Effect of icariin on the transformation efficiency of induced pluripotent stem cells into sperm cells in vitro

Abstract

Objective

To investigate the effect of icariin on the transformation efficiency of germ cell-like cells from mouse induced pluripotent stem cells into sperm cells in vitro.

Methods

Firstly, mouse induced pluripotent stem cells were induced and cultured to transform into germ cell-like cells, and the primordial germ cell-like cells were identified by Western blot and RT-PCR. Then, different concentrations of icariin (0.1 μg/mL, 1 μg/mL, 10 μg/mL and 100 μg/mL) were added into the culture medium, and the obtained primitive germ cell-like cells were cultured, Western blot and RT-PCR were used to identify the obtained sperm cells, the transformation efficiency was compared.

Results

The primordium germ cell-like cells obtained from mouse induced pluripotent stem cells in vitro specially expressed Oct-4 protein, C-kit protein, Mvh mRNA, Fragilis mRNA and Stella mRNA. The sperm cells were specially expressed VASA, SCP3 and γH2AX proteins. RT-PCR showed that the sperm cells were specially expressed Ddx4, Tp2 and Prm1 mRNA. Compared with the control group, the expression level of VASA protein (1.744 ± 0.283, 2.882 ± 0.373, 6.489 ± 0.460), SCP3 protein (2.250 ± 0.306, 7.058 ± 0.521, 8.654 ± 0.804), γH2AX protein (4.304 ± 0.433, 5.713 ± 0.339, 9.268 ± 0.545), Ddx4 mRNA (1.374 ± 0.145, 2.846 ± 0.194, 4.021 ± 0.154), Tp2 mRNA (1.358 ± 0.130, 3.623 ± 0.326, 5.811 ± 0.390) and Prm1 mRNA (1.326 ± 0.162, 3.487 ± 0.237, 4.666 ± 0.307) in 0.1 μg/mL, 1 μg/mL, 10 μg/mL icariin experimental groups were all lower than that of VASA protein (10.560 ± 0.413), SCP3 protein (13.804 ± 0.642), γH2AX protein (11.874 ± 0.464), Ddx4 mRNA (6.4005 ± 0.361), Tp2 mRNA (7.314 ± 0.256) and Prm1 mRNA (7.334 ± 0.390) in 100 μg/mL icariin experimental group.

Conclusions

Icariin can promote the transformation of mouse induced pluripotent stem cells into sperm cells in vitro, and it is concentration-dependent manner in a certain concentration range.

Resumen

Objetivo

Investigar el efecto de icariina en la eficiencia de la conversión in vitro inducida en espermatozoides de cultivos de células germinativas derivadas de la transformación de células madre pluripotentes inducidas de ratón.

Métodos

Primero se indujeron y cultivaron células madre pluripotentes inducidas de ratón para transformarlas en células similares a las células germinales, y las células similares a las células germinales primordiales se identificaron mediante Western blot y RT-PCR. A continuación, se añadieron diferentes concentraciones de icariina (0,1 μg/mL, 1 μg/mL, 10 μg/mL and 100 μg/mL) al medio de cultivo, y se cultivaron las células primitivas similares a células germinales obtenidas, se utilizaron Western blot y RT-PCR para identificar las células espermáticas obtenidas, y se comparó la eficacia de la transformación.

Resultados

Las células germinales primitivas obtenidas in vitro a partir de células madre pluripotentes inducidas de ratón expresaron especialmente la proteína Oct-4, la proteína C-kit, el ARNm de Mvh, el ARNm de Fragilis y el ARNm de Stella. Los espermatozoides expresaban especialmente las proteínas VASA, SCP3 y γH2AX. La RT-PCR mostró que los espermatozoides expresaban especialmente los ARNm Ddx4, Tp2 y Prm1. En comparación con el grupo de control, el nivel de expresión de la proteína VASA (1,744 ± 0,283; 2,882 ± 0,373; 6,489 ± 0,460), la proteína SCP3 (2,250 ± 0,306; 7,058 ± 0,521; 8,654 ± 0,804), proteína γH2AX (4,304 ± 0,433; 5,713 ± 0,339; 9,268 ± 0,545), ARNm Ddx4 (1,374 ± 0,145; 2,846 ± 0,194; 4,021 ± 0,154), ARNm Tp2 (1,358 ± 0,130; 3,623 ± 0,326; 5,811 ± 0,390) y ARNm Prm1 (1,326 ± 0,162; 3,487 ± 0,237; 4,666 ± 0,307) en grupos experimentales de 0,1 μg/mL, 1 μg/mL, 10 μg/mL de icariina fueron todos más bajos que los de la proteína VASA (10,560 ± 0,413), proteína SCP3 (13,804 ± 0,642), proteína γH2AX (11,874 ± 0,464), ARNm Ddx4 (6,4005 ± 0,361), ARNm Tp2 (7,314 ± 0,256) y ARNm Prm1 (7,334 ± 0,390) en 100 μg/mL icariina grupo experimental.

Conclusiones

La icariina puede promover la transformación de células madre pluripotentes inducidas de ratón en células de esperma in vitro, y es de manera dependiente de la concentración en un determinado rango de concentración.