Gloria Hoffman, Ph.D. - US grants

LHRH regulates pituitary and non-pituitary systems. An understanding of the projections of the LHRH systems and the specific systems with which they interact aids in the understanding of the actions of the peptide. From initial studies of the interactions of the LHRH system, the tuberoinfundibular dopamine system and the magnocellular oxytocin or vasopressin network appear to be two systems that receive input from LHRH. In this proposal immunohistochemical techniques will be used to further characterize the anatomical relationship of LHRH containing processes with these systems and also define other possible systems on which LHRH may exert an influence. The role of LHRH in alerting the activity or function of the targeted system will then be determined by utilizing functional explants and measuring effects of LHRH on the release of vasopressin, or oxytocin by radioimmunoassay; a co-culture system which includes the tuberoinfundibular dopamine system and the anterior pituitary will be used to test LHRH effects on dopamine-mediated prolactin release. Since gonadal steroids influence the magnocellular systems and the tuberoinfundibular dopamine system, assessment of the influence of estrogen and/or testosterone on patterns of LHRH terminals and effects of LHRH on function of these systems will be examined in vitro with tissue from normal adult animals and in subjects with altered reproductive states such as gonadectomy, pregnancy and lactation. This knowledge is of importance for elucidation of the processes that regulate the reproductive system or are synchronized with the reproductive cycle.

The gonadal steroids can influence both brain function and organization. Organizational effects of the gonadal steroids are determined during a critical period of development (although the manifestation of their effect may not be evident until adulthood); the nascent steroid need not be present for the effect to be preserved. In contrast, activational effects require the presence of the steroid, and are terminated when the steroid is removed. Both classes of steroid effect have been observed in regions of the brain that play a role in the reproductive process. In one of these, the preoptic area (POA), axons containing an opiate peptide, metenkephalin, (m-ENK), in the periventricular region of the POA (pePOA) show sex differences in density, and the m-ENK fiber pattern can be manipulated by alteration of the gonadal steroids. Preliminary studies indicate that this peptide system shows both organizational and activational effects of steroids. This proposal is designed to further examine the sexual dimorphism of the m-ENK system. Specifically, experiments will be designed to: 1) Characterize the anatomical organization of the sexually dimorphic pePOA and its m-ENK fiber system at light microscopic and ultrastructural levels by examining the organization of pePOA and its m-ENK fiber distribution at the light microscopic (LM) and ultrastructural (EM) levels, 2) determine the degree to which the expression of this sexually dimorphic patterna in m-ENK immunoreactivity is determined by activational actions of gonadal steroids by exploring the changes in m-ENK immunoreactivity produced by gonadal steroids, the time course of this effect, and the selectivity of this activational process for different steroids, and 3) determine the degree to which the expression of this sexually dimorphic pattern in m-ENK immunoreactivity and neuronal density is determined by organizational actions of gonadal steroids by exploring the effects of gonadal steroids administered during the neonatal period on the sexually dimorphic patterns of the pePOA.

Oncogenes, originally found in viruses, code for proteins which transform healthy cells into tumor cells. In the course of this work, it was found that normal cells contain non-mutagenic homologs of oncogenes, called proto-oncogenes. C-fos, is a proto-oncogene, and its expression appears to be one of the initial events resulting from stimulation of a cell. Thus, the determination of c-fos in neuron reactivity represents a promising tool to determine with high resolution and high sensitivity the pattern of activity at the cellular level. Dr. Hoffman will develop and validate the use of c-fos immunocytochemistry as a molecular tool to map hypothalamic neuronal activation. She has selected the magnocellular neurons within the supraoptic and paraventricular nuclei of the hypothalamus since quantifiable physiological stimuli activate these neurons to synthesize and release vasopressin and oxytocin. Vasopressin is involved in the regulation of water balance and blood volume and oxytocin is necessary for lactation and parturition. Using immunocytochemical double-labelling techniques and appropriate physiological stimulation, Dr. Hoffman will dissect the relationship between c-fos expression and hormone secretion and/or release. These studies will specifically distinguish active from inactive neurons and thus will add a new dimension to our understanding of the relationship between structure and function in the mammalian central nervous system. These data are likely to have a significant impact on the understanding of the functional physiology of the hypothalamus.

