The effect of 12-myristate 13-acetate phorbol ester on human sperm hyperactivation

Objective: To determine whether 12-myristate 13-acetate phorbol ester (PMA) can increase hyperactivated motility of human sperm.

Design: A controlled pharmacological study using computer-assisted semen analysis.

Setting: Andrology laboratory in a medical research institution.

Patient(s): Normal semen was obtained from 48 men.

Intervention(s): Washed sperm were exposed to different concentrations of PMA alone or with P and pentoxifylline (PTX) for up to 2 hours and sperm motility measured by a computerassisted semen analyzer.

Main Outcome Measure(s): The percentage of sperm with hyperactivated motility was determined from the motility parameters: curvilinear velocity, linearity, and maximum amplitude of lateral head displacement.

Result(s): Phorbol ester PMA increased hyperactivated motility in a dose- and time-dependent manner. At 1 hour, the average increases in hyperactivated motility were as follows: 2 μM, 4.8% ± 1.5%; 6 μM, 9.6% ± 1.5%; and 20 μM, 11.3% ± 2.2%. The PMA effect was not altered when P or PTX were added although each separately had a positive effect on hyperactivation.

Conclusion(s): Phorbol ester PMA stimulates human sperm hyperactivated motility, indicating the involvement of protein kinase C in the signal transduction pathway.     

Interferon-gamma enhances mRNA and surface expression of class I antigen on human extravillous trophoblast

Human trophoblast cells with immunocytochemical characteristics of the extravillous population have been isolated from 1st trimester placentae. Treatment of these cells with IFN-gamma increases the expression of Class I antigens at both the cell surface and mRNA level. A similar increase in Class I antigens is also found in JEG-3 choriocarcinoma cells after treatment with IFN-gamma. The possibility that aberrant production of IFN-gamma may upset the fetal-maternal equilibrium in vivo is discussed.     

GPR30在妊娠期胎盘中的表达及其对滋养细胞侵袭能力影响的研究

目的:探讨G-蛋白偶联受体30(GPR30)在妊娠期胎盘中的表达,以及其对人胎盘滋养细胞侵袭力的影响。方法:免疫组化法检测早孕期绒毛、正常产妇胎盘和重度子痫前期患者胎盘组织中GPR30表达。用17-β-雌二醇(17β-E2)、GPR30激动剂G1和阻滞剂G15预处理体外培养绒毛组织和人绒毛外滋养细胞株HTR8/SVneo。显微镜下观察体外绒毛组织滋养细胞生长侵袭范围,Transwell侵袭实验检测HTR8/SVneo细胞侵袭能力。免疫荧光法检测绒毛组织和HTR8/SVneo细胞中GPR30蛋白表达。Western blot法检测HTR8/SVneo细胞中GPR30和MMP-9蛋白表达。结果:GPR30在早期绒毛组织和正常末期胎盘滋养细胞上都有表达,且早孕期的表达水平高于正常末期胎盘,但在重度子痫前期胎盘上GPR30蛋白表达明显减少。E2及GPR30激动剂G1可增加滋养细胞的侵袭能力,阻滞剂G15则可下调其侵袭性;E2、G1可诱导滋养细胞GPR30蛋白表达上调,而G15则下调其表达。GPR30蛋白水平与侵袭相关蛋白MMP-9表达水平有相关性。结论:GPR30可能参与人类滋养细胞侵袭力的调节,对滋养细胞的侵袭力有正性促进作用。     

Regulation by hypoxia of adrenomedullin output and expression in human trophoblast cells

Objectives

Plasma adrenomedullin concentrations are increased in the fetal circulation in acute and chronic hypoxic conditions. The effect of hypoxia in regulating adrenomedullin synthesis and secretion was investigated in human placental trophoblast cells.

Study design

Human trophoblast cells obtained from term placentas (n = 7) were cultured in hypoxic condition (3% oxygen). Cytotrophoblast cells were cultured for up to 48 h and syncytiotrophoblasts for 2, 8 and 24 h. Changes in adrenomedullin output compared to normoxic conditions were measured by radioimmunoassay. Protein expression was evaluated with Western blot and immunocytochemistry.

Results

Hypoxia induced a time-dependent increase in adrenomedullin output and protein expression by placental trophoblast cells.

Conclusions

Hypoxia regulates adrenomedullin secretion and expression by human placenta, thereby promoting increased adrenomedullin concentration in the fetal circulation in clinical circumstances characterized by reduced oxygen levels.     

