Leptin and melanocortin signaling control ingestive behavior, energy balance, and substrate utilization, but only leptin signaling defects cause hypothalamic hypogonadism and infertility. Although GnRH neurons do not express leptin receptors, leptin influences GnRH neuron activity via regulation of immediate downstream mediators including the neuropeptides neuropeptide Y and the melanocortin agonist and antagonist, α-MSH, agouti-related peptide, respectively. Here we show that modulation of melanocortin signaling in female db/db mice through ablation of agouti-related peptide, or heterozygosity of melanocortin 4 receptor, restores the timing of pubertal onset, fertility, and lactation. Additionally, melanocortin 4 receptor activation increases action potential firing and induces c-Fos expression in GnRH neurons, providing further evidence that melanocortin signaling influences GnRH neuron activity. These studies thus establish melanocortin signaling as an important component in the leptin-mediated regulation of GnRH neuron activity, initiation of puberty and fertility.

Leptin may program abnormal AMH signaling, thereby resulting in ovarian dysfunction. This study opens a new perspective for understanding the low ovarian reserve seen in obese women and provides new insights into potential mechanisms that explain the lower AMH seen in obese women. Whether our findings explain the worse response to ovulation induction observed in obese women needs to be further elucidated.

Mammary gland development is critically dependent on the interactions between the stromal and the epithelial compartments within the gland. These events are under the control of a complex interplay of circulating and locally-acting hormones and growth factors. To analyze the temporal and quantitative contributions of stromal adipocytes, we took advantage of the FAT-ATTAC mice (“Apoptosis Through Triggered Activation of Caspase-8”), a model of inducible and reversible loss of adipocytes. This loss can be achieved through the induced dimerization of a caspase-8 fusion protein. In the context of female mice, we can achieve ablation of mammary adipocytes relatively selectively without affecting other fat pads. Under these conditions, we find that adipocytes are essential for the formation of the extended network of ducts in the mammary gland during puberty. Beyond their role in development, adipocytes are also essential to maintain the normal alveolar structures that develop during adulthood. Loss of adipose tissue initiated 2 weeks after birth triggers fewer duct branching points, fewer terminal end buds (TEBs), and also triggers changes in proliferation and apoptosis in the epithelium associated with the TEBs. The reduced developmental pace that adipocyte-ablated glands undergo is reversible, as the emergence of new local adipocytes, upon cessation of treatment, enables the ductal epithelium to resume growth. Conversely, loss of local adipocytes initiated at 7 weeks of age resulted in excessive lobulation, indicating that adipocytes are critically involved in maintaining proper architecture and functionality of the mammary epithelium. Collectively, using a unique model of inducible and reversible loss of adipocytes, our observations suggest that adipocytes are required for proper development during puberty and for the maintenance of the ductal architecture in the adult mammary gland.

The mechanism(s) by which vitamin D(3) regulates female reproduction is minimally understood. We tested the hypothesis that peripubertal vitamin D(3) deficiency disrupts hypothalamic-pituitary-ovarian physiology. To test this hypothesis, we used wild-type mice and Cyp27b1 (the rate-limiting enzyme in the synthesis of 1,25-dihydroxyvitamin D(3)) null mice to study the effect of vitamin D(3) deficiency on puberty and reproductive physiology. At the time of weaning, mice were randomized to a vitamin D(3)-replete or -deficient diet supplemented with calcium. We assessed the age of vaginal opening and first estrus (puberty markers), gonadotropin levels, ovarian histology, ovarian responsiveness to exogenous gonadotropins, and estrous cyclicity. Peripubertal vitamin D(3) deficiency significantly delayed vaginal opening without affecting the number of GnRH-immunopositive neurons or estradiol-negative feedback on gonadotropin levels during diestrus. Young adult females maintained on a vitamin D(3)-deficient diet after puberty had arrested follicular development and prolonged estrous cycles characterized by extended periods of diestrus. Ovaries of vitamin D(3)-deficient Cyp27b1 null mice responded to exogenous gonadotropins and deposited significantly more oocytes into the oviducts than mice maintained on a vitamin D(3)-replete diet. Estrous cycles were restored when vitamin D(3)-deficient Cyp27b1 null young adult females were transferred to a vitamin D(3)-replete diet. This study is the first to demonstrate that peripubertal vitamin D(3) sufficiency is important for an appropriately timed pubertal transition and maintenance of normal female reproductive physiology. These data suggest vitamin D(3) is a key regulator of neuroendocrine and ovarian physiology.

