Comparative proteomic analysis of human placenta derived from assisted reproductive technology

The aim of this study was to use proteomics-based approach to examine differences in protein expression in placenta derived from assisted reproductive technology (ART) and normal pregnancy. Using 2-DE we found that, compared with the control group, 12 spots in standard in vitro fertilization group and 18 spots in intracytoplasmic sperm injection group were identified as significantly differentially expressed proteins. Among them, six spots were differentially expressed in both standard IVF and ICSI groups with the same change tendency. Totally, 20 proteins were successfully identified by MALDI TOF/TOF MS, including proteins involved in the membrane traffic, metabolism, nucleic acid processing, stress response and cytoskeleton. Notably, five proteins detected to be differentially expressed in both ART groups were identified as annexin A3, hnRNP C1/C2, alpha-SNAP, FTL and ATP5A. Some of the proteins were confirmed by Western blot and immunohistochemistry analysis. Our study allowed for the initial identification of these proteins related to various functions in placentation with significantly altered abundance in ART groups. The present results reveal that abnormal protein profiles are involved in ART placenta and these differentially expressed proteins may be valuable for the evaluation of potential association between ART treatment and offspring outcome.     

Bovine endometrial legumain and TIMP-2 regulation in response to presence of a conceptus

The precise mechanism of placentation in the bovine species where a restricted trophoblast invasion occurs to form the synepitheliochorial placenta is not fully understood. This study initially investigated the conceptus-maternal interactions in the peri-attachment period by comparing the proteins present at Days 16 and 18 in uterine luminal fluid (ULF) of pregnant with nonpregnant cows using 2-D gel electrophoresis. Nine protein spots were identified that were present in greater amounts in pregnant compared to nonpregnant ULF: carbonic anhydrase, ezrin, heat shock protein 70, isocitrate dehydrogenase, nucleoside diphosphate kinase, peroxiredoxin 1, purine nucleoside phosphorylase, thioredoxin and triosephosphate isomerase and four proteins that were less abundant in ULF from the gravid compared to the nongravid horns or nonpregnant uteri: cystatin E/M, legumain, retinol-binding protein (RBP) and tissue inhibitor of matrix metalloproteinase 2 (TIMP-2). Successful placentation requires the remodelling of the endometrial surface therefore uterine mRNA and protein expression of legumain, a protease activator, and TIMP-2, a protease inhibitor, was examined in detail during the oestrous cycle and from Days 13 to 31 of pregnancy. Both mRNAs were up-regulated in the endometrium during the luteal phase of the oestrous cycle and during early pregnancy. Although legumain and TIMP-2 mRNA expression levels were similar between uterine horns at the same day of pregnancy, the amount of protein differed between gravid and nongravid horns possibly modulated by interferon-tau or by other factors produced by the conceptus. These events at the conceptus-maternal interface may provide localised control of protease activity necessary for controlling trophoblast invasion of the endometrium.     

The Role of Pro- and Antioxidant Systems in the Development of Placental Dysfunction in Human Pregnancy

The pro-and antioxidant systems of the human placenta have been studied in its central and peripheral areas in the state of dysfunction. It has been shown that the intensity of free-radical oxidation (FRO) is 24% higher (p < 0.05) in mitochondria isolated from peripheral placental areas in the case of preterm termination of pregnancy than in placental mitochondria of women with normal pregnancy ending in delivery on due dates. The values of total antioxidant activity in mitochondria isolated from the central and peripheral areas of placentae of women with preterm labor exceeded 1.5-and 1.8-fold the respective values for the placental mitochondria of women with the normal duration of pregnancy. The rate of glutathione peroxidase activity in placental mitochondria of women with preterm labor was lower than in patients with normal duration of pregnancy terminated on due dates. Higher values of intensity of both the FRO processes and the active components of thiobarbituric acid (TBA) were recorded (higher by 42% and 62%, respectively) in the postmitochondrial fraction in the peripheral area of placentae of women with spontaneous termination of pregnancy, compared with the placentae of women with uncomplicated duration of pregnancy with labor on due date. No differences have been observed in the intensity of oxidative modification of placental proteins in both the periphery and the center in the placentae of women from the studied groups. The rate of glutathione peroxidase activity in the placenta of women with spontaneous termination of pregnancy was more than twice as high as the activity of this enzyme during the first trimester of normal pregnancy and remained high during the second and third trimesters. The activity of the enzyme did not depend on its localization (center or periphery) in placentae of women participating in the study. The values of glutathione transferase activity in the placentae increased in the course of normal pregnancy but remained at the level of the first trimester in the central and peripheral areas in the case of a miscarriage at different gestational terms. Our findings allow us to suggest that oxidative stress developing in placenta from its center to periphery plays a key role in the pathogenesis of placental dysfunction, mainly, due to the glutathione-dependent component of the placental antioxidant defense.     

