p38丝裂原活化蛋白激酶在小鼠围植入期子宫内膜及胚胎中的表达及作用
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摘要
目的:
胚胎植入是指在特定的植入窗口期,胚泡附着并侵入子宫内膜的全过程。在这个复杂的过程中,激素、细胞因子、黏附分子、蛋白、肽及酶类等介导了一系列分子生物学事件,在这些因素的精细调控下,某些细胞内信号转导通路被激活,调控特定基因的表达,使胚泡滋养层和子宫内膜发生同步协调变化,从而使得发育正常且具有侵入性的胚泡顺利植入。
近年来,尽管关于胚胎植入机理的研究取得了长足的进展,但仍存在许多问题亟待深入探讨。如在植入过程中哪些信号转导通路被激活?激活这些信号转导通路的信号分子来自母体还是来自胚胎、或是两者共同作用?各通路间的关系如何?等等。
丝裂原活化蛋白激酶(Mitogen-Activated Protein Kinase,MAPK)通路是真核细胞调节增殖、分化、凋亡及细胞间功能同步化的基本信号通路。目前,至少鉴定出了4种MAPK超家族成员,包括细胞外信号调节蛋白激酶(extracellularsignal-regulated kinases,ERK)、p38MAPK、C-jun氨基末端/应激活化蛋白激酶(thec-Jun N-terminal kinase or stress-activated protein kinases,JNK/SAPK)和大丝裂原活化蛋白激酶1(big MAP kinase 1,BMK1/ERK5)通路。这些蛋白及酶构成相互交织的信号级联通路,在细胞内信号传递中起重要作用。
近几年国外研究发现,MAPK通路在早期胚胎发育、子宫内膜蜕膜化、胚泡黏附、滋养层细胞增殖以及侵入子宫内膜过程中都起着重要作用。其中p38MAPK信号通路参与了细胞的生长发育及细胞间功能同步等多种生理过程,并与炎症、应激反应的调控密切相关,被认为是细胞信息传递的中枢。最近又有实验显示,p38MAPK在小鼠植入前胚胎中激活,且其激活是子宫内膜蜕膜化过程中所必需的,但国内尚未见这方面的报道。关于围植入期子宫内膜中p38MAPK的表达状况及在体注射p38MAPK特异性抑制剂干扰植入的研究国内外尚未见报道。
本实验以妊娠昆明小鼠为动物模型,用免疫组化法检测围植入期胚胎和子宫内膜中p38MAPK的表达及其变化规律;用动物在体宫腔内注射p38MAPK特异性抑制剂和体外胚胎培养技术,观察p38MAPK对早胚发育及植入的影响,从而探讨p38MAPK与胚胎植入的相关关系,以进一步揭示植入的机制,为提高体外受精-胚胎移植(IVF-ET)等辅助生殖技术的成功率以及研究和开发新型抗植入避孕药物提供理论依据和动物模型。
研究方法:
1.取真孕及假孕1～8天小鼠子宫,石蜡包埋切片,用免疫组化及图象分析法检测围植入期子宫内膜中p38MAPK的表达及变化规律,同时取未孕动情期子宫作为对照组。
2.取未经产雌性小鼠常规促排卵,收集2细胞,4细胞,8细胞,桑葚胚,胚泡等不同发育阶段的早胚,用免疫组化及免疫荧光法测定早胚中p38MAPK的表达及变化情况。
3.p38MAPK抑制剂干预实验
体内实验:取正常妊娠第4天小鼠,剖腹在两侧子宫角处分别注射等剂量的p38MAPK特异性抑制剂和生理盐水,于妊娠第8天颈椎脱臼处死小鼠,检查植入胚胎数。同时取子宫作切片,观察胚胎及子宫内膜的组织学变化。
体外实验:取经过PMSG、HCG超排卵后孕2天小鼠,分离输卵管,获取2细胞期胚卵,体外培养。将胚卵随机分为5组,接种到添加不同成分的培养板中培养。组Ⅰ仅含基础培养液(阴性对照);组Ⅱ含基础培养液和DMSO(溶剂对照);组Ⅲ、组Ⅳ、组Ⅴ的基础培养液内含不同浓度的p38MAPK特异性抑制剂SB203580,其剂量分别为:0.2μmol/L;2μmol/L;20μmol/L。将培养板置37℃、5%CO_2、100%湿度的培养箱内培养。每12小时观察一次,看其发育情况。48小时后观察其发育到胚泡的情况,用台盼蓝检测胚胎细胞存活率,用免疫荧光法检测各组胚泡中p38MAPK的表达情况。
结果:
1.在小鼠子宫内膜中,p38MAPK主要表达于腔上皮、腺上皮和蜕膜细胞的胞浆内,偶尔在胞核中。真孕组孕3d,腔上皮中p38MAPK呈阳性表达,孕4～5天呈强阳性表达,此后p38MAPK在腔上皮的表达逐渐减弱;腺上皮中p38MAPK的表达从孕4天开始一直维持在阳性水平,从孕5d开始蜕膜细胞中p38MAPK的表达逐渐增强。假孕组只有孕4天呈弱阳性表达。
2.p38MAPK在小鼠胚植入前各细胞期均呈阳性表达,随着妊娠进展,其表达逐渐加强。
3.p38MAPK抑制剂干预实验
体内实验显示,抑制剂组(10μM)平均胚胎植入数为2.6250±0.5175,明显低于空白对照(7.1250±0.8345)和生理盐水组(6.2500±0.8864),与两者相比,差异十分显著(P＜0.01)。组织学观察结果:抑制剂组胚胎组织呈退化坏死状态,胚胎周围蜕膜细胞呈明显的空泡样变性,蜕膜及胚胎中均可见大量血细胞浸润。
体外实验显示,抑制剂组与对照组胚卵以同样的速率发育到8细胞阶段,但是抑制剂组的胚卵发育却停滞在8-16细胞之间,不能发育到胚泡阶段,比对照组发育晚1-2个细胞期。但抑制剂组胚卵仍具存活能力,将其移入无抑制剂的培养基中,它们仍能发育到胚泡阶段。
结论:
1.p38MAPK在小鼠植入前胚胎发育过程中起作用。
2.p38MAPK参与胚泡植入过程,并可能间接参与植入后的胚泡滋养层扩展、分化及子宫内膜蜕膜化反应。
3.p38MAPK在子宫内膜中的表达不仅受母体因素的调控,还可能与胚泡的刺激有关。
4.p38MAPK特异性抑制剂可抑制在体小鼠胚泡植入及植入后胚胎和蜕膜细胞的发育。
Objective:
Implantation is a key event of the human and mammals reproductive process,it is a complicated process involving the blastocyst adheres to and invades the maternal endometrium gradually at the specific step of implantation window.In the complicated process,a number of factors mediate a series of molecular biology events which include steroid hormones(estrogen,progesterone),growth factors,cytokines,adhesion molecules,proteinums,peptides and enzymes.At the subtle regulation of these factors,some intracellular signal conducting pathways were activated,and regulate the expression of special genes,to coordinate the development of embryo trophblast and the maternal endometrium, accordingly to make the well-developed blastocyst invade the endometrium successfully.
