Sergio R. Ojeda - US grants

The overall goal of this application is to elucidate the neuroendocrine mechanisms involved in the developmental regulation of ovarian function. The concept is proposed that the developing ovary is subjected to a "neuroendocrinotrophic" control effected by the interactive participation of the extrinsic innervation, an intragonadal source of neurotransmitters, and a family of neurotrophic genes previously believed to target only the nervous system for their biological actions. This concept stems from a series of observations demonstrating: a) the contribution of the extrinsic innervation to the regulation of ovarian steroidogenesis and follicular development, b) the involvement of nerve growth factor (NGF) in regulating ovarian development indirectly via its neurotrophic effects on the ovarian innervation, c) the ability of neurotransmitters contained in ovarian nerves to initiate the molecular differentiation of early granulosa cells at the onset of follicular growth, and d) the existence in the primate ovary of an intrinsic source of catecholamine biosynthesis. Further studies suggested that an enhanced activity of the ovarian sympathetic innervation may contribute to the etiology of polycystic ovarian syndrome, the most common ovarian pathology affecting women of reproductive age. While studying the role of NGF in supporting the ovarian innervation, it became apparent that the developing ovary not only synthesizes several members of the NGF family of neurotrophins, but unexpectedly, also expresses the tyrosine kinase receptors that mediate the biological actions of neurotrophins in the nervous system. The presence of such receptors in endocrine cells of the ovary implies that neurotrophins can directly affect ovarian function without the intermediacy of the innervation, and raises the intriguing possibility of an hitherto unsuspected role for neurotrophins in the control of ovarian development. The present application proposes a combination of physiological, cellular and molecular approaches to address this issue, and to define the contribution of each of the proposed "neuroendocrinotrophic" components to the regulation of specific developmental events in ovarian maturation. To this end, the following specific aims are proposed: 1. To examine the hypothesis that two neurotrophins, NGF and neurotrophin-4, play different, but complementary roles in the cytodifferentiation/ organizational process underlying the period of definitive ovarian histogenesis, at the time of follicular formation. 2. To examine the hypothesis that neurotransmitters and neurotrophins interact positively in promoting the molecular differentiation of granulosa cells during early follicular development. 3. To examine the hypothesis that activation of trkA receptors during the hours preceding the first ovulation contributes to the cytodifferentiation process leading to follicular rupture. 4. To examine the hypothesis that activation of ovarian NGF synthesis and that of its low-affinity receptor is a key component in the etiology of polycystic ovarian syndrome. 5. To characterize the molecular forms and cellular sources of tyrosine hydroxylase and dopamine-beta hydroxylase, the two key enzymes in catecholamine synthesis recently found to be expressed in the developing primate ovary.

The proposal is aimed at gaining new insight into the developmental processes that underlie the maturation of the reproductive hypothalamus and, hence, the onset of female puberty. Utilizing the rat as the animal model, experiments are proposed to examine the hypothesis that brain neuronotrophic factors regulate the development of the hypothalamic LHRH- secreting system, which culminates when a discharge of LHRH release triggers the first preovulatory surge of gonadotropins. To this end, the following specific aims are proposed. 1. To document the hypothesis that nerve growth factor (NGF) and transforming growth factor alpha (TGFalpha), as well as their respective receptors, are expressed in the hypothalamus, and to examine their contribution to the developmental regulation of LHRH secretion. 2. To examine the hypothesis that TGFalpha facilitates the morphological and functional differentiation of LHRH neurons and to determine whether NGF is trophic for LHRH neurons and/or a neuronal system regulatory to LHRH function. 3. To examine the hypothesis that hypothalamic trophic factors, and in particular the epidermal growth factor receptor EGFr/TGFalpha ligand system can influence LHRH synthesis by directly regulating transcriptional activity of the LHRH gene. 4. To examine the hypothesis that TGFalpha is one of the neuronotrophic factors that contribute to the peripubertal, gonadal-independent activation of LHRH release. The accomplishment of these aims depends on the team participation of one neuroendocrinologist, one molecular biologist, and two morphologists, and on the integrated use of neuroendocrine, neurobiology and molecular biology approaches.

We have previously described the presence of catecholaminergic neurons expressing low affinity nerve growth factor receptors (NGFR) in the rhesus monkey ovary. We are now assessing the postnatal ontogeny of these NGFR-positive cells, as well as their catecholaminergic capabilities in ovaries from neonatal to senescent animals. Immunocytochemistry (ICC) for NGFR revealed that the greatest number of these neurons were present in neonatal ovaries, with the numbers gradually declining throughout postnatal life, so that these were only occasional neurons by 20 years of age. ICC for tyrosine hydroxylase (TH), revealed that the capability of the neurons to produce catecholamines was lowest in neonatal (2 mo) ovaries, then increased dramatically during juvenile (8 mo) and again during peripubertal (3.5 yr) development. Our most recent data, although preliminary, suggests that the number of TH-positive neurons decline gradually until approximately 12 years of age. Subsequently, the number of these neurons declined markedly by 17 years of age, becoming virtually absent in monkeys reaching 20-27 years of age. We have also initiated experiments to determine the origin and prenatal ontogeny of ovarian neurons. We have positively identified NGFR-neurons in monkey ovaries from fetuses as early as 100 days. Unfortunately, we have had difficulty in obtaining younger fetuses. We have, however, recently discovered the presence of NGFR/TH-positive neurons in adult pig ovaries, and are now collecting pig fetuses at various ages in order to continue the prenatal ontogeny study. To date, we have obtained evidence that the neurons (some of which are TH-positive) are migrating from the developing neural tube and are already within the genital ridge and the undifferentiated gonad by fetal day 23. Earlier ages are currently being collected.

