Development And Regulation Of The Gonadotropin Releasing Hormone System

National Institute Of Neurological Disorders And Stroke

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Abstract

Gonadotropin hormone releasing hormone -1 (GnRH) neurons, which are critical for reproduction, are derived from the nasal placode and migrate into the brain where they become integral members of the hypothalamic-pituitary-gonadal axis. Disruption of either the development or regulation of the GnRH system can result in changes in puberty (delayed, absent or precocious), infertility, polycystic ovarian syndrome, and altered cognitive function. Our laboratory studies the mechanisms critical for successful reproduction to occur: 1) GnRH neuronal development (differentiation, migration and axonal targeting) and 2) regulation of GnRH neuronal activity (gene expression, peptide synthesis, and GnRH secretion). Our current experimental models include normal, transgenic and genetically engineered mice, and in vitro model systems incuding organotypic explants and GnRH derived cell lines from mouse and human. State of the art techniques are used to identify and understand the multitude of molecules and factors which play a role in directing the GnRH neurons to their final location in the CNS. These include differential screening of libraries obtained from migrating versus non-migrating cells, examination of molecules differentially expressed at key locations along the migratory route, morphological examination of the development of the GnRH system in knockout mice, and perturbation of molecules in vitro (explants and cell lines) and subsequent monitoring of GnRH neuronal development and physiological properties. To investigate the maturation of GnRH neurons, we use calcium imaging, electrophysiology, and biochemical measures to examine GnRH neuronal activity and peptide secretion. In addition, we collaborate with labs performing human genetic screening of patients with reproductive dysfunction. Once a mutation is identified, we analyze the expression pattern and perform biological assays to determine the outcome of the mutated gene on the GnRH system. Over the past year, 2 primary article and 2 collaborative articles have been published, with a 3rd collaborative article in revision. Our first paper addressed the effect of mild gestational hypothyroidism of the GnRH system and reproduction. Thyroid hormones (TH) play a key role in fetal brain development. While severe thyroid dysfunction has been shown to cause neurodevelopmental and reproductive disorders, the rising levels of TH disruptors in the environment in the past few decades have increased the need to assess effects of subclinical (mild) TH insufficiency during gestation. Since embryos do not produce their own TH before mid-gestation, early development processes rely on maternal production. Notably, the reproductive network governed by GnRH neurons develops during this critical period. The risk of mild maternal hypothyroidism on 1) the development of GnRH neurons and 2) long-term effects on neuroendocrine function in the offspring was investigated using a mouse model of gestational hypothyroidism induced by methimazole (MMI) treatment. We report that MMI treatment during gestation led to reduced litter size, consistent with increased miscarriages in humans due to hypothyroidism. Embryos collected from MMI-treated dams, had a decreased number of GnRH neurons, but the migration of the remaining GnRH neurons was normal. Cell proliferation was reduced in the region in which the GnRH arise, correlating with the reduced number of GnRH neurons detected. Using a GnRH cell line confirmed attenuated proliferation in the absence of T3. Pups born from hypothyroid mothers had normal postweaning growth and estrus cycles, yet adult offspring had significantly more cells expressing the estrogen receptor alpha in the arcuate nucleus, an area rich in neuroendocrine cells that modulate feeding and reproduction. Notably, by adulthood, GnRH cell number and distribution was comparable with nontreated controls indicating that compensatory mechanisms occurred during mid-gestation. Overall, this work shows that mild TH disruption during early gestation transiently affects proliferation of the pool of GnRH neurons prior to migration in the brain yet has a long-term impact on neuroendocrine systems. Although many clinical studies have identified correlations between thyroid dysfunction and reproductive issues, the underlying mechanisms behind this interaction remained poorly understood. As such, the second paper from our laboratory investigated the effect of T3 on the activity of GnRH