Part 2 Echinacoside Increases Sperm Quantity in Rats By Targeting The Hypothalamic Androgen Receptor

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Discussion

Ethnopharmacological records and our previous study13 confirmed that Cistanche tubulosa extracts effectively increased sex hormone levels and improved sperm quality. ECH as one of the main compounds in Cistanche tubulosa19,20, can augment sperm counts and increase the secretion of LH and T (Fig. 1). In this study, we demonstrated that ECH was distributed to the hypothalamus instead of the testes suggesting an indirect effect of ECH on the testes. Indeed, ECH directly inhibited hypothalamic AR activity, AR translocation to the nucleus, and HPG-axis related gene expression and also provided protection against BPA-induced reproductive damage.

In studies of human gonadotrophin production, sex hormone levels are tightly regulated by the HPG-axis and a negative feedback loop21. Namely, T secretion is markedly increased in response to consistent and sustained increases in blood LH levels in response to a lack of negative androgenic feedback to the pituitary and hypothalamus. In this study, ECH increased the secretion of LH in encephalon+pituitary and production of testosterone in testis and mRNA levels of LHR and Gnrhr in encephalon+pituitary (Fig. 1 and Fig. 4), suggesting that ECH may affect HPG axis negative feedback. Not surprisingly, AR itself plays an important role in the feedback regulation of T and LH levels22,23. The male HPG axis paradigm is centered on testosterone providing negative feedback repression at the level of both the hypothalamus and pituitary gland24,25. Previous studies used Foxg1-Cre to ablate genes of interest in the pituitary but not the hypothalamus to determine the relative importance of the two regions. The results demonstrated that AR signaling in the pituitary of the male mouse is dispensable in regards to testosterone-dependent regulation of LH secretion26. For this reason, we chose to focus on hypothalamic AR for further study.

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As a steroid nuclear hormone receptor, the activity of AR is regulated by the steroid ligand T, the binding of which initiates nuclear translocation and the transcriptional regulatory function of AR27. In this study, we showed that ECH treatment inhibited AR translocation from the cytoplasm into nuclei in the hypothalamus, (Fig. 3). Moreover, the PK study showed that ECH can markedly penetrate to the hypothalamus rather than to the testes, suggesting that more ECH crosses the Blood-Brain Barrier, possibly via endocytosis, rather than the Blood Testis Barrier. Furthermore, we demonstrated by indirect ELISA the combining capacity of ECH and AR suggesting ECH can directly bind to the AR. This was further supported by results of the molecule docking assay that indicated that putative binding sites for ECH exist at Met-894 and Val-713 in the AR pocket (Fig. 5D). This supports the findings of others that AR participates in the regulation of a negative feed loop and that a reduction in AR-T interactions in the brain results in increased LH secretion23,28,29

Steroidogenesis is a tightly controlled, essential process for the development of spermatogenesis with the involvement of steroidogenic protein (StAR)30 and steroidogenic enzymes such as CYP11A1, CYP17A1, 3β-hydroxysteroid dehydrogenase (HSD3β), and 17β-hydroxysteroid dehydrogenase (HSD17β) which convert cholesterol into testosterone31. We showed that the mRNA levels of the genes encoding these enzymes significantly increased with ECH treatment whereas the expression of StAR mRNA did not change. This suggests that ECH had no effect on the transfer of cholesterol to the inner membrane of the mitochondria, whereas it may play a role in promoting cholesterol conversion into testosterone. The conclusion can then be drawn that ECH treatment significantly increases sperm count by augmenting testosterone synthesis and secretion.

To investigate the protective effect of ECH on sperm injury, bisphenol A (BPA), a typical exogenous endocrine disruptor, was used as a model of male reproductive damage in mice32. ECH treatment effectively recovered sperm counts, sperm motility, T and LH levels diminished by BPA (Fig. 6A, B), suggesting that ECH can attenuate sperm injury.

Compared to the artificially synthesized chemicals for hormone replacement therapy, ECH is a natural non-hormonal compound that affects negative feedback on the HPG axis thereby increasing the secretion of LH, T, and sperm quality. This mechanistic pathway may protect the male reproductive system and thus ECH can be considered as a potential natural reproductive protective agent. However, hormone regulations are complex and often lead to systemic effects. As indicated in Fig. 7, ECH increased T and LH secretions and inhibited AR activation, which may also lead to consequences on other health or disease-associated pathways such as osteoporosis33 and depression in men34, therefore, the comprehensive effects of ECH should be further investigated.

