Pubertal timing after neonatal diethylstilbestrol exposure in female rats: Neuroendocrine vs peripheral effects and additive role of prenatal food restriction
Introduction
The neuroendocrine control of female reproduction through the preovulatory gonadotrophin surge and its alteration following exposure to sex steroids during fetal or perinatal life has been known for decades [1]. Although this finding provided a rationale for studies on neuroendocrine effects of endocrine disrupting chemicals, this issue received relatively little attention compared to the direct peripheral effects. This was illustrated in a recent review about neuroendocrine disruption where we found, among 17 in vivo studies on neuroendocrine disruption, three dealing with the central control of the preovulatory surge and two dealing with sexual maturation [2]. Only one study looked at diethylstilbestrol (DES) effects on vasotocin expression in the anterior hypothalamus and copulatory behavior in the quail [3]. As shown in Table 1, vaginal opening and estrous cyclicity were assessed after early DES exposure in 8 previous studies [4], [5], [6], [7], [8], [9], [10], [11] with no specific neuroendocrine endpoint. However, the neuroendocrine functions including centrally mediated onset of puberty and ovulation are potentially affected by early events such as exposure to endocrine disrupting chemicals (EDCs).
DES is a potent synthetic estrogen that was prescribed to prevent miscarriages during the 1950s and 1960s. Its prescription was then forbidden because of the increased risk of vaginal cancer amongst the female offspring who was exposed to DES in utero [12]. DES is still used as a paradigmatic EDC in animal models because of its well-described disruptive activity as a potent estrogen. Animal studies reported that, beyond neoplasia and genital malformations, fetal or neonatal exposure to DES lead to early puberty, ovulatory disorders, obesity in adulthood and metabolic syndrome [13], [14], [15]. Strikingly, fetal malnourishment leads to a similar spectrum of disorders of human reproduction and energy balance, including sexual precocity [16], [17], polycystic ovarian syndrome [18], increased risk of obesity and metabolic syndrome [19], [20]. Because those environmental changes occurring in the perinatal period appeared to possibly result in disturbed homeostasis later in life, they could share common hypothalamic targets. Following this observation and because DES and prenatal food restriction lead to early onset of puberty [15], [16], [17], we hypothesized that neonatal exposure to DES could result in subsequent alterations of the neuroendocrine control of GnRH secretion and that prenatal food restriction could have additive effects.
We aimed at studying pubertal development and estrous cyclicity after neonatal exposure to two different doses (1 and 10 μg/kg/d) of DES in normally fed animals. In parallel, we studied spontaneous GnRH secretion using hypothalamic explants obtained from immature female rats. Some changes could be expected since, in our laboratory, the greater sensitivity of female hypothalamic explants (vs males) to the potentiating effects of E2 on pulsatile GnRH secretion was reduced after neonatal exposure to estrogens. This indicated that sex steroids, with estradiol as the final effector, were responsible for perinatal masculinization of GnRH secretion in the hypothalamus [21]. Kisspeptin is a key regulator in the hypothalamic control of puberty and energy balance [22]. Since both prenatal food restriction [23] as well as neonatal exposure to bisphenol A [24], an estrogenic EDC were shown to cause subsequent reduction in hypothalamic expression of KiSS1 mRNAs, we studied those hypothalamic transcripts in the period preceding puberty. In addition, we used a human KiSS1 promoter construct in order to determine whether KiSS1 promoter activity in GnRH neuronal cell line could be affected by DES. The option of neonatal exposure from day 1 to 5 of postnatal life was consistent both with the observation that this was a critical period for DES causing possible metabolic syndrome in adulthood [25] as well as BPA causing persisting reduction in hypothalamic KiSS1 mRNA levels in adulthood [24].
We also paid attention to the possible mechanistic role of leptin. This hormone has been shown to have an important neuroendocrine stimulatory effect on GnRH secretion in prepubertal animals that could be evidenced after in vivo administration in our experimental rat model [26]. In addition, we have previously shown that leptin was able to accelerate pulsatile GnRH secretion from hypothalamic explants in vitro in prepubertal female rats [27]. Further arguments to study leptin effects came from its organizing role in the early life set up of hypothalamic neuronal connections important for control of energy balance [28]. Here, we studied the effects of neonatal exposure to DES and prenatal food restriction on hypothalamic sensitivity to leptin as assessed through GnRH secretion.
Section snippets
Animal care and exposure
Female Wistar rats were purchased from the University of Liège. They were housed in standardized conditions (22.8 °C, lights on from 6.30 am to 6.30 pm) and mated. Litters were standardized for size and sex ratio on the first postnatal day of life in order to have 8–12 pups per litter and an identical number of males and females. The day of birth was considered as the age of day 1. Weaning occurred at 21 days of age. In order to avoid a litter effect, a maximum of two pups coming from the same
Effects of DES and prenatal food restriction on age at vaginal opening and estrous cycling
DES led to opposite effects on vaginal opening depending on the dose (1 or 10 μg/kg/d) while both doses led to alterations of estrous cycle. Neonatal exposure to 1 μg/kg/d of DES from day 1 to day 5 of postnatal life resulted in delayed vaginal opening (Fig. 1, panel A). The average age ± SEM at vaginal opening was 34.1 ± 0.3 days in the control group and 38.0 ± 0.4 days in the exposed group. After vaginal opening, 75% of the females exposed to 1 μg/kg/d of DES, displayed a permanent estrus as confirmed
Discussion
In this study, we confirm that neonatal exposure to DES, a reference and potent estrogenic EDC that can affect both the reproductive system and the control of energy balance, has lifelong consequences on the reproductive system. We provide the first evidence that neuroendocrine effects are involved and that a lower dose of DES causes delayed puberty as opposed to the sexual precocity caused by a higher dose of DES. In addition, it appears that prenatal food restriction could have additive
Conflict of interest
None of the co-authors have any actual or potential conflict of interest to disclose.
Acknowledgments
This work was supported by grants from the European Society for Paediatric Endocrinology (ESPE Research Unit), from the “Fonds National de la Recherche Scientifique”, the University of Liège and the Belgian Study Group for Paediatric Endocrinology. We are grateful to the laboratory team of Pr Philippe Delvenne for assistance in technical processing of vaginal smear.
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