Recently,although much progress on the mechanism of blastocyst implantation have been made,many key questions are still to be further explored.For example,what intracellular signal conductiong pathways were activated? The signals activating these conductiong pathways come from maternal or embryonic body,or from both ? How is a relationship between various intracellular signal conductiong pathways during implantation? and etc.
MAPK pathways mediate some cellular processes,including cell proliferation,growth,differentiation,apoptosis and death.
The MAPK superfamily of proteins consists of four separate signaling cascades:the c-Jun N-terminal kinase or stress-activated protein kinases (JNK/SAPKs),the extracellular signal-regulated kinases(ERKs),the ERK5 or big MAP kinase 1,and the p38 MAPK group of protein kinases,all of which are highly conserved throughout eukaryotic systems.These proteins and enzymes make a signaling cascades which is interlace each other, and play an important role in the intracellular signal transmission.
In recent years,studies oversea showed that MAPK pathway play important roles in many process such as embroygenesis,uterus decidualization,blastocyst adhesion,trophoblastic cell proliferation and invades the maternal endometrium.
The p38 MAPK signaling cascade regulates a variety of cellular activities in different cells and tissue types.It is influenced by a variety of stimulus including growth factors,cytokines,and pathogens. So it is considered as the center of the intracellular signal transmission.
Lately,there was study showing that p38MAPK is activated in mammalian perimplantation development,and its activation is required by uterus decidualization,This idea is not yet reported in domestic literature. Studies both about the expression of p38MAPK in peri-implantation endometrium and injection inhibitor SB203580(specific inhibitors of p38 MAPKα/β)in vivo to interfere with implantation are not reported at home and abroad.
To further explore the effect of p38 MAPK on mouse embryo development and implantation,the methods including immunohistochemistry,embryo culture in vitro and intrauterine injection in vivo were used in our experiments in order to further understand the mechanism of implantation, enrich the theory of implantation in reproductive biology,facilitate the successful rate of assisted reproductive technology such as IVF-ET and offer reliable experimental evidence and animal models for developing new types of contraceptives.
Methods:
1.Pregnant and pseudopregnant uterus of 1～8 days in mice were obtained.The specimens were sliced into section(of 5um) Immunohistochemistry method and image analysis were employed to explore the expression and change rules of p38MAPK in peri-implantation mouse endometrium,uterus of nonpregnancy as control.
2.Early embryos were obtained at the following stages:two-cell, four-cell,eight-cell,morulae and blastocyst.The expression and changes of p38MAPK in early embryos were examined by immunohistochemistry and immunofluorescence.
3.Interfering experiment by p38MAPK inhibitors
In vivo:
Mice were obtained on day 4 of pregnant,intrauterine in jection of p38 MAPK inhibitors were used and morphological and histological methods were employed to observe the embryo number of implantation on day 8 and the variances in the embryo and the endometrium.
