Regulatory Mechanisms Of Second Messenger CAMP/cGMP Signaling On Mammalian Ovarian Functions

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.