The present findings clearly show that prepubertal exposure to T-2 toxin causes a delay of puberty onset in infantile female rats without showing any visible toxicity. Vaginal opening and the first ovulation are decisive markers of puberty in female rats [23
], and the indirect marker of the first ovulation is the emergence of the first diestrus. Age of the first estrus, which occurs the day after the first preovulatory surge of gonadotropins, is an endpoint used to assess the completion of the pubertal process in females [24
]. Many studies have used the age of vaginal opening and the emergence of the first diestrus as efficient indicators of puberty in female rats [25
]. A previous study indicated that the day of vaginal opening of female rats was around the 34th day [27
], which is consistent with our current result. In the present study, the days of vaginal opening, first diestrus, and first estrus were all postponed by T-2 toxin, suggesting that prepubertal exposure to T-2 toxin can delay the onset of puberty. The development of reproductive organs is usually adopted as an adequate indicator of the appearance of secondary sexual characteristics in female rats. The present study revealed that T-2 toxin treatment for five consecutive days at a daily dose of 187.5 μg/kg bw diminished the relative weights of uterus, ovaries, and vagina (Table 2
), and reduced the incidence of corpora lutea (Table 2
). These above results combined with the abnormal histological findings of the reproductive organs (Figure 2
, Figure 3
and Figure 4
), indicated that prepubertal exposure to T-2 toxin induced a delay of reproductive organ development.
The physical changes associated with puberty are caused by prior activation of hypothalamic-pituitary-gonadal (HPG) axis. The initiation of HPG axis requires an increase in pulsatile release of GnRH from the hypothalamus. Thereafter, GnRH binds with high affinity to its specific receptor GnRHR on the surface of gonadotrope pituitary cells, leading to the synthesis and release of gonadotrophins such as LH and FSH from the anterior pituitary gland [28
]. LH and FSH, which in turn stimulates GnRH secretion [29
], eliciting the secretions of gonadal hormones estrogen and testosterone, which are responsible for the development of reproductive function and secondary sexual characteristics. Therefore, the levels of serum gonadotrophins are also effective targets to determine the onset of female maturation. In the present study, prepubertal exposure to T-2 toxin reduced the serum levels of FSH, LH and estradiol of female rats (Table 3
), indicating that T-2 toxin could delay the onset of maturation. These hormone changes were well consistent with delayed reproductive organ development.
GnRH, the pulsatile release of which initiates HPG axis, is located mainly in the hypothalamus. Studies have proved that the onset of puberty in humans and rodents is highlighted by the augmentation of pulsatile GnRH secretion [30
]. More recently, our team finds that T-2 toxin can regulate GnRH secretion in hypothalamic GT1-7 cells in vitro
]. In that study, GT1-7 cells were treated with a series of low concentrations of T-2 toxin (0.01, 0.05, 0.1, 0.5 and 1 ng/mL) for 3 h and 6 h, respectively. Moderate increases in GnRH level were observed for 6 h of T-2 toxin treatment at the concentrations of 0.05, 0.1 and 0.5 ng/mL, but not for 3 h of treatment and higher concentration. However, in our present study in vivo
, prepubertal exposure to T-2 toxin decreased mRNA expressions of hypothalamic GnRH and pituitary GnRHR (Figure 5
), which were well consistent with the alterations of serum gonadotrophins (Table 3
), delayed reproductive organ development (Table 2
), and the manifestations of delayed puberty onset (vaginal opening and estrous cycle) (Figure 1
). There seems to be a discrepancy between the stimulated GnRH secretion in vitro
reported by Liu et al.
] and the decreased hypothalamic GnRH mRNA expression and delayed vaginal opening (a marker of puberty) in vivo
in our present study. To our opinion, this discrepancy may be attributed to several factors. On the one hand, T-2 toxin had been reported to possess a bi-directional modulatory activity. In a study of cultured human lymphocytes, enhanced immunoglobulin production was observed in cells exposed to lower doses of T-2 toxin, whereas decreased immunoglobulin production was noted at higher doses [32
]. A different modulatory effect of T-2 toxin on the secretion of steroid hormone in granulosa cells was also observed. Maruniakova et al.
