Yangjing capsule (YC), a traditional Chinese compound herbal preparation, has been proven as an effective drug to improve spermatogenesis in clinical practice. However, its pharmacological mechanisms were not fully clarified. This study was designed to investigate the protective effects of YC on spermatogenesis in the mouse model of spermatogenesis dysfunction induced by cyclophosphamide (CP). The administration of YC significantly increased the epididymal index, sperm count, and sperm motility of model mice. Histopathological changes demonstrated that CP caused obvious structural damage to testis, which were reversed by the administration of YC. Results from TUNEL assay showed that treatment with YC dramatically decreased the apoptosis of spermatogenic cell induced by CP. Moreover, YC treatment could inhibit the mRNA and protein expression of Bax to Bcl-2 and also raised expression of AR at both mRNA and protein levels. These data suggest that YC might ameliorate spermatogenesis in male mice exposed to CP through inhibiting the apoptosis of spermatogenic cell and enhancing the actions of testosterone in spermatogenesis.
Infertility affects 15% of couples worldwide. It is estimated that roughly half of the infertility cases are due to male factors [
Spermatogenesis is a sophisticated multistep process involving three major phases: mitotic, meiotic, and postmeiotic phases. Other cellular events such as apoptosis of spermatogenic cell, migration, and differentiation also play vital roles in the process of spermatogenesis [
The Yangjing capsule (YC), a traditional Chinese compound herbal preparation, has been used for over ten years for the treatment of male reproductive diseases, in China, including male infertility and sexual dysfunction. Clinical practice showed that YC could improve the density of sperm, its motility, and DNA integrity in infertile men [
In recent years, mouse models of spermatogenesis dysfunction are used widely to investigate the mechanisms of spermatogenesis. Alkylating agents such as CP are the most common agents implicated in inducing the mouse model. Furthermore, it has been demonstrated that CP can inhibit testosterone synthesis and induce apoptosis of spermatogenic cells [
CP was purchased from Pude Medicine Co. Ltd. (Shanxi, China). Iodine [125I] Testosterone Radioimmunoassay Kit was supplied by Beijing North Institute of Biological Technology (Beijing, China). The primers were synthesized by Invitrogen Life Tech (Carlsbad, CA). The mouse monoclonal anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) and horseradish peroxidase-conjugated secondary antibodies were purchased from Bioworld (St. Louis Park, MN). The rabbit monoclonal anti-tubulin and rabbit polyclonal anti-Bax antibodies were procured from Abcam (Cambridge, MA). The rabbit polyclonal anti-AR and rabbit polyclonal anti-B-cell lymphoma 2 (anti-Bcl-2) antibodies, goat anti-rabbit immunoglobulin G (
The YC was provided by Nanjing General Hospital of Nanjing Military Region (Nanjing, China). YC is composed of 11 types of traditional Chinese drugs: 13.3% Herba Epimedii Brevicornus, 6.7% Rhizoma Polygonati Sibirici, 6.7% Radix Rehmanniae Preparata, 10% Radix Astragali Mongolici, 6.7% Placenta Hominis, 6.7% Semen Astragali Complanati, 10% Radix Angelicae Sinensis, 6.7% Hirudo, 6.7% Semen Litchi, 13.3% Semen Vaccariae Segetalis, and 13.3% Concha Ostreae (calcined). In this study, contents of the dried capsule were diluted into different concentrations using distilled deionized water, and the suspensions were stored at 4°C.
Mature male (8-week) Balb/c mice (22–25 g) were procured from Yangzhou University Comparative Medical Center (Yangzhou, China). Mice were housed under standard laboratory conditions and were provided with free access to standard food and water ad libitum. This study was approved by the Animal and Human Ethics Board of Southeast University. After adapting to the environment for a week, mice were randomly divided into four groups: control (
The animals were weighed and anesthetized with pentobarbital sodium (50 mg/kg, i.p.) at the end of the treatment. Blood samples were collected by extirpation of eyeball and allowed to clot, and the serum was separated at 3000 rpm for 15 min and stored at −80°C to analyze the testosterone level.
The testes and epididymis were rapidly excised and weighed. The relative testicular or epididymal weight (the testis index or the epididymal index) was calculated by dividing the weight of testis or epididymis by body weight. One testis and one epididymis from a mouse were fixed in Bouin’s solution for pathological examination and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis. The other testis was freshly washed in ice-cold saline and immediately stored in liquid nitrogen. The fresh testis frozen in liquid nitrogen was then divided into two parts for the isolation of RNA and extraction of protein.
The epididymal sperm concentrations and motility were evaluated as previously described with some modifications [
For histological studies, tissues were initially fixed in Bouin’s fluid for 16 h and they were then washed three times with 70% ethanol followed by dehydration in graded ethanol. They were finally embedded in paraffin. Five-micrometer-thick sections were stained using hematoxylin-eosin and then examined under a light microscope at 200x or 400x magnification. A total of 200 seminiferous tubules from each testis were examined. Damaged tubules which showed severe hypocellularity (reduction in number of spermatogenic cells) and thinned seminiferous epithelium were recorded.
