Induction Of Mouse Embryonic Stem Cells Into Germ Cell Like Cells By Kidney Tonic Chinese Traditional Medicine

Objective:To investigate the influences of monomer of kidney-supplementing herbs and rat serum containing herbal formulas on mouse embryonic stem cells induction to committed oocyte differentiation.Methods:1、The influence of OA on the differentiation of mESC to the ooctyeMouse embryonic stem cells (mESC) of three strains of mice were applied, including R1/E,1B10and D3. They were cultivated and induced to embryoid bodies (EB), with intervention of1%o oleanolic acid, and1‰retinoic acid as the control, and1‰dimethyl sulfoxide as the blank. All cells were cultured under the condition of37℃and5%CO2. Morphological alterations were observed by the microscope. Then cellular RNA was extracted three days later and cDNA was synthesized. Finally, expressions of reproduction associated genes, including Oct4, Gdf9, Scp3, Mvh, Zp1, Zp2and Zp3were measured by Real-time Quantitative PCR (qPCR).2、The influence of medicated serum on the differentiation of mESC to the ooctyemESC cells of1B10were cultured. The EB were induced after the proliferation and intervened with5%1#,2#,3#,5#,10#medicated serum, with5%normal serum as the blank and non serum as the control. They were cultured under the condition of37℃and5%CO2. The medium was changed every three days and cells in all groups were collected. Their morphological alterations were observed by the microscope. Finally, cellular immunofluorescence technique was applied to reveal if the expressions of oocyte marking proteins including GDF9, SCP3and ZP3did existed in cells after medicated serum intervention. Gene expressions including Oct4, Nanog, Prdm14, Blimp1, Tfap2c, Gdf9, Scp3and Zp3were measured by qPCR, three, five, eight, eleven days later each after interventions by herbal serums. Meanwhile, values of germ cell markers of GDF9, SCP3and ZP3were tested by Enzyme Linked Immunosorbent Assay (ELISA), five, eight, eleven, fourteen days later each after interventions by herbal serums.All data were processed by SPSS17.0x±s was applied when data was indicated either normal distribution or homogeneity of variance, and data was tested by Chi-square method. Results:1、The experimental results of direction induction of OACompared to DMSO, oleanolic acid monomer increased Scp3, Mvh, Zpl, Zp2gene expressions in Rl/E (P<0.01), Gdf9, Mvh, Zp2, Zp3gene expressions in1B10(P <0.05), Oct4, Gdf9,Scp3, Mvh, Zpl, Zp3gene expressions in D3(P<0.05), and promoted the differentiation of R1/E,1B10and D3to germ cells. While, retinoic acid monomer increased Mvh gene expressions in R1/E (P<0.01), Gdf9, Mvh, Zp3gene expressions in1B10(P<0.01), and decreased all gene expressions in D3.2、The experimental results of direction induction of medicated serum2.1The appearance of oocyte-like structures was found in all five herbal groups, blank group and control group.2.2After intervention by rat serum containing kidney-supplementing herbs, expressions of oocyte marker GDF9and ZP3as well as meiotic marker SCP3were tested in EB. Otherwise, there was no expression in the blank and control groups.2.3Compared to blank group, gene expressions including Prdm14, Blimpl, Tfap2c and Scp3were significantly raised three days later after differentiation by the intervention of1#medicated serum (P<0.01). The elevated gene expressions including Gdf9, Scp3and Zp3were manifested by active differentiated stage five to eight days later after differentiation by interventions of3#,5#,10#medicated serums (P<0.05). All gene expressions were significantly raised to peak values, and Prdm14, Blimpl, Gdf9, Scp3gene expressions were promoted (P<0.01), eight days later after differentiation by the intervention of2#medicated serum. All gene expressions were reduced more or less with prolonged medicated serum interventions.2.4Compared to blank group, tests of protein values of intercellular GDF9, SCP3, and ZP3in various intervals indicated that GDF9decreased significantly at5d except in2#group (P<0.05), SCP3cellular protein content decreased significantly at8d in3#group and8d,11d,14d in2#group (P<0.01), ZP3cellular protein content increased significantly in3#group and at14d in5#group.Conclusion:1. Both OA and medicated serum of herbal formulas have actions to induct differentiation of mESC to ooctye.2. OA monomer might not be suitable for long-term intervention in vitro since the direction induction by OA is working rapidly in the beginning but with rapid cell apoptosis. However, direction induction by medicated serum containing kidney-supplementing herbs is working slowly but with stable action. Therefore, the Chinese herbal application might be significant.