The Activity And Experssion Of Protein Kinase B In Early Development Of Mouse Fertilized Eggs

Protein phosphorylation/dephosphorylation modification is a key event in the regulation of cell division. In dividing eukaiyotic cells, entry into mitosis is governed by the M - phase - promoting factor ( MPF). This factor consists of the cdc2 protein kinase and cyclin B and acts by phosphorylating substrates that are essential for the execution of mitotic progression. Before mitosis, the activity of cdc2/cyclinB Histone H1 kinase is suppressed through inhibitory phosphoryla-tion of Tyr15 and Thr14 residues of cdc2 by the Weel and Myt kinase, at mitosis , the complex is dephosphorylated and activated by Cdc25 , a phosphatase responsible for dephosphorylation of Cdc2 at Tyrl5. Cdc25 undergoes serine/thre-onine phosphorylation at mitosis and enhances its phosphatase activity. MPF phosphorylates and actives Cdc25, which in turn dephosphorylates and further activates preMPF.Protein kinase B ( PKB, also named kinase AKT) is newly found as a ser-ine/threonine protein kinase. Its well established PKB play an important role in cellular processes such as glucose metabolism, cell proliferation, apoptosis, and transcription and cell migration. There are recent indications that PKB could have a wide spread function in cell cycle regulation, but little is known about the effect of PKB on the regulation of mammalian cell cycle, especially the mouse fertilized eggs. The mouse fertilized egg is the most simple and natural mammalian model of cell cycle, there are few reports about the mechanism on the regulation of early development of mouse fertilized eggs especially the events of entry into M - phase.Its the first time we report here that PKB exists and effects on G2/M phase transition in mouse one - cell stage fertilized eggs. The activity and expression of PKB increased in G2/M phase, the mRNA level also detectable contract to M II oocytes. Microinjection of LY294002 and SAODN oligodeoxynucleotide of PKB inhibits the embryo cleavage. Both cdc2 and cdc25 kinase activity were inhibi-ted by LY294002 and SAODN oligodeoxynucleotide of PKB, which indicate that PKB may act as an initiator of G2/M phase transition through the phosphoryla-tion of cdc25c at Ser216.MaterialsAnimalKunming genealogy mouse were provided by the laboatory animal department of China Medical University. Female with 4W (18g) and male with 8W (30g).AbtibidiesGoat anti - PKB polyclonal antibodies, antiphospho - Tyr15 of cdc2 and antiphospho - Ser216 of cdc25C polyclonal antibodies, alkaline phosphatase conjugated anti-goat secondary antibodies were purchased from Santa Cruz Biotechnology.Polyclonal anti - cdc2 antibodies, anti - cdc25C monoclonal antibodies and peroxidase - conjugated horse anti goat IgG were purchased from sigma chemical Co. (Stlouis, MO)Reagents[a-32P] dCTP and [r-32P] ATP were purchased from YaHui Biotech-nology Co. Ltd, China. UNA extraction kit was purchased from Promega. RT -PCR kit was from Takara technology Co. Ltd.The sequence of the primer of PKB are shown below, together with all the SODN and SAODN oligodeoxynucleotide were synthesized in Takara technology Co. Ltd.PKB primers; forward primer 5'CGA GAA GCC GCG ACC CAA CAC3'and reverse primer 5ATG CAC TAC CGC CGG A3'.b-actin primers: Forward 5TCC TAT CGG CCT CCT CCT AC 3' and reverse 5AGA ATG TAG TCC GCC AGG TC 3', designed for 200bps.SAODN oligodeoxynucleotide of PKB: STAC AGC CTG AGA GGA CCT ACC TG3'.SODN oligodeoxynucleotide of PKB: 5CAC ATG GAC GTC AAA GCT TCC AA3'each base of both SODN and SAODN oligodeoxynucleotide was sulfurated.Methods1. Superovulation, natural mating and eggs collection.Female mice 4-6 weeks old were intraperitoneally injected with 10IU of pregnant mares serum gonadotropin (PMSG) , and 48h later with 10IU human chorionic gonadotropin (HCG). A single female was placed with a single male for fertilization. One - cell embryos were collected with M2 medium the next day (24h after HCG injection) from oviduct of females possessing a vaginal plug. Then embryos were transferred to M16 for culture...