Complementary effects of coenzyme Q10 and Lepidium sativum supplementation on the reproductive function of mice: An experimental study

Abstract Background Coenzyme Q10 (CoQ10) and Lepidium sativum (LS) have therapeutic effects on infertility. Objective To evaluate the combined effects of LS and CoQ10 on reproductive function in adult male NMRI mice. Materials and Methods Eighty three-months-old male mice (35–40 gr) were divided into four groups (n = 10/each): control (treated with water), CoQ10-treated (200, 300, and 400 mg/kg/body weight), LS-treated (200, 400, 600 mg/kg/body weight), and co-treated (LS [600 mg/kg/body weight] + CoQ10 [200 mg/kg/body weight]) groups. Serum testosterone, luteinizing hormone, follicle-stimulating hormone, and gonadotropin realizing hormone (GnRH) levels were measured using ELISA method. The sperm quality was assessed using Sperm Class AnalyzerⓇ (SCA) CASA system and GnRH mRNA expression levels were evaluated by real-time polymerase chain reaction. Results The number of sniffing and following behavior was significantly higher in LS-treated (400 and 600 mg/ml/body weight) groups than the control group (p = 0.0007 and p = 0.0010, respectively). The number of mounting and coupling behaviors was significantly higher in the CoQ10 (300 and 400 mg/ml/body weight)-treated animals than the control group (p = 0.0170 and p = 0.0006, respectively). Co-treatment of CoQ10 (200 mg/ml/body weight) and LS (600 mg/ml/body weight) significantly increased all aspects of sexual behaviors as well as the levels of serum testosterone (p = 0.0011), luteinizing hormone (p = 0.0062), and follicle-stimulating hormone (p = 0.0001); sperm viability (p = 0.0300) and motility (p = 0.0010); and GnRH mRNA levels (p = 0.0016) compared to the control group. Conclusion The coadministration of CoQ10 and LS significantly improves the activity of the hypothalamic-pituitary-gonadal axis and enhances the reproductive parameters in adult male mice.


Introduction
Chemical medicines used to treat infertility problems are usually followed by side effects, however, herbal medicines have shown to improve sperm quantity and quality as well as testicular function with low side effects (1)(2)(3).
Lepidium sativum (LS), sometimes referred to as garden cress is a herbal medicine with antiinfertility activity ( 4). It is a fast-growing annual herb from the Brassicaceae family that can reach a height of 50 cm ( 5). LS seeds oil contains a considerable amount of sinapic acid and sinapine, which modulate sex steroids metabolism and act on male reproductive system to improve semen parameters and sperm function ( 6). LS extract are used in many countries for the treatment of various medical conditions including diabetes ( 7), hypertension ( 8), and renal diseases ( 9). Other species of Lepidium such as Lepidium meyenii has been reported to have improving effects on sperm count and motility and on sexual behavior in male animals ( 10,11). It has been shown that plants in the Brassicaceae family can elevate sexual desire in healthy menopause women by acting on the hypothalamus-pituitary-gonad axis ( 12).
Experimental data have revealed the improving effects of LS on the epididymis morphology in a diabetic rat model ( 4) and gonadotropin secretion in rabbits ( 10). Other key factors affecting male fertility are reactive oxygen species (ROS). Under normal physiologic conditions, a slight amount of ROS is produced by sperm cells. However, the excess production of ROS by sperms can cause DNA damage, motility decrease, and sperm membrane dysfunction ( 13). The balance between production and destruction of ROS is essential for sperm motility and male fertility ( 14) and hence, the medicines and nutrients that are able to improve this balance can improve male fertility.
Antioxidants such as coenzyme Q10 (CoQ10) have been shown to inhibit ROS and prevent oxidative DNA damage (15,16).
Although a large body of experimental studies have been carried out to investigate the antioxidant, antimicrobial, and anti-inflammatory characteristics of oil extracted from the seeds of LS, few studies have reported the anti-infertility effects of LS seed oil. There are also very few, if any, reports on the synergistic effects of LS seed extract and CoQ10 on improving the male reproductive system function. Therefore, this study was designed to determine the combined effects of LS and CoQ10 on the reproductive function in adult male NMRI mice and the findings will add new approach in the treatment of male infertility by using combined LS and CoQ10 administration.

Preparation of herbal extract
In this laboratory experimental study, to prepare the aqueous LS seed extract, the Moroccan traditional method was followed ( 17). Briefly, LS seeds were obtained from regional botanical shops, dried in the shade, and kept away from light (to prevent light-dependent reactions in seed cells) in closed containers until use. A mixture of 100-ml distilled water and 1 gr of powdered seeds was simmered for 10 min and left to be cooled for 15

Drug
Purified CoQ10 powder (Pure bulk, US) was used in this study.

Animals and experimental design
Eighty three-months-old NMRI male mice

Evaluation of male sexual behavior
The evaluation of male sexual behavior was carried out using the method described by Schrader and Lemasters ( 20). In brief, sexual behavior (sniffing, following, mounting, and coupling) of male mice was evaluated
Intra-assay and inter-assay coefficients of variation were < 10%, according to the competitive binding principle and the manufacturer's instructions.

Evaluation of epididymal sperm count
The animals were anesthetized with a combination of ketamine and xylazine, then

Statistical analysis
Data are presented as mean ± SEM and were analyzed using the unpaired t test and oneway analysis of variance followed by Tukey's post hoc test. P-values < 0.05 were considered as statistically significant. Statistical analysis was performed using GraphPad Prism 7 software (GraphPad Software Inc., San Diego, USA) and SPSS20 (SPSS Inc., Chicago, USA).

