The eggs were collected 0.5, 1, 1.5, 2, 4, and 6 h, respectively, after Iodixanol insemination and prepared for confocal microscopy. Microinjection of Pyk2 siRNA In order to investigate the role Pyk2 plays during the development of preimplantation embryos, small strings of siRNA were microinjected into the cytoplasm of the fertilized eggs (obtained as described above). in the pre-blastula cytoplasm. Pyk2 and its activated form then disappeared from the blastula nuclei and localized to the perinuclear regions, where blastula cells come into contact with each other. Pyk2 knockdown via microinjection of siRNA into the zygote did not inhibit early embryo development. Our results suggest that Pyk2 plays multiple functional roles in mouse oocyte fertilization as well as Iodixanol throughout early embryo development. reported Pyk2 activation during the period of anaphase resumption after fertilization. Furthermore, the study used Pyk2 suppression with a highly specific inhibitor, reducing the ability of mouse oocytes to incorporate sperm and proceed into anaphase [20]. These data suggest the involvement of Pyk2 in the both oocyte maturation and fertilization. In this report, antibodies against Pyk2 and tyrosine 402-phosphorylated Pyk2 (p-Pyk2) were used to determine both the distribution and activation patterns of Pyk2 during mouse oocyte fertilization and early embryo development. Both Pyk2 and p-Pyk2 exhibited complex dynamic changes throughout the sperm head, the male and female pronucleus, the cytoplasm, the cell junctions, and the perinuclear region. However, a Pyk2 knockdown via injection of small-interfering RNA into the zygote did not inhibit early embryo development. Materials and Methods Reagents and antibodies Media (M2, M16, and KSOM) were purchased from Sigma Chemical Company (St. Louis, MO). siRNA duplex oligoribonucleotides targeting the coding region of Pyk2 (GenBank Accession no: “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_001162365.1″,”term_id”:”241982786″,”term_text”:”NM_001162365.1″NM_001162365.1) and non-silencing siRNA were obtained from Invitrogen (Carlsbad, CA, USA). Polyclonal rabbit anti-mouse Pyk2 antibodies were purchased from Abcam (Cambidge, England). Polyclonal anti-phosphorylated Tyr402 Pyk2 antibodies were obtained from Sigma (St. Louis, MO) and Abcam. Mito-Tracker Green was obtained from Beyotime Biotechnology (NanTong, China). Pregnant mare serum gonadotropin (PMSG) and human chronic gonadotropin (hCG) were purchased from Sansheng Company (Ningbo, China). Unless otherwise mentioned, all other chemicals used in this study were purchased from Sigma Chemical Company. Oocyte and embryo collection We conducted all Iodixanol experiments using protocols proved by and in accordance with the Animal Research Committee Guidelines of the Shandong Normal University. 10 IU PMSG was injected intraperitoneally into Kunming female mice aged 4 to 6 6 weeks (purchased from Shandong University experimental animal center, Jinan, China). 48 h later, 10 IU hCG was injected using the same procedure. After hCG injection, mice were killed and oviducts were removed at 14 to 16 h. The cumulus-oocyte complexes were collected from the oviducts into M2 medium, supplemented with 60 g/ml penicillin, and 50 g/ml streptomycin. To remove all cumulus cells, MII-arrested oocytes were briefly exposed to 300 IU/ml hyaluronidase followed by three subsequent washes in M2 medium. In order to collect fertilized eggs and embryos, females were superovulated with PMSG and mated with Kunming mature male mice following hCG injection. 19 h after mating, fertilized eggs were obtained from oviducts. Embryos were collected at the following stages of development: time post-hCG: 22 h: zygote; 40 h: 2-cell; 60 h: 4-cell; 68 h: 8-cell; 80 h: morula; 96 h: blastocyst. Early-stage embryos were released from the oviduct and blastocysts were obtained from the uterus. Fertilized eggs were used for siRNA microinjection, while embryos were used for confocal microscopy. In vitro fertilization fertilization was conducted using 1 106/ml motile cauda epididymal sperm that Col11a1 was previously capacitated for 1 h with 2.5 mM taurine in M16 medium. We used ZP-free and MII-arrested oocytes to achieve a more synchronous time of fertilization. Oocytes were inseminated in a 50 l drop of M16 medium at 37oC and 5% CO2. The eggs were collected 0.5, 1, 1.5, 2, 4, and 6 h, respectively, after insemination and prepared for confocal microscopy. Microinjection of Pyk2 siRNA In order to investigate the role Pyk2 plays during the development of preimplantation embryos, small strings of siRNA were microinjected into the cytoplasm of the fertilized eggs (obtained as described above). A minimum of three repetitions was conducted per experiment, and 50 to 80 eggs were used per experimental group. To minimize damage to the eggs, the diameter of the microinjection needle was smaller than 1 m. Throughout all experiments, we used a microinjection volume of about 10 pl diluted siRNA (10 M) per egg. The same amount of non-silencing siRNA was injected as control. Microinjections were performed using an Olympus IX71 (Olympus, Tokyo, Japan) inverted microscope equipped with Narishige IM-9C hydraulic three-dimensional micromanipulators (Narishige, Tokyo, Japan). All microinjections were completed within 30 min. After microinjection, fertilized eggs of the RNAi treatment group and those of the control group were thoroughly washed and cultured in KSOM under mineral oil at 37oC and with 5% CO2. Subsequently, some of the injected eggs were collected after 19 h for RNA extraction, reverse transcription, and real-time.
