Trophoblast Apoptosis in Human Placenta at Term as Detected by Expression of a Cytokeratin 18 Degradation Product of Caspase

Abstract Context.—Apoptosis occurs in the normal placenta. The monoclonal antibody M30 is directed against a novel epitope of cytokeratin 18 (CK18) that is formed by caspase cleavage early in the apoptotic cascade, and this antibody may therefore be useful for evaluating trophoblast apoptosis. Objective.—We undertook the present study to evaluate the use of monoclonal antibody M30 to assess trophoblast apoptosis in placenta at term. Methods.—We stained paraffin-embedded placental tissues from 15 deliveries at term with M30. We compared positive M30 staining and CK18 staining (as detected by a monoclonal antibody directed against CK18) of trophoblasts in serial slides. We also compared apoptotic rates as detected by M30 and TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling) in 7 of the placentas. Results.—In fields of villous tissue, most M30-positive cells were CK18-positive syncytiotrophoblasts. Approximately half of M30-positive cells occurred as focal positive staining in the syncytial layer, and half occurred as abundant staining of syncytiotrophoblasts in areas with increased intervillous or perivillous fibrinoid. We found very few M30-positive cells in villous stroma. In decidual/basal plate tissues, most (two thirds) of the M30-positive cells were CK18-positive extravillous trophoblasts, whereas one third were syncytiotrophoblasts of anchoring villi. Since TUNEL detects apoptosis in both epithelial and nonepithelial cells, more cells were positively stained with TUNEL than with M30 in some tissue fields. However, our observations suggest that M30 was more sensitive than TUNEL in recognizing apoptotic trophoblasts and had less nonspecific staining than TUNEL. Conclusion.—We recommend the use of monoclonal antibody M30 for apoptosis studies in placental tissues. This antibody is easy to handle, the staining obtained seems specific, and the nonspecific staining seems negligible.

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PURIFICATION AND CHARACTERIZATION OF REACTIVE AND NON-REACTIVE PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1) FROM HUMAN PLACENTA

10.1055/s-0038-1642806 ◽

1987 ◽

Author(s):

M Philips ◽

A G Juul ◽

S Thorsen ◽

J Selmer ◽

L Thim

Keyword(s):

Monoclonal Antibody ◽

Plasminogen Activator ◽

Human Placenta ◽

Solid Phase ◽

Plasminogen Activator Inhibitor ◽

Tissue Type ◽

Single Chain ◽

Plasminogen Activator Inhibitor 1 ◽

Sds Page ◽

Pai 1

Reactive and non-reactive forms of PAI-1 have been identified in various biological materials. The structural differences between these forms remain to be determined.A monoclonal antibody specific for a non-reactive PAI-1 and a monoclonal antibody reacting with both the reactive and nonreactive form of the inhibitor were obtained by immunization with a tissue-type plasminogen activator (t-PA)-PAI-1 complex (Philips et al., Thromb Haemostas 1986; 55:213-7). These antibodies were used for the isolation of reactive and non-reactive PAI-1 by solid-phase immunoadsorption from extracts of human placenta. The inhibitor preparations were further purified by HPLC. Reactive and non-reactive PAI-1 both migrated with a Mr ∼ 52,000 when analyzed by SDS-PAGE. Furthermore, the two inhibitor forms were indistinguishable by N-terminal sequence analysis. Two N-terminal sequences were found in about equal ammounts for both the reactive and non-reactive PAI-1. They were Ser-Ala-Val-His-His-Pro-Pro- and a two residues shorter sequence (Val-His-His-Pro-Pro-). These sequences are in agreement with the published cDNA sequence of PAI-1 and shows that the inhibitor is N-terminally heterogeneously processed. The second order rate constant (ki) for the reaction between reactive PAI-1 and single-chain t-PA was about 6 106 M-1s-1. Treatment with 4 M guanidinium-HCl partially converted the non-reactive PAI-1 to a reactive form exhibiting a similar k1 for inhibition of single-chain t-PA. SDS-PAGE showed that the t-PA-PAI-1 complex could be dissociated by 1,5 M NH4OH/ 39 mM SDS resulting in the release of a PAI-1 with approximately the same Mr as native PAI-1. This indicates either that t-PA does not cleave the inhibitor or that it cleaves a peptide bond close to the C-terminus.In conclusion a non-reactive and a reactive form of PAI-1 can be purified from placenta. The two forms are distinguishable by monoclonal antibodies but they show similar Mr′ls and the same N-terminal sequences.