Taken together, our results indicate that ECH binds to the AR in the hypothalamus and inhibits the transfer of the AR from the cytoplasm to the nucleus. Our data suggest that ECH binds to sites in the AR pocket at amino acids Met-894 and Val-713. This may explain the increase in T and AR levels as a systemic response to balance hormone levels by promoting LH secretion and a subsequent increase in T production and sperm counts (Fig. 8).

Methods

Ethics Statement.

This study was carried out in strict compliance with the Guidelines for Experimental Animals established by the Ministry of Science and Technology (Beijing, China). All the experimental protocols were reviewed and approved by the Ethics Committee of Anyang Institute of Technology.

Experimental Animals.

Four-week-old inbred Kunming mice (22 g–25 g) were obtained from the animal center of the fourth military medical university (Xi’an, China). Te animals were kept under controlled conditions at a temperature of 22 °C ± 2 °C, 70% ± 4% humidity with 12 h light-dark-cycling, and free access to feed and water.

Experiment treatment.

Experiment 1. Mice were randomly divided into six groups, seven mice in each group (n=7). ECH (CAS: 82854-37-3; Chengdu Preferred Biotechnology Co., Ltd, Sichuan, China) was administered using an intragastric tube, and testosterone propionate (TP, CAS: 57-85-2; KingYork, Tianjin, China) were given by intramuscular injection once daily for 14 days as per the following experimental design: Control group: normal saline (10mL/kg), ECH(L) group (5mg/kg), ECH(M) group (20mg/kg), ECH(H) group (80mg/kg), TP (15mg/kg) and enzalutamide (AR inhibitor, once daily every other day for 14 days; 20mg/kg; CAS: 915087-33-1; Aladdin, Shanghai, China).

After 2 weeks of treatments, the mice were anesthetized with diethyl ether to collect blood samples for analyses of hormone levels. The mice were then dissected to separate the hypothalamus, encephalon+pituitary, testis, and cauda epididymis. Samples of cauda epididymis were put in normal saline with 5% BSA for sperm quality assessment, and the hypothalamus, encephalon+pituitary, and testes were frozen in liquid nitrogen and stored at −80 °C for further investigations.

Experiment 2. Mice were randomly divided into 10 groups, five animals per group. ECH (20mg/kg) was administered orally and the mice in each group were anesthetized with diethyl ether at the time points 0.5 h, 1h, 1.5h, 2h, 2.5h, 3h, 4h, 6h, 9h, and 12h after administration for sample collection. Plasma was collected and the heart was exposed. Perfusion of normal saline into the left ventricles was conducted by an infusion apparatus until the liver and lungs were blanched. Ten hypothalami and testis were collected to determine the tissue concentrations of ECH.

Experiment 3. Mice were randomly divided into 4 groups, seven in each group. ECH and BPA (CAS: 80-05-7; Aladdin, Shanghai, China) were administered using an intragastric tube once daily for 6 weeks as per the following experimental design: normal group (corn oil, 10 mL/kg, bw/d), model group: BPA group (BPA 200 mg/kg, bw/d, corn oil), and experimental group: BPA+ECH group (BPA 200mg/kg; ECH 20mg/kg). After 42 days of treatment, the mice were anesthetized with diethyl ether and the blood samples were collected for hormone level analyses. Te testis and cauda epididymis were separated and collected. The cauda epididymis was used for sperm quality assessment and the testis was frozen in liquid nitrogen and stored at −80 °C for further investigation.

Determination of sperm quality. Sperm suspension preparation. Cauda epididymides were minced into 5 mL of normal saline with 5% BSA and incubated for 5 min at 37 °C to allow their contents to spread into the medium. Sperm number: As per the method described by Yokoi (2003), the diluted sperm suspension (1:10; v/v; sperm suspension/10% methanol) was transferred to each counting chamber of a hemocytometer and was allowed to stand for 5min and then counted under a light microscope (Nikon, Instruments Inc., Japan) at X200 magnification.Sperm viability: A total of 20μL of sperm suspension was mixed with an equal volume of eosin-nigrosin stain for 2min; the un-stained sperms were counted under a light microscope at 200x magnification.Sperm motility: A total of 10μL of sperm suspension was put onto a glass slide and were recorded as either mobile or immobile under a light microscope at 200x magnification.

