Progesterone and human placental lactogen inhibit leptin secretion on cultured trophoblast cells from human placentas at term.

The placenta is an important source of leptin production that contributes to the state of hyperleptinemia observed in pregnant women. Moreover, the synthesis of leptin and its receptors by syncytiotrophoblast cells suggests a potential paracrine or autocrine action of leptin in the placenta. In the present study we examined the effect of gestational hormones, human chorionic gonadotropin (hCG), human placental lactogen (hPL), progesterone and estradiol, on in vitro leptin release by human term trophoblast cells in culture. Placentas at term were obtained immediately after delivery from mothers with uncomplicated pregnancies. Leptin levels were measured by enzyme-linked immunosorbent assay in culture media of trophoblasts maintained in monolayer culture for 24, 48 and 72 h with different hormonal treatments or placebo. Treatment with hPL and progesterone led to a time- and dose-dependent decrease in leptin release that was statistically significant after 24 h, with a maximal effect after 72 h of incubation. In contrast, incubation with estradiol and hCG did not have exhibit any effect on leptin secretion at any of the doses and times assayed in this work. The results obtained in this study support that leptin can be considered a gestational hormone implied in the endocrine function of the placenta and that its secretion is at least partially regulated by steroid and peptidic reproductive hormones in trophoblast cells in vitro.     

Progesterone and human placental lactogen inhibit leptin secretion on cultured trophoblast cells from human placentas at term

The placenta is an important source of leptin production that contributes to the state of hyperleptinemia observed in pregnant women. Moreover, the synthesis of leptin and its receptors by syncytiotrophoblast cells suggests a potential paracrine or autocrine action of leptin in the placenta. In the present study we examined the effect of gestational hormones, human chorionic gonadotropin (hCG), human placental lactogen (hPL), progesterone and estradiol, on in vitro leptin release by human term trophoblast cells in culture. Placentas at term were obtained immediately after delivery from mothers with uncomplicated pregnancies. Leptin levels were measured by enzyme-linked immunosorbent assay in culture media of trophoblasts maintained in monolayer culture for 24, 48 and 72?h with different hormonal treatments or placebo. Treatment with hPL and progesterone led to a time- and dose-dependent decrease in leptin release that was statistically significant after 24?h, with a maximal effect after 72?h of incubation. In contrast, incubation with estradiol and hCG did not have exhibit any effect on leptin secretion at any of the doses and times assayed in this work. The results obtained in this study support that leptin can be considered a gestational hormone implied in the endocrine function of the placenta and that its secretion is at least partially regulated by steroid and peptidic reproductive hormones in trophoblast cells in vitro.     

A fibrin matrix modulates the proliferation, hormone secretion and morphologic differentiation of cultured human placental trophoblast.

Term placental trophoblast epithelialize fibrin deposits attached to villi in vitro and trophoblast cultured on a fibrin matrix form an epithelial bilayer typical of the trophoblast layer on term villi. We compared the morphology of cells grown on fibrin with cells grown on substrates of type IV collagen, laminin, type I collagen, or Matrigel. We also used autoradiography, hormone assays, electron microscopy, and immunofluorescence to determine what functional activities were influenced by trophoblast-fibrin interactions. Cultured cellular trophoblast from term placentae differentiated to form syncytial trophoblast and to secrete estrogen, progesterone, and hCG in the presence or absence of matrices. Trophoblast proliferation was lower in cells grown on matrices and was inversely related to cell height after 24 h in culture. Cells grown on fibrin remained the tallest and had the lowest labelling index. Cells grown for 72 h on fibrin had the most dilated rough endoplasmic reticulum but the lowest media hormone levels. Only cells grown on a fibrin matrix formed a basal lamina-like structure at the trophoblast-substrate interface, and only a fibrin matrix facilitated trophoblast to form an epithelial bilayer in culture. However, this histology was not accompanied by a change in the amount of syncytial trophoblast formed by the cells grown on fibrin. The results suggest that a fibrin matrix uniquely modulates the trophoblast phenotype, away from the secretion of placental specific products like hCG in favour of a repair-oriented phenotype that forms basement membrane and a trophoblast bilayer.     