Involvement of matrix metalloproteinases 2 and 9, tissue inhibitor of metalloproteinases and apoptosis in tissue remodelling in the sheep placenta

Placental growth and development is crucial for successful pregnancy. The aim of this study was to characterize the activity and localization of the matrix metalloproteinase 2 (MMP-2) and MMP-9, which are capable of degrading basement membrane collagen (predominantly collagen type IV), and their endogenous tissue inhibitor of matrix metalloproteinases (TIMPs), in amniotic fluid and in the developing ovine placenta. Cell deletion by apoptosis during placental development was also examined. Zymography with gelatin as substrate indicated that MMP-2 (72 kDa gelatinase A; predominantly latent form) was present in increasing amounts in amniotic fluid from day 70 of gestation to labour (days 140-145), and MMP-9 (92 kDa gelatinase B; predominantly latent form) was detectable from day 125 to labour; there was no increase in MMP-2 or -9 in labour. A broad range of TIMPs was detected in amniotic fluid; the molecular masses corresponded to TIMP-1, -2 and -3. Immunohistochemical techniques localized MMP-2, MMP-9 and TIMP-3 in the sheep placenta, predominantly in the trophoblast layer in uninucleate, but not binucleate, cells. However, MMP-2 and -9 activated proteins in placental homogenates were low throughout pregnancy. Apoptosis was identified by morphological criteria and also by TdT-mediated dUTP nick end labelling. Apoptosis was present in discrete regions in the placenta, predominantly in trophoblast cells near the tips and the basal regions of the fetomaternal interdigitations. During pregnancy the sheep placenta becomes more complex and the area of the fetomaternal interface increases. MMP-2 and -9 are likely to be involved in breaking down basement membranes to allow cell migration during this process. It is suggested that digestion of supporting extracellular matrix may trigger apoptosis and in some way increase the branching pattern in the villi.     

Glypican-3 (GPC3) expression in human placenta: localization to the differentiated syncytiotrophoblast

The expression of glypican-3 (GPC3), a heparan-sulfate proteoglycan associated with the Simpson-Golabi-Behmel fetal overgrowth syndrome, was studied in normal human placental tissue and cell lines derived from human placentae. Cytotrophoblasts derived from term placentae expressed GPC3 mRNA at low levels in culture. GPC3 mRNA expression increased markedly during trophoblast differentiation. By contrast, fibroblast cell lines derived from normal placentae did not express GPC3 in culture. Similarly, choriocarcinoma cell lines derived from human placentae (BeWo, JAR, and JEG) failed to express GPC3 mRNA. In situ hybridization confirmed the localization of GPC3 mRNA to the syncytiotrophoblast. Furthermore, immunohistochemical staining of paraffin imbedded placental tissue demonstrated intense staining of the syncytiotrophoblast cell layer and less intense staining of cytotrophoblasts. No staining of mesenchymal elements was noted. These data confirm the presence of GPC3 in human placenta and suggest it is expressed by the differentiated syncytiotrophoblast at term.     

Epigenetic characterization of the PBEF and TIMP-2 genes in the developing placentae of normal mice

Reprogramming errors, which appear frequently in cloned animals, are reflected by aberrant gene expression. We previously reported the aberrant expression of TIMP-2 and PBEF in cloned placenta and differential expression of PBEF genes during pregnancy. To examine the epigenetic modifications that regulate dynamic gene expression in developing placentae, we herein analyzed the mRNA and protein expression levels of PBEF and TIMP-2 in the placentae of normal mice during pregnancy and then examined potential correlations with epigenetic modifications. DNA methylation pattern analysis revealed no difference, but ChIP assays using antibodies against H3-K9/K14 and H4-K5 histone acetylation revealed that the H3-K9/K14 acetylation levels, but not the H4-K5 acetylation levels, of the TIMP-2 and PBEF loci were significantly correlated with their gene expression levels during placentation in normal mice. These results suggest that epigenetic changes may regulate gene expression level in the developing placentae of normal mice and that inappropriate epigenetic reprogramming might be one cause of the abnormal placentae seen in cloned animals.     

