【作者】 王正朝；

【导师】 石放雄；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 在哺乳动物卵巢中,卵泡的生长发育及分化需要颗粒细胞和卵母细胞特异性基因的协同表达。这一程序的执行依赖于卵母细胞与颗粒细胞之间交换的局部信号,以及促性腺激素的调控。因此,用信号转导通路来解释促性腺激素对哺乳动物卵巢卵泡生长发育的调控作用,可以很好的解释在这些过程中那些基因的适时表达与调控。卵巢卵泡的生长发育以及卵母细胞的成熟与排卵是一个复杂的内分泌、旁分泌和自分泌的作用过程,其都可以转为对环核苷酸水平的调控。胞内cAMP的变化可以调控促性腺激素对颗粒细胞与膜细胞功能的调节作用。由此可见,第二信使cAMP在细胞信号转导中起着重要作用,但为什么相同的cAMP信号可以诱导不同的生理反应。20年前,cAMP信号区域化的提出似乎解答了这一疑惑。哺乳动物卵巢产生可用的卵母细胞主要取决于促性腺激素对卵泡发育、颗粒细胞成熟,排卵及黄体化的调节作用。LH和FSH的作用很大程度上取决于cAMP依赖性信号转导,现在也已经发现许多其他因子可以通过激活其他信号转导通路来调节促性腺激素对卵巢的作用。因此,目前研究表明第二信使cGMP对卵巢功能有重要的调节作用。1第二信使cAMP/cGMP通路对大鼠颗粒细胞体外成熟的调控及其机制研究颗粒细胞中第二信使cAMP/cGMP的失活很大程度上取决于一系列相应的PDE的表达及其活性的变化。为了研究第二信使通路在大鼠颗粒细胞体外成熟过程中的调控作用,本试验体外培养了未成熟大鼠卵巢颗粒细胞,利用FSH处理颗粒细胞,并检测了E2,cAMP和cGMP浓度的变化,同时分析了PDE4蛋白的表达及cAMP-PDE活性的变化。结果清楚地表明颗粒细胞在48h成熟时,试验组cAMP浓度显著高于对照组,而cGMP没有明显变化;蛋白印迹试验发现试验组出现两条PDE4条带,约80和70KDa;cAMP-PDE活性分析结果表明对照组在整个48h过程中的活性都较低,而试验组在6到12h时,cAMP-PDE活性显著升高。综上所述,在大鼠颗粒细胞体外成熟过程中FSH使cAMP-PDE活性升高主要与PDE4蛋白的表达有关,同时颗粒细胞中cAMP的累积不是通过cAMP-PDE活性的变化来实现的。2第二信使cAMP/cGMP通路在大鼠胚胎早期卵巢发育过程中的作用为了研究第二信使cAMP/cGMP通路在哺乳动物早期卵巢生长发育过程中的作用,本试验通过组织学观察确定胎鼠卵巢发育,通过RIA研究胎鼠卵巢中cAMP/cGMP水平及其相应PDE活性变化。结果发现胎鼠卵巢cAMP水平在12日龄时最低,到15日龄之后显著升高,直到出生后1日龄没有明显变化;而胎鼠卵巢cAMP-PDE活性呈相反的变化,12日龄时最高,其他时期差异不显著。胎鼠卵巢cGMP水平变化在整个妊娠后期,卵巢分化形成之后,一直没有明显变化;而cGMP-PDE活性变化在12日龄时最高,15日龄时显著降低,之后虽然有所下降,但不显著。这些结果表明cAMP信号通路可能参与雌性胎鼠卵巢的生长发育,同时表明cAMP/cGMP信号通路中PDE在这个过程中作用方式也是不同的。3第二信使cAMP/cGMP通路在新生大鼠卵巢卵泡发育过程中的作用为了研究第二信使cAMP/cGMP通路在哺乳动物卵巢卵泡生长启动与发育过程中的作用,本试验通过组织学观察确定新生大鼠卵巢卵泡发育,通过RIA研究新生大鼠卵巢中cAMP/cGMP水平和相应PDE活性变化。结果发现新生大鼠在10日龄前cAMP浓度逐步升高,与卵巢卵泡发育是一致的,21日龄时显著降低;而cAMP-PDE活性在10日龄前没有明显变化,21日龄时则显著升高。cGMP水平在出生时较高,在5日龄时显著降低,然后到10日龄时升高到最高水平,紧接着在21日龄时降低;而cGMP-PDE活性在1到10日龄期间没有显著性变化,但在21日龄时显著升高了。这些结果表明cAMP信号通路可能参与大鼠卵巢卵泡生长的启动与发育,而PDE在卵巢卵泡的早期发育中没有显著性作用;21日龄cAMP/cGMP水平和PDE活性变化同时表明cAMP/CGMP信号通路之间存在着复杂的对话。4第二信使cAMP/cGMP通路在未成熟大鼠卵巢卵泡发育过程中的作用为了研究第二信使cAMP/cGMP通路在哺乳动物卵巢卵泡发育过程中的作用,本试验通过组织学观察确定未成熟大鼠卵巢卵泡发育模型,通过RIA研究未成熟大鼠卵巢cAMP/cGMP水平和相应PDE活性变化。结果发现未成熟大鼠卵巢cAMP水平在eCG处理后显著升高,hCG处理后24h继续显著性升高,到72h时则显著性下降;而cAMP-PDE活性在eCG处理后没有明显变化,hCG处理后则先是升高,紧随着开始下降。cGMP水平的变化与cAMP变化相似,eCG处理后显著升高,hCG处理后24h继续显著性升高,到72h时则显著性下降;而cGMP-PDE活性在eCG处理后显著下降,hCG处理后则显著升高。这些结果表明cAMP/cGMP信号通路可能参与大鼠卵巢卵泡生长与发育,而PDE在卵巢卵泡发育中的作用呈阶段特异性。5第二信使cAMP/cGMP通路在成年大鼠卵巢卵泡发育过程中的作用为了研究第二信使cAMP/cGMP通路在哺乳动物发情周期中对卵巢卵泡发育的调控作用,本试验通过阴道涂片的方法确定成年大鼠的发情周期,通过组织学观察确定成年大鼠卵巢卵泡发育模型,通过RIA研究成年大鼠卵巢cAMP/cGMP水平和相应PDE活性变化。结果发现成年大鼠卵巢cAMP水平除发情期(E)外的其他三个发情阶段之间没有显著差异,其中发情期(E)卵巢cAMP水平则显著低于发情周期的其他各个阶段;而AMP-PDE活性在整个发情周期中都没有显著性的变化。cGMP水平在发情期(E)和乏情期(D)较低,在间情期(M)和发情前期(P)较高;而cGMP-PDE活性与cGMP水平相比呈现相反的变化趋势,cGMP-PDE活性在发情期(E)和乏情期(D)较高,在间情期(M)和发情前期(P)较低;相关性分析发现卵巢cGMP水平与cGMP-PDE活性之间存在显著性负相关(r=-0.7715,n=16,P＜0.05)。这些结果表明cAMP/cGMP信号通路可能参与大鼠卵巢卵泡生长与发育,其中cAMP-PDE在发情周期中对cAMP信号的调控受多种因素的影响,而在发情周期中cGMP-PDE可能是调控cGMP信号的主要影响因素。6第二信使cAMP/cGMP通路在老年大鼠卵巢卵泡发育过程中的作用为了研究第二信使cAMP/cGMP通路在老年哺乳动物卵巢功能中的作用,本试验通过组织学观察确定老年大鼠卵巢卵泡发育模型,通过RIA研究老年(PE)大鼠卵巢cAMP/cGMP水平和相应PDE活性变化。结果发现老年(PE)大鼠卵巢cAMP水平与成年大鼠发情期(E)卵巢cAMP水平相比没有明显差异,而老年(PE)大鼠卵巢cAMP-PDE活性则显著低于大鼠发情期(E)卵巢cAMP-PDE活性。发现老年(PE)大鼠卵巢cGMP水平虽然高于成年大鼠发情期(E)卵巢cGMP水平,但差异不显著,而老年(PE)大鼠卵巢cGMP-PDE活性则显著低于大鼠发情期(E)卵巢cGMP-PDE活性。这些结果表明cAMP/cGMP信号通路可能与雌性老年(PE)大鼠卵巢囊肿直接相关,尤其是cAMP-PDE与cGMP-PDE活性的降低可能就是由于雌性老年(PE)大鼠卵巢囊肿的形成。7第二信使cAMP/cGMP通路对哺乳动物妊娠期卵巢功能的调节及相关机制研究为了研究第二信使cAMP/cGMP通路对哺乳动物妊娠期卵巢功能的调控作用,本试验通过组织学观察确定不同妊娠日龄时大鼠卵巢卵泡的发育情况,并通过RIA研究了妊娠奶牛血液雌激素(E2)、孕酮(P)和cAMP/cGMP水平。结果发现在大鼠妊娠期间,其卵巢卵泡有不同程度的发育,与妊娠阶段有关。奶牛血液雌激素检测结果发现其在不同的妊娠时期没有显著变化;而孕酮随着妊娠日龄的增加呈逐步升高的趋势,且差异显著。奶牛血液cAMP/cGMP水平变化相似,在妊娠中期最高,在妊娠晚期最低。这些结果表明第二信使cAMP/cGMP信号通路可能调节生殖激素的方式参与维持哺乳动物妊娠期卵巢的功能。