Zearalenone (ZEA) is a naturally occurring estrogenic mycotoxin produced by numerous species of Fusarium(F.) (including F. roseum, F. tricinctum, F. sporotrichioides, F. oxysporum, and F. moniliforme) growing on grains, mainly corn and hay exposed to high moisture during storage [1]. ZEA causes alterations in the reproductive tract of animals such as decreased fertility, increased fetal resorption, reduced litter size, changed weight of endocrine glands and changed serum hormone levels [16,21]. In an NTP cancer bioassay a dose-related incidence of hepatocellular adenomas and endometrial adenocarcinomas were seen in female mice [18]. Despite the potential toxicity of ZEA, only limited information is currently available in regard to the spermatogenesis impairment effects of ZEA.

In our previous study, ZEA induced apoptosis of germ cells in rats [10]. Testicular germ cell apoptosis occurs normally and continuously throughout life [25] and when the testicular environment cannot support spermatogenesis, e.g. during alterations of hormonal support, heat exposure, radiation, or exposure to environmental and chemotherapeutic compounds. Though many different apoptosis-related elements have been identified in the testis, the molecular and cellular mechanisms by which testicular toxicants induce germ cell apoptosis are not thoroughly understood.

The Fas signaling system is composed of the interacting proteins Fas (CD95, APO-1) and Fas ligand (Fas-L; CD95L, APO-1L) [12]. In the testis, the Fas system plays a role in germ cell apoptosis following ischemia- reperfusion, stress, and exposure to testicular toxicants [3,11]. However, the Fas signaling system may be dispensable for nitrobenzene [19] or heat-induced germ cell apoptosis in the testis [9].

Cellular homeostasis in higher organisms is maintained by balancing cell growth, differentiation, and death. Estrogen receptors (ERs) induce expression of genes that control cell fates, including proliferation and cell death via apoptosis [2]. The high expression of ERs in the testis has been demonstrated by Heikinheimo et al. [8], suggesting that these receptors may have some important direct roles in modulating the spermatogenic process by estrogenic chemicals. However, possible roles of ERs underlying the spermatogenesis impairment by estrogenic chemicals are still largely unclear.

In our previous study, ZEA induced apoptosis of germ cells in rats [10]. It is likely that apoptosis by ZEA is controlled in a cell type specific and spermatogenic stage specific fashion, but the basic elements of the death machinery are not thoroughly understood. In the present study, we assessed germ cell apoptosis in rats treated with ZEA in the short term, and possible involvement of Fas, Fas-L, and ERα.

ZEA is a naturally occurring mycotoxin and a potent estrogenic. It is quickly transformed within the liver to α- and β-zearalenol by 3 α-hydroxysteroid dehydrogenase. These products are comparatively much more estrogenic than ZEA [26]. In this study, apoptotic cells were detected maximally at 12 h after ZEA-treatment, and then decreased gradually; these findings were similar to those observed in a previous study [10]. ZEA may have exerted its cytotoxity on target germ cells within only a short period immediately after dosing. In stage specific analysis of apoptosis by ZEA, apoptotic cells were increased in stages I-VI following ZEA-treatment; however, no significant difference was observed between the control and ZEA-treated groups in stage VII-XIV seminiferous tubules. Stages IV-VI correspond to meiotic divisions of primary spermatocytes and mitotic proliferation of B1 and B2 spermatogonia [22]. In contrast, stages V-VI and most importantly VII-VIII (androgen-dependent stages) have been shown to be relatively protected from programmed cell death [15]. Administration of exogenous estradiol affected the hypothalamus pituitary gonadal axis with a dose-dependent concomitant increase in intratesticular estrogen levels and reduced serum gonadotropins and intratesticular testosterone [6]. Selective deprivation of gonadotropins and testicular testosterone by gonadotropin-releasing hormone agonist treatment is followed by a stage-specific increase in germ cell apoptosis in stages VII-VIII [15]. Thus, of the 14 stages in the seminiferous epithelial cycle, stages VII-VIII are the most sensitive to estrogen treatment.

ZEA is a xenoestrogen and binds to ERs, generating an oestrogen-like response [13]. It passively crosses the cell membrane to the cytosolic ER. The receptor-ZEA complex is rapidly transferred to the nucleus, where it binds to the oestrogen-responsive element, thereby activating gene transcription [20]. Two subtypes of ER exist, namely ERα and ERβ, which are distributed differently in the body. An in vitro study demonstrated that 17 β-oestradiol and ZEA acted as competitive agonists for ERα and mixed agonist-antagonists for ERβ [13]. Endogenous estrogens physiologically inhibit steroidogenesis via ERα by acting directly on the testis early in fetal and neonatal development [5]. Also, neonatal administration of the weak estrogenic compound, genistein, affects male reproductive organs at the molecular levels of ERα and AR in adulthood [24]. In the present study, ZEA did not change ERα levels in the testis. This result indicates that spermatogenesis impairments by ZEA in short-term exposure are not closely associated with estrogenic activity of ZEA. Meanwhile, ERα and ERβ levels in testes of mice exposed to tributyltin for 30 days were decreased in one study [4]. However, in another study, long-term treatment of cimetidine induced ERβ overexpression and apoptosis in the rat testis [23]. Clearly, a more thorough understanding of the molecular changes of ERs induced by estrogenic chemicals is needed to understand the pathophysiology of impairment of spermatogenesis.

The mechanisms of how ZEA induces germ cell apoptosis are not well understood. The Fas system was originally characterized as a key mechanism for inducing apoptosis in immune cells [12]. As an immune privileged organ, the testis is known to express Fas. In spermatogenic cell death, it is generally accepted that a direct interaction between Sertoli cell-expressed Fas-L and germ cell-Fas led to the elimination of injured germ cells via apoptosis [27]. In the present study, we confirmed the expression of Fas and Fas-L in testes treated with ZEA as well as the frequency of apoptosis in germ cells. One pathway of testis toxicity may therefore be through apoptosis involving the Fas/Fas-L system. However, while a maximum number of apoptotic cells and a peak in Fas expression were seen at 12 h, Fas-L expression levels increased in a time-dependent manner. In another study, apoptosis and Fas/Fas-L expression levels were not matched [7]. One explanation for this may lie in differences in other molecular mechanisms involved in germ cell apoptosis. In the testis, p21, p53, cytochrome C and the bcl-2 family of proteins, which contains both proapoptotic (such as Bax) and antiapoptotic (such as Bcl-2) family members, play a critical role in regulating the process of apoptosis [11,14,17]. Further studies should be done to determine the precise mechanism of this derangement.

In conclusion, acute exposure of ZEA induces apoptosis in germ cells of male rats and this toxicity is partially mediated through modulation of Fas and Fas-L systems though ERα may not play a significant role.