Effects of human growth hormone upon term placental hormone secretion in vitro

The role of human growth hormone (hGH) on placental hormone secretion at term was investigated in two in vitro models: placental explants and cultured trophoblastic cells. Physiological concentrations of hGH caused a significant dose-dependent increase in placental lactogen and progesterone secretion. In the explant model it stimulated estradiol secretion. In order to determine whether this stimulatory effect on estradiol is exerted via aromatase, an isolated cell culture was utilized where androstenedione was supplied as substrate. In this model, hGH exerted a mild inhibitory effect. In conclusion, hGH at levels present in the fetal circulation exerts a significant stimulatory effect upon placental function as reflected by both peptide and steroid hormone production and secretion. The effect of estradiol secretion is the end result of an inhibitory effect on androgen aromatization and a stimulatory effect on earlier steps.     

Hormonal regulation of neonatal weight: placental leptin and leptin receptors

Objective To examine whether umbilical and maternal leptin levels correlate with birthweight, placental weight, and maternal weight; and to detect membrane-bound leptin receptors in placental tissue as well as soluble leptin receptors in umbilical and maternal blood.
Design Prospective observational study.
Setting University teaching hospital.
Methods Serum levels of leptin and soluble leptin receptors were analysed in 31 randomly selected mother/newborn pairs at delivery. In addition, placental tissue was assayed for leptin receptors using immunocytochemistry and Western blot.
Results The mean [SD] leptin level in umbilical cord venous blood (7.1 ng/mL [4.0]) was significantly lower (   P < 0.001  ) than in maternal blood (22.5 ng/mL [10.8]). Umbilical cord leptin concentrations correlated significantly with birthweight (   P < 0.001  ), placental weight (   P < 0.005  ) but not with maternal leptin. Maternal leptin concentrations correlated only with maternal weight (   P < 0.001  ). In chorionic villous tissue, trophoblasts stained strongly positive for leptin receptor-like immunoreactivity. Two membrane-bound isoforms of the leptin receptor were also detected in placental tissue. In both umbilical and maternal serum, a soluble leptin receptor was found migrating as broad band at M_r 97,000 D.
Conclusion The present data strongly reinforce the idea that circulating leptin levels may provide a growth-promoting signal for fetal development during late pregnancy. While membrane-bound leptin receptors may be involved in autocrine regulation of placental leptin production, the soluble receptor form may serve as a transport vehicle for leptin to fetal tissues.     

地塞米松对人类胎盘糖皮质激素代谢酶11β-HSD2的调节

目的:研究地塞米松对人类胎盘糖皮质激素受体前代谢酶11β-HSD2活性的调节作用,探讨产前应用地塞米松对妊娠和胎儿的影响,为临床防治早产和合理应用糖皮质激素提供理论依据。方法:利用改良的Kliman法分离提纯胎盘滋养细胞,原代培养3天后药物处理,用放射性酶活性结合薄层层析法(TLC)测定地塞米松对11β-HSD2氧化酶活性的调节作用。结果:体外实验条件下,培养的胎盘滋养层细胞具有11β-HSD2氧化酶活性,且与时间呈依赖关系。地塞米松作用于细胞4h、8h后,胎盘合体滋养细胞11β-HSD2的氧化酶活性明显降低,但孵育24h后,酶活性无明显变化。结论:地塞米松可显著降低胎盘合体滋养细胞11β-HSD2的氧化酶活性,可能是地塞米松、对胎儿有不良影响的重要机制之一,因此产前应用地塞米松必须慎重。     

Influence of serotonin on progesterone and estradiol secretion of cultured human granulosa cells.

