopy. Prior to dissection of salivary glands, exposed ticks were held at 25 C for 15 to 18 days, followed by a 3-day incubation at 37 C. Ticks of 2 species transmitted A marginale to calves; the third tick species was confirmed infected by demonstration of typical colonies in tick gut cells, but transmission was not attempted; Colonies of A marginale were seen with light microscopy in salivary glands of all 3 species of ticks; they were located in acinar cells that contained simple granules. Colonies varied morphologically from small, compact ones to larger structures that contained distinct organisms and often were adjacent to the host cell nucleus. Electron microscopy confirmed that the colonies were rickettsial organisms. Morphologic features of A marginale varied and included reticulated forms, forms with electron-dense centers, and small particles; these various forms were similar to those described previously in midgut epithelial cells of ticks. We believe that the organism seen within tick salivary glands may replicate in the glands before its transmission to the vertebrate host.  相似文献   

12.
Some studies in mammalian species recently demonstrated that tumor necrosis factor (TNF)-alpha plays an important role for corpus luteum (CL) function by way of apoptosis during the estrous cycle. The objectives of this study were to clarify the induction of apoptosis in cultured porcine luteal cells by TNF-alpha treatment. Luteal cells prepared from porcine ovaries collected from crossbred mature gilts on Days 10-14 of the estrous cycle were isolated and examined as follows: 1) Flow cytometric analysis was carried out to determine apoptosis in cultured luteal cells. 2) Single-cell gel electrophoresis (comet assay) was performed to investigate apoptotic DNA fragmentation in luteal cells. The results of the flow cytometric analysis and comet assay demonstrated coincidentally that TNF-alpha induces DNA fragmentation in luteal cells causing apoptosis. These results revealed that TNF-alpha is an inducing factor of apoptosis in luteal cells.  相似文献   

13.
Contents In this experiment, the possibility that the follicular-wall cells' death during ovarian follicular atresia occurs as a result of apoptosis was examined. Programmed cell death or apoptosis is a process whereby cells die in a controlled fashion, triggered by changes in levels of specific physiological stimuli. Morphological transformations of the cells are preceded by endo- nuclease-mediated genomic-DNA cleavage. The analysis of DNA from the theca and granulosa layers of follicles indicated that internucleosomal fragmentation of DNA occurred in atretic granulosa cells but not in atretic theca cells. The healthy granulosa and theca cells in all classes of follicles showed no apoptosis. This paper demonstrates that the death of porcine ovarian-follicle walls can be caused by different processes and, contrary to granulosa cells' apoptosis, either does not or only partly concerns the internal theca layer.  相似文献   

14.
以体外培养的小鼠睾丸间质细胞系TM3 为材料,加入不同质量浓度的钼酸钠溶液（0,10,20,40,80,160mg/L）染毒培养,分别在干预4,8,12,24,48h采用MTT法检测细胞的增殖。干预48h后,采用流式细胞术检测细胞周期和凋亡,单细胞凝胶电泳检测DNA损伤的变化。结果表明：与对照组相比,不同剂量钼酸钠作用24h后,睾丸间质细胞的增殖活性均受到抑制;不同质量浓度的钼酸钠作用48h后,细胞周期阻滞于G0/G1期,20mg/L及其以上剂量组G0/G1期细胞百分率与对照组相比显著升高（P〈0.05或P〈0.01）;与对照组相比,各剂量组TM3 小鼠睾丸间质细胞凋亡率显著升高,差异极显著（P〈0.01）;细胞尾部DNA含量及细胞尾长随着钼剂量的增加呈不同程度的增加,且存在着剂量—效应关系。结论说明钼能够引起睾丸间质细胞周期的紊乱,并诱导睾丸间质细胞发生DNA损伤和凋亡。  相似文献   

