Cell-specific expression of the human gonadotropin-releasing hormone gene in transgenic animals.

We have previously demonstrated that 1131 base pairs (bp) of the human gonadotropin-releasing hormone (hGnRH) gene promoter can target simian virus 40 T antigen expression to GnRH neurons in transgenic mice. In these animals, GnRH neurons were transformed before they migrated to their final location in the rostral hypothalamus, complicating an analysis of cell-specific expression. To localize regions of the hGnRH promoter that are important for cell-specific expression, we created transgenic mice with various 5'-flanking regions of the hGnRH gene fused to the luciferase reporter gene. When 3828 or 1131 bp of the hGnRH promoter 5'-flanking DNA were used (-3828/+5LUC and -1131/+5LUC, respectively), luciferase expression in adult transgenic mice was observed in the rostral hypothalamus and olfactory tissues, regions which have been shown to be loci of GnRH-expressing neurons. Luciferase expression was not observed in other brain or peripheral tissues. Double-labeled in situ hybridization further demonstrated that luciferase expression was invariably colocalized with GnRH expression. When transgenic animals were created with a construct consisting of 484 bp of the hGnRH 5'-flanking DNA fused to the luciferase gene (-484/+5LUC), luciferase expression was not observed in the hypothalamus or in olfactory tissues. This is the first report localizing DNA sequences responsible for cell-specific expression of the GnRH gene in vivo.

Amplitude and frequency modulation of pulsatile luteinizing hormone-releasing hormone release.

1. A variety of neuroendocrine approaches has been used to characterize cellular mechanisms governing luteinizing hormone-releasing hormone (LHRH) pulse generation. We review recent in vivo microdialysis, in vitro superfusion, and in situ hybridization experiments in which we tested the hypothesis that the amplitude and frequency of LHRH pulses are subject to independent regulation via distinct and identifiable cellular pathways. 2. Augmentation of LHRH pulse amplitude is proposed as a central feature of preovulatory LHRH surges. Three mechanisms are described which may contribute to this increase in LHRH pulse amplitude: (a) increased LHRH gene expression, (b) augmentation of facilitatory neurotransmission, and (c) increased responsiveness of LHRH neurons to afferent synaptic signals. Neuropeptide Y (NPY) is examined as a prototypical afferent transmitter regulating the generation of LHRH surges through the latter two mechanisms. 3. Retardation of LHRH pulse generator frequency is postulated to mediate negative feedback actions of gonadal hormones. Evidence supporting this hypothesis is reviewed, including results of in vivo monitoring experiments in which LHRH pulse frequency, but not amplitude, is shown to be increased following castration. A role for noradrenergic neurons as intervening targets of gonadal hormone negative feedback actions is discussed. 4. Future directions for study of the LHRH pulse generator are suggested.

Neuropeptide Y stimulates luteinizing hormone-releasing hormone release from superfused hypothalamic GT1-7 cells.

The effects of neuropeptide Y (NPY) on LHRH release from an immortalized cell line were investigated using a flow-through cell culture superfusion system. Immortalized hypothalamic GT1-7 cells were cultured for 72 h and superfused for a total of 180 min. In initial experiments, discrete 5-min pulses of NPY (10(-12)-10(-5) M) were administered to the cells. A clear dose-dependent stimulatory effect on NPY on LHRH release from the cells was observed with a calculated 50% effectiveness concentration of 33 nM. The stimulatory effects of brief NPY exposure were rapid and robust, e.g. reaching and maintaining levels of 173% over baseline for 20 min at the 10(-7) dose. The lowest dose of NPY that showed a significant effect was 10(-10) M; maximal responses were observed at 10(-6) M and reached a plateau thereafter. Control pulses of Dulbecco's modified Eagle's medium (DMEM) and 10(-6) M substance P or arg-vasopressin were also presented to the cells to serve as controls for our pulse protocol, and these challenges produced no significant LHRH responses. The NPY receptor antagonists, PYX1 and PYX2, at 10(-8) M, completely blocked the observed NPY responses in these cells. To assess the NPY receptor subtypes that mediate the NPY effects pharmacologically, GT1-7 cells were challenged with a Y1 receptor agonist, (Leu31Pro34)NPY, a Y2 receptor agonist, NPY(13-36), or peptide YY, at doses 10(-12)-10(-5) M. All four peptides stimulated LHRH release from GT1-7 cells with a rank-ordered potency of NPY = peptide YY > Y1 agonist = Y2 agonist. To examine possible signal transduction mechanism(s) involved in mediating this effect, pertussis toxin, RpcAMPs (cyclic adenosine-3'5'-monophosphothioate Rp diastereomer), Ca(2+)-free DMEM and TMB-8 (3, 4, 5-trimethoxybenzoic acid 8-(diethylamino) octylester) were used to treat the cells before and during superfusion with NPY. Treatment with pertussis toxin, RpcAMPs, and Ca(2+)-free DMEM did not significantly alter NPY-stimulated LHRH release responses to 10(-7) M NPY. However, the addition of 100 microM and 250 microM TMB-8 to Ca(2+)-free DMEM almost completely blocked this NPY effect, as did 10 microM ryanodine. Finally, the locus of action for this NPY effect was examined using tetrodotoxin to reduce action potential propagation in the GT1-7 cells. Tetrodotoxin treatment blocked the LHRH response to NPY by more than 50%.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuroendocrine regulation of the luteinizing hormone-releasing hormone pulse generator in the rat.

We have analyzed the mechanisms by which several known regulators of the LHRH release process may exert their effects. For each, we have attempted to determine how and where the regulatory input is manifest and, according to our working premise, we have attempted to identify factors which specifically regulate the LHRH pulse generator. Of the five regulatory factors examined, we have identified two inputs whose primary locus of action is on the pulse-generating mechanism--one endocrine (gonadal negative feedback), and one synaptic (alpha 1-adrenergic inputs) (see Fig. 29). Other factors which regulate LHRH and LH release appear to do so in different ways. The endogenous opioid peptides, for example, primarily regulate LHRH pulse amplitude (Karahalios and Levine, 1988), a finding that is consistent with the idea that these peptides exert direct postsynaptic or presynaptic inhibition (Drouva et al., 1981). Gonadal steroids exert positive feedback actions which also result in an increase in the amplitude of LHRH release, and this action may be exerted through a combination of cellular mechanisms which culminate in the production of a unique, punctuated set of synaptic signals. Gonadal hormones and neurohormones such as NPY also exert complementary actions at the level of the pituitary gland, by modifying the responsiveness of the pituitary to the stimulatory actions of LHRH. The LHRH neurosecretory system thus appears to be regulated at many levels, and by a variety of neural and endocrine factors. We have found examples of (1) neural regulation of the pulse generator, (2) hormonal regulation of the pulse generator, (3) hormonal regulation of a neural circuit which produces a unique, punctuated synaptic signal, (4) hormonal regulation of pituitary responsiveness to LHRH, and (5) neuropeptidergic regulation of pituitary responsiveness to LHRH. While an attempt has been made to place some of these regulatory inputs into a physiological context, it is certainly recognized that the physiological significance of these mechanisms remains to be clarified. We also stress that these represent only a small subset of the neural and endocrine factors which regulate the secretion or actions of LHRH. A more comprehensive list would also include CRF, GABA, serotonin, and a variety of other important regulators. Through a combination of design and chance, however, we have been able to identify at least one major example of each type of regulatory mechanism.