| PrefaceCell multiplication is an elementary character of cell life. In eukary-otic cell, MPF is a key factor of G2/M transition for mitosis and meiosis. Cyclic AMP activating PKA pathway is one of the most important signal transduction pathways, which have an effect on metabolic regulation and cell proliferation. The Cyclic AMP— dependent protein kinase A, as a downstream signaling element of cAMP pathway, plays an important negative role in cell proliferation. In Xenopus embryonic extracts, oscillation in cAMP concentration and PKA activity accompany cell cycle progression, they are low during mitosis, increase at M/G1 transition, and then remain at high level until the next mitosis. High PKA activity depresses MPF activity, while inhibition of PKA activiation can enhance the MPF activity.Our team found that, in one—celll stage mouse fertilized eggs, MPF activity increased during M phase and decreased during interphase. In another hand,cAMP concentration and PKA activity also oscillated , high in G2 phase and reduced during mitosis. But little is known about the regulation of cAMP level in mouse fertilized eggs. The level of cAMP is maintained by synthesis, via adenylate cyclase, and degradation via phosphod-iesterases. In this article, we examined the expression of PDE3A gene in different level by RT—PCR and Western blotting respectively, and investigated the effect of cilostamide , a specific phosphodiesterase inhibitor, on the division percentage of 1 — cell stage fertilized eggs.Materials1. Kuming strain mice were supplied from the Department of Laboratory Animals China Medical University2. Pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) were obtained from Tianjin Huafu Biological Products Research Institute and Shanghai Products Research Insititute, respectively.M2 medium, Ml6 medium, Hyaluronidase, DEPC and Cilostamide from Sigma BiotechnologyAnti—PDE3A mouse polyclonal antibody from Santa Cruz BiotechnologyAnti—goat IgG secondary antibody from Santa Cruz Biotechnology RNA PCR (AMV) Kit from TAKARA Biotechnology QuickPrepTM Micro mRNA Purification Kit from Amersham DMSO was purchased from Beijing Zhongshan BiotechnologyMethods1. Superovulation and collection of eggsFor superovulation, female Kunming strain mice 3 — 5 weeks old were injected with pregnant mare serum gonadotropin (PMSG) and after 46 — 48 hours with human chorionic gonadotropin (hCG). One— cell embryos were collected with M2 medium from the oviducts of females possessing a vaginal plug. The embryos were then cultured in M16 medium at 37 °C in a humidified atmosphere of 5% CO2 in air. According to the time point, embryos of different phase were collected.2. Reverse Transcription—Polymerase Chain ReactionPoly (A)+RNAs from mouse fertilized eggs were extracted using QuickPrep? Micro mRNA Purification Kit following the manufacturer's instructions. First — strand cDNA synthesis and reverse transcription —polymerase chain reactions were performed using RNA PCR(AMV)Kit following the manual . Products were analyzed on 1. 2% angarose gels stained with ethidium bromide.3. Collected embryos remained at the one—cell stage, 27hr after human chorionic gonadotropin,mRNA and cultured in Ml6 medium with or without cilostamide for another 3 hours. Then the percentage of division was calculated by observing the embryos under a dissecting microscope.Results1. We employed RT—PCR to identify the presence of PDE3A and to investigate relative quantity of PDE3A mRNA expressed in each phase of one—cell stage mouse embryos. Using primer pairs specific for PDE3A, we confirmed the presence of PDE3A mRNA in one — cell stage mouse fertilized eggs. By contrast, RNA from G2 phase and M phase embryos consistently revealed strong RT — PCR bands for PDE3A. Clear significant differences (P |
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