in situ hybridization; immunocytochemistry; biomedical facility; tissue resource /registry; cooperative study; female reproductive system; reproductive hormone;

luteinizing hormone; gonadotropin releasing factor; gene induction /repression; hormone regulation /control mechanism; ovulation; protooncogene; preoptic areas; galanin; alpha adrenergic receptor; tyrosine 3 monooxygenase; antisense nucleic acid; neurons; regulatory gene; estrogens; afferent nerve; neuronal transport; dopamine; neurotensin; tissue /cell culture; in situ hybridization; immunocytochemistry;

To provide the prolactin necessary for lactation, the suckling stimulus releases pituitary lactotropes from tonic inhibition. Adaptive changes in the tuberoinfundibular dopamine (TIDA) neurons regulating prolactin appear to promote this process by producing a shift in their transmitter from dopamine (DA) to enkephalin (Enk). This "phenotype" switch is accomplished by curbing synthesis of mRNA for the rate limiting enzyme of DA synthesis tyrosine hydroxylase (TH) and by inducing enkephalin (Enk) synthesis. The stimulus to suppress the TH appears to be principally neural, but the pathways that provide that suppression are not known. This proposal will determine if the pathways from the nipples to the hypothalamus initially regulate the TIDA neurons through a stimulatory relay in the peripenduncular n. that extends to the ventral arcuate before inhibiting TIDA cells. The next set of experiments will test the hypothesis that the inhibitory signal is dynorphin. Experiments will then focus on the question of what distinguishes intermittent suckling form weaning; particularly,, can the up-regulation of TH expression be interrupted if pups are returned to their damns before TH fully up-regulates, and does Enk persist after suckling cases? Lastly while hyperprolactinemia is adaptive for lactation, it can be maladaptive at other times. To understand this process, experiments will test the hypothesis that intermittent prolactin administration causes the same transmitter changes as lactation for Enk (but not TH) and that non- habituating stress induces transmitter phenotype changes in TIDA neurons that resemble those of lactation for both TH and Enk. These studies will not only provide understanding of the neural plasticity that supports lactation, but will also provides a basis for understanding neuroendocrine dysregulation of the prolactin regulating system during stress.

Description (provided by applicant): The objective of the Immunocytochemistry Core is to support the three research projects of this SCCPRR with high quality, rapid, efficient, and uniform histology of and immunocytochemical localization/expression of specific proteins in the baboon fetal ovary (Project I) and testes (Project II) and human/baboon endometrium (Project III). The immunocytochemistry Core will also provide training for SCCPRR graduate students, postdoctoral fellows, investigators, and technicians in histology and immunocytochemistry.

Description (provided by applicant): The objective of the Immunocytochemistry Core is to support the three research projects of this SCCPRR with high quality, rapid, efficient, and uniform histology of and immunocytochemical localization/expression of specific proteins in the baboon fetal ovary (Project I) and testes (Project II) and human/baboon endometrium (Project III). The immunocytochemistry Core will also provide training for SCCPRR graduate students, postdoctoral fellows, investigators, and technicians in histology and immunocytochemistry.