Study on PGDH activity in human term placenta--relationship between PGDH activities and perinatal factors

The high activity of 15-hydroxyprostaglandin dehydrogenase (PGDH) which catalyzes the first step in prostaglandin metabolism has been demonstrated in the human placenta. To investigate the role of placental PGDH (p-PGDH) in the perinatal period, PGDH activity was measured in 167 placentas of gestational ages 37 to 41 weeks. The activity was expressed as n mole/min/non heme protein of 15-keto-PGE2 which was produced by incubation of PGE2 and placental homogenate supernatant. The following data were obtained. p-PGDH activity was significantly lower at the 40 weeks of gestation than at other weeks. There was no difference in placental PGDH activity between cesarean section and vaginal delivery, and between the periods before and after onset labor of cesarean section. p-PGDH activity in the PGs administered group was higher than that in the non PGs group. p-PGDH activity of a male fetus was higher than that of a female. This suggests that p-PGDH activity is under the control of hormones. No relationship was observed between p-PGDH activity and other perinatal factors such as toxemia of pregnancy, parity, Apgar score, birth weight, placental weight, blood loss, and duration of labor.     

Neogenin对人滋养细胞RGMa、DAPK、Neogenin mRNA表达及细胞凋亡的影响

目的:观察不同浓度Neogenin对人滋养细胞RGMa、DAPK、Neogenin表达的影响,以阐明Neogenin在滋养细胞凋亡过程中的作用。方法:先用含0,0.5,1,5,10及50ng/ml的Neogenin无血清培养基预处理TEV-1细胞24h,用噻唑蓝(MTT)比色、流式细胞术(FCM)检测细胞增殖指数及凋亡率;用实时定量PCR检测TEV-1细胞RGMa、DAPK、Neogenin mRNA的变化。结果:MTT、FCM检测表明Neogenin能诱导TEV-1凋亡,且呈一定的浓度依赖性,Neogenin对TEV-1细胞增殖无明显影响。TEV-1细胞RGMa mRNA的表达水平依次为空白对照组(0ng/ml Neogenin)的0.62±0.04,0.90±0.04,0.97±0.04,0.90±0.03及0.75±0.03;DAPK mRNA的表达水平依次为空白对照组的0.68±0.02,0.35±0.01,0.74±0.01,0.51±0.01及0.33±0.01;Neogenin mRNA的表达水平依次为空白对照组的1.45±0.03,1.80±0.05,1.22±0.08,1.47±0.00及1.14±0.00。即随着Neogenin浓度增加,TEV-1细胞内DAPK mRNA表达下调,Neogenin mRNA表达上升,RGMa mRNA的表达水平基本不变。结论:Neogenin可促进TEV-1细胞凋亡,这一过程可能是经DAPK途径调控的。     

Differential expression of complement regulatory proteins on subpopulations of human trophoblast cells.

Trophoblast cells forming the reactive interface between the mother and her semiallogeneic fetus risk attack by cellular and humoral elements of the maternal immune system. Biochemical, molecular, and immunohistologic studies have identified membrane cofactor protein (MCP) and decay accelerating factor (DAF) on trophoblast cells, which could assist in preventing lysis of the cells by complement-activating maternal antibodies. In this immunocytochemical study, differential expression of these two members of the family of complement regulatory proteins on subpopulations of human trophoblast cells and other types of cells in first and third trimester placentas was demonstrated. Staining with anti-MCP was particularly strong on villous cytotrophoblast cells and giant cells in first trimester tissues in comparison with other types of cells. In contrast, staining with anti-DAF was strong on proliferating cytotrophoblast in first trimester tissues, and on basal plate cytotrophoblast and decidual cells in term tissues. Placental villous mesenchymal cells but not trophoblast cells expressed a third regulatory protein, complement receptor 1. These observations support the postulate that complement regulatory proteins are critical to protection of the fetal allograft, and suggest specific requirements for trophoblast cells according to stage of differentiation and anatomic location.     

Terbutaline inhibits corticotropin-releasing hormone (CRH) expression in human trophoblast cells

Objective.?To evaluate the effect of beta-adrenergic agonists on the regulation of the expression of the placental corticotropin-releasing hormone (CRH) gene.

Study design.?Term placentae were collected at the time of elective cesarean section, and trophoblast cells were harvested, isolated, and cultured. The isolated trophoblasts were plated, cultured and subsequently treated with cortisol, terbutaline, RU486, or vehicle control. CRH expression, mRNA abundance of CRH, and the housekeeping gene beta-actin were evaluated by Northern blot analysis.