8第二信使cAMP/cGMP信号通路元件蛋白在中国荷斯坦奶牛卵巢中的定位研究应用七种特异性抗体(sGCα、sGCβ、nNOS、FOXO1、PDE4、PKB/Akt和Inhibinα)对中国荷斯坦奶牛卵巢卵泡细胞进行免疫组织化学定位。结果表明PKB主要存在于原始卵泡、腔前卵泡和有腔卵泡颗粒细胞,黄体细胞中没有;FoxO1主要位于原始卵泡、腔前卵泡和有腔卵泡颗粒细胞;PDE4仅位于部分有腔卵泡的颗粒细胞;抑制素α、nNOS、sGCα和β存在于各级卵泡的颗粒细胞层,其中sGCα和β主要存在于原始卵泡和腔前卵泡的颗粒细胞。这七种蛋白的阶段/细胞特异性表达表明它们在中国荷斯坦卵巢卵泡的发育、闭锁和黄体化过程中起非常重要的作用。综上所述,本试验研究利用一个颗粒细胞体外培养模型和六个大鼠卵巢发育模型系统地研究了第二信使cAMP/cGMP信号通路在整个卵巢卵泡发育过程中作用,结果表明在大鼠颗粒细胞体外成熟过程中FSH使cAMP-PDE活性升高主要与PDE4蛋白的表达有关,颗粒细胞中cAMP的累积不是通过cAMP-PDE活性的变化来实现的;同时表明cAMP-PDE在卵巢卵泡发育中的作用呈阶段特定性;另外,我们还研究了cGMP-PDE在哺乳动物卵巢卵泡发育过程中的作用;以及第二信使cAMP/cGMP信号通路元件蛋白在中国荷斯坦奶牛卵巢中的定位研究,验证了本文中提出的cAMP区域化及cGMP作用的潜在意义,并提出了第二信使cAMP/cGMP对哺乳动物卵巢功能调控的主要信号通路模式图。更多还原

【Abstract】 In the mammalian ovary,the growth and terminal differentiation of the follicle require the coordinated expression of specific genes in granulosa cells and the oocyte.The execution of the programs in a timely and coordinated fashion is dependent on local signals exchanged between the gamete and somatic cells and on gonadotropin regulation. Therefore,signaling pathways can be used to interpret the regulatory role of gonadotropin on the development of mammalian ovarian follicles,and suggest how to control the switching on and off of genes at the appropriate time during these phases of growth and differentiation.The development of the ovarian follicle,oocyte maturation,and ovulation require a complex set of endocrine,paracrine,and autocrine inputs that are translated into the regulation of cyclic nucleotide levels.Changes in intracellular cAMP mediate the gonadotropin regulation of granulose and theca cell functions.Thus it can be seen that cyclic AMP(cAMP),as a second messenger,plays a critical role in cellular signaling transduction.However,it is not clear how this apparently identical cAMP signal induces divergent physiological re-sponses.The potential explanation that cAMP signaling is compartmentalized was proposed by Buxton and Brunton twenty years ago.The production of a viable oocyte is dependent upon the critical influences of gonadotrophins in follicular development,granulose cell maturation,ovulation,and luteinizafion.While the effects of LH and FSH are due in large part to cyclic AMP dependent signaling mechanisms,it is clear that a number of other factors modulate the actions of gonadotrophins on the ovary via activation of alternative signaling pathways.In this regard,recent studies indicate that the second messenger guanosine 3’,5’-cyclic monophosphate(cGMP) mediates a wide range of influences on the ovary.1 Regulation and Its Mechanism of the Second Messenger cAMP/cGMP Signaling in the Rat Granulose Cell Maturation in vitroInactivation of the cyclic nucleotide signal in granulosa cells depends on a complex array of cyclic nucleotide phosphodiesterases(PDE).In order to examine the role of PDE in cyclic AMP(cAMP) signaling in granulosa cells,the present study cultured rat ovarian granulosa cells treated with FSH,examined the changes of E2,cAMP and cGMP,and analysis the the expression of PDE4 proteins and regulation of cAMP-PDE activities.The results showed that cAMP level in the experimental group was significantly higher than that in control group,while cGMP level has no difference between two groups,when granulose cells were cultured 48 hours for maturation.The results of immunoblot analyses showed that two predominant PDE4D subtypes of approximately 80 and 70 kDa appeared when immature