Infertility often results from disruption of menstrual cyclicity and/or ovulation: processes known to be driven by specific hypothalamic signals. By monitoring neurosecretions in either naturally ovulating or ovariectomized (OVX) rhesus macaques, we observed a 10-fold increase in gonadotropin-releasing hormone (GnRH) secretion during an estradiol- 17beta (E2)-induced luteinizing hormone (LH) surge. No less than 14 neurotransmitter/neuropeptide(s) are implicated in the regulation of GnRH secretion. Because adrenergic receptors are found on GnRH neurons, we initially focus on norepinephrine (NE) and neuropeptide Y (NPY), which are colocalized in noradrenergic cells and stimulate GnRH gene expression and secretion in E2-conditioned animals, including macaques.
Aim 1 a will resolve if the preovulatory rise in ovarian E2 stimulates NE and NPY secretion in conjunction with the E2-induced GnRH/LH surge.
Aim 1 b will decipher if blockade of either NE or NPY receptor activity by selective antagonists blocks the GnRH/LH surge. Continuous push-pull perfusion or microdialysis will be performed in the mediobasal hypothalamus (MBH) of both intact and OVX monkeys with or without treatment of prazosin (NE antagonist) and (Ac[3-(2,6 dichlorobenzyl)tyr(27,36)D-Thr(32)]NPY-(27- 36)amide (NPY antagonist). Changes in perfusate levels of NE, NPY and GnRH will be analyzed by high-performance liquid chromatography and radioimmunoassays, respectively.
Aim 2 addresses the morphometric and intracellular evidence of how E2 influences NPY/NE/GnRH secretion.
Aim 2 a will determine if estrogen receptors are localized in NE and/or NPY neurons, if NPY is produced in NE neurons, and if NE/NPY terminals synapse with hypothalamic GnRH neurons. Histological sections of the MBH and brainstem (i.e., locus coeruleus/lateral tegmentum) will be examined for the localization of neurons that express estrogen receptor mRNAs (by in situ hybridization) and NE (i.e., dopamine-beta-hydroxylase), NPY and GnRH (by immunocytochemistry).
Aim 2 b will quantitate E2-induced changes in NPY and NE (i.e., tyrosine hydroxylase) mRNA contents in identified loci of brainstem and hypothalamus by ribonuclease protection assay.
Aims 3 and 4 examine how synaptic NE secretion interacts with other putative neuromodulators to regulate GnRH release.
Aim 3 a will determine if blockade of NE transporter activity (presynaptic NE reuptake) by the antidepressant, desipramine, acutely stimulates NE, hence GnRH, release.
Aim 3 b will show that continuous desipramine infusion suppresses NE/GnRH secretion, and establish if GnRH pulses can be reinitiated by pulses of NE or neuroexcitatory amino acids (i.e., N-methyl-D,L-aspartate). Microdialysis cannulae implanted in the MBH of OVX monkeys will be used for acute (3a) or continuous (3b) desipramine treatment, pulsatile NE or N-methyl-D,L-aspartate infusion (3b) and microdialysate collection for NE and GnRH measurements (3a,b).
Aim 4 a will examine by in situ hybridization whether chronic intrahypothalamic desipramine can suppress retrogradely NE transporter, tyrosine hydroxylase, or NPY mRNAs in brainstem NE cells.
Aim 4 b will search for evidence of E2 downregulation of NE transporter message in NE neurons. These studies will indicate the importance of specific neurochemical on GnRH secretion thereby increasing the knowledge of biological and pathological events in fertile and neurally depressed, infertile female primates.
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