Brian P. Kenealy

Recent Discoveries on the Control of Gonadotrophin-Releasing Hormone Neurones in Nonhuman Primates

Since Ernst Knobil proposed the concept of the gonadotrophin‐releasing hormone (GnRH) pulse‐generator in the monkey hypothalamus three decades ago, we have made significant progress in this research area with cellular and molecular approaches. First, an increase in pulsatile GnRH release triggers the onset of puberty. However, the question of what triggers the pubertal increase in GnRH is still unclear. GnRH neurones are already mature before puberty but GnRH release is suppressed by a tonic GABA inhibition. Our recent work indicates that blocking endogenous GABA inhibition with the GABAA receptor blocker, bicuculline, dramatically increases kisspeptin release, which plays an important role in the pubertal increase in GnRH release. Thus, an interplay between the GABA, kisspeptin, and GnRH neuronal systems appears to trigger puberty. Second, cultured GnRH neurones derived from the olfactory placode of monkey embryos exhibit synchronised intracellular calcium, [Ca2+]i, oscillations and release GnRH in pulses at approximately 60‐min intervals after 14 days in vitro (div). During the first 14 div, GnRH neurones undergo maturational changes from no [Ca2+]i oscillations and little GnRH release to the fully functional state. Recent work also shows GnRH mRNA expression increases during in vitro maturation. This mRNA increase coincides with significant demethylation of a CpG island in the GnRH 5′‐promoter region. This suggests that epigenetic differentiation occurs during GnRH neuronal maturation. Third, oestradiol causes rapid, direct, excitatory action in GnRH neurones and this action of oestradiol appears to be mediated through a membrane receptor, such as G‐protein coupled receptor 30.

Neuroestrogen, rapid action of estradiol, and GnRH neurons ☆

Estradiol plays a pivotal role in the control of GnRH neuronal function, hence female reproduction. A series of recent studies in our laboratory indicate that rapid excitatory actions of estradiol directly modify GnRH neuronal activity in primate GnRH neurons through GPR30 and STX-sensitive receptors. Similar rapid direct actions of estradiol through estrogen receptor beta are also described in mouse GnRH neurons. In this review, we propose two novel hypotheses as a possible physiological role of estradiol in primates. First, while ovarian estradiol initiates the preovulatory GnRH surge through interneurons expressing estrogen receptor alpha, rapid direct membrane-initiated action of estradiol may play a role in sustaining GnRH surge release for many hours. Second, locally produced neuroestrogens may contribute to pulsatile GnRH release. Either way, estradiol synthesized in interneurons in the hypothalamus may play a significant role in the control of the GnRH surge and/or pulsatility of GnRH release.

STX, a novel nonsteroidal estrogenic compound, induces rapid action in primate GnRH neuronal calcium dynamics and peptide release

Previously, we reported that 1 nM 17ß-estradiol (E(2)) induces a rapid action, which is, in part, mediated through the G protein-coupled receptor GPR30 in primate GnRH neurons. Because it has been reported that the diphenylacrylamide compound, STX, causes estrogenic action in the mouse and guinea pig hypothalamus, the present study examined effects of STX in primate GnRH neurons and whether there is an action independent of GPR30. Results are summarized as follows. STX (10 nM) exposure increased 1) the oscillation frequency of intracellular calcium concentration ([Ca(2+)](i)), 2) the percentage of cells stimulated, and 3) the synchronization frequency of [Ca(2+)](i) oscillations. STX (10-100 nM) also stimulated GnRH release. The effects of STX on both [Ca(2+)](i) oscillations and GnRH release were similar to those caused by E(2) (1 nM), although with less magnitude. STX (10 nM)-induced changes in [Ca(2+)](i) oscillations were not altered by GPR30 small interfering RNA transfection, indicating that STX-sensitive receptors differ from GPR30. Finally, a higher dose of E(2) (10 nM) induced a larger change in [Ca(2+)](i) oscillations than that with a smaller dose of E(2) (1 nM), and the effects of 10 nM E(2) were reduced but not completely blocked by GPR30 small interfering RNA transfection, indicating that the effects of 10 nM E(2) in primate GnRH neurons are mediated by multiple membrane receptors, including GPR30 and STX-sensitive receptors. Collectively, the rapid action of E(2) mediated through GPR30 differs from that mediated through STX-sensitive receptors. The molecular structure of the STX-sensitive receptor remains to be identified.

