Jenny Clarkson

Kisspeptin–GPR54 Signaling Is Essential for Preovulatory Gonadotropin-Releasing Hormone Neuron Activation and the Luteinizing Hormone Surge

Kisspeptin and its receptor GPR54 have recently been identified as key signaling partners in the neural control of fertility in animal models and humans. The gonadotropin-releasing hormone (GnRH) neurons represent the final output neurons of the neural network controlling fertility and are suspected to be the primary locus of kisspeptin–GPR54 signaling. Using mouse models, the present study addressed whether kisspeptin and GPR54 have a key role in the activation of GnRH neurons to generate the luteinizing hormone (LH) surge responsible for ovulation. Dual-label immunocytochemistry experiments showed that 40–60% of kisspeptin neurons in the rostral periventricular area of the third ventricle (RP3V) expressed estrogen receptor α and progesterone receptors. Using an ovariectomized, gonadal steroid-replacement regimen, which reliably generates an LH surge, ∼30% of RP3V kisspeptin neurons were found to express c-FOS in surging mice compared with 0% in nonsurging controls. A strong correlation was found between the percentage of c-FOS-positive kisspeptin neurons and the percentage of c-FOS-positive GnRH neurons. To evaluate whether kisspeptin and/or GPR54 were essential for GnRH neuron activation and the LH surge, Gpr54- and Kiss1-null mice were examined. Whereas wild-type littermates all exhibited LH surges and c-FOS in ∼50% of their GnRH neurons, none of the mutant mice from either line showed an LH surge or any GnRH neurons with c-FOS. These observations provide the first evidence that kisspeptin–GPR54 signaling is essential for GnRH neuron activation that initiates ovulation. This broadens considerably the potential roles and therapeutic possibilities for kisspeptin and GPR54 in fertility regulation.

Distribution of Kisspeptin Neurones in the Adult Female Mouse Brain

Kisspeptin‐GPR54 signalling is essential for normal reproductive functioning. However, the distribution of kisspeptin neuronal cell bodies and their projections is not well established. The present study aimed to provide a detailed account of kisspeptin neuroanatomy in the mouse brain. Using a polyclonal rabbit antibody AC566, directed towards the final ten C‐terminal amino acids of murine kisspeptin, three populations of kisspeptin‐expressing cell bodies were identified in the adult female mouse brain. One exists as a dense periventricular continuum of cells within the rostral part of the third ventricle, another is found within the arcuate nucleus, and another is identified as a low‐density group of scattered cells within the dorsomedial nucleus and posterior hypothalamus. Kisspeptin‐immunoreactive fibres were abundant within the ventral aspect of the lateral septum and within the hypothalamus running in periventricular and ventral retrochiasmatic pathways. Notable exclusions from the kisspeptin fibre innervation were the suprachiasmatic and ventromedial nuclei. Outside of the hypothalamus, a small number of kisspeptin fibres were identified in the bed nucleus of the stria terminalis, subfornical organ, medial amygdala, paraventricular thalamus, periaqueductal grey and locus coerulus. All kisspeptin cell body and fibre immunoreactivity was absent in brain tissue from Kiss1 knockout mice. These observations provide a map of kisspeptin neurones in the mouse brain and indicate that a limited number of mostly medial hypothalamic and lateral septal brain regions are innervated by the three hypothalamic kisspeptin cell populations; the functions of these projections remain to be established.

Postnatal Development of an Estradiol-Kisspeptin Positive Feedback Mechanism Implicated in Puberty Onset