Results.?Exposure of the trophoblasts to terbutaline (10^?8 M) inhibited the expression of the CRH gene as depicted by Northern blot analysis. Co-addition of terbutaline (10^?8 M) and RU486 (10^?6 M) did not block the stimulatory effects of RU486 on placental trophoblast cells.

Conclusion.?The beta-adrenergic agonist terbutaline inhibits the expression of CRH in human trophoblasts. This finding may provide insight into the mechanism of action of terbutaline as a tocolytic agent.     

Corticotropin releasing hormone modulates endotoxin-induced inflammatory cytokine expression in human trophoblast cells

Recent studies have suggested a significant increase in corticotropin releasing hormone (CRH) in maternal plasma and placenta during the course of maternal infection. The aim of this study was to examine the possible role of CRH in lipopolysaccharide (LPS)-induced pro-inflammatory cytokine expression using the well-established human extravillous trophoblast cell line HTR-8/SVneo. Exposure of the HTR-8/SVneo cells to LPS resulted in increased secretion of tumour necrosis factor alpha (TNF-alpha) and interleukin (IL)-8. Pre-treatment of the cells with CRH prior to LPS exposure significantly enhanced LPS induced TNF-alpha and IL-8 secretion. This effect was inhibited by the CRH antagonist astressin. Stimulation of the cells with CRH caused a rapid and transient phosphorylation of p38/MAPK while CRH had no effect on ERK1/2 activation. The effect of CRH on p38/MAPK activation was suppressed by astressin and by the p38/MAPK inhibitor SB203580. Exposure of the cells to CRH resulted in increased expression of TLR-4 and this effect was also inhibited by astressin. Taken together, these findings suggest that CRH augments LPS induced cytokine secretion in human trophoblast cells. Modulation of LPS induced immune responses by CRH may be mediated through regulation of TLR-4 and selective activation of the p38/MAPK signalling pathway.     

Wnt2在植入胎盘组织中的表达及其对滋养细胞迁移和侵袭功能的影响

目的检测Wnt2在植入胎盘组织中的表达,分析Wnt2对滋养细胞凋亡、迁移和侵袭功能的影响。方法收集2020年7月至2021年1月于武汉大学中南医院妇产科行剖宫产的胎盘植入孕产妇胎盘组织15例(植入组)和正常孕产妇胎盘组织15例(对照组)。RT-PCR法和Western blot法检测胎盘组织中Wnt2表达水平,采用siRNA敲低HTR-8/Svneo细胞中Wnt2表达水平,流式细胞学法检测细胞凋亡,Transwell侵袭和迁移实验评估细胞的迁移和侵袭能力,RT-PCR法和Western blot法检测β-catenin、Bcl-2、Caspase-3、MMP-2和MMP-9表达水平。结果与对照组相比,植入组胎盘组织中Wnt2、MMP-2和MMP-9 mRNA和蛋白表达量均显著升高(均P<0.05)。转染敲低HTR-8/Svneo细胞Wnt2水平后,细胞凋亡率显著增加(P<0.05),细胞迁移和侵袭能力显著抑制(P<0.01),β-catenin、Bcl-2、MMP-2和MMP-9表达显著下降(均P<0.05),而Caspase-3表达显著升高(P<0.05)。结论植入胎盘组织中Wnt2蛋白表达显著升高,且Wnt2可能通过wnt/β-catenin信号通路参与了胎盘滋养细胞的凋亡、迁移和侵袭。     

Selective cholesteryl ester uptake from high density lipoprotein by human first trimester and term villous trophoblast cells

