A pilot study on the relationship between thrombospondin-1 (THBS1) and transforming growth factor beta1 (TGFß1) in the bovine placenta during early mid-pregnancy as well as parturition with normally released and retained placenta.

During pregnancy, it is necessary to create appropriate conditions for the development of the placenta and the fetus. However, during parturition, the placenta must be separated and subsequently removed as soon as possible to not expose the female to the possibility of infection. In this study, the relationship between thrombospondin-1 (THBS1) and transforming growth factor beta1 (TGFß1) concentrations was described during bovine pregnancy (second, fourth, and sixth months; n = 3/each month), at normal parturition (NR) and parturition with fetal membrane retention (R). The presence of THBS1 and TGFß1 was confirmed in bovine placental tissues of both maternal and fetal parts. Enzyme-linked immunosorbent assay showed statistically significant differences (p < 0.05) in THBS1 concentrations (pg/mg protein) between examined parturient samples (maternal part: 5.76 ± 1.61 in R vs. 2.26 ± 1.58 in NR; fetal part: 2.62 ± 1.94 in R vs. 1.70 ± 0.23 in NR). TGFß1 concentrations (pg/mg protein) were significantly lower (p < 0.05) in the retained fetal membranes compared to the released fetal membranes in the maternal part of the placenta (26.22 ± 7.53 in NR vs. 17.80 ± 5.01 in R). The participation of THBS1 in the activation of TGFß1 in parturient bovine placental tissues leading to the normal release of fetal membranes may be suggested.

Changes in plasma PLAC-1 concentration and its expression during early-mid pregnancy in bovine placental tissues - a pilot study.

BACKGROUND: Placenta-specific protein 1 (PLAC1) is a small secreted protein considered to be a molecule with a significant role in the development of the placenta and the establishment of the mother-foetus interface. This study aimed to confirm the presence of bovine PLAC1 and to examine its profile in the placenta and plasma in the first six months of pregnancy. The expression pattern of PLAC1 was analysed by RT-qPCR and Western Blotting. Quantitative evaluation was carried out using ELISA. RESULTS: PLAC1 concentrations in the plasma of pregnant cows were significantly higher (p < 0.05) than those obtained from non-pregnant animals. PLAC1 protein concentrations in the placental tissues of the foetal part were significantly (p < 0.05) higher than in the tissues of the maternal part of the placenta. PLAC1 transcripts were detected in both placental tissue samples and epithelial cell cultures. CONCLUSIONS: In conclusion, the results of the present preliminary study suggest that PLAC1 is involved in the development of bovine placenta. The presence of this protein in the plasma of pregnant animals as early as the first month may make it a potential candidate as a pregnancy marker in cows. Further studies on exact mechanisms of action of PLAC1 in bovine placenta are necessary.

Proteomic profile in plasma as a biomarker of pregnancy in cows.

Pregnancy has its own protein dynamics, reflecting the hormonal profile. Quantitative and qualitative changes in plasma protein profile may provide useful information about this condition. Any alterations may be a signal heralding clinical or subclinical pathology. The objective of our study was to compare the plasma protein profile between selected months of pregnancy in cows for a better understanding gestation course. For this purpose, we collected blood from healthy pregnant (n = 30; n = 6 for each pregnancy stage) and non-pregnant (C; n = 6) Holstein-Friesian cows during a routine veterinary examination. Collected samples were selected according to pregnancy month (first, second, third, sixth, and ninth), prepared, and separated by two-dimensional electrophoresis. The Delta-2D program compared and statistically evaluated scanned gel images from the appropriate months. The mean volume of the spots was considered. The MALDI TOF/TOF spectrometer was used to identify statistically significant proteins. There were 11 distinct proteins found, including peptidyl-prolyl cis-trans isomerase F, oligoribonuclease, and PRELI domain-containing protein 3B (all of them have the lowest abundance in the C group), alpha-1B-glycoprotein, L-gulonolactone oxidase, hemopexin (first month with higher abundance than control), alpha-2-HS-glycoprotein (significantly higher abundance in the first month than in remaining groups), ermin (absent in the first month and lower abundance in the third and sixth months than in the remaining groups and control), endophilin-A2 (significant differences between the control and the second, third, sixth, and ninth months), apolipoprotein A-I (significant difference between control and the first and sixth months), alpha-1-antiproteinase (significant difference between control and the ninth month). The study demonstrated the distinctions between plasma protein composition and alterations during the pregnancy course which may potentially serve as diagnostic tools.

