Influence of mycotoxin zearalenone and its derivatives (alpha and beta zearalenol) on apoptosis and proliferation of cultured granulosa cells from equine ovaries

Many different mycotoxins have been recognized and isolated from a variety of Fusarium moulds and some of the disease states, caused by consumption of cereals containing these toxins in domestic animals as well as in humans, have been called fusariotoxicoses. Zearalenone (ZEA) and related compounds α and β zearalenol (α and β-ZOL) and α and β zearalanol (α and β-ZAL) are synthesized by a number of species of Fusarium such as F. graminearum, F. tricinctum, F. moniliforme and F. oxysporum [1].

Sensitivity to the effects of mycotoxins is related to species-dependent biotransformation pathways. Zearalenone is metabolized via two pathways in hepatocytes and intestinal cells, namely conjugation with glucuronic acid and reduction to α and β-ZOL by 3 α(β)-hydroxysteroid dehydrogenase (HSD) [2‐4]. These reactions show similarities to processes in steroid metabolism because HSDs catalyse oxidation/reduction reactions in the synthesis and inactivation of steroid hormones [4].

The influences of ZEA on reproductive structural and functional parameters are well recognized. Of all domestic species, swine is the most sensitive species, followed by ruminants. Birds are the most resistant species [3]. The high sensitivity of pigs to ZEA-mediated oestrogenic effects can be related to ZEA bio-activation prevalently into α-ZOL. The higher estrogenicity of α-ZOL is related to its stronger relative binding affinity to the cytoplasmic oestrogenic receptor than β-ZOL, as described in the rat uterus [5].

Of all stages of maturity, the pre-pubertal gilt is the most sensitive to ZEA [6]. The changes induced by ZEA depend on the time of administration, in relation to the oestrous cycle, and on the administered dose [6]. A vulvovaginitis syndrome in young female swine and anaestrus induction in the mature sow, have been reported [7]. In extreme cases, rectal and vaginal prolapses occurred. Several atresic follicles are present on ovaries and degeneration of oocytes occurs [8‐10].

Concerning the equine species, the effects of ZEA have only been demonstrated in a few cases. A field outbreak of ZEA mycotoxicosis in horses was associated with corn screenings containing approximately 2.6 mg/Kg of ZEA [11]. Raymond et al. [12, 13] reported the influence of ZEA, administered at a low dose, on performance and hematological parameters. No disturbances on reproductive functions were reported for cycling mares after exposure of 1 mg/Kg of ZEA in the feed [14]. However, these levels are also ineffective on sensitive species, such as swine [7]. The limited number of reports on ZEA-related clinical symptoms in the horse could not exclude the involvement of this feed contaminant in hypo-fertility cases. No data have been reported to date concerning the pharmaco-kinesis and metabolism of ZEA in the horse.

In relation with the high incidence of ZEA-related reproductive failures in other species [15] and considering the difficulties in conducting in vivo studies on reproductive influence of ZEA on system in the horse, the purpose of this paper was to investigate the effects of ZEA and its derivatives, α and β-ZOL, on granulosa cells (GCs) collected from mare ovaries during the breeding season. It has been demonstrated that mural GCs are strictly involved in the control of oocyte growth and maturational competence [16] and are a sensitive target of xenoestrogen substances [17].

Premature follicular atresia is a recognized ZEA-induced consequence associated with either natural or experimental contamination [8‐10]. The aim of this work was to use flow cytometric analysis in order to evaluate the atretic process in cultured GCs after treatment with zearalenones. Analysis of DNA content by flow cytometry provides a means of looking at early apoptosis and atresia as well as cell cycle perturbation and accumulation or depletion of cells in particular phases. With this method, apoptotic cells, containing less DNA than normal diploid cells, exhibit reduced fluorescence and are recorded as a sub-G₀ peak. This method has been reported to date in porcine, bovine, ovine and rat follicles [18‐22]. Atresia consists of a dynamic equilibrium between GC division, differentiation and death and simultaneous apoptosis and mitosis processes occur [23]. For this reason, an evaluation of GC proliferation was carried out using colorimetric (MTT test) and flow cytometric methods.

An increase in cell proliferation of equine GCs was observed using the MTT test in the presence of 1 × 10^-4 and 1 × 10^-3 μM of ZEA. High doses of ZEA and its metabolites demonstrated a trend towards down-regulation of GC proliferation indices. This increased proliferation could be the consequence both of the reduction of ZEA into α-ZOL and the modification of ovarian steroidogenesis with accumulation of the active hormonal components related to the involvement of 3α(β)-HSD, enzymes present in the ovary and GCs [3, 29]. The HSDs have also been claimed to be important factors in ovarian follicular development and their inhibition may promote follicular atresia, since abnormalities in the androgen, progestin and oestrogen metabolism can lead to ovarian dysfunction [3, 28]. Zearalenone and its derivatives induced apoptotic processes within equine CGs at different concentrations. The mean intensity of apoptosis induced by ZEA was greater than that caused by α-ZOL, probably derived from the combined effect exerted by both mycotoxins metabolized by HSD in GCs, as reported previously [29]. Beta-zearalenol was less active than other mycotoxins because it induced apoptosis only at the highest concentration.

