Neuroendocrine mechanisms of puberty in non-human primates

Abstract

An increase in pulsatile release of gonadotropin releasing hormone (GnRH) initiates puberty in mammalian species. While mutations in KISS1 and TAC3 and their receptors, KISS1R and NK3R, respectively, result in the absence or abnormal timing of puberty, the neurocircuitry and precise role of kisspeptin and neurokinin B (NKB) in regulation of the GnRH neurosecretory system in primate puberty remain elusive. This review discusses how kisspeptin and NKB signaling contributes to the pubertal increase in GnRH release in non-human primates and how remodeling of the NKB and kisspeptin signaling circuitry controlling GnRH neurons takes place during the progress of puberty. Importantly, the pubertal remodeling of kisspeptin and NKB signaling ensures efficient functions of the GnRH neurosecretory system that regulates sex-specific reproduction in primates.

Introduction

Puberty is one of the most significant developmental events in life. After puberty one can attain the capacity to reproduce the next generation. Puberty starts when pulsatile GnRH release in the pituitary portal circulation is persistently elevated, as documented by direct measurement of the GnRH peptide in the hypothalamus (1). In fact, experimentally precocious puberty is induced by pulsatile administration of GnRH into sexually immature, prepubertal monkeys (2). At the time of puberty onset the GnRH neuron in the hypothalamus-preoptic area becomes active and stimulates synthesis and release of pituitary LH and FSH, which gives a signal to the ovaries or testes to facilitate steroidogenesis and gametogenesis. In primates, a brief increase in GnRH release also occurs during the neonatal period resulting in so-called “mini-puberty”, but this elevation does not last long, as “central inhibition” takes place, suppressing GnRH neurosecretory activity in the hypothalamus (3–5). Although the makorin ring finger protein 3 (mKRN3) gene has been reported as a gene involved in central inhibition of GnRH release (6), the mechanism and neuronal substrates involved in central inhibition are presently little known. In contrast, a significant number of recent studies have shown how the stimulatory genes play a role in the pubertal increase in GnRH release, after the dissipation of central inhibition. The stimulatory genes are KISS1 (kisspeptin) and its receptor KISS1R (7,8) and TAC3 (neurokinin B, NKB) and its receptor NK3R (9). This article summarizes the mechanism through which kisspeptin and neurokinin B neurons regulate the pubertal increase in GnRH release in non-human primates.

1. Neonatal period (Mini-puberty)

GnRH neurons originate from the olfactory placode of embryos in the middle of the first trimester in humans and rhesus monkeys and complete migration in the preoptic area and hypothalamus by the early second trimester (10–13). Although during the mid to late gestational period GnRH neurons exhibit some activity (10,14), further heightened activity of GnRH neurons is seen in the first week of the neonatal period through several weeks and several months of life in rhesus monkeys and humans, respectively, by assessment of LH release (15,16). The period shortly after birth when GnRH neurons are transiently active is commonly called “Mini-Puberty”. Further studies under the condition with the absence of the gonads by neonatal gonadectomy in rhesus monkeys (15) or agonadal children due to genetic disorders (17) indicate that both LH and FSH levels are significantly elevated over gonadally intact individuals, suggesting the presence of the gonadal steroids negative feedback influences on the hypothalamo-pituitary axis during the neonatal period. Importantly, an immunohistochemical study by Plant and his collaborators (18) indicates that the number of kisspeptin neurons and peptide expression level in the hypothalamus in infantile monkeys resemble those in adult. A similar role of kisspeptin neurons in human late gestation has also been reported (19). Therefore, it appears that the GnRH neurosecretory system is regulated by kisspeptin neurons during the late gestational and mini-puberty periods. The physiological significance of mini-puberty in adult testicular function (20,21) and importance of the transient increase in testosterone during mini-puberty for maturation of cortical neurons and subsequent adult behavior (22) have been reviewed recently.

2. Prepubertal (Juvenile) period

After the neonatal period, central inhibition takes over the GnRH neurosecretory system. In primates, circulating LH and FSH levels are very low (Fig. 1) regardless of the presence or absence of gonads, and the hypothalamo-pituitary axis does not respond to gonadal steroid hormone challenge. In fact, a longitudinal study shows that LH levels of neonatally gonadectomied male and female monkeys are clearly suppressed until the age of puberty onset (15,23). Similar low circulating gonadotropin levels during the prepubertal (juvenile) period with gonadal dysgenesis in humans (17,24). During the prepubertal period no gonadal steroid feedback system is operative, as shown by the observation that injection of estradiol benzoate (EB) in ovariectomized prepubertal female monkeys induces neither negative feedback nor positive feedback effects on LH release (25,26).