OBJECTIVE: To explore the direct action of serotonin on progesterone (P) and estradiol (E2) secretion of human granulosa cells cultured in serum-free medium. DESIGN: Progesterone and E2 production was measured in the presence and absence of serotonin, propranolol, or cycloheximide using radioimmunoassays; statistical analysis of the data was performed by ANOVA. SETTING: In vitro fertilization and embryo transfer (IVF-ET) for infertility treatment at the University Women's Hospital, University of Tübingen, Germany. PATIENTS, PARTICIPANTS: Fourteen women, 30 +/- 3 years old, undergoing IVF-ET. RESULTS: Serotonin stimulated a dose-related increase in P secretion with a maximal stimulatory effect at 10(-4) M. This response was blocked specifically by the beta-receptor antagonist propranolol (10(-4) M). Estradiol secretion in response to serotonin was dose-independent stimulation, which was highest at 10(-6) M and was inhibited by 10(-4) M propranolol. The protein synthesis inhibitor cycloheximide markedly reduced the stimulatory effect of serotonin on P secretion. Estradiol production in the presence of cycloheximide was significantly reduced; serotonin had no stimulatory effect under these conditions. CONCLUSION: Serotonin may have a physiological role in the corpus hemorrhagicum, when luteinization is initiated.     

Effect of human corticotropin-releasing hormone on gonadotropin secretion in cycling and postmenopausal women.

OBJECTIVE: To test the hypothesis that corticotropin-releasing hormone (CRH) is linked to stress-associated reproductive dysfunction in the human by determining if the administration of human corticotropin-releasing hormone (hCRH) results in an inhibition of gonadotropin secretion. DESIGN: Twenty-four-hour prospective study with frequent (every 10 minutes) blood sampling. SETTING: University Clinical Research Center. INTERVENTIONS: Sequential 8-hour infusions of normal saline, hCRH (1 to 5 micrograms/kg per hour), and hCRH plus naloxone (2 mg/h). SUBJECTS: Four normal cycling women and four postmenopausal women. MAIN OUTCOME MEASURES: Plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PRL), and adrenal and ovarian steroids. RESULTS: In response to hCRH, a prompt and sustained rise in cortisol (F) was noted in both normal cycling women and postmenopausal women. No inhibition of LH or FSH was noted during either the hCRH or hCRH plus naloxone infusion in either group of women. Unexpectedly, elevations in the mean LH peak amplitude and the transverse mean LH concentration were noted in the postmenopausal women during the infusion of hCRH as compared with saline. The infusion of hCRH had no apparent effect on concentrations of PRL, FSH, and gonadal and adrenal steroids (except for F). CONCLUSIONS: Under these conditions, intravenously administered hCRH has no inhibitory effect on gonadotropin secretion in either premenopausal or postmenopausal women. The mechanism by which stress exerts its deleterious effect on reproductive function in the human remains unknown.     

瘦素对人子宫内膜癌细胞雌激素原位合成的影响

目的探讨瘦素对子宫内膜癌(EC)细胞中芳香化酶表达和原位雌激素合成的影响,以及瘦素在子宫内膜癌发病机制中的作用。方法 2009年4月至2010年6月在辽宁医学院附属第一医院采用EC间质细胞与Ishikawa细胞共培养模式;MTT法检测瘦素对EC细胞增殖的影响;RT-PCR法及免疫印迹法分别检测间质细胞和Ishikawa细胞中芳香化酶mRNA及蛋白的表达,并观察瘦素对共培养细胞中芳香化酶表达的影响;放免法检测瘦素对共培养细胞生成雌二醇的影响。结果瘦素明显促进共培养的子宫内膜癌间质细胞和Ishikawa细胞的增殖,在0～100μg/L范围内呈剂量依赖性,100μg/L时增殖效应最大,自(121.14±5.50)%至(274.24±6.03)%;芳香化酶在间质细胞中的表达水平高于Ishikawa细胞,瘦素增加EC细胞芳香化酶的表达及雌激素的合成,当100μg/L瘦素时,E2(1.35±0.17)pmol/L,当加入100μg/L瘦素+1μmol/L来曲唑时E2(0.47±0.18)pmol/L,来曲唑可抑制此作用。结论瘦素可能通过促进EC细胞芳香化酶的表达,增加原位雌激素合成进而刺激EC细胞增殖。     

Histologic characteristics of cells cultured from rat placental tissue.