The regulation of GnRH neurons by kisspeptin is critical for normal puberty onset in mammals. In the rodent the kisspeptin neurons innervating GnRH neurons are thought to reside in the rostral periventricular area of the third ventricle (RP3V). Using kisspeptin immunocytochemistry we show that kisspeptin peptide expression in the RP3V of female mice begins around postnatal d 15 (P15) and rapidly increases to achieve adult-like levels by P30, the time of puberty onset. Ovariectomy of female pups at P15 resulted in a 70-90% reduction (P < 0.01) in kisspeptin peptide expression within the RP3V of P30 or P60 mice. Replacement of 17-beta-estradiol (E2) in P15-ovariectomized mice from P15-30 or P22-30 resulted in a complete restoration of kisspeptin peptide expression in the RP3V (P < 0.01). Kisspeptin-immunoreactive fibers throughout the hypothalamus, including the arcuate nucleus, followed the same pattern of estrogen-dependent expression. To test the absolute necessity of estrogen for kisspeptin expression in the RP3V, aromatase knockout mice were examined. Kisspeptin-immunoreactive cells were detected in the arcuate nucleus, but there was a complete absence of kisspeptin peptide in RP3V neurons of aromatase knockout adult females. These results demonstrate that E2 is essential for the prepubertal development of kisspeptin peptide within RP3V neurons and suggest that an E2-kisspeptin positive feedback mechanism exists before puberty. This implies that RP3V kisspeptin neurons are E2-dependent amplifiers of GnRH neuron activity in the prepubertal period.

Oestrogen, Kisspeptin, GPR54 and the Pre‐Ovulatory Luteinising Hormone Surge

Ovulation is central to mammalian fertility, yet the precise mechanism through which oestrogen triggers the gonadotrophin‐releasing hormone (GnRH) surge that generates the pre‐ovulatory luteinising hormone (LH) surge has remained elusive. The recent discovery that kisspeptin‐GPR54 signalling is an essential regulator of the neuroendocrine axis at puberty has led investigators to evaluate the role of kisspeptin in the pre‐ovulatory GnRH surge mechanism. Kisspeptin neurones are known to express oestrogen and progesterone receptors and have their cell bodies located in brain regions implicated in the positive‐feedback mechanism in several mammalian species. In rodents, kisspeptin neurones located in the rostral periventricular area of the third ventricle (RP3V) are positively regulated by oestrogen and most likely are activated by oestrogen at the time of positive feedback. A similar scenario appears to exist for a sub‐population of kisspeptin neurones located in the mediobasal hypothalamus of sheep and primates. The majority of GnRH neurones express GPR54, and kisspeptin causes an intense electrical activation of these cells. In concordance with this, kisspeptin administration in vivo results in an abrupt and prolonged release of LH in all mammalian species examined to date. Functional evidence from immunoneutralisation and knockout studies suggests that RP3V kisspeptin neurones projecting to GnRH neurones are an essential component of the surge mechanism in rodents. Taken together, the studies undertaken to date provide substantial evidence in support of a key role of kisspeptin‐GPR54 signalling in the generation of the oestrogen‐induced pre‐ovulatory surge mechanism in mammals.

Development of GABA and glutamate signaling at the GnRH neuron in relation to puberty

The gonadotropin-releasing hormone (GnRH) neurons represent the critical cell type activated to induce puberty in mammals. However, the mechanisms underlying their activation remain unclear. As the principal amino acid neurotransmitters in the brain, GABA and glutamate are known to have critical roles in the development of neuronal networks. This review provides an update on what is known about GABA and glutamate signaling at the GnRH neuron across development. An examination of morphological, receptor subunit expression, and electrophysiological data suggest that GABAA receptor signaling develops in advance of glutamatergic signaling. However, compared with other networks, the switch from GABAA receptor depolarization to hyperpolarization of GnRH neurons is delayed until the time of puberty. These observations suggest that developing GnRH neurons exhibit a sequence of GABA-->glutamate signaling similar to that of other neuronal networks but that it is significantly elongated so as to only be complete by the time of puberty onset.

Dependence of fertility on kisspeptin–Gpr54 signaling at the GnRH neuron

Signaling between kisspeptin and its receptor, G-protein-coupled receptor 54 (Gpr54), is now recognized as being essential for normal fertility. However, the key cellular location of kisspeptin-Gpr54 signaling is unknown. Here we create a mouse with a GnRH neuron-specific deletion of Gpr54 to assess the role of gonadotropin-releasing hormone (GnRH) neurons. Mutant mice are infertile, fail to go through puberty and exhibit markedly reduced gonadal size and follicle-stimulating hormone levels alongside GnRH neurons that are unresponsive to kisspeptin. In an attempt to rescue the infertile phenotype of global Gpr54⁻/⁻ mutants, we use BAC transgenesis to target Gpr54 to the GnRH neurons. This results in mice with normal puberty onset, estrous cyclicity, fecundity and a recovery of kisspeptins stimulatory action upon GnRH neurons. Using complimentary cell-specific knockout and knockin approaches we demonstrate here that the GnRH neuron is the key site of kisspeptin-Gpr54 signaling for fertility.