We recently identified neuron-like cells as a novel source of catecholamines in the rhesus monkey ovary, and observed that these cells express the gene encoding tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. Thus, neuron-like cells and the extrinsic innervation appear to be two sources of norepinephrine and dopamine (DA) in the ovary. Although high levels of DA have been found in follicular fluid of the primate ovary, the intraovarian functions of DA are not known. A direct action of DA on ovarian cells requires specific DA receptors. The present study is addressing this issue. Using reverse transcription-PCR amplification and specific oligonucleotide primers, we isolated a 275 bp cDNA that corresponded to the monkey DA1 receptor (D1-R) mRNA. Semiquantitative RT-PCR studies followed by Southern blotting showed that the D1-R mRNA levels were lower in prepubertal than in the adult ovaries. Immunohistochemistry with a D1-R antiserum revealed that D1-R immunoreactivity was predominantly present in cells of the corpus luteum (CL). RT-PCR experiments supported this finding by showing a greater abundance of D1-R mRNA in luteal cells than in freshly isolated GC from preovulatory follicles and in luteinizing GC (derived form monkeys undergoing IVF). D1-R mRNA levels were more abundant during the second half of the luteal phase. Incubation of either isolated midphase luteal cells, or luteinized, cultured GC, with DA in the absence or presence of hCG failed to consistently affect P release. Thus, activation of ovarian D1-R does not appear to be coupled to P secretion. Taken together, our results suggest the existence of a novel regulatory complex within the ovary involving DA and DA type 1 receptors, which are present primarily in the CL. The luteal functions affected by activation of these receptors remain to be identified.

Ovarian endocrine cells form functional syncytia, due to extensive intercellular gap junctional (GJ) communication. Identification of mechanisms underlying intragonadal GJ communication may be a key to the understanding of ovarian physiology. The relative paucity of information concerning the protein makeup of GJs in the primate ovary prompted us to identify the Cx type(s) expressed in the rhesus monkey and human ovary and to define the cellular sites of their synthesis. In immunohistochemical studies we used specific antisera against Cx43, Cx32, and Cx26. There was strong staining for Cx43 in thecal and interstitial cells, GCs and luteal cells of the monkey and human ovary. No Cx26 staining was detected. While Cx32 immunoreactivity was present in thecal cells, it was noted only in a subpopulation of GCs in both human and monkey ovaries. Western blots revealed that while Cx43 protein was abundant in luteinized human GCs obtained from IVF patients, Cx26 was absent, and Cx32 was present in small amounts. Immunocytochemical staining of human cultured GCs showed strong Cx43 immunoreactivity at the contact zones of virtually every cell, but Cx32 immunoreactivity was seen in only some of the cells. RT-PCR of monkey ovarian mRNA and human luteinized GC mRNA demonstrated that the Cx32 gene is indeed expressed in the monkey ovary and human GCs. Using the monkey Cx32 PCR fragment as a probe for in situ hybridization experiments, Cx32 mRNA was detected in thecal cells and in a subpopulation of GCs. The presence of two Cx subtypes, Cx43 and Cx32 in granulosa and in thecal cells, suggests that follicular cells of the primate ovary utilize GJs with different Cx compositions. Identification of the factors regulating the presumed homo- and, perhaps, heterotypic GJ channels, will lead to a better understanding of cell-cell interactions affecting follicular growth, ovulation and atresia.

In recent years, alcohol (ALC) use and abuse by adolescents has been rising at an alarming rate. Whether ALC consumption during prepubertal years affects specific hormones and the process of sexual maturation is not known. This study used immature female rhesus macaques to assess the effects of ALC consumption on the circulating levels of critical hormones known to be involved in the pubertal process. Ten monkeys averaging 20.28 (?0.3) months of age were bled by saphenous vein puncture at 8:30 a.m. and 8:30 p.m. each day for five consecutive days to determine the baseline, initial levels of growth hormone(GH), insulin-like growth factor-1 (IGF-1), follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), and leptin. For the next 12 months, each day at 1:40 p.m., five monkeys were administered ALC (2g/kg) and five monkeys were administered an isocaloric sucrose solution via a nasogastric approach. Blood samples were collected again at 24, 28 and 32 mo nths for subsequent hormone analysis. Food consumption and weight gain were similar for ALC-treated and control animals. The night-related increase in serum GH levels that occurs during late juvenile development (28-32 months of age) in control animals was suppressed in the ALC-treated animals. This action was paralleled by a depression in the serum levels of IGF-1, a peptide recently shown to facilitate the onset of puberty in both rodents and non-human primates. Importantly, serum levels of LH and E2 were so depressed during development by the ALC, with the lowest levels at 32 months. Serum FSH and leptin levels were not altered by ALC. These results demonstrate the detrimental effects of ALC on the activation of hormone secretion that accompanies the initiation of puberty in female rhesus monkeys. Also, they suggest the subsequent growth spurt and normal timing or progression of puberty may be at risk in human adolescents and teenagers consuming even relatively moderate amoun ts of ALC on a regular basis. FUNDING Subcontract with Texas A&M University PUBLICATIONS None

