Robert L. Goodman - US grants

DESCRIPTION (provided by applicant): The long-term goal of this research is to understand the structural and functional changes in the hypothalamus that are responsible for the reversible suppression of ovarian function, such as occurs prior to puberty. It is well recognized that increases in response to estradiol (E2) negative feedback play an important role in producing these periods of infertility (e.g., gonadostat theory of puberty). Using seasonal breeding as a model, we have identified a neural circuit (E2 responsive-neurons in the ventromedial POA [vmPOA] and the retrochiasmatic area stimulate A15 dopamine neurons to inhibit GnRH secretion) that is activated to induce the non-breeding (anestrous) season. We have recently developed strong evidence that glutamate and 3-amino butyric acid (GABA) are important controllers of A15 neural activity in anestrus. The experiments in Aim 1 will identify the GABA and glutamatergic neurons afferent to the A15 and test two specific hypotheses to account for seasonal alterations in E2 negative feedback: 1) there is a decrease in E2 receptors in GABAergic neurons during the breeding season, and 2) there is a decrease in input from E2-responsive glutamatergic afferents to the A15 during the breeding season. Studies in Aim 2 will test two alternate hypotheses to account for A15 inhibition of GnRH secretion in anestrus: 1) A15 neurons project directly to GnRH terminals in the median eminence, or 2) A15 neurons stimulate gonadotropin-inhibitory hormone (GnIH) neurons or inhibit a set of kisspeptin-containing neurons to suppress GnRH. Aim 3 is based on evidence that thyroid hormone (T4) actions in the premammillary region and vmPOA are required for the structural changes that lead to activation of this circuit at the transition to anestrus. Specifically, we will test the hypothesis that brain-derived neurotrophic factor mediates the action of T4 during the transition to anestrus and begin to identify the down- stream targets of T4 by determining if it is necessary for seasonal changes in the number of glutamatergic synapses on to A15 neurons. We will use a combination of molecular, anatomical, pharmacological, and physiological approaches to test specific hypotheses relevant to each aim. The results of these studies will provide fundamental information on how the brain changes to shut down, and restart, fertile ovarian cycles. This information may provide novel treatments for pathological conditions, such as precocious puberty or hypothalamic ammenorrhea, in which these changes occur inappropriately. This may also provide insight into the converse situation of insufficient activity of inhibitory neural systems that appears to contribute to polycystic ovarian syndrome.

The long-term goal of this project is to elucidate the mechanisms controlling the occurrence of ovulatory cycles in female mammals. The working hypothesis to be tested is that anovulation during seasonal anestrus in the ewe is due to activation of inhibitory neural systems. Two such systems are proposed: a steroid-sensitive catecholaminergic system and a steroid-insensitive serotonergic system. One major specific aim of this project is to determine the anatomical location of these inhibitory neurons. This will be done using immunohistochemical staining, neurotransmitter receptor localization by in vitro incubation with 3H-ligand and autoradiography, knife cuts of specific neural tracts, and microinjection of neurotransmitter antagonists into specific hypothalamic areas. Experiments in the third specific aim will also make use of the lesioning techniques to test the possibility that one of these inhibitory neural systems activates the other.

dopamine receptor; neuroendocrine system; estrus; hypothalamus; brain electrical activity; estradiol; estrogen receptors; neurons; gonadotropin releasing factor; photostimulus; histology; sheep;

