Anne M. Etgen - US grants

The objective of the proposed research is to investigate the relationship between steroid hormone action at the cellular and molecular levels within the central nervous system and the hormonal regulation of behavior. More specifically, the experiments to be described are concerned with the role of neural receptors for one class of steroid hormones, the progestins, in the mediation of female sexual behavior in rodents. After a sufficient period (24-48 hr) of estrogen priming, an injection of progesterone synergizes with estrogen to induce high levels of sexual receptivity in female rodents. This same period of estrogen priming also induces the synthesis of progestin receptors in the hypothalamus of rats and guinea pigs, a brain region known to be intimately involved in the activation of female sexual behavior. The research outlined in this proposal will examine the interactions of progestins with the neural receptor system to assess whether (a) estrogen induction of progestin receptors, (b) progestin-specific binding to cytosol receptors, (c) depletion of cytosol receptors and translocation to the nucleus, (d) binding of the nuclear progestin-receptor complex to the chromatin material, and (e) progestin induction of specific protein synthesis are positively correlated with the capacity of various progestins to facilitate estrous behavior in estrogen-primed rodents of several species. The experiments will rigorously test the relevance of steroid-receptor interactions to progestin activation of a specific brain response and will thus provide information regarding potential molecular mechanisms of steroid regulation of neural functions.

DESCRIPTION (provided by applicant): The ovarian hormones estradiol (E2) and progesterone (P) act in the hypothalamus (HYP) and preoptic area (POA) to ensure that the preovulatory luteinizing hormone (LH) surge coincides with reproductive behavior (lordosis), thereby maximizing reproductive success. We propose that E2 and P-dependent changes in norepinephrine (NE) synaptic activity in the HYP and POA are key mediators of the neuroendocrine integration of reproduction. We propose further that insulin-like growth factor-I (IGF-1) and its receptor (IGF-1R) mediate certain reproductive actions of E2 in the HYP-POA and engage in cross talk with NE receptors in these brain areas. The proposed research will elucidate the molecular mechanism(s) by which E2 and P interact with IGF-1 R and a1-adrenergic receptor signaling pathways in the HYP and POA and will relate these to lordosis behavior. Specific Aim 1 tests the hypothesis that IGF-1 Rs mediate E2 actions in the HYP-POA that are involved in female reproductive function via activation of phosphoinositide 3-kinase (PI3-K) or mitogen-activated protein kinase (MAPK). We will assess (a) E2 facilitation of lordosis behavior; (b) E2-induced increases in IGF-1 R density in the HYPPOA; (c) E2+P-induced coupling of a1-adrenoceptors to cGMP synthesis in the HYP-POA; and (d) E2-induced increases in spine density on ventromedial hypothalamic neurons. Specific Aim 2 tests the hypothesis that E2+P-dependent linkage of a1-adrenoceptors to cGMP also results in activation of MAPK signaling in the HYP and POA. Specific Aim 3 tests the hypothesis that in the HYP-POA of E2-treated rats, P switches a1-adrenoceptor signaling to synthesis of cGMP, which mediates NE facilitation of lordosis, by elevating intracellular calcium. We will determine whether a1-adrenoceptors stimulate cGMP formation by activating: (a) extracellular calcium influx via N and/or L-type calcium channels; (b) intracellular calcium mobilization from the endoplasmic reticulum; (c) release of ryanodine-sensitive calcium stores; and/or (d) growth factor-regulated kinases such as src or P13-K. Specific Aim 4 will determine how IGF-1 acutely potentiates a1-adrenoceptor signaling in the HYP-POA of E2-treated females and whether this is important for reproductive behavior.

The long-term objective of this research is to understand how brain[unreadable] cells produce specific behavioral responses. Because lordosis, a[unreadable] component of female reproductive behavior, is strictly dependent on the[unreadable] ovarian steroid hormones estradiol (E2) and progesterone (P),[unreadable] examination of mechanisms that control lordosis allow us to ask how[unreadable] hormonal signals are recognized by brain cells and translated into[unreadable] specific changes in behavior. The research proposed in this application[unreadable] combines behavioral, biochemical, and pharmacological approaches to[unreadable] address specific questions raised by our prior work regarding the neural[unreadable] mechanisms of E2 regulation of lordosis behavior in female rats.[unreadable] [unreadable] Specific Aim 1 uses in vivo microdialysis to test the hypotheses: (1)[unreadable] that somatosensory (flank/perineal and/or vaginocervical) rather than[unreadable] olfactory stimuli associated with copulation are responsible for[unreadable] norepinephrine (NE) release from the hypothalamus of hormone-treated,[unreadable] sexually receptive female rats, and (2) that steroid priming potentiates[unreadable] somatosensory stimulus-evoked NE release. Specific Aim 2 uses[unreadable] biochemical pharmacological approaches to test the hypothesis that E2[unreadable] modifies opioid receptor signaling in the hypothalamus such that[unreadable] pathways inhibitory to lordosis (e.g., mu receptor-linked) are[unreadable] attenuated while pathways that facilitate lordosis (e.g., kappa and[unreadable] delta receptor-linked) are enhanced. Specific Aim 3 tests the[unreadable] hypothesis that the second messenger cGMP and its cellular effectors are[unreadable] hormone-regulated mediators of the facilitatory actions of E2 and P on[unreadable] lordosis behavior in female rats. Neuropharmacological studies will[unreadable] examine whether inhibition of cGMP-dependent protein kinase (PKG)[unreadable] reduces lordosis, and microdialysis studies will evaluate whether there[unreadable] is efflux of cGMP in the hypothalamus during mating tests in hormone-[unreadable] primed female rats. Immunological, biochemical and molecular biological[unreadable] methods will determine whether hormones regulate the expression of[unreadable] guanylyl cyclase, of PKGs, and/or of a specific PKG substrate in brain[unreadable] areas that regulate reproduction. These studies will provide novel[unreadable] insights into the cellular and molecular mechanisms by which ovarian[unreadable] sterioids act in the brain and to produce predictable behavioral[unreadable] changes.