As villous trophoblast does represent the contact zone between foetal and maternal tissues, the present in vitro study was aimed at investigating cholesterol supply from human high density lipoprotein subclass 3 (HDL(3)) to trophoblast cells isolated from human first trimester and term placenta. Binding of (125)I-HDL(3) was specific and saturable with similar K(d)-values for first trimester (54 microg HDL(3)-protein/ml) and term villous trophoblast cells (29 microg HDL(3)-protein/ml). The cell-association of (125)I-HDL(3) was 3-fold higher for term trophoblast cells while the specific cell-association of [(3)H]cholesterol ester(CE)-labelled HDL(3) was higher for first trimester trophoblast preparations. As a consequence, first trimester trophoblast cells have a pronounced capacity for selective CE-uptake from HDL(3). Competition experiments with native and oxidized low-density lipoprotein as well as cAMP-mediated stimulation of cell-association of [(3)H]CE-HDL(3) in both trophoblast preparations suggested the scavenger receptor class B, type I (SR-BI) as a likely receptor mediating this pathway. SR-BI m RNA could be identified by RT-PCR and Northern blot experiments in both trophoblast preparations. Western blot analysis and immunocytochemistry revealed high expression of SR-BI in first trimester trophoblast. A polyclonal antiserum raised against murine SR-BI significantly decreased cell-association of [(3)H]CE-HDL(3) in trophoblast cells. We conclude that human first trimester and term trophoblast cells express SR-BI which could serve as an efficient route for supplying cholesterol esters from maternal lipoproteins to foetal tissues.     

Terbutaline inhibits corticotropin-releasing hormone (CRH) expression in human trophoblast cells.

OBJECTIVE: To evaluate the effect of beta-adrenergic agonists on the regulation of the expression of the placental corticotropin-releasing hormone (CRH) gene. STUDY DESIGN: Term placentae were collected at the time of elective cesarean section, and trophoblast cells were harvested, isolated, and cultured. The isolated trophoblasts were plated, cultured and subsequently treated with cortisol, terbutaline, RU486, or vehicle control. CRH expression, mRNA abundance of CRH, and the housekeeping gene beta-actin were evaluated by Northern blot analysis. RESULTS: Exposure of the trophoblasts to terbutaline (10(-8) M) inhibited the expression of the CRH gene as depicted by Northern blot analysis. Co-addition of terbutaline (10(-8) M) and RU486 (10(-6) M) did not block the stimulatory effects of RU486 on placental trophoblast cells. CONCLUSION: The beta-adrenergic agonist terbutaline inhibits the expression of CRH in human trophoblasts. This finding may provide insight into the mechanism of action of terbutaline as a tocolytic agent.     

Effects of phorbol ester and forskolin on steroidogenesis and protein phosphorylation in cultured rat granulosa cells

Granulosa cells obtained from immature estradiol-treated SD rats (10 rats/experiment) were employed in elucidating the control mechanism of steroid secretion. Phorbol 12-myristate 13-acetate (PMA) inhibited estradiol production by cultured rat granulosa cells with IC50 less than 1 nM. PMA, however, stimulated small but significant increases in progesterone production in a dose-dependent manner with ED50 of 14 nM to 3.5-fold above the basal control level. These effects could not be induced by calcium ionophore A23187. Forskolin-stimulated progesterone production was inhibited by the concomitant addition of PMA with IC50 less than 1 nM. The phosphorylation of proteins by [32P] orthophosphate-labelled cells was examined by two-dimensional polyacrylamide gel electrophoresis and autoradiography. Treatment of cells with forskolin altered the intensity of 40 kDa acidic phosphoprotein compared to that of the control. On the other hand, treatment of cells with PMA altered the intensity of 78 and 32 kDa acidic phosphoproteins. These results suggest, therefore, that PMA can modulate steroidogenesis in rat granulosa cells, presumably through activation of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C).     

Alpha-fetoprotein gene expression in early and full-term human trophoblast

Alpha-fetoprotein (AFP) is a major serum glycoprotein synthesized during fetal life mainly by the yolk sac and the fetal liver. At term, it reaches high concentrations in the maternal intervillous blood, which is in direct contact with the placental trophoblastic microvillous membrane, and this suggests the placental origin of the AFP at the fetal-maternal interface. We used several experimental approaches to investigate the expression of AFP gene and fetal protein production in early gestation and term placentas. RT-PCR and immunological studies clearly identified AFP messenger RNA and AFP protein in the placental villi from first trimester of pregnancy. The AFP gene was also expressed in highly purified cytotrophoblasts from early placentas, and enzymo-immunoassay showed that AFP protein was synthesized and secreted by early cytotrophoblasts. AFP was also detected in the cytoplasm of these cells by immuno-cytochemistry. However, none of these methods detected any expression of the AFP gene in full-term placental villi or in cultured trophoblasts.These findings demonstrate that both AFP mRNA and protein are present in trophoblastic cells early in pregnancy. The absence of AFP gene expression in term placental villi also suggests, that the AFP at the fetal-maternal interface is attributable to a notable transplacental passage of AFP from fetal blood in late pregnancy.