Effects of Perilla frutescens Var. Acuta in Busulfan-Induced Spermatogenesis Dysfunction Mouse Model

It is known that approximately 15% of all couples have difficulty conceiving and estimated that approximately 20%–70% of these cases are due to male infertility [1, 2]. Overexposure to certain environmental factors and lifestyle behaviors are associated with a decrease in men’s reproductive function and has recently generated some amount of interest [3]. Unfortunately, however, there are more cases of no identifiable reason than cases where a clear cause can be known and treatment can be selected accordingly.

A new term called Male Oxidative Stress Infertility (MOSI) has emerged due to identification of oxidative stress (OS) as an important factor in idiopathic male infertility [4, 5, 6]. In a healthy state, pro-oxidants and antioxidants are balanced, but when the antioxidant defense mechanism is weakened, oxidative damage to gonadal cells and mature spermatozoa is unavoidable [7]. It has been reported that elevated reactive oxygen species (ROS) levels in seminal plasma are found in approximately 30%–40% of patients with male infertility [8]. OS has also been linked to poor fertilization, poor embryonic development, pregnancy loss, birth defects (including autism), and childhood cancer [9, 10]. In a situation where there is no effective treatment for idiopathic male infertility, research to develop antioxidants to lower the OS and use them for treatment has been continuously conducted [11].

Drugs with various antioxidant effects and herbal extracts have been developed in mono- or poly-formulations, and several research results from basic to clinical studies have been published [12, 13]. Antioxidants currently widely used clinically include vitamin A, vitamin C, vitamin E, carnitine, N-acetyl cysteine, coenzyme Q10, and lycopene, along with important antioxidant cofactors, such as zinc, selenium, and folic acid [14]. However, since the effects of these antioxidants are not consistent between reported studies, it is necessary to continuously try to develop new antioxidants [11, 15]. Antioxidants extracted from herbs are relatively safe and have high drug compliance; therefore, they can be high-value candidates for development.

The leaves of Perilla frutescens var. acuta (PFA) are an herbal medicine that has been used for various medical purposes in Asia [16, 17]. It is also widely used as a food ingredient [18]. Studies on PFA have revealed that PFA has antioxidant, anti-glycosuria, anti-inflammatory, anti-allergic, antimicrobial, anti-dementia, antidepressive, antitumor, and anti-obesity properties [16, 17, 18, 19]. The known chemical compounds of this plant include phenolic compounds, flavonoids, anthocyanins, essential oils, and phenylpropanoids [17, 18]. Despite the antioxidant effects of PFA, there have been no reports on the effect of PFA on male fertility. Therefore, we conducted this study to evaluate the therapeutic effects of PFA on spermatogenesis in busulfan-induced spermatogenesis dysfunction in mice.

In the present study, busulfan-induced testicular injury in mice was characterized by decreased testis/body weight, decreased epididymal sperm count and motility, and disorganized seminiferous epithelium, which may be a consequence of elevated OS in the testis and abnormal hormone levels. To the best of our knowledge, this is the first study to demonstrate the beneficial effects of PFA in busulfan-related testicular injury by attenuating OS in the male reproductive system. The results of this study showed that sperm concentration and motility were significantly increased in male mice with busulfan-induced testicular toxicity when administered with 200 mg/kg of PFA compared with those with busulfan alone. The level and expression of SOD and GPx, the diameter of the germinal epithelium, and testosterone hormone levels were increased in busulfan-induced male infertile mice administered 100 and 200 mg/kg of PFA compared with the busulfan alone group.

The incidence of cancer continues to increase and one of the most prevalent cancers during reproductive age is leukemia [21, 22]. Despite the development of cancer treatments such as chemotherapy regimens, radiotherapy therapy, and surgical options, busulfan is known for its effective management of leukemia, especially in children. However, because busulfan is very effective in spermatogonial stem cells (SSCs), it affects the male reproductive system and causes loss of fertility, especially in young cancer survivors [20]. In addition, busulfan-induced male sterility in mice is very similar to that in humans [20].

Among various approaches as the animal model in infertility studies, 5 to 40 mg/kg of busulfan was the most commonly used for induction of infertility mice due to its easy administration by intraperitoneal injection [23]. Zohni et al [24] reported that single injections of busulfan at 15, 30, or 45 mg/kg were direct consequences of SSC loss. Wang et al [23] reported that a busulfan dose of up to 30 mg/kg was optimal in terms of reducing lethality in BALB/c mice. Dobrinski et al [25] reported that the intraperitoneal injection of busulfan at 50 mg/kg was lethal in BALB/c mice due to hematopoietic toxicity. Xie et al [20] reported that testes showed dynamic changes at 9 days after the administration of 40 mg/kg of busulfan but partially recovered 90 days after busulfan administration. To date, there has not yet been a systematic study to determine the optimal dose of busulfan for the induction of infertility in experimental animals such as mice. In previous studies, the intraperitoneal injection of busulfan at 40 mg/kg in C57BL/6 mice resulted in decreased testis/body weight, atrophied STs, reduced sperm function, and abnormal hormone levels than 10 to 20 mg/kg. Consistent with previous studies, the results of this study also showed lower sperm quantity and quality in mice treated with higher busulfan doses than in those treated with lower busulfan doses. However, the mortality rate of a single intraperitoneal injection of 40 mg/kg of busulfan was up to 15% for 1 week due to the possibility of the inhibition of hematopoiesis by absorption into other organs via blood (data not shown). In the current investigation, we selected two intraperitoneal injections of busulfan at a total dose of 40 mg/kg at 3-hour interval to establish a model of azoospermia with low hematopoietic toxicity and zero mortality. In the present study, with no mortality, significantly lower body and testicular weights were observed in the busulfan-treated group than in the control group (Fig. 1).