DESCRIPTION (provided by applicant): Alterations in the hypothalamic-pituitary-ovarian axis in perimenopausal women are associated with multi- organ risk factors for disease, yet the biological mechanisms underlying this increased disease risk are largely unknown. This proposal addresses unanswered questions regarding the vulnerability of the middle- aged brain to global ischemia. In young female rats, the presence of physiological levels of estradiol before and after global ischemia, as might occur during cardiac arrest, reduces hippocampal CA1 neuron loss and associated cognitive impairments. Whether estradiol retains its neuroprotective actions in middle-aged females, and whether the age-related decline in insulin-like growth factor-l (IGF-I) increases vulnerability to ischemia-induced neurodegeneration and cognitive impairment, are unknown. This proposal aims examines the roles of age, estrogen and IGF-I in the survival and function of hippocampal neurons in a rat model of global ischemia. The underlying hypotheses are (1) that the middle-aged brain retains its responsiveness to the neuroprotective actions of estradiol if the duration of estrogen withdrawal is brief ("critical period hypothesis") or circulating levels of IGF-I are maintained, and (2) that estrogen acts in the middle-aged brain to activate specific cell survival pathways and thereby intervenes in apoptotic cascades to prevent death of neurons otherwise "destined to die". Specific Aim 1 uses stereological cell counting and behavioral tests to evaluate the outcome of global ischemia in middle-aged female rats that are intact, ovariectomized at various intervals prior to insult, or ovariectomized and treated with estradiol at various intervals after ovariectomy. If estradiol does not preserve neurons and cognitive function in older hormone-deprived animals, we, will also determine if IGF-I can reinstate estrogen protection. Specific Aim 2 examines the apoptotic death cascades triggered by global ischemia and identifies the site at which estrogen intervenes in these cascades. We will examine 1) mitogen-activated protein kinase and cAMP response element binding protein at early times after ischemia;2) the anti-apoptotic gene Bcl-2 and activation of caspase 3 at later times after ischemia;3) inactivation of Akt and subsequent activation of the forkhead transcription factor FKHRL1 at early times after ischemia. These experiments will provide new information on the potential for hormone therapy instituted during the perimenopausal transition to protect the brain from damage due to global ischemia.

This project tests the hypothesis that insulin-like growth factor-1 (IGF-1) and its receptor are essential comediators of critical reproductive actions of estradiol (E2) in the neuroendocrine hypothalamus (HYP). We have shown that chronic intracerebroventricular infusion of an IGF-1 receptor antagonist suppresses estrous cycles, an effect that is not attributable to impaired food intake or reduced body weight. Therefore, the proposed experiments test the hypothesis that IGF-1 receptor signaling in the HYP is essential for neuroendocrine regulation of female reproductive function by E2. We will identify the mechanisms and neural sites of IGF-1 regulation of the hypothalamic-pituitary-gonadal axis by determining whether IGF-1 acts directly on the gonadotropin releasing hormone (GnRH) neurons, their afferent inputs, and/or their responsiveness to E2. Finally, we will test the hypothesis that the delayed and attenuated luteinizing hormone (LH) surge that characterizes female rats making the transition to reproductive senescence is causally related to reduced IGF-1 receptor signaling in the aging brain. Specific Aim 1 tests the hypothesis that IGF-1 regulation of the E2-dependent LH surge is mediated at the level of the HYP rather than the pituitary. Specific Aim 2 tests the hypothesis that IGF-1 regulates E2-dependent afferent signals to GnRH neurons. We will determine the effects of brain IGF-1 receptor blockade on GnRH neuronal activation under hormonal conditions that should generate LH surges. We will also determine the effects of brain IGF-1 receptor blockade on E2 regulation of progestin receptors and kisspeptin gene expression in the HYP and on the release of excitatory (glutamate) and inhibitory (GABA) neurotransmitters in medial preoptic area (HYP site of GnRH cell bodies). Specific Aim 3 tests the hypothesis that IGF-1 regulates GnRH neuronal responsiveness to E2 afferent input. We will determine the effects of brain IGF-1 receptor blockade on glutamate, kisspeptin and a1-adrenergic activation of LH release in hormone-primed females. Specific Aim 4 tests the hypothesis that declining levels of bioavailable IGF-1 are causally related to the delayed and attenuated LH surges in middle-aged females undergoing the transition to reproductive senescence. We will determine whether elevating brain IGF-1 restores hormone-dependent LH surges in middle-aged rats, and if so, whether this manipulation also restores E2 regulation of amino acid neurotransmission and of kisspeptin gene expression. These findings may provide insight into the mechanisms underlying premature ovarian failure and reproductive neuroendocrine dysfunction that accompanies diabetes and polycystic ovarian syndrome (PCOS). This could suggest new therapeutic strategies for treating reproductive disorders associated with altered IGF-1 levels, such as PCOS. They could also identify factors whose manipulation might prolong exposure of middle-aged women to the physiological benefits of ovarian steroids