It has been known for decades that nutritional state affects puberty and the reproductive status in many species, including humans (1
). Sexual maturation is usually initiated when energy stores meet the demands of the reproductive function including the production of hormones and gametes, copulation, pregnancy, and lactation. If excessive leanness occurs in young women, puberty is often delayed (2
). Moreover, the existence of a link between the advance of childhood obesity and the increasing rates of precocious puberty has also been postulated. A previous study published in 1997 reported that, in the USA, 6.7% of girls had clinical evidence of puberty at age 7 years and 14.7% at age 8 years. Authors also described the youngest population age at puberty onset as 9.9 ± 1.8 years (8
). In the last decade, an increment of 5%–8% in the number of girls with clinical evidence of puberty at age 7 and 8 years has been reported (5
). The cause for this alarming phenomenon is not quite understood. One possibility is the increased levels of circulating leptin in obese children. Increased adiposity is often correlated to high leptin levels (67
), a condition known to accelerate pubertal development (69
). However, the sites of leptin action on puberty initiation and therefore the mechanisms by which this physiological event is regulated have been difficult to determine.
The role played by the Kiss1/Kiss1r system in puberty and fertility has been well described. Loss-of-function mutations of Kiss1
genes in rodents and humans result in hypogonadism, abnormal sexual maturation, and decreased circulating levels of sex steroids and gonadotropins (71
). Administration of Kisspeptin to juvenile rodents induces vaginal opening, increases LH secretion, and induces ovulation (75
). Hypothalamic levels of Kiss1
mRNAs and electrophysiological responses of GnRH neurons to Kisspeptin increase across puberty (76
). Collectively, these findings indicate that Kiss1 neurons play an essential role in pubertal development, but what triggers the Kiss1/Kiss1r system is still unsettled. Leptin has been a plausible candidate, and therefore, leptin-responsive Kiss1 neurons would be well positioned to relay leptin’s effect on puberty initiation. To test this model, we generated mice with selective deletion of LepR from Kiss1 neurons and assessed their sexual maturation. We found that male and female mice progress through puberty normally and are fertile, suggesting that leptin signaling in Kiss1 neurons is not required for pubertal development and fertility. Studies from different laboratories have shown that kisspeptin induces LH secretion via direct stimulation of GnRH neurons (75
). But the estrogen-induced LH surge still occurs in Kiss1r
-knockout mice, suggesting the existence of an additional relevant pathway in the regulation of GnRH-induced LH secretion (82
). Our findings are in agreement with this concept and indicate that leptin’s effect in the onset of puberty is relayed by an alternative route outside Kiss1 neurons. Nevertheless, the physiologic relevance of direct leptin signaling in Kiss1 neurons in different paradigms is yet to be determined, as developmental adaptations may have occurred in our mouse model.
The role played by PMV neurons in mediating leptin’s effects has been largely ignored. The PMV neurons express sex steroid receptors (83
) and are activated following copulation or exposure to sexually relevant odors (85
), but its role in pubertal development had not been anticipated. In fact, the lack of mouse models in which to selectively manipulate LepR in the PMV represents an additional difficulty in assessing the physiological role played by PMV neurons. In this study, we employed stereotaxic techniques to lesion PMV neurons of ob/ob
mice or to reactivate endogenous expression of LepR in PMV neurons of LepR-null mice. These techniques inherently produce heterogeneous experimental subjects due to variable injection sizes and injection sites. Moreover, in order to precisely determine the functional reactivation of LepR or lesion of neurons that express LepR, mice were fasted to potentiate leptin-induced pSTAT3-ir. This precluded a systematic analysis of their hormonal profile and evaluation of differential reproductive parameters. However, our results are unambiguous. We identified the PMV as a key site of leptin action in the onset of puberty. Reexpression of LepR in adjacent sites outside the PMV did not trigger pubertal development in LepR-null mice. Likewise, partial lesion of the PMV or of neurons outside the PMV did not blunt leptin’s effect of inducing sexual maturity in ob/ob
Previous studies have suggested that leptin plays a small or virtually no role in the progress of pregnancy (92
). Therefore, we established the copulatory plugs as the endpoint for the lesion experiment. In this experimental design, we also assessed leptin’s effect on LH secretion. Leptin treatment prevents the fall in LH levels during states of negative energy balance, when leptin levels are low (34
). As previously demonstrated (14
), all intact ob/ob
female mice exhibited low levels of LH, which were increased following leptin administration. However, leptin was ineffective at increasing LH levels in PMV-lesioned ob/ob
mice. These findings confirm and extend previous data (34
) indicating that PMV is also required for leptin’s stimulatory effect on LH secretion in leptin-deficient ob/ob
mice. During proestrus, the increase in LH levels also stimulates progesterone production, which in turn is essential for copulatory behavior (97
). Following copulation, female rodents display a sustained increase in progesterone as a result of neuroendocrine reflex generated by vaginal-cervical stimulation (99
). Therefore, the low levels of progesterone observed in PMV-lesioned mice may be a consequence of the decreased LH secretion and/or may indicate a deficient copulatory behavior in these mice.