In vitro:
Murine embryos were flushed from oviducts of timed pregnant mice at the two-cell stage(48 h post-hCG),pooled,washed,and divided into five groups.They were cultured with different culture treatments:①1:1DMEM/F12 alone,②1:1DMEM/F12 + DMSO(inactive analogue),③1:1DMEM/F12 + 0.2μmol/L SB203580,④1:1DMEM/F12 + 2.0μmol/L SB203580, and⑤1:1DMEM/F12 + 20μmol/L SB203580.Embryos were assessed for morphology and progression through cleavage divisions at 24 h postdrug treatment(72 h post-hCG),at which point half of the embryos in groups 3,4,and 5 were removed from culture,washed,and placed in fresh medium culture drops for the remainder of the experiment.All embryos were then assessed at 48 h postdrug treatment(96 h post-hCG)to observe the developmental information(from cleavage to blastocyst).Cell viability and cell numbers were determined at this time by assaying for uptake of a vital dye(trypan blue).The expression of p38MAPK in blastocysts of each treated group were examined by immunofluorescence.
Results:
1.The p38MAPK-positive staining was found in the cytoplasm of the lumina epithelium,glandular epithelium and decidual cell.In the pregnant group,p38MAPK-positive expression in lumina epithelium was weak at 3 days and peak at 4～5 days in the endometrium of pregnant mouse, and then declined:in glandular epithelium,p38MAPK-positive expression is observed from 4~(th)day and then keep at this level,p38MAPK-positive expression in decidual cell is increased gradually from the 5~(th)day of gestation.In the pseudopregnant group,weak staining of p38MAPK was only observed on the 4~(th)day of gestation,and negative staining were observed on the other days.p38MAPK was expressed weakly in the endometrium of nonpregnant mouse.
2.The p38MAPK-positive staining was found in the pre-implanted embryos and gradually increased with development;
3.Interfering experiments:
In vivo:
The average of embryo-implantation(2.6250±0.5175)in p38MAPK inhibitor(10μM)treated uterus were significantly lower than that of control uterus given saline solution(6.2500±0.8864),P＜0.01.The microscopic observation demonstrated that the embryo degenerated,and even necrosed in some parts.In the decidual cells there was obvious adipose degeneration,while many blood cells invaded the embryos and the decidua.
In vitro:
Two-cell stage embryos cultured in the presence of SB203580(specific inhibitors of p38 MAPKα/β),progressed to the eight-cell stage with the same frequency as controls;however,treated embryos halted their development at the 8- to 16-cell stage.In inhibitor-treated groups,the embryos were composed of a mixture of compacting and noncompacting cells, and the embryo development were delayed for one to two cell cycle than that of controls.Treated embryos remained viable as the developmental blockade was rescued by removing embryos from the drug treatment and placing them in drug-free medium until they progressed to the blastocyst stage.
Conclusions:
1.p38MAPK activity may be required to support embryo development through the murine preimplantation interval.
2.p38MAPK,in addition to correlating to blastocyst implantation,may participate indirectly in the process of endometrial decidualization.
3.The expression of p38MAPK in mouse endometrium is regulated not only by maternal factors(steroid hormones),but also by the implanted embryos.
4.p38MAPK inhibitors can inhibit the blastocyst implantation and the development of embryos and decidual cells in vivo.
胚胎植入是指在特定的植入窗口期,胚泡附着并侵入子宫内膜的全过程。在这个复杂的过程中,激素、细胞因子、黏附分子、蛋白、肽及酶类等介导了一系列分子生物学事件,在这些因素的精细调控下,某些细胞内信号转导通路被激活,调控特定基因的表达,使胚泡滋养层和子宫内膜发生同步协调变化,从而使得发育正常且具有侵入性的胚泡顺利植入。
