In present work, we investigated whether maternal chronodisruption is related to changes on kisspeptin and irisin plasma levels in female offspring and if there is relationship with the time of vaginal opening. Our findings support a role of gestational chronodisruption in kisspeptin levels at time of vaginal opening that it cannot be reversed by melatonin treatment. However, our experimental model showed that maternal exposure to continuous light during pregnancy, promotes a delay on vaginal opening that can be reversed by melatonin treatment.
It is clearly demonstrated that the absence of the maternal melatonin rhythm by chronodisruption affects the postnatal reproductive axis development both in males and females. In this way, previously we have demonstrated that maternal pinealectomy alters LH secretion mainly at juvenile period in female rats [19], also that maternal pineal gland participates in cellular and nuclear volumes of prepubertal oocytes development [20], even that exogenous melatonin treatment to mothers during pregnancy altered the maturation of the gonadotropin and prolactin feedback system to estradiol in female offspring [21].
Alterations in several neuropeptides levels in hypothalamus, pituitary or striatum, through development, in rat offspring from pinealectomized or melatonin treated mother were also observed by our group [22,23].
In addition, other authors had been suggested a role for maternal photoperiod on offspring development. The absence of maternal melatonin rhythm during pregnancy had a marked effect on the newborn temperature rhythm, one of the main circadian rhythms, and melatonin treatment to mothers synchronized this newborn rhythm [24]. On the other hand, It has been proposed that maternal melatonin acts on the foetus hypothalamus-pituitary unit to sets the trajectory of reproductive and metabolic development in pups and has a persistent effect on their subsequent sensitivity to the photoperiod through thyroid metabolism [25].
Vaginal opening is an external marked of puberty onset. This event depends on the adequate functional status of the reproductive neuroendocrine system. In this sense, the discovery that the neuropeptide kisspeptin play a critical role in regulating the hypothalamus reproductive axis and shed new perspectives on the understanding of puberty. The essential role of kisspeptin in the onset of puberty can be attributed to the critical role of hypothalamic arcuate nucleus kisspeptin neurons to generate the pulsatile GnRH release required for pubertal activation of the reproductive axis [26].
Our results show increased plasma kisspeptin levels at puberty, in female offspring from control mothers, according to results of previous authors from female animals [27,28,29,30].
Despite the scientific evidence about the role of the neurohormone melatonin on Kisspeptin system, there is not studies about the possible role of the maternal melatonin on developmental offspring kisspeptin levels. In this way, we found that both the absence of melatonin caused by continuous light exposure and the excess, affects plasma kisspeptin levels at puberty. It is known that melatonin is the pivotal cronobiotic to the circadian system and light/dark cycle is the most important synchronizer of the system [31]. It seems clear that the cronodisruption to which the mothers were exposed during gestation exerts its influence on developmental female offspring reproductive axis. Melatonin can cross placental barrier [32], then during intrauterine life foetuses were exposed to maternal circadian signals. In addition, newborns were also exposed to maternal melatonin rhythm [33]. Alteration of the maternal melatonin levels has been associated with disruption of the brain programming and developmental long effects [34]. Based on our results, we deduce that maternal chronodisruption by constant light exposure exert an excitatory influence on kisspeptin hypothalamic neurons at puberty since significant high kisspeptin values were found in female offspring from continuous light exposed mothers, so affecting the onset of puberty in the offspring but it is striking that those animals showing increased kisspeptin values are correlated to delayed vaginal opening. In any case, one of the strength of our study is that shows in an animal model that continuous light exposure at night during fetal life affect the postnatal neuroendocrine-reproductive axis affecting the pivotal process of puberty. In today`s society pregnant mothers are exposed to bright light at night for various reasons: night work, shift work, or constant abuse of electronic devices at night, as it was verified in several studies [35,36,37,38]. Then, we considered that the present results allow us to infer the danger of future mothers' exposure to night light during pregnancy for an event as capital as puberty for their offspring.
Further, exogenous melatonin treatment during pregnancy was not able to restore plasma kisspeptin values to those found in control group, even values are higher in female offspring from melatonin treated mother rats exposed to continuous light.
Most of the studies looking for a relation between melatonin and kisspeptin at puberty have been carried out in seasonal breeders in which it has been demonstrated that kisspeptin levels are influenced by photoperiod changes related to changes in melatonin secretion to reduce kisspeptin levels [39,40]. Some authors suggest that in those animals, melatonin may alter the negative feedback effects of sex steroids on kisspeptin expression. These data are not agreeing with our results but It should be noted that our study was carried out in a non-seasonal animal, also that exposure to the hormone melatonin in our model occurred not during postnatal life but during intrauterine life. However, we propose that the ineffectiveness of exogenous melatonin to act as a chronobiotic and repair the effects of chronodisruption caused by continuous exposure to light may be due that longer exposure to melatonin may cause that the neuroendocrine-reproductive axis escape to its influence. In this way, it was demonstrated that acute exogenous melatonin induces a reduction in kisspeptin gene expression, but longer effects lead to an increase in kiss gene expression [41].