Serum testosterone was examined using Iodine [125I] Testosterone Radioimmunoassay Kit according to the protocol described by the manufacturer. The amount of testosterone was determined from a calibration curve.
Five-micrometer-thick sections of the testis were used for TUNEL staining to examine cell apoptosis. The TUNEL method was performed according to the manufacturer’s instructions. The numbers of positive and total cells were recorded, and the apoptosis index [(number of positive cells/total cells) × 100] of the tissue was calculated as described by Yuan et al. [
Bax, Bcl-2, and AR mRNA expression levels in the testes were determined using real-time quantitative PCR. The total RNA of the testes samples was extracted using Trizol reagent (TaKaRa Biotechnology, Dalian, China). Sample cDNAs were used as templates for amplification to quantify the mRNA levels of target genes using SYBR Green PCR Master Mix reagent kits (TaKaRa Biotechnology). GAPDH was selected as the control. The primer sequences were as follows: GAPDH: forward, 5′-TGG CCT TCC GTG TTC CTA C-3′; reverse, 5′-GAG TTG CTG TTG AAG TCG CA-3′; AR, forward, 5′-TCC AAG ACC TAT CGA GGA GCG-3′; reverse, 5′-GTG GGC TTG AGG AGA ACC AT-3′; Bax, forward, 5′-AGA CAG GGG CCT TTT TGC TAC-3′; reverse, 5′-AAT TCG CCG GAG ACA CTC G-3′; Bcl-2, forward, 5′-GCT ACC GTC GTG ACT TCG C-3′; reverse, 5′-CCC CAC CGA ACT CAA AGA AGG-3′. Relative quantification of expression of target genes was estimated using the
Total proteins extracted from testis were prepared following standard procedures and quantified using the bicinchoninic acid protein assay (Beyotime, Shanghai, China). The proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred onto nitrocellulose membranes. The membranes were blocked with 5% skim milk in Tris-buffer saline containing 0.1% Tween 20 at 37°C for 1 h and then incubated at 4°C overnight with the primary antibodies of Bax (1 : 200 dilution), Bcl-2 (1 : 300 dilution), and AR (1 : 300 dilution). Tubulin or GAPDH was used as an internal control. Densitometric analysis of the scanned immunoblotting images was performed using a Quantity One image system.
All quantitative data derived from this study were analyzed using the SPSS 20.0 statistical package. The results were expressed as the mean ± standard deviation. The one-way analysis of variance was used to analyze the difference between groups, and post hoc least significant difference test was used for intergroup comparisons.
The relative weights of reproductive organ and the sperm parameters were used to monitor the damage to the male reproductive system. As shown in Table
Effects of YC on characteristics of sperm, seminiferous tubules, serum testosterone, body weight, and the relative testicular and epididymal weights.
Parameters | Control | CP | CP + YC (630 mg/kg) | CP + YC (1260 mg/kg) |
---|---|---|---|---|
Sperm count (×106 mL−1) | 82.56 ± 7.49 | 43.94 ± 4.70 |
51.31 ± 2.67 |
65.11 ± 8.18 |
Sperm motility (%) | 69.00 ± 6.22 | 45.76 ± 7.28 |
62.22 ± 4.68 |
65.25 ± 7.06 |
Proportion of damaged tubules (%) | 2.9 ± 2.3 | 75.1 ± 5.3 |
20.5 ± 7.6 |
13.3 ± 6.1 |
Body weight (g) | 29.11 ± 1.54 | 26.0 ± 1.41 |
26.50 ± 1.41 | 28.11 ± 1.76 |
Relative testicular weight (mg/g bodyweight) | 4.02 ± 0.28 | 2.75 ± 0.54 |
3.13 ± 0.29 | 3.32 ± 0.39 |
Relative epididymal weight (mg/g bodyweight) | 1.47 ± 0.20 | 1.10 ± 0.11 |
1.44 ± 0.13 |
1.37 ± 0.12 |
Testosterone (mol/L) | 1.81 ± 0.86 | 0.56 ± 0.22 |
0.61 ± 0.26 | 0.76 ± 0.35 |
Data are expressed as the mean ± SD (
CP: cyclophosphamide; SD: standard deviation; YC: Yangjing capsule.
The histopathology of the testis and epididymis was shown in Figure
The effects of treatment with YC on the testicular ((a), ×400) and epididymal ((b), ×400) damage in the spermatogenesis dysfunction mice induced by CP (lesion site labeled with arrow heads). (A) control; (B) CP (50 mg/kg); (C) CP + YC (630 mg/kg); (D) CP + YC (1260 mg/kg).