近年来,尽管关于胚胎植入机理的研究取得了长足的进展,但仍存在许多问题亟待深入探讨。如在植入过程中哪些信号转导通路被激活?激活这些信号转导通路的信号分子来自母体还是来自胚胎、或是两者共同作用?各通路间的关系如何?等等。
丝裂原活化蛋白激酶(Mitogen-Activated Protein Kinase,MAPK)通路是真核细胞调节增殖、分化、凋亡及细胞间功能同步化的基本信号通路。目前,至少鉴定出了4种MAPK超家族成员,包括细胞外信号调节蛋白激酶(extracellularsignal-regulated kinases,ERK)、p38MAPK、C-jun氨基末端/应激活化蛋白激酶(thec-Jun N-terminal kinase or stress-activated protein kinases,JNK/SAPK)和大丝裂原活化蛋白激酶1(big MAP kinase 1,BMK1/ERK5)通路。这些蛋白及酶构成相互交织的信号级联通路,在细胞内信号传递中起重要作用。
近几年国外研究发现,MAPK通路在早期胚胎发育、子宫内膜蜕膜化、胚泡黏附、滋养层细胞增殖以及侵入子宫内膜过程中都起着重要作用。其中p38MAPK信号通路参与了细胞的生长发育及细胞间功能同步等多种生理过程,并与炎症、应激反应的调控密切相关,被认为是细胞信息传递的中枢。最近又有实验显示,p38MAPK在小鼠植入前胚胎中激活,且其激活是子宫内膜蜕膜化过程中所必需的,但国内尚未见这方面的报道。关于围植入期子宫内膜中p38MAPK的表达状况及在体注射p38MAPK特异性抑制剂干扰植入的研究国内外尚未见报道。
本实验以妊娠昆明小鼠为动物模型,用免疫组化法检测围植入期胚胎和子宫内膜中p38MAPK的表达及其变化规律;用动物在体宫腔内注射p38MAPK特异性抑制剂和体外胚胎培养技术,观察p38MAPK对早胚发育及植入的影响,从而探讨p38MAPK与胚胎植入的相关关系,以进一步揭示植入的机制,为提高体外受精-胚胎移植(IVF-ET)等辅助生殖技术的成功率以及研究和开发新型抗植入避孕药物提供理论依据和动物模型。
研究方法:
1.取真孕及假孕1～8天小鼠子宫,石蜡包埋切片,用免疫组化及图象分析法检测围植入期子宫内膜中p38MAPK的表达及变化规律,同时取未孕动情期子宫作为对照组。
2.取未经产雌性小鼠常规促排卵,收集2细胞,4细胞,8细胞,桑葚胚,胚泡等不同发育阶段的早胚,用免疫组化及免疫荧光法测定早胚中p38MAPK的表达及变化情况。
3.p38MAPK抑制剂干预实验
体内实验:取正常妊娠第4天小鼠,剖腹在两侧子宫角处分别注射等剂量的p38MAPK特异性抑制剂和生理盐水,于妊娠第8天颈椎脱臼处死小鼠,检查植入胚胎数。同时取子宫作切片,观察胚胎及子宫内膜的组织学变化。
体外实验:取经过PMSG、HCG超排卵后孕2天小鼠,分离输卵管,获取2细胞期胚卵,体外培养。将胚卵随机分为5组,接种到添加不同成分的培养板中培养。组Ⅰ仅含基础培养液(阴性对照);组Ⅱ含基础培养液和DMSO(溶剂对照);组Ⅲ、组Ⅳ、组Ⅴ的基础培养液内含不同浓度的p38MAPK特异性抑制剂SB203580,其剂量分别为:0.2μmol/L;2μmol/L;20μmol/L。将培养板置37℃、5%CO_2、100%湿度的培养箱内培养。每12小时观察一次,看其发育情况。48小时后观察其发育到胚泡的情况,用台盼蓝检测胚胎细胞存活率,用免疫荧光法检测各组胚泡中p38MAPK的表达情况。
结果:
1.在小鼠子宫内膜中,p38MAPK主要表达于腔上皮、腺上皮和蜕膜细胞的胞浆内,偶尔在胞核中。真孕组孕3d,腔上皮中p38MAPK呈阳性表达,孕4～5天呈强阳性表达,此后p38MAPK在腔上皮的表达逐渐减弱;腺上皮中p38MAPK的表达从孕4天开始一直维持在阳性水平,从孕5d开始蜕膜细胞中p38MAPK的表达逐渐增强。假孕组只有孕4天呈弱阳性表达。
2.p38MAPK在小鼠胚植入前各细胞期均呈阳性表达,随着妊娠进展,其表达逐渐加强。
3.p38MAPK抑制剂干预实验
体内实验显示,抑制剂组(10μM)平均胚胎植入数为2.6250±0.5175,明显低于空白对照(7.1250±0.8345)和生理盐水组(6.2500±0.8864),与两者相比,差异十分显著(P＜0.01)。组织学观察结果:抑制剂组胚胎组织呈退化坏死状态,胚胎周围蜕膜细胞呈明显的空泡样变性,蜕膜及胚胎中均可见大量血细胞浸润。
体外实验显示,抑制剂组与对照组胚卵以同样的速率发育到8细胞阶段,但是抑制剂组的胚卵发育却停滞在8-16细胞之间,不能发育到胚泡阶段,比对照组发育晚1-2个细胞期。但抑制剂组胚卵仍具存活能力,将其移入无抑制剂的培养基中,它们仍能发育到胚泡阶段。
结论:
1.p38MAPK在小鼠植入前胚胎发育过程中起作用。
2.p38MAPK参与胚泡植入过程,并可能间接参与植入后的胚泡滋养层扩展、分化及子宫内膜蜕膜化反应。
3.p38MAPK在子宫内膜中的表达不仅受母体因素的调控,还可能与胚泡的刺激有关。
4.p38MAPK特异性抑制剂可抑制在体小鼠胚泡植入及植入后胚胎和蜕膜细胞的发育。
Objective:
Implantation is a key event of the human and mammals reproductive process,it is a complicated process involving the blastocyst adheres to and invades the maternal endometrium gradually at the specific step of implantation window.In the complicated process,a number of factors mediate a series of molecular biology events which include steroid hormones(estrogen,progesterone),growth factors,cytokines,adhesion molecules,proteinums,peptides and enzymes.At the subtle regulation of these factors,some intracellular signal conducting pathways were activated,and regulate the expression of special genes,to coordinate the development of embryo trophblast and the maternal endometrium, accordingly to make the well-developed blastocyst invade the endometrium successfully.
Recently,although much progress on the mechanism of blastocyst implantation have been made,many key questions are still to be further explored.For example,what intracellular signal conductiong pathways were activated? The signals activating these conductiong pathways come from maternal or embryonic body,or from both ? How is a relationship between various intracellular signal conductiong pathways during implantation? and etc.
MAPK pathways mediate some cellular processes,including cell proliferation,growth,differentiation,apoptosis and death.