Despite the clear influence of the maternal cronodisruption on kisspeptin levels at puberty, we did not find a direct relationship between these values and vaginal opening since as we described above, we found delayed vaginal opening in female offspring from mother exposed to continuous light with increased plasma kisspeptin values compared to control but the female offspring from melatonin treated mother rats exposed to continuous light even with the highest kisspeptin values show vaginal opening at the same time that control offspring. All this leads us to think that other factors in addition to kisspeptin are necessary for the beginning of puberty. In relation to this approach other authors have proposed that the time of puberty is dictated by kisspeptin independent mechanism controlling the ontogeny of GnRH pulse generation. These authors propose that the impact of loss of function by mutation of genes encoding kisspeptin or its receptor on the onset of puberty can be attributes to the critical role of the arcuate kisspeptin neurons in the generation of GnRH pulses, necessary for pubertal activation of the neuroendocrine reproductive axis but kisspeptin neurons do not determine the timing of puberty. Rather this event is achieved by upstream neuronal mechanism [26].
The onset of puberty is a very complex phenomenon in which a lot of different signalling systems both inhibitory or excitatory participates. Among them, metabolic signals as leptin, ghrelin or insulin have been already investigated [42,43,44].
In recent years it has been reported irisin changes in different stages of puberty [15,45]. Also, it has been hypothesized [13] that irisin serves as metabolic trigger for the onset of puberty based on the increase in the expression of irisin gene (FNDCS) mRNA levels in mice during postnatal development and the systemic irisin levels increase close to puberty in humans as it was previously described by others [15,46].
We study the irisin levels throughout development during the juvenile period in female offspring rats from control mothers and we found a progressive increase from 25 to 30 days of age and to the day of vaginal opening being values at vaginal opening day 3-fold-times higher than those found at the beginning of the pre-pubertal period. Furthermore, we found a similar developmental pattern, to one found for control group, both in offspring of continuous light exposed mother rats and in offspring of continuous light exposed mother rats plus melatonin treatment during pregnancy, at 25 and 30 days of age. But different response was found at vaginal opening day since irisin concentration was lower in offspring from continuous light mother rats compared to controls, which allows us to suggest that the lack of maternal melatonin caused by chronodisruption during pregnancy affects to the secretion of isirin at the critical time of the pubertal onset. On the other hand, unlike what we found for kisspeptin, it seems that muscle or adipose tissue are not as sensitive to the refractory effect of melatonin. Since, exogenous melatonin received throughout pregnancy seems to be able to reverse the effects of continuous light on muscle and/or adipose tissue on irisin synthesis.
To our knowledge this is the first study to evaluate the effect of maternal chronodisruption on developmental irisin in the female offspring. Given the importance of this molecule for issues related to reproduction as the onset of puberty, as well as its importance on other fields such as health of the musculoskeletal system, metabolic diseases, inflammatory processes, bone formation and functioning the nervous system [46,47,48], we posit that this result contribute one more with a novel reason to consider exposure to light at night as a health risk factor especially during pregnancy in view of the consequences for the offspring.
In relation to melatonin influence on irisin levels, scientific evidence is very scarce although recently it was reported that pharmacological concentration of melatonin may modulate irisin signalling pathways on remote ischemic preconditioning after myocardial ischemia-reperfusion injury in rats [49, 50]. On the other hand, chronic continuous melatonin administration reduces weight gain and the serum total cholesterol levels and additionally, it enhances the circulating irisin [51]. But in any case there is no scientific evidence about the direct influence of melatonin on irisin at the onset of puberty and much less in relation to the influence of maternal melatonin on the onset of puberty. Our results allow us to affirm not only that the absence of melatonin during intrauterine life may alter the secretion of irisin at the onset of puberty but treatment with melatonin can restore levels to those observed in offspring from control mother rats.
Further, looking at our results, we infer that it is possible that irisin plays an important role in the timing of the onset of puberty along with kisspeptin because as we previously exposed the altered kisspeptin levels at the onset of puberty in the two experimental groups does not seem to explain the different behaviour in terms of timing of vaginal opening, but it is possible that the decrease in plasma levels of the irisin hormone is related to the delay in vaginal opening observed in the group of offspring of mothers exposed to continuous light, which is reinforced by the fact that the treatment of mothers with melatonin allows a recovery in the plasma concentration of irisin in their offspring, while they do not show a delay in vaginal opening.
In conclusion, our results demonstrated that maternal chronodisruption alters kisspeptin and irisin plasmatic levels at the critical phase of the onset of puberty, however pharmacological melatonin influence it is not the same on the kisspeptin hypothalamic neurons as on muscle and/or adipose cells that synthesize irisine.