Effect of Sex Steroids and PGF2α on the Expression of Their Receptors and Decorin in Bovine Caruncular Epithelial Cells in Early-Mid Pregnancy.

Changes in the expression of various genes, including pregnancy-associated hormone receptors and extracellular matrix proteins, have been suggested to play a significant role in bovine placental development. This study aimed to examine the influence of sex steroids and PGF2α on decorin (DCN) expression in the epithelial cells of bovine caruncle in early−mid pregnancy in cows. The expression patterns of DCN, PTGFR, PGR and ESR1 were analyzed by RT-qPCR and Western blotting in primary caruncular epithelial cell cultures (PCECC) and placental tissue homogenates derived from the 2nd and 4th months of pregnancy. PCECC were found to express DCN, PTGFR, PGR and ESR1. The intensity of PGR staining was higher in cells derived from the 4th month of pregnancy (p < 0.05). The 17ß-estradiol, progesterone and PGF2α have not been shown to affect DCN expression. PGF2α decreased PTGFR expression in cells derived from the 4th month of gestation (p < 0.05). In conclusion, the results of the present preliminary study showed that the expression of the PTGFR, ESR1, PGR and DCN in PCECC does not vary throughout early−mid pregnancy. Further studies should be carried out to observe the relationship between hormonal status and cellular adhesion to determine their importance for properly developing placentation and pregnancy in cows.

The comparison of pro- and antioxidative parameters in plasma and placental tissues during early phase of placental development in cows.

Physiological balance between pro- and antioxidative processes is crucial for placentation and further development of fetus and placenta. Parameters of pro- and antioxidative profile may serve as markers of proper course of pregnancy. The aim of study was to assess whether the balance between pro- and antioxidative parameters during placentation phase in bovine placenta is maintained. Placental and blood samples were collected from healthy, HF, pregnant (2nd-3rd month) cows (n = 8) in slaughterhouse and in farm, respectively. Formylokinurenine and bityrosine content were measured spectrofluorimetrically in blood plasma and tissue homogenates while metabolites of lipid peroxidation, total antioxidant capacity, SH groups and activity of antioxidative enzymes (glutathione peroxidase and superoxide dismutase) were determined in examined tissues by spectrophotometry. Western blotting was used to confirm the presence of enzymatic proteins in placenta. Results: Local profile in tissues was more pronounced than general profile in blood plasma. Activities of antioxidative enzymes were significantly (p < 0.05) higher in 2nd compared to 3rd month of pregnancy in maternal part of placenta while prooxidant parameters showed opposite relationship. Obtained results showed significant differences when compared to data from non-pregnant animals or time of parturition. Further studies are necessary for elucidation of placentation phase in cows.

The influence of progesterone and prostaglandin F2α on decorin and the adhesion of caruncular epithelial cells of bovine placenta at early-mid pregnancy-Part II.

One of the most important processes determining the proper course of gestation and its physiological termination in cows is the adhesion of epithelial cells allowing for direct contact of maternal and foetal parts of the placenta. Throughout pregnancy, placental cells are under strict hormonal control, which among others regulates the concentration and activity of specific proteins participating in the extracellular matrix remodelling of foetal membranes. The aim of the study was to evaluate the influence of progesterone and prostaglandin F2α on the adhesion of epithelial cells at early-mid pregnancy in cows. Additionally, the impact of selected hormones on anti-adhesive properties of decorin was evaluated. Caruncular epithelial cells were isolated from healthy cows during pregnancy, immediately after slaughter. Primary cell cultures derived from the 2nd and 4th month of gestation were used in the experiments. The viability of cells was assessed by MTT assay. The adhesion of cells to fibronectin was measured spectrophotometrically. The activity of metalloproteinases was confirmed by the metalloproteinase assay. Progesterone (10-5 and 10-7  mol/L) and prostaglandin F2α (10-4 , 10-5 and 10-7  mol/L) increased the viability of bovine caruncular epithelial cells in the 2nd month of pregnancy. The treatment with prostaglandin F2α significantly reduced the number of adherent cells from the 2nd month of gestation at the doses of 10-4 and 10-5  mol/L. Both progesterone and prostaglandin F2α were shown to have an effect of decorin resulting in both a decrease in metalloproteinase activity and an increase in adhesion of cells to fibronectin.