Selective reexpression of LepR in PMV neurons induced puberty and sexual maturation in a high percentage of LepR-null female mice. Remarkably, this effect was independent of changes in body weight or food intake. Thus, endogenous reexpression of LepR in the PMV did not correct the obese phenotype and likely did not rescue most of the endocrine dysfunctions of LepR-null mice. This is worth emphasizing, as most of the pregnant females underwent miscarriages. It is now well documented that obesity as well as diabetes may increase the probability of miscarriage and malformations in rodents and humans (100
). Therefore, the miscarriages and the absence of pregnancy in some of the reactivated mice may be caused by the remaining metabolic or endocrine abnormalities following reactivation of LepR in PMV neurons of LepR-null mice.
Deficits in gonadal development of leptin-deficient ob/ob
male mice are well described (14
). In contrast, and in agreement with our findings, the absence of a clear morphological abnormality in the reproductive tract of LepR-deficient male mice has also been reported by others (53
). But intriguingly, reexpression of LepR in the PMV of LepR-null mice did not ameliorate male fertility. This may indicate that the sites of leptin action in the reproductive physiology of rodents are sexually dimorphic. The generation of mouse models to specifically manipulate genes of interest in PMV neurons will allow further evaluation of the contribution of leptin signaling in PMV neurons to male sexual maturation and also to female pregnancy.
In the present study, we demonstrate that leptin’s effect in pubertal development is relayed by PMV neurons expressing the excitatory neurotransmitter glutamate. Leptin directly depolarizes PMV neurons (89
), which may, in turn, “activate” their downstream neuronal targets, including GnRH cell bodies in the preoptic area (89
) and/or GnRH terminals in the median eminence/mediobasal hypothalamus. This model is in agreement with a recent study that reported that glutamate stimulates GnRH secretion in the absence of the Kiss1/Kiss1r system (106
). Thus, our findings indicate that PMV neurons may serve as an alternative pathway in this circuitry, conveying inputs on changing levels of leptin directly to GnRH neurons. As observed in db/db
), LepR-null mice displayed no deficits in GnRH neuronal migration, GnRH
gene expression, or peptide production. We also noticed that LepR-null mice show modest or undetectable changes in Kiss1
gene expression compared with control littermates. Together, these data suggest that the infertility phenotype of the LepR-null mice is not caused by deficient expression of Gnrh
genes. Rather, the leptin-signaling deficient mice display increased GnRH content in the median eminence/mediobasal hypothalamus, strengthening the model that GnRH secretion is inhibited in females with leptin-signaling deficiency (53
). This elevated GnRH content is not observed in the infertile Kiss1
gene knockout mice (73
), suggesting that the disruption in the normal pubertal development observed in Kiss1 and in leptin-signaling deficiencies is caused by dissociated mechanisms. Importantly, reexpression of LepR in PMV neurons of LepR-null mice normalizes their GnRH content in the median eminence/mediobasal hypothalamus. This is in concert with the well-established concept acquired from studies conducted in vivo and in vitro, in different species and sexes, that leptin acts in the brain stimulating GnRH secretion (107
). Although the bulk of evidence collected in the present study favors a direct action of PMV neurons in GnRH terminals, we cannot rule out the existence of additional and/or alternative relays. In many physiological systems and, in particular, in the reproductive function, redundant pathways are essential for species survival. Here, we demonstrate that a direct action of leptin on PMV neurons is required and sufficient for leptin’s effect in the onset of puberty. But it is not clear whether glutamatergic PMV neurons exert their role by directly stimulating GnRH secretion or by acting in an additional relay (e.g., kisspeptin neurons) that impinges on GnRH neurons. In any case, the hypothalamic circuitry (i.e., GnRH and Kiss1 neurons) must be in place to provide the underlying substrate for leptin’s effect as a “permissive” factor in the onset of puberty.
The role played by leptin as a permissive factor for the onset of puberty is well determined. But the efforts to dissect the effects of leptin and the mechanisms by which puberty is initiated have been obstructed by the lack of information on where in the brain this effect takes place. Our study establishes the PMV as a key site for leptin action in the onset of puberty and demonstrates that direct leptin signaling in Kiss1 neurons is not required for pubertal development and fertility. Our findings also add to the growing literature suggesting that leptin’s multiple actions to control metabolism and reproduction are anatomically dissociated.