Determination of hormone levels. Levels of testosterone (T) and LH were quantified in serum, encephalon+pituitary, and testis homogenates using radioimmunoassay (RIA) kits (Beijing Sino-UK Institute of Biological Technology, Beijing, China). Briefly, anti-testosterone or anti-LH IgG antibody (100 μL) and 125-I-conjugated anti-mouse antibody were added to samples or the standard (100 μL), and mixed on a rocker overnight at 4 °C. After washing three times with PBS-Tween 20 (500 μL), the mixtures were centrifuged (3500rpm/min) at 4 °C for 15min. The CPM values of the precipitates were assessed by radioimmunoassay instrument (Beijing Sino-western Technology Co. Ltd, CN202M/KZ4GC-1200, Beijing, China) and the concentrations of T and LH were calculated according to the formula of a standard curve. Te T and LH coefficient of variation is 2.8% and 3.2% in sample groups, and 2.1% and 2.8% in standard groups, respectively.

Determination of gene expressions by real-time quantitative PCR. Total RNA was isolated from frozen testicular and encephalon+pituitary tissues using an RNA Simple Total RNA kit (Tiangen, Beijing, China). Quantitative real-time PCR (q RT-PCR) was carried out for the application of cDNA using 2×SYBR Green I PCR Master Mix (Vazyme, Nanjing, China). The PCR procedure consisted of 95 °C for 30 seconds followed by 35 cycles of 95 °C for 15 seconds, 58 °C for 30 seconds, and 72 °C for 30 seconds. The melting curve analysis was performed on the PCR products to verify primer specificity and product purity. A dissociation curve was performed for each plate to confirm the production of a single product. The relative abundance of each mRNA was calculated. The PCR primers used for the study are shown in Table 2.

Western blot. For AR expression analysis, the extraction and isolation of cytoplasmic and nuclear protein were performed using a Cytoplasmic and Nuclear Protein Extraction Kit (Beyotime, Nanjing, China) according to the manufacturer’s instructions. Te concentrations of cytoplasmic and nuclear proteins were assessed using a Bradford Protein Assay Kit (Beyotime, Jiangsu, China). Protein samples (80μg) were run on a 12% and 5% SDS-PAGE gel and transferred onto PVDF membranes. After blocking, the membranes were incubated with anti-AR IgG antibodies (1:1,000; Bioss; Beijing China), and mouse polyclonal anti-GAPDH antibodies (1:1,000; Wuhan Boster Biological

Technology, Wuhan, China) or mouse polyclonal anti-Lamin b1 antibodies for 2h. The membrane was washed three times with TBST and incubated with an HRP-conjugated rabbit anti-mouse IgG antibody (1:5,000; Bioss; Beijing China). The signal was visualized using the ChemiDoc Imaging System (Tanon-3,500, Shanghai, China).

High-Performance Liquid Chromatography (HPLC) assay.

Blood was collected in heparinized glass tubes. The plasma was separated by immediate centrifugation at 6,000 rpm for 10 min and stored at −20 °C for further experiment. The hypothalamus and testis samples from each time point were pooled and homogenized with methanol. After centrifugation at 10,000rpm at 4 °C for 10min, the supernatant was concentrated by N2 and the residue was dissolved in 50μL of methanol and filtered through a 0.45 μm filter. Ten μL of the sample filtered liquid was injected into the HPLC system for analysis. The standard curve consisted of samples containing 50, 100, 250, 500, and 750 ng/mL of the ECH (Chengdu Pufei De Biotech Co., Ltd, Sichuan, China). Plasma quality control samples spiked with 75ng/mL (low), 150 ng/mL (medium), and 300 ng/mL (high) of the ECH were accordingly prepared to measure the accuracy and precision of the method.

Chromatography was performed with an HPLC system (D2000 Elite series, Hitachi, Japan) coupled to a UV detector (L-2400, Hitachi, Japan). Separation was performed on a Termo-C18 (250mm ×4.6 mm i.d., 4.6 μm particles) column kept at 25 °C. The mobile phase was a gradient prepared from 0.1% phosphoric acid containing 0.04% trimethylamine (component A) and methanol (component B). The linear gradient was as follows: 70–90% A over 0-2 min, 60–70% A over 2–6 min, 55–60% B over 6–8 min, and then returned to 90% A at 8 min immediately. The flow rate was 0.8 mL/min. The UV detector was operated at 332 nm. The Peak area was evaluated as the analytical measurement.

