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.     

The expression of Fas/FasL and apoptosis in yak placentomes

To clarify the status and distribution of Fas and Fas-Ligand (FasL) in yak's placentomes, immunohistochemistry (IHC) was carried out to analyze the expression and location of Fas and FasL in paraffin embedded sections. The area of positive stained sites was selected and measured using image analyses software (Image Pro-Plus 6.0). So the positive index (PI) was calculated to estimate the intensity of protein expression according to the percentage of positive area in corresponding compartment of the placentomes. In cotyledonary villi, Fas mainly presented on the villous trophoblast cells in early pregnancy. The positive index reached a maximum of 20.7±8.8 at the third month of pregnancy. Then Fas was declined rapidly along with the progress of gestation and the value was 2.8±1.3 after the 7th month of pregnancy. However, in caruncular crypts, Fas was mainly localized to isolated cells or clustered cells of the uterine stroma underlying the caruncular epithelium. The intensity was lower and the positive index was changed between 4.7±0.9 and 8.5±1.6 throughout gestation. For FasL, it gave a distinct immunostained distribution. In cotyledonary villi, FasL was localized dominantly and strongly in the cytoplasm of binuclear, mononuclear and trinuclear trophoblast giant cells (TGC). The positive index of FasL maintained a moderate level all through the gestation. In caruncular crypts, the expression of FasL was weak and the positive index was declined. Only in the first two months, maternal uterine epithelial cells intensely expressed FasL and the index reached to the maximum of 19.8±5.2. The result of subcellular localization of Fas ligand using immunoelectron microscopy technology indicated that FasL was subcellular located in some intracellular vesicles of TGC. This means the vesicles of trophoblast giant cells itself can express FasL. By the TUNEL method, apoptosis was detected in yak placentomes. The amount of apoptotic cells was rare. The fetal chorionic trophoblast cells and caruncular crypt epithelium cells demonstrated higher percentage of apoptosis in middle pregnancy, which suggested that apoptosis plays an important role in placental cellular regeneration. In addition, the apoptosis of maternal caruncular stromal cells provides a local mechanism for maternal immunotolerance to the fetus and this mechanism was mediated by Fas-FasL pathway.     

To hydrolyze or not to hydrolyze: the dilemma of platelet-activating factor acetylhydrolase

Mounting ambiguity persists around the functional role of the plasma form of platelet-activating factor acetylhydrolase (PAF-AH). Because PAF-AH hydrolyzes PAF and related oxidized phospholipids, it is widely accepted as an anti-inflammatory enzyme. On the other hand, its actions can also generate lysophosphatidylcholine (lysoPC), a component of bioactive atherogenic oxidized LDL, thus allowing the enzyme to have proinflammatory capabilities. Presence of a canonical lysoPC receptor has been seriously questioned for a multitude of reasons. Animal models of inflammation show that elevating PAF-AH levels is beneficial and not deleterious and overexpression of PAF receptor (PAF-R) also augments inflammatory responses. Further, many Asian populations have a catalytically inert PAF-AH that appears to be a severity factor in a range of inflammatory disorders. Correlation found with elevated levels of PAF-AH and CVDs has led to the design of a specific PAF-AH inhibitor, darapladib. However, in a recently concluded phase III STABILITY clinical trial, use of darapladib did not yield promising results. Presence of structurally related multiple ligands for PAF-R with varied potency, existence of multi-molecular forms of PAF-AH, broad substrate specificity of the enzyme and continuous PAF production by the so called bi-cycle of PAF makes PAF more enigmatic. This review seeks to address the above concerns.     

Assessment of expression of the receptor for colony-stimulating factor-1 (fms) in bovine trophoblast.