The MAPK superfamily of proteins consists of four separate signaling cascades:the c-Jun N-terminal kinase or stress-activated protein kinases (JNK/SAPKs),the extracellular signal-regulated kinases(ERKs),the ERK5 or big MAP kinase 1,and the p38 MAPK group of protein kinases,all of which are highly conserved throughout eukaryotic systems.These proteins and enzymes make a signaling cascades which is interlace each other, and play an important role in the intracellular signal transmission.
In recent years,studies oversea showed that MAPK pathway play important roles in many process such as embroygenesis,uterus decidualization,blastocyst adhesion,trophoblastic cell proliferation and invades the maternal endometrium.
The p38 MAPK signaling cascade regulates a variety of cellular activities in different cells and tissue types.It is influenced by a variety of stimulus including growth factors,cytokines,and pathogens. So it is considered as the center of the intracellular signal transmission.
Lately,there was study showing that p38MAPK is activated in mammalian perimplantation development,and its activation is required by uterus decidualization,This idea is not yet reported in domestic literature. Studies both about the expression of p38MAPK in peri-implantation endometrium and injection inhibitor SB203580(specific inhibitors of p38 MAPKα/β)in vivo to interfere with implantation are not reported at home and abroad.
To further explore the effect of p38 MAPK on mouse embryo development and implantation,the methods including immunohistochemistry,embryo culture in vitro and intrauterine injection in vivo were used in our experiments in order to further understand the mechanism of implantation, enrich the theory of implantation in reproductive biology,facilitate the successful rate of assisted reproductive technology such as IVF-ET and offer reliable experimental evidence and animal models for developing new types of contraceptives.
Methods:
1.Pregnant and pseudopregnant uterus of 1～8 days in mice were obtained.The specimens were sliced into section(of 5um) Immunohistochemistry method and image analysis were employed to explore the expression and change rules of p38MAPK in peri-implantation mouse endometrium,uterus of nonpregnancy as control.
2.Early embryos were obtained at the following stages:two-cell, four-cell,eight-cell,morulae and blastocyst.The expression and changes of p38MAPK in early embryos were examined by immunohistochemistry and immunofluorescence.
3.Interfering experiment by p38MAPK inhibitors
In vivo:
Mice were obtained on day 4 of pregnant,intrauterine in jection of p38 MAPK inhibitors were used and morphological and histological methods were employed to observe the embryo number of implantation on day 8 and the variances in the embryo and the endometrium.
In vitro:
Murine embryos were flushed from oviducts of timed pregnant mice at the two-cell stage(48 h post-hCG),pooled,washed,and divided into five groups.They were cultured with different culture treatments:①1:1DMEM/F12 alone,②1:1DMEM/F12 + DMSO(inactive analogue),③1:1DMEM/F12 + 0.2μmol/L SB203580,④1:1DMEM/F12 + 2.0μmol/L SB203580, and⑤1:1DMEM/F12 + 20μmol/L SB203580.Embryos were assessed for morphology and progression through cleavage divisions at 24 h postdrug treatment(72 h post-hCG),at which point half of the embryos in groups 3,4,and 5 were removed from culture,washed,and placed in fresh medium culture drops for the remainder of the experiment.All embryos were then assessed at 48 h postdrug treatment(96 h post-hCG)to observe the developmental information(from cleavage to blastocyst).Cell viability and cell numbers were determined at this time by assaying for uptake of a vital dye(trypan blue).The expression of p38MAPK in blastocysts of each treated group were examined by immunofluorescence.
Results:
1.The p38MAPK-positive staining was found in the cytoplasm of the lumina epithelium,glandular epithelium and decidual cell.In the pregnant group,p38MAPK-positive expression in lumina epithelium was weak at 3 days and peak at 4～5 days in the endometrium of pregnant mouse, and then declined:in glandular epithelium,p38MAPK-positive expression is observed from 4~(th)day and then keep at this level,p38MAPK-positive expression in decidual cell is increased gradually from the 5~(th)day of gestation.In the pseudopregnant group,weak staining of p38MAPK was only observed on the 4~(th)day of gestation,and negative staining were observed on the other days.p38MAPK was expressed weakly in the endometrium of nonpregnant mouse.
2.The p38MAPK-positive staining was found in the pre-implanted embryos and gradually increased with development;
3.Interfering experiments:
In vivo:
The average of embryo-implantation(2.6250±0.5175)in p38MAPK inhibitor(10μM)treated uterus were significantly lower than that of control uterus given saline solution(6.2500±0.8864),P＜0.01.The microscopic observation demonstrated that the embryo degenerated,and even necrosed in some parts.In the decidual cells there was obvious adipose degeneration,while many blood cells invaded the embryos and the decidua.
In vitro:
Two-cell stage embryos cultured in the presence of SB203580(specific inhibitors of p38 MAPKα/β),progressed to the eight-cell stage with the same frequency as controls;however,treated embryos halted their development at the 8- to 16-cell stage.In inhibitor-treated groups,the embryos were composed of a mixture of compacting and noncompacting cells, and the embryo development were delayed for one to two cell cycle than that of controls.Treated embryos remained viable as the developmental blockade was rescued by removing embryos from the drug treatment and placing them in drug-free medium until they progressed to the blastocyst stage.