[unreadable] DESCRIPTION (provided by applicant): We have identified a group of neurons in the arcuate nucleus of the sheep that co-express two neuropeptides: the endogenous opioid peptide, dynorphin A (DYN), and the tachykinin, neurokinin B (NKB). There is strong evidence that DYN mediates the negative feedback actions of progesterone or gonadotropin-releasing hormone (GnRH) and indirect evidence for a similar role for NKB. We have also recently demonstrated that many of these neurons also contain kisspeptin, an important newly discovered stimulator of GnRH. Specifically over 80% of the DYN-NKB neurons in the caudal arcuate also contain kisspeptin, but that this falls to less than 50% in the rostral arcuate. Although all three peptides are found in the same neurons that appear to mediate steroid negative feedback, they have different effects on GnRH neurons: DYN inhibits GnRH secretion, while kisspeptin and most likely NKB, stimulate it. Therefore, we postulate that the negative feedback actions of ovarian steroids have differential effects on expression and release of these three neuropeptides, stimulating DYN and inhibiting Kisspeptin and NKB. In this proposal, we will test this hypothesis at three different levels: 1) integrative release into the CSF, 2) regionally within the arcuate nucleus, and 3) at the single cell level in the caudal and rostral portions of the arcuate. First we use liquid chromoatograph and mass spectrometry to monitor all three peptides in CSF collected from luteal phase, ovariectomized and steroid-treated ovariectomized ewes. We will then collect hypothalamic tissue from these ewes and cut alternating thick (100 Cm) and thin (20 mm) sections with a cryostat. The region containing the arcuate nucleus will be microdissected from the thick sections and pooled into rostral, middle, and caudal regions. The right side pools will be used to measure mRNA levels of the appropriate prepropeptide by RT-PCR and the left side pools used to monitor protein concentrations be Western analysis. Finally, the thin frozen sections will be used for either single cell RT-PCR or dual in situ hybridization to determine if the differential actions of ovarian steroids occur in the same neurons. The results of this study will provide novel information on control of three transmitters that have been implicated in the negative feedback actions of steroids in both sheep and women. It is also clear that abnormalities in steroid negative feedback occur in a number of pathologies, including polycystic ovarian syndrome (PCOS) and anorexia nervosa. For example, the increased episodic LH secretion in PCOS is due, in part, to decreased response to progesterone negative feedback. Thus, these studies may provide information important for the development of better treatments of these pathological disruptions of reproductive function. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): GnRH secretion is critical for normal human reproduction and defects in GnRH/LH pulses and the preovulatory GnRH/LH surge contribute to many common reproductive pathologies, including amenorrhea, polycystic ovarian syndrome (PCOS), and infertility associated with anorexia nervosa. Despite the long-standing recognition of the importance and functions of the GnRH neuroendocrine system, a detailed knowledge of the pathways by which endogenous gonadal steroid hormones are conveyed to GnRH neurons and control their secretory activity during the ovarian cycle remains elusive. Understanding of the neural substrates responsible for GnRH secretion has been revolutionized in the last few years by identification of the key role of a neuronal subpopulation in the arcuate nucleus that co-expresses kisspeptin, neurokinin B (NKB) and dynorphin (termed KNDy neurons). Genetic mutations in two of the three KNDy peptides, kisspeptin and NKB, lead to human infertility, and there is strong evidence that the third peptide conveys the inhibitory influence of progesterone on GnRH neurons. A number of features of the KNDy neurons suggest that they may play a key role in episodic GnRH secretion, but this hypothesis and the precise role of each of the KNDy peptides remains to be tested. In the current proposal, we will use a combination of physiological, pharmacological, molecular and neuroanatomical approaches to address this and other questions. First, we will determine if the KNDy cell network is critical for episodic GnRH secretion, examining the effects of disrupting each of the KNDy peptides on GnRH pulse shape. Second, we will address the gap in knowledge concerning the normal physiological role of NKB by determining its role in steroid feedback control of GnRH/LH pulses and the GnRH/LH surge. Finally, we will address key unresolved questions about the anatomy of KNDy cells, including the presence of direct synaptic connections between KNDy and GnRH neurons, and the possibility of synaptic plasticity in these connections during the normal estrous cycle. These questions will be addressed using the female sheep, a model that possesses several unique advantages including the ability to directly monitor GnRH in hypophysial portal blood in awake, unanesthesized animals, and an estrous cycle whose neuroendocrine control closely resembles that of the human menstrual cycle. The proposed studies will provide new information on the mechanisms underlying GnRH secretion and the role of NKB that may lay the foundation for the development of better treatments for pathological disruptions of reproductive function, and the design of novel contraceptive techniques

? DESCRIPTION (provided by applicant): GnRH secretion is critical for normal human reproduction and defects in GnRH secretion contribute to many common reproductive pathologies, including amenorrhea, polycystic ovarian syndrome (PCOS), and infertility associated with anorexia nervosa. Despite the long-standing recognition of the importance and functions of the GnRH neuroendocrine system, a detailed knowledge of the pathways by which the feedback actions of endogenous gonadal steroid hormones are conveyed to GnRH neurons during the ovarian cycle remains elusive. Understanding of the neural substrates responsible for GnRH secretion has been revolutionized in the last few years by identification of two key neuropeptides that are essential for fertility in humans: kisspeptin and neurokinin B (NKB). Work in many laboratories over the last decade has provided a wealth of information on the role of kisspeptin, but it remains unclear whether NKB is also important for either the negative or positive feedback effects of estradiol and progesterone. There is also only rudimentary information on the pathways by which NKB acts to control GnRH secretion. In the current proposal, we will use a combination of physiological, pharmacological, molecular and neuroanatomical approaches to determine if NKB contributes to the control of GnRH secretion by ovarian steroids and to begin identification of the pathways involved. First, we will test the role of NKB in negative feedback by determining which ovarian steroid inhibits NKB expression and the effects of disrupting NKB synthesis or action on episodic LH secretion. Experiments in Aim 2 are based on recent data that NKB actions in the retrochiasmatic area (RCh) contribute to the estrogen-induced LH surge and are mediated by kisspeptin neurons in the arcuate nucleus; they will further test this hypothesis and identify the phenotype of these neurons and the location of the NKB neurons projecting to them. Finally, we will assess the possibility that sexually-dimorphic aspects of NKB input to, or action in, the RCh contribute to the inability of estrogen to induce an LH surge in rams. These questions will be addressed using male and female sheep, an animal model that possesses several unique advantages including the ability to administer reagents to local anatomical areas in the hypothalamus, and an ovarian cycle whose neuroendocrine control closely resembles that of the human menstrual cycle. The proposed studies will provide new information on the physiological roles of NKB in control of LH secretion that may lay the foundation for the development of better treatments for pathological disruptions of reproductive function, and the design of novel contraceptive techniques.