rat granulosa cells were treated with FSH.Immature rat granulosa cells treated with medium alone displayed low cAMPPDE activity throughout 48 h of culture while those treated with FSH(2 ng·mL-1) showed a marked increase in cAMP-PDE activity between 6 and 12 h of culture,followed by a decline.The findings from the present study indicate that the increased cAMP-PDE activity by FSH is mainly related to the changes of PDE4D protein levels,and that cAMP accumulation in rat granulosa cells are not via the increased cAMP-PDE activity.2 Role of the Second Messenger cAMP/cGMP Signaling during the Ovarian Early Development in the Rat EmbryoIn order to examine the role of the second messenger cAMP/cGMP signaling during the ovarian early development in mammalian,the present study was conducted to investigate the fetal ovarian development through histology and to analysis the ovarian cGMP levels and cGMP-PDE activities in the fetal female rat model by radioimmunoassay (RIA).The results showed ovarian cAMP levels is low at Day 12,increased significantly after Day 15,then no difference until birth in the fetal rats.While the contrary changes were discovered in the ovarian cAMP-PDE activity,which was the highest at Day 12 and there was no difference between other stages.Ovarian cGMP levels changed in the whole late pregnancy,then no difference was found after ovarian differentiation;while cGMP-PDE activity was high at Day 12,and decreased significantly at Day 15,then no marked decline. Together,the results of our present study have indicated that cAMP signaling may take part in the development of female fetal ovary,and that PDE in the cAMP/cGMP signaling have different roles during this development process. 3 Role of the Second Messenger cAMP/cGMP Signaling during the Ovarian Follicular Development in the Postnatal RatIn order to examine the role of the second messenger cAMP/cGMP signaling during the ovarian follicular initial development in mammalian,the present study was conducted to investigate the ovarian follicular development through histology and to analysis the ovarian cGMP levels and cGMP-PDE activities in the postnatal rat model by radioimmunoassay(RIA).The results of the present study showed in postnatal rats,cAMP level was very high in ovaries of 10-day-old females,while cAMP-PDE activity was the highest at 21 days of age.cGMP levels were high at birth day and decreased dramatically at Day 5,then increased to the highest level at Day 10 and followed by a decline at Day 21. While,the cGMP-PDE activity was not significantly changed during Day 1 to 10,but increased significantly at Day 21.Together,these findings indicate that ovarian cAMP signaling may be involved in ovarian follicular invitation and development in the rat,while PDE seems no significant roles in the early development of ovarian follicles.The changes of cAMP/cGMP levels and PDE activity at Day 21 implied that there are complex crosstalking between cAMP and cGMP signaling pathways.4 Role of the Second Messenger cAMP/cGMP Signaling during the Ovarian Follicular Development in the Immature RatIn order to examine the role of the second messenger cAMP/cGMP signaling during the ovarian follicular development in mammalian,the present study was conducted to investigate the ovarian follicular development through histology and to analysis the ovarian cGMP levels and cGMP-PDE activities in the immature