Poly(ADP-Ribose) Glycohydrolase in Bovine Retained and Not Retained Placenta

Poly(ADP-ribose) glycohydrolase (PARG) is the enzyme which degrades poly(ADP-ribose) polymers synthesized by poly(ADP-ribose) polymerase (PARP). Both enzymes are activated in response to different stimuli like oxidative stress and are involved in DNA repair processes. The retention of bovine foetal membranes (RFM) is supposed to be connected with oxidative stress conditions. The aim of the study was to detect the presence of PARG protein in bovine placenta in order to find the relationship between the process of releasing, retaining placenta and DNA repair. Placentomes, collected alter spontaneous delivery or caesarian section were divided into maternal as well as foetal part of placenta, homogenized and subjected to electrophoresis. Animals were divided into six groups as follows: A--caesarian section before term with RFM; B--caesarian section before term without RFM; C--spontaneous delivery at term with RFM; D--spontaneous delivery at term without RFM; E--caesarian section at term with RFM; F--caesarian section at term without RFM. PARG protein was detected in nitrocellulose membranes using commercially available bovine anti-PARG antibody and Western blotting technique. Single bands referred to bovine PARG standard were observed in all examined tissues as well as in human placenta used as the control of procedure. In addition, the intensity of staining was stronger in retained than properly released term placenta and in foetal than in maternal part of the placenta. These results may suggest the differences in enzyme protein content and careful conclusions can be drawn that the activities of PARG may be altered between compared groups of animals. It may confirm the presence of oxidative stress conditions and their consequences on metabolic pathways, the content of biologically active substances and processes of proper releasing placenta. Further experiments on PARG activity in bovine foetal membranes with respect to proper and improper placental release are necessary.     

Non-enzymatic Antioxidative Defence Mechanisms Against Reactive Oxygen Species in Bovine-retained and not-retained Placenta: Vitamin C and Glutathione

Vitamin C and glutathione (GSH) are water-soluble antioxidants which take part in defence mechanisms against reactive oxygen species (ROS). They may also be involved in processes of releasing/retaining bovine fetal membranes. Hence vitamin C, reduced (GSH) and oxidised (GSSG) glutathione levels were determined in retained and not-retained bovine fetal membranes in order to describe the non-enzymatic antioxidative status. Placental samples were collected immediately after spontaneous delivery or during caesarean section before term and at term, and 6 groups were formed as follows: (A) pre-term caesarean section without retained placenta; (B) pre-term caesarean section with retained placenta; (C) term caesarean section without retained placenta; (D) term caesarean section with retained placenta; (E) spontaneous delivery without retained placenta and (F) spontaneous delivery with retained placenta. Homogenates of maternal and fetal placental tissues were prepared, and vitamin C, GSH and GSSG were measured spectrophotometrically. Vitamin C levels were significantly higher in the maternal part than in the fetal part of the placenta in all groups examined. In retained placenta cases the levels were significantly lower than in control cows, except in pre-term groups. GSH concentrations were significantly higher in placentas without retention than with retention. GSSG levels showed the opposite relationship and were significantly higher in samples with retention of fetal membranes than in controls. Further experiments on antioxidative as well as oxidative status in bovine placenta are necessary.     

Macrophages in bovine term placenta: An ultrastructural and molecular study

Retention of foetal membranes (RFM) is a major reproductive disorder in dairy cows. An appropriate immune response is important for a physiological expulsion of the foetal membranes at parturition. Our study aims to provide a deeper insight into characteristics of foetal and maternal macrophages in bovine term placenta. We used transmission electron microscopy (TEM), immunohistochemistry and semi-quantitative RT-PCR to provide a deeper insight into characteristics of foetal and maternal macrophages in bovine term placenta. Semi-quantitative RT-PCR was used to define macrophage polarization in foetal and maternal compartments of normal term placenta. Gene expression of factors involved in M1 polarization [interferon regulatory factor-5 (IRF5), interleukin (IL)-12A, IL12B] and in M2 polarization (IL10) were studied. Ultrastructurally, foetal macrophages showed an irregular shape and large vacuoles, whereas the maternal macrophages were spindle shaped. By immunohistochemistry, macrophages were identified by a strong staining with the lysosomal marker Lysosome-associated membrane glycoprotein 1 (LAMP-1), while myofibroblast in the maternal stroma was positive for alpha-smooth muscle actin. We used the LAMP-1 marker to compare the density of foetal stromal macrophages in placentas of cows with RFM and in controls, but no statistically significant difference was observed. RT-PCR showed a higher expression of all studied genes in the maternal compartment of the placenta and generally a higher expression of M1-, compared to M2-associated genes. Our results indicated that at parturition placental macrophages predominantly show the pro-inflammatory M1 polarization. The higher expression of all the target genes in the maternal compartment may denote that maternal macrophages in bovine term placenta are more frequent than foetal macrophages.     