Conclusions:
1.p38MAPK activity may be required to support embryo development through the murine preimplantation interval.
2.p38MAPK,in addition to correlating to blastocyst implantation,may participate indirectly in the process of endometrial decidualization.
3.The expression of p38MAPK in mouse endometrium is regulated not only by maternal factors(steroid hormones),but also by the implanted embryos.
4.p38MAPK inhibitors can inhibit the blastocyst implantation and the development of embryos and decidual cells in vivo.
引文
1 田永强,赵兴绪,段恩奎.胚泡植入的分子机理[J].甘肃农业大学学报.2002,37(1):1-9.
2 范衡宇,白玉妍,杨增明.胚泡着床窗口的分子调控[J].生物化学与生物物理进展,2001,28(1):11-16.
3 Paria BC,Lim H,DasSK,et al.Molecular signaling in uterine receptivity for implantation[J].Semin Cell Dev Biol,2000,Ii:67-76.
4 Ono,K,Han,J.The p38 signal transduction pathway:activation and function[J].Cell.Signal.2000,12:1-13.
5 Kyriakis J.M,Avruch J.Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation[J].Physiol Reviews,2001,81(2):807-869.
6 Madan P,Calder MD,Watson AJ.Mitogen-activated protein kinase (MAPK)blockade of bovine preimplantation embryogenesis requires inhibition of both p38 and extracellular signal-regulated kinase(ERK)pathways[J].Reproduction,2005,130(1):41-51.
7 Pearson G.,Robinson F,Beers Gibson T,et al.Mitogen-activated protein(MAP)kinase pathways:regulation and physiological functions[J].Endocr Rev,2001,22(2):153-183.
8 Natale DR,Paliga AJ,Beier F,et al.p38 MAPK signaling during murine preimplantation development[J].Dev.Biol,2004,268(1):76-88.
9 Paliga AJ,Natale DR,Watson AJ,eL al.P38 mitogen activated protein kinase(MAPK)first regulates filamentous actin at the 8-16 cell stage during preimplantation development[J].Biol Cell,2005,97(8):629-640.
10 Fan HY,Bai YY,Yang ZM.Molecular control of implantation window of balstocyst [J]. Progress in Biochemistry, 2001, 28(1):11-16.
11 Wilcox A J,Baird DD, Weinberg CR, et al.Time of implantation of the conceptus and loss of pregnancy [J].N Engl J Med, 1999,340(23): 1796-1799.
12 Seger R, Krebs EG. The MAPK signaling casade [J]. FASEB, 1995,9(9): 726-735.
13 Downey JS,Han J. Cellular activation mechanisms in septic shock [J]. Front Biosci,1998 , 3(2) :468-476.
14 Cano E, Mahadevan LC. Parallel signal processing among mammalian MAPKS [J]. TIBS, 1995, 20(3)117-112.
15 Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 Protein kinases [J]. Science, 2002, 298 (12): 1911-1912.
16 Jiang Y, Chen CH, Li ZJ, et al . Characterization of the structure and function of a new mitogen-activated protein kinase (p38 β) [J]. Biol Chem. 1996, 271 (30): 17920-17926.
17 Lazou A, Sugden PH, Clerks A. Activation of mitogen activated protein kinase (p28MAPK, SAPKS/JNKS and ERKS) by the G-protein-coupled receptor agonist phenylephrine in the perfused rat heart [J].Biochem , 1998, 332 (Pt2): 459-465.
18 Alessi DR, Gomez N, Moorhead G, et al. Inactivation of p42 MAP kinase by protein phosphatase 2A and a protein tyrosine phosphatase, but not CL100, in various cell lines [J].Curr Biol, 1995, 5 (3): 283-295.
19 Brewster JL, De Valoir T, Dwyer N , et al .An osmosensing signal transduction pathway in yeast [J]. Science, 1993,259(5102:1760-1763.
20 Han J, Lee JD, Bibbs L et al.A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells[J]. Science ,19 , 265(5173):808-811.
21 Herskowitz. MAP kinase pathways in yeast : for mating and more [J].Cell, 1995, 80 (2) : 187-197.
22 Gargova G, Deri jard ZB, Wu IH, et al. An osmosening signal transduction pathway in mammalian cells [J]. Science, 1994, 265 (5173) : 806-808.
23 JiangY, Gram H , Zhao M , et al. Characterization of the structure and function of the fourth member of p38 group mitogen-activated protein kinases, p38 delta [J]. Biol Chem, 1997, 272(48):30122-30128.
24 Li Z, Jiang Y, Ulevitch RJ, et al. The primary structure of p38 gamma:A new member of p38 group of MAP kinases [J]. Biolchem Biophys Res Commun,1996, 228(2): 334-340.
25 Jiang Y, Li Z, Schwarz EM, et al. Structure-functiong studies of p38 mitogen-activated protein kinase: Loop-12 influence substrate specif ity and autophosphorylation, but not upstream kinase selection [J]. Biol Chem, 1997, 272 (17) : 11096-11102.
26 Tebar F, Villalonga P, Sorkina T, et al. Calmodulin regulates intracellular trafficking of epidermal growth factor receptor and the MAPK signaling pathway [J]. Mol Biol Cell , 2002, 13 (6): 2057-2068.
