β-Elemene Inhibits Human Sperm Function by Affecting Sperm Vitality and Intracellular Calcium

β-Elemene, a sesquiterpene compound in zedoary turmeric oil extracted from Curcuma zedoaria, has been reported to have multiple forms of bioactivity, including anti-microbial, anti-fibrosis, and anti-tumor effects [1, 2]. Because of its potent anti-tumor activity and low toxicity β-elemene has been approved by the China Food and Drug Administration for treatment of tumors, such as cancer of the brain, ovary, prostate, breast, lung, liver, colon, and other tissues [1-7]. Previous studies revealed that β-elemene affects tumors by arresting the cell cycle, inhibiting cell invasion, and inducing apoptosis [3, 6-12]. Hence, intensive investigations into the pharmacology and clinical applications of β-elemene have been ongoing in China for the past decade.

The long-term use of β-elemene for treating various types of cancer in patients is considered to be relatively safe [2, 13, 14]. However, several side effects have been reported. For example, injections may lead to phlebitis, local pain, shock, fever, and asthma [2, 15, 16]. Oral administration of β-elemene has been associated with relatively minor adverse effects, including nausea, emesis, diarrhea, and anorexia [15, 17]. To date, investigations on the adverse effects of β-elemene have mainly focused on the hemic, digestive, and renal systems. Nevertheless, some traditional Chinese herbs with components that are similar to β-elemene in terms of bioactivity and biochemical structure have been observed to affect sperm vitality and function in vitro [18, 19]. Therefore, determining the effects of β-elemene on the reproductive system is essential for a more comprehensive understanding of the side effects of β-elemene in clinical applications.

In this study, we evaluated the effects of β-elemene on the characteristics of human sperm essential for fertilization, including vitality, motility, capacitation, acrosome reaction, and responsiveness to progesterone. The intracellular free Ca²⁺ concentration ([Ca²⁺]_i) was examined to explore the underlying mechanism. Our results may represent a new reference for understanding the side effects of β-elemene in clinical applications and may be relevant for developing new guidelines for the use of β-elemene in clinical practice.

The three cell types were used in this study, MCF-10A (normal cell control), MDA-MB-231 (breast cancer cells), and A540 (lung cancer cells), were all obtained from ATCC. MCF-10A cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) / F12 Ham medium supplemented with 5% horse serum, 20 ng/mL epidermal growth factor, 0.5 mg/mL hydrocortisone, 100 ng/mL cholera toxin, and 10 μg/mL insulin. Additionally, MDA-MB-231 and A549 cells were maintained in DMEM medium containing 10% FBS.

Cells were seeded at a density of 1, 000 (MCF-10A and MDA-MB-231) or 2, 000 (A549) cells/well in 96-well plates. The next day, cell cultures were treated with β-elemene (0, 40, 80, 160, 320, or 640 μM) or docetaxel (20 or 200 nM; dissolved in DMSO). All cell cultures contained 0.1% DMSO. The docetaxel concentrations used in this study are reported to be cytotoxic [20]. Thus, they served as positive controls. Cell viability was determined using the Cell Proliferation Assay Kit (Abcam, Cambridge, MA) according to the manufacturer’s instructions. The SpectraMax M2 plate reader (Molecular Devices, LLC., San Jose, CA) was used to measure the optical density of solutions (492 nm).

Cells were seeded at a density of 500 (MCF-10A and MDA-MB-231) and 1, 000 (A549) cells/well in 6-well plates. The next day, cells were treated with the same concentrations of β-elemene and docetaxel as in the cell viability assay. All cell cultures contained 0.1% DMSO. Surviving colonies were counted at 6-7 days after seeding.