In this study, ZEA and its derivatives did not induce statistically different modifications of cell distribution in different phases of the cell cycle. However, it should be noted a trend in growth increasing, evaluated as S+G₂/M proliferation peak, that was affected by 1 × 10^-2 and 1 × 10^-3 μM of ZEA and α-ZOL, respectively. The observed variability could be related to the different levels of HSDs in GS cultured in each experiment, as reported by other authors [30]. In addition, seasonal (light, temperature), environmental (stress, diet) and hormonal factors (including the enzymatic contents) as well as the holding time and temperature can influence the variability observed during experiments carried out with equine GCs [31]. Though no statistical differences were found in our experiments, simultaneous increases in the sub-G₀ and S+G₂/M peaks indicate a simultaneous occurrence of apoptosis and the proliferation process after exposure with ZEA and α-ZOL. This dynamic equilibrium between division, differentiation and death was found by Pedersen et al. [32] in atretic follicles. The simultaneous occurrence of proliferation and apoptosis was studied by Quirk et al [33, 34] in bovine GCs. These authors reported that oestradiol, an important intraovarian growth, differentiation and survival factor, increases the progression from G₀/G₁ to S phase, the percentage of cells in S phase and, consequently, the percentage of DNA synthesizing cells and that this cell cycle progression is mediated by the oestrogen receptor [33]. Granulosa cells may be most susceptible to apoptosis at the transition from G₁ to S phase [34]. We hypothesize that zearalenones, oestrogen-like substances, can act with a similar mechanism on equine GCs and can induce proliferation and apoptotic process.

Follicular degeneration, or atresia, is the result of an apoptotic process defined as a sequence of programmed intracellular events leading to cell death [20]. The first signs of atresia are evidenced as the degeneration of the antral GCs, whereas the oocyte is affected in the last stages of atresia [35, 36]. Various techniques have been adopted to measure the degree of follicular degeneration in ovaries. Few reports are available to date on apoptosis of GCs in horse. Okolski et al. [37] evaluated equine GCs for the presence of nuclear fragments they termed atretic bodies. The presence of these bodies was correlated with reduced oestrogen levels in the follicular fluid but was not correlated with the proportion of oocytes that matured in vitro [37]. Pedersen et al. [32], the first group using DNA laddering to study apoptosis in the mares, found that small follicles were more likely than large follicles to be apoptotic. These authors reported that the histological method used for determining the presence of atresia was not reliable because atretic GCs were not uniformly distributed but were observed in focal points, within areas of healthy GCs [32]. Albrizio et al. [38] investigated follicular wall apoptosis in the mare by comparing DNA laddering and the incidence of apoptotic bodies (AB) analyzed by Hoechst 33258 staining. These authors found that high rates (80%) of typical masses of AB were observed in GCs of small follicles from anoestrous mares and, with a lower incidence, in pools from regressing follicles collected from the ovaries of transitional, oestrous and dioestrous mares (30–40%). Large pre-ovulatory follicles were totally lacking AB. Correspondingly, the presence of DNA fragments forming the characteristic banding pattern (ladder) of apoptosis was revealed in the GCs from anoestrous mares; these fragments were not detected in the GCs from large pre-ovulatory follicles while they had an intermediate incidence in the follicles from ovaries of transitional and dioestrous mares. Dell'Aquila et al. [39] reported that GC apoptosis in the mare is related to cumulus expansion and to an increase in oocyte meiotic competence but has no effect on the proportion of meiotically competent oocytes that activate after ICSI. Subsequently, Pedersen et al. [40] detected GC apoptosis by comparing two biochemical methods, 3'-end labelling and staining with ethidium bromide then microscopic examination of GC morphology, with the established method of detecting atresia by histology. These authors concluded that the more parameters used in conjunction with each other, the more accurate was the analysis of the state of the follicle. Many studies have shown that GCs with hypodiploid DNA content are apoptotic, may represent a biochemical marker for atretic follicles in several animal species and that this method is sensitive enough to detect apoptosis in its very early stages [18‐22]. Granulosa cells that have initiated chromatin cleavage are definitively non-functional and flow cytometry enables the rapid detection of cells that are undergoing the early events of apoptosis, as previously reported in porcine, bovine, ovine and rat follicles [18‐22]. In our experimental conditions, flow cytometry was effective and rapid for evaluating the in vitro effects of the mycotoxin on GC apoptosis in the mare. This is the first report on the use of flow cytometry to study the atretic process in mare cultured GCs.

A "mare reproductive loss syndrome (MRLS)", which occurred in Kentucky in 2001, was characterized by non etiologically defined cases of early and late-term abortions [41]. Different etiological agents were thought to be responsible for this pathology, such as ergot alkaloids, phytoestrogens or mycotoxins, caterpillar and Actinobabacillus spp[41]. The possible role of the mycotoxin ZEA as a cause of MRLS has been reviewed by Newman, 2003 [42] and Newman and Raymond, 2005 [43]. Our data integrate previous in vitro observations conducted on both the germinal and somatic compartments of ovarian follicles in animal models as well as in humans. In sows, ZEA and its hydroxylated metabolites (α and β-ZOL) exerted negative effects on oocyte maturation [44] and inhibited the FSH-, forskolin- and pregnenolone-stimulated progesterone synthesis in GCs [45]. In cattle, ZEA and its derivatives inhibited the maturation of oocytes to metaphase II and high levels of 17 β-estradiol production by mural GCs were found [46]. The negative influence of ZEA was reported by Skorska-Wyswynska et al. [26] on cellular viability and morphology of cultured granulosa and internal theca cells of ovarian follicles in bitches. In human granulosa-luteal cells, ZEA was a potent inhibitor of aromatase/17 β HSD up to 10^-7 M with consequent inhibition of the conversion of androstenedione to oestradiol [17]. No in vitro studies have been previously carried out in the mare reproductive tract.

Equine GCs are sensitive to the effects of zearalenones, especially to α-ZOL and ZEA. This rank of activity was in agreement to that observed in vitro for other farm animals [45, 46]. This sensitivity of equine GCs to oestrogenic effects induced by ZEA and its derivatives emphasizes the importance of ZEA in reproductive disorders in this species. Studies are in progress to deepen understanding the mode of action of zearalenones on apoptosis and cell proliferation in GCs and the influence of zearalenones on reproductive function in the mare.