Bovine trophoblast was employed to assess the questions of whether the receptor for CSF-1 is expressed by noninvasive trophoblast and whether expression changes with differentiation within placentomes. Bovine placental poly(A) mRNA contained sequences cross-reactive with cDNA probes to c-fms and v-fms. Using a monoclonal antibody to v-fms, immunohistochemistry of postattachment bovine trophoblast showed expression of an fms-like protein between Day 29 and term. Expression occurred in both the intercotyledonary and cotyledonary trophoblast. Reactivity that was fms-like was also demonstrated on preattachment conceptuses flushed at Days 14 and 7 of gestation and on Day 7 embryos derived from in vitro oocyte maturation and fertilization. Unexpectedly, in the second half of pregnancy some cells, including binucleate cells, showed nuclear rather than cytoplasmic reactivity to the antibody. These data indicate expression of an fms-like protein in bovine placenta that does not correlate with properties of trophoblast cell invasiveness or major morphological differentiation. The data do support a universal role for this protein during mammalian placental development.     

Hemochorial placentation in the primate: expression of vascular endothelial growth factor, angiopoietins, and their receptors throughout pregnancy

Vascular development and its transformation are necessary for successful hemochorial placentation, and vascular endothelial growth factor (VEGF), angiopoietins, and their receptors may be involved in the molecular regulation of this process. To determine the potential role of these putative regulators in a widely studied primate, the common marmoset, we investigated their mRNA expression and protein location in the placenta throughout pregnancy using in situ hybridization, Northern blot analysis, and immunocytochemistry. VEGF was localized in decidual and cytotrophoblast cells, and its highest expression was found in the maternal decidua. The Flt receptor was exclusively detected in the syncytial trophoblast with increasing expression in placentae from 10 wk to term. Soluble Flt (sFlt) was also detectable by Northern blot analysis. KDR receptor expression was restricted to mesenchymal cells during early placentation and to the fetoplacental vasculature during later placentation. KDR expression increased throughout pregnancy. Angiopoietin-1 (Ang-1) was localized in the syncytial trophoblast, being highly expressed in the second half of gestation. Ang-2 mRNA localized exclusively to maternal endothelial cells, and was highly expressed in 10-wk placentae. The Tie-2 receptor was found in cytotrophoblast cells and in fetal and maternal vessels. High Tie-2 levels were detected in the wall of chorion vessels at 14-wk, 17-wk, and term placentae. These results suggest that the processes of trophoblast invasion, maternal vascular transformation, and fetoplacental vascular differentiation and development are regulated by the specific actions of angiogenic ligand-receptor pairs. Specifically, 1) VEGF/Flt and Ang-1/Tie-2 may promote trophoblast growth, 2) VEGF/KDR and Ang-1/Tie-2 may support fetoplacental vascular development and stabilization, 3) sFlt may balance VEGF actions, and 4) Ang-2/Tie-2 may remodel the maternal vasculature.     

Platelet-activating factor (PAF)-dependent transacetylase and its relationship with PAF acetylhydrolases

Platelet-activating factor (PAF)-dependent transacetylase (TA) is an enzyme that transfers an acetyl group from PAF to acceptor lipids such as lysophospholipids and sphingosine. This enzyme is distributed in membrane and cytosol of the cells. We previously revealed that TA purified from rat kidney membrane showed an amino acid sequence similarity to that of bovine PAF-acetylhydrolase (AH) (II). In the present study, we purified TA from the rat kidney cytosol and analyzed its amino acid sequence. The amino acid sequence of the cytosolic TA is similar to that of bovine PAF-AH (II) and membrane TA. To clarify the relationship between TA and PAF-AH (II), we isolated cDNA of rat PAF-AH (II). The predicted amino acid sequence of rat PAF-AH (II) from isolated cDNA included all the sequences found in TAs purified from the membrane and cytosolic TAs. In addition, monoclonal antibody to recombinant PAF-AH (II) cross-reacted with both cytosolic and membrane TAs. Consistent with sequence identity, recombinant PAF-AH (II) showed TA activity, whereas recombinant PAF-AH Ib, which is a different subtype of intracellular PAF-AHs, did not possess TA activity. Analysis of a series of site-directed mutant PAF-AH (II) proteins showed that TA activity was decreased, whereas PAF-AH activity was not affected in C120S and G2A mutant proteins. Thus, Cys(120) and Gly(2) are implicated in the catalysis of TA reaction in this enzyme. Furthermore, the transfer of acetate from PAF to endogenous acceptor lipids was significantly increased in a time-dependent manner in CHO-K1 cells transfected with PAF-AH (II) gene. These results demonstrate that PAF-AH (II) can function, as a TA in intact cells, and PAF-AH (II) and TA are the same enzyme.     