Laboratory animals, especially rats, have provided a comparative model for extensive research on maternal-fetal relationships. To identify individual placental cells for subsequent immunologic analysis, cultures of rat placentas were studied. Five cell types were observed, including: two histologically distinct giant cells, mononucleate and multinucleate, apparently formed by amitosis and fusion, respectively; small round cells; polygonal cells, probably cytotrophoblasts. Of particular interest are the small round cells which are phagocytic and morphologically similar to macrophages, cells known to be important in the immune response.     

GnRH-a体外对人卵巢黄素化颗粒细胞雌二醇和孕酮分泌量的影响

目的 :观察促性腺素释放激素激动剂 (GnRH -a)在体外对人卵巢黄素化颗粒细胞雌二醇 (E2 )和孕酮 (P)分泌量的影响。方法 :培养人卵巢黄素化颗粒细胞 ,分别用终浓度为 1.0、10 .0、10 0 .0ng ml的GnRH -a刺激 ,同时设对照组 ,培养时间为 2、4、6d。用放射免疫法检测黄素化颗粒细胞E2 和P的分泌量。结果 :培养 2d ,GnRH -a中、高浓度组E2 和P分泌量分别为 (12 2± 37) %、(12 8± 2 4 ) % ;(143± 32 ) %、(137± 2 9) %对照组为10 0 % ,均显著高于对照组 (P <0 .0 5) ;低浓度组E2 和P分泌量与对照组差异无显著性。随着细胞培养天数的增加 ,GnRH -a中、高浓度组E2 分泌量明显低于对照组 ,高浓度组E2 分泌量与培养天数呈负相关 (r =- 0 .75,P <0 .0 5)。结论 :GnRH -a对黄素化颗粒细胞分泌甾体激素功能的影响随着作用时间和浓度的不同而变化     

Effects of cyclic adenosine monophosphate on human chorionic gonadotropin and estradiol output by cultured human placental cells

We have previously shown that adenosine-3',5'-cyclic monophosphate (cAMP) inhibits basal estradiol output in human trophoblast cells. The objective of this study was to investigate further the effect of 8-bromo-cAMP on the conversion of C19 androgens to estradiol by placental cells. Trophoblast cells were prepared from human term placenta and maintained in monolayer culture. On days 3 and 4 of culture, these cells were treated with dehydroepiandrosterone sulfate, androstenedione, or testosterone with or without the concomitant presence of 8-Br-cAMP. 8-Br-cAMP markedly enhanced human chorionic gonadotropin secretion into the culture medium. On the other hand, the concomitant addition of 8-Br-cAMP with the androgen precursors led to an inhibition of estradiol output. The concentrations of androstenedione and dehydroepiandrosterone in the culture medium after treatment with dehydroepiandrosterone sulfate were elevated by the concomitant presence of 8-Br-cAMP. From these results we conclude that 8-Br-cAMP enhances human chorionic gonadotropin output in human term placental cells, whereas the presence of 8-Br-cAMP in cells given androgen precursors inhibits estradiol output, probably at the level of aromatization.     

Effects of diacylglycerol and gonadotropin-releasing hormone on human chorionic gonadotropin release by cultured trophoblast cells.

The effect of activation of calcium- and phospholipid-dependent protein kinase (protein kinase C) on human chorionic gonadotropin (hCG) release by cultured trophoblast cells was studied and a role of protein kinase C in the GnRH-mediated hCG release was also evaluated. Both GnRH and 1-oleoyl-2-acetylglycerol (OAG), a protein kinase C activator, stimulated hCG release after 3 h incubation in a dose-dependent manner with ED50 of 55 nmol/l and 4.0 nmol/l, respectively. A tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) also stimulated hCG release while two non-tumor-promoting compounds, phorbol and 4 alpha-phorbol, failed to stimulate hCG release. hCG release by maximal effective dose of GnRH (10 mumol/l) or OAG (1 mumol/l) was further stimulated when cells were incubated with same concentrations of GnRH and OAG. OAG-stimulated hCG release was completely inhibited by a protein kinase C inhibitor, H-7, with ID50 of 23 nmol/l while H-7 did not affect GnRH-mediated hCG release. These results indicate that GnRH-stimulated hCG release is not mediated by protein kinase C pathway, however, the secretion of hCG is also regulated by the mechanism that involves protein kinase C activation.