27 Strakova Z , Srisuparp S , Fazleabas AT, et al . IL-lbeta during in vitrodecidualization in primate [J]. Reprod Immunol , 2002 , 55 (1-2) : 35-47.
28 Scherle PA , Ma W , Lim H , et al . Regulation of cyclooxygenase-2 induction in the mouse uterus during decidualization. An event of early pregnancy [J]. Biol Chem , 2000, 275 (47): 37086-37092.
29 Adams RH , Porras A , Alonso G, et al. Essential roleof p38alpha MAP kinase in placental but not embryonic cardiovascular development [J]. Mol. Cell 2000, 6: 109- 116.
30 Allen M, Svensson L, Roach M, et al. Deficiency of the stress kinase p38alpha results in embryonic lethality: characterization of the kinase dependence of stress responses of enzyme-deficient embryonic stem cells[J].Exp.Med.2000,191:859-870.
31 Mudgett JS,Ding J,Guh-Siesel L,et al.Essential role for p38alpha mitogen-activated protein kinase in placental angiogenesis[J].Proc.Natl.Acad.Sci.U.S.A.2000,97:10454-10459.
32 Yang J,Boerm M,McCarty M,et al.Mekk3 is essential for early embryonic cardiovascular development[J].Nat.Genet.2000,24:309-313.
33 Cirillo PF,Pargellis C,Regan J.The non-diaryl heterocycle classes of p38 MAP kinase inhibitors[J].Curr.Top.Med.Chem.2002,2:1021-1035.
34 Davidson SM,Morange M.Hsp25 and the p38 MAPK pathway are involved in differentiation of cardiomyocytes[J].Dev.Biol.2000,218:146-160.
35 Huot J,Houle F,Marceau F,et al.Oxidative stress-induced actin reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascular endothelial cells [J].Circ.Res.1997,80:383-392.
36 Zayzafoon M,Botolin S,McCabe LR.p38 and activating transcription factor-2 involvement in osteoblast osmotic response to elevated extracellular glucose[J].Biol.Chem.2002,277:37212-37218.
37 蔡理全 曹宇静 段恩奎 整合素ανβ3在小鼠胚胎植入中的作用科学通报[J].2000,45(15):1639-1643.
38 武玉玲、邮鲁军、王富武等宫腔注射纤连蛋白抗体和层粘蛋白抗体对小鼠胚泡着床的影响[J].解剖学杂志,2000,23(4):313-316.
39 李英,杨京京,龙洪清等 小鼠子宫角注射层粘连蛋白及其抗血清对胚泡着床的影响[J].北京医科大学学报1996,28(1):11-13.
40 Holmes PV,Rachoyel AR,Svalander PC.The antibody-neurealisation of PDGF,CSF-1,TGF β2,3 EGF and EGF-receptor utero in pre-implantation [J].Ups.Med Sci,1997,102(1):41-48.
2 范衡宇,白玉妍,杨增明.胚泡着床窗口的分子调控[J].生物化学与生物物理进展,2001,28(1):11-16.
3 Paria BC,Lim H,DasSK,et al.Molecular signaling in uterine receptivity for implantation[J].Semin Cell Dev Biol,2000,Ii:67-76.
4 Ono,K,Han,J.The p38 signal transduction pathway:activation and function[J].Cell.Signal.2000,12:1-13.
5 Kyriakis J.M,Avruch J.Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation[J].Physiol Reviews,2001,81(2):807-869.
6 Madan P,Calder MD,Watson AJ.Mitogen-activated protein kinase (MAPK)blockade of bovine preimplantation embryogenesis requires inhibition of both p38 and extracellular signal-regulated kinase(ERK)pathways[J].Reproduction,2005,130(1):41-51.
7 Pearson G.,Robinson F,Beers Gibson T,et al.Mitogen-activated protein(MAP)kinase pathways:regulation and physiological functions[J].Endocr Rev,2001,22(2):153-183.
8 Natale DR,Paliga AJ,Beier F,et al.p38 MAPK signaling during murine preimplantation development[J].Dev.Biol,2004,268(1):76-88.
9 Paliga AJ,Natale DR,Watson AJ,eL al.P38 mitogen activated protein kinase(MAPK)first regulates filamentous actin at the 8-16 cell stage during preimplantation development[J].Biol Cell,2005,97(8):629-640.
10 Fan HY,Bai YY,Yang ZM.Molecular control of implantation window of balstocyst [J]. Progress in Biochemistry, 2001, 28(1):11-16.
11 Wilcox A J,Baird DD, Weinberg CR, et al.Time of implantation of the conceptus and loss of pregnancy [J].N Engl J Med, 1999,340(23): 1796-1799.
12 Seger R, Krebs EG. The MAPK signaling casade [J]. FASEB, 1995,9(9): 726-735.
13 Downey JS,Han J. Cellular activation mechanisms in septic shock [J]. Front Biosci,1998 , 3(2) :468-476.
14 Cano E, Mahadevan LC. Parallel signal processing among mammalian MAPKS [J]. TIBS, 1995, 20(3)117-112.
15 Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 Protein kinases [J]. Science, 2002, 298 (12): 1911-1912.