Platelet-activating factor (PAF)-like activity, localization of PAF receptor (PAF-R) and PAF-acetylhydrolase (PAF-AH) activity in bovine endometrium at different stages of the estrous cycle and early pregnancy

PAF-like activity in the endometrium increased from days 2-4 to day 12 and day 20 in both cyclic and pregnant cows. There was an increase in platelet aggregation induced by PAF-like activity in the endometrium of pregnant animals on day 20 as compared to cyclic animals at the same point in time. Two major bands of PAF-R protein at 67 kDa and 97 kDa were detected by Western blot analysis. PAF-R was localized mainly in luminal and glandular epithelium of the endometrium, but the staining was markedly increased in the endometrium of pregnant cows on day 20 compared to cyclic animals on the same day. The purified PAF-AH from the endometrium is similar to in plasma. In cyclic cattle, no changes in PAF-AH activity of endometrium were observed, whereas a decrease in enzyme activity occurred in pregnant cows on day 20 as compared to cyclic animals on the same day. We suggest that the bovine endometrium produces PAF-like activity, expresses the PAF-R and possesses a PAF-AH activity which varies during pregnancy.     

Role of platelet-activating factor in pulmonary vascular remodeling associated with chronic high altitude hypoxia in ovine fetal lambs

Platelet-activating factor (PAF) is implicated in pathogenesis of chronic hypoxia-induced pulmonary hypertension in some animal models and in neonates. Effects of chronic hypoxia on PAF receptor (PAF-R) system in fetal pulmonary vasculature are unknown. We investigated the effect of chronic high altitude hypoxia (HAH) in fetal lambs [pregnant ewes were kept at 3,801 m (12,470 ft) altitude from approximately 35 to 145 days gestation] on PAF-R-mediated effects in the pulmonary vasculature. Age-matched controls were kept at sea level. Intrapulmonary arteries were isolated, and smooth muscle cells (SMC-PA) were cultured from HAH and control fetuses. To determine presence of pulmonary vascular remodeling, lung tissue sections were subjected to morphometric analysis. Percentage medial wall thickness was significantly increased (P < 0.05) in arteries at all levels in the HAH lambs. PAF-R protein expression studied by immunocytochemistry and Western blot analysis on lung tissue SMC-PA demonstrated greater PAF-R expression in HAH lambs. PAF-R binding (femtomoles per 10(6) cells) in HAH SMC-PA was 90.3 +/- 4.08 and 66% greater than 54.3 +/- 4.9 in control SMC-PA. Pulmonary arteries from HAH fetuses synthesized >3-fold PAF than vessels from controls. Compared with controls SMC-PA of HAH lambs demonstrated 139% and 40% greater proliferation in 10% FBS alone and with 10 nM PAF, respectively. Our data demonstrate that exposure of ovine fetuses to HAH will result in significant upregulation of PAF synthesis, PAF-R expression, and PAF-R-mediated effects in pulmonary arteries. These findings suggest that increased PAF-R protein expression and increased PAF binding contribute to pulmonary vascular remodeling in these animals and may predispose them to persistent pulmonary hypertension after birth.     

Immunohistochemical localization of prostaglandin G/H synthase 1 and 2 in sheep placenta after glucocorticoid-induced and spontaneous labour