Pharmacokinetic studies in mice. Te penetration of ECH to the hypothalamus and testis were subjected to pharmacokinetic analysis with Drug and Statistics software (Drug and Statistics, Mathematical Pharmacology Professional Committee of China). Pharmacokinetic parameters were determined using the non-compartmental method based on statistical moment theory. The preliminary pharmacokinetic parameters including the time to peak constant (Tmax), peak concentration (Cmax), elimination rate constant (Ke), elimination half-life (T0.5), the area under the curve (AUC0–12), apparent distribution volume (Vc), and clearance (CL) were obtained for further analysis. Brain/plasma ratios were calculated based on AUC0-t values for plasma and brain.

ECH-Ovalbumin (ECH-OVA) synthesis and identification. A total of 9.8 mg of ECH and 1.0 mg of butanediol anhydride were dissolved in 2mL pyridine, mixed for 12 h with stirring at room temperature. The mixture was concentrated by N2 and the residue was combined with 10.8 mg of N-hydroxysuccinimide (NHS) and 19.3mg of dicyclohexylcarbodiimide (DCC) and dissolved in 4mLN, N-dimethylformamide (DMF) for 12h with stirring at room temperature. After centrifugation at 2,000 g for 5min at 4 °C, the supernatant was added to 5mL of PBS in which 14.4mg ovalbumin (OVA) was dissolved. The new mixture was stirred for 24h at 4 °C. The reaction solution was then dialyzed against PBS for three days. The presence of ECH-OVA was confirmed by UV spectra (Shimadzu Scientifc Instruments, Inc. Columbia, MD USA) at a wavelength ranging from 190 to 400nm as well as by denaturing PAGE.

Indirect ELISA (iELISA). A microtiter plate was coated with the ECH-OVA (2 μg/100 μL) and incubated overnight at 4 °C. The plate was washed three times with PBST and two times with PBS, and 5% PBSM (PBS containing 5% skimmed milk) was added to block the unbound sites at 37 °C for 2 h. After the plates were washed with PBST and PBS, wells were divided into 4 groups; experimental group, 1 μg AR total protein/100μL; positive group, rabbit anti-OVA antibody (1:1,000); negative group, 1 μg of bovine serum albumin; and a blank group containing PBS. After incubation for 1.5h, the plates were washed, and 100μL/well of rabbit HRP-conjugated IgG (1:1,000) was added to the positive group, 100 μL/well of anti-AR was added (1:1,000) to the other groups. After incubation for 1.5 h, rabbit HRP-conjugated IgG (1:5,000) dilution was added to the plates. After incubation at 37 °C for 1 h, the plates were washed three times with PBST and two times with PBS. TMB was then added and incubated for 10min in the dark at room temperature and 2M H2SO4 was added to stop the reaction. The absorbance was read at a wavelength of 450nm.

Molecular docking. A molecular docking study was performed to investigate the binding mode of the compound ECH to the human androgen receptor (AR) using Autodock vina 1.1.2 (http://vina.scripps.edu). The three-dimensional (3D) structure of AR (PDB ID: 2YHD) was downloaded from Protein Data Bank (http://www. rcsb.org/pdb/home/hone.do). The 3D structure of ECH was obtained by ChemBioDraw Ultra 14.0 and ChemBio 3D Ultra 14.0 software. The AutoDockTools 1.5.6 package (http://mgltools.scripps.edu) was employed to generate the docking input files. The search grid of AR was identified as center x: 36.141, center y: 8.513, and center z: 21.304 with dimensions size x: 15, size y: 15, and size z: 15. The value of exhaustiveness was set to 20. For Vina docking, the default parameters were used if it was not mentioned. The best-scoring pose as judged by the Vina docking score was chosen and visually analyzed using PyMOL 1.7.6 software (http://www.pymol.org). Data analysis and statistical methods.

Data analysis and statistical methods. The data were analyzed using statistical software SPSS 19.0 (SPSS Inc., Chicago, IL, USA). A one-way ANOVA was employed for comparison among the groups. Tukey’s comparison tests of significant differences among groups were determined. The results were expressed as mean±standard deviation (SD) using Graph Pad Prism software v.7 (GraphPad Sofware, Inc, California, USA).

Ethics approval and consent to participate. Ethical approval for this study was obtained from the Ethics Committee of Anyang Institute of Technology