Nuclear receptor LRH-1 induces the reproductive neuropeptide kisspeptin in the hypothalamus.

The differential expression and secretion of the neuropeptide kisspeptin from neurons in the arcuate (Arc) and anteroventral periventricular (AVPV) nuclei of the hypothalamus coordinate the temporal release of pituitary gonadotropins that control the female reproductive cycle. However, the molecular basis for this differential regulation is incompletely understood. Here, we report that liver receptor homolog-1 (LRH-1), a member of the nuclear receptor superfamily, is expressed in kisspeptin neurons in the Arc but not in the AVPV in female mice. LRH-1 binds directly to the kisspeptin (Kiss1) promoter and stimulates Kiss1 transcription. Deletion of LRH-1 from kisspeptin neurons in mice decreased Kiss1 expression in the Arc, leading to reduced plasma FSH levels, dysregulated follicle maturation, and prolongation of the estrous cycle. Conversely, overexpression of LRH-1 in kisspeptin neurons increased Arc Kiss1 expression and plasma FSH concentrations. These studies provide a molecular basis for the differential regulation of basal kisspeptin expression in Arc and AVPV neurons and reveal a prominent role for LRH-1 in hypothalamus in regulating the female reproductive axis.

Leptin signaling in Kiss1 neurons arises after pubertal development.

The adipocyte-derived hormone leptin is required for normal pubertal maturation in mice and humans and, therefore, leptin has been recognized as a crucial metabolic cue linking energy stores and the onset of puberty. Several lines of evidence have suggested that leptin acts via kisspeptin expressing neurons of the arcuate nucleus to exert its effects. Using conditional knockout mice, we have previously demonstrated that deletion of leptin receptors (LepR) from kisspeptin cells cause no puberty or fertility deficits. However, developmental adaptations and system redundancies may have obscured the physiologic relevance of direct leptin signaling in kisspeptin neurons. To overcome these putative effects, we re-expressed endogenous LepR selectively in kisspeptin cells of mice otherwise null for LepR, using the Cre-loxP system. Kiss1-Cre LepR null mice showed no pubertal development and no improvement of the metabolic phenotype, remaining obese, diabetic and infertile. These mice displayed decreased numbers of neurons expressing Kiss1 gene, similar to prepubertal control mice, and an unexpected lack of re-expression of functional LepR. To further assess the temporal coexpression of Kiss1 and Lepr genes, we generated mice with the human renilla green fluorescent protein (hrGFP) driven by Kiss1 regulatory elements and crossed them with mice that express Cre recombinase from the Lepr locus and the R26-tdTomato reporter gene. No coexpression of Kiss1 and LepR was observed in prepubertal mice. Our findings unequivocally demonstrate that kisspeptin neurons are not the direct target of leptin in the onset of puberty. Leptin signaling in kisspeptin neurons arises only after completion of sexual maturation.

Shift in Kiss1 cell activity requires estrogen receptor α.

Reproductive function requires timely secretion of gonadotropin-releasing hormone, which is controlled by a complex excitatory/inhibitory network influenced by sex steroids. Kiss1 neurons are fundamental players in this network, but it is currently unclear whether different conditions of circulating sex steroids directly alter Kiss1 neuronal activity. Here, we show that Kiss1 neurons in the anteroventral periventricular and anterior periventricular nuclei (AVPV/PeN) of males and females exhibit a bimodal resting membrane potential (RMP) influenced by K(ATP) channels, suggesting the presence of two neuronal populations defined as type I (irregular firing patterns) and type II (quiescent). Kiss1 neurons in the arcuate nucleus (Arc) are also composed of firing and quiescent cells, but unlike AVPV/PeN neurons, the range of RMPs did not follow a bimodal distribution. Moreover, Kiss1 neuronal activity in the AVPV/PeN, but not in the Arc, is sexually dimorphic. In females, estradiol shifts the firing pattern of AVPV/PeN Kiss1 neurons and alters cell capacitance and spontaneous IPSCs amplitude of AVPV/PeN and Arc Kiss1 populations in an opposite manner. Notably, mice with selective deletion of estrogen receptor α (ERα) from Kiss1 neurons show cellular activity similar to that observed in ovariectomized females, suggesting that estradiol-induced changes in Kiss1 cellular properties require ERα. We also show that female prepubertal Kiss1 neurons are under higher inhibitory influence and all recorded AVPV/PeN Kiss1 neurons were spontaneously active. Collectively, our findings indicate that changes in cellular activity may underlie Kiss1 action in pubertal initiation and female reproduction.

Leptin's effect on puberty in mice is relayed by the ventral premammillary nucleus and does not require signaling in Kiss1 neurons.

Studies in humans and rodents indicate that a minimum amount of stored energy is required for normal pubertal development. The adipocyte-derived hormone leptin is a key metabolic signal to the neuroendocrine reproductive axis. Humans and mice lacking leptin or the leptin receptor (LepR) (ob/ob and db/db mice, respectively) are infertile and fail to enter puberty. Leptin administration to leptin-deficient subjects and ob/ob mice induces puberty and restores fertility, but the exact site or sites of leptin action are unclear. Here, we found that genetic deletion of LepR selectively from hypothalamic Kiss1 neurons in mice had no effect on puberty or fertility, indicating that direct leptin signaling in Kiss1 neurons is not required for these processes. However, bilateral lesions of the ventral premammillary nucleus (PMV) of ob/ob mice blunted the ability of exogenous leptin to induce sexual maturation. Moreover, unilateral reexpression of endogenous LepR in PMV neurons was sufficient to induce puberty and improve fertility in female LepR-null mice. This LepR reexpression also normalized the increased hypothalamic GnRH content characteristic of leptin-signaling deficiency. These data suggest that the PMV is a key site for leptin's permissive action at the onset of puberty and support the hypothesis that the multiple actions of leptin to control metabolism and reproduction are anatomically dissociated.