Growth of the mammalian ovary is regulated by both the endocrine and nervous systems. During the past year of support we have gathered additional evidence in support of the concept that ovarian development is subjected to the regulatory influence of a "neuroendocrinotrophic" complex. This complex consists of three basic components the extrinsic innervation to the gland, an intragonadal source of catecholamines, and growth factors of the neurotrophin family. We have found that neurotransmitters acting via the cyclic AMP generating system contribute to the early differentiation of newly formed ovarian follicles by facilitating formation of FSH receptors, before the ovary becomes responsive to gonadotropins. Using mice carrying null mutations of genes encoding neurotrophins (nerve growth factor [NGF], neurotrophin-4 [NT-4], brain derived neurotrophic factor [BDNF]) or the receptor that mediates the actions of NT-4 and BDNF (trkB), we found that neurotrophins are r equired for the initial development of primordial follicles. The nonhuman primate gland contains an intrinsic network of neuron-like cells, some of which are catecholaminergic. These neurons derive from the neural crest, migrate into the ovary during fetal life, become more abundant as the animal approaches puberty, and all but disappear during aging. In addition to this intrinsic source of catecholamines, monkey oocytes are able to uptake dopamine (DA) via a DA transporter and convert it into norepinephrine (NE). In turn, NE activates ?-adrenoreceptors in adjacent granulosa cells, which respond with cAMP formation. Since cAMP inhibits oocyte maturation, oocyte-derived catecholamines may represent one of the cell-to-cell mechanisms involved in the autoregulation of oocyte maturation. In addition to their role in early follicular development, neurotrophins also participate in the ovulatory process. The expression of trkA, the tyrosine kinase receptor of NGF, increases in the ovary during the preovulatory surge of gonadotropins of both rodents and primates. Purified bovine thecal cells, transfected with a trkA expression vector to simulate a periovulatory condition, respond rapidly to NGF with prostaglandin E2 formation and proliferation. Thus, NGF may contribute to the ovulatory process by facilitating the formation of inflammatory mediators and the differentiation of thecal cells into their luteal counterparts. FUNDING NIH HD-24870. PUBLICATIONS Lara HE, Leyton V, Fiedler JL, Dissen GA, Ojeda SR. An increase in ovarian neurotrophins contributes to the development of polycystic ovary. Soc Neurosci Abstr 24:2005, 1998 (abstract #801.9). Mayerhofer A, Smith GD, Danilchik M, Levine JE, Wolf DP, Dissen GA, Ojeda SR. Oocytes are a source of catecholamines in the primate ovary Evidence for a cell-cell regulatory loop. Proc Natl Acad Sci USA 95:10990-10995, 1998.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. These studies examine the role that cell-cell communication within the brain plays in the control of female puberty. The concept is being developed that reciprocal communication between neurons (one of the two main functional and structural units of the central nervous system) and astroglial cells (the other main building block of the nervous system) is critical for the timely acquisition of female sexual maturity and reproductive competence. We have identified several components of this glia-neuron regulatory system and elucidated some of the intercellular mechanisms they employ to transfer information from astroglial cells to the neurons that secrete luteinizing hormone-releasing hormone (LHRH), the hormone controlling female sexual development. A family of growth factors related to a protein known as epidermal growth factor (EGF) was found to be produced by astroglial cells and to function interactively to facilitate LHRH secretion and, thus, regulate the initiation of the pubertal process. Using mutant mice in which the normal function of these pivotal recognition molecules mediating the actions of EGF-related proteins was impaired, we demonstrated that these astroglial-derived growth factors are required for normal female sexual development. We have also used genomic and proteomic approaches to identify new genes that, expressed in the hypothalamus, participate in the control of the pubertal process. Altogether these results provide support for the concept that the syndromes of sexual precocity and delayed sexual development of central origin in humans may be related to abnormalities affecting these major regulatory pathways.

This application proposes the use of a genetic approach to define the potential roles of luteinizing hormone releasing hormone (LHRH) and its receptors in the brain. Specifically, the investigator proposes to employ the bacteriophage P1-derived Cre-loxP recombination system in a gene targeting approach to selectively delete the LHRH receptor gene from brain neurons and/or astrocytes, without affecting expression of the gene in endocrine tissues, such as the pituitary gland and ovaries.

