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.     

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.     

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.     

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.     

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.     

Role of antioxidants in the protection against oxidative stress in cattle--trace elements and enzymatic mechanisms (Part 3)

The most important enzymatic mechanisms which protect an organism against oxidative stress are superoxide dismutase (SOD), peroxidase (Px), e.g. glutathione peroxidase (GSH-Px) and ascorbate peroxidase, catalase and glutathione reductase. Their activity depends on many trace elements. Enzymatic mechanisms, functioning under physiological conditions, prevent the spread of free radical reactions. New and reoccurring metabolic and infectious diseases of cattle emerge when there is a disproportion in the balance between reactive oxygen species and antioxidative enzymatic barrier.     

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.     

Activity of selected glycosidases and availability of their substrates in bovine placenta during pregnancy and parturition with and without retained foetal membranes

The activity of glycosidases is crucial for the function and biological activity of proteins conjugated with sugar moieties, which play an important role in adhesion of cells during attachment and detachment of the foetal membranes. The aim of study was to describe the ability of bovine placental tissues to break down O-glycosidic bonds in different glycoproteins by the determination of activity of β-galactosidase, α-l-fucosidase, β-N-acetyl-hexosaminidase and sialidase in early–mid-pregnancy as well as at parturition with released and retained foetal membranes. Moreover, the availability of substrates for these glycosidases in placental homogenates was evaluated. Placental samples were collected from pregnant (2–4 months) cows in slaughterhouse (n = 8) as well as during Caesarean section and divided into released foetal membranes (n = 8) and retained foetal membranes (n = 8). Tissue homogenates were subjected to spectrofluorimetric and spectrophotometric determinations of enzyme activities as well as electrophoretic separations. Enzyme activities expressed changes within examined time with significant (p < .05) differences between pregnancy and physiological parturition in β-N-acetyl-hexosaminidase and α-l-fucosidase in foetal part of placenta while in maternal part only in the latter one. Decreasing tendency in enzyme activity was noticed in foetal part of retained samples in comparison with released ones with significant (p < .05) differences in α-l-fucosidase activity. The analysis of staining of sugar moieties attached to selected proteins depicted availability of sugar molecules in examined tissues, but their patterns differed between samples. In conclusion, sugar moieties in conjugated proteins express changes in the course of pregnancy which is reflected by the alterations in activities of placental glycosidases.     

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

Contents Poly(ADP-ribose) polymerase (PARP) synthesizes poly(ADP-ribose) in response to DNA strand breaks using NAD⁺ as a substrate. It leads to consequences for metabolism not only on a cellular level, but also on a tissue level, among others: NAD⁺ and ATP depletion. Retention of foetal membranes (RF) in cows is supposed to be connected with the imbalance between production and neutralization of reactive oxygen species, leading to oxidative damage to DNA, lipids and proteins. The aim of this preliminary study was to detect the presence of PARP in bovine placenta and to describe the enzyme with respect to type of placental tissue, time and mode of delivery. Placentomes, collected after spontaneous delivery or caesarian section, were divided into maternal and foetal parts of placenta, homogenized, and subjected to electrophoresis. Cows were divided into six groups as follows: (A) caesarian section before term with RF, (B) caesarian section before term without RF, (C) spontaneous delivery at term with RF, (D) spontaneous delivery at term without RF, (E) caesarian section at term with RF, (F) caesarian section at term without RF. PARP was detected by Western blotting using commercially available bovine anti PARP antibody. Bands referred to as bovine PARP standard were present in all examined tissues as well as the products of its cleavage. However, the patterns of bands were different with respect to type of tissue, time, and mode of delivery. Further experiments on detailed relationship between PARP activity and the process of releasing and retaining of bovine placenta are necessary.     