neurons. Dual labeling confirmed GnRH neurons express thyroid receptor (TR)α and integrin αVβ3 receptors mediating genomic and non-genomic effects of thyroid hormones, respectively. Using calcium imaging in an ex vivo model, we show that T3 induces a rapid and sustained increase of calcium oscillation frequency in GnRH neurons. The T3 stimulatory effect was not inhibited by a TR-specific antagonist but was mimicked by membrane-impermeable T3-BSA, indicating a mechanism independent of nuclear TR signaling. In contrast, the blockade of membrane αVβ3 integrins prevented the T3-induced increase in calcium peak frequency in GnRH neurons. Using modulators of intracellular and calcium entry revealed that binding to αVβ3 integrin can induce distinct calcium responses depending on the ligand, with T3 triggering a complex response involving multiple channels and calcium sources, possibly with compensatory mechanisms. In sum, this paper demonstrated direct effect of thyroid hormones on GnRH neuronal activity, with T3 stimulating calcium oscillations through the non-genomic αVβ3 integrin pathway. Understanding this thyroid-reproductive axis interaction will help clarify the mechanisms linking thyroid dysfunction to reproductive disorders and pave the way for targeted therapeutic interventions. Our collaborative work relies on our expertise in the development and regulation of the GnRH system. One paper focuses on a new mouse model for Polycystic ovary syndrome (PCOS) which is a heterogenous disorder characterized by reproductive and metabolic abnormalities. PCOS etiology remains poorly understood. Human genetic studies have shown an association with the transcription factor-coding gene GATA4, but without providing a functional link. This paper shows that adult Greywick female mice phenocopy PCOS with partial penetrance, due to serendipitous insertion of a Gata4 promoter-driven transgene in a strong enhancer region. Resulting robust transgene expression in subsets of hypothalamic neurons and glia impairs endogenous Gata4 expression, resulting in misexpression of genes linked to the control of fertility and food intake. Overall, this study sheds light on both PCOS etiology as well as the role of GATA4 in the central control of reproduction. The second collaboration paper identified immunoglobulin superfamily member 10 (IGSF10) as a RET antagonist. RET is a receptor tyrosine kinase that plays important roles in development, cancers, and Parkinson’s disease. The paper shows that Ewing sarcoma, a cancer of bone and soft tissue in children and young adults, depends on IGSF10 and that IGSF10 prevents RET-mediated activation of a Rho family G protein (cdc42) that is a key regulator of Ewing sarcoma growth as well as cell migration. Experiments showed that IGSF10 assembles an inhibitory complex, preventing the stimulatory complex activation. IGSF10 mutations are also associated with delayed puberty and IGSF10 was shown to be necessary for proper migration of gonadotropin-releasing hormone (GnRH) neurons. The data in this paper show that this same IGSF10-RET pathway regulates migration of GnRH neurons and that IGSF10 mutants linked to delayed puberty are defective in RET-cdc42 regulation. These results reveal a critical role for IGSF10 as a RET antagonist in Ewing sarcoma and GnRH neurons, two cell types that under go migration. During the past year I have trained 2 new postdoctoral fellows, who are now both working on first author papers, 1 new postbaccalaureate student and 2 new college students. My senior postdoctoral fellow obtained a job in her home country (France) and ended her 3 year postdoc with 3 first author papers, a single author methods unit and will be a co-author on 2 future papers. My postbaccalaureate trainee was accepted to graduate school and left with 2 published co-authorships and will be a first co-authorship on a future paper. I was an invited to 3 meetings: workshop chair for AAAS (had to abruptly decline due to travel ban), Keynote Speaker at the International Regulatory Peptide Society and a speaker at Rhythms, networks and slow-fast analysis in neural and endocrine systems at McGill University, Montreal. Due to budget freezes and changes in personnel that started January 2025, much time was occupied by meetings: looking for budget changes for the Porter Building Animal Facility, the Building 49 Central Animal Facility, as well as my own lab. In addition, I was one of the 11 senior scientist at NINDS that was RIFed on April 1st. This event disrupted myself, my personnel and the entire NINDS faculty. My perspective is that research at the NIH will never recover from the events of this year.