The goal of Project 29 is to obtain preliminary evidence for the development of a nonhuman primate model of polycystic ovarian syndrome (PCOS). The 4 monkeys used for the experiment had cycled consistently over the previous 12 months (12 or 13 menses recorded, at regular intervals). They were provided with an intraovarian graft of genetically modified baby hamster kidney cells, encapsulated in a polymer of poly [acrylonitrile vinyl chloride, P(AN-VC)]. The surgical implants were performed as close to the first day of the new menses cycle as possible. Two of the monkeys received devices which contained NGF-secreting cells and two of the monkeys received control devices containing unmodified cells. The implants were 0.7 mm outside diameter and 7 mm long. Each monkey received two implants in each ovary. Following laparatomy to expose the ovaries, the implants were inserted into the ovary using a large bore needle and plunger (a suture in the ovarian capsule prevented migration of the device). Following implantation, the monkeys that received the control cells have continued to cycle. After 3 cycles one monkey has had cycle lengths of 26, 28, and 27 days while the other control monkey has had cycle lengths of 30, 31, and 29 days. Measuring serum progesterone levels every third day confirmed the occurrence of normal ovulatory menstrual cycles. Thus far, the menstrual cycles of the monkeys receiving the NGF implants appear altered as judged by their progesterone profiles. The active life span of the corpus luteum appears shortened in both animals. While in one monkey the follicular phase is also shortened, thus decreasing the overall cycle length to 23 days, the second monkey is showing extended follicular phase (22 days), thereby resulting in cycle lengths that are either normal or extended in length. The data thus far collected suggest that overproduction of NGF in the ovary may disrupt cyclic ovarian function. A firmer conclusion will be obtained upon termination of the project in three more months.

Gamma aminobutyric acid (GABA), the dominant inhibitory neurotransmitter in the central nervous system plays a prominent role in the control of hypothalamic LHRH secretion. It now appears that a GABAergic control exerted via GABA/A receptors (GABA-AR) is established during early LHRH neuronal development and continues to operate throughout the natural history of the LHRH neuronal network. It is likely that part of this regulatory influence is exerted directly on LHRH neurons, as they express all of the receptor subunits required for the assembly of functional GABA-AR. Although recent studies have demonstrated a modulatory effect of GABA-AR activation on LHRH neuronal migration, it is not known if such an effect is directly exerted on LHRH neurons. Likewise, nothing is known about the physiological impact that the GABAergic innervation on LHRH neurons may have on the control of adult reproductive function. Resolution of these issues by conventional neuroendocrine experimentation is difficult, because of the intricacy of the neuronal circuities affected by GABA and the molecular complexity of the GABA-AR system. The recent development of genetic approaches to modify the expression of genes in a cell-specific and temporally- restricted manner, and the identification of some of the key components involved in GABA-AR-mediated signaling, provide us with a unique opportunity to unravel some of the basic mechanisms underlying the GABAergic control of reproductive function. In this study, we propose a combination of genetic and neuroendocrine approaches to define the contribution of GABA to the embryonic development of LHRH neurons and to the functional competencies of the LHRH neuronal network during adulthood. To this end, the following aims are proposed to test the hypotheses that: 1) Direct GABAergic excitatory inputs play a role in the migration and developmental rate of LHRH neurons. 2) The direct GABA-AR-mediated input received by adult LHRH neurons is a regulatory component of the LHRH neuronal network required for normal reproductive cyclicity. 3) Selective disruption of the GABA-AR beta/3 subunit in LHRH neurons results in hypothalamic hypogonadism, and 4) A site- and time- specific, reversible activation of GABA release suffices to disrupt menstrual cyclicity in non-human primates, thus re-creating in an experimental setting the human syndrome of hypothalamic amenorrhea. We anticipate that these studies will lead to a better understanding of the cellular mechanisms underlying the central loss of reproductive competence in human syndromes such as hypothalamic amenorrhea and idiopathic hypothalamic hypogonadism.

In these studies we are using a genetic approach to define the potential roles of luteinizing hormone releasing hormone (LHRH) and its receptors in brain. LHRH is the hypothalamic neuropeptide that controls sexual development and mature reproductive function. The first phase of the research plan is now complete. We have obtained two hemizygous transgenic mice (one male and one female) of the 671-Bx/Gal Nac (T8) strains carrying the Cre recombinase gene under the control of the neural cell-specific synapsin I promoter (Syn-Cre). They have been crossed to produce a first generation (F1) of mice carrying the Syn-Cre transgene. Seventy-five percent of the F1 pups (6 of 8) were positive for Syn-Cre, indicating that the transgene is transmitted in a Mendelian fashion. Two of these mice (one male and one female) were tentatively identified as homozygotes by semiquantitative PCR, and confirmed as such by backcrossing to wildtype mice. We then crossed a F1 Syn-Cre pos itive mal e with F1 Syn-Cre positive females to produce additional homozygous animals. As before, we used semiquantitative PCR and back crossing to identify homozygous mice of the F2 generation. The brains of two of these mice were fixed by transcardiac perfusion and subjected to in situ hybridization. The results of this analysis demonstrated the presence of Cre mRNA in neurons containing LHRH receptor mRNA. This colocalization was especially evident in the hippocampus, a region of the brain involved in learning and memory. We are currently using three pairs of young adult hemizygous animals to maintain this transgenic line. The second, and more complex phase of the proposal is well under way. We have prepared the targeting construct to Aflox@ exon 1 and proximal promoter of the LHRH receptor gene, and worked out a screening strategy to identify, first those ES clones carrying a homologously recombined targeting construct, and secondly, those clones in which Cre-mediated recombination was effective in generating the intended recombination events. ES cells have now been transfected with the targeting construct and grown under neomycin selection. A total of 112 clones were selected; they are currently being analyzed by PCR/ Southern blot analysis to identify those carrying an homologously integrated targeting construct. FUNDING NIH HD35958 PUBLICATIONS None