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Modulation of Wolframin Expression in Human Placenta during Pregnancy: Comparison among Physiological and Pathological States

BioMed Research International ◽

10.1155/2014/985478 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7 ◽

Cited By ~ 2

Author(s):

Angela Lucariello ◽

Angelica Perna ◽

Carmine Sellitto ◽

Alfonso Baldi ◽

Alessandro Iannaccone ◽

...

Keyword(s):

Human Placenta ◽

Autosomal Recessive Disorder ◽

Cell Loss ◽

Wolfram Syndrome ◽

First Trimester ◽

Third Trimester ◽

Gene Encoding ◽

Cytotrophoblast Cell ◽

The Third ◽

Normal Placenta

TheWFS1gene, encoding a transmembrane glycoprotein of the endoplasmic reticulum called wolframin, is mutated in Wolfram syndrome, an autosomal recessive disorder defined by the association of diabetes mellitus, optic atrophy, and further organ abnormalities. Disruption of theWFS1gene in mice causes progressiveβ-cell loss in the pancreas and impaired stimulus-secretion coupling in insulin secretion. However, little is known about the physiological functions of this protein. We investigated the immunohistochemical expression of wolframin in human placenta throughout pregnancy in normal women and diabetic pregnant women. In normal placenta, there was a modulation of wolframin throughout pregnancy with a strong level of expression during the first trimester and a moderate level in the third trimester of gestation. In diabetic women, wolframin expression was strongly reduced in the third trimester of gestation. The pattern of expression of wolframin in normal placenta suggests that this protein may be required to sustain normal rates of cytotrophoblast cell proliferation during the first trimester of gestation. The decrease in wolframin expression in diabetic placenta suggests that this protein may participate in maintaining the physiologic glucose homeostasis in this organ.

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Evidence of extrahepatic replication of hepatitis E virus in human placenta

Journal of General Virology ◽

10.1099/vir.0.063602-0 ◽

2014 ◽

Vol 95 (6) ◽

pp. 1266-1271 ◽

Cited By ~ 57

Author(s):

Purabi Deka Bose ◽

Bhudev Chandra Das ◽

Rajib Kishore Hazam ◽

Ashok Kumar ◽

Subhash Medhi ◽

...

Keyword(s):

Human Placenta ◽

Hepatitis E Virus ◽

Placental Tissue ◽

Hepatitis E ◽

Fetal Mortality ◽

Positive Staining ◽

Structural Protein ◽

Tissue Sections ◽

Extrahepatic Replication ◽

Immunohistochemical Studies

The incidence and severity of hepatitis E virus (HEV) infection in pregnant women is high in developing countries. Transplacental transmission of HEV in the third trimester of pregnancy has been found to be associated with high fetal mortality. Based on this evidence and in the absence of reports on HEV replication in extrahepatic sites, this study was carried out to investigate if HEV replication occurs in the placenta of infected mothers. The study included 68 acute viral hepatitis (AVH) and 22 acute liver failure (ALF) pregnant patients. Viral RNA was extracted from blood and placenta. HEV replication in placenta was confirmed by a replicative negative-strand-specific reverse transcriptase PCR. Viral load was estimated by real-time PCR. Immunohistochemical studies were also carried out for in situ detection of HEV in placental tissue sections. Replicative HEV RNA was detectable only in the placenta in ALF and AVH cases and not in blood samples. Positive staining of placental tissue sections with HEV antibody against the viral structural protein ORF3 was observed. HEV replication in placenta also correlated with fetal and maternal mortality in ALF patients. It is demonstrated for the first time that HEV replication occurs in human placenta and that placenta may be a site of extrahepatic replication of HEV in humans.

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Evidence That Relaxin Inhibits Apoptosis in the Cervix and the Vagina during the Second Half of Pregnancy in the Rat*

Endocrinology ◽

10.1210/endo.142.6.8182 ◽

2001 ◽

Vol 142 (6) ◽

pp. 2221-2229 ◽

Cited By ~ 16

Author(s):

Shuangping Zhao ◽

P. A. Fields ◽

O. D. Sherwood

Keyword(s):

