Evaluation of novel mouse specific germ cell gene expression in embryonic stem cell- derived germ cell-like cells in vitro with retinoic acid treatment

Background: Defects in natural and complete meiosis are one of the obstacles in achieving functional gametes. Retinoic acid (RA) induced intracellular signals also play an important role in germ cell differentiation.Objective: The goal of the present study was to evaluate an in vitro differentiation model of mouse embryonic stem cells into germ cells, using RA.Materials and Methods: There are three groups in this experimental study. 1-ESCs differentiated using EB method for 7 days then EB aggregation singled and cultured with RA for 7 days as (+RA) 2- EB aggregation in day 7 singled and cultured without RA for 7 days as control group (-RA) 3- EB aggregation in day 7 of culture as (EB7). We designed a study to induce differentiation of Oct4-GFP (expression of Green Fluorescent Protein of oct4) embryonic stem cells (ESCs) by embryoid body (EB) culture system into germ cells using RA and evaluated the expression level of (Fkbp6, Mov10l1, 4930432K21Rik, Tex13) in differentiated cells. The expression levels of 4 GC-related genes, Oct4, Mvh, Scp3 and Stra8 was determined by q-RT-PCR. Immunostaining and Flowcytometry used as additional test to confirm q-RT-PCR findings.Results: Significant increase occurred in expression of meiotic markers and specific genes Fkbp6 (p=0.00), Mov10l1 (p=0.01) and Tex13 (p=0.00) in ESCs treated with Retinoic Acid (+RA) compared to the controls (-RA). Oct4 expression was decreased in all studied groups. The expression levels of 4930432K21Rik, Mvh, Stra8 and Scp3 in the +RA group was higher than that of the -RA group. Flowcytometry analysis showed that mean number of Mvh-positive cells in the +RA group was greater as compared with ESCs, -RA and EB7 groups (p=0.00). Immunofluorescence staining showed that higher Mvh expression staining was observed in the +RA group, compared to that of the -RA group, which confirmed the data from the q-RT-PCR. The cells characterizing GCLCs with round nuclei are shown by Hoechst nuclei counterstaining.Conclusion: These 4 specific germ cells genes are expressed in the testis, but not the ovary, which indicates their role in the development of male gametes. According to the findings, down regulation of Oct4 as a pluripotency factor as well as the expression of meiosis markers this hypothesis is raised that ESCs are differentiated by RA, and have been introduced into the zygote / pachytene of first meiosis as germ cell-like cells. So induction of mESCs by RA may cause differentiation towards GCLCs. Improving in vitro germ cell differentiation with high efficiency may simplify the generation of mature gametes for understanding of biology of gametogenesis.

کلیدواژه ها:

Embryonic stem cells ، Primordial germ cells ، Retinoic acid ، Embryoid body