Several pharmacological studies have examined the leaves of PFA for their antioxidant, anti-inflammatory, anti-allergic, antimicrobial, and antitumor effects [17, 18]. Kim et al [17] reported the efficacy of the antioxidant activity of PFA in polyphenolic components such as rosmarinic acid, luteolin-7-O-glucuronide, and apigenin-7-O-glucuronide. With reference to previous studies on the antioxidant function of PFA, our experimental study demonstrated the relationship between the antioxidant effects of PFA and busulfan-induced testicular damage due to OS, which is very important in sperm fertility potential. Building on previous studies on the antioxidant functions of PFA, our experimental study investigated the antioxidant effects of PFA and the restoration of sperm quality in mice with busulfan-induced testicular injury as an ex vivo effect of antioxidant defense. The present study demonstrated, for the first time, the therapeutic effects of PFA on busulfan-induced testicular damage by suppressing OS. According to the histomorphometric findings, the control group showed complete spermatogenic activity, and in the busulfan group, the thickness of the germinal epithelium decreased due to the decrease in the proportion of spermatogonia, spermatocytes, and spermatids (Fig. 2). PFA administration in busulfan-treated mice significantly increased the proportion of spermatogonia and spermatocytes compared with busulfan alone. Based on our data, we suggest that PFA administration protects germ cells from busulfan-induced testicular toxicity. Zhengwei et al [26], reported that a direct relationship between the germinal cell number and testis weight in primates. Our findings are consistent with previous reports showing that busulfan-induced testicular weight loss is associated with loss of germ cells and that testicular weight gain with PFA administration is associated with germ cell recovery (Fig. 1). Quantitative recovery of germ cells can be evidenced by an increase in the total diameter and cellular diameter of the ST, which comprise a substantial portion of the testes. In addition, our results clearly showed that PFA administration increased sperm concentration and motility compared with busulfan alone, which suggests that PFA rescues spermatogenesis (Fig. 3).

OS has also emerged as a major cause of male fertility in at least 40% of the patients with potential impacts on the developmental capacity of embryos and the health and well-being of the offspring [27, 28]. Busulfan is known to partially eliminate stem cells due to its alkylating properties and inhibit the spermatogenesis process, especially by oxidative damage [23, 24]. Testicular OS is known to increase the MDA levels in testicular tissue, while decreasing the activity of protective antioxidant enzymes (SOD and GPx). In this study, busulfan exposure induced increased MDA production along with a decreased activity of the antioxidant enzymes SOD and GPx in the testes of mice, presumably due to testicular damage caused by overproduction of ROS and lack of antioxidant defenses. However, the increased formation of MDA and decreased activities of SOD and GPx were significantly restored by simultaneous treatment with PFA, suggesting that PFA has an ameliorating effect on busulfan-induced reproductive and developmental toxicity in mice. Our results also showed that PFA administration upregulated the expression of genes encoding the antioxidant enzymes Sod1 and Gpx1 (Fig. 5). However, the mechanisms involved in PFA-mediated OS suppression are not yet clear; therefore, further mechanistic studies of the relationship between PFA and transcription factors affecting antioxidants are needed.

In this study, the lowered serum testosterone and FSH levels in mice treated with busulfan injection in male mice were consistent with previous reports [29]. It is now recognized that Leydig cells produce testosterone in response to LH, which diffuses into the STs and acts as a paracrine factor necessary to maintain spermatogenesis [30]. Although the mechanism underlying the detrimental effects of busulfan injection is not yet clear, previous studies have shown that injection of high-dose busulfan inhibits testosterone production by affecting the exocrine and endocrine compartments of the testes with decreased antioxidant enzyme activity, increased testicular lipid peroxidation, and persistent impairment of Leydig cell function [30]. The oral administration of PFA in busulfan-treated mice significantly increased the serum testosterone levels compared with the busulfan-treated group (Fig. 4). Therefore, the amelioration in the serum testosterone levels observed after PFA administration in busulfan-treated mice may be related to the recovery of Leydig cells by ROS inhibition.

In the present study, the body and testicular weights, sperm count, sperm motility, serum testosterone levels, increased SOD levels, and decreased MDA levels occurred in a dose-dependent manner in the PFA- and busulfan-treated groups compared with those in the busulfan alone group. These effects of the high dose of PFA were better than those of low doses during the administration period.

The present study has some limitations. First, there are no existing standards for optimal oral antioxidant supplementation regimens and duration of administration; therefore, further investigation of various dosages or long-term administration of PFA is needed. Second, it is necessary to evaluate the ability of PFA to inhibit apoptosis in busulfan-induced testicular tissue because spermatogenesis is a complex process and germ cell apoptosis is essential. Third, we could not investigate the effects of each of the individual components of PFA. An in-depth study of the active ingredients of PFA should follow.

The oral administration of PFA restored busulfan-induced disrupted spermatogenesis by improving testicular germ cell development and sperm quality through the attenuation of OS. These benefits of PFA can be used to improve male reproduction in patients treated with busulfan or other cancer medications or in patients with idiopathic failed gametogenesis. We propose that PFA may affect these infertile couples through improved spermatogenesis.

In conclusion, PFA exhibited a protective effect against busulfan-induced testicular damage in male mice, showing decreased sperm count and motility, testosterone levels, and antioxidant status and increased MDA levels. Our results also showed that the aforementioned changes would be dose-dependent in PFA. Our results suggest that PFA helps improve the reproductive parameters in busulfan-induced male reproductive toxicity.