Electron Microscopy ◽

Monoclonal Antibody ◽

Stromal Cells ◽

Hormonal Control ◽

Terminal Deoxynucleotidyl Transferase ◽

Blood Levels ◽

Apoptotic Cells ◽

Labeling Method

Abstract The growth of the cervix and vagina that occurs during the second half of rat pregnancy is accompanied by an increase in both epithelial and stromal cells. Neither the mechanism(s) that regulates this accumulation of cells nor its hormonal control is known. To test the hypothesis that the rate of apoptosis declines during the second half of pregnancy, cervices and vaginas were collected on days 5, 10, 15, 18, and 21 of pregnancy. Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5′-triphosphate nick end-labeling was used to detect apoptotic cells. The rate of apoptosis declined (P < 0.05) in epithelial and stromal cells in both the cervix and vagina during the second half of pregnancy, when blood levels of relaxin are increasing. To test the hypothesis that relaxin inhibits apoptosis, cervices and vaginas were collected 6, 12, 24, 48, and 72 h after the neutralization of endogenous relaxin, on days 19–21 of pregnancy, with a monoclonal antibody for rat relaxin. Both the terminal deoxynucleotidyl transferase-mediated deoxyuridine 5′-triphosphate nick end-labeling method and electron microscopy were used to detect apoptotic cells. Withdrawal of relaxin caused an increase in the rate of apoptosis in both the cervix and the vagina (P < 0.05). It is concluded that the rate of apoptosis declines in the cervix and the vagina during the second half of rat pregnancy, and that relaxin likely contributes to this process.

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Vasopressin inhibits apoptosis in renal collecting duct cells

AJP Renal Physiology ◽

10.1152/ajprenal.00431.2012 ◽

2013 ◽

Vol 304 (2) ◽

pp. F177-F188 ◽

Cited By ~ 14

Author(s):

R. Lance Miller ◽

Pablo C. Sandoval ◽

Trairak Pisitkun ◽

Mark A. Knepper ◽

Jason D. Hoffert

Keyword(s):

Arginine Vasopressin ◽

Collecting Duct ◽

Critical Role ◽

Terminal Deoxynucleotidyl Transferase ◽

Cortical Collecting Duct ◽

Caspase Cleavage ◽

Mammalian Kidney ◽

Duct Cells ◽

V2 Receptor ◽

Collecting Duct Cells

The peptide hormone arginine vasopressin (AVP) plays a critical role in regulating salt and water transport in the mammalian kidney. Recent studies have also demonstrated that AVP can promote cell survival in neuronal cells through V1 receptors. The current study addresses whether AVP can inhibit apoptosis in kidney collecting duct cells via V2 receptors and also explores the downstream signaling pathways regulating this phenomenon. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling analysis and caspase cleavage assays demonstrated that 1-desamino-8-d-arginine vasopressin (dDAVP) inhibited apoptosis induced by various agents (staurosporine, actinomycin D, and cycloheximide) in cultured mouse cortical collecting duct cells (mpkCCD). Incubation with dDAVP also inhibited apoptosis induced by the phosphatidylinositol 3-kinase (PI3K) pathway inhibitor LY294002, suggesting that the antiapoptotic effects of dDAVP are largely independent of PI3K signaling. The V2 receptor antagonist SR121463 completely abolished the antiapoptotic effects of dDAVP. In addition, incubation with 8-cpt-cAMP, a cell-permeable analog of cAMP, reproduced the antiapoptotic effects of dDAVP. Both dDAVP and 8-cpt-cAMP increased phosphorylation of proapoptotic Bcl-2 family members Bad and Bok. Bad phosphorylation at Ser-112 and Ser-155 is known to inhibit its proapoptotic activity. Preincubation with H89 blocked dDAVP-induced phosphorylation of both Bad and Bok, suggesting dependence on protein kinase A (PKA). This study provides evidence that AVP can inhibit apoptosis through the V2 receptor and downstream cAMP-mediated pathways in mammalian kidney. The antiapoptotic action of AVP may be relevant to a number of physiological and pathophysiological conditions including osmotic tolerance in the inner medulla, escape from AVP-induced antidiuresis, and polycystic kidney disease.

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Changes in the expression and cytological localization of betacellulin and its receptors (ErbB-1 and ErbB-4) in the trophoblasts in human placenta over the course of pregnancy

Acta Endocrinologica ◽

10.1530/eje.0.1510093 ◽

2004 ◽

pp. 93-101 ◽

Cited By ~ 7

Author(s):

K Tanimura ◽

S Nakago ◽

H Murakoshi ◽

S Takekida ◽

T Moriyama ◽

...