Biology of the prolactin family in bovine placenta. II. Bovine prolactin-related proteins: Their expression, structure and proposed roles

The placenta produces various peptides and steroid hormones that regulate placental function and fetal growth. Prolactin‐related proteins are peptides that are produced by the placenta and belong to the growth hormone/prolactin family, and have structural similarity to prolactin and placental lactogen. Although several prolactin‐related protein genes have been detected in bovine placenta, their expression profiles and functions are not clear. The main difficulties in examining their biological function is the similarity between their genes and the lack of information about their proteins. Recently, molecular biology methods have been used to detect some new bovine prolactin‐related proteins, and elucidate their biological functions. This review focuses on the structures, expression profiles and conceivable functions of prolactin‐related proteins in bovine placenta. With respect to their expression profiles, bovine prolactin‐related proteins fall into four groups: (i) those expressed around the implantation period; (ii) those that reach peak expression in the middle of gestation; (iii) those that increase with the progress of gestation, reaching a peak in late gestation; and (iv) those that reach a plateau in early gestation and are maintained at that level throughout gestation. Data indicate that bovine prolactin‐related proteins have different biological roles in different periods of gestation. In situ monitoring suggests that bovine prolactin‐related protein‐I has a role in the attachment of trophoblast cells to endometrium during the early implantation period.     

Effect of decorin and selected glycosylation inhibitors on the adhesion of caruncular epithelial cells of pregnant cows—part I

Adhesion process ensures the formation of the appropriate connection between mother and foetus during placentation and further placental development, which determines physiological pregnancy course. Extracellular matrix of foetal membranes are a rich source of biologically active proteins, the synthesis of which is regulated by hormones. Depending on the stage of pregnancy, the protein profile of the placenta changes, thanks to which its remodelling is possible. The aim of the study was to evaluate the effect of decorin, as well as selected glycosylation inhibitors on the adhesion of caruncular epithelial cells derived from cows during pregnancy. Placental cells were isolated from healthy, pregnant (2nd and 4th month) cows after slaughter, which allowed for the establishment of 4 primary cell cultures without visible cells of fibroblast morphology. The presence of decorin in cell monolayer and cell lysates was determined by the use of immunocytochemistry and Western blotting, respectively. The viability of cells was evaluated by MTT assay. The adhesion of cells to fibronectin was measured spectrophotometrically. Protein N-glycosylation and O-glycosylation have a modulating effect on the adhesion and viability of placental cells during early–mid pregnancy. Decorin and tunicamycin were shown to have anti-adhesive properties with respect to caruncular cells of the pregnant bovine uterus.     

银杏叶提取物对热应激致鸡心肌细胞氧化损伤的保护作用研究

本试验旨在研究银杏叶提取物（Ginko biloba extract,GBE）对热应激致鸡心肌细胞氧化损伤的保护作用。取12日龄AA肉鸡心脏制备鸡原代心肌细胞,向原代心肌细胞培养板中分别加入0、5、10、50、100、150 mg/mL GBE,CCK-8试剂盒检测心肌细胞存活率;将原代心肌细胞以1×10⁴个/孔接种于96孔板,培养48 h,分别加入0、5、10、50、100、200、500、1 000 μg/mL GBE,Hoechst 33258检测心肌细胞凋亡率,确定最适GBE浓度。42℃热应激处理（0、0.5、1、2和4 h）建立心肌细胞损伤模型,ELISA试剂盒检测乳酸脱氢酶（LDH）、细胞丙二醛（MDA）和谷胱甘肽过氧化物酶（GSH-Px）含量,以及超氧化物歧化酶（SOD）活性;Western blotting检测Hsp72蛋白表达。结果显示,高浓度GBE具有细胞毒性,50、100、150 mg/mL GBE能极显著抑制细胞生长（P<0.01）,50 μg/mL GBE能够极显著提高细胞增殖率（P<0.01）。热应激后细胞发生凋亡,细胞内MDA、LDH含量增加,SOD活性、GSH-Px含量减少,产生氧化损伤;GBE可以降低热应激时细胞LDH和MDA的含量,增加SOD活性和GSH-Px的含量,热应激2、4 h,GBE处理效果明显。此外,GBE可增加心肌细胞中Hsp72蛋白的表达。综上所述,GBE能降低热应激状态下心肌细胞的氧化损伤,其机制可能与提高抗氧化酶活性及提高Hsp72的表达有关。     

