| Since Mattioli et al researched on pig oocytes in vitro maturation (IVM) for the first time in1988, in the efforts of many researchers, porcine oocyte IVM system has been greater improved and optimized, but the effect is still poor.There are many reasons which influence the effect of oocyte IVM, such as:the basal culture solution component of IVM;The added components in IVM system:hormones and their concentrations, cytokines, follicular fluid, antioxidants and vitamins, etc; follicle diameter size; the presence and the number of cumulus cells around oocytes; the temperature, gas condition and the time of IVM,and so on.The maturation of oocyte nucleus and cytoplasm is often not at the same time, which is one of the key factors leads to IVM less effective.In order to promote the porcine oocyte nucleus and cytoplasm synchronized maturation process, improve the efficiency of porcine oocyte IVM, this study for the first time designs FSH receptor overexpressed in granulosa cells(GCs) in IVM,uses Lentiviral vector with new fluorescent protein gene mediate FSH receptor gene transfection in GCs, attains that FSH receptor overexpression stably and long-term in GCs cytomembrane.Transfected GCs co-cultured with oocytes to IVM, by the analysis of the nucleoplasm mature process, clarify the adjustment mechanism of FSH receptor overexpression in GCs membrane in pig oocyte IVM, and to improve the IVM effect of pig oocytes. Specific steps are as follows.1. Clone of pig FSHR and the construction of CSII-EF-FSHR-IRES2-VenusBy the application of Trizol reagent method, we successfully extract swine ovarian tissue total RNA, reverse transcript cDNA and amplify FSHR. First connect FSHR to pMD19-T vector, Then FSHR gene is digested down use incomplete digestion method and connect to the lentiviral vector CS II-EF-MCS-IRES2-Venus. Successfully construct lentiviral recombinant expression vector CS II-EF-FSHR-IRES2-Venus.2. Porcine granulosa cells isolated and incubated in vitro Extract pig ovarian2-6mm diameter follicles’follicular fluid to a sterile centrifuge tube, after centrifugation, digested with0.2%hyaluronidase PBS, washed with PBS and digested with0.25%trypsin steps,put the cells to the cell culture dish. After4days, Granulosa cells can completely covered the dish. Enlarged by passage culture once, frozen in liquid nitrogen for use.In this experiment, We successfully isolate and purify pig primary granulosa cells.The granulosa cells grew strong, in line with the requirements of the next step experiment.3. Lentivirus packagingCSⅡ-EF-FSHR-IRES2-Venus, pCAG-HIVgp and pCMV-VSV-G-RSV-Rev plasmids were co-transfected293T cells, and packaged in293T cells with infectious Lentiviral Particles. The cell suspension is filtered and infect293T cells, to detect the infection effect.4. Electroporation method transfect CS Ⅱ-EF-FSHR-IRES2-Venus to porcine follicular granulosa cells and in vitro co-culture with porcine oocytesElectroporation method transfect CSⅡ-EF-FSHR-IRES2-Venus to porcine follicular granulosa cells and in vitro co-culture with porcine oocytes, by counting the first polar body discharge rate and fertilized oocyte cleavage rate compared with the control groups, clarify the adjustment mechanism of FSH receptor overexpression in GCs membrane in pig oocyte IVM.5. Result:when FSH and LH at a low concentration (0.01U/ml),the effect of Experimental group (granulosa cells transfected CS Ⅱ-EF-FSHR-IRES2-Venus co-culture with oocytes) is significantly higher than control groups (transfected CS Ⅱ-EF-MCS-IRES2-Venus granulosa cells group and no granulosa cells co-cultured group).But in high concentrations of the hormone (O.1U/ml), the oocytes maturation rate is less effective than control groups. In addition, oocytes co-cultured with granulosa cells,the maturation rate is higher than that without granulosa cell co-culture group.This experiment explores a new culture system of porcine oocyte IVM, designed to improve the maturation rate and cleavage rate in porcine oocyte IVM.The implementation of this project have certain promotion role on oocyte IVM,embryo engineering, animal cloning and transgenic animal production. |
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