rats during gonadotropin-primed follicular development,ovulation and luteinization by radioimmunoassay(RIA).The results of the present study showed in immature female rats,the cAMP level increased remarkably with treatment with equine chorionic gonadotropin(eCG),while cAMP-PDE activity was similar to cAMP change;induction of ovulation and luteinization by an ovulatory dose of human chorionic gonadotropin(hCG) increased ovarian cAMP level and cAMP-PDE activity,then followed a significant decline,cGMP levels decreased remarkably with treatment of equine chorionic gonadotropin(eCG),while cGMP-PDE activity did not show any significant changes;however,ovarian cGMP level and cGMP-PDE activity increased after injection of an ovulatory dose of human chorionic gonadotropin(hCG) for induction of ovulation and luteinization.Together,these findings indicate that ovarian cAMP/cGMP signaling may take part in the growth and development of ovarian follicles,while roles of PDE during the development of ovarian follicles were in stage-specific manner.5 Role of the Second Messenger cAMP/cGMP Signaling during the Ovarian Follicular Development in the Adult RatIn order to examine the role of the second messenger cAMP/cGMP signaling during the ovarian follicular development in mammalian,the present study was conducted to investigate the ovarian follicular development through histology and to analysis the ovarian cGMP levels and cGMP-PDE activities in the adult rats during normal estrous cycling by radioimmunoassay(RIA).The results of the present study showed in young adult rats during normal estrus cycle,cAMP levels were low at estrus day,while cAMP-PDE activities had no significant change in each day of estrus cycle,cGMP levels were high at proestrus and metestrus days,while cGMP-PDE activities were high at estrus day.In addition,there was a significant negative relationship between ovarian cGMP contents and cGMP-PDE activities during normal estrous cycles in the adult rat(r=-0.7715,n=16, P＜0.05),but not in the postnatal rat(r＝-0.1055,n=20,P＞0.05).Together,these findings indicate that ovarian cAMP/cGMP signaling may take part in the growth and development of ovarian follicles,especially that cAMP-PDE regulation of cAMP signal was affected by many kinds of factors,while cGMP-PDE may be a major factor to mediate cGMP during normal estrus cycle.6 Role of the Second Messenger cAMP/cGMP Signaling during the Ovarian Follicular Development in the Persistent Estrus RatIn order to examine the role of the second messenger cAMP/cGMP signaling during the ovarian follicular development in mammalian,the present study was conducted to investigate the ovarian follicular development through histology and to analysis the ovarian cGMP levels and cGMP-PDE activities in the persistent estrus(PE) rats by radioimmunoassay(RIA).The results of the present study showed in persistent estrus(PE) rats,ovarian cAMP levels were similar to that in young adult rats at estrus day but lower than that at the other estrus cycle days;ovarian cAMP-PDE activities were lower than that at every estrus cycle day.ovarian cGMP levels were similar to that in adult rats at estrus and diestrus days but lower than that at proestrus and metestrus days;ovarian cGMP-PDE activities were lower than that at estrus day but similar to the other estrous cycle days. Together,these findings indicate that ovarian cAMP/cGMP