16 Jiang Y, Chen CH, Li ZJ, et al . Characterization of the structure and function of a new mitogen-activated protein kinase (p38 β) [J]. Biol Chem. 1996, 271 (30): 17920-17926.
17 Lazou A, Sugden PH, Clerks A. Activation of mitogen activated protein kinase (p28MAPK, SAPKS/JNKS and ERKS) by the G-protein-coupled receptor agonist phenylephrine in the perfused rat heart [J].Biochem , 1998, 332 (Pt2): 459-465.
18 Alessi DR, Gomez N, Moorhead G, et al. Inactivation of p42 MAP kinase by protein phosphatase 2A and a protein tyrosine phosphatase, but not CL100, in various cell lines [J].Curr Biol, 1995, 5 (3): 283-295.
19 Brewster JL, De Valoir T, Dwyer N , et al .An osmosensing signal transduction pathway in yeast [J]. Science, 1993,259(5102:1760-1763.
20 Han J, Lee JD, Bibbs L et al.A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells[J]. Science ,19 , 265(5173):808-811.
21 Herskowitz. MAP kinase pathways in yeast : for mating and more [J].Cell, 1995, 80 (2) : 187-197.
22 Gargova G, Deri jard ZB, Wu IH, et al. An osmosening signal transduction pathway in mammalian cells [J]. Science, 1994, 265 (5173) : 806-808.
23 JiangY, Gram H , Zhao M , et al. Characterization of the structure and function of the fourth member of p38 group mitogen-activated protein kinases, p38 delta [J]. Biol Chem, 1997, 272(48):30122-30128.
24 Li Z, Jiang Y, Ulevitch RJ, et al. The primary structure of p38 gamma:A new member of p38 group of MAP kinases [J]. Biolchem Biophys Res Commun,1996, 228(2): 334-340.
25 Jiang Y, Li Z, Schwarz EM, et al. Structure-functiong studies of p38 mitogen-activated protein kinase: Loop-12 influence substrate specif ity and autophosphorylation, but not upstream kinase selection [J]. Biol Chem, 1997, 272 (17) : 11096-11102.
26 Tebar F, Villalonga P, Sorkina T, et al. Calmodulin regulates intracellular trafficking of epidermal growth factor receptor and the MAPK signaling pathway [J]. Mol Biol Cell , 2002, 13 (6): 2057-2068.
27 Strakova Z , Srisuparp S , Fazleabas AT, et al . IL-lbeta during in vitrodecidualization in primate [J]. Reprod Immunol , 2002 , 55 (1-2) : 35-47.
28 Scherle PA , Ma W , Lim H , et al . Regulation of cyclooxygenase-2 induction in the mouse uterus during decidualization. An event of early pregnancy [J]. Biol Chem , 2000, 275 (47): 37086-37092.
29 Adams RH , Porras A , Alonso G, et al. Essential roleof p38alpha MAP kinase in placental but not embryonic cardiovascular development [J]. Mol. Cell 2000, 6: 109- 116.
30 Allen M, Svensson L, Roach M, et al. Deficiency of the stress kinase p38alpha results in embryonic lethality: characterization of the kinase dependence of stress responses of enzyme-deficient embryonic stem cells[J].Exp.Med.2000,191:859-870.
31 Mudgett JS,Ding J,Guh-Siesel L,et al.Essential role for p38alpha mitogen-activated protein kinase in placental angiogenesis[J].Proc.Natl.Acad.Sci.U.S.A.2000,97:10454-10459.
32 Yang J,Boerm M,McCarty M,et al.Mekk3 is essential for early embryonic cardiovascular development[J].Nat.Genet.2000,24:309-313.
33 Cirillo PF,Pargellis C,Regan J.The non-diaryl heterocycle classes of p38 MAP kinase inhibitors[J].Curr.Top.Med.Chem.2002,2:1021-1035.
34 Davidson SM,Morange M.Hsp25 and the p38 MAPK pathway are involved in differentiation of cardiomyocytes[J].Dev.Biol.2000,218:146-160.
35 Huot J,Houle F,Marceau F,et al.Oxidative stress-induced actin reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascular endothelial cells [J].Circ.Res.1997,80:383-392.
36 Zayzafoon M,Botolin S,McCabe LR.p38 and activating transcription factor-2 involvement in osteoblast osmotic response to elevated extracellular glucose[J].Biol.Chem.2002,277:37212-37218.
37 蔡理全 曹宇静 段恩奎 整合素ανβ3在小鼠胚胎植入中的作用科学通报[J].2000,45(15):1639-1643.
38 武玉玲、邮鲁军、王富武等宫腔注射纤连蛋白抗体和层粘蛋白抗体对小鼠胚泡着床的影响[J].解剖学杂志,2000,23(4):313-316.
39 李英,杨京京,龙洪清等 小鼠子宫角注射层粘连蛋白及其抗血清对胚泡着床的影响[J].北京医科大学学报1996,28(1):11-13.
40 Holmes PV,Rachoyel AR,Svalander PC.The antibody-neurealisation of PDGF,CSF-1,TGF β2,3 EGF and EGF-receptor utero in pre-implantation [J].Ups.Med Sci,1997,102(1):41-48.