Enhanced prostaglandin production and release by the placenta is an essential element in the normal transition to labour in many animal species. In sheep, expression of prostaglandin G/H synthase (PGHS) is the central enzyme regulating this process. In this study immunohistochemistry was used to examine the distribution of cells expressing PGHS-1 and PGHS-2 in ovine placenta in association with spontaneous parturition (n = 6) and glucocorticoid-induced labour (n = 5). Labour was induced in ewes after the intrafetal injection of betamethasone on day 131 of gestation. Animals administered an intrafetal injection of isotonic saline (n = 5) acted as non-labour controls. In placentomes collected from all groups, immunoreactive PGHS-1 was present in the mononuclear trophoblast cells of the fetal placenta. Cells in the maternal mesenchyme and epithelial syncytium were weakly immunopositive for this enzyme. PGHS-1 immunoreactivity was also demonstrated in the endothelial cells of the chorionic vessels. The PGHS-2 isozyme was localized exclusively to the trophoblast epithelial cells. Immunoreactive PGHS-2 was not detectable in the maternal epithelial syncytium or the stroma of the cotyledons. The binucleate cells of the fetal placenta were consistently immunonegative for both PGHS isozymes. These results indicate that the cellular localization of PGHS-1 and PGHS-2 in ovine placenta does not change during the last 15 days of pregnancy. Co-localization of these isozymes indicates that the source of arachidonic acid and the site of prostanoid formation are the same. Quantitation of the percentage area of positive staining for PGHS-1 and PGHS-2 using image analysis software demonstrated a significant increase in PGHS-2 in the fetal trophoblast after glucocorticoid-induced labour and spontaneous parturition. This finding indicates that increased formation of the PGHS-2 isozyme is responsible for the large increase in prostaglandin production by the ovine placenta at term labour.     

Epidermal growth factor (EGF) induces motility and upregulates MMP‐9 and TIMP‐1 in bovine trophoblast cells

Differentiation and restricted invasion/migration of trophoblast cells are crucial for feto‐maternal communication in the synepitheliochorial placenta of cattle. EGF is expressed in the bovine placenta and likely regulates these cell properties. As cell migration and motility rely on the degradation of extracellular matrix we hypothesize that EGF is involved in the regulation of the MMP‐9/TIMP‐1 balance and thus could influence trophoblast migration, tissue remodeling, and the release of the fetal membranes after parturition. The aim of this in vitro study was to examine EGF‐mediated effects on cell motility, proliferation, and MMP‐9 and TIMP‐1 expression in cultured bovine trophoblast cells. We used a trophoblast cell line (F3) derived from bovine placentomes to examine the influence of EGF on MMP‐9 and TIMP‐1 expression by semiquantitative RT‐PCR and MMP activity by zymography. Migration assays were performed using a Boyden chamber and cell motility was measured by time‐lapse analyses. To identify the involved signaling cascades, phosphorylation of mitogen‐activated protein kinase (MAPK) 42/44 and Akt was detected by Western blot. EGF treatment increased both the abundance of MMP‐9 and TIMP‐1 mRNAs and the proteolytic activity of MMP‐9. Furthermore, EGF stimulated proliferation and migration of F3 cells. Addition of specific inhibitors of MAPK (PD98059) and/or PI3K (LY294002) activation abolished or reduced EGF‐induced effects in all experiments. In conclusion, EGF‐mediated effects stimulate migration and proliferation of bovine trophoblast cells and may be involved in bovine placental tissue remodeling and postpartum release of fetal membranes. Mol. Reprod. Dev. 77: 622–629, 2010. © 2010 Wiley‐Liss, Inc.     

Platelet-activating factor (PAF)-like activity, localization of PAF receptor (PAF-R) and PAF-acetylhydrolase (PAF-AH) activity in bovine endometrium at different stages of the estrous cycle and early pregnancy*

PAF-like activity in the endometrium increased from days 2-4 to day 12 and day 20 in both cyclic and pregnant cows. There was an increase in platelet aggregation induced by PAF-like activity in the endometrium of pregnant animals on day 20 as compared to cyclic animals at the same point in time. Two major bands of PAF-R protein at 67 kDa and 97 kDa were detected by Western blot analysis. PAF-R was localized mainly in luminal and glandular epithelium of the endometrium, but the staining was markedly increased in the endometrium of pregnant cows on day 20 compared to cyclic animals on the same day. The purified PAF-AH from the endometrium is similar to in plasma. In cyclic cattle, no changes in PAF-AH activity of endometrium were observed, whereas a decrease in enzyme activity occurred in pregnant cows on day 20 as compared to cyclic animals on the same day. We suggest that the bovine endometrium produces PAF-like activity, expresses the PAF-R and possesses a PAF-AH activity which varies during pregnancy.     

