Investigation of the control mechanism of gene coding glucose metabolic protein in preimplantation embryo

2002 Fiscal Year Final Research Report Summary

• Project/Area Number: 12671632
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Obstetrics and gynecology
• Research Institution: Teikyo University, School of Medicine
• Principal Investigator: AYABE Takuya Teikyo University School of Medicine,Dept of Obstetrics and Gynecology Associate Professor, 医学部, 助教授 (00272568)
• Co-Investigator(Kenkyū-buntansha): ANDO Noriyuki Teikyo University School of Medicine Dept. of Obstetrics and Gynecology Instructor, 医学部, 助手 ; TAKAHASHI Shin-ichiro Teikyo University School of Medicine Dept. of Obstetrics and Gynecology Instructor, 医学部, 助手 ; KIDO Koichiro Teikyo University School of Medicine Dept. of Obstetrics and Gynecology Instructor, 医学部, 助手 (40312003) ; ARIKI Saori Teikyo University School of Medicine Dept of Obstetrics and Gynecology Instructor, 医学部, 助手
• Project Period (FY): 2000 – 2002
• Keywords: in situ PCR / in situ RT-PCR / follicle growth / polycystic ovary syndrome

Research Abstract

We intended, at first, to investigate the control mechanism of gene coding protein which play a role on glucose metabolism in preimplantation embryo. As a method of analysis, we utilized in situ hybridization. In order to analyze a sample for this method, an egg should be prepared with cryostat. This step was, however, difficult and could not achieved because an egg was so small. So, we changed our object from an egg to a follicle. In a menstrual cycle, many follicles are recruited at once but only one follicle matures, and remaining go into atresia by apoptosis.
Up to now gene working on apoptosis of follicles were analyzed using a whole ovary. In this study, we investigated the gene in a follicle level. Mouse ovary was collected, then was frozen in OCT compound Cryostat sections of 4-10 micrometer were collected onto pre-coated slideglass. Samples were pre-treated with proteinase K in order to get penetration of the PCR primers into the cell and allow the target sequences to be accessed for amplification. The permeabilization that allows the primers to penetrate the cell must be carefully controlled so that tissue morphology is maintained and the larger products of amplification do not diffuse out Primers for Fas receptor gene were used and a target in 374 bp was amplified by in situ PCR. Two-step reaction program was used and 10 cycles achieved. Digoxygenin(DIG) was incorporated into dNTP, and the amplified product was detected using anti-DIG antibody. Fas receptor gene was detected, and it gathered in inner layer of a follicle. This result may be achieved by in situ hybridization method, but through in situ PCR method we may be able to achieve an investigation of messenger RNA by in situ RT-PCR method.
In polycystic ovary syndrome, not one but many follicles grow up at once, so we suppose apoptosis mechanism is deteriorated in this syndrome, Further study may reveal the pathogenesis of this syndrome.