PART ONE Echinacoside Increases Sperm Quantity in Rats By Targeting The Hypothalamic Androgen Receptor
Mar 09, 2022
How does echinacoside stimulate estrogen and increase sperm count?
Zhihui Jiang1,2, Bo Zhou2, Xinping Li2, Gordon M. Kirby3 & Xiaoying Zhang1,2
Male infertility is a major health issue with an estimated prevalence of 4.2% of male infertility worldwide. Our early work demonstrated that Cistanche extracts protect against sperm damage in mice and that echinacoside (ECH) is one of the major active components. Here we report an essential role for ECH, a natural product that reverses or protects against oligo as the aspermia in rats. ECH was assayed by HPLC, the quantity and quality of sperm were evaluated and hormone levels were determined by radioimmu sorbent assay. ECH reduced levels of androgen receptor (AR) and key steroidogenic-related genes as determined by Western blot and qPCR analysis. The interaction between ECH and AR was evaluated by indirect ELISA and molecular docking. The results show that ECH combined with hypothalamic AR in the pocket of Met-894 and Val-713 inhibits the transfer of AR from the cytoplasm to nuclei in the hypothalamus. While negative feedback of sex hormone regulation was inhibited, positive feedback was stimulated to increase the secretion of luteinizing hormone and testosterone subsequently enhancing the quantity of sperm. Taken together, these data demonstrate that ECH blocks AR activity in the hypothalamus to increase the quantity of sperm and protect against oligo aspermia in rats.
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The production of testosterone in testicular cells is strongly regulated by the hypothalamic-pituitary-gonadal axis (HPG) by forming a homeostatic feedback loop1. Gonadotrophin-releasing hormone (GnRH), secreted by the hypothalamus, can stimulate the secretion of luteinizing hormone (LH) from the pituitary gland, which further stimulates testosterone production in testicular Leydig cells. Testosterone is biosynthesized by a series of steroidogenic enzymes. As one of the major pathways, steroidogenic acute regulatory (StAR) protein can transport cholesterol from intracellular sources into the mitochondria2 where it is exposed to cholesterol side-chain cleavage enzyme (CYP11A1), 3β-hydroxysteroid dehydrogenase (HSD3β), 17α-hydroxylase (CYP17A1), and 17βhydroxysteroid dehydrogenase (HSD 17β) that catalyze the conversion of cholesterol to testosterone3,4. Testosterone then negatively feeds back to the HPG element to down-regulate further LH secretion in a dose-dependent manner.
The effect of testosterone on the HPG axis feedback loop occurs by binding to the androgen receptor (AR), found in both the hypothalamus and the pituitary gland5. In the mice, ablation of the AR and minimal testosterone production causes levels of LH and follicle-stimulating hormone (FSH) to increase6, suggesting that AR participates in the regulation of the negative feed loop. The classical genomic mechanism of testosterone signaling occurs when testosterone diffuses into the cell and binds to the AR. This ligand-receptor complex then translocates to the nucleus where it binds to androgen response elements (AREs) in the regulatory regions of testosterone-responsive genes to modify their translocation. Testosterone also induces the non-classical pathway of steroid hormone action, characterized by rapid events that lead to the activation of cytosolic signaling cascades normally triggered by growth factors7,8. Classical and non-classical testosterone pathways both contribute to maintaining spermatogenesis and fertility. However, the function of the AR is more important in the classical pathway as testosterone acts to increase sperm quality.
Echinacoside (ECH) is one of the bioactive components derived from the medicinal plant species of Echinacea9 and Cistanche10. With a broad spectrum of pharmacological activities, extracts of the Echinacea are one of the most popular herbal supplements in Europe and the US mainly due to their antioxidant properties11 and their ability to prevent the common cold12. Interestingly, Cistanche extracts and ECH have been traditionally used as a tonic agent to cure reproductive dysfunction and to boost male sexual activity in traditional Chinese medicine10. Some OTC products of Cistanche extraction have been developed as nourishing supplements and are gaining popularity in the health food markets of China and some other Asian countries (China Food and Drug Administration)13. However, the underlying mechanisms of ECH action remain unclear. Male infertility is a major health issue with an estimated prevalence of 4.2% of male infertility worldwide14. The diagnosis of male infertility is currently based on the study of sperm quality including the analysis of seminal parameters such as sperm concentration, motility, and morphology15. Te estrogen-mimic Bisphenol A (BPA) is a widespread environmental contaminant that has been studied for its impact on male fertility in several species of animals and humans16. BPA disrupts the hypothalamic-pituitary-gonadal axis, inhibits the expressions of testicular steroidogenic enzymes and the synthesis of testosterone in the male pups17, causing a state of hypogonadotropic hypogonadism18. In this study, we investigate the effects of ECH on sperm quality and hormone levels. In addition, BPA was chosen as a sperm injury model agent to further study the protective effect of ECH against poor sperm quality.
Results ECH enhances the sperm quantity. Te sperm numbers in cauda epididymis, sperm viability, and sperm motility are presented in Table 1. Treatment with 80mg/kg of ECH and 15mg/kg of testosterone propionate (TP) significantly increased the epididymal sperm counts. However, there was no significant difference in sperm viability and sperm motility.