Semen samples were collected from healthy donors following masturbation after 3-5 days of sexual abstinence. Fertility and normal sperm quality were confirmed for all donors according to the criteria in the World Health Organization (WHO) laboratory manual for examining and processing human semen (WHO, 2010). Sample collection was approved by the Institutional Ethics Committee on Human Subjects of the Jiangxi Provincial Maternal and Child Health Hospital. Sperm was harvested based on the direct swim-up technique involving human tubal fluid (HTF) medium (93.8 mM NaCl, 4.69 mM KCl, 0.2 mM MgSO₄, 0.37 mM KH₂PO₄, 2.04 mM CaCl₂, 21.4 mM lactic acid, 2.78 mM glucose, 21 mM HEPES, 4 mM NaHCO₃, and 0.33 mM C₃H₃NaO₃; pH adjusted to 7.35 with NaOH) as described previously [21]. Sperm samples were incubated in HTF medium (0.1% DMSO) containing 0, 10, 20, 40, 80, 160, or 320 μM β-elemene at 37 °C in a 5% CO₂ incubator for various periods of time according to the experimental protocol. The β-elemene (purity > 99%) used in this study was purchased from the National Institutes for Food and Drug Control (China).

Sperm vitality was examined using the eosin-nigrosin staining method, and sperm motility was assessed with the WLJY-9000 computer-assisted sperm analysis (CASA) system (WeiLi Co., Ltd., Beijing, China) as described previously [21].

A sperm penetration assay involving a 1% (w/v) methylcellulose solution mimicking the viscous environment in the female reproductive tract was performed as described previously [21].

The capacitation and acrosome reaction of human sperm were evaluated with the chlortetracycline (CTC) staining method as described [21].

Changes in human sperm [Ca²⁺]_i were measured using the Fluo-4 AM fluorescent Ca²⁺ indicator (Molecular Probes, Eugene, OR) with the EnSpire® Multimode Plate Reader (Perkin Elmer, Waltham, MA) as described [21].

Sperm responsiveness to progesterone was examined in terms of enhancement of penetrating ability, induction of capacitation and the acrosome reaction, and increase in [Ca²⁺]_i. To evaluate the effects of β-elemene on the progesterone-induced penetrating ability and acrosome reaction, human sperm were first capacitated for 4 h in HTF medium containing 25 mM NaHCO₃ and 0.4% human serum albumin (HSA; Vitrolife Corporation, Göteborg, Sweden). Next, 20 mM progesterone and different concentrations of β-elemene were added, after which samples were incubated for 1 h. To evaluate the effect of β-elemene on progesterone-induced [Ca²⁺]_i increase, human sperm were first incubated with different concentrations of β-elemene for 15 min and then with 1 µM progesterone.

Data are expressed as the mean ± standard error of mean (SEM). Differences between the controls and treated samples were assessed using two-way analysis of variance (ANOVA). Significant differences (P < 0.05) were determined using the GraphPad Prism program (version 5.01; GraphPad Software, San Diego, CA).

We first examined the anti-tumor effects of β-elemene. The three cell lines, MCF-10A (normal cell control), MDA-MB-231 (breast cancer cells), and A540 (lung cancer cells), were treated with different concentrations of β-elemene or docetaxel (positive control). The cytotoxicity of β-elemene toward these cells was evaluated using MTS and colony formation assays. The results showed obvious cytotoxic effects of β-elemene against both cancer cell lines at β-elemene concentrations of up to 160 μM (Fig. 1B, C, E, and F). These β-elemene concentrations were non-toxic or minimally toxic to MCF-10A (Fig. 1A and D). Moreover, a dose of ≥ 320 μM β-elemene was required to produce the same cytotoxic effect as docetaxel (Fig. 1B, C, E, and F). However, 640 μM β-elemene was obviously toxic to MCF-10A cells (Fig. 1A and D). These results are consistent with those of previous studies in which β-elemene was toxic to tumor cells at high concentrations, but was non-toxic or minimally toxic to normal cells [2, 5, 6, 11, 12, 14, 17, 22].