The placental growth factor (PGF) – a positional and functional candidate gene influencing calving ease and stillbirth in German dairy cattle

The bovine placental growth factor‐encoding gene (PGF) was analysed as a positional and functional candidate gene for the maternal effect on stillbirth and calving ease in first parity. Prominent levels of PGF expression have been reported for the whole human placenta and umbilical vein endothelial cells. Modulation of angiogenesis, vessel remodelling and vascular permeability during implantation and placentation suggest an influence on trophoblast function during pregnancy. Changes of expression or protein function may therefore be crucial to pregnancy and parturition. By comparative sequencing of bulls with extreme approximate daughter yield deviations for calving traits, we identified 37 SNPs and two insertions/deletions within the PGF gene. Seventeen of the identified polymorphisms were genotyped in 368 selected bulls and tested for association with approximate daughter yield deviations for calving traits. In a single marker analysis, all SNPs were significantly associated with maternal stillbirth and calving ease first parity. The allele substitutions of the significant SNPs explain 8% to 14% and 8% to 15% of the additive genetic variance for maternal stillbirth and maternal calving ease first parity, respectively. There is no evidence that any of the polymorphisms identified within this study could be the causal mutation underlying the QTL, which is likely to be a regulatory mutation. In summary, we report polymorphisms in the bovine PGF gene significantly associated with the maternal effect on stillbirth and calving ease in animals under selection. These results should be confirmed and extended in further studies to identify the causal mutation underlying the QTL analysed.     

Differential expression of VEGF isoforms and VEGF(164)-specific receptor neuropilin-1 in the mouse uterus suggests a role for VEGF(164) in vascular permeability and angiogenesis during implantation

The mechanism(s) by which localized vascular permeability and angiogenesis occur at the sites of implantation is not clearly understood. Vascular endothelial growth factor (VEGF) is a key regulator of vasculogenesis during embryogenesis and angiogenesis in adult tissues. VEGF is also a vascular permeability factor. VEGF acts via two tyrosine kinase family receptors: VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). Recent evidence suggests that neuropilin-1 (NRP1), a receptor involved in neuronal cell guidance, is expressed in endothelial cells, binds to VEGF(165) and enhances the binding of VEGF(165) to VEGFR2. We examined the spatiotemporal expression of vegf isoforms, nrp1 and vegfr2 as well as their interactions in the periimplantation mouse uterus. We observed that vegf(164) is the predominant isoform in the mouse uterus. vegf(164) mRNA accumulation primarily occurred in epithelial cells on days 1 and 2 of pregnancy. On days 3 and 4, the subepithelial stroma in addition to epithelial cells exhibited accumulation of this mRNA. After the initial attachment reaction on day 5, luminal epithelial and stromal cells immediately surrounding the blastocyst exhibited distinct accumulation of vegf(164) mRNA. On days 6-8, the accumulation of this mRNA occurred in both mesometrial and antimesometrial decidual cells. These results suggest that VEGF(164) is available in mediating vascular changes and angiogenesis in the uterus during implantation and decidualization. This is consistent with coordinate expression of vegfr2, and nrp1, a VEGF(164)-specific receptor, in uterine endothelial cells. Their expression was low during the first 2 days of pregnancy followed by increases thereafter. With the initiation and progression of implantation (days 5-8), these genes were distinctly expressed in endothelial cells of the decidualizing stroma. Expression was more intense on days 6-8 at the mesometrial pole, the presumptive site of heightened angiogenesis and placentation. However, the expression was absent in the avascular primary decidual zone immediately surrounding the implanting embryo. Crosslinking experiments showed that (125)I-VEGF(165) binds to both NRP1 and VEGFR2 present in decidual endothelial cells. These results suggest that VEGF(164), NRP1 and VEGFR2 play a role in VEGF-induced vascular permeability and angiogenesis in the uterus required for implantation. genesis 26:213-224, 2000.