Keyword(s):

Growth Factor ◽

Human Placenta ◽

Southern Blotting ◽

Cell Function ◽

Soluble Form ◽

Rt Pcr ◽

Epidermal Growth ◽

Cytological Localization ◽

Design And Methods ◽

Extravillous Trophoblasts

OBJECTIVE: Betacellulin (BTC), purified and cloned from mouse beta cell tumor (BTC-JC10), is regarded as a new member of the epidermal growth factor family. The present study was conducted to clarify the expression of BTC and its receptors, ErbB-1 and ErbB-4, in the trophoblasts in the human placenta over the course of pregnancy. DESIGN AND METHODS: Human placental tissues were obtained from 4 pregnant women at the 4th to 5th week of pregnancy (very early placentas), 10 women at the 6th to 12th week (early placentas), 5 women at the 18th to 21st week (mid placentas) and 8 women at the 38th and 40th week (term placentas). The mRNA expressions of BTC, erbB-1 and erbB-4 were evaluated by quantitative RT-PCR with Southern blotting and the expression of the soluble form of BTC was determined by western immunoblot with a specific antibody to BTC protein. Immunohistochemical staining of BTC, ErbB-1 and ErbB-4 was also performed. RESULTS: The levels of BTC mRNA expression in early and mid placentas were significantly higher than those in term placentas. The soluble form of BTC protein with an estimated molecular mass of 9.5 kDa was expressed in early and mid placentas, whereas the soluble form was not detected in term placentas. BTC from very early placentas until mid placentas was immunolocalized in syncytiotrophoblasts (S-cells), and was most abundant in early placentas. In contrast, BTC was immunolocalized in extravillous trophoblasts (EVTs), but not in villous trophoblasts in term placentas. The levels of erbB-1 mRNA in the early and mid placentas were significantly higher than those in term placentas, whereas the levels of erbB-4 mRNA in early placentas were significantly lower than those in mid and term placentas. ErbB-1 was immunolocalized in cytotrophoblasts in very early placentas, whereas it was immunolocalized in S-cells from early until term placentas. ErbB-4 from very early placentas until mid placentas was immunolocalized in S-cells, whereas ErbB-4 in the term placentas was detected in EVTs, but not in villous trophoblasts. CONCLUSIONS: These findings provide evidence for changes in expression and cytological localization of BTC and its receptors in the trophoblasts in human placenta over the course of pregnancy. BTC may play a pivotal role as a local growth factor in promoting the differentiated villous trophoblastic function via ErbB-1 in early placentas and in contributing to placental growth through the maintenance of EVT cell function via ErbB-4 in term placentas.

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The Extent to which Relaxin Promotes Proliferation and Inhibits Apoptosis of Cervical Epithelial and Stromal Cells Is Greatest during Late Pregnancy in Rats

Endocrinology ◽

10.1210/en.2004-0796 ◽

2005 ◽

Vol 146 (1) ◽

pp. 511-518 ◽

Cited By ~ 30

Author(s):

Hyung-Yul Lee ◽

Shuangping Zhao ◽

P. A. Fields ◽

O. D. Sherwood

Keyword(s):

Electron Microscopy ◽

Monoclonal Antibody ◽

Cell Proliferation ◽

Epithelial Cells ◽

Stromal Cells ◽

Late Pregnancy ◽

Terminal Deoxynucleotidyl Transferase ◽

Apoptotic Cells ◽

Cervical Cells

Relaxin promotes marked growth of the cervix during the second half of rat pregnancy, and this growth is accompanied by an increase in both epithelial and stromal cells. The objective of this study was to test the hypothesis that the extent to which relaxin promotes proliferation and inhibits apoptosis of cervical cells is greatest during late pregnancy in rats. The influence of neutralization of circulating relaxin by iv injection of 5 mg monoclonal antibody against rat relaxin (MCA1) was examined at 3-d intervals throughout the second half of pregnancy. Controls were injected with either 5 mg monoclonal antibody against fluorescein or 0.5 ml PBS vehicle. To evaluate cell proliferation, 5′-bromo-2-deoxyuridine was injected sc 8 h before cervixes were collected. Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5′-triphosphate nick end-labeling and electron microscopy were used to detect apoptotic cells. Neutralization of relaxin with MCA1 decreased the rate of proliferation and increased the rate of apoptosis of cervical cells by d 13. However, the extent to which relaxin influenced these processes was greatest and dramatic by late pregnancy. In MCA1-treated rats on d 22 of pregnancy, the rates of proliferation of both epithelial and stromal cells were less than 20% those in controls, and the rates of apoptosis in epithelial cells and stromal cells were more than 10- and 3-fold, respectively, greater than those in controls. In conclusion, this study provides evidence that the extent to which relaxin promotes proliferation and inhibits apoptosis of cervical epithelial and stromal cells is greatest during late pregnancy.

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