signaling may be involved in ovarian cysts,especially the decline of cAMP/cGMP-PDE activity may be caused by the formation of ovarian cysts in PE rats.7 Regulation and Its Mechanism of Ovarian Functions via Second Messenger cAMP/cGMP Signaling in Mammalian GestationIn order to examine the regulation of cAMP/cGMP signaling on ovarian functions in mammalian gestation,the present study was conduct to investigate the ovarian follicular development in different-day gestation rat via histology,and analysis the blood E2,P and cAMP/cGMP levels in pregnancy daily cows by radioimmunoassay(RIA).The results of the present study showed ovarian follicles have developed in a different degree,which is related to different stages of gestation during the rat pregnancy.There was no significant difference in blood E2 levels of daily cows,while P levels increased significantly with the day of pregnancy.There were similar changes between cAMP and cGMP in daily cow blood,which were high in the middle of pregnancy and were low during the late pregnancy. Together,these findings indicate that second messenger cAMP/cGMP signaling maybe play a critical role in maintaining ovarian functions via mediating reproduction hormones in mammalian gestation.8 Study on the Localization of Element Proteins of cAMP/cGMP Signaling Pathway in Ovaries from Chinese Holstein CowsExperiments were conducted to examine the cellular localization of PKB/Akt,FoxO1, PDE4,inhibinα-subunit,nNOS,sGCαandβsubunit proteins in the ovarian follicular cells of Chinese Holstein Cows by immunohistochemistry.The results indicated that PKB was localized in the granulosa cells of primordial,preantral and antral follicles,but were not localized in corpora lutea.FoxO1 was mainly localized in the granulosa cells of of primary, preantral and antral follicles.PDE4 was only localized in the granulosa cells of some antral follicles.Inhibinα-subunit nNOS,sGCαandβsubunits were localized in the granulosa cells of follicles at all stages but were not localized in corpora lutea,but sGCαandβsubunit staining were intense in the granulosa cells of primordial and preantral follicles. Together,the stage- and cell- specific expression patterns of PKB/Akt,FoxO1,PDE4, inhibinα-subunit,nNOS,sGCαandβsubunits suggest that these proteins might play potential roles in follicular development,atresia,and luteinization in the Chinese Holstein Cow.In conclusion,we use a kind of granulose cell model cultured in vitro and six models of rat ovarian development to examine the roles of second messenger cAMP/cGMP signaling pathways during the development of rat ovarian follicles.The results of the present study have indicated that the increased cAMP-PDE activity by FSH is mainly related to the changes of PDE4D protein levels,and that cAMP accumulation in rat granulosa cells is not via the increased cAMP-PDE activity.The results of animal models suggested that roles of cAMP-PDE during the development of ovarian follicles are in a stage-specific manner.Furthermore,we investigated the roles of cGMP-PDE in these physical processes and the localization of element proteins of cAMP/cGMP signaling pathway in ovaries from Chinese Holstein cows,which validated cAMP compartmentalization and cGMP potential effects showed in the present thesis and developed a signaling pathway model of regulations of second messengers cAMP/cGMP on ovarian functions in mammalian.更多还原