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. 2017 Sep 12;7(1):11441.
doi: 10.1038/s41598-017-11790-0.

A Phytochemical Approach to Promotion of Self-renewal in Murine Spermatogonial Stem Cell by Using Sedum Sarmentosum Extract

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Free PMC article

A Phytochemical Approach to Promotion of Self-renewal in Murine Spermatogonial Stem Cell by Using Sedum Sarmentosum Extract

Sang-Eun Jung et al. Sci Rep. .
Free PMC article

Abstract

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis, which is dependent on the ability to self-renew and differentiation. Controlling self-renewal and differentiation of SSCs could apply to treatment of disease such as male infertility. Recently, in the field of stem cell research, it was demonstrated that effective increase in stem cell activity can be achieved by using growth factors derived from plant extracts. In this study, our aim is to investigate components from natural plant to improve the self-renewal of SSCs. To find the components, germ cells were cultured with comprehensive natural plant extracts, and then the more pure fraction, and finally single compound at different concentrations. As a result, we found 5H-purin-6-amine at 1 µg/mL, originated from Sedum sarmentosum, was a very effective compound induced SSCs proliferation. Our data showed that germ cells cultured with 5H-purin-6-amine could maintain their stable characteristics. Furthermore, transplantation results demonstrated that 5H-purin-6-amine at 1 µg/mL increased the activity of SSCs, indicating the compound could increase true SSC concentration within germ cells to 1.96-fold. These findings would be contributed to improve further reproductive research and treat male infertility by using natural plant extracts.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Evaluation of germ cell proliferation cultured with natural plant-derived extracts. Total 11 natural plant derived extract were used in cell culture medium at concentrations of 0.1, 1, or 10 µg/mL to measure the proliferation of cultured germ cells after 1 week of exposure. Values are mean ± SEM (n = 3 established independent cultures for each treatment). Asterisk indicates significant difference (P < 0.05) compared to the control.
Figure 2
Figure 2
Comparison of germ cell proliferation rates between groups treated with Sedum sarmentosum fractions. Relative proliferation rates were evaluated compared to the control by counting the cells after 1 week culture with different S. sarmentosum fractions. Proliferation effect on germ cells after culture with four fractions from S. sarmentosum at concentrations of 0.1, 1, or 10 µg/mL. Values are mean ± SEM (n = 4). Cont, control; He, n-hexane fraction; MC, methylene chloride fraction; EA, ethyl acetate fraction; Bu, n-butanol fraction. A statistically significant difference (P < 0.05) is displayed as different letters at the top of the column.
Figure 3
Figure 3
Effect of Sedum sarmentosum compounds on germ cell proliferation. (A) Chemical structure of compounds from n-butanol fraction of Sedum sarmentosum. (B) Proliferation effect on germ cells after culture with compounds at concentrations of 0.01, 0.1, 1, or 10 µg/mL. (C) Proliferation capacity of germ cells treated by 5H-purin-6-amine 1 μg/mL (Blue, DAPI; Green, GFP; Red, Ki67). Values are mean ± SEM (n = 5). (B) n = 5; (C) n = 3. A statistically significant difference (P < 0.05) is displayed as asterisk at the top of the column. Scale bars; (C) = 100 μm.
Figure 4
Figure 4
Characterization of germ cells cultured with n-butanol fractions or 5H-purin-6-amine. (A) EGFP-positive germ cells enriched for SSCs cultured in a serum-free medium containing 10 ng/mL GDNF, 75 ng/mL GFRα − 1, and 1 ng/mL bFGF in the presence of 1 µg/mL 5H-purin-6-amine. The cells were examined using bright field and fluorescence microscopy. (B) Germ cells cultured in a serum-free medium with 5H-purin-6-amine were immunostained with the markers for undifferentiated spermatogonia (PLZF and GFRα1), germ cell lineage (VASA), and differentiating germ cells (c-Kit), and isotype IgG negative control (Alexa Fluor 568-conjugated goat anti-mouse, Alexa Fluor 568-conjugated donkey anti-rabbit, and Alexa Fluor 568-conjugated donkey anti-goat). (C) Effects of 1 µg/mL Bu and 1 µg/mL 5H-purin-6-amine on Lhx1 (undifferentiated spermatogonia or spermatogonial stem cell marker) and Pgk2 (from meiotic spermatocytes to post meiotic spermatid marker) expression were evaluated using real-time PCR analysis. Bu, n-butanol fraction. Values are mean  ±  SEM (n = 3 established independent cultures for each treatment). A statistically significant difference (P < 0.05) is displayed as different letters at the top of the column. Scale bars: (A,B) = 100 µm.
Figure 5
Figure 5
Analysis of functional activity of SSCs cultured with S. sarmentosum through germ cell transplantation. (A) The dark-field fluorescence image in a recipient testis after transplantation of germ cells cultured with 1 μg/mL 5H-purin-6-amine isolated from S. sarmentosum. In the recipient seminiferous tubules, the colonies derived from donor SSCs were distinctly green. (B) Cryosection of recipient testis showed GFP expressing colonies derived from donor SSCs, indicating complete spermatogenesis in the lumen of seminiferous tubules. Complete spermatogenesis is evidenced by the presence of sperm (white arrow). (C) The number of colonies per 105 cells transplanted with 1 µg/mL Bu or 1 µg/mL 5H-purin-6-amine. (D) The relative number of SSCs recovered after culture with 1 µg/mL Bu or 1 µg/mL 5H-purin-6-amine. The total number of mice/testes used for testis analysis was 8/13, 9/13, and 8/13 in the control (cultured only with GDNF at 10 ng/mL), Bu (cultured with GDNF at 10 ng/mL and 1 µg/mL n-butanol fraction), and 5H-purin-6-amine (cultured with GDNF at 10 ng/mL and 1 µg/mL 5H-purin-6-amine), respectively. Bu, n-butanol fraction. Scale bars: (A) = 2 mm; (B) = 50 µm.

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