Schisandrin B for the treatment of male infertility

The decline of male fertility and its consequences on human populations are important public-health issues. However, there are limited choices for treatment of male infertility. In an attempt to identify a compound that could promote male fertility, we identified and characterized a library of small molecules from an ancient formulation Wuzi Yanzong-Pill, which was used as a folk medicine since the Tang dynasty of China. We found that SB enabled evident repairs in oligoasthenospermia-associated testicular tissue abnormality and in spermatogenesis disruption, resulting in significant improvements of sperm count, mobility, and reproductive ability in oligoasthenospermia mice. Furthermore, SB could alter substantial testicular genes (2033), among which, upregulation of Fst while downregulation of Inhba involved in reproductive signaling pathway could explain its role in enhancing spermatogenesis. The encouraging preclinical data with pharmacokinetics warranted a rapid development of this new class of therapeutic agent. Our finding provides a strong potent drug for treatment of male infertility.


Dear Editor,
The decline of male fertility and its consequences on human populations are severe public-health issues, and oligoasthenospermia is a common cause of male infertility. 1,2 However, the treatment choices for male infertility are limited. 3 Here, we first report that schisandrin B (SB) was screened from Wuzi Yanzong-Pill (WP), which enabled the treatment of male infertility, and uncover the underlying mechanism.
The ancient prescription WP has been widely used for treating oligoasthenospermia since the Tang dynasty of China. However, its active component(s) are still not clear. To find active component(s), we had identified 106 major compounds in WP using UPLC-ESI-LTQ-Orbitrap-MS, 4 and their similarity scores of drug molecular structures were evaluated using MedChem Studio, 22 compounds have higher similarity scores of drug molecular structures, and subsequently, the relative abundances of 22 components were assessed by factor analysis with SPSS ( Figure 1A and B; Supplementary Dataset S1-S3). SB had the highest comprehensive score. To determine its oral availability according to site of action, 3 h after oral administration SB, SB was identified in the plasma and testicular tissues of normal male mice (Figure 1C-I; Figure S1), demonstrating the SB availability in plasma and testicular tissue of mice upon oral administration. Different drugs used to treat a disease usually produce similar gene-profiling signatures, 5 and to verify whether SB had testicular gene (TG) expression similar to WP, we investigated SB involvement in the regulation of TG expression by comparing it with that of WP in an established model of oligoasthenospermia mice (OM). 6 In mice, the expression of 100 of the most upregulated and downregulated TGs (50:50) by WP was compared with the corresponding TGs regulated by SB. Both heatmap and Pearson's correlation analysis revealed that SB and WP had similar TGs signature and were highly correlated (r = 0.735) ( Figure 1J and K; Supplementary Dataset S4), suggesting that SB could be used to treat male infertility.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. To observe the spermatogenic effect of SB, testicular tissues and sperm samples from OM were sampled after oral administration at 2 week, SB had similar spermatogenic effects to WP, and could repair damaged seminiferous tubules and spermatogenic cells in OM testis ( Figure 2A). Moreover, SB and WP could increase the sperm number (concentration), sperm-activity (sperm mobility and progressive mobile sperm), sperm-motion velocities (curvilinear velocity, straight-line velocity, and average path velocity), and improve sperm-motion parameter (straightness, beat cross frequency, amplitude of lateral head displacement, and linearity) (Figure 2B-D; Videos S1-S5; Supplementary Dataset S5). Furthermore, to investigate the reproductive ability, normal mice, OM, OM after treatment with SB or WP for 2 weeks and male mice were mated with female mice at a 1:2 ratio, respectively. Treatment of OM with SB or WP increased the number of pups in the first litter and average number of births, showing fertility close to that of normal mice, respectively ( Figure 2E; Supplementary Dataset S6).
We wished to reveal the mechanism of action of SB. Hence, RNA sequencing was done on the testicular tissues of OM after SB treatment for 2 weeks. SB could alter substantial TGs (2033) ( Figure 3A), and directly regulated three reproductive pathways: gamete generation, meiotic cell cycle, and spermatid development, and enriched 137 TGs in the three pathways ( Figure 3B and C; Supplementary Dataset S7 and S8). Among 137 TGs, Fst showed the remarkable upregulation of expression upon oral administration of SB (Supplementary Dataset S8). Based on follistatin, protein (encoded by Fst) promotes the growth and development of spermatogenic cells by blocking the action of activin-A protein. 7 Overexpression of activin-A protein (encoded by Inhba) can induce apoptosis of spermatogenic cells and lead to spermatogenic blockage, and downregulation of Inhba expression could contribute directly to the decrease in activin A-expression, thereby attenuating spermatogenic blockage. 8   of the action of overexpressed activin-A, thereby repairing spermatogenic blockage. 9 Therefore, we further investigated the expression of Inhba in testicular tissues, and Inhba expression was downregulated markedly in the testicular tissue of OM after SB treatment ( Figure 3D, Supplementary Dataset S9). Furthermore, the upregulation of Fst expression and downregulation of Inhba expression were verified by RT-qPCR ( Figure 3E, Supplementary Dataset S10). These results indicated that SB could treat OM by regulating the expression of Fst and Inhba.
With regard to potential clinical use, we investigated the plasma and testicular pharmacokinetics of SB in normal mice after oral administration using UPLC-QqQ-MS/MS. The measurement was validated 10 and consisted of specificity, calibration curves, correlation coefficients, linear ranges, and lower limit of quantifications; intra/interday precisions and accuracies; recovery stability; and measurement stability ( Figure S2A-C, E; Supplementary Dataset S11-S14). After oral administration, plasma and testicular concentration-time profiles for SB were plotted (Figure S2D and F; Supplementary Dataset S15 and S16), and the corresponding pharmacokinetic parameters were calculated ( Figure S2G and H; Supplementary Dataset S17 and S18). Plasma parameters demonstrated that oral administration led to rapid absorption and an effective exposure of SB in blood, and SB could be eliminated from blood within 1 day (seven-fold half-life washing-out period about 21 h). After absorption, SB was distributed effectively into testicular tissue but with a delay, and SB in testicular tissue had comparable pharmacokinetic behavior to that in blood. These results revealed that SB could be absorbed rapidly after oral administration, and became fully available at the intended action site, indicating a remarkable potential for clinical application.
In conclusion, SB as an active component was screened from WP, which enabled the repairs of spermatogenesis arrest and male infertility. The action mechanism could be explained by the repaired spermatogenesis via upregulation of Fst, while downregulation of Inhba genes involved in the reproductive signaling pathway. The encouraging preclinical data with pharmacokinetics warranted a rapid development of this new class of therapeutic agent. Our study provides a promising drug for treatment of male infertility and a novel strategy for discovery of new small-molecule drugs from vast plant-based medicinal resources.