In these studies we are using a genetic approach to define the potential roles of luteinizing hormone releasing hormone (LHRH) and its receptors in brain. LHRH is the hypothalamic neuropeptide that controls sexual development and mature reproductive function. The first phase of the research plan is now complete. We have obtained two hemizygous transgenic mice (one male and one female) of the 671-Bx/Gal Nac (T8) strains carrying the Cre recombinase gene under the control of the neural cell-specific synapsin I promoter (Syn-Cre). They have been crossed to produce a first generation (F1) of mice carrying the Syn-Cre transgene. Seventy-five percent of the F1 pups (6 of 8) were positive for Syn-Cre, indicating that the transgene is transmitted in a Mendelian fashion. Two of these mice (one male and one female) were tentatively identified as homozygotes by semiquantitative PCR, and confirmed as such by backcrossing to wildtype mice. We then crossed a F1 Syn-Cre pos itive mal e with F1 Syn-Cre positive females to produce additional homozygous animals. As before, we used semiquantitative PCR and back crossing to identify homozygous mice of the F2 generation. The brains of two of these mice were fixed by transcardiac perfusion and subjected to in situ hybridization. The results of this analysis demonstrated the presence of Cre mRNA in neurons containing LHRH receptor mRNA. This colocalization was especially evident in the hippocampus, a region of the brain involved in learning and memory. We are currently using three pairs of young adult hemizygous animals to maintain this transgenic line. The second, and more complex phase of the proposal is well under way. We have prepared the targeting construct to Aflox@ exon 1 and proximal promoter of the LHRH receptor gene, and worked out a screening strategy to identify, first those ES clones carrying a homologously recombined targeting construct, and secondly, those clones in which Cre-mediated recombination was effective in generating the intended recombination events. ES cells have now been transfected with the targeting construct and grown under neomycin selection. A total of 112 clones were selected; they are currently being analyzed by PCR/ Southern blot analysis to identify those carrying an homologously integrated targeting construct. FUNDING NIH HD35958 PUBLICATIONS None

DESCRIPTION (Provided by applicant): This application seeks support for a workshop that will identify and discuss new integrative approaches to maximize the contribution of NHP species to the ongoing efforts to unravel the fundamental principles governing human brain function in normalcy and disease. The overall topic of the workshop is based on the belief that research using nonhuman primates (NHPs) will exert its greatest impact on five major areas of Neuroscience Research: Brain Development, Aging and Neurodegenerative Diseases, Cognition and Behavior, Mental Illness, and Gene Therapy of the Nervous System. The underlying premise of this meeting is that the contribution that NHPs may make to these areas of neuroscience research will inextricably depend on the effective use of new technological advances in the fields of genomics, imaging, and cell and molecular biology that are revolutionizing biomedical research. The Aims of the Workshop are to: 1) Assess the current state of Neuroscience Research employing NHP with special attention to the current use of new technologies. 2) Identify strategies to allow Regional Primate Research Centers (RPRCs) to most effectively incorporate newly developed technologies into their research programs. 3) Identify mechanisms to foster collaborative efforts involving the RPRCs, other Academic Institutions, and the Biotechnology Industry. 4) Make recommendations to the National Center for Research Resources (NCRR) as to the resources and mechanisms that should be instituted to facilitate (via the use of newly available technologies) the contribution of NHPs to the much anticipated leap in knowledge already gaining momentum in the neuroscience field.

Abstract Not Provided

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This ongoing project is examining the hypothesis that growth factor molecules known as neurotrophins, and chemical messengers required for the development and mature function of the nervous system are produced by the mammalian ovary and contribute to regulating critical developmental phases in the natural history of the gland. The analysis of mutant mice carrying deletions of the genes encoding either neurotrophins or their recognition molecules (i.e., the neurotrophin receptors) showed that these growth factors are required for early development of ovarian follicles (the structural and functional unit of the ovary). In addition to neurotrophins, the ovary produces a variety of the same chemical messengers required for neuron-to-neuron communication in the nervous system. These messengers, known as neurotransmitters, appear to contribute to the process by which follicles become differentiated during early development and acquire the capability of responding to those hormones that -- secreted by the pituitary gland -- control the growth of mature follicles before puberty and during adult reproductive life. We are currently carrying out studies aimed at defining the cell types able to recognize neurotrophins during ovarian development, and are using genetic means to define the importance of these recognition molecules in ovarian development, including ovulation. We are also investigating the hypothesis that an excess of neurotrophins is deleterious, instead of beneficial, to ovarian function.