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.

Influence of two different feeding strategies in the dry period on dry matter intake and plasma protein peroxidative and antioxidative profile during dry period and early lactation.

BACKGROUND: Dairy cows undergo dramatic changes in endocrine and metabolic status around parturition and in early lactation. Meeting the nutritional requirements of transition dairy cows is important for animal health, production and animal wellbeing. Dry cow feeding and managing play an essential role in this. The changes in metabolism of periparturient cows also lead to a rise in the production of oxidising agents, leading to oxidative stress. The relationship between dry cow diet composition and oxidative stress has received little research attention so far. In the present study, the influence of two different dry cow feedings (single diet with medium energy content over the whole dry period versus traditional two-phase diet with a low-energy "far-off" ration and a high energy "close-up" ration) on dry matter intake, energy intake and plasma protein peroxidative and antioxidative profile was investigated. RESULTS: The examined parameters revealed a dynamic profile within the experimental period. Dry matter intake (DMI) did not differ between groups. However, there was a time and a group x time interaction effect: Group 1 ("one-phase") had a very constant DMI with a slow and even decrease until calving. In Group 2 ("two-phase"), an initial increase in DMI two weeks antepartum (a.p.) was followed by a sharp drop at week 1 a.p.. The highest total antioxidant capacity and sulfhydryl residue concentration was noted at partus. In contrast, concentration of formylokinurenine and bityrosine bridges as representatives of protein peroxidation were lowest at parturition. The time course of formylokinurenine and bityrosine bridges showed parallels to the DMI. The contents of sulfhydryl groups, formylokinurenine and total antixoxidant capacity did not differ between groups. In contrast, concentration of bityrosine bridges was always higher in Group 2 compared with Group 1 and these differences were statistically significant at week 3 a.p., week 2 a.p., week 1 a.p. and at parturition. CONCLUSION: The results of our study suggest time-related changes of pro- and antioxidative plasma parameters. Different dry cow feeding affected antepartal DMI. Furthermore, DMI and diet compositions seemed to have an influence on plasma protein peroxidative profile and activity of antioxidative defence.

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.

Decorin concentrations in canine normal and neoplastic mammary gland tissues.

In the present study, the concentration of decorin in canine normal and neoplastic mammary gland tissues was examined to understand the potential role of decorin in development and progression of canine mammary tumours. The homogenates of 48 mammary gland tumours (10 benign and 38 malignant) and 10 samples of normal canine mammary gland tissue were used in the study. The presence and quantification of decorin was examined in the homogenates using Western blot and specific canine ELISA. Western blotting confirmed the presence of decorin both in the normal mammary gland tissues and in the mammary gland tumours. The concentration of decorin was significantly higher (p < .05) in the benign tumours and non-metastatic malignant tumours than in the normal mammary gland. The concentration of decorin was significantly lower (p < .05) in the malignant tumours with metastasis to regional lymph nodes compared with benign tumours and non-metastatic malignant tumours. No significant differences were found in the level of decorin between the benign and the non-metastatic malignant tumours. Both the histological type of malignant tumours and the histological grade did not significantly affect the concentration of decorin. These findings suggest that neoplastic transformation in the canine mammary gland leads to increase in the decorin protein synthesis. The reducing decorin concentration in canine malignant mammary tumours appears to facilitate the metastatic spread of these tumours.

Identification of protein patterns in bovine placenta at early-mid pregnancy - Pilot studies.