A C K N O W L E D G M E N T S
We are grateful to the biological expertise provided by Chong Tang at the BGI Genomics Co., Ltd.

C O M P E T I N G I N T E R E S T S
The authors declare no competing interests in relation to publication of this study.  (Suiplus, BeiJing, China). The samples were obtained from normal mice (n = 6), OM (n = 6), and TP-treated OM (n = 6; i.p. TP 0.2 mg/kg/twice a week for 2 weeks), WP-treated OM (n = 6; i.g. WP 1.56 g/kg/day for 2 weeks), or SB-treated OM (n = 6; i.g. SB 20 mg/kg/day for 2 weeks). The results directly demonstrate that SB enables to increase the sperm number of OM. The dynamic videos of this study are available in Videos S1-S5. (C) Sperm motion track images of mouse cauda epididymidis samples under Suiplus Semen Analysis Automatic Detection System (Suiplus). The samples were obtained from the same as above ( Figure 2B). The observation displays that SB increases the sperm mobile activity of OM. The analyses were performed for evaluating the quality of sperms in OM after oral treatment with SB. (D) Quality of spermatogenesis. D1, sperm concentrations; D2, sperm mobility; D3, progressive mobile sperms; D4, curvilinear velocity (VCL); D5, straight-line velocity (VSL); D6, average path velocity (VAP); D7, straightness (STR); D8, linearity (LIN); D9, beat cross frequency (BCF); D10, amplitude of lateral head displacement (ALH). The studies (E and F) were performed for evaluating the male reproductive ability by comparing the number of pups in the first litter of female mice, and the average number of births (ANB; = total number of births/birth females). Each male mouse was placed in one cage, and mated with two females. (E) Efficacy in enhancing reproductive ability (n = 3). E1, pups in the first litter of female mice; E2, average number of births (ANB; = total number of births/birth females). These data demonstrate that SB significantly increases male reproductive ability, leading to an enhanced ability of male mice to make female mice pregnant and the mean number of offspring