Dual Phenotype Kisspeptin-Dopamine Neurones of the Rostral Periventricular Area of the Third Ventricle Project to Gonadotrophin-Releasing Hormone Neurones

The neuropeptide kisspeptin and its G‐protein‐coupled receptor, Gpr54, are critical regulators of fertility. Two major populations of kisspeptin neurones exist in the rodent: one in the rostral periventricular area of the third ventricle (RP3V) and another in the arcuate nucleus. The RP3V population of kisspeptin neurones is crucial for the generation of the luteinising hormone surge that drives ovulation in females. The RP3V kisspeptin neurones are sexually dimorphic, with many more neurones in females than males, and they project to gonadotrophin‐releasing hormone (GnRH) neurones. Tyrosine hydroxylase (TH) expressing neurones in the RP3V are also sexually dimorphic and are assumed to project to GnRH neurones. In the present study, we examined the coexpression of kisspeptin and TH peptides in the RP3V of dioestrous and pro‐oestrous female mice. We also investigated whether kisspeptin and TH peptides colocalised in terminal appositions with GnRH neurones in the rostral preoptic area (rPOA). Approximately half of the kisspeptin neurones in the RP3V were found to also express TH and vice versa, although there was no difference between mice in dioestrus or pro‐oestrus. The majority (95%) of GnRH neurones in the rPOA exhibited a close apposition from a kisspeptin fibre, whereas only one quarter exhibited a close apposition from a TH fibre. Many of the TH close appositions with GnRH neurones coexpressed kisspeptin (62–86%), although these dual‐labelled appositions comprised < 20% of all kisspeptin appositions on GnRH neurones. The percentage of GnRH neurones with kisspeptin, TH and double‐labelled appositions did not differ between dioestrous and pro‐oestrous mice. These findings indicate that a subpopulation of kisspeptin neurones expressing dopamine innervate GnRH neurones in the rPOA.

Development of a methodology for and assessment of pulsatile luteinizing hormone secretion in juvenile and adult male mice.

Current methodology to monitor pulsatile LH release in mice is limited by inadequate assay sensitivity, resulting in the need for collection of large blood volumes. Thus, assessment of pulsatile LH secretion in mice remains highly challenging, and observations are limited to adult mice. To address this, we developed a highly sensitive ELISA for assessment of mouse LH concentrations in small fractions of whole blood. We demonstrate that this assay is capable of reliably detecting LH down to a theoretical limit of 0.117 ng/mL in a 2-μL fraction of whole blood. Using an established frequent blood collection procedure, we validated the accuracy of this method by determining the pulsatile LH secretion in early-adult (10 weeks old) C57BL6/J male mice. Data demonstrate regular pulsatile release of LH, with peaks in LH secretion rarely exceeding 3 ng/mL. Moreover, assessment of LH release in Gpr54 knockout mice demonstrates the lack of pulsatile LH release after the loss of kisspeptin-mediated pubertal maturation. We next determined age-associated changes in pulsatile LH secretion by assessment of LH secretion in prepubertal (28 days old) C57BL6/J male mice and repeated assessment in the same mice in adulthood (120 days old). Data demonstrate that the rise in total LH secretion in mice after pubertal maturation occurs along with an overall rise in the pulsatile LH secretion rate. This was coupled with a significant increase in the number of LH secretory events (number of pulses). In addition, we observed a decrease in the clearance (increased half-life) and a decrease in the regularity (approximate entropy) of LH release. This method will be of wide general utility within the field of reproductive biology.

