Marie-Claude Hofmann - US grants

DESCRIPTION (provided by applicant): Our long-term goal is to study the biology of spermatogonial stem cells, the A/single spermatogonia. There is great interest in the A/single spermatogonia because of their importance for understanding basic mechanisms of cell self-renewal versus differentiation, for the treatment of infertility, for the development of contraceptives and for the understanding of the etiology of testicular cancer, in particular seminoma. However, studies on the biology of these cells have been severely hampered because their number is low, no unique marker exists and it has been difficult to physically separate them from the other more mature spermatogonia. We recently developed a method that allows us to isolate pure populations of A/single spermatogonia, and we have started to study their behavior in vitro. Using microarrays, we also have analyzed the transcriptional program of the A/single spermatogonia in the presence of glial cell line-derived neurotrophic factor (GDNF), a growth factor produced by Sertoli cells. In the first aim, we will isolate Asingle spermatogonia and evaluate their clonogenic potential by germ cell transplantations. In the second aim, we will determine which signaling pathways are induced by GDNF, leading to Asingle spermatogonia self-renewal or differentiation. In the third aim, we will study how GDNF interacts with other growth factors/receptors to promote self-renewal or differentiation. In summary, this line of work will expand our knowledge of the functional characteristics of the A/single spermatogonia and give us better insight into the molecular mechanisms that drive the first steps of spermatogenesis. In particular, the role of the growth factor GDNF in regulating self-renewal and/or differentiation will be elucidated.

DESCRIPTION (provided by applicant): This Career Development Plan will ensure Dr. Marie-Claude Hofmann's success as an independent research scientist in the Department of Biology, University of Dayton. Dr. Hofmann's general area of interest is the biology of spermatogonial stem cells (SSCs), the Asingle spermatogonia. There is great interest in the Asingle spermatogonia because of their importance for understanding basic mechanisms of cell self-renewal versus differentiation, for the treatment of reproductive dysfunction, and for the understanding of the etiology of testicular cancer, in particular seminoma. However, studies on the biology of these cells have been severely hampered because their number is low, no unique marker exists and it has been difficult to physically separate them from the other more mature spermatogonia. Dr. Hofmann's team recently developed a method that allows the isolation of pure populations of SSCs, and has started to study their behavior in vitro. Using microarrays, they also have analyzed the transcriptional program of SSCs in the presence of glial cell line derived neurotrophic factor (GDNF), a growth factor produced by Sertoli cells. One of Dr. Hofmann's research objectives is to elucidate the signaling pathways induced by GDNF that promote SSCs renewal and differentiation. In the first aim, Asingle spermatogonia will be isolated and their clonogenic potential evaluated by germ cell transplantations. In the second aim, they will determine which signaling pathways are induced by GDNF, leading to Asingle spermatogonia self-renewal or differentiation. In the third aim, they will study how GDNF interacts with other growth factors/receptors to promote self-renewal or differentiation. This work will expand the knowledge of the functional characteristics of the Asingle spermatogonia and lend better insight into the molecular mechanisms that drive the first steps of spermatogenesis. In particular, the role of the growth factor GDNF in regulating self-renewal and/or differentiation will be elucidated. This Career Development Plan will assist Dr. Hofmann in accomplishing her goals. Specifically, it will release her from substantial teaching, thereby allowing her to devote full attention to research, increasing her skills in specific areas of biomedical science and technology. In addition, Dr. Hofmann will not be required to serve on administrative committees, and be released from undergraduate advising duties. At the University of Dayton, the Department of Biology, the College of Arts &Sciences and the Graduate School will provide a supportive environment for the development of Dr. Hofmann's research career. The University of Dayton has recently committed considerable resources to promote interdisciplinary bioscience and engineering research, and Dr. Hofmann is a vital member of our core group of lead investigators. This award will allow her to further collaborations with other departments at our University as well as collaborations with other institutions.

DESCRIPTION (provided by applicant): In the mammalian testis, spermatogenesis starts from a small population of spermatogonial stem cells (SSCs) that self-renew and differentiate to ultimately produce sperm. Failure of any of the molecular events underlying spermatogenesis will result in non-obstructive azoospermia in men. Non-obstructive azoospermia affects approximately 1% of men. Upon testis biopsy, azoospermia presents with Sertoli cell-only pattern, maturation arrest (MA), or hypospermatogenesis. In studies examining patients with non obstructive azoospermia, approximately 30% of the cases were identified with MA. In two studies that examined only patients with MA, all were found to be deficient in either the Notch-1 receptor or its ligand Jagged. Thus, 1 in 300-400 men may have a form of testicular failure resulting from a deficiency in Notch signaling. However to date, the exact role of Notch signaling in the testis is unknown. The Notch proteins (Notch1-4) are large cell-surface receptors that are activated by contact with membrane-bound ligands on neighboring cells, such as Jagged and Delta (Dll). Upon activation, the Notch intracellular domain (NICD) is cleaved and translocates to the nucleus where it associates with DNA-binding proteins and upregulates the expression of target genes, most notably the Hes/Hey family of transcriptional repressors. Hes/Hey proteins are bHLH transcription factors that form homo or heterodimers to function properly. In addition to interacting with other bHLH proteins, mainly co-repressors, and binding to specific DNA promoter sequences, they also can recruit chromatin modifiers such as histone deacetylases (HDACs). The presence of a number of Notch receptors and ligands in the testis has been established, but to date no functional studies have been attempted to identify their exact role. We have evidence that Jagged1 triggers Notch1 activation and upregulates Hes1 in undifferentiated spermatogonia, while it triggers Notch3 activation and upregulation of HeyL in pachytene spermatocytes, specifically at the XY body. Therefore, the effects of Jagged1 depend on the target cell. In this application, we will test the hypothesis that Notch signaling, through up- regulation of Hes1 and HeyL, plays a crucial role at 2 critical steps of spermatogenesis, 1) spermatogonial differentiation and 2) XY silencing at meiosis. PUBLIC HEALTH RELEVANCE: Spermatogenesis is the process of sperm formation in the testis. It starts with a spermatogonial stem cell that self-renew or differentiate into more mature spermatogonia. These cells develop into spermatocytes that will undergo meiosis to become haploid spermatids and sperm cells. Failure of any of the molecular events underlying spermatogenesis will result in infertility. Recent studies indicate that a number of infertility cases might be linked to a defect in the Notch signaling pathway. Notch is a large transmembrane receptor at the surface of germ cells, which receives signals from the somatic nursing Sertoli cells. This proposal seeks to understand the function of Notch and its downstream intracellular targets at 2 critical steps of spermatogenesis: spermatogonial differentiation, and meiosis. )