Is poly(ADP-ribose) polymerase involved in bovine placental retention?

Poly(ADP-ribose) polymerase (PARP) is the enzyme which utilises NAD to synthesise poly(ADP-ribose) polymers. This process appears in response to DNA lesions. Oxidative stress, which might be involved in bovine placental retention, is the reason for oxidative DNA injury. In this mini-review, the relationship between PARP activity and bovine placental retention is discussed. The results of our experiments on PARP activity in placental tissues showed that the enzyme of 113 kDa and its cleavage products were present in retained as well as released fetal membranes. Western blotting technique showed different intensities in the staining of bands which might suggest different activities of the enzyme.     

The Presence of SOD 1 and GSH‐Px in Bovine Retained and Properly Released Foetal Membranes

The maintenance of antioxidative/oxidative balance is crucial for cellular and extracellular environment. That is why antioxidative enzymes express their activity in different isoforms in different cell compartments and extracellular space. The aim of study was to verify the results of previous experiment on activities of antioxidative enzymes by the determination of their enzymatic proteins in bovine placental tissues by Western blotting technique. Moreover, the presence of particular isoenzymes was detected and differentiated. Homogenates of maternal and foetal part of both properly released and retained bovine placenta were subjected to PAGE electrophoresis in non‐reducing and reducing conditions and Western blotting with appropriate antibodies against superoxide dismutase (SOD) and glutathione peroxidase (GSH‐Px). Electrophoresis allowed for the detection of protein bands of molecular weight related to CuZn‐SOD as well as cGSH‐Px isoenzymes. The reaction with appropriate antibodies confirmed this. Densitometric analysis, although semi‐quantitative, allowed for the observation of trends in differences in antioxidative enzyme proteins, which may partly confirm previously described results in cases of retained and released placenta. Local antioxidative enzymatic mechanisms in bovine placental tissues are represented by CuZn‐SOD and cGSH‐Px, which show the changes in their expression during improper placental release.     

Post-partum leucocytic activity and its relationship to caesarian section and retained placenta

The activity of the leucocytes of 30 cows during 10 days after parturition is described. A comparison is made between the activity of leucocytes of 10 cows after normal parturition, 10 cows after caesarian section and 10 cows with retained placenta. When compared with normal parturition, caesarian section does not have a negative influence on the activity of the leucocytes during the puerperal period, during which time however the activity of leucocytes of cows with retained placenta is strongly decreased.     

Post‐partum leucocytic activity and its relationship to caesarian section and retained placenta

Summary

The activity of the leucocytes of30 cows during 10 days after parturition is described. A comparison is made between the activity of leucocytes of 10 cows after normal parturition, 10 cows after caesarian section and 10 cows with retained placenta. When compared with normal parturition, caesarian section does not have a negative influence on the activity of the leucocytes during the puerperal period, during which time however the activity of leucocytes of cows with retained placenta is strongly decreased.     

Profile of Bovine Proteins in Retained and Normally Expelled Placenta in Dairy Cows

Tissue‐specific protein profile is determined by its function, structure, intensity of metabolism and usefulness. This profile remains under hormonal control. Any disturbance in the general metabolism may be reflected in changes in both protein quantity and quality. These changes can be of low or high specificity, and some can be used as clinical markers of pathological conditions. The aim of this study was to describe and to compare the protein profile of caruncle and foetal villi of bovine placenta that was either properly released or retained. Placental tissues were collected from healthy cows, divided into releasing and retaining foetal membranes, homogenized and subjected to 1D and 2D electrophoresis. Computer‐aided analysis of gel images showed essential qualitative and quantitative alterations in protein profile between tissues that were properly released and retained. Alterations concerned both the number of fractions and spots as well as the intensity of staining. This preliminary study provides a general overview of the differences in the protein profile between released and retained foetal membranes. It may allow for selecting the group of proteins or single molecules, which should be further analysed in detail as possible markers differentiating the retention of foetal membranes in cows from placentas that were released spontaneously. The continuation of the study for the identification of particular spots detected in 2D gels is necessary.     