Sex differences in hypothalamic astrocyte response to estradiol stimulation

BackgroundReproductive functions controlled by the hypothalamus are highly sexually differentiated. One of the most dramatic differences involves estrogen positive feedback, which leads to ovulation. A crucial feature of this positive feedback is the ability of estradiol to facilitate progesterone synthesis in female hypothalamic astrocytes. Conversely, estradiol fails to elevate hypothalamic progesterone levels in male rodents, which lack the estrogen positive feedback-induced luteinizing hormone (LH) surge. To determine whether hypothalamic astrocytes are sexually differentiated, we examined the cellular responses of female and male astrocytes to estradiol stimulation.MethodsPrimary adult hypothalamic astrocyte cultures were established from wild type rats and mice, estrogen receptor-α knockout (ERKO) mice, and four core genotype (FCG) mice, with the sex determining region of the Y chromosome (Sry) deleted and inserted into an autosome. Astrocytes were analyzed for Sry expression with reverse transcription PCR. Responses to estradiol stimulation were tested by measuring free cytoplasmic calcium concentration ([Ca2+]i) with fluo-4 AM, and progesterone synthesis with column chromatography and radioimmunoassay. Membrane estrogen receptor-α (mERα) levels were examined using surface biotinylation and western blotting.ResultsEstradiol stimulated both [Ca2+]i release and progesterone synthesis in hypothalamic astrocytes from adult female mice. Male astrocytes had a significantly elevated [Ca2+]i response but it was significantly lower than in females, and progesterone synthesis was not enhanced. Surface biotinylation demonstrated mERα in both female and male astrocytes, but only in female astrocytes did estradiol treatment increase insertion of the receptor into the membrane, a necessary step for maximal [Ca2+]i release. Regardless of the chromosomal sex, estradiol facilitated progesterone synthesis in astrocytes from mice with ovaries (XX and XY-), but not in mice with testes (XY-Sry and XXSry).ConclusionsAstrocytes are sexually differentiated, and in adulthood reflect the actions of sex steroids during development. The response of hypothalamic astrocytes to estradiol stimulation was determined by the presence or absence of ovaries, regardless of chromosomal sex. The trafficking of mERα in female, but not male, astrocytes further suggests that cell signaling mechanisms are sexually differentiated.

Mitochondrial involvement in transhemispheric diaschisis following hypoxia-ischemia: Clomethiazole-mediated amelioration.

Mitochondria play a central role in both the physiological and pathophysiological regulation of cell survival/death. Increasing evidence places mitochondrial dysfunction at the center of many neuropathological conditions. The present study investigates the extent of mitochondrial dysfunction in cortical, hippocampal and cerebellar tissues in a rat model of hypoxia-ischemia (HI). We hypothesized that; mitochondrial dysfunction in situ may be prevented by treatment with clomethiazole (CMZ), a GABA(A) receptor agonist. Assessment of mitochondrial FAD-linked respiration at both 1- and 3-day post-HI revealed a marked decrease in activity from ipsilateral cortical and hippocampal regions (P<0.001). In addition, small changes were seen in contralateral cortical and hippocampal tissues as well as in the cerebellum at 3-days (P<0.05). Assessment of the mitochondrial electron transport chain (complexes I-V), and mitochondrial markers of integrity (citrate synthase) and oxidative stress (aconitase) confirmed mitochondrial impairment in ipsilateral regions following HI. Complexes I, II-III, V and citrate synthase were also impaired in contralateral regions and cerebellum 3-days post-HI. Treatment with CMZ (414 mg/kg/day via minipumps) provided marked protection to all aspects of neuronal tissue assessed. Circulating cytokine (interleukin [IL]-1alpha, IL-1beta, tumor necrosis factor [TNF]-alpha, granulocyte macrophage colony-stimulating factor [GM-CSF], IL-4 and IL-10) levels were also assessed in these animals 3-days post-HI. Plasma IL-1alpha, IL-1beta, TNF-alpha and GM-CSF levels were significantly increased post-HI. Treatment with CMZ ameliorated the increases in IL-1alpha, IL-1beta, TNF-alpha and GM-CSF levels while increasing plasma IL-4 and IL-10 levels. This study provides evidence of the extent of mitochondrial damage following an HI-insult. In addition, we have shown that protection afforded by CMZ extends to preservation of mitochondrial function and integrity via anti-inflammatory mediated pathways.