Subsequent examination of the cytotoxicity of β-elemene toward human sperm revealed that 40-320 μM β-elemene dose-dependently reduced human sperm vitality, and there were no viable sperm following treatment with 320 μM β-elemene (Fig. 2A). To assess whether β-elemene cytotoxicity β-elemene affects sperm function, we examined sperm motion parameters (total motility, progressive motility, and curvilinear velocity) and penetrating ability, which represented a comprehensive evaluation of sperm motility, especially hyperactivated motility. The total motility (Fig. 2B), progressive motility (Fig. 2C), curvilinear velocity (Fig. 2D), and penetrating ability (Fig. 2E and F) of human sperm decreased considerably at β-elemene concentrations of ≥ 40 mM. These results suggest that β-elemene causes injury to sperm at relatively low concentrations, which are non-toxic to cancer cells.

Concentrations of 10 and 20 μM β-elemene did not affect human sperm vitality and motility. Thus, we assessed whether these two concentrations β-elemene affects other aspects of sperm function. Although they did not affect capacitation (Fig. 3A), 10 and 20 μM β-elemene dose-dependently enhanced the spontaneous acrosome reaction (Fig. 3B), which is an exocytotic process important for the penetration of the oocyte by sperm. Because the acrosome reaction is a Ca²⁺-dependent process, we evaluated whether β-elemene affects the [Ca²⁺]_i of human sperm. Concentrations of 10 and 20 μM β-elemene initially increased human sperm [Ca²⁺]_i in a dose-dependent manner, followed by a gradual reduction in [Ca²⁺] _i to almost resting levels (Fig. 3C and D). These results indicate that β-elemene induces the spontaneous acrosome reaction by increasing [Ca²⁺]_i in human sperm.

Progesterone facilitates fertilization by enhancing sperm function [23]. Thus, we also examined whether β-elemene affects progesterone-induced human sperm function. The ability of human spermatozoa to penetrate artificial viscous media, which mimics the viscous environment in the female reproductive tract is influenced by sperm motility, especially hyperactivated motility. The 20 µM progesterone treatment increased the mean cell numbers density by approximately 60% at 1 cm (Fig. 4A, P4) and 2 cm (Fig. 4B, P4) from the base of the tube compared with the control levels, while the addition of 10 and 20 µM β-elemene to the progesterone treatment resulted in smaller increases in cell density (P4 + 10 and P4 + 20, Fig. 4A and B). Moreover, 20 µM progesterone significantly enhanced the acrosome reaction (Fig. 4C, P4) and capacitation (Fig. 4D, P4) of human sperm. Concentrations of 10 and 20 µM β-elemene significantly inhibited the progesterone-induced acrosome reaction (P4 + 10 and P4 + 20, Fig. 4C), but did not affect progesterone-induced capacitation (P4 + 10 and P4 + 20, Fig. 4D).

Progesterone-induced human sperm function is are closely correlated with progesterone-stimulated Ca²⁺ influx [23]. To clarify whether β-elemene modifies progesterone-induced Ca²⁺ signaling, we pre-treated human spermatozoa with different concentrations of β-elemene for 15 min and then added 1 µM progesterone. The increase in progesterone-induced [Ca²⁺]_i was significantly inhibited by 10 and 20 µM β-elemene (Fig. 5A and B). Furthermore, we compared the [Ca²⁺]_i amplitudes induced by β-elemene and progesterone alone or in combination. Ca²⁺ response induced by 20 µM β-elemene and 1 µM progesterone were 18.4% and 100.1%, respectively (Fig. 5C and D). Meanwhile, the Ca²⁺ response induced by the combination of β-elemene and progesterone was 93.5% (Fig. 5C and D). Lower Ca²⁺ response due to the combined treatment compared with progesterone alone was observed, suggesting that β-elemene and progesterone may use identical pathways to increase human sperm [Ca²⁺]_i. These results imply that β-elemene affects progesterone-induced human sperm function by inhibiting progesterone-stimulated Ca²⁺ influx.

