Agouti-related peptide plays a critical role in leptin's effects on female puberty and reproduction.

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(-/-) 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.

Peripubertal vitamin D(3) deficiency delays puberty and disrupts the estrous cycle in adult female mice.

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.

Effects of leptin and melanocortin signaling interactions on pubertal development and reproduction.

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.

Morphogenesis of the developing mammary gland: stage-dependent impact of adipocytes.

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 and 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.

Leptin receptor modulation of adiposity and fertility.

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 of obesity/diabetes mutations in rodents and humans. Genetic manipulations that target Lepr expression in specific neurons or hypothalamic areas have generated insights into the modes by which body composition and reproductive function are modulated by the 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.

EP(3) prostanoid receptor isoforms utilize distinct mechanisms to regulate ERK 1/2 activation.

Prostaglandin-E(2) (PGE(2)) is a hormone derived from the metabolism of arachidonic acid whose functions include regulation of platelet aggregation, fever and smooth muscle contraction/relaxation. PGE(2) mediates its physiological and pathophysiological effects through its binding to four G-protein coupled receptor subtypes, named EP(1), EP(2), EP(3) and EP(4). The EP(3) 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 EP(3) receptor isoforms and their effects on gene regulation. We generated HEK cell lines expressing the human EP(3-Ia), EP(3-II) or EP(3-III) isoforms. Using immunoblot analysis we found that nM concentrations of PGE(2) strongly stimulated the phosphorylation of ERK 1/2 by the EP(3-II) and EP(3-III) isoforms; whereas, ERK 1/2 phosphorylation by the EP(3-Ia) isoform was minimal and only occurred at muM concentrations of PGE(2). Furthermore, the mechanisms of the PGE(2) mediated phosphorylation of ERK 1/2 by the EP(3-II) and EP(3-III) isoforms were different. Thus, PGE(2) stimulation of ERK 1/2 phosphorylation by the EP(3-III) isoform involves activation of a Galpha(i)/PI3K/PKC/Src and EGFR-dependent pathway; while for the EP(3-II) isoform it involves activation of a Galpha(i)/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 EP(3-II) and EP(3-III) prostanoid receptor isoforms.

Activation of EP2 prostanoid receptors in human glial cell lines stimulates the secretion of BDNF.

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 to be 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.

PGF(2alpha) stimulates FP prostanoid receptor mediated crosstalk between Ras/Raf signaling and Tcf transcriptional activation.

Prostaglandin-F(2alpha) (PGF(2alpha)) is a product of the cyclooxygenase pathway and is a local signaling molecule that activates a G-protein coupled prostanoid receptor named FP. FP receptors can stimulate T-cell factor (Tcf) transcriptional activation by stabilization of beta-catenin and can upregulate the expression of mRNA encoding cysteine-rich protein 61 (Cyr61), a secreted extracellular matrix protein that stimulates angiogenesis. We now show in both HEK cells and human microglial cells that the induction of Cyr61 protein expression by the human FP receptor utilizes a novel mechanism involving the activation of Ras and Raf followed by a MEK/ERK independent activation of Tcf signaling. The upregulation of Cyr61 in microglial cells may contribute to glioma tumorigenesis and could be a potential therapeutic target.

Human EP3(I) prostanoid receptor induces VEGF and VEGF receptor-1 mRNA expression.

A critical event in tumor development is the formation of new blood vessels to provide oxygen, nutrients and growth factors to the rapidly growing cancer cells. This process of angiogenesis is complex, however, it is well established that vascular endothelial growth factor (VEGF)-mediated signaling is an important early event. Knockout mice studies have implicated the EP3 receptor in tumor development and angiogenesis; however, the signaling mechanism involved with this effect is unclear. We now show that stimulation of the EP3(I) isoform of the human EP3 receptor with prostaglandin E(2) increases the mRNA expression of both VEGF and its cognate receptor VEGF receptor-1 (VEGFR-1). These inductions by the EP3(I) receptor involve the sequential activation of phosphatidylinositol 3-kinase and the extracellular signal-regulated kinases. Up-regulation of VEGF and VEGFR-1 mRNA by the human EP3(I) receptor has not been previously reported and further strengthen the role of this receptor in tumor-associated angiogenesis.