RATIONALE: Placenta is a crucial tissue for an appropriate development of the fetus and the course of pregnancy. Its composition and structure change dynamically along pregnancy but the full pattern of these changes is not fully described in cows yet. The aim of the present study was to detect qualitative and quantitative protein profiles of bovine placenta during early-mid pregnancy at the time of placental formation. METHODS: Placental tissues from healthy cows (n = 3) in early pregnancy (3-5 months) were collected at the slaughterhouse. Maternal and fetal parts were manually divided prior to homogenization. Further analysis was done in triplicates on the maternal and fetal parts separately and subjected to one-dimensional (1D) electrophoretic separation, followed by identification of peptide maps by nanospray liquid chromatography/tandem mass spectrometry (nanoLC/MS/MS). Proteins were identified by use of the MASCOT software with the SwissProt database. RESULTS: Proteomic analysis showed more than 4000 differentially expressed proteins in maternal and fetal parts of placenta. Each part expressed around 900 proteins, of which ca. 90 were common. The identified proteins were analyzed in accordance to molecular function and their participation in biological processes. CONCLUSIONS: The obtained results provide new insight into the knowledge about biochemical characteristics of placenta (new proteins) and serve for further studies on the possible markers of physiological/pathological pregnancy or function of placenta. Moreover, our data can be a good starting point for further studies on the processes underlying the attachment of placenta.

Is it useful to use several "omics" for obtaining valuable results?

The integration of cell communication and the transfer of signals from stimuli via transcription to translation and further to activation of new protein is crucial for appropriate metabolism and function of living organisms. The overall elucidation and the examination of these complex processes require multistep laboratory approaches in order to obtain results which will not only detect particular stage but also indicate the mechanisms lying upon this process. Such results will be reliable because they will cover multidirectional methods and approaches. The analysis of currently available results already provided with the conclusion that often single omics approach does not correspond with other expected information and may bring misinterpretations. That is why the integration of several "omics" is useful for searching entire explanations and answers as well as appropriate interpretation of obtained complex results. The hypothesis was stated that "from transcriptomics can not be concluded to proteomics". This review focuses on the reasons for the integration of transcriptomic, proteomic and other-omics analysis. Moreover it also describes the examples of clinical meanings and mentions some methods used in these approaches.

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.

The preliminary studies on protein profile in retained and not retained foetal membranes in heavy draft mares.

Protein profile of the placenta expresses its function and maintenance. Any alterations can be reflected in qualitative and quantitative changes in this profile. The aim of the present study was the evaluation of protein profile in the placenta of mares suffering from the retention of foetal membranes (FMR) by two separation methods and the comparison with physiologically released tissues. Placentas from 14 healthy, heavy draft mares were collected immediately after the expulsion of newborn. Tissues after homogenization and staining with fluorescent dyes were subjected to electrophoretic as well as chromatographic separation. Electrophoretic gels were statistically analysed for the presence and abundance of examined proteins, while some proteins were identified in chromatographic fractions. Out of 248 spots detected in endometrium, 38 differed significantly between FMR and control animals, while in allantochorion, respective values reached 241 and 27 spots (p < .05). Among identified proteins that expressed higher abundance in endometrium of FMR mares than control animals were prostaglandin reductase, dehydrogenase/reductase SDR family, and placental growth factor. These proteins are involved in regulation of parturition. Additionally, the following proteins responsible for physiological activity of a cell-guanine methyl transferase, aspartyl/asparaginyl beta-hydroxylase and GTP-binding protein, were identified. These proteins expressed higher abundance in allantochorion of FMR mares than in controls. This preliminary study confirmed the disturbances in protein pattern between foetal membranes in FMR and healthy mares. Further qualitative and quantitative experiments are necessary to deepen our knowledge on the mechanisms of the retention of foetal membranes in mares.

Total antioxidant capacity and protein peroxidation intensity in seminal plasma of infertile and fertile dogs.