Patterns of protein glycosylation in bovine placentomes as a function of gestational age and in retained versus non‐retained placenta

The formation of placenta at the beginning of pregnancy and its separation at parturition require not only deep remodelling of extracellular matrix, which mainly consists of proteins conjugated with sugar moieties, but also the cooperation with cells from both maternal and foetal parts of placenta. The aim of the study was to compare the patterns of selected conjugated proteins with sugar moieties between pregnant and term placenta as well as between released and retained placenta in cows. Placental samples from healthy pregnant cows (3–5 months of pregnancy) were collected at a slaughterhouse (n = 6), and parturient samples were collected during caesarean section at term and retrospectively divided into retained (n = 6) and released (n = 6). The pattern of selected sugar moieties conjugated with proteins was detected by use of lectin blotting with Phaseolus Vulgaris leucoagglutinin, Maackia Amurensis and Sambucus Nigra (Elderberry). The comparison and analysis of obtained band patterns showed differences between their number, molecular weight and abundance related to the intensity of staining. Samples from 3 to 4 months showed similarities, while at the 5th month, clear differences were visible in all 3 lectins, which were used in this study. Samples from retained/released placenta expressed significant differences in PHA‐L and SNA pattern in the foetal part. Obtained results indicate that the development of placenta related to extracellular matrix and accompanying cells from both sides of placenta shows dynamic changes during pregnancy. Moreover, in the case of animals with the retention of foetal membranes the patterns of proteins conjugated with sugar moieties are altered, suggesting that the changes in extracellular matrix metabolism can be involved in the attachment and detachment of the placenta in cows.     

Abnormal Expression of TIMP-2, SOD, Vimentin and PAI Proteins in Cloned Bovine Placentae

Cloned mammals suffer from high rates of placental abnormality and foetal loss during pregnancy. We previously used 2-D gel electrophoresis and mass spectrometry for global proteomic analysis of cloned and normal bovine placentae to identify differential protein expression patterns. Here, we used Western blot analysis to confirm the expression levels of several pregnancy-related proteins putatively identified as being differentially expressed in somatic cell nuclear transfer (SCNT) vs normal bovine placentae. The expression levels of tissue inhibitor of metalloproteinase-2 (TIMP-2), its downstream protein, matrix metalloproteinase-2 (MMP-2), superoxide dismutase (SOD), vimentin and plasminogen activator inhibitor-1 (PAI) were analysed in the placentae of SCNT cloned Korean native cattle that died immediately after birth and in normal placentae obtained by AI. Our results revealed that TIMP-2 and SOD were up-regulated in SCNT placenta compared with normal placenta, whereas MMP-2 levels were comparable in cloned and normal placentae, and vimentin and PAI were significantly down-regulated in SCNT compared with normal placentae. Our results suggest that key proteins of placental development are abnormally expressed in SCNT cloned bovine placentae, probably resulting in abnormal placental function and clonal mortality.     

Bacterial Isolates Associated With Dystocia And Retained Placenta In Iraqi Buffaloes

The present study was conducted on 50 recently calved Iraqi Buffalo cows. Depending on the kind of parturition, buffalo cows were divided into two main groups, the first group had normal unassisted parturition (NP) (26 animals) and the second group with certain periparturent complications (PPC) (24 animals). After 24 h of parturition, these two groups were further subdivided into two groups as cows expel their foetal membranes in <24 h postpartum and referred as non‐retained placenta (NRP) while cows that did not expel their foetal membrane after 24 h referred as retained placenta (RP). Sampling for bacteriology, uterine discharge for polymorphonuclear cells per cent and blood samples for polymorphonuclear neutrophil (PMN) and the enzyme creatine kinase activity were performed at 6, 24 and 48 h postpartum. In PPC group, the most prevalent bacteria after 6 h of calving were Escherichia coli, β‐haemolytic Streptococci and Lactobacillus acidophilus. Total bacterial isolates in the uterus of buffaloes with RP in PPC group after 24 and 48 h were 129 and 183 respectively. Among the isolates, Archanobacterium pyogenes, Fusobacterium necrophorum, Prevotella melaninogenicus and Staphylococcus aureus were the most prevalent isolates after 48 h of RP buffaloes in PPC group. Polymorphonuclear neutrophil were significantly (p < 0.01) increased in the uterine discharge than in blood in buffaloes with RP in both PPC and NP groups. In conclusion, uterine contamination occurs as a result of postpartum ascending contamination by non‐specific environmental organisms. The presence of Lactobacillus sp. in the uterus indicated a healthy uterus. Peripartum complications followed by retention of foetal membranes with the dominance of E. coli in the uterine lumen might favour the colonization of other bacteria including facultative anaerobic and strictly anaerobic in the uterine wall of buffaloes.     

Antigenic similarity between squamous cell carcinoma of horn (horn cancer) and normal bovine foetal tissues.

