Sheng Jing Decoction, as a Traditional Chinese Medicine Prescription, Can Promote Spermatogenesis and Increase Sperm Motility


 BackgroundSheng Jing Decoction (SJD), as a traditional Chinese medicine prescription, is mainly be used to treat male infertility. It has been applied in many hospitals in China, and the clinical effect of patients' reaction is satisfactory. However, pharmacological function and molecular mechanism of SJD are poorly understood. In this study, mainly investigated the function of SJD on spermatogenesis and sperm motility, and explored the potential mechanisms.MethodsThe oligozoospermia model of ICR mice was induced by injecting intraperitoneally (ip) with 60 mg/kg dose of cyclophosphamide. At the same time of modeling, high, medium and low doses of SJD were given orally for treatment respectively. Sperm vitality and motility were detected and analyzed by CASA, and sperm morphology was tested by Papanicolaou staining. Sperm mitochondrial membrane potential (MMP) was tested by JC-1 staining. Sperm plasma membrane integrity was determined by SYBR-14/PI double staining. Histopathological changes of testis were analyzed by HE staining and Immunohistochemistry. Genes expression related to spermatogenesis and sperm development were detected by real-time RT-PCR.Results High, medium and low doses of SJD are effective to recover from the impairment of the whole body and testicular tissue by cyclophosphamide inducing, and to rescue the damage of testicular tissue cells including sertoli cells and germ cells by cyclophosphamide inducing. SJD can all partly restore the decrease in sperm concentration, sperm vitality, sperm motility and normal sperm morphology rate by cyclophosphamide inducing. Ki67 staining analyses confirm SJD can promoted testicular tissue cells proliferation. Real-time RT-PCR analyses reveal SJD can up-regulates the expression of proliferation-associated gene Lin28a, and differentiation-associated genes Kit, Sohlh2 and Stra8. SJD can also reduce the impairment of MMP and sperm plasma membrane integrity by cyclophosphamide inducing.ConclusionSJD is effective to support sperm quantity and quality by increasing sperm concentration, sperm vitality, sperm motility and normal sperm morphology rate. SJD can promote spermatogenesis by up-regulating the expression of the proliferation-associated gene Lin28a, and the differentiation-associated genes (Kit, Sohlh2 and Stra8). SJD can sustain MMP and sperm plasma membrane integrity to increase sperm motility.


Abstract
Background Sheng Jing Decoction (SJD), as a traditional Chinese medicine prescription, is mainly be used to treat male infertility. It has been applied in many hospitals in China, and the clinical effect of patients' reaction is satisfactory. However, pharmacological function and molecular mechanism of SJD are poorly understood. In this study, mainly investigated the function of SJD on spermatogenesis and sperm motility, and explored the potential mechanisms.

Methods
The oligozoospermia model of ICR mice was induced by injecting intraperitoneally (ip) with 60 mg/kg dose of cyclophosphamide. At the same time of modeling, high, medium and low doses of SJD were given orally for treatment respectively. Sperm vitality and motility were detected and analyzed by CASA, and sperm morphology was tested by Papanicolaou staining. Sperm mitochondrial membrane potential (MMP) was tested by JC-1 staining. Sperm plasma membrane integrity was determined by SYBR-14/PI double staining. Histopathological changes of testis were analyzed by HE staining and Immunohistochemistry. Genes expression related to spermatogenesis and sperm development were detected by real-time RT-PCR.

Results
High, medium and low doses of SJD are effective to recover from the impairment of the whole body and testicular tissue by cyclophosphamide inducing, and to rescue the damage of testicular tissue cells including sertoli cells and germ cells by cyclophosphamide inducing. SJD can all partly restore the decrease in sperm concentration, sperm vitality, sperm motility and normal sperm morphology rate by cyclophosphamide inducing. Ki67 staining analyses con rm SJD can promoted testicular tissue cells proliferation. Real-time RT-PCR analyses reveal SJD can up-regulates the expression of proliferationassociated gene Lin28a, and differentiation-associated genes Kit, Sohlh2 and Stra8. SJD can also reduce the impairment of MMP and sperm plasma membrane integrity by cyclophosphamide inducing.