Reduced Phagocytotic Activity of Macrophages in the Bovine Retained Placenta

This study was carried out to investigate the distribution of immune cells in the bovine placenta during the postpartum period and to compare these cells between normal and retained placenta. Within 1 h after normal calving, biopsy samples of placentomes were collected from 10 cows. The occurrence of retention of fetal membranes was monitored for more than 8 h post-calving, and the samples obtained were divided into two groups: normally discharged and retained placenta (n = 5 each). Immunohistochemical procedures were utilized to detect macrophages and T lymphocytes. Numerous CD14-positive macrophages were found in the stroma of both normal placenta and retained placenta whereas only a few CD3-positive T lymphocytes were found in both cases. However, histochemical staining for acid phosphatase, a predominant lysozomal enzyme, revealed that almost all macrophages showed strong enzyme activity in the normally discharged placentas, whereas in retained placenta the activity of acid phosphatase was conspicuously decreased in intensity. These results indicate that there are functional differences in placental macrophages between normal and retained placenta.     

Messenger RNA expression of angiotensin-converting enzyme, endothelin, cyclooxygenase-2 and prostaglandin synthases in bovine placentomes during gestation and the postpartum period

The bovine placenta contains local vasoactive-related systems, including angiotensin-converting enzyme (ACE), endothelin-1 (ET-1), ET-A receptor (ETAR) and ET-B receptor (ETBR), as well as arachidonic acid (AA) cascade-related enzymes, such as cyclooxygenase-2 (Cox-2), prostaglandin E-synthase (PGES) and prostaglandin F-synthase (PGFS). The mRNA expression of these molecules was examined in bovine placentomes (caruncles and cotyledons) collected immediately (0 h) and 6h after spontaneous parturition from 15 cows with early (fetal membranes released within 6 h of parturition) or late (fetal membranes released 6-12 h after parturition) detachment, as well as from 15 pregnant cows at a slaughterhouse. Significant differences were observed in expression of ET-1, ETAR and ETBR mRNAs between gestation and the postpartum period in both caruncles and cotyledons. Significant differences were also found between 0 and 6 h postpartum in the expression of ETBR mRNA in the early detachment group and PGES mRNA in the early and late detachment groups. Compared to PGFS, both Cox-2 and PGES exhibited opposite mRNA expression patterns during gestation and the postpartum period. The vasoactive-related peptide systems and AA cascade-related enzymes may mediate placental development and fetal membrane detachment after parturition in cattle.     

The Comparison of Antioxidative/Oxidative Profile in Colostrum,Milk and Blood of Early Post‐Partum Cows During their First and Second Lactation

The aim of the study was to compare antioxidative/oxidative profile in blood, colostrum and milk of early post‐partum cows during their first and second lactation. A total of 19 healthy, primiparous cows were included in experiment and samples were collected during 2 years from the same animals immediately after parturition, 24, 48 h as well as 6 and 12 days later. All parameters including the activity of glutathione peroxidase (GSH‐Px), total antioxidant capacity (TAC), the content of vitamin A, C as well as the contents of products of lipid and protein peroxidation were determined by the use of spectrophotometric methods. Comparing the profile within lactation, TAC and GSH‐Px activity in blood showed decreasing trends, while parameters of lipid and protein peroxidation fluctuated. All examined parameters in colostrum and milk except from intermediates of lipid peroxidation exhibited increasing trends. These results which showed dynamic changes of antioxidative/oxidative profile not only in blood but also in colostrum/milk within examined period of time suggested appropriate answer of organism to current challenge. Moreover, not uniform but detectable changes between first and second lactation suggested that two consecutive lactations are not the same. Comparing first and second lactation, TAC and parameters of lipid and protein peroxidation in blood showed increasing tendency in second as first lactation while GSH‐Px activity was opposite. The content of antioxidative vitamins and SH groups in colostrum/milk showed increasing tendency in second as first lactation, while TAC and content of end products of lipid peroxidation showed opposite trend, and GSH‐Px together with intermediates of lipid peroxidation remained stable. Molecular and biochemical background for it require further elucidation.     