DESCRIPTION (provided by applicant): This proposal is in response to RFA-HD-09-008 Cooperative Research Partnerships to Promote Workforce Diversity in the Reproductive Sciences (U01), entitled, Kisspeptin Regulation and GPR54 Signaling in Reproduction. The application is a research partnership among Drs. Sally Radovick and Andrew Wolfe at Johns Hopkins University School of Medicine and Dr. Gloria Hoffman at Morgan State University. Undergraduate students from Morgan State University and Johns Hopkins University will be recruited and mentored under this collaborative award mechanism. The curriculum will include didactic lectures, a wide breadth of laboratory based studies and career counseling. A Steering Committee comprised of the investigators will guide the process. The release of LH is under the regulation of the neurohormone, luteinizing hormone releasing hormone (LHRH). Recently, the peptide kisspeptin (KP) that signals through a G-protein coupled receptor, GPR54, was found to be a key component in the regulation of LHRH. Evidence for a direct role for KP at the level of the LHRH neuron comes from anatomical and in vitro studies in LHRH expressing ceil lines. Two principal populations of KP neurons are described in the hypothalamus: one in the arcuate nucleus (Arc) and one in the AVPV. The AVPV KP neurons project directly to LHRH neurons and stimulate LHRH neurons at the time of an LH surge. The Arc population is not sexually dimorphic and is implicated in basal LHRH release and negative feedback in both sexes, but how it does so is unknown. While some aspects of KP regulation are preserved in primates, there is little resolution as to whether the AVPV population is present, and whether connections of the KP neurons to LHRH and other systems are preserved across species. This proposal will examine the connectivity of KP neurons with the LHRH system in rodents vs. primates and will also generate a floxed GPR54 mouse line to specifically ablate GPR54 in the LHRH neuron to study pubertal onset and estrogen feedback regulation in addition to cellular changes in LHRH neurons. In summary, this proposal will delineate both the role of KP-GPR54 signaling in estrogen feedback regulation and its locus in the central reproductive axis. PUBLIC HEALTH RELEVANCE: The goal is to provide selected undergraduates the unique resources available from two institutions focused on a broad spectrum of didactic and laboratory experiences in the field of reproductive biology. Partnering to learn with academic researchers, the students will study regulatory mechanisms controlling mammalian reproduction.

DESCRIPTION (provided by applicant): This proposal is in response to RFA-HD-09-008 Cooperative Research Partnerships to Promote Workforce Diversity in the Reproductive Sciences (U01), entitled, Kisspeptin Regulation and GPR54 Signaling in Reproduction. The application is a research partnership among Drs. Sally Radovick and Andrew Wolfe at Johns Hopkins University School of Medicine and Dr. Gloria Hoffman at Morgan State University. Undergraduate students from Morgan State University and Johns Hopkins University will be recruited and mentored under this collaborative award mechanism. The curriculum will include didactic lectures, a wide breadth of laboratory based studies and career counseling. A Steering Committee comprised of the investigators will guide the process. The release of LH is under the regulation of the neurohormone, luteinizing hormone releasing hormone (LHRH). Recently, the peptide kisspeptin (KP) that signals through a G-protein coupled receptor, GPR54, was found to be a key component in the regulation of LHRH. Evidence for a direct role for KP at the level of the LHRH neuron comes from anatomical and in vitro studies in LHRH expressing ceil lines. Two principal populations of KP neurons are described in the hypothalamus: one in the arcuate nucleus (Arc) and one in the AVPV. The AVPV KP neurons project directly to LHRH neurons and stimulate LHRH neurons at the time of an LH surge. The Arc population is not sexually dimorphic and is implicated in basal LHRH release and negative feedback in both sexes, but how it does so is unknown. While some aspects of KP regulation are preserved in primates, there is little resolution as to whether the AVPV population is present, and whether connections of the KP neurons to LHRH and other systems are preserved across species. This proposal will examine the connectivity of KP neurons with the LHRH system in rodents vs. primates and will also generate a floxed GPR54 mouse line to specifically ablate GPR54 in the LHRH neuron to study pubertal onset and estrogen feedback regulation in addition to cellular changes in LHRH neurons. In summary, this proposal will delineate both the role of KP-GPR54 signaling in estrogen feedback regulation and its locus in the central reproductive axis. PUBLIC HEALTH RELEVANCE: The goal is to provide selected undergraduates the unique resources available from two institutions focused on a broad spectrum of didactic and laboratory experiences in the field of reproductive biology. Partnering to learn with academic researchers, the students will study regulatory mechanisms controlling mammalian reproduction.