Deficient leptin signaling causes infertility via reduced activity of GnRH neurons, causing a hypogonadal state in both rodents and humans. Because GnRH neurons do not express leptin receptors, leptin's effect on GnRH neurons must be indirect. Neurons within the hypothalamic arcuate nucleus that coexpress AGRP and NPY are considered to be important intermediate neurons involved in leptin regulation of GnRH neurons. Previously, we reported that the absence of AGRP and haploinsufficiency of MC4R in leptin receptor mutant (Lepr db/db ) females result in restoration of fertility and lactation despite the persistence of obesity and insulin resistance. The overarching hypothesis in the present study is that the absence or reduction of leptin's inhibition of AGRP/NPY neurons leads to suppression of GnRH release in cases of leptin signaling deficiency. Since TAC2 (NKB)-TAC3R signaling plays a role in puberty maturation and is modulated by metabolic status, the other aim of this study is to test whether TAC2/NKB neurons in ARC regulated by melanocortinergic signals herein affect leptin's action on puberty and reproduction. Our data showed that AGRP deficiency in Lepr db/db females restores normal timing of vaginal opening and estrous cycling, although uterine weight gain and mammary gland development are morphologically delayed. Nonetheless, Agrp Ϫ/Ϫ Lepr db/db females are fertile and sustain adequate nutrition of pups with lactation to weaning age. AGRP deficiency results in advanced vaginal opening in wild-type female mice. The postpubertal increase in hypothalamic TAC2 mRNA was not observed in Lepr db/db females, whereas AGRP deficiency restored it in Lepr db/db females. Additionally, MC4R activation with MTII induced FOS expression in TAC2 neurons, supporting the concept of melanocortinergic regulation of TAC2 neurons. These studies suggest that AGRP imposes an inhibitory effect on puberty and that TAC2 neurons may transmit melanocortinergic inhibition of GnRH neurons.agouti-related peptide; leptin; puberty; reproduction THE ONSET OF PUBERTY is the culmination of a subtle shift in balance of stimulatory and inhibitory neurotransmission within a complex neural network. Whereas the intrinsic pulsatility of GnRH neurons points toward a cell-autonomous function of gonadotropin-releasing hormone (GnRH) secretion, extrinsic signals are likely to be required to trigger activity in quiescent GnRH neurons. There is undoubtedly a link between metabolic status and GnRH activity (34). In the context of normal leptin signaling, a role has been implicated on the basis of a direct correlation between puberty onset and body weight (8, 16). Additionally, cases of defective leptin signaling in both humans and rodents result in hypogonadotropic hypogonadism (13). In a case report of a girl with leptin deficiency, treatment with leptin appeared to reverse the hypogonadal state (12). In cases of lipodystrophy with attendant hypoleptinemia, leptin therapy normalized pituitary hormone release (29) and restored menstrual cycles to all treat...

The leptin receptor was discovered as a leptin binding protein, which is highly expressed in the choroid plexus. Mapping of the gene's chromosomal locations in rodents revealed that mutations in Lepr were the basis for obesity/diabetes mutations in rodents and humans. Genetic manipulations that target Lepr expression in specific neurons or hypothalamic areas have generated insights to the modes by which body composition and reproductive function are modulated by leptin receptor. These animal models have also been instrumental in identifying diabetes susceptibility genes. In this review, we discuss the evidence that supports the concept of networked functions of leptin receptor as it pertains to feeding, substrate utilization and reproduction.