Luteinizing hormone-releasing hormone (LHRH) secretion is controlled by transsynaptic inputs of both excitatory and inhibitory nature, in addition to gila-to-neuron signaling pathways. While neurons that utilize gamma aminobutydc acid (GABA) for synaptic communication provide the major inhibitory input to the LHRH neuronal network, the bulk of the excitatory control of LHRH release is furnished by neuronal circuitries that use glutamate for neurotransmission. During the past period of support we focused our attention on the GABAergic system, and demonstrated that - contrary to the prevailing dogma - the direct GABAA receptor (R)-mediated input to LHRH neurons is excitatory, and not inhibitory. Using gene transfer-cell grafting techniques and transgenic approaches we demonstrated that a GABAergic tone is required for the normalcy of both LHRH neuronal migration and adult female reproductive capacity. We also identified the cellular mechanisms underlying the GABAAR-mediated excitation of LHRH neurons, and prepared the molecular and genetic reagents to define the importance of such mechanisms in the control of adult LHRH neuronal function. In addition, we used gene discovery approaches to identify genes that appear to be upstream components of the dual inhibitory/excitatory transsynaptic control of LHRH neurosecretion. Studies are now proposed to define the impact that each of these regulatory components may exert on the functional competence of LHRH neurons during female adulthood. To this end, the following aims are proposed: 1) to test the hypothesis that excitatory GABAAR-mediated inputs exerted directly on LHRH neurons are required for normal reproductive cyclicity, 2) to determine the role that members of the novel FXYD family of ion transport-controlling proteins, play in the regulation of LHRH secretion, 3) to test the hypothesis that Nell2, a novel gene specifically expressed in glutamatergic neurons, is an upstream regulatory element required for the glutamatergic control of reproduction, and 4) to define the role that a novel gene known as C14ORF4 may play in coordinating the dual excitatory/inhibitory transsynaptic control of reproductive cyclicity. We anticipate that the concepts derived from these studies will lead to a better understanding of the cellular mechanisms underlying the loss of reproductive competence in human syndromes such as hypothalamic amenorrhea and idiopathic hypothalamic hypogonadism.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This is a subcontract aimed at understanding the physiological mechanisms responsible for the onset of puberty in humans. Studies performed in Dr. Ojeda's lab are those proposed under Specific Aim 3 of the main application, which is: "To determine, in the absence of interfering testicular hormone feedback, the global profile of hypothalamic gene expression at the time of 1) the suppression of pulsatile GnRH release during infancy, and 2) the resurgence in pulsatile GnRH release at the end of the juvenile phase of development". The rhesus monkey is being used as an experimental paradigm.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of this project, now completed, was to define the role played by a gene termed FXYD1 in the neuropathology of Rett syndrome (RTT). This syndrome is an X-linked neurodevelopmental disorder linked to heterozygous de novo mutations in the MECP2 gene. In the absence of MECP2, the FXYD1 gene is overexpressed in both humans and Mecp2-null mice. FXYD1 encodes a trans-membrane modulator of Na+, K+-ATPase activity. These and other observations suggest that FXYD1 is a MeCP2 target gene whose de-repression may directly contribute to RTT neuropathogenesis. The results obtained indicate that decreasing FXYD1 expression by genetic means rescues learning and memory defects seen in the absence of MECP2, in addition to morphological deficiencies affecting neurons of the frontal cortex.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The objective of this Pilot project was to test the hypothesis that genetic variation in a novel gene termed EAP1 gene, manifested in the form of unique single nucleotide polymorphisms (SNPs), is a mechanism underlying the disorder of functional hypothalamic amenorrhea (FHA). The PI and his colleagues recently reported the functional characterization of this gene (termed EAP1, Enhanced at Puberty 1) and provided evidence supporting the concept that EAP1 is an upstream transcriptional regulator of neuronal networks controlling female reproductive function. These results also suggested that EAP1 plays an essential role in the control of female reproductive cyclicity. FHA is a disorder of the neuroendocrine brain that affects 3% of amenorrhea cases in women of reproductive age, and accounts for over 30% of all cases of amenorrhea. A natural nonhuman primate model of FHA exists in the ONPRC colony, with as many as 10% of adult females failing to cycle regularly or not cycling at all. The molecular underpinnings of this deficiency remain unknown, but they may be related to detrimental sequence defects in genes, such as EAP1, that control the rhythmic output of GnRH secretion from the hypothalamus. The results of this study, now completed, howed a significant association between a SNP located in the EAP1 promoter region and the incidence of amenorrhea in adult rhesus monkeys.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The objective of this study project is to test the hypothesis that short hairpin RNA (shRNA) and micro (RNA) constructs delivered to the brain by viral vectors will be efficacious in decreasing mutant gene expression and improving motor function in rodent models of Huntington's disease (HD). HD is a neurodegenerative brain disease that is caused by a trinucleotide (CAG) repeat expansion in the IT locus on chromosome 4. The resulting mutant protein contains a polyglutamine stretch at the N terminus of the protein. HD is characterized by cell death throughout the brain, with the cortex and striatum exhibiting the largest amount of degeneration. Patients experience severe motor dysfunction, cognitive decline and psychiatric disturbances and normally die within 10-15 years of disease onset (usually in the fourth decade of life). Currently, there is no cure for HD and treatments aim at alleviating the symptoms of the disease, rather than preventing its development. RNA interference (RNAi) is a phenomenon naturally occurring in cells to regulate gene expression and scientists have co-opted this biological mechanism as a means by which to decrease the expression of disease-related genes. Our lab has previously identified several shRNAs and miRNAs that bind to mutant huntingtin (htt) and reduce its expression by approximately 50-60% upon expression in the mouse striatum. The goal of the current studies are to investigate whether this approach will be efficacious in long-term knockdown of htt and whether this treatment can prevent the development of behavioral deficits in a mouse model of HD.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall goal of the present study is to develop a system for long[unreadable]lasting inhibition of gene expression in the brain or ovary that can be used to cause permanent infertility in dogs and cats. This application is a collaborative effort between the University of Iowa (UI) and the Oregon National Primate Research Center (ONPRC). During the past six months we have performed several experiments aimed at achieving the objectives of the original application. These experiments involve the use of a bio-panning strategy to identify peptide epitopes that can be delivered via the blood stream and specifically home to cells in either the hypothalamus or the ovary of the rat to deliver RNAi. To date, we have performed three rounds of bio-panning and recovered phage clones from both the hypothalamus and the ovary that can now be subjected to additional rounds of bio-panning to finally select candidates that specifically home to the hypothalamus, ovary or both, and that can therefore be used for the next phase of the study: re-directing viral tropism of AAV based on these selected epitopes to deliver RNA interference to those particular cell populations.

