Role of MT transposon like-element, clone 7 (MTi7) in the mouse oocytes and embryos studied by RNA Interference (RNAi)

Abstract

Objective: Specific mRNA degradation by sequence specific double-stranded RNA (dsRNA), namely RNAi is a useful method with which to study gene function. Previously, we found that the MTi7 was one of the highly expressed genes in the primary follicles, and the MTi7 mRNA expression was oocyte-specific. To determine the role of MTi7 in the folliculogenesis, dsRNA targeting MTi7 was produced. To evaluate whether the MTi7 dsRNA is working, it was microinjected into the mouse oocytes and embryos. The present study was conducted to determine the role(s) of MTi7 in the mouse oocytes and embryos by RNAi.Design: Experimental study using RNAi, semi-quantitative RT-PCR, and immunofluoresence staining.Materials and Methods: The dsRNAs were prepared by in vitro transcription and annealing of the equimolar amounts of the sense and antisense single stranded RNAs followed by purification of the dsRNA. A gene with known function, c-mos dsRNA was used as a control for injection into the oocytes with germinal vesicle (GV). Purified MTi7 dsRNA was microinjected into the GV, zygotes with pronucleus (PN), and 2-cell embryos. Changes in the mRNA expression after dsRNA injection was measured by semi-quantitative RT-PCR 18–20 hours after microinjection. Changes in morphology were observed under the microscope, and changes in the intracellular organelles were analyzed by immunofluoresence staining.Results: MTi7 expressed in GV, MII, and 2-cell embryos and reduced thereafter. The relative amount of c-mos or MTi7 compared to that of control was determined after microinjection. We found 60%-90% decrease in c-mos and MTi7 mRNA, in control and experiment group, respectively. The MTi7 dsRNA-microinjected GV oocytes were alive and showed several shapes such as GVBD, PN, and uneven cytoplasmic distribution. Some oocytes had polar body-like structure in the perivitelline space. By propidium iodide and tubulin staining, arrested chromosomes before division, several nuclei, and uneven distribution of tubulin was observed. However, almost all MTi7 dsRNA-microinjected PN and 2-cell embryos were arrested at that stage, while medium-injected control embryos developed to the next stages.Conclusion: We report here that RNAi provides an outstanding approach to study the function of a gene with unknown functions. We concluded that the MTi7 is a gene related to the cell division in the early stage embryos, but it seems likely that the MTi7 has different roles in the oocytes compared to the embryos. Objective: Specific mRNA degradation by sequence specific double-stranded RNA (dsRNA), namely RNAi is a useful method with which to study gene function. Previously, we found that the MTi7 was one of the highly expressed genes in the primary follicles, and the MTi7 mRNA expression was oocyte-specific. To determine the role of MTi7 in the folliculogenesis, dsRNA targeting MTi7 was produced. To evaluate whether the MTi7 dsRNA is working, it was microinjected into the mouse oocytes and embryos. The present study was conducted to determine the role(s) of MTi7 in the mouse oocytes and embryos by RNAi. Design: Experimental study using RNAi, semi-quantitative RT-PCR, and immunofluoresence staining. Materials and Methods: The dsRNAs were prepared by in vitro transcription and annealing of the equimolar amounts of the sense and antisense single stranded RNAs followed by purification of the dsRNA. A gene with known function, c-mos dsRNA was used as a control for injection into the oocytes with germinal vesicle (GV). Purified MTi7 dsRNA was microinjected into the GV, zygotes with pronucleus (PN), and 2-cell embryos. Changes in the mRNA expression after dsRNA injection was measured by semi-quantitative RT-PCR 18–20 hours after microinjection. Changes in morphology were observed under the microscope, and changes in the intracellular organelles were analyzed by immunofluoresence staining. Results: MTi7 expressed in GV, MII, and 2-cell embryos and reduced thereafter. The relative amount of c-mos or MTi7 compared to that of control was determined after microinjection. We found 60%-90% decrease in c-mos and MTi7 mRNA, in control and experiment group, respectively. The MTi7 dsRNA-microinjected GV oocytes were alive and showed several shapes such as GVBD, PN, and uneven cytoplasmic distribution. Some oocytes had polar body-like structure in the perivitelline space. By propidium iodide and tubulin staining, arrested chromosomes before division, several nuclei, and uneven distribution of tubulin was observed. However, almost all MTi7 dsRNA-microinjected PN and 2-cell embryos were arrested at that stage, while medium-injected control embryos developed to the next stages. Conclusion: We report here that RNAi provides an outstanding approach to study the function of a gene with unknown functions. We concluded that the MTi7 is a gene related to the cell division in the early stage embryos, but it seems likely that the MTi7 has different roles in the oocytes compared to the embryos.