15.
We examined the effectiveness of saline, Euro-Collins solution (EC), and ET-Kyoto solution (ET-K) as preservation media for the cold storage of feline ovaries. Ovaries were maintained in these media at 4°C for 24, 48, or 72 h until oocyte retrieval. The ET-K group exhibited a higher oocyte maturation rate than the saline group after 72 h of storage. Moreover, ET-K could sustain the competence of the feline oocytes to cleave after 48 h, and the morula formation rate of the ET-K group was higher than that of the other groups after 24 and 48 h. Furthermore, the ET-K group exhibited a higher blastocyst formation rate than the other groups after storage for 24 h, and only ET-K retained the developmental competence in blastocysts after 48 h of storage. In addition, regarding the cell numbers of the blastocysts, there was no significant difference among the tested groups. In conclusion, our results indicate that ET-K is a suitable preservation medium for feline ovaries.  相似文献   

16.
OBJECTIVE: To determine the effect of pamidronate disodium on the in vitro viability of osteosarcoma cells and non-neoplastic cells from dogs. SAMPLE POPULATION: 3 osteosarcoma and 1 fibroblast cell lines derived from dogs. PROCEDURE: Cell counts and cell viability assays were performed in cultures of osteosarcoma cells (POS, HMPOS, and COS31 cell lines) and fibroblasts after 24, 48, and 72 hours of incubation with pamidronate at concentrations of 0.001 to 1000 microM or with no drug (control treatment). Percentage viability was determined in cell samples for each concentration of pamidronate and each incubation time. A DNA fragmentation analysis was performed to assess bisphosphonate-induced apoptosis. RESULTS: Osteosarcoma cell viability decreased significantly in a concentration- and time-dependent manner at pamidronate concentrations ranging from 100 to 1000 microM, most consistently after 48 and 72 hours' exposure. In treated osteosarcoma cells, the lowest percentage cell viability was 34% (detected after 72 hours' exposure to 1000 microM pamidronate). Conversely, 72 hours' exposure to 1000 microM pamidronate did not significantly reduce fibroblast viability (the lowest percentage viability was 76%). After 72 hours of exposure, pamidronate did not cause DNA fragmentation in POS or HMPOS cells. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that pamidronate may have the potential to inhibit osteosarcoma growth in dogs, possibly through a nonapoptotic mechanism. The clinical relevance of these in vitro findings remains to be determined, but administration of pamidronate may potentially be indicated as an adjuvant treatment in chemotherapeutic protocols used in dogs.  相似文献   

17.
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18.
Sperm DNA fragmentation is one of the major causes of infertility; the sperm chromatin dispersion test (SCDt) evaluates this parameter and offers the advantage of species‐specific validated protocol and ease of use under field conditions. The main purpose of this study was to evaluate sperm DNA fragmentation dynamics in both fresh and post‐thaw bottlenose dolphin sperm using the SCDt following different cryopreservation protocols to gain new information about the post‐thaw differential sperm DNA longevity in this species. Fresh and cryopreserved semen samples from five bottlenose dolphins were examined for sperm DNA fragmentation dynamics using the SCDt (Halomax®). Sperm DNA fragmentation was assessed immediately at collection and following cryopreservation (T0) and then after 0.5, 1, 4, 8, 24, 48 and 72 h incubation at 37°C. Serially collected ejaculates from four dolphins were frozen using different cryopreservation protocols in a TES‐TRIS‐fructose buffer (TTF), an egg‐yolk‐free vegetable lipid LP1 buffer (LP1) and human sperm preservation medium (HSPM). Fresh ejaculated spermatozoa initially showed low levels of DNA fragmentation for up to 48 h. Lower Sperm DNA fragmentation (SDF) was found in the second fresh ejaculate compared to the first when more than one sample was collected on the same day (p < 0.05); this difference was not apparent in any other seminal characteristic. While there was no difference observed in SDF between fresh and frozen–thawed sperm using the different cryopreservation protocols immediately after thawing (T0), frozen–thawed spermatozoa incubated at 37°C showed an increase in the rate of SDF after 24 h. Sperm frozen in the LP1? buffer had higher levels (p < 0.05) of DNA fragmentation after 24‐ and 48‐h incubation than those frozen in TTF or HSPM. No correlation was found between any seminal characteristic and DNA fragmentation in either fresh and/or frozen–thawed samples.  相似文献   