Extracellular matrix in epitheliochorial,endotheliochorial and haemochorial placentation and its potential application for regenerative medicine

A placenta is defined as structural approximation of maternal and foetal tissues to perform physiological exchange. Associated processes of differentiation and the establishment of its cells take place within the extracellular matrix (ECM) that provides a rich environment of collagens, fibronectins, cytokines and other components. Placental ECM is promising for tissue regeneration purposes, because it has immune tolerance capacities that may cause only minimal rejections of transplants with immunological differences between donor and recipient. However, specific characteristics of ECM during evolution of the structurally very diverse mammalian placenta are not yet revealed. We here address the major aspects of placental types, that is non‐invasive (epitheliochorial), medium (endotheliochorial)‐to‐high (haemochorial) invasive nature of the interhemal barrier between the foetal and maternal blood system as well as their main components of ECM with special reference to species that are commonly used as animal models for human placentation and in the potential applications for regenerative medicine.     

Effect of dietary antioxidant supplementation on the oxidative status of plasma in broilers

In this study, the effect of dietary antioxidants on the plasma oxidative status of growing birds fed a diet rich in polyunsaturated fatty acids was investigated. One‐day‐old broilers were fed for 42 days a diet containing 4% linseed oil and supplemented with single plant extracts rich in antioxidants (natural tocopherols, rosemary, grape seed, green tea, tomato) or a combination of some of these plant extracts, in two different total doses (100 and 200 mg product/kg feed). A diet with synthetic antioxidants with and without α‐tocopheryl acetate (200 mg/kg feed) were also included. The plasma oxidative status was evaluated measuring the ferric reducing ability of plasma (FRAP), the superoxide dismutase (SOD) and glutathione peroxidase (GSH‐Px) activity. Lipid peroxidation was measured by thiobarbituric acid‐reactive substances (TBARS). No significant effect of the dietary treatments was observed for FRAP as well as for TBARS. However, diet affected GSH‐Px activity (p = 0.002) and a trend for an effect on SOD activity was observed (p = 0.084). A higher GSH‐Px activity was found for 200 mg/kg tomato extract and natural α‐tocopherol in relation to the corresponding 100 mg/kg treatment, and the lowest GSH‐Px activity was measured for the synthetic antioxidants treatment. The lowest and highest SOD activity were found for the 200. and 100 mg/kg treatment with tomato extract respectively. In conclusion, the oxidative status and lipid oxidation of plasma in broilers was not affected by feeding natural antioxidant extracts at the doses in the present study, but some changes in antioxidant enzyme activities were observed, of which the implication remains to be elucidated.     

Influence of the duration of calving and obstetric assistance on the placental retention index in Holstein dairy cows

The aim of this study is to evaluate the influence of duration of calving and obstetric assistance on retained placenta incidence of high milk production Holstein cows. Experimental groups were determined according to the duration of the expulsive phase of calving: 2 h (n = 16), 2‐4 h (n = 16) and >4 h (n = 12), and additionally allocated in two sub‐groups: spontaneous calving (n = 22) and intervention calving (n = 22). Diagnosis of retained placenta was considered with a threshold of 8 h after the expulsive phase. Cows without obstetric intervention, with labor duration of 2 h, presented reduced time needed for placental release, in comparison to those with obstetric assistance. In the 2–4 h group and >4 h, there was no statistical difference. The 2 h and 2–4 h groups with intervention and the spontaneous >4 h group were considered retained placenta groups. On the other hand, performing obstetric intervention when calving period was superior to 4 h nulled the occurrence of retention of fetal membranes. As a conclusion, obstetric assistance predisposes placental retention to calving with <2 h of duration in dairy cows. Conversely, when calving is more than 4 h, performing fetal extraction has a beneficial influence on preventing retained placenta.