] found that high dose of T-2 (1000 ng/mL) combined with 10 ng/mL insulin-like growth factor I (IGF-I) significantly stimulated progesterone release in porcine ovarian granulosa cells. On the contrary, a significant inhibition of progesterone release by low dose of T-2 toxin (100 ng/mL) combined with 10 ng/mL IGF-I was observed. In the study of Liu et al.
, the stimulated GnRH secretion in GT1-7 cells in vitro
was only observed in a relative low concentration range with the maximal effective concentration of 0.5 ng/mL. Considering the bi-directional modulatory activity of T-2 toxin mentioned above, we think the probability that higher doses of T-2 toxin exerts inhibitory activity on GnRH secretion in GT1-7 cells can not be excluded. And this hypothesis will also be tested in our future in vitro
studies. Furthermore, T-2 toxin had been revealed to cause the impairment of the blood-brain barrier function [34
], and to increase the blood-brain barrier permeability [35
], both probably promote the entry of T-2 toxin into brain regions especially hypothalamus, resulting in the influence on HPG axis function. However, the metabolism and distribution of T-2 toxin in the brain following oral exposure are really unknown. Therefore, the concentration of T-2 toxin or its metabolites in different brain regions especially hypothalamus is unpredictable. Accordingly, it is hard to correlate the in vitro
concentrations that stimulate GnRH secretion in GT1-7 cells with the in vivo
exposure dose that delays puberty onset in female rats. On the other hand, unlike the regulation of GnRH secretion in cell cultures in vitro
, the GnRH secretion and puberty onset in vivo
are regulated by a complex network. The onset of puberty derives from the complex interplay among neuropeptides, neurotransmitters, and neurosteroids that occurs in the awakening of HPG axis [36
]. It had been revealed that acute or repeated exposure to T-2 toxin could induce changes in the levels of brain neurotransmitters such as serotonin, dopamine and norepinephrine, which play important modulatory role in the onset of puberty [37
]. Therefore, this difference between in vitro
and in vivo
regulatory mechanisms may at least in part explain the discrepancy between the observed in vitro
and in vivo
effects. Taken these points together, the delayed puberty onset in female rats induced by prepubertal exposure to T-2 toxin might be ascribed to its regulatory activity on hypothalamic function.
It is well confirmed that GnRH neurons play a pivotal role in regulating pubertal development and reproduction which are triggered by GnRH pulsatile secretion. The synthesis and secretion of GnRH is regulated by a complex and systematic network in hypothalamus, and kisspeptins the product of KiSS-1 gene and their G protein-coupled receptor 54 (GPR54) are recognized an essential gatekeeper in control of GnRH secretion and reproductive function [40
]. In recent years, increasing studies have revealed the key role of kisspeptins in xenobiotics especially chemicals with endocrine disruption activity induced adverse effects on puberty onset and reproductive function [43
]. As described above, our team recently finds that T-2 toxin has regulatory activity on GnRH secretion in hypothalamic GT1-7 cells. The further study reveals that exogenous kisspeptin-10 pretreatment elevates the reactivity of GT1-7 cells to T-2 toxin in light of GnRH secretion [22
]. In that study, in the presence of 10 nM kisspeptin-10 pretreatment for 1 h that shows maximal GnRH release, the GnRH levels in GT1-7 cells were further increased after 3 h and 6 h of treatment with different concentrations of T-2 toxin. In addition, the change trends of protein expression of kisspeptins ligand GPR54 following T-2 toxin treatment, were consistent with GnRH changes in the absence or presence of exogenous kisspeptin-10. As discussed above, although there exists a discrepancy between the in vitro
stimulatory effect on GnRH secretion and the reduced GnRH mRNA expression and delayed puberty onset in vivo
, these in vitro
findings provide a clue for us that T-2 toxin may disturb puberty onset in immature female rats by the perturbation of kisspeptins/GPR54 signaling pathway. Other central regulatory mechanisms such as the alterations of important neurotransmitters in brain should also be considered and investigated.