Figure 1.

Schematic illustration of postnatal changes in GnRH release in association with puberty in male and female non-human primates. The GnRH neurosecretory system is active during the infantile period, but it is suppressed by a central inhibition during the juvenile period, which can be seen as a low frequency and low amplitude of GnRH release. At the time of puberty, the pulse amplitude, pulse frequency and mean release of GnRH start to increase, and those changes are further augmented through puberty. A higher nocturnal level of GnRH release, shown by hatched bars (stippled bars indicate morning levels) becomes prominent at the time of puberty onset and the nocturnal increase in GnRH release continues until first ovulation, after which GnRH release is reduced to the adult level. Modified from Terasawa E and Fernandez DL (2001). Neurobiological mechanisms of the onset of puberty in primates. End Rev 22 (2001) 111–151.

a). Central inhibition over GnRH release

Presently, the neuronal substrate responsible for “central inhibition” is little known. It has been clearly shown that endogenous opioids are not involved in primates (see ref. 4 for more discussions). The Plant lab hypothesized a possible role of neuropeptide Y (NPY) based on the reciprocal relationship between GnRH mRNA and NPY mRNA expressions in male rhesus monkeys (27). These authors describe that while GnRH mRNA levels in the hypothalamus during the juvenile period are higher than that of the pubertal period, NPY mRNA levels are lower at the corresponding period.

A series of studies in my lab, however, indicate that the gamma-aminobutyric acid (GABA) neuron is a component of the central inhibition. First, GABA levels in the median eminence (ME) of prepubertal female monkeys in which GnRH release is suppressed, are much higher than those of pubertal monkeys, in which pulsatile GnRH release is accelerated (28). Second, while infusion of the GABA_A antagonist bicuculline into the ME of prepubertal female monkeys stimulates GnRH release, the same treatment in pubertal females is ineffective (28). Recently, we further found a parallel bicuculline effect on GnRH release in prepubertal and pubertal male monkeys (29). Third, pulsatile infusion of bicuculline into the ME of prepubertal female monkeys resulted in precocious menarche (30). Fourth, infusion of bicuculline into the ME stimulates not only GnRH release, but also kisspeptin release in prepubertal females. In addition, bicuculline’s stimulatory effect on GnRH release in prepubertal females does not occur in the presence of the kisspeptin antagonist, Peptide 234 (31), indicating that GABA inhibition on GnRH release occurs at the kisspeptin signaling level. Finally, a recent preliminary observation showing that the stimulatory bicuculline effect on GnRH release in prepubertal males are greatly attenuated by the presence of the NKB antagonist, SB222200 (29), suggests that GABA inhibition on GnRH release also appears to occur at the level of NKB-kisspeptin-GnRH signaling in males. Therefore, the GABA neuron is a part of “central inhibition”, although the precise mechanism including neurocircuitry remains unclear.

b). Kisspeptin and NKB signaling

Since discoveries that inactivating mutations in kisspeptin and NKB and their respective receptors resulted in delayed puberty or no puberty in humans (7–9), these peptides have been implicated in the mechanism of puberty. Recent studies in several laboratories including this lab show both kisspeptin and NKB signaling contributes to the pubertal increase in GnRH release (32,33). However, as discussed above, during the prepubertal/juvenile period activities of kisspeptin and NKB signaling are suppressed by “central inhibition” and they do not reveal until after puberty onset. As such, in this article we will discuss the pubertal changes in kisspeptin and NKB signaling by comparing their activities during the prepubertal and the pubertal periods.

3. Pubertal period

Puberty is initiated by an increase in the frequency and amplitude of GnRH release from the hypothalamus (1,34). In primates, GnRH release during the juvenile/prepubertal period consists of low baseline levels with low pulse amplitude and pulse frequency (Fig. 1). As puberty progresses into the early pubertal stage (there are signs of puberty but before first menstruation) mean release, pulse frequency, and pulse amplitude all increase. As puberty further progresses into the midpubertal stage (between menarche and first ovulation), the mean levels and pulse amplitude of GnRH release keep increasing to the patterns observed in adulthood (Fig. 1). Importantly, the initial phase of the pubertal increase in GnRH release is independent of circulating estradiol levels, as the pubertal increase in GnRH release occurs in ovariectomized monkeys with similar timing (34,35). A similar pubertal increase in LH release in normal girls and girls with gonadal dysgenesis has also been reported (17,24). Thus, after puberty onset, a drive to increase GnRH release plays a significant role in the pubertal progression.

