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 ß-galactosidase, α-l-fucosidase, ß-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 ß-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.

Differences in extracellular matrix remodeling in the placenta of mares that retain fetal membranes and mares that deliver fetal membranes physiologically.

INTRODUCTION: In mammals, placenta separation at term may involve degradation of the extracellular matrix by matrix metalloproteinases (MMPs). The activity of MMPs is modulated by TIMPs. We hypothesized that the placentas of mares that deliver fetal membranes physiologically and those that retain fetal membranes (FMR) differ in terms of histology; mRNA expression of MMP-2 and MMP-9; protein expression of MMP-2, MMP-9, and TIMP-2; and the potential activity of both MMPs. METHODS: Placenta biopsies were taken from mares (n = 9; 4 FMR, 5 controls) immediately after foal expulsion. Retention was defined as failure to expel all fetal membranes within 3 h of expulsion. All mares were monitored for time of expulsion. The degree of allantochorial/endometrial adhesion was determined in FMR mares, and biopsies from all mares were histologically examined. mRNA expression, protein immunolocalization, protein amount and potential enzyme activity were determined with RT-PCR, immunohistochemistry, Western Blotting and zymography, respectively. RESULTS: FMR mares had strong to extremely strong allantochorial/endometrial adhesion, and significantly more connective tissue in the allantochorial villi than controls. The range of MMP-2 mRNA expression levels was more than 13 times greater in FMR mares than in controls. Protein content of both MMPs and TIMP-2 differed significantly between groups. The range of potential MMP-2 and MMP-9 activity was larger in FMR mares, and MMP-2 potential activity was 1.4 times higher in controls (P = 0.02). DISCUSSION: These results indicate differences in extracellular matrix remodeling in FMR mares and controls, and suggest dysregulation of MMP expression and activation in FMR mares.

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.

Expression of matrix metalloproteinase (MMP)-2, MMP-14 and tissue inhibitor of matrix metalloproteinase (TIMP)-2 during bovine placentation and at term with or without placental retention.

Matrix metalloproteinases (MMPs) and counteracting tissue inhibitors of metalloproteinases (TIMPs) are balancing extracellular matrix (ECM) formation and degradation. The latter is believed to be an important aspect for the detachment of fetal membranes postpartum when loosening the feto-maternal connection which is a prerequisite to avoid placental retention a common disease in cows leading to considerable economic loss. Membrane-type (MT) MMPs have been suggested as potential activators controlling ECM remodelling. In particular, MT1-MMP (MMP-14) is able to degrade ECM substrates and activate MMP-2 through binding TIMP-2 at the cell surface. Since the connection between the trophoblast and the maternal caruncular epithelium is supported by integrin receptors bound to ECM, we hypothesize that impaired modulation of the ECM by TIMPs/MMPs participates in the aetiology of bovine retained fetal membranes. To analyse this involvement, placentomes were collected from cows after term parturition and timely release of fetal membranes (n = 4) and cows with retained fetal membranes after various treatments for the induction of parturition using progesterone antagonist (aglepristone), PGF(2α) analogue, glucocorticoid, and after elective caesarean sections (each group n = 3). The expression of MMP-14, MMP-2 and of TIMP-2 was examined by real-time-PCR, immunohistochemistry, Western blot and zymography. The relative mRNA expression levels of MMP-14 remained unchanged, while the expression levels of TIMP-2 and MMP-2 partly increased in animals with induced parturition and retention of fetal membranes compared to animals without placental retention. MMP-14 protein was expressed in cells of the uninucleated trophoblast, the fetal mesenchyme and maternal stroma. TIMP-2 was present exclusively in trophoblast giant cells, while MMP-2 could be detected in uninucleated trophoblast cells and the fetal mesenchyme. The presence of the activated enzyme was confirmed by zymography. In conclusion, MMP-14, MMP-2 and TIMP-2 are co-localized in the fetal compartment and therefore could influence the timely release of fetal membranes in cattle.

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.

Prostaglandin E(2) 9-keto reductase activity in bovine retained and not retained placenta.

Prostaglandin E(2) 9-keto reductase (9-KPR) activity shifts reversibly PGE(2) into PGF(2 alpha) and may be responsible for the control of prostaglandins (PGs) levels in, among others, placental tissues. The retention of fetal membranes in cows is the postpartum disorder where the disturbances in PGs metabolism have been reported. It has been argued whether these disturbances are due to alterations in 9-KPR activity. In this study, the activity of the enzyme was determined in maternal and fetal bovine placental tissues which were divided into 6 groups as follows: (A) caesarian section before term without retained fetal membranes (n=10), (B) caesarian section before term with retained fetal membranes (n=10), (C) caesarian section at term without retained fetal membranes (n=12), (D) caesarian section at term with retained fetal membranes (n=12), (E) spontaneous delivery at term without retained fetal membranes (n=12), (F) spontaneous delivery at term with retained fetal membranes (n=12). The enzyme activity was measured spectrophotometrically and expressed in nanokatals (nkat) per protein content. The activity increased towards parturition and was significantly higher in maternal than in fetal part of placenta in all groups examined. The significantly higher values in retained than in not retained placental tissues were observed in the samples examined. The present results indicate that the disturbances in 9-KPR activity in bovine retained placenta exist but their reasons still require further experiments.