Acute Influences of Bisphenol A Exposure on Hypothalamic Release of Gonadotropin-Releasing Hormone and Kisspeptin in Female Rhesus Monkeys

Bisphenol A (BPA) is an industrial compound with pervasive distribution in the environments of industrialized countries. The U.S. Centers for Disease Control recently found that greater than 90% of Americans carry detectable levels of BPA, raising concern over the direct influences of this compound on human physiology. Epidemiologic evidence links elevated BPA serum concentrations to human reproductive dysfunction, although controlled studies on the acute effect of BPA exposure on reproductive function are limited, particularly in primates. We evaluated the effect of direct BPA exposure on female primate hypothalamic peptide release. Specifically, using a microdialysis method, we examined the effects of BPA (0.1, 1, and 10nM) directly infused to the stalk-median eminence on the release of GnRH and kisspeptin (KP) in mid to late pubertal ovarian intact female rhesus monkeys. We found that the highest level of BPA exposure (10nM) suppressed both GnRH and KP release, whereas BPA at lower concentrations (0.1 and 1nM) had no apparent effects. In addition, we measured BPA in plasma and hypothalamic dialysates after an iv bolus injection of BPA (100 μg/kg). We found a relatively stable distribution of BPA between the blood and brain (plasma:brain ≅ 5:1) persists across a wide range of blood BPA concentrations (1-620 ng/mL). Findings of this study suggest that persistent, high-level exposures to BPA could impair female reproductive function by directly influencing hypothalamic neuroendocrine function.

Neuroestradiol in the Stalk Median Eminence of Female Rhesus Macaques Decreases in Association With Puberty Onset

In primates, despite the fact that GnRH neurons are mature at birth, a gonadal steroid independent central inhibition restrains the initiation of puberty. The neural substrates responsible for this central inhibition, however, are unclear. In this study, we tested the hypothesis that neuroestradiol release in the hypothalamus decreases prior to the pubertal increase in GnRH release. We found that in female monkeys at the prepubertal stage, when GnRH release was low, estradiol (E2) levels in the stalk-median eminence of the hypothalamus were higher than those in older, early pubertal females in which nocturnal GnRH release begins to increase. Furthermore, estrone (E1) levels were higher in the stalk-median eminence of prepubertal and early pubertal monkeys compared with midpubertal monkeys, which have the highest GnRH release. The elevated E2 and E1 levels at the prepubertal stage are likely hypothalamic in origin because circulating E2 and E1 levels in prepubertal and early pubertal monkeys were much lower than those in midpubertal monkeys. Heightened synthesis and release of neuroestradiol during the prepubertal period and subsequent reduction at puberty onset indicate possible roles for neuroestradiol in central inhibition of GnRH release. The mechanism governing the reduction in neuroestradiol synthesis at puberty onset remains to be determined.

Prolonged Infusion of Estradiol Benzoate Into the Stalk Median Eminence Stimulates Release of GnRH and Kisspeptin in Ovariectomized Female Rhesus Macaques

Our recent study indicates that a brief infusion (20 min) of estradiol (E2) benzoate (EB) into the stalk-median eminence (S-ME) stimulates GnRH release with a latency of approximately 10 minutes. In contrast to the effect induced by a brief infusion of EB, it has previously been shown that systemic EB administration suppresses release of GnRH, kisspeptin, and LH with a latency of several hours, which is known as the negative feedback action of E2. We speculated that the differential results by these 2 modes of EB administration are due to the length of E2 exposure. Therefore, in the present study, the effects of EB infusion for periods of 20 minutes, 4 hours, or 7 hours into the S-ME of ovariectomized female monkeys on the release of GnRH and kisspeptin were examined using a microdialysis method. To assess the effects of the EB infusion on LH release, serum samples were also collected. The results show that similar to the results with 20-minute infusion, both 4- and 7-hour infusions of EB consistently stimulated release of GnRH and kisspeptin from the S-ME accompanied by LH release in the general circulation. In contrast, sc injection of EB suppressed all 3 hormones (GnRH, kisspeptin, and LH) measured. It is concluded that regardless of the exposure period, direct E2 action on GnRH and kisspeptin neurons in the S-ME, where their neuroterminals are present, is stimulatory, and the E2-negative feedback effects do not occur at the S-ME level.