DESCRIPTION (provided by applicant): The primary goal of this training program is to provide multidisciplinary predoctoral and postdoctoral training in reproductive biology for those who wish to pursue an academic career in research and teaching in this area of biological science. This application requests funds to continue a training grant which has been funded for the past 30 years. Trainees will be selected based on their academic merit and potential for a productive research career in reproductive biology. The training faculty have expertise in many areas of reproductive biology including: neuroendocrine regulation of sexual development and mature reproductive function, hormonal control of the pituitary, the molecular biology of GnRH action, endocrine and paracrine control of gonadal function, regulation of the reproductive tract, and identification of genes involved in ovarian function and early embryogenesis. Many of the training faculty have a strong molecular orientation, and virtually all use molecular techniques into their research. Predoctoral students will be trained in one of three graduate programs at the Oregon Health and Science University: the Program in Molecular and Cellular Biosciences, the Dept. of Behavioral Neuroscience, or the Neuroscience Graduate Program. They will meet the course, seminar, and laboratory research requirements of the graduate council of OHSU and the specific requirements of the program in which they are training. Postdoctoral fellows will have access to the same courses and seminars, but emphasis will be placed on laboratory research. The unique features of this training program reside in the roster of faculty who study a wide range of areas within reproductive biology;the close ties between the research laboratories within the training program which facilitate co-mentoring of students;free access by students to expertise and equipment in all of the laboratories of the training program;regular meetings of journal clubs and research discussion groups;and the accessibility of performing reproductive research with nonhuman primates at the Oregon National Primate Research Center, and the presence of a number of collaborative interactions that include clinicians and basic scientists interested in reproductive biology. Programs for recruitment of minority trainees and formal instruction in the principles of scientific conduct and practice are in place. PUBLIC RELEVANCE: This grant application is to continue training pre- and postdoctoral fellows in reproductive biology. Strengths of the 30-year program include faculty with expertise in a wide range of areas in reproductive biology, strong interdisciplinary links between labs, the ability to work with nonhuman primate models and undertake translational research, and a number of collaborative centers encompassing reproductive biology research.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This application addresses broad Challenge Area (15): 15-NS-102: Translation of Gene Silencing Therapeutics. The proposed challenge set forth in the RFA is to extend the current understanding of the feasibility and safety of RNA interference (RNAi) therapeutics for the treatment of chronic neurological disorders from rodent models of disease to a more clinically relevant species. The current proposal outlines a systematic approach to translate work we, and others, have undertaken to investigate RNAi as a potential therapy for the neurological disorder, Huntington's disease (HD) in cell culture and rodent models and apply these findings to the non-human primate (NHP). The proposed studies are a collaboration between the University of Iowa and the Oregon National Primate Research Center (ONPRC). University of Iowa has experience developing and testing RNAi therapeutics in rodents, while the ONPRC has expertise in stereotaxic delivery of viral vectors to the NHP brain. Also, the ONPRC has methodologies, equipment and personnel in place that can evaluate if application of HTT suppression, or RNAi in general, induces neuropathology or neurological symptoms after delivery of RNAi expression vectors to NHP brain. To date, we have shown efficacy of reducing HTT expression in the rhesus macaque putamen by 45%. This suppression of HTT is not associated with the manifestation of any behavioral abnormalities nor neuropathological changes in the putamen.

During the last several years, investigators have observed an earlier initiation of human female puberty, a worrisome trend because early puberty is associated with increased risk for cancer, obesity, diabetes, and cardiovascular disease. It has also become clear that the timing of puberty is under genetic control, and likely determined by a host of regulatory genes. Yet, few attempts have been made to identify the genetic networks controlling this critical milestone in mammalian development. Drs. Sergio Ojeda (a neuroscientist) and Kemal Sönmez (a computer/electrical engineer) have combined expertise and developed a partnership between biology and engineering to test the hypothesis that a repressive gene network operating within the brain controls the initiation of female puberty. Using a combination of computational methods, systems biology approaches and molecular strategies, they performed preliminary studies revealing the existence of such a network and showing that it represses subordinate genes, which if left unchecked, would unleash the pubertal process prematurely. To disseminate their findings, Ojeda and Sönmez will partner with the Institute for Systems Biology in Seattle to generate a database resource of Web-based tools, links and protocols for analyses accessible via Internet. They will also make their raw data, results, and codes available on their website (www.sonmezsysbio.org), and deposit their data and results in public databases. Because the research plan includes the participation of undergraduate students and high-school students, The project will encourage young investigators to use similar integrative approaches for the study of many other complex physiological processes in mammalian systems. Theses studies will significantly enhance the understanding of basic systems-wide mechanisms underlying the neuroendocrine control of mammalian puberty.