Conclusion
SJD is effective to support sperm quantity and quality by increasing sperm concentration, sperm vitality, sperm motility and normal sperm morphology rate. SJD can promote spermatogenesis by up-regulating the expression of the proliferation-associated gene Lin28a, and the differentiation-associated genes (Kit, Sohlh2 and Stra8). SJD can sustain MMP and sperm plasma membrane integrity to increase sperm motility.
In China, Traditional Chinese medicine (TCM) has been used to treat male infertility for nearly 2000 years, and increasingly appealing in surrounding areas and western countries. Holistic treatment is the essence and basic feature of TCM, and utilizing this theory for treating male infertility achieved satisfactory results [24,25]. Sheng Jing Decoction (SJD) is one of traditional Chinese medicine prescription mainly be used to treat male infertility. SJD consists of several herbs such as Rehmannia glutinosa (Gaertn.) DC., Astragalus membranaceus (Fisch.) Bunge, Pseudostellaria heterophylla (Miq.) Pax, Dipsacus acaulis (A.Rich.) Napper, Lycium arenicolum Miers, Astragalus complanatus Bunge and Gleditsia sinensis Lam.. SJD has been applied in many hospitals in China, and patients' reaction is effective. However, the mechanism of action underlying the therapeutic effect of SJD is poorly understood. In this study, we used oligozoospermia model of ICR mice to verify the function of SJD and explore its mechanism. We identify that SJD can increase sperm concentration, sperm vitality, sperm motility and normal sperm morphology rate. To further con rm that SJD enhance sperm vitality and motility by keeping sperm mitochondria's function and sperm plasma membrane integrity. SJD can promote spermatogenesis by up-regulating the expression of the proliferation-associated gene Lin28a, and the differentiation-associated genes (Kit, Sohlh2 and Stra8).   Table 1.

Experimental design
The oligozoospermia model mice were constructed by cyclophosphamide inducing. Cyclophosphamide was dissolved in PBS and injected intraperitoneally (ip) to the mice (60mg/kg), once in a day for a period of 5 days. High (33g/kg), medium (16.5 g/kg) and low (8.25 g/kg) doses of SJD were given orally to mice, once in a day for a period of 35 days. The medium dose is converted from the therapeutic dose of human. Body weights were measured every week. Testes were quickly dissected out and weighed. One half of testes were collected and stored at -70 •C for real-time RT-PCR analysis, and the other half of testes were xed in Bouin's xative (0.2% picric acid/2% (v/v) formaldehyde in PBS) for histological evaluation.

Sperm motility and count
The epididymis was placed in 1ml normal saline, and then a deep hole was cut in each caudate nucleus with micro scissors. Sperm was allowed to release for 5 minutes in an incubator with a temperature of 34°C and 5% CO2. The sperm suspension was incubated in CO2 incubator for 30 min and a drop of sperm suspension was uniformly smeared on clean glass slide. Sperm motility and count were analyzed by CASA system and bright-eld microscopy (BX43 Olympus, Tokyo, Japan). and DAB (ZSGB-bio, China) were used for staining. The results were obtained by uorescence microscope (BX51 Olympus, Tokyo, Japan) and Color digital camera (DFC425 Leica, Wetzlar, Germany).
JC-1 staining JC-1 staining kit was purchased from Beyotime (Shanghai, China), Equal volume sperm and JC-1 staining working uid were mixed. Incubated at 37℃ with 5%CO2 for 30min. Then the sperm were washed twice with JC-1 staining buffer. The results were observed under uorescence microscope (BX51 Olympus, Tokyo, Japan).

Sperm plasma membrane integrity test
The Sperm (1×10 /mL) suspension was added syBR-14 and PI dye kit(Thermo Fisher Scienti c, Waltham, MA) successively. Mixed and incubated in dark for 5 min. After washing, stained sperm were observed by uorescence microscope (BX51 Olympus, Tokyo, Japan).

Statistics and data analyses
Data are expressed as the mean ± SEM, and statistical evaluation was performed using the Student's ttest for independent groups. Values of p < 0.05 were considered statistically signi cant.