Proteomic analysis of the extraembryonic tissue from cloned porcine embryos

Cloned animals developed from somatic cell nuclear transfer (SCNT) embryos are useful resources for agricultural and medical applications. However, the birth rate in the cloned animals is very low, and the cloned animals that have survived show various developmental defects. In this report, we present the morphology and differentially regulated proteins in the extraembryonic tissue from SCNT embryos to understand the molecular nature of the tissue. We examined 26-day-old SCNT porcine embryos at which the sonogram can first detect pregnancy. The extraembryonic tissue from SCNT embryos was abnormally small compared with the control. In the proteomic analysis with the SCNT extraembryonic tissue, 39 proteins were identified as differentially regulated proteins. Among up-regulated proteins, Annexins and Hsp27 were found. They are closely related to the processes of apoptosis. Among down-regulated proteins, Peroxiredoxins and anaerobic glycolytic enzymes were identified. In the Western blot analysis, antioxidant enzymes and the antiapoptotic Bcl-2 protein were down-regulated, and caspases were up-regulated. In the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay with the placenta from SCNT embryos, apoptotic trophoblasts were observed. These results demonstrate that a major reason for the low birth rate of cloned animals is due to abnormal apoptosis in the extraembryonic tissue during early pregnancy.     

铅致胎盘损伤机制的研究进展(英文)

铅是一种嗜神经和嗜胎盘的毒性重金属,本综述主要是关于铅的胎盘毒性。孕期铅暴露可以导致胎盘重量减轻,滋养层增生,血管堵塞,细胞间隙增宽,血管周围大量的纤维蛋白沉积,以及粗面内质网扩张,膜上核糖体数量减少。当孕期铅暴露在一定范围内时,一氧化氮(NO),一氧化氮合酶(NOS)水平升高,以保证胎盘组织器官的正常结构和功能;进一步加重时,NO及NOS反而降低,导致胎儿-胎盘循环阻力增高,胎盘灌注量下降;孕期铅暴露时,丙二醛(MDA)升高,说明存在胎盘氧化与抗氧化系统平衡失调;基质金属蛋白酶-9(MMP-9)表达降低,而基质蛋白酶组织抑制因子-1(TIMP-1)表达增强,胎盘MMP-9/TIMP-1的表达失衡,导致滋养细胞浸润能力减弱,胎盘着床过浅,血管重铸障碍,从而影响胎盘发育及胎儿生长;染铅胎盘NF-kB的表达及血栓调节蛋白(TM)的表达明显高于对照组。NF-kB的激活又可以反式激活表皮生长因子,血小板生长因子等的表达,促进血管平滑肌细胞的增殖,细小动脉胶原纤维增加,血管痉挛性收缩;TM表达异常,说明孕期铅暴露可致胎盘血管内皮细胞损伤。总之,孕期铅暴露可引起胎盘病理及一系列的分子化学改变,从而影响胎盘功能和胎儿发育,可引起子代早产、出生体重低、智力障碍等。     

Ultrastructural studies of the equine uterus and placenta following parturition.

Post-partum placentae and uterine biopsy samples from mares after normal and abnormal foalings are described. After normal delivery there is little damage to fetal or maternal tissues. The villous epitheliochorial palcenta separates cleanly at the maternal-fetal interface and the afterbirth consists almost exclusively of fetal tissue. Uterine involution is well advanced by the 3rd and 4th days post partum and the changes are usually complete by the oestrus 7--10 days after parturition. Placental separation and involution of the uterus appear to proceed normally in malpresented foals and in otherwise viable foals with musculoskeletal defects. In aborted, stillborn or dysmature foals there are obvious signs of damage to both fetal and maternal tissues. It is generally accepted that the growth and development of the fetus is dependent upon a placenta of adequate functional capabilities. The observations suggest that the placenta is similarly dependent upon its association with a normal healthy fetus.     

Extracellular nucleic acids in maternal circulation as potential biomarkers for placental insufficiency

Since the placenta is being continuously remodeled during normal placental development, extracellular nucleic acids of both fetal and placental origin, packed into either trophoblast-derived apoptotic bodies or shedding syncytiotrophoblast microparticles, may be detected in maternal circulation during the course of normal gestation. Placental-insufficiency-related pregnancy complications have been shown to be associated with excessive placental trophoblast apoptosis and shedding of placenta debris. Recent advances in the field are reviewed with a focus on the diagnostic potential of particular molecular biomarkers and their eventual implementation in the currently used predictive and diagnostic algorithms for placental-insufficiency-related pregnancy complications.     