Normal bovine foetal (liver and skin) and horn cancer tissue antigens were examined using double diffusion agar gel precipitation and immuno-electrophoretic tests to detect any cross reactivity among them. Rabbit horn cancer antisera absorbed with normal bovine liver, skin and horn core epithelium antigens, when tested with foetal skin and liver (4 to 6 months of gestation), revealed the presence of 2 foetal antigens in horn cancer. Immuno-chemically 2 of the horn cancer antigens were found to be identical to the bovine foetal antigens.     

Analysis and Validation of the Differentially Expressed ENO1 in the Maternal Placenta of Cow with Retained Foetal Membrane

The paper aimed to analyze and validate the function of differentially expressed ENO1 in the cow with retained foetal membrane.In our research,we chose three healthy Holstein dairy cows and three Holstein dairy cows with retained foetal membrane of similar age,foetal times,weight and milk yield and divided them into two groups.The total protein of maternal placenta was extracted in the control and retained foetal membrane of cow.The differential expression of proteins were found out by the 2-DIGE,and the differential expression of ENO1 was validated by the Western blotting and Real-time quantitative PCR.The results showed that ENO1 was significant expression and large multiple in the two groups,and it was significant increased expression in the retained foetal membrane of cow after Western blotting and Real-time quantitative PCR (P=0.015<0.05;P=0.001<0.01).The ENO1 participated in the energy homeostasis,immune and fibrinolysis process which related to the retained foetal membrane of cow.It suggested that ENO1 was likely connected with the retained foetal membrane.     

胎衣不下奶牛母体胎盘α-烯醇化酶异常表达分析及验证

试验通过对胎衣不下奶牛母体胎盘组织中差异α-烯醇化酶(enolase,ENO1)的分析验证,探讨ENO1在胎衣不下中的作用。本研究选取了年龄、胎次、体重和泌乳量均相近的产后胎衣不下和产后胎衣正常排出奶牛各3头,分为两组,提取了胎衣不下组与胎衣正常排出组母体胎盘组织中的总蛋白,采用双向凝胶电泳的方法筛选出差异蛋白,并利用Western blotting及实时荧光定量PCR的方法对其中差异表达量大的ENO1进行验证。结果发现,胎衣不下组和胎衣正常组母体胎盘组织中ENO1差异表达量大且倍数较大,Western blotting及实时荧光定量PCR验证发现ENO1在胎衣不下奶牛母体胎盘中的表达量升高,t检验结果分别为P=0.015<0.05,差异显著;P=0.001<0.01,差异极显著。该基因参与机体的能量调节、免疫和纤溶等过程,而这些过程与奶牛胎衣不下密切相关,提示ENO1可能参与该病的发生发展。     

Engraftment of severe combined immune deficient/beige mice with bovine foetal lymphoid tissues.

To develop a model of bovine thymus and lymph node growth in vivo, we have implanted bovine foetal tissues (16-23 weeks gestation) under the renal capsule of severe combined immune deficient (SCID)/beige (BG) mice and assayed for graft growth and characteristics 2-18 weeks after engraftment. Bovine foetal thymus and lymph node grew considerably following engraftment of SCID/BG mice. Growth was optimal if bovine foetal tissues were used before gestation Week 17. Bovine-mouse chimerism was confirmed using glucose phosphate isomerase analysis. Bovine thymus grew during the entire 18 weeks of study. Growth of bovine lymph node was initially rapid, reaching a maximum at 2 weeks after transplantation followed by a progressive decrease in size. Transplanted bovine lymph node and thymus were morphologically similar to age-matched bovine foetal tissue for a limited time period. Fibrosis, degeneration and depletion of lymphocytes were evident 6 weeks after engraftment; changes were more severe in lymph node than in thymus whereas increases in lymphocytes, lymphopoiesis and follicle formation were evident in age-matched bovine foetal tissue. Despite growth and morphological similarities of the transplanted tissue, blood counts suggested there was no peripheralization of bovine leucocytes. Bovine immunoglobulins (IgG1 and IgG2) were detected in serum of some SCID/BG chimeric mice for a limited time. The appearance of bovine immunoglobulins at 2 weeks in SCID/BG chimeric mice depended on the age of the foetal donor (> 18 weeks) and coincided with the appearance of morphologically mature lymphocytes in the donor foetus lymph nodes. The ability to produce bovine immunoglobulins decreased 8 weeks after engraftment, coinciding with the depletion of lymphocytes in the engrafted lymph node. Lymphocyte depletion and loss of function of engrafted tissues appear the result of a lack of lymphoid progenitors normally derived from hematopoietic stem cells in the bone marrow.     