Results
SJD can accelerate recovery from impairment caused by cyclophosphamide inducing in the whole body and testicular tissue To investigate the function of SJD, we constructed oligozoospermia model of ICR mice induced by injecting intraperitoneally (ip) with 60 mg/kg dose of cyclophosphamide [26]. At the same time of modeling, high, medium and low doses of SJD were given orally for treatment respectively. We measured the body weight of mice after ip cyclophosphamide every week and testis weight for SJD treatment 35 days. The high, medium and low doses of SJD all can speed up the recovery from weight loss of whole body by cyclophosphamide inducing (Fig. 1A). The relative weight of the testes of oligozoospermia model mice signi cantly increased after SJD treatment (Fig. 1B). H&E staining of analysis of fresh testicular tissue found that testicular tissue was severely damaged by cyclophosphamide inducing, and SJD is helpful to restoration from the damage of testicular tissue (Fig. 1C). Testicular tissue cells mainly include Sertoli cells, germ cells Leydig cells, we can determine them by their respective molecular marker (GATA4, TRA98 and 3β-HSD). Immunohistochemistry data showed sertoli cells and germ cells were remarkably damaged oligozoospermia model mice, and could be partly recovered by SJD treatment (Fig.   1C). Whereas Leydig cells had no signi cant differences during different groups (Fig. 1C).
SJD can partly restore the defects of sperm quantity and quality in oligozoospermia model mice To evaluate the concentration, vitality and motility of sperm by computer-aided sperm analysis (CASA).
CASA reports revealed sperm concentration, vitality and motility of oligozoospermia model mice without SJD treatment were signi cantly decreased compare with control group, whereas it had a signi cant improvement of sperm concentration, vitality and motility after SJD treatment ( Fig. 2A, B and C). Normal sperm morphology rate was markedly increase in oligozoospermia model mice by SJD treatment compared to without SJD treatment (Fig. 2D).

SJD can promote spermatogenesis and up-regulate the expression of the proliferation-associated gene
Lin28a and the differentiation-associated genes (Kit, Sohlh2 and Stra8) The above data showed SJD can signi cantly increase the number of sertoli cells and germ cells of oligozoospermia model mice. In addition, Ki67 staining analysis also con rmed that SJD has the function of promoting proliferation of testicular tissue cells in oligozoospermia model mice (Fig. 3A). To further investigate the mechanism of SJD, we examined the mRNA expression of the proliferationassociated gene Lin28a, and the differentiation-associated genes (Kit, Sohlh2 and Stra8) by performing quantitative RT-PCR. The results suggested that the expression of genes Lin28a,Kit, Sohlh2 and Stra8 have no difference in oligozoospermia model mice without SJD treatment compare with normal mice, but signi cantly up-regulated in oligozoospermia model mice by SJD treatment (Fig. 3B, C, D and E).
SJD has the effect of sustaining sperm mitochondrial membrane potential (MMP) and sperm plasma membrane integrity Sperm mitochondrial membrane potential (MMP) and sperm plasma membrane integrity were two important factors involved in sperm motility. MMP was tested and analyzed by JC-1 staining. The results showed MMP was remarkably decreased by cyclophosphamide inducing, whereas SJD treatment could offset the loss of MMP (Fig 4A). SYBR-14/PI double staining analysis showed that sperm plasma membrane integrity was less impaired in oligozoospermia model mice by SJD treatment compared with without SJD treatment (Fig. 4 B).