In the YC (630 mg/kg) group, in spite of thinned seminiferous epithelium, many mature spermatids appeared in the seminiferous tubules (Figure
The mean percentage of damaged seminiferous tubules in the CP-treated mice was significantly higher,
The administration of CP caused a significant decrease in the serum testosterone level, from
TUNEL assay was performed to analyze the apoptosis of spermatogenic cell. After administration of CP, apoptotic cells were observed more frequently in the testis of the CP group mice (Figure
The effects of treatment with YC on apoptosis of spermatogenic cell in the spermatogenesis dysfunction mice induced by CP. (a) The representative apoptosis of spermatogenic cell in the testes using TUNEL assay (×400). (b) The spermatogenic cell apoptotic index. (A) Control; (B) CP (50 mg/kg); (C) CP + YC (630 mg/kg); (D) CP + YC (1260 mg/kg) (
To further study the potential mechanism of antiapoptosis of YC, the expressions of Bax and Bcl-2, at mRNA and protein levels, in the testes of mice were examined using the quantitative real-time PCR and Western blotting. As shown in Figure
The effects of YC on the ratio of Bax to Bcl-2 mRNA expression (a) and protein expression (b) in the testes of spermatogenesis dysfunction mice induced by CP. Data given are the mean ± SD (
Testosterone is the major androgen in the testis that regulates spermatogenesis. Effects of androgen are mediated by the AR. The lowered testosterone or dysfunctional AR will result in the impaired spermatogenesis. In this study, the expressions of AR mRNA and protein in the testes of different groups were compared. As shown in Figure
The effects of YC on AR mRNA (a) and protein expressions (b) in the testes of spermatogenesis dysfunction mice induced by CP. Data are expressed as mean ± SD (
Male infertility causes significant duress to couples. Defects in spermatogenesis are the most common reasons for male infertility. At present, hormonal treatment or empiric medical treatment (e.g., aromatase inhibitor or selective estrogen receptor modulator) is used to treat infertile men with spermatogenic defects, but clinical results are limited especially for patients with idiopathic failure of spermatogenesis [
CP, as an alkylating agent, is the most common agent implicated in causing dysfunction of spermatogenesis. In this study, mature male Balb/c mice were injected with CP (50 mg/kg) i.p. for 7 days to induce the dysfunction of spermatogenesis. For the next 30 days, the mice received treatment with YC. Animals were killed at 30 days after the last injection. We found that administration of CP significantly decreased the sperm count and motility as well as the relative testicular weights and body weights. The administration of CP also caused histologic lesions of the testis and epididymis. Testicular tissues from the CP-treated mice showed atrophied seminiferous tubules, thinned seminiferous epithelium, and reduced spermatogenic cells and interstitial cells. We also found that the cauda epididymis from the CP-treated mice illustrated a lower density of sperm and an increase in the number of premature sperm cells.
Interestingly, we found that treatment with YC significantly restored these CP damage instances. YC (630 and 1260 mg/kg) treatment not only notably increased the sperm count and motility but also increased seminiferous epithelial layers and the number of mature spermatids and interstitial cells in testis. YC treatment also increased the density of sperm and decreased the deciduous premature sperm cells in the ductus epididymis when compared with the CP group. These results indicated that YC could ameliorate spermatogenesis in male mice exposed to CP.
Mammalian spermatogenesis depends on the balance among proliferation, differentiation, and apoptosis of germ cells. Apoptosis of spermatogenic cell occurs during various stages of mammalian spermatogenesis to remove abnormal spermatogenic cells [
Testosterone is a major regulator in spermatogenesis, which works by binding to AR. In the absence of testosterone or the dysfunctional AR, spermatogenesis is halted during meiosis so that few germ cells develop to the haploid spermatid stage and the elongated spermatid can not form [
Previous studies have shown that administration of CP could inhibit the synthesis of testosterone [
As we know, traditional Chinese medicine, especially those compound recipes, plays a role through multiway and multitarget with its multicomponents. YC is composed of 11 traditional Chinese drugs. Of necessity, YC contains many active ingredients. It has been demonstrated that total flavonoids of Herba Epimedii Brevicornus and polysaccharides could not only enhance testosterone actions [
In conclusion, the present study suggests that treatment with YC can ameliorate the impairment of spermatogenesis induced by CP in male mice. The effects of YC on improving spermatogenesis are likely due to the inhibition of apoptosis of spermatogenic cell and enhancement in the actions of testosterone in spermatogenesis. Therefore, YC might be considered as an alternative therapeutic remedy for male infertility.
Yangjing capsule
Cyclophosphamide
Terminal deoxynucleotidyl transferase dUTP nick end labeling
Androgen receptor
Steroidogenic acute regulatory protein
Cytochrome P450, family 11, subfamily A, polypeptide 1
3
Bcl-2 Associated X Protein.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This study was supported by the National Natural Science Foundation of China (Grants nos. 81273760 and 81302969) and Postgraduate Innovation Program of the Jiangsu Province Education Department (SJLX_0430). The authors thank the State Key Laboratory of Reproductive Medicine, the Clinical Center of Reproductive Medicine, Nanjing Medical University, for providing laboratory and technological assistance.