a). Kisspeptin release:

A series of studies in rhesus monkeys indicate that the pattern of developmental changes in kisspeptin release is parallel to those described for GnRH release in females (33,34,36). Mean release, pulse amplitude and pulse frequency in pubertal female monkeys are all higher than those in prepubertal females and the pubertal increase in kisspeptin during the early part of puberty is independent of the pubertal increase in estradiol, as the pubertal increase in kisspeptin release is also seen in ovariectomized early pubertal females (36,37). Importantly, however, the kisspeptin increase in the later part of puberty appears to be due to the pubertal increase in estradiol (see below for further Discussions, 32,36). In males, an increase in mean release of kisspeptin parallels the pubertal increase in GnRH release across puberty has also been observed (33,37). Although we have not systematically studied the developmental changes in the pulse frequency and pulse amplitude in males, we can assume that there is no sex-difference in this context. Collectively, kisspeptin release in the ME, where their neuroterminals are concentrated, increases along with pubertal progression.

b). NKB release:

Preliminary data from this lab indicate that release of NKB in the ME increases at puberty in males (38), similar to those of the pubertal increase in release of GnRH and kisspeptin.

c). GnRH response to kisspeptin challenge:

Infusion of human kisspeptin-10 (KP10) into the ME of female and male rhesus monkeys at both the prepubertal and pubertal stages stimulates GnRH release in a dose-responsive manner (39,40). Importantly, within the same sex the GnRH neurons in pubertal animals are more sensitive to KP10 than in prepubertal animals, as GnRH response to KP10 at the same dose in pubertal animals is larger than in prepubertal animals. Interestingly, female GnRH neurons are ten-fold more sensitive than male GnRH neurons, as 1) the minimum effective dose is 10-fold less in female than male and 2) KP10 at 0.1 μM induces a larger response in females than in males (33). Pubertal amplification of KP10 action on GnRH release in females is due to the pubertal increase in estradiol, as there is no GnRH response to KP10 in ovariectomized pubertal females (39). Unlike in females, however, the GnRH response to KP10 in males is less dependent on the pubertal increase in androgens, as orchidectomy in pubertal males does not alter the GnRH response to KP10 (33). Therefore, the contribution of kisspeptin signaling to the pubertal increase in GnRH release in both males and females is two folds: 1) a larger amount of endogenous kisspeptin output after puberty onset and 2) an increase in kisspeptin receptor sensitivity of GnRH neurons.

d). GnRH response to senktide challenge:

GnRH neurons also release the GnRH peptide in response to the NKB agonist, senktide. Interestingly, however, in males there is a little dose-dependent response at both the prepubertal and pubertal stages, and unlike that seen with KP10 there is no developmental amplification by senktide (33,38). In females, however, GnRH response to senktide is dose-dependent, although dissimilar to seen with KP10, there is no developmental amplification with senktide. Additionally, unlike what is seen with the GnRH response to kisspeptin, there is no sex difference in the sensitivity of senkide-induced GnRH release (38). We have not examined how gonadectomy influences the senktide-induced GnRH release in both sexes. Nevertheless, the role of NKB on the pubertal increase in GnRH release in males appears to be limited to the increased NKB signaling. In females, however, dose dependent stimulation of kisspeptin release and NKB’s pubertal amplification on kisspeptin release (see below) suggest that NKB signaling contributes to the pubertal increase in GnRH release directly or indirectly through kisspeptin release.

e). Kisspeptin response to senktide challenge:

Kisspeptin neurons release the kisspeptin-54 peptide in response to senktide in both developmental stages in both sexes. Importantly, while there is neither dose related response nor developmental change in males, the kisspeptin response to senktide in females is dose dependent and the responses in pubertal females is greater than in prepubertal female (33). In pubertal males, the senktide-induced kisspeptin release is not affected by the absence of gonadal androgens, as in orchidectomized males senktide induced kisspeptin release in a dose-responsive manner (J. Garcia and E. Terasawa, unpublished observation). The effects of ovariectomy on the senktide-induced kisspeptin release in pubertal females have not been examined.

f). Changes in the kisspeptin and NKB signaling pathways during puberty:

In order to clarify the interaction between kisspeptin and NKB signaling, we have conducted a series of experiments, in which we examine 1) whether senktide can stimulate GnRH release under the condition where KISS1R is blocked by the kisspeptin antagonist peptide (P) 234, and 2) whether KP10 can stimulate GnRH release under the condition where NK3R is blocked by the NKB antagonist SB222200. The results show that whereas in prepubertal males the senktide-induced GnRH release is attenuated, but not blocked, under the condition where kisspeptin signaling is eliminated, in pubertal males the senktide-induced GnRH release is completely blocked. This suggests that NKB signaling to GnRH release is only in part mediated through kisspeptin neurons in prepubertal males, but in pubertal males NKB signaling is mediated through kisspeptin neurons (29). Similarly, kisspeptin signaling to GnRH release is also in part mediated through NKB neurons in prepubertal males, but in pubertal males kisspeptin signaling does not require the NKB neurons (29). These observations suggest that in prepubertal males there are reciprocal pathways between NKB and kisspeptin signaling, but when they reach puberty, NKB signaling mediated through kisspeptin neurons becomes dominant and kisspeptin signaling through NKB neurons is no longer available. These data are interpreted to mean that the presence of reciprocal pathways in prepubertal male might reflect that active GnRH neurons during the neonatal period were suppressed by the central inhibition (29). As puberty progresses, the NKB signaling pathway mediated through kisspeptin becomes dominant to scalp the need in adult male reproductive function. In contrast, while in prepubertal females kisspeptin signaling and NKB signaling are independently operative, as senktide effect is neither mediated through kisspeptin neurons, nor are KP10 effects mediated through NKB neurons. In pubertal females in addition to those independent pathways, NKB signaling is in part mediated through kisspeptin neurons and kisspeptin signaling is in part mediated through NKB neurons (40). The establishment of the reciprocal pathways gives more power and flexibility to regulate GnRH neurons in adult females, such that cyclic ovulations and pregnancy can be achieved.

4. Conclusions, Perspectives, and Future Directions

In this article we have discussed after the reduction in “Central Inhibition” how kisspeptin and NKB signaling contributes to the pubertal increase in GnRH. The response of GnRH neurons to kisspeptin is a dose dependent and kisspeptin release increases after puberty onset in both sexes. Additionally, in females the pubertal increase in circulating estradiol further amplifies the GnRH response to kisspeptin. NKB signaling as well stimulates GnRH release directly or through kisspeptin neurons. Our observations further suggest that during the pubertal period there is a major remodeling of the NKB and kisspeptin network. In males, collaborative kisspeptin and NKB signaling to GnRH neurons existed in prepuberty, while after initiation of puberty a kisspeptin dominant signaling mechanism regulates GnRH release in adulthood (Fig. 2A). This is quite different from females: Reciprocal signaling pathways between kisspeptin and NKB neurons are necessary to provide efficiency and flexibility for GnRH stimulation to ensure complex reproductive functions in adulthood (Fig. 2B). Our series of in vivo microdialysis experiments revealed similarities and differences in the role of kisspeptin and NKB signaling in regulation of GnRH neurons at the time of puberty. Because of the collection site, kisspeptin and NKB signaling to GnRH neurons takes place at their neuroterminals in the ME in non-human primates. There are many questions remain to be answered. Among them are: Why female GnRH neurons are more sensitive to KP10 than in males? What is the underlying mechanism of the sex-specific neurocircuitry remodeling during puberty? How are opioid neurons involved in the NKB-kisspeptin-GnRH neurocircuitry? We hope that a series of future experiments will provide answers to these questions.

Figure 2.

Schematic illustration summarizing developmental changes in the neuroendocrine mechanism of puberty. Possible interactions between kisspeptin (red), NKB (blue), Opioid (gray) and GnRH (black) neurons in the hypothalamus in prepubertal males and females monkeys are shown. The number of colored dots reflects estimated amount of neuropeptide release at the neuroterminals, based on the results from a series of experiments. Note that, major remodeling of kisspeptin and NKB signaling pathways takes place during puberty, such that their regulation of the GnRH neurosecretory system is most effective in adult reproductive function. A black X indicates signaling pathways are not present. The role of opioid neurons is rather hypothetical at this point. Recreated from Garcia, J.P., K.A. Guerriero, K.L. Keen, B.P. Kenealy, S.B. Seminara, E. Terasawa, Kisspeptin and neurokinin B signaling network underlies the pubertal increase in GnRH release in female rhesus monkeys, Endocrinology 158 (2017) 3269–3280; Garcia J.P., K.L. Keen, B.P. Kenealy, S.B. Seminara, E. Terasawa, Role of kisspeptin and neurokinin B signaling in male rhesus monkey puberty, Endocrinology 159 (2018) 3048–3060; and Garcia, J.P., K.L. Keen, S.B. Seminara, E. Terasawa, Role of Kisspeptin and NKB in Puberty in Nonhuman Primates: Sex Differences. Semin Reprod Med. 37 (2019) 47–55.