DESCRIPTION (provided by applicant): Gonocytes (or prospermatogonia) are the precursors to spermatogonial stem cells (SSCs), which provide the foundation for spermatogenesis through their ability to both self-renew and generate daughter cells. Despite their relative importance, th regulatory mechanisms that govern gonocyte maintenance in the fetus and transition to SSCs after birth are poorly understood. Using transgenic mice, we established that constitutive activation of NOTCH1 signaling in Sertoli cells causes gonocyte loss-the first suggestion of the potential role of this signaling pathway in the testis. We then inhibited NOTCH activation in mouse Sertoli cells and observed an increase in germ cell numbers and testicular size. Therefore dysregulation of NOTCH signaling induces either sterility (NOTCH overactivation) or hyperplasia that could enhance predisposition to testicular cancer (NOTCH downregulation). This proposal will test the hypotheses that NOTCH activity, through its target effectors HEY1 and HEYL, downregulates two crucial molecules that maintain the undifferentiated states of germ cells: GDNF and CYP26B1. We will use NOTCH overactivation, NOTCH lack of function and wild type mouse models to test whether the transcriptional repressors HEY1 and/or HEYL directly influence the expression of GDNF and CYP26B1 when NOTCH is activated. In Aim1, we will investigate the temporal expression of Hey1 and HeyL transcription factors by qPCR, and use ChIP-PCR to demonstrate direct binding of these repressors to the Gdnf promoter. Further, we will test whether failure of maintaining gonocyte quiescence in our NOTCH lack-of-function model leads to a carcinoma-in-situ-like (CIS-like) phenotype. In Aim 2, we will investigate the role of NOTCH signaling on the expression of CYP26B1, an enzyme that blocks germ cell differentiation. Using ChIP-PCR analysis, we will demonstrate that HEY1/HEYL transcription factors directly bind to the Cyp26b1 promoter to downregulate its expression. Finally, we will test whether overexpression of NOTCH signaling truly leads to a Sertoli cell-only syndrome through inhibition of CYP26B1. In Aim 3, using germ cell-Sertoli cells co-cultures, we will test the hypothesis that germ cells regulate NOTCH activity in Sertoli cells and therefore can regulate their own numbers. Altogether, this proposal will demonstrate for the first time that NOTCH signaling modulates the expression of two molecules essential for germ cell proliferation and maintenance of the undifferentiated state, and is a component of normal germ cell homeostasis. Dysregulation of this pathway will induce sterility or germ cell hyperplasia.

PROJECT SUMMARY Spermatogenesis is a highly coordinated process that starts with spermatogonial stem cells (SSCs), which self-renew and differentiate into more mature germ cells to sustain male fertility throughout life. Within the seminiferous epithelium, germ cell development depends on a specific environment created by somatic cells, which provide a number of growth and differentiation factors. The interplay between microenvironment and germ cells is crucial for sperm production, as dysregulation of this process will lead to sterility. Our work focuses on the function and regulation of the transcription factor RBPJ in Sertoli cells. RBPJ is the main mediator of NOTCH signaling, and belongs to a repressor/activator complex at the promoter of target genes. Depending on the cellular context, RBPJ indirectly represses or allows the expression of genes such as Gdnf and Cyp26b1, which are critical for the maintenance of undifferentiated spermatogonia. Through a yeast-2-hybrid assay, we have discovered a novel RBPJ-interacting protein in Sertoli cells, called BHC80. BHC80 is a histone reader that presumably stabilizes KDM1A (a H3K4me2 demethylase) at the repressor complex. BHC80 is encoded by the gene Phf21a and is highly expressed in the brain and testis. In humans, mutations (usually microdeletions) in the Phf21a gene cause Potocki-Saffer syndrome, which is associated with severe intellectual disabilities and craniofacial anomalies. In order to understand how BHC80 regulates RBPJ activity, and to study the function of BHC80 and its target genes beyond NOTCH signaling in Sertoli cells, we will establish a mouse Sertoli cell-specific knockout of Phf21a and characterize its testicular phenotype. To produce the floxed Phf21a mouse, we will employ a novel CRISPR/Cas9 technique called DECAI, which is predicted to generate a higher ratio of pups containing the insert. This small research project aligns with NICHD?s Fertility and Infertility?s mission and its high-priority research area of genetic basis of idiopathic male infertility. It is intended for the development of a research methodology, and the established floxed model will be an important resource for research in the areas of reproductive biology, intellectual disabilities and craniofacial anomalies. !