Effect of LS on sexual behavior
To evaluate the sexual behavior of the experimental and control groups of mice, receptive female mice were placed in the males' home cages. Our observation revealed that the numbers of sniffing (p = 0.0007, Figure 1A) and following (p = 0.0010, Figure 1B) behaviors were significantly higher in the LS-treated groups (400 and 600 mg/kg/body weight) than the control group, while there was no significant difference in the numbers of mounting (p = 0.3635, Figure 1C) and coupling (p = 0.4178, Figure 1D) behaviors between the experimental and control groups.

Effect of CoQ10 on sexual behavior
The numbers of sniffing (p = 0.6003, Figure 2A) and following (p = 0.7602, Figure 2B) behaviors did not show a significant difference compared to the control group. The monitoring of sexual behavior in the experimental and control groups showed that CoQ10-treated groups (300 and 400 mg/kg/body weight) exhibited significantly more frequent mounting (p = 0.017, Figure 2C) and coupling (p = 0.0006, Figure 2D) behaviors than the control group.

Combination effect of CoQ10 and LS on sexual behavior
Compared to the data obtained from the control group, the co-treatment of CoQ10 (200 mg/kg/body weight) and LS (600 mg/kg/body weight) caused a significant increase in all aspects of sexual behaviors: sniffing (p = 0.0022, Figure 3A), following (p = 0.0082, Figure 3B), mounting (p = 0.0124, Figure 3C), and coupling (p = 0.0267, Figure 3D) behaviors.

Co-treatment effect of CoQ10 and LS on the serum levels of testosterone, LH, and FSH
The mean values of serum testosterone (p = 0.0011), LH (p = 0.0062), and FSH (p = 0.0001) were revealed to be significantly higher for the co-treatment group than the control group ( Figure 4).

Co-treatment effect of CoQ10 and LS on semen parameters
The co-treatment of CoQ10 and LS intensified sperm parameters. The percentage of sperm viability increased significantly compared to the pretreatment sperm viability (p = 0.03). The total sperm motility increased significantly by 5% (p = 0.001) after the co-treatment.
There was no significant difference in the total sperm count (p = 0.6532) and the number of healthy sperms between the control and co-treated groups (p = 0.7322) (Table II).  Data are presented as Mean ± SEM. *Significant difference compared with the control group (*p < 0.05 and **p < 0.01). Data were analyzed using unpaired t test. LS: Lepidium sativum

Discussion
Our findings indicate that while LS treatment increases the sniffing and following behaviors, Indeed, male infertility accounts for 30-50% of infertility cases. Generally, male fertility depends on the quality, motility, and sperms morphology, therefore any disorder in these factors can lead to a dysfunction in reproductive system ( 2).
In line with our findings, it has been shown that reduced antioxidant capacity caused by decreased cellular CoQ10 function or content can lead to an increase in free radicals' adverse effects on sexual behavior and fertility ( 22). CoQ10 is an androgenic-soluble benzoquinone compound found throughout the body in cell membranes, especially in the mitochondrial membranes, and is abundant in the heart, lungs, and adrenal glands and has improving effects on male reproductive organs ( 23). CoQ10 induces antioxidant activity in rat testes ( 24). In addition, it has been reported that CoQ10 supplementation can boost fertility factors such as sperm count and motility ( 16) and elevates the sexual hormones levels and spermatogenesis in rats ( 25).
Many medicinal herbs have been traditionally administrated to treat infertility due to their potential beneficial effects on male fertility parameters ( 2). One strategy for the evaluation of couples with male infertility is the identification of sexual behavior parameters. LS is one of the most popular herbs used to elevate sexual pleasure and sexual behaviors ( 11). It has been reported to increase dopamine and acetylcholine levels and regulate gamma-aminobutyric acid (GABA) secretion in rats, both of which are related to sexual behavior ( 26). Moreover, LS increases the blood supply and consequently erection via nitric oxide synthesis induction in the endothelial cells ( 27). LS can also enhance sex steroid levels in male and female reproductive system ( 28 CoQ10 has beneficial effects on the reproductive hormones metabolism and secretion ( 30).
The fundamental effect of CoQ10 on sperm parameters may be mediated through multiple pathways. CoQ10 is concentrated within the mitochondrial midpiece of sperm and provides the energy for sperm movement (31). It can also have additive neuroprotective effects and reduces lipid peroxidation of the sperm membrane, which increases the membrane fluidity and motility ( 32).
On the other hand, LS has antioxidant properties ( 8) and a positive impact on sexual behaviors and fertility ( 4,12). Taken together, these facts suggest that LS and CoQ10 can synergistically increase spermatogenesis, sex hormones production and release, and antioxidant activity. Accordingly, all aspects of sexual behavior in male mice can be enhanced by co-administration of LS and CoQ10 via inducing sexual hormones secretions.
However, the exact cellular and molecular basis for the improving effects of LS and CoQ10 on male reproductive system of male mice remains unknown and further research focusing on LS and CoQ10 effects on HPG axis and investigating the cellular and molecular mechanisms involved in LS and CoQ10 effects on testicular tissue as well as sperm cells will reveal many mysteries behind their action on target cells and tissues.

Conclusion
Our