The functional significance of the endometrial hCG/ LH receptors has been related to a rapid release of prostaglandins. However, as compared to gonads and myometrium, in-endometrium mechanisms of transmembrane signall...The functional significance of the endometrial hCG/ LH receptors has been related to a rapid release of prostaglandins. However, as compared to gonads and myometrium, in-endometrium mechanisms of transmembrane signalling of the hCG/LH receptors are probably not conventional and remain unclear. Here we investigated, in vivo, the potential of hCG to interact with, and stimulate the membrane effector enzyme, adenylyl cyclase (AC), in human endometrium. Hormonal and nonhormonal activation of AC was tested in membrane fractions prepared from endometrial biopsies obtained from patients undergoing evaluation cycles for hormone replacement therapy (HRT) and controlled ovarian hyperstimulation (COH). AC activity was determined by the direct conversion of the substrate ATP into cAMP under unstimulated conditions and in the presence of the non-hormonal activators guanyl nucleotide and forskolin. Also AC activity was tested in the presence of hCG under conditions allowing maximal enzyme stimulation. Isoproterenol and prostaglandin E2 (PGE2) were included for comparison. Immunoblot analyses demonstrated the presence of hCG/LH receptors and Gsα protein and other members of the G protein family in the membrane fractions. Endometrial membranes also exhibited high levels of AC activity compared to luteal membranes used as control. Stimulation by GMP-P(NH)P alone was 196 ± 63 (n = 8) (pmol/mg/ min ± SD). Neither hCG nor isoproterenol showed stimulation of endometrial AC (210 ± 65, and 197 ± 53, respectively;n = 66 assays). But PGE2 stimulated the enzyme system significantly (264 ± 63, p < 0.05;n = 66 assays). These data show that membrane fractions from human endometrium express all the AC system components, namely, hCG/LH receptors, Gsα protein and AC;however, hCG does not stimulate the endometrial AC system. Our data indicate that, in great contrast to gonadal receptors, endometrial hCG/ LH receptors are not coupled to the transmembrane AC effector. The well known release of eicosanoids in response to hCG suggests that these receptors are functional in human endometrium but throughout a signalling system different from AC. This enzyme is certainly coupled to and directly activated by eicosanoids and other embryonic signals.展开更多
目的:探讨人子宫内膜细胞体外培养模型中,HCG对VEGF表达的调控作用和机制。方法:采用机械分离加梯度离心法分别获得腺上皮与基质细胞进行体外原代细胞混合培养;实验设对照组、HCG组(对照组使用常规DF/12培养液提取总蛋白;HCG组在DF/12...目的:探讨人子宫内膜细胞体外培养模型中,HCG对VEGF表达的调控作用和机制。方法:采用机械分离加梯度离心法分别获得腺上皮与基质细胞进行体外原代细胞混合培养;实验设对照组、HCG组(对照组使用常规DF/12培养液提取总蛋白;HCG组在DF/12培养液中加终浓度为20U/ml HCG)。采用Q-PCR测定子宫内膜细胞中VEGF mRNA表达水平。免疫组化或荧光法进行LH-R和VEGF蛋白质定位表达,Western blot法测定蛋白半定量表达。结果:子宫内膜腺上皮与基质细胞均存在LH/HCG-R和VEGF蛋白表达。HCG对子宫内膜细胞VEGF蛋白的峰值作用时间点为24h。与对照组相比,20IU/ml HCG体外干预24h后,子宫内膜细胞VEGF mRNA和蛋白质水平均显著上调(Q-PCR:1 vs 2.0132±0.6024;WB:0.4069±0.1897 vs 0.7781±0.1651)(P均<0.05)。结论:成功建立了人子宫内膜细的体外培养模型,并完成HCG的干预实验。HCG与HCG/LH-R结合后上调子宫内膜细胞VEGF的基因和蛋白质水平表达。展开更多
文摘The functional significance of the endometrial hCG/ LH receptors has been related to a rapid release of prostaglandins. However, as compared to gonads and myometrium, in-endometrium mechanisms of transmembrane signalling of the hCG/LH receptors are probably not conventional and remain unclear. Here we investigated, in vivo, the potential of hCG to interact with, and stimulate the membrane effector enzyme, adenylyl cyclase (AC), in human endometrium. Hormonal and nonhormonal activation of AC was tested in membrane fractions prepared from endometrial biopsies obtained from patients undergoing evaluation cycles for hormone replacement therapy (HRT) and controlled ovarian hyperstimulation (COH). AC activity was determined by the direct conversion of the substrate ATP into cAMP under unstimulated conditions and in the presence of the non-hormonal activators guanyl nucleotide and forskolin. Also AC activity was tested in the presence of hCG under conditions allowing maximal enzyme stimulation. Isoproterenol and prostaglandin E2 (PGE2) were included for comparison. Immunoblot analyses demonstrated the presence of hCG/LH receptors and Gsα protein and other members of the G protein family in the membrane fractions. Endometrial membranes also exhibited high levels of AC activity compared to luteal membranes used as control. Stimulation by GMP-P(NH)P alone was 196 ± 63 (n = 8) (pmol/mg/ min ± SD). Neither hCG nor isoproterenol showed stimulation of endometrial AC (210 ± 65, and 197 ± 53, respectively;n = 66 assays). But PGE2 stimulated the enzyme system significantly (264 ± 63, p < 0.05;n = 66 assays). These data show that membrane fractions from human endometrium express all the AC system components, namely, hCG/LH receptors, Gsα protein and AC;however, hCG does not stimulate the endometrial AC system. Our data indicate that, in great contrast to gonadal receptors, endometrial hCG/ LH receptors are not coupled to the transmembrane AC effector. The well known release of eicosanoids in response to hCG suggests that these receptors are functional in human endometrium but throughout a signalling system different from AC. This enzyme is certainly coupled to and directly activated by eicosanoids and other embryonic signals.
文摘目的:探讨人子宫内膜细胞体外培养模型中,HCG对VEGF表达的调控作用和机制。方法:采用机械分离加梯度离心法分别获得腺上皮与基质细胞进行体外原代细胞混合培养;实验设对照组、HCG组(对照组使用常规DF/12培养液提取总蛋白;HCG组在DF/12培养液中加终浓度为20U/ml HCG)。采用Q-PCR测定子宫内膜细胞中VEGF mRNA表达水平。免疫组化或荧光法进行LH-R和VEGF蛋白质定位表达,Western blot法测定蛋白半定量表达。结果:子宫内膜腺上皮与基质细胞均存在LH/HCG-R和VEGF蛋白表达。HCG对子宫内膜细胞VEGF蛋白的峰值作用时间点为24h。与对照组相比,20IU/ml HCG体外干预24h后,子宫内膜细胞VEGF mRNA和蛋白质水平均显著上调(Q-PCR:1 vs 2.0132±0.6024;WB:0.4069±0.1897 vs 0.7781±0.1651)(P均<0.05)。结论:成功建立了人子宫内膜细的体外培养模型,并完成HCG的干预实验。HCG与HCG/LH-R结合后上调子宫内膜细胞VEGF的基因和蛋白质水平表达。