Antioxidative defence mechanisms against reactive oxygen species in bovine retained and not-retained placenta: activity of glutathione peroxidase, glutathione transferase, catalase and superoxide dismutase.

Glutathione peroxidase (GSH-Px), glutathione transferase (GSH-Tr), catalase (CAT) and superoxide dismutase (SOD)-the members of enzymatic antioxidative defence mechanisms against reactive oxygen species-may play an important role in proper or improper release of bovine fetal membranes. The aim of the following study was the determination of GSH-Px, GSH-Tr, CAT and SOD activity in order to define antioxidative status of bovine placenta during retention of fetal membranes (RFM) in cows. Placental samples were collected immediately after spontaneous parturition or during caesarean section before term and at term and divided into six groups as follows: A: caesarean section before term without RFM; B: caesarean section before term with RFM; C: caesarean section at term without RFM; D: caesarean section at term with RFM; E: spontaneous delivery at term without RFM; F: spontaneous delivery at term with RFM. The enzyme activities in placental homogenates were measured spectrophotometrically. GSH-Px activity was statistically significantly higher in fetal than in maternal placenta in all examined groups, increased towards parturition and was higher in caesarean section groups than spontaneous delivery groups. Statistically significantly higher activities were noticed in retained than not-retained placentae. GSH-Tr activity was significantly lower in fetal than in maternal placenta. In preterm groups, the activity was statistically significantly higher in retained than not retained placenta. In term groups, the opposite relationship was observed, higher values in caesarean section groups than spontaneous delivery were noticed. CAT activity was statistically significantly higher in fetal than in maternal part of placenta in all groups examined. The highest values in C and D groups and the differences between retained and not-retained placenta were observed. SOD exhibited the highest values in preterm placenta and alterations between retained and not-retained fetal membranes. In conclusion, the activities of GSH-Px, GSH-Tr, CAT and SOD are altered in cases of retained fetal membranes which may suggest the activation of antioxidative mechanisms caused by the imbalance between production and neutralization of reactive oxygen species.

Matrix metalloproteinases (MMP-2 and MMP-9) and tissue inhibitor-2 of matrix metalloproteinases (TIMP-2) in the placenta and interplacental uterine wall in normal cows and in cattle with retention of fetal membranes.

Matrixmetalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) play a key role in tissue re-modelling in the placenta. In the present study, distribution of MMP-2, MMP-9 and TIMP-2 was demonstrated immunohistochemically in the bovine placenta and interplacentomal tissue. Specimens representing the whole gestation until parturition were processed. Additionally, materials from cows with and without retention of fetal membranes were compared. MMP-2 expression was abundant in the maternal septae of the placentome in early gestation, with ongoing pregnancy immunoreactivity was restricted to the stromal tissue at the openings of maternal crypts. The chorionic epithelium opposite to these regions was also positive for MMP-2. MMP-9 expression was observed in the chorionic epithelium, except in the giant binucleate cells. In addition, the maternal epithelium and stroma showed immunoreactivity for MMP-9. No differences in MMP-2 and MMP-9 distribution could be observed between cows with proper release of fetal membranes and cows with retained fetal membranes. Giant binucleate cells expressed TIMP-2 during the whole gestation. Immunostaining for alpha-smooth muscle actin revealed contractile elements in the bovine placentome. Balance between proteolytic enzymes and their activators and inhibitors is essential for regular development of the placenta. The expression of TIMP-2 in the giant binucleate cells indicates an essential role of inhibitory factors during gestation. It is likely that less TIMP-2 is produced at the end of pregnancy as the number of binucleate cells is diminished.

Acyloxyacyl hydrolase activity of neutrophil leukocytes in normal early postpartum dairy cows and in cows with retained placenta.