The aim of this study was to evaluate the total antioxidant capacity and protein peroxidation intensity in seminal plasma of infertile and fertile dogs. The study was conducted on 10 infertile and 10 fertile dogs of various breeds. Infertility was defined as conception failure at least three matings with different bitches. Semen was collected by manual manipulation. The sperm concentration and motility parameters were evaluated using CASA Hamilton Thorne, Vers. IVOS 12.3. The morphology of spermatozoa and the percentage of live and dead sperm cells were assessed microscopically, total antioxidant capacity and the content of SH-groups in seminal plasma were determined spectrophotometrically, the contents of protein peroxidation markers in seminal plasma, bityrosine and formylokinurenine, were determined using spectrofluorimetric methods. Sperm concentration and total sperm count were significantly (p < 0.05) lower in infertile dogs than in fertile dogs (99.92 ± 3 0.05 × 106 /ml vs. 282.07 ± 48.27 × 106 /ml; 214.19 ± 114.74 × 106 vs. 747.57 ± 210.94 × 106 , respectively). The percentage of spermatozoa with normal morphology and the most determined motility parameters differed significantly (p < 0.05) between both groups. The mean values of total antioxidant capacity in the seminal plasma were significantly (p < 0.05) lower (19.95 ± 20.94 vs. 25.66 ± 23.18 µmol/g protein), whereas the mean contents of bityrosine and formylokinurenine in seminal plasma were significantly (p < 0.05) higher in infertile dogs than in fertile dogs (3.71 ± 4.83 µg/mg protein vs. 1.55 ± 2.00 µg/mg protein and 0.37 ± 0.45 µg/mg protein vs. 0.14 ± 0.08 µg/mg protein, respectively). In conclusion, the obtained results suggest that the poor semen quality and infertility in dogs could be associated with lowered total antioxidant capacity and increased protein peroxidation in seminal plasma as a consequence of oxidative stress.

Preliminary results of the placental decorin profile in bovine pregnancy and parturition.

Decorin is a small leucine-rich proteoglycan which is involved in multiple biological functions mainly as a structural and signaling molecule. Due to its biological properties in connective tissue, decorin may participate in remodeling of ECM during attachment and detachment of placenta within the course of pregnancy and at parturition in cows. The aim of the study was to detect the presence of decorin protein in bovine placental tissues and to evaluate its profile during pregnancy and at parturition. Placental tissues from healthy pregnant cows (2-5 month) were collected in abattoir (n = 10), while parturient tissues were obtained during caesarian section at physiological term (n = 6). Maternal and fetal parts were separated manually and subjected to homogenization and to quantitative ELISA and verification by Western blotting with anti-decorin antibodies. ELISA test showed that the concentration of decorin during pregnancy was higher in the fetal part of placenta compared with the maternal part (p < 0.001). Similar pattern was noted regarding to maternal and fetal samples derived from parturient cows. Our preliminary results demonstrate that the concentration of decorin is gestation time-dependent in healthy bovine placenta. Possible confirmation of the involvement of decorin in early pregnancy attachment and detachment of the placenta during parturition requires further research.

Impact of Virtual Patients as Optional Learning Material in Veterinary Biochemistry Education.

Biochemistry and physiology teachers from veterinary faculties in Hannover, Budapest, and Lublin prepared innovative, computer-based, integrative clinical case scenarios as optional learning materials for teaching and learning in basic sciences. These learning materials were designed to enhance attention and increase interest and intrinsic motivation for learning, thus strengthening autonomous, active, and self-directed learning. We investigated learning progress and success by administering a pre-test before exposure to the virtual patients (vetVIP) cases, offered vetVIP cases alongside regular biochemistry courses, and then administered a complementary post-test. We analyzed improvement in cohort performance and level of confidence in rating questions. Results of the performance in biochemistry examinations in 2014, 2015, and 2016 were correlated with the use of and performance in vetVIP cases throughout biochemistry courses in Hannover. Surveys of students reflected that interactive cases helped them understand the relevance of basic sciences in veterinary education. Differences between identical pre- and post-tests revealed knowledge improvement (correct answers: +28% in Hannover, +9% in Lublin) and enhanced confidence in decision making ("I don't know" answers: -20% in Hannover, -7.5% in Lublin). High case usage and voluntary participation (use of vetVIP cases in Hannover and Lublin >70%, Budapest <1%; response rates in pre-test 72% and post-test 48%) indicated a good increase in motivation for the subject of biochemistry. Despite increased motivation, there was only a weak correlation between performance in final exams and performance in the vetVIP cases. Case-based e-learning could be extended and generated cases should be shared across veterinary faculties.