Discussion
In recent years, male infertility has been attracting more attention from public with the decline of semen quality. The majority of male infertility cases are because of poor sperm quality low sperm quantity or both [27,28]. There are many factors leading to the male infertility including abnormal spermatogenesis [29,30]; anomalies or obstruction of reproductive tract [31]; abnormal sexual and ejaculatory functions [32]; and inadequate sperm motility [33], imbalance in hormone levels [34], and genetic defects [4]. In addition, idiopathic infertility accounts for 30% of infertile men [35]. Although a great progression has been made in treating male infertility, including intrauterine insemination, in vitro fertilization (IVF) and even intracytoplasmic sperm injection (ICSI), but these treatments have disadvantages such as unsatisfactory e cacy, strong invasiveness, high risk, or excessive cost. Therefore, it is necessary to seek effective natural therapy to increase fertility. TCM has been used to treat male infertility in China for a very long time and has now been using increasingly in neighboring and western countries [24,36]. SJD, as a traditional Chinese medicine prescription, has been used to treat male infertility in many Chinese hospital and helped many men with fertility problems achieve clinical pregnancy. However, its biological function and the relevant mechanism is poorly understood.
To investigate the role of SJD in treating male infertility, we established the oligozoospermia model of ICR mice by cyclophosphamide inducing. Cyclophosphamide caused temporary impairment of normal male reproductive system by low dose treatment. The weight of the whole body and testis signi cantly decreased after cyclophosphamide inducing ( Fig. 1A and B), and testicular tissue was severely damaged (Fig. 1C). There is a great impairment of sperm and its fertilizing ability in oligozoospermia mice model ( Fig. 2). At the same time of modeling, SJD were given orally for treatment. SJD is helpful to restoration from the damage of testicular tissue (Fig. 1C). It had a signi cant increase of sperm concentration, vitality and motility and normal sperm morphology rate after SJD treatment in oligozoospermia model mice (Fig. 2), which veri ed SJD treats male infertility mainly by enhancing sperm quantity and quality.
Male fertility is maintained through complex cellular and molecular interactions, which ensure that spermatogonial stem cells (SSCs) self-renewal and differentiation into mature sperm [6]. Spermatogenesis is a highly organized and complex process, which can continuously produce millions of haploid sperm in the whole adult male life, and keep the complete genome and appropriate epigenome from generation to generation [30,37]. Our data showed that the expression of sertoli cells marker GATA4 [38] and germ cells marker TRA98 [39] increased by SJD treatment in oligozoospermia model mice (Fig. 1C), and sperm concentration and vitality signi cantly increased by SJD treatment ( Fig. 2A and B). In addition, SJD has the function of enhancing the ability of proliferation of testicular tissue cells in oligozoospermia model mice (Fig. 3A). These data demonstrated that SJD plays promoting roles on selfrenewal, proliferation and differentiation process during which diploid SSCs produce haploid spermatozoa.
Sperm motility is an important factor involved in the overall sperm quality, critical to ensure fertilization success [40]. The mitochondrion is the major energy provider to sustain sperm motility. Previous studies focused on the time regulation of mitochondrial membrane potential (MMP) on sperm motility. MMP can be used as a potential regulator and indicator of sperm motility, which is directly related to male fertility [41][42][43]. Our data showed that SJD returned MMP of sperm impaired by cyclophosphamide inducing to normal (Fig. 4A). These data demonstrated that SJD play an important role to sustain mitochondrial function and sperm motility. As a dynamic platform, many components of membrane participate in cell motility process, including force generation, adhesion, signal transmission and regulation [44]. Sperm plasma membrane integrity plays a crucial role in sperm motility and fertilization [45,46]. Our results showed that sperm plasma membrane integrity was less impaired in oligozoospermia model mice by SJD treatment compared to without SJD treatment (Fig. 4B). These data provided strong evidences that SJD is an important factor involved in supporting sperm plasma membrane integrity and functions.

Conclusions
In summary, SJD, as a traditional Chinese medicine prescription, has been popularly used to treat male infertility in many Chinese hospital. SJD can increase sperm concentration, sperm vitality, sperm motility and normal sperm morphology rate, to support sperm quantity and quality. SJD has the effect of promoting the spermatogenesis, and further demonstrated that it up-regulates the proliferationassociated gene Lin28a, and differentiation-associated genes Kit, Sohlh2 and Stra8. In addition, our data con rmed SJD increases sperm motility by sustaining MMP and sperm plasma membrane integrity.

Declarations
Ethics approval and consent to participate All mice were purchased from Shanghai Experimental Animal Center and the experiments were approved by the Research Ethics Committee, Seventh People's Hospital of Shanghai University of TCM (Shanghai, China).

Consent to publish
All the authors were concerned and agreed to publish before the submission.

Availability of data and materials
Details of data mining, selection, extraction and assessment carried out to support the ndings of this study are available from the corresponding author upon request.

Competing interests
The authors declare that there are no competing interests regarding the publication of this paper.

Supplementary Files
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