Prostaglandin E 2 (PGE 2 ) is produced at high levels in the injured central nervous system, where it is generally considered a cytotoxic mediator of inflammation. The cellular actions of PGE 2 are mediated by G-protein signaling activated by prostanoid receptors termed EP 1 , EP 2 , EP 3 and EP 4 . Recent studies have implicated the EP 2 prostanoid receptor in apparently conflicting roles promoting neuronal death in some model systems and the survival of neurons in others. Here we show that treatment of immortalized human microglia and CCF-STTG1 astrocytes with either PGE 2 or the EP 2 selective agonist butaprost stimulates the release of brain-derived neurotrophic factor (BDNF). Both cell lines express mRNA for the EP 2 receptor, whereas transcripts for the other subtypes are not detected. Pharmacological studies using PGE 2 and modulators of cyclic AMP signaling implicate this pathway in PGE 2 -stimulated BDNF release. These results indicate that EP 2 prostanoid receptor activation induces BDNF secretion through stimulation of cyclic AMP dependent signaling. Our findings provide a mechanism by which endogenous PGE 2 might contribute to either neurotoxicity or neuroprotection in the injured brain via the induction of BDNF release from microglial cells and astrocytes. KeywordsProstaglandin E 2 ; microglia; astrocytes; BDNF; G-protein coupled receptor; brain derived neurotrophic factor; cyclic AMP Prostaglandin E 2 (PGE 2 ) is a prominent lipid autocrine/paracrine signaling mediator produced by the sequential metabolism of arachidonic acid by cyclooxygenase (COX) and PGE 2 synthase. PGE 2 is a major agent in local intercellular signaling associated with inflammation, pain, fever and immune responses (reviewed by Matsuoka & Narumiya, 2007;Khanapure et al., 2007;Harris et al., 2002). The cellular actions of PGE 2 are mediated by the activation of a group of G-protein coupled receptors that includes subtypes termed the EP 1 , EP 2 , EP 3 and EP 4 prostanoid receptors. These receptor subtypes are distinguished from one another by their selectivity for different ligands and by their unique coupling to intracellular signaling #Corresponding author (E-mail: regan@pharmacy.arizona.edu). * These authors contributed equally to this work Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. (Funk et al., 1993) while stimulation of the EP 2 receptor promotes the production of intracellular cyclic AMP (Regan et al., 1994a). Activation of the EP 3 receptor, which is expressed as multiple isoforms through alternative mRNA splicing, inhibits cyclic AMP signaling (Regan et al., 1994b) ...

Summary Prostaglandin-E2 (PGE2) is a hormone derived from the metabolism of arachidonic acid whose functions include regulation of platelet aggregation, fever and smooth muscle contraction/relaxation. PGE2 mediates its physiological and pathophysiological effects through its binding to four G-protein coupled receptor subtypes, named EP1, EP2, EP3 and EP4. The EP3 prostanoid receptor is unique in that it has multiple isoforms generated by alternative mRNA splicing. These splice variants display differences in tissue expression, constitutive activity and regulation of signaling molecules. To date there are few reports identifying differential activities of EP3 receptor isoforms and their effects on gene regulation. We generated HEK cell lines expressing either the human EP3-Ia, EP3-II or EP3-III isoforms. Using immunoblot analysis we found that nM concentrations of PGE2 strongly stimulated the phosphorylation of ERK 1/2 by the EP3-II and EP3-III isoforms; whereas, ERK 1/2 phosphorylation by the EP3-Ia isoform was minimal and only occurred at μM concentrations of PGE2. Furthermore, the mechanisms of the PGE2 mediated phosphorylation of ERK 1/2 by the EP3-II and EP3-III isoforms were different. Thus, PGE2 stimulation of ERK 1/2 phosphorylation by the EP3-III isoform involves activation of a Gαi/PI3K/PKC/Src and EGFR-dependent pathway; while for the EP3-II isoform it involves activation of a Gαi/Src and EGFR-dependent pathway. These differences result in unique differences in the regulation of reporter plasmid activity for the downstream effectors ELK1 and AP-1 by the EP3-II and EP3-III prostanoid receptor isoforms.