Acyloxyacyl hydrolase (AOAH) is an enzyme of bovine polymorphonuclear neutrophil leukocytes (PMN) that is capable of detoxifying endotoxin (25). The activity of AOAH in PMN isolated from the blood was investigated in dairy cows that expelled the fetal membranes normally (Group NFM) and in cows with retained fetal membranes (Group RFM) to obtain better insight into the role of the AOAH enzyme of neutrophils in endotoxin-related diseases, which occur frequently in dairy cows during the early postpartum period, especially in RFM cows. Twenty early postpartum dairy cows were used in the study: 13 NFM cows and 7 RFM cows. In the RFM cows, the percentage of PMN in blood (29+/-4%) was significantly (P<0.05) lower than in NFM cows (43+/-4%). The average AOAH activity in RFM cows (mean +/- SEM = 89+/-13 pmol fatty acid/10(7) PMN/h) was lower than in NFM cows (107+/-6 pmol fatty acid/10(7) PMN/h), but the difference in neutrophil AOAH activity between the 2 groups was not significant. There was also a higher percentage of immature neutrophils in isolated leukocyte suspensions from RFM cows (22+/-8%) than from NFM cows (15+/-4%), so that impairment of AOAH activity in early postpartum cows could be explained, in part, by immaturity of the neutrophils. These results suggest that the decreased AOAH activity of PMN could play a role in the pathogenesis of endotoxin-related diseases in dairy cows during the early postpartum period.

Activity of hyaluronidase in placental tissues from cows with and without retained fetal membranes.

Hyaluronidase, a proteoglycan-degrading enzyme, may have an influence on collagenolysis in bovine placenta and take part in the separation processes of the placenta in cows. There was, however, no evidence concerning the activity of this enzyme in those tissues. The experiment was performed on cows divided into two groups as follows: A, placenta not retained (n = 16); and B, placenta retained (n = 9). The activity of the enzyme was measured using a spectrophotometric method and zymography. The results showed statistically significant higher activity of hyaluronidase in the maternal and fetal parts of the placenta in cows with a retention of fetal membranes. Further experiments require substrate specificity studies and the presence of regulatory factors of this enzyme in bovine placental tissues.

Activity of 72-kDa and 92-kDa matrix metalloproteinases in placental tissues of cows with and without retained fetal membranes.

This study compared the activity of matrix metalloproteinases (MMP-2 and MMP-9) in retained and non-retained bovine placenta. The activities of MMPs and their zymogens were measured in fetal and maternal placental tissues from control cows (group B) and animals affected with retention of fetal membranes (group A) using a zymography technique on 10 per cent SDS polyacrylamide gels. The activity of proMMP-9 detected only in the maternal part of the placenta was lower in group A than in group B. ProMMP-2 activity was higher in group A than in group B in both tissues. The active forms of MMP-2 were observed in the maternal and fetal part of placenta in group B, but only the 68-kDa form was detected in the placental tissues of group A. The differences in enzyme activity between the groups and the lack of 64- and 60-kDa active forms of MMP-2 in the maternal and fetal parts of the retained placenta may have influenced the hydrolysis of collagen and the proper release of fetal membranes.

Enzyme activities in placental tissues from cows with and without retained fetal membranes.

The aim of this study was the determination and the comparison of activities of AST, ALT, GGT, 5'-NU and CK in the placental tissues of cows with and without retained fetal membranes. Placental samples were obtained from 16 cows immediately after parturition and divided into 2 groups: A-not retained (n = 10, NRF), B-retained (n = 6, RF). The activities of the examined enzymes were determined in supernatants of homogenized tissues using spectrophotometric methods and ready kits. The activity of AST was statistically significantly lower in the maternal part of the placenta in group B than in group A. There were no differences in ALT activity. The activity of GGT was statistically significantly higher in the maternal part of the placenta in group B than in group A. The activity of 5'-NU was statistically significantly higher in the maternal part than in the fetal part of placenta in both groups examined. The activity of CK did not differ, except for statistically significant lower activity in the fetal part of the placenta in group B. The results can suggest that the metabolism of amino acids is altered to some extend in cases of the retained placenta. Changes in GGT my indicate on imbalance in free radicals generation and neutralisation. Energetic status may not be influenced by the retention of the fetal membranes. Further experiments concerning more frequent sample collecting during whole periparturient period are necessary.

Activity of placental glutathione peroxidase and superoxide dismutase in cows with and without retained fetal membranes.

Changes in the levels of the activity of two enzymes that neutralize free radicals-glutathione peroxidase [(EC.1.11.1.9), GSH-Px] and superoxide dismutase--[(EC.1.15.1.1), SOD]--in cows with fetal membranes retention (RFM) were studied. The activities were measured in maternal and fetal placental tissues after spontaneous parturition without (group A) and with (group B) retained placenta. GSH-Px activities were higher in group B than in group A (P < 0.05). The activity of this enzyme was lower in the maternal than in the fetal part of the placenta in both groups. The activity of SOD was significantly (P < 0.05) lower in maternal part, and higher in fetal part, of placenta in group B compared with group A. Experiments on defence mechanisms against free radicals in periparturient cows in connection with more frequent sampling, prostaglandins levels, oxidative stress, nutrition and retained placenta are required to further elucidate the role of these enzymes.