Over-expression and secretion of angiogenin in intrauterine growth retardation placenta

Human angiogenin is a potent inducer of neovascularization. There is a strong evidence to suggest that it might be involved in morphological and angiogenic changes in the placenta, that are necessary for a successful fetal outcome during pregnancy. However, its precise role in the pathogenesis of abnormal pregnancies is yet unknown. Intrauterine growth retardation (IUGR), an abnormal pregnancy is not a specific disease entity per se, but rather a manifestation of many possible fetal and maternal disorders. In this study, we demonstrated, for the first time, that placental explants in vitro secrete significantly elevated levels of angiogenin in placental tissues from patients with IUGR. We also observed enhanced mRNA expression in placenta from these patients. In addition, using the immunohistochemical methods, we observed identical staining of angiogenin to villous syncytiotrophobalst and fetal endothelial cells in both IUGR and normal placenta. Functionally active placental explants were used to detect immunoreactive angiogenin in conditioned media of all the samples from IUGR placenta and normal term group. The mean levels of angiogenin secreted by IUGR placenta were 1.4-, 1.6-, and 1.3-fold higher (P < 0.01) than normal term samples at 24, 48, and 72 hr of culture, respectively. Expression profiles of angiogenin from term and IUGR cases are in agreement with its mRNA levels and immunoblot analysis. In conclusion, the significant elevated levels of angiogenin in IUGR placenta may provide a molecular mechanism for the abnormal placental development.     

Diagnosis and monitoring of pregnancy in mice: correlations between maternal weight, fetal and placental mass and the maternal serum levels of progesterone, pregnancy-associated murine protein-2 and alpha-fetoprotein

Outbred Bom:NMRI mice were weighed daily for 18 days from observation of a vaginal plug. In a separate experiment, fetuses and placentae were weighed on each day of pregnancy. Pregnancy can be determined with 99% certainty on day 12 of gestation by the maternal body weight increase from day 1. The pregnancy-specific proteins alpha-fetoprotein (m-AFP) and pregnancy-associated murine protein-2 (PAMP-2), of fetal and placental origin respectively, were detectable on days 8 and 10 in the maternal circulation. Significant correlations were observed between m-AFP and fetal weight and PAMP-2 and placental weight. These markers may therefore be useful in the monitoring of fetal growth and placental growth respectively.     

Dynamics of activities of matrix metalloproteinases-9 and -2, and the tissue inhibitors of MMPs in fetal fluid compartments during gestation and at parturition in the mare

During late gestation in the mare, rapid fetal growth is accompanied by considerable placental growth and further invasion of the endometrium by microvilli. This growth requires extensive remodeling of the extracellular matrix (ECM). In early pregnancy, we know that matrix metalloproteinase (MMP)-9 and -2 are involved in the endometrial invasion during endometrial cup formation. The present study investigated whether MMPs are found in fetal fluids later in gestation and during parturition, and if there was a difference in their activities between normal and preterm delivery. Amniotic fluids were collected from pony mares during the latter half of gestation, and amniotic and allantoic fluids from pony and thoroughbred mares at foaling. The fluids were analysed for the activity of MMP-9 and -2, and TIMPs using zymography techniques. There was an increase (P = 0.002) in activity of latent MMP-9 when approaching normal foaling, and a decrease (P < 0.001) during foaling. MMP-2 activity did not change through gestation, or during foaling. When comparing samples from pregnancies resulting in preterm deliveries with samples from foaling mares, the activity of MMP-9 was lower (P < 0.001) and MMP-2 activity was higher (P = 0.004) during foaling than preceding preterm delivery. The activity of MMP-9 was lower (P = 0.002) prior to preterm delivery than before delivery of a live foal at term, whereas no difference (P = 0.07) was demonstrated for latent MMP-2 activity when comparing the same groups. The activity of TIMP-2 was higher (P < 0.001) in the pre-parturient period before normal foaling than preceding preterm delivery. These results suggest that MMPs may have a role as markers for high risk pregnancy in the mare.     

A proteomics study of in vitro cyst germination and appressoria formation in Phytophthora infestans

A proteomics study using two-dimensional gel electrophoresis (2-DE) and mass spectrometry was performed on Phytophthora infestans. Proteins from cysts, germinated cysts and appressoria grown in vitro were isolated and separated by 2-DE. Statistical quantitative analysis of the protein spots from five independent experiments of each developmental stage revealed significant up-regulation of ten spots on gels from germinated cysts compared to cysts. Five spots were significantly up-regulated on gels from appressoria compared to germinated cysts and one of these up-regulated spots was not detectable on gels from cysts. In addition, one spot was significantly down-regulated and another spot not detectable on the gels from appressoria. The corresponding proteins to 13 of these spots were identified with high confidence using tandem mass spectrometry and database searches. The functions of the proteins that were up-regulated in germinated cysts and appressoria can be grouped into the following categories: protein synthesis (e.g. a DEAD box RNA helicase), amino acid metabolism, energy metabolism and reactive oxygen species scavenging. The spot not detected in appressoria was identified as the P. infestans crinkling- and necrosis-inducing protein CRN2. The identified proteins are most likely involved in the establishment of the infection of the host plant.