19.
The involvement of apoptosis was evaluated in lesions of endotoxemic piglets. A single injection with E. coli O111:B4 lipopolysaccharide (LPS) induced foci of coagulative necrosis in the liver and kidneys. No significant change was observed in these organs at 1.5 hr after LPS injection, but at 6 hr, epithelial cells with chromatin condensation or fragmentation and apoptotic bodies were visible. Foci of coagulative necrosis were formed within 24 hr after LPS inoculation. In and adjacent to the necrotic foci, dead hepatocytes with nuclear condensation or fragmentation were scattered. These dead cells were positively stained by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) methods. Electronmicroscopy revealed apoptotic cells with condensed or fragmented homogeneous nuclear chromatin, and necrotic cells with irregularly destroyed nuclei and cytoplasmic membranes. Apoptotic cell death were also observed in parietal cells of the stomach and lymphocytes in the lymphatic system. DNA ladders with approximately 200-bp multimers were observed in hepatic, renal and thymic samples prepared after 6 and 24 hr of LPS injection by agarose gel electrophoresis. These results suggest that apoptosis is involved in the pathology of swine endotoxemia.  相似文献   

20.
More than 99% of follicles undergo a degenerative process known as "atresia", in mammalian ovaries, and only a few follicles ovulate during ovarian follicular development. We have investigated the molecular mechanism of selective follicular atresia in mammalian ovaries, and have reported that follicular selection dominantly depends on granulosa cell apoptosis. However, we have little knowledge of the molecular mechanisms that control apoptotic cell death in granulosa cells during follicle selection. To date, at least five cell death ligand-receptor systems [tumor necrosis factor (TNF)alpha and receptors, Fas (also called APO-1/CD95) ligand and receptors, TNF-related apoptosis-inducing ligand (TRAIL; also called APO-2) and receptors, APO-3 ligand and receptors, and PFG-5 ligand and receptors] have been reported in granulosa cells of porcine ovaries. Some cell death ligand-receptor systems have "decoy" receptors, which act as inhibitors of cell death ligand-induced apoptosis in granulosa cells. Moreover, we showed that the porcine granulosa cell is a type II apoptotic cell, which has the mitochondrion-dependent apoptosis-signaling pathway. Briefly, the cell death receptor-mediated apoptosis signaling pathway in granulosa cells has been suggested to be as follows. (1) A cell death ligand binds to the extracellular domain of a cell death receptor, which contains an intracellular death domain (DD). (2) The intracellular DD of the cell death receptor interacts with the DD of the adaptor protein (Fas-associated death domain: FADD) through a homophilic DD interaction. (3) FADD activates an initiator caspase (procaspase-8; also called FLICE), which is a bipartite molecule, containing an N-terminal death effector domain (DED) and a C-terminal DD. (4) Procaspase-8 begins auto-proteolytic cleavage and activation. (5) The auto-activated caspase-8 cleaves Bid protein. (6) The truncated Bid releases cytochrome c from mitochondrion. (7) Cytochrome c and ATP-dependent oligimerization of apoptotic protease-activating factor-1 (Apaf-1) allows recruitment of procaspase-9 into the apoptosome complex. Activation of procaspase-9 is mediated by means of a conformational change. (8) The activated caspase-9 cleaves downstream effector caspases (caspase-3). (9) Finally, apoptosis is induced. Recently, we found two intracellular inhibitor proteins [cellular FLICE-like inhibitory protein short form (cFLIPS) and long form (cFLIPL)], which were strongly expressed in granulosa cells, and they may act as anti-apoptotic/survival factors. Further in vivo and in vitro studies will elucidate the largely unknown molecular mechanisms, e. g. which cell death ligand-receptor system is the dominant factor controlling the granulosa cell apoptosis of selective follicular atresia in mammalian ovaries. If we could elucidate the molecular mechanism of granulosa cell apoptosis (follicular selection), we could accurately diagnose the healthy ovulating follicles and precisely evaluate the oocyte quality. We hope that the mechanism will be clarified and lead to an integrated understanding of the regulation mechanism.  相似文献   

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