The progression and completion of puberty thus appears to be governed by a positive feedback loop, in which rising levels of circulating estrogens in early puberty stimulate kisspeptin expression, which further elevates GnRH release and hence, prompts additional gonadotropin and steroid production

The progression and completion of puberty thus appears to be governed by a positive feedback loop, in which rising levels of circulating estrogens in early puberty stimulate kisspeptin expression, which further elevates GnRH release and hence, prompts additional gonadotropin and steroid production. nervous system, where gonadotropin-releasing hormone (GnRH) neurons are activated to release high-frequency pulses of the neurohormone, stimulating pituitary gonadotropic hormone secretions that in turn direct gonadal steroid hormone production and maturation of gametes (13). An ill-defined developmental clock, as well as permissive somatic and environmental signals, govern the onset, progression, and completion of the pubertal acceleration of GnRH release. However, the cellular signaling pathways that mediate the pubertal stimulation and/or disinhibition of GnRH neurons have remained obscure. In rodents, sheep, and other animals, a gonadostat hypothesis has held that this pace of puberty is usually a function of changing sensitivity of the GnRH neurosecretory system to the inhibitory feedback actions of gonadal steroids. In females, the hypothesis stipulates that this sensitivity of the central GnRH pulse generator to inhibition by circulating gonadal steroids, principally estradiol-17 (E2), is usually reduced in the late juvenile stage of development (46). The concept has been strongly supported in female mice, where classic analyses revealed that a 10-fold increase in the dose of E2is usually required to fully suppress LH secretion in adult vs. juvenile animals (7). This mechanistic model has been revised to apply largely to the later stages of puberty in monkeys (8). A second steroid-dependent mechanism has also been proposed, in which E2s positive neurotropic effects contribute to the maturation of the GnRH pulse generator during puberty (9). We sought to determine the specific neuronal populations in which estrogen receptors may mediate either the juvenile feedback suppression of GnRH release or the pubertal stimulation of the GnRH releasing system, or both. PI4KIIIbeta-IN-10 The identification of kisspeptins, neuropeptides expressed by theKiss1gene, and their receptor, GPR54, has provided an opportunity for understanding how the timing and progression of puberty are controlled. Inactivating mutations of the humanGPR54gene are associated PI4KIIIbeta-IN-10 with hypogonadotropic hypogonadism and an absence of puberty (10,11). Ablation of theGPR54(12,13) or theKiss1(13,14) gene renders mice of either sex hypogonadotropic and infertile. Kisspeptin is usually a powerful GnRH secretagogue (15,16), and kisspeptin administration advances puberty onset (17,18), whereas GPR54 antagonists delay puberty onset (19). These data provide compelling support for the idea that activation of kisspeptin neurons precipitates the pubertal stimulation of GnRH neurosecretion. Estrogens are key regulators of kisspeptin expression in pre- and postpubertal female mice (20,21) and rats (22), as they potently exert inhibitory effects on hypothalamic arcuate nucleus (ARC) neurons and stimulatory actions in anteroventral periventricular (AVPe) neurons. Furthermore, the majority of kisspeptin neurons in both hypothalamic regions coexpress ER (23), raising the possibility that these receptors in kisspeptin neurons directly modulate the progression of puberty. To test this hypothesis, we generated mice with a conditional knockout of theER(Esr1) gene in kisspeptin neurons, and assessed the tempo and completeness of their reproductive maturation. == Results and Discussion == == Generation of Kisspeptin Cell-Specific ER Knockout Mice. == We first used gene targeting in embryonic stem cells to express Cre recombinase under the control of the mouse kisspeptin promoter. We inserted an internal ribosome entry site (IRES) sequence followed by aCre recombinasecDNA downstream of theKiss1coding region (Fig. 1A). Transcription of Kit the recombinantKiss1allele yields a bicistronic messenger RNA, from which kisspeptin and Cre recombinase are independently translated. == Fig. 1. == Conditional ER knockout in kisspeptin cells. (A) Targeting strategy to express Cre recombinase under control of theKiss1promoter. The targeting vector, the WT alleles, and the targeted allele before (neo+) and after (neo) removal of the neomycin selection cassette are shown from top to bottom. Restriction sites used for Southern blot analysis, as well as the location of the probes, are indicated. Black boxes represent exons. The inserted cassette is composed of an internal ribosomal entry site (IRES) followed by the coding sequence for Cre recombinase (Cre), PI4KIIIbeta-IN-10 and aphosphoglycerate kinasepromoter-driven neomycin resistance cassette flanked by Flp recombinase recognition sites (FRT). Southern blot analysis of ES-cell DNA after digestion with SacI. The expected fragment sizes detected by the probe are indicated (WT, 11.4 kb; mutant, 9.6 kb). Clone 2 carries the mutant KissI allele (KissICneo+). Southern blot analysis of DNA digested with PI4KIIIbeta-IN-10 Bsu36I from WT, and heterozygous mutant mice before and after removal of the neomycin selection cassette. The expected fragment sizes detected by the probe are indicated (WT,.