Incorporation of bovine bone marrow stromal cells into porcine foetal tissues after xenotransplantation

Bovine bone marrow stromal cells (BMSCs) were injected into the liver of foetal pigs at about 40 days of gestation to test whether these cells could populate developing tissue, and if so, which ones. Approximately 40 days after injection, the foetuses were harvested and tissue sections from many areas of the body were analysed for the presence of bovine cells using two different methods. First, using PCR, bovine repetitive DNA was found to be present in DNA extracted from foetal pig tissues. Secondly, using oligonucleotide primed in situ synthesis (PRINS), the in situ presence of bovine cells was found within porcine tissue sections. PRINS-labelled cells were found within cartilage, perichondrium, connective tissue and smooth muscle. These data suggest that bovine BMSCs integrate throughout the foetal pig.     

Intra-operative cisplatin for the treatment of canine extremity soft tissue sarcomas

Nineteen dogs with histologically confirmed soft tissue sarcomas of the extremities were treated with a combination of marginal surgery and intra-operative chemotherapy in the form of cisplatin in a biodegradable implant delivery system (Atrigel^®; Atrix Laboratories, Fort Collins, Co, USA). None of the dogs had evidence of metastasis at time of treatment. The median dose of cisplatin was 52.1 mg/m² (mean 55.4 mg/m², range 18.5–108.6 mg/m²). Wound complications were noted in 16 dogs (84.2%). Median follow-up time was 874 days (mean 777 days, range 125–1463 days). Nine dogs (47.3%) were alive at the time of analysis. Local recurrence occurred in three dogs (16.6%). The time to recurrence was 214, 264 and 874 days.     

Apoptosis induction in BEFV-infected Vero and MDBK cells through Src-dependent JNK activation regulates caspase-3 and mitochondria pathways

Our previous report demonstrated that bovine ephemeral fever virus (BEFV)-infected cultured cells could induce caspase-dependent apoptosis. This study aims to further elucidate how BEFV activates the caspase cascade in bovine cells. BEFV replicated and induced apoptosis in Vero and Madin-Darby bovine kidney (MDBK) cells, and a kinetic study showed a higher efficiency of replication and a greater apoptosis induction ability of BEFV in Vero cells. Src and c-Jun N-terminal kinase (JNK) inhibitor, but not extracellular signal-regulated kinase (ERK) or p38 inhibitor, alleviated BEFV-mediated cytopathic effect and apoptosis. In BEFV-infected Vero and MDBK cells, BEFV directly induced Src tyrosine-418 phosphorylation and JNK phosphorylation and kinase activity, which was inhibited specifically by SU6656 and SP600125, respectively. The caspase cascade and its downstream effectors, Poly (ADP-ribose) polymerase (PARP) and DFF45, were also activated simultaneously upon BEFV infection. In addition, cytochrome c, but not Smac/DIABLO, was released gradually from mitochondria after BEFV infection. SU6656 suppressed Src, JNK, and caspase-3 and -9 activation, as well as PARP and DFF45 cleavage; SP600125 reduced JNK and caspase-3 and -9 activation, as well as PARP and DFF45 cleavage. Taken together, these results strongly support the hypothesis that a Src-dependent JNK signaling pathway plays a key role in BEFV-induced apoptosis. The molecular mechanism identified in our study may provide useful information for the treatment of BEFV.     

Intrauterine Idiopathic Amputation of the Head of a Porcine Foetus

An anencephalic full‐term porcine foetus accompanied by a mummified head was submitted for examination. The neck almost entirely lacked skin and was covered by granulation tissue as were the exposed parts of the spine and spinal cord. The case represents a rare case of intrauterine amputation. A definitive cause could not be established because the placenta was not available. The most likely cause is strangulation of the neck. Such strangulation could be due to a defect of the allantoamnion with herniation of the foetal head or entanglement by amniotic constriction bands.     

Enzymatic activity in the placenta in cows in relation to the course of parturition

The activity of aminopeptidases and cathepsins was determined in placentoma homogenates; placentomas of cows were extirpated immediately after parturition and in four and eight hours. In cows with afterbirth retention (a. r.) following induced parturition, the activity of these enzymes was always higher than in cows without a. r., no matter if after induced and spontaneous parturitions; it was at a similar or slightly higher level than in the eighth month of pregnancy. The content of total proteins in placentomas of cows with a. r. was also higher. These findings point to the insufficient ripening, or aging of placental tissue, which is related to a release of lysosomal enzymes. The enzymes are active through hydrolysis in the separation of foetal placenta from the maternal one and in the modification of proteins participating in intercellular linkages.