The dependence between glycodelin and selected metalloproteinases concentrations in bovine placenta during early gestation and parturition with and without retained foetal membranes.

Pregnancy course depends on the appropriate connection between the mother and the developing foetus. Pregnancy is completed when the placenta is timely expelled. Placental retention is one of the possible pregnancy complications. Extracellular matrix, including adhesive proteins and enzymes that can break down collagens, seems to be responsible for it. The aim of the present study was to examine the impact of one of the adhesive proteins - glycodelin (Gd) - on selected metalloproteinases degrading collagens (MMP2, MMP3, MMP7). Placental tissues from healthy pregnant cows collected during early-mid pregnancy (2nd month n = 7, 3rd month n = 8, 4th month n = 6) and in cows that properly released placenta (NR; n = 6) and cows with retained foetal membranes (R; n = 6) were experimental material. The concentrations of glycodelin and protein content of selected metalloproteinases were measured by ELISA in the maternal and foetal placental homogenates as well as in the culture of epithelial cells derived from the maternal part of the placenta. The presence of these protein molecules was confirmed by Western Blotting. In the bovine placenta, the concentrations of examined proteins exhibit significant changes during placental formation. Gd, MMP3 and MMP7 concentrations decrease with pregnancy progress (between the 2nd and 4th month), while MMP2 concentrations were on the same level in this period. During parturition, concentrations of Gd and MMP3 were significantly higher in the R group compared to the NR group. In parallel, MMP2 concentrations did not show significant differences between the groups (NR vs R), and MMP7 concentrations decreased significantly in the maternal part of the placenta in cows with retained foetal membranes (R). Obtained results show correlations between the gestational age and proteins' (Gd, MMP3, MMP7) concentration, both in the maternal and foetal part of the placenta.

The influence of age and gender on antioxidant enzyme activities in humans and laboratory animals.

Antioxidative/oxidative balance is one of the important factors for homeostasis. Antioxidative systems which protect from peroxidative damage are supposed to be under the influence of steroid hormones. The implications of this influence are age and gender as well as tissue dependent alterations in antioxidative enzyme activities. Apart from hormonal influence, antioxidative enzymes require the presence of microelements in their active centers as well as concerted action of non enzymatic antioxidants which support enzymes in their scavenging action. The aim of this review is to analyze and compare existing knowledge about the changes in activity of antioxidant enzymes in human and animal females and males of different age. Evidence as regards participation of oxidative stress in senescence are specific diseases which, to some extent, are gender dependent and appear more frequently in males or females. Several experiments in laboratory animals revealed that changes in enzyme activities are reflected in histopathological pictures of cells. The alterations observed during perimenopausal period provide with additional evidence of the participation of steroid hormones in the regulation of antioxidative system activity. Moreover, estrogens themselves exhibit antioxidative activity which is receptor independent. In conclusion, apart from genetic-related influences, also diet and style of life may have an impact on the antioxidative system which requires appropriate supplementation in microelements and vitamins for its effective function of scavenging excess of free radicals.

Reference gene selection in bovine caruncular epithelial cells under pregnancy-associated hormones exposure.

Examination of transcriptional regulation occurring during pregnancy establishment and maintenance requires the identification of endogenous reference genes characterized by high expression stability. Since the expression of some reference genes may be modulated by pregnancy-associated hormones, the goal of our study was to identify suitable reference genes unaffected by hormonal treatment. In our study bovine caruncular epithelial cells were subjected to progesterone, estrogen and prostaglandin F2α treatment. Ten candidate reference genes (ACTR1A, CNOT11, HDAC1, HPRT1, RPL19, RPS9, SDHA, SUZ12, UXT and ZNF131) were evaluated with the use of four approaches (geNorm, NormFinder, BestKeeper, delta Ct). We found that RPS9 and SUZ12 displayed the highest expression stability in the tested material. Moreover, HPRT1 and SDHA were found inappropriate for RT-qPCR data normalization as they demonstrated the highest expression variability out of all candidates analysed. Hence geNorm calculations shown that the use of just two best-performing genes would be sufficient for obtaining reliable results, we propose that RPS9 and SUZ12 be used as suitable endogenous controls in future studies investigating gene expression in normal and compromised pregnancies.