Kisspeptin and Neurokinin B Signaling Network Underlies the Pubertal Increase in GnRH Release in Female Rhesus Monkeys

&NA; Loss‐of‐function or inactivating mutations in the genes coding for kisspeptin and its receptor (KISS1R) or neurokinin B (NKB) and the NKB receptor (NK3R) in humans result in a delay in or the absence of puberty. However, precise mechanisms of kisspeptin and NKB signaling in the regulation of the pubertal increase in gonadotropin‐releasing hormone (GnRH) release in primates are unknown. In this study, we conducted a series of experiments infusing agonists and antagonists of kisspeptin and NKB into the stalk‐median eminence, where GnRH, kisspeptin, and NKB neuroterminal fibers are concentrated, and measuring GnRH release in prepubertal and pubertal female rhesus monkeys. Results indicate that (1) similar to those previously reported for GnRH stimulation by the KISS1R agonist (i.e., human kisspeptin‐10), the NK3R agonist senktide stimulated GnRH release in a dose‐responsive manner in both prepubertal and pubertal monkeys; (2) the senktide‐induced GnRH release was blocked in the presence of the KISS1R antagonist peptide 234 in pubertal but not prepubertal monkeys; and (3) the kisspeptin‐induced GnRH release was blocked in the presence of the NK3R antagonist SB222200 in the pubertal but not prepubertal monkeys. These results are interpreted to mean that although, in prepubertal female monkeys, kisspeptin and NKB signaling to GnRH release is independent, in pubertal female monkeys, a reciprocal signaling mechanism between kisspeptin and NKB neurons is established. We speculate that this cooperative mechanism by the kisspeptin and NKB network underlies the pubertal increase in GnRH release in female monkeys.

Obligatory role of hypothalamic neuroestradiol during the estrogen-induced LH surge in female ovariectomized rhesus monkeys

Significance The concept that ovarian cycles in female mammals are regulated by gonadal steroids released from the ovary is well-established dogma. The findings of this study, however, showing that the removal of neuroestradiol synthesis in the median eminence of the hypothalamus by the aromatase inhibitor, letrozole, greatly attenuates estrogen-induced gonadotropin releasing hormone and luteinizing hormone surges in ovariectomized female monkeys, indicate that neuroestradiol locally synthesized and released in the hypothalamus is an integral part of the ovary-initiated positive feedback mechanism of estradiol. Negative and positive feedback effects of ovarian 17β-estradiol (E2) regulating release of gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH) are pivotal events in female reproductive function. While ovarian feedback on hypothalamo–pituitary function is a well-established concept, the present study shows that neuroestradiol, locally synthesized in the hypothalamus, is a part of estrogen’s positive feedback loop. In experiment 1, E2 benzoate-induced LH surges in ovariectomized female monkeys were severely attenuated by systemic administration of the aromatase inhibitor, letrozole. Aromatase is the enzyme responsible for synthesis of E2 from androgens. In experiment 2, using microdialysis, GnRH and kisspeptin surges induced by E2 benzoate were similarly attenuated by infusion of letrozole into the median eminence of the hypothalamus. Therefore, neuroestradiol is an integral part of the hypothalamic engagement in response to elevated circulating E2. Collectively, we will need to modify the concept of estrogen’s positive feedback mechanism.

Role of Kisspeptin and Neurokinin B Signaling in Male Rhesus Monkey Puberty

Despite the well-established concept that an increase in pulsatile GnRH release triggers puberty, the precise signaling mechanism responsible for the pubertal increase in GnRH release remains unclear. A recent study indicates that developmental changes in the network formation between kisspeptin and neurokinin B (NKB) signaling greatly contribute to the pubertal increase in GnRH release in female monkeys. It is, however, unknown whether similar developmental changes in the kisspeptin and NKB network are involved in male puberty. In the current study, we first characterized the pubertal stages in male rhesus monkeys by assessing physiological and hormonal changes during sexual development. Subsequently, we examined the role of the kisspeptin and NKB signaling network in the pubertal increase in GnRH release. Results suggest that while collaborative kisspeptin and NKB signaling to GnRH neurons was active before puberty onset, after initiation of puberty the role of NKB signaling in GnRH neurons diminished and kisspeptin signaling assumed the primary stimulatory role in the regulation of GnRH release in male monkeys. These findings in males differ from those seen in females.