Study Of The Activity Of PDE During Early Development Of Mouse Zygote And Distribution Of PDE3A In Mouse Ovaries Tissue

INTRODUCTIONThe discovery of cyclic nucleotides as second messengers has paved the way to much of what we know about signal transduction and the mechanisms of horm- one action. It is now established that protein kinase As (PKAs) are not the only intracellular effectors of cAMP. Cyclic nucleotide gated channels and cAMP- regulated guanine nucleotide exchange factors (cAMP — GEFs or EPACs) allow branching of the cyclic nucleotide signals。 Hormones bind to receptors that are coupled via G proteins to cyclases, which synthesize cAMP/cGMP. In the cyclic nucleotide cascade, phosphodiesterases (PDEs) are the enzymes that hy-drolyze cAMP and cGMP, inactivating these second messengers. Together with phosphatases, PDEs are negative steps in the signaling pathway, and signal termination was thought to be their only function. In recent years, aroused scholar 's widespread interest about the cell cycle regulation research. Because of the limit of subject, The research of related cell cycle regulation mechanism are much in vertebrate or the amphibians animal egg, the somatic cell and so on as the experimental object for it is easy to get, but studies in the higher mammal egg early growth machine are extremely limitedly. The mouse is mammal quite close to the humanity and provided the good mammal model to us, therefore the study of mouse fertilized egg during early development is very important for us to understood the mechanism of signal transduction of mammalian zygote. The experimental technology progress finally enables us take the limited mouse fertilized egg as an object, Carries on the related phosphodiesterase (PDE) research of the active change and this will be helpful for us to understand mammal (including human) cell cycle regulation mechanism. As we all known internal ferti-lization occurs after injects hCG about 11 h, and then provide evidencd in times of fertilized egg first cell cycle . It has become clear that at least 11 different families of genes encoding cyclic nucleotide PDEs are present in mammals The rat and the mouse ovocyte main PDE form is PDE3A also discovered PDE the special inhibitor prevents the mature of ovocyte in vitro and in vivo, and therefore confirmed that this enzyme play plays an important role in arrested of oocyte o The experiment confirmed in the oocyte along with PDE active elevating, oocyte meiosis resumption o This experiment carries on the detection of PDE in mouse fertilized egg first cell cyclr and has invested expression of PDE3 A in mouse ovary .MATERIAZLS AND METHODS1. KUNMING mice were supplied from Department of Laboratory Animals , China Medical University The2. PMSG were obtained from Tianjin Huafu High and new technology biology centerHCG from ShangHai Biological biochemistry pharmaceutical manufacturing plantPDE3A from Santa CruzHyalaseN BSA^3H - cAMP^snake venomNMl6 medium from SigmaPPC\POPOP from FlukaSP Ultrasensitive kit ^ DAB kit from FUZhou Maixin Biotechnology devlop company limited3. Ovaries were removed from Kunming pregnant female mice (3 -4w) after inject PMSG and HCG and transferred to prewarmed (37T1) M2medium. Cumulus cells were removed by 300fxg/ml hyaluronidase. Zygote were cultured in M,6 medium and collected at appointed time.4. PDE activity assayThe collected zygotes frozed them for 3 times. The reaction was started by adding 400 [xL assay buffer to the samples and the samples were incubated for lOmin at 30T1. boil 2. 5 min stop reaction ,then adding snake venom lOmin at30°C boil again . Activity of PDE are test by count cpm in Beckman liquid scintillation analysis meter.5. MPF activity assayAdd 5[xl lysis buffer to the collected zygotes and frozed them for 3 times. The reaction was started by adding 20 ui assay buffer to the samples and the samples were incubated for 30min at 30 Ti. Quantity of MPF activity was detected by scintillation analysis meter.6. Analysis of mouse ovaries PDE3A expressMouse ovaries was treat with HE ang Immunohistochemistry dyeing and carry on analysis of PDE3A express .ResultsThe activity of PDE in zygote was fluctuated during the early development of mouse zygote, and PDE3A was expressed in primary oocyte of primordial follicle but not in oocyte of growing follicle Nvesicula graafianae and granular cell in mouse ovaries tissue .DiscutionThe PDE gene superfamily apparently encodes more than 50 PDE proteins . Some cells are relatively enriched in specific PDEs;these include oocytes and retinal rods and cones , in which PDE3A and PDE6 , respectively, are the predominant PDEs. Most cells, however, contain representatives of at least several PDE gene families and different subfamily isoforms from the same gene family, but in different amounts, proportions, and subcellular locations. In the female reproductive system, differential cellular expression of PDE4 and PDE3 isoforms in granulosa cells and oocytes, respectively, seems to be important in the integrating signals involved in luteinizing hormone ( LH) induction of oocyte maturation , which apparently involves opposing effects of cAMP in somatic and germ cells . Actions of PDE4 inhibitors in follicle cells enhance effects of LH on oocyte maturation, whereas PDE3 inhibitors act directly in oocytes, increasingcAMP and suppressing meiotic progression. MPF controls both the entry and exist from M - phase and is a complex of Cdc2 and cyclinB. The activity of MPF is regulated not only by formation of the complex but also by phosphatization and de phosphatization of the Cdc2 subunit.Our study shows that PDE axtivity exist fluctuate during mouse zygote development step up in G2 phase and decrease in M phase o we investigated PDE3A distribute in mouse ovaries by immunohistochemistry method , the result shows that PDE3A was expressed in primary oocyte of primordial follicle but not in oocyte of growing follicle ^vesicula graafianae and granular cell in mouse ovaries tissue .ConclutionThe PDE activity step up at G2 - phase and decreases rapidly on existing from M - phase during the first mitosis.The PDE3A was expressed in primary oocyte of primordial follicle but not in oocyte of growing follicle ^vesicula graafianae and granular cell in mouse ovaries tissue .