Hypothalamic sites of leptin action linking metabolism and reproduction.

A critical amount of energy reserve is necessary for puberty initiation, for normal sexual maturation and maintenance of cyclicity and fertility in females of most species. Therefore, the existence of circulating metabolic cues which directly modulate the hypothalamus-pituitary-gonad axis is predictable. The adipocyte-derived hormone leptin is one of these cues having been studied extensively in the context of regulating the reproductive physiology. Humans and mice lacking leptin (ob/ob) or leptin receptor (LepR, db/db) are infertile. Leptin administration to leptin-deficient subjects and ob/ob mice induces puberty and restores fertility. LepR is expressed in brain, pituitary gland and gonads, but studies using genetically engineered mouse models determined that the brain plays a major role. Recently, it has been made clear that leptin acts indirectly on gonadotropin-releasing hormone (GnRH)-secreting cells via actions on interneurons. However, the exact site(s) of leptin action has been difficult to determine. In this review, we discuss the recent advances in the field focused on the identification of potential site(s) or specific neuronal populations involved in leptin's effects in the neuroendocrine reproductive axis.

Insights into the organization of dorsal spinal cord pathways from an evolutionarily conserved raldh2 intronic enhancer.

Comparative studies of the tetrapod raldh2 (aldh1a2) gene, which encodes a retinoic acid (RA) synthesis enzyme, have led to the identification of a dorsal spinal cord enhancer. Enhancer activity is directed dorsally to the roof plate and dorsal-most (dI1) interneurons through predicted Tcf- and Cdx-homeodomain binding sites and is repressed ventrally via predicted Tgif homeobox and ventral Lim-homeodomain binding sites. Raldh2 and Math1/Cath1 expression in mouse and chicken highlights a novel, transient, endogenous Raldh2 expression domain in dI1 interneurons, which give rise to ascending circuits and intraspinal commissural interneurons, suggesting roles for RA in the ontogeny of spinocerebellar and intraspinal proprioceptive circuits. Consistent with expression of raldh2 in the dorsal interneurons of tetrapods, we also found that raldh2 is expressed in dorsal interneurons throughout the agnathan spinal cord, suggesting ancestral roles for RA signaling in the ontogenesis of intraspinal proprioception.

ALDH1A2 (RALDH2) genetic variation in human congenital heart disease.

BACKGROUND: Signaling by the vitamin A-derived morphogen retinoic acid (RA) is required at multiple steps of cardiac development. Since conversion of retinaldehyde to RA by retinaldehyde dehydrogenase type II (ALDH1A2, a.k.a RALDH2) is critical for cardiac development, we screened patients with congenital heart disease (CHDs) for genetic variation at the ALDH1A2 locus. METHODS: One-hundred and thirty-three CHD patients were screened for genetic variation at the ALDH1A2 locus through bi-directional sequencing. In addition, six SNPs (rs2704188, rs1441815, rs3784259, rs1530293, rs1899430) at the same locus were studied using a TDT-based association approach in 101 CHD trios. Observed mutations were modeled through molecular mechanics (MM) simulations using the AMBER 9 package, Sander and Pmemd programs. Sequence conservation of observed mutations was evaluated through phylogenetic tree construction from ungapped alignments containing ALDH8 s, ALDH1Ls, ALDH1 s and ALDH2 s. Trees were generated by the Neighbor Joining method. Variations potentially affecting splicing mechanisms were cloned and functional assays were designed to test splicing alterations using the pSPL3 splicing assay. RESULTS: We describe in Tetralogy of Fallot (TOF) the mutations Ala151Ser and Ile157Thr that change non-polar to polar residues at exon 4. Exon 4 encodes part of the highly-conserved tetramerization domain, a structural motif required for ALDH oligomerization. Molecular mechanics simulation studies of the two mutations indicate that they hinder tetramerization. We determined that the SNP rs16939660, previously associated with spina bifida and observed in patients with TOF, does not affect splicing. Moreover, association studies performed with classical models and with the transmission disequilibrium test (TDT) design using single marker genotype, or haplotype information do not show differences between cases and controls. CONCLUSION: In summary, our screen indicates that ALDH1A2 genetic variation is present in TOF patients, suggesting a possible causal role for this gene in rare cases of human CHD, but does not support the hypothesis that variation at the ALDH1A2 locus is a significant modifier of the risk for CHD in humans.

The evolutionary origin of cardiac chambers.

Identification of cardiac mechanisms of retinoic acid (RA) signaling, description of homologous genetic circuits in Ciona intestinalis and consolidation of views on the secondary heart field have fundamental, but still unrecognized implications for vertebrate heart evolution. Utilizing concepts from evolution, development, zoology, and circulatory physiology, we evaluate the strengths of animal models and scenarios for the origin of vertebrate hearts. Analyzing chordates, lower and higher vertebrates, we propose a paradigm picturing vertebrate hearts as advanced circulatory pumps formed by segments, chambered or not, devoted to inflow or outflow. We suggest that chambers arose not as single units, but as components of a peristaltic pump divided by patterning events, contrasting with scenarios assuming that chambers developed one at a time. Recognizing RA signaling as a potential mechanism patterning cardiac segments, we propose to use it as a tool to scrutinize the phylogenetic origins of cardiac chambers within chordates. Finally, we integrate recent ideas on cardiac development such as the ballooning and secondary/anterior heart field paradigms, showing how inflow/outflow patterning may interact with developmental mechanisms suggested by these models.