ECH increases testosterone and LH levels in serum, encephalon+pituitary, and testis. ECH treatment notably increased LH levels in the serum, encephalon+pituitary, and testis (Fig. 1A). In the ECH(H) group, LH levels significantly increased by 1.1-fold in serum, 1.4-fold in encephalon+pituitary, and 1.2-fold in testis. Testosterone was significantly increased by 1.5 and 1.3-fold in encephalon+pituitary and testis, respectively, but not in serum after ECH(H) treatment. After TP treatment, the testosterone levels were significantly increased by 1.6, 1.8, and 1.4-fold in serum, encephalon+pituitary, and testis.
ECH increased steroidogenic enzyme gene expression. ECH significantly increased the expressions of key steroidogenic enzymes in the testis (Fig. 1B). The mRNA levels of CYP11A1, CYP17A1, HSD3β1/2, and HSD17β in the ECH(H) group were increased more than 3-fold, while the levels of StAR mRNA showed no significant differences with all ECH treatments (p>0.05). TP did not significantly alter the expression of CYP11A1, CYP17A1, HSD3β1/2, and HSD17β. This is reflected in a comprehensive heatmap analysis of the effect of ECH on steroidogenic enzyme expression indicating that ECH significantly increased mRNA levels of CYP11A1, CYP17A1, HSD3β1/2, and HSD17β, and particularly HSD3β1/2 (Fig. 1C).
ECH can cross the blood-brain barrier and to a lesser extent the blood-testis barrier. To evaluate the androgen feedback loop at the HPG-axis, the ability of ECH to enter the hypothalamus was evaluated in a series of PK studies. ECH concentrations were detected in the hypothalamus after 12h single oral administration of 30mg/kg of ECH, and ECH showed marked hypothalamus penetrance with the mean hypothalamus/plasma ratio of 33.92% (Fig. 2A). Te T1/2 values were 2.61±0.42 h and 1.88±0.22 h in plasma and the hypothalamus, respectively (Fig. 2B). However, very limited access of ECH into the testis was detected using HPLC. Te concentrations of ECH in testis were 0.083 μg/mL, 0.043 μg/mL and 0.028 μg/mL at 0.5h, 1h and 1.5h respectively, after treatment.
ECH reduces hypothalamic AR translocation to the nucleus. To test the effect of ECH on the AR translocation, the expression of AR in the cytoplasm and nucleus of testis and hypothalamus was determined using western blot analysis. As shown in Fig. 3A, ECH down-regulated AR protein in hypothalamic nuclei by five-fold compared to the control group. Treatment with enzalutamide, an AR inhibitor, decreased levels of AR protein in hypothalamic nuclei by 4.8-fold. Notably, cytoplasmic AR was higher with ECH treatment and enzalutamide treatment than controls, suggesting that ECH blocks AR transport from the cytoplasm to the nucleus in the hypothalamus. As expected, ECH does not inhibit AR transport from the cytoplasm to the nucleus in the testis (Fig. 3B).
ECH increased the expressions of HPG-axis related genes. ECH significantly increased the expressions of LHβ, LHR, GnRH 1, and Gnrhr in the mixture pools of encephalon+pituitary, however, there was no significant difference in GnRH 1 levels after TP treatment. The heatmap analysis shows that the expressions of GnRH 1 and LH β were highest among HPG-axis related genes.
AR is a target of ECH. To confirm the relationships between ECH and AR, ECH-Ovalbumin (ECH-OVA) was successfully synthesized (Fig. 5A, B). The molecular weight of ECH-OVA is larger than OVA, so they have different rates of mobility (Fig. 5A), and the absorbance peak of ECH-OVA is between ECH and OVA (Fig. 5B),
suggesting that ECH was successfully conjugated to OVA. Indirect ELISA results (Fig. 5C), show that anti-AR antibody is able to detect AR protein present in wells containing ECH-OVA (indicated by anti-OVA antibody) suggesting that ECH can bind to AR protein. ECH combines with AR pocket of Met-894 and Val-713. Compound ECH was docked to the AF2 site on the surface of the human AR, and the theoretical binding mode of ECH and AR was shown in Fig. 5D. Compound ECH adopted a compact conformation binding at the pocket of the human AR. Te two phenyl groups of ECH bind at the hydrophobic domain of the AR pocket and maintained close hydrophobic contacts with the residues Leu-712, Val-716, Met-734, Ile-737, Met-894, and Ile-898, whereas the other two sides of ECH were positioned at the entrance of the pocket and made only a few contacts. Detailed analysis showed that both Met-894 (bond length: 2.4Å) and Val-713 (bond length: 2.7Å) formed hydrogen bonds with the hydroxy1 groups of the ECH, which was the main interaction between ECH and human AR. ECH attenuates BPA-induced reproductive damage. To investigate the effect of ECH on reproductive damage, the sperm quality and levels of sex hormones and steroidogenic enzymes were examined in BPA-induced mice. As shown in Fig. 6, BPA treatment significantly decreased the sperm count and sperm motility by 26.5% and 39.2%, respectively. ECH administration prevented the decrease in sperm count and sperm motility by 35.5% and 30.1%, respectively. BPA administration also resulted in a significant decrease in LH and T secretion, and ECH considerably increased the levels of LH and T by 24.1% and 18.3%, respectively compared to BPA treatment alone (Fig. 6A, B). The mRNA levels of StAR, CYP17A1, 3β-HSD, and 17β-HSD in BPA-ECH treatment were significantly increased compared to BPA treatment (Fig. 6C, D).