DESCRIPTION (provided by applicant): To date, manipulating hypothalamic function mostly relies on the use of conditional knockout mice or transgenic overexpression. The major limitation to these approaches is that they do not take into account the complexities in the development of neuroendocrine neurons and their projections, and the compensatory adaptations that occur when these neurons are manipulated during early life. Alternatively, microinjections of adeno-associated viruses (AAV) delivering siRNAs have been used to modify hypothalamic function in adulthood. The greatest limitation of this technique is the invasiveness and relative inefficiency of the procedure. The present application intends to circumvent these limitations by developing a novel, minimally invasive method to manipulate hypothalamic neuronal function in a temporally defined and cell-specific manner. Among the hypothalamic systems that can be used as a prototype for these studies, the melanocortin system of the arcuate nucleus (ARC) stands out as an ideal candidate. It has been extensively studied and shown to play a critical role in regulating energy balance through modulation of food intake, body weight and glucose homeostasis. It is composed of two major populations of neurons with opposite functions; neurons containing pro-opiomelanocortin (POMC) inhibit the drive to eat and stimulate energy expenditure, neurons containing neuropeptide Y/Agouti-related peptide (NPY/AgRP) stimulate feeding behavior and inhibit energy expenditure. The consequences of altering the functions of either neuronal subset can be reliably assessed non-invasively, by measuring food intake and body weight. From the human health standpoint, developing new tools to study this system has an enormous value; the dramatic increase in childhood and adult obesity resulting from nutritional alterations during early life makes it urgent to develop novel methods to better understand the central mechanisms underlying the control of feeding behavior and energy homeostasis. This is a particularly important issue because energy balance can be permanently affected by nutritional challenges taking place during the critical period of developmental programming that in humans occurs during late gestation and in rodents, during the early postnatal period. A major advantage of the technology we propose to develop is that it can be used to modify ARC function after the developmental programming of energy balance is complete. We propose to silence the POMC and AgRP genes by delivering RNA interference (RNAi) to the ARC via the intravascular administration of modified AAV2 particles engineered to transduce hypothalamic cells. We anticipate that the successful execution of these studies will pave the way to the eventual application of similar approaches to treat disorders of the neuroendocrine brain. We also anticipate that these studies will provide the basis for new delivery strategies to the brain for basic research purposes and emerging therapies.

? DESCRIPTION (provided by applicant): It has been known for years that energy balance can be permanently affected by nutritional challenges taking place during a critical period of developmental programming, which in humans occurs during late gestation and in rodents during early post-natal life. It is also well established that these alterations affect female neuroendocrine reproductive development; increased nutritional availability advances the timing of puberty, and nutritional deficiency delays it. Perhaps due to the complexity of the systems involved and the lack of definitive candidates, neither the molecules linking nutritional programming to pubertal development nor the puberty-related genes they may regulate have been identified. We recently discovered that female puberty is regulated by an epigenetic mechanism that involves lifting of a transcriptional repressive tone exerted by the Polycomb group (PcG) of transcriptional silencers, and that this repression is imposed on downstream genes involved in the stimulatory control of GnRH secretion (epitomized by the Kiss1 gene).By discovering a novel epigenetic mechanism controlling the timing of puberty and identifying its basic components, we have now unveiled the existence of a regulatory system that may not only fulfill the long-sought out role of linking nutrition to neuroendocrine reproductive development, but is also amenable to experimental scrutiny. Accordingly, this proposal will test the hypothesis that alterations in the developmental programming of energy balance affect the timing of puberty by regulating the mechanism of epigenetic silencing that keeps GnRH secretion in check during prepubertal maturation. To this end, the following hypotheses will be tested: 1) That altering nutrient availability during early postnatal life affects puberty by regulating an epigenetic repressive tone imposed by the PcG complex on puberty-activating (PA) genes. 2) That additional PcG target genes potentially relevant to the timing of puberty and to the nutritional regulation of this process can be identified by epigenome-wide anlaysis using RNA-and ChIP- massively parallel sequencing technology; and 3) That one of the epigenetic link connecting nutrition to pubertal development is SIRT1, a fuel-sensing molecule that according to our hypothesis would function as a biological rheostat to silence/derepress PA genes in response to early nutritional unbalance. We anticipate that a successful outcome of the proposed studies will provide major insights into the integrative mechanisms linking energy homeostasis, the neuroendocrine brain and the control of puberty. We also anticipate that these studies will significantly enhance our understanding of how disorders in energy balance influence the timing and progression of puberty, and will make researchers and clinicians aware of the epigenetics contribution to these disorders.