β-Elemene exhibits various types of bioactivity and represents a promising compound for the development of cancer treatment. It is a liposoluble compound that can cross the blood–brain and blood–testis barriers, enabling its distribution throughout the body via blood. Also, β-elemene can accumulate in the testes at maximum concentrations of up to approximately 100 µM [24, 25]. However, whether β-elemene is toxic to the human reproductive system remains unclear. Although many phytogenic anti-tumor agents have been reported to be toxic to the human male reproductive system, several of these agents have been approved for use because most patients have no desire to preserve their fertility. However, the incidence of cancer among adolescents has recently been increasing and thus preserving the fertility of adolescents while they undergo cancer treatment is an important concern. Consequently, the reproductive toxicity of anti-tumor agents must be thoroughly considered.

Our data indicate that 40-320 µM β-elemene is nontoxic or minimally toxic to cancer and normal somatic cells (Fig. 1), but is cytotoxic to human sperm; treatment with 320 μM β-elemene left no viable sperm (Fig. 2). These results suggest that human sperm are more sensitive to β-elemene than cancer cells or somatic cells. Thus, the cytotoxic effect of β-elemene on human sperm should be emphasized in clinical application.

Although 10 and 20 µM β-elemene treatments were not cytotoxic to human sperm, these concentrations can affect the spontaneous acrosome reaction (Fig. 3A and B), a physiological process that occurs in less than 20% of sperm after capacitation in normal males. Thus, enhancing the spontaneous acrosome reaction with β-elemene treatment may influence the penetrative ability of sperm because sperm undergo this reaction before encountering the egg. Additionally, 10 and 20 µM β-elemene treatment also inhibits progesterone-induced sperm function (Fig. 4). The acrosome reaction and progesterone-induced sperm function are Ca²⁺-dependent processes. Therefore, we examined whether β-elemene affects sperm [Ca²⁺]_i and progesterone-induced [Ca²⁺]_i increases. Treatment with 10 and 20 µM β-elemene caused a transient increase in sperm [Ca²⁺]_i, followed by a gradual decrease until reaching almost resting levels (Fig. 3C and D). Moreover, pre-incubating human sperm with β-elemene for 15 min ([Ca²⁺]_i had already decreased to almost resting levels) inhibited the progesterone-induced [Ca²⁺]_i increaseβ-elemene (Fig. 5). This discrepancy may be explained by the fact that that β-elemene and progesterone possibly use identical pathways to increase human sperm [Ca²⁺]_i. During the pre-incubation of β-elemene and human sperm, the β-elemene may occupy the binding site(s) of the target protein(s) and affect the progesterone responsiveness of human sperm. This possibility is supported by the Ca²⁺ response induced by the combination of β-elemene and progesterone (Fig. 5C and D). These results imply that the non-cytotoxic β-elemene concentrations influence human sperm function via a Ca²⁺-dependent mechanism.

The present study examined the toxicity of β-elemene toward human sperm and conclude that in vitro exposure to 40-320 µM β-elemene significantly inhibits sperm motility, while 10-20 µM β-elemene affects the acrosome reaction and progesterone-induced sperm function through a Ca²⁺-dependent mechanism. This study has revealed the toxicity of β-elemene toward the human male reproductive system. Therefore, caution is required when administering β-elemene to cancer patients who wish to preserve their fertility.

This study was supported by the National Natural Science Foundation of China (31170202 and 31570195), the Basic Working Special Project of the Ministry of Science and Technology of China (2013FY111500), and the Scientific Research Fund of Hongda Zhang, Sun Yat-Sen University. This study was also funded by the Science and Technology Program of Guangzhou (201704020095), the Program for Guangdong Introducing Innovative and Entrepreneurial Teams (2016ZT06S252), Fundamental Research Funds for the Central Universities (17ykjc14), and the Guangdong Provincial Natural Science Foundation (no.2016A030313312).

The authors declare that they have no conflicts of interest.