DESCRIPTION (provided by applicant): The release of LH is under regulation by the neurohormone gonadotropin releasing hormone (GnRH). Recently, the peptide kisspeptin (KP) which signals through a G-protein coupled receptor, GPR54, was found to be a key component in the regulation of GnRH. Evidence for a direct role for KP at the level of the GnRH neuron comes from anatomical and in vitro studies in GnRH expressing cell lines. Two principal populations of KP neurons are described in the hypothalamus: one in the arcuate nucleus (AN) and one in a region of the preoptic area, the AVPV. The AVPV KP neurons project directly to GnRH neurons and stimulate GnRH neurons at the time of the estrogen induced LH surge, while the AN population is implicated in negative feedback. This proposal will explore the role of estrogen in the neuroendocrine regulation of the gonadotropin surge in three aims. Aim 1 will determine if the negative and positive feedback actions of estradiol (E2) are mediated by activation of ERa in KP neurons. The responses of KP cell-specific ERa knockout (KERaKO) mice to treatments that evoke negative and positive feedback actions on LH neurosecretion will be assessed using a newly developed microdialysis system. Aim 2 will determine if prenatal androgen exposure (PNA), which induces features of polycystic ovary disease, programs resistance to E2 feedback actions in KP neurons. KP cell-specific AR null mutant (KARKO) mice will be generated and proposed studies will determine if they are refractory to androgenic programming of altered negative and positive feedback actions of E2. Aim 3 will determine the role of E2 in modulating KP GPR54 signaling in the regulation of the GnRH neuron. To determine if GPR54 activation in GnRH neurons mediates the physiological regulation of GnRH expression by KP neurons, the responsiveness of GnRH neuron-specific GPR54 knockout (GnRH-GPR54KO) mice to E2 negative and positive feedback actions will be assessed. The role of ER¿ in mediating E2 negative feedback and KP mediated synthesis and secretion of GnRH will be determined. Overall, these studies will provide a systematic, hierarchical assessment of the effects of E2 in the neuroendocrine control of the gonadotropin surge, and determine the mechanisms by which early androgen exposure may program resistance to E2 feedback actions.

DESCRIPTION (provided by applicant): FMR1 is a gene on the X chromosome that contains an expansion-prone CGG CCG repeat in its 5' untranslated region. Alleles with 55-200 repeats are considered to be Fragile X premutation (PM) alleles. The frequency of such alleles in women ranges from 1 in 113 to 1 in 250. Carriers of such alleles are at risk for Fragile X Primary Ovarian Insufficiency (FXPOI) that is seen in up to 28% of women who carry such alleles. FXPOI accounts for ~11.5% of familial cases of infertility and 3.5% of idiopathic cases. Even without a diagnosis of FXPOI, the average age at menopause of all women with the premutation is ~5 yrs earlier than their siblings without the PM. Thus, female PM carriers not only have increased fertility problems, but are at greater risk of cardiovascular disease, Alzheimer disease, osteoporosis and other problems that are seen at higher frequency in menopausal women. Hormonal assessment of PM carriers indicates that FSH is elevated, inhibins and anti-Mullerian hormone (both made by granulosa cells) are reduced. Preliminary data from a knock-in Fragile X PM mouse model containing ~130 CGG repeats suggest a number of ovarian problems. These include early losses of immature follicles and reductions in corpora lutea. Advanced follicles were smaller in PM mice and showed a higher rate of atresia. This was associated with a smaller than normal number of granulosa cells (GCs). In addition, PM oocytes showed abnormal nuclear morphology, reduced levels of gap junction proteins, abnormal staining of their zona pellucida, a primarily nuclear location of the normally cytoplasmic fragile X mental retardation protein (FMRP) and high levels of ubiquitinated proteins that often accumulated in the nucleus or perinuclear region. A high incidence of large cysts was also seen in PM mice. Thus our mice exhibit signs consistent with ovarian insufficiency and have unusual ovarian changes that could contribute to this ovarian dysfunction. These mice may therefore provide a good model of FXPOI. Aim 1 uses transplantation approaches to address whether the PM in the ovary alone is sufficient to produce the FXPOI-like and aberrant ovarian features or instead requires the PM in the hypothalamic/pituitary unit. Aim 2 focuses on the abnormal FMRP, ubiquitin, and gap junctions as well as the abnormalities in zona pellucida composition to attempt to uncover the mechanism(s) responsible for the follicle decline.