Aconitine primarily exists in aconite tubers

onstrated that xRic8 is maternally expressed in amphibian’s oocytes where it participates in the maintenance of meiotic arrest. However, the role of mammalian RIC8 in these complicated processes is unknown so far. The present study addressed the potential function of RIC8 in mammalian oogenesis by characterizing its expression and localization pattern during the oocyte growth and meiotic maturation, as well as fertilization and first zygotic cleavage process. We demonstrate that the localization of maternally expressed RIC8 protein is highly dynamic and is dependent on the stage of folliculogenesis, oogenesis and cleavage. In addition, downregulation of Ric8 expression by siRNA in maturing oocytes leads to reduced translocation of Gi to cortical region of cells. Our findings imply that RIC8 may have a regulatory function in mammalian gametogenesis. Materials and Methods Animals Throughout the present study wild-type C57Bl/6J mice were used. Animals were maintained under a 12 h light/12 h dark cycle and at temperature of 21C with food and water available ad libitum. The permission for the present study was given by the Estonian National Board of Animal Experiments in accordance with Directive of the Council of the European Communities of 24 November 1986. Immunohistochemistry Dissected ovaries and reproductive tracts of sacrificed adult female mice were fixed in 4% paraformaldehyde in PBS for 20 min and cryoprotected in 20% sucrose in PBS. Tissue sections with a thickness of 10 m were cut from frozen specimens embedded in tissue freezing medium Jung. Cryosections were mounted on Superfrost plus slides, dried PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19705642 overnight, permeabilized with 0.1% Triton X-100 in PBS for 20 min and blocked for 60 min at room temperature with 1% BSA and 5% goat serum in PBS. Proteins were revealed by incubation with rabbit polyclonal anti-RIC8 and anti-beta tubulin antibody overnight at 4C followed by Alexa Fluor 594 goat anti-rabbit or Alexa Fluor 633 goat anti-mouse secondary antibody for 60 min at room temperature. Cell nuclei were counterstained with DAPI and specimens mounted in Fluoromount G. For negative controls, primary antibodies were omitted PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19704080 and no staining was observed. Harvesting of Oocytes and One-cell Embryos To analyze progression of meiosis I, 4 week-old female mice were superovulated by injecting 5 IU of equine chorionic gonadotropin and 5 IU of human chorionic gonadotropin at an interval of 48 hours. The females were sacrificed and the ovaries were dissected in 4 to 10 hours after hCG injection. Oocytes were MedChemExpress 518303-20-3 harvested by puncturing ovarian follicles with sterile needle. Surrounding cumulus cells were removed with hyaluronidase solution by gentle pipetting. To study meiosis II and first zygotic division, female mice at age of 1011 weeks were mated. Fertilized oocytes were harvested from the oviducts at approximately 40 min to 20 h after the detection of the vaginal plug and were treated with hyaluronidase. 3 / 19 Dynamics of RIC8 in Oogenesis Harvesting of Oocytes and Microinjection 4 week-old female mice were superovulated by injecting 5 IU of eCG. The females were sacrificed and ovaries were dissected in 48 hours after eCG injection. Ovaries were placed in M2 medium and punctured several times with sewing needles that are fastened together. Oocytes were collected and cumulus cells were removed by pipetting several times. Cumulus free oocytes were transferred to KSOM medium that contained 0,2 mM 3-isobutyl1-methylxanthine , an inhibitor of c