Leslie L. Heckert - US grants

Sertoli and granulosa cells form an important somatic component of the gonads. These cells help regulate germ cell development by providing essential nutrients and regulatory signals that support their maturation. In turn, Sertoli and granulosa cells are influenced by a host of cellular associations and intricate sets of endocrine and paracrine signals. The complexities of this system serve to underscore the need to develop animal models to study how Sertoli and granulosa cell factors impact gonadal function. Such studies will ultimately improve approaches for birth control and for treating gonadal abnormalities and infertility. SF-1 and CREB emerge as proteins critical for Sertoli and granulosa cell function. These proteins, like many others, have been characterized within these cells but their role in mediating germ cell development remains obscure. This proposal tests the importance of these proteins, while developing a new transgenic approach that will specifically over- express proteins in Sertoli and granulosa cells. This work will not only provide valuable animal models to elucidate gonadal functions of SF-1 and CREB but will yield important technology that is advantageous to many, as it provides a means to create animal models that express elevated levels of proteins while using a weak but highly specific promoter. Four lines of transgenic mice will be created. Two will contain transgenes in which the Cre recombinase gene is expressed from the follicle-stimulating hormone receptor promoter. This promoter is active only in Sertoli and granulosa cells of the gonads and therefore Cre recombinase will reside only in these cells of transgenic mice. Two other lines f mice will contain targeting transgenes designed to over- express either SF-1 or a dominant negative form of CREB from a strong ubiquitous promoter. The genes for these proteins will not be active unless a recombination event occurs which is facilitated by Cre recombinase when Cre expressing mice are mated with mice harboring the targeting transgenes. Recombination and therefore over-expression of SF-1 and CREB will occur only in Sertoli and granulosa cells of doubly transgenic mice.

DESCRIPTION: (adapted from the applicant's abstract) The objective of this application is to analyze the promoter for the FSHR gene for cis-acting elements and, ultimately, transcription factors that interact to mediate hormonal responsiveness and Sertoli cell-specific expression of this gene. In aim 1, scanning mutagenesis of the proximal promoter of the FSHR gene will be used to identify DNA response elements important for expression in a Sertoli cell line and primary cultures of Sertoli cells. In aim 2, the effects of FSH, EGF, and TGF-beta on the expression of the FSHR gene will be studied. First, the mRNA encoding the FSHR following treatment of primary cultures of Sertoli cells with either FSH, EGF, or TGFbeta will be analyzed. Second, the transcriptional response of the FSHR promoter to these same substances will be addressed by transient expression assays in Sertoli cells. Third, co-expression of FSHR promoter constructs with constitutively active forms of intracellular kinases that are involved in either FSH, EGF, or TGFbeta signal transduction will be performed.

Steroidogenic factor 1 (SF-1), or Ad4bp, is a member of the nuclear hormone receptor family that is important for normal development and steroid hormone production. The transcription factor regulates a variety of genes important to the endocrine system and is essential for formation of adrenal glands and gonads. Expression and gene targeting studies indicated that SF-1 acts early in adrenal and gonad formation, is regulated in a sexually dimorphic manner, and is important for the function of these tissues after birth. The critical nature of this transcription factor reveals the need to examine mechanisms important to its regulation and function. The major goal of this proposal is to identify and characterize transcription factors regulating SF-1 in the gonads. This will ultimately lead to the discovery of new proteins important for gonadal development and function, proper endocrine regulation, and the enhancement of our understanding of factors controlling fertility. Using transient transfection analysis (aim I) and transgenic mouse studies (aim III), we will uncover all the needed elements for correct spatial and temporal expression of SF- 1. Protein/DNA interactions will be examined using electrophoretic mobility shift assays and in vivo footprinting, leading to identification and cloning of important proteins. In addition, studies are proposed (aim II) to investigate two proteins, USF1 and USF2, that we have shown interact with an important SF-1 promoter element (E-box) in the testis, and thus are implicated in its regulation. Immunohistochemistry and in situ hybridization analysis will be used to examine the expression of these proteins, while co-transfection studies will investigate the direct regulation of the SF-1 promoter by these transcription factors.

Dmrt1 is a recently described gene encoding a putative transcription factor whose structural features, expression profile, and chromosomal localization suggest it is important for testis development and function. Furthermore, male mice lacking Dmrt1 are infertile due to defects in Sertoli cell differentiation and germ cell survival. The goals of the proposed research are to identify both the transcriptional mechanisms responsible for testis-specific expression of Dmrt1 and the means by which Dmrt1 regulates testis differentiation. Aim I, proposes to identify the regulatory elements and binding proteins necessary for cell-specific expression and hormonal regulation of Dmrt1. Elements will be identified and characterized by transient transfection analysis followed by protein binding studies to identify relevant regulatory proteins. Transgenic mice carrying a LacZ reporter driven by various lengths of the Dmrt1 promoter will be generated and the expression profile for betagalactosidase determined with respect to temporal, spatial, and hormonal regulation. Aim I also proposes to evaluate FSH regulation of the Dmrt1 promoter and identify the responsible regulatory elements and proteins. This will involve characterization of promoter mutants by transient transfection analysis in the presence and absence of added hormone and evaluation of relevant DNA-protein interactions. Aim II, will examine the DNA binding and transcriptional properties of Dmrt1 using in vitro binding assays and transient transfection analysis. This will employ a PCR-based assay to identify randomized DNA sequences to which Dmrt1 binds and co-transfection experiments to evaluated Dmrt1 transcriptional activity. Finally, downstream target genes will be identified by both searching sequence databases for candidate genes using an identified high-affinity binding site for Dmrt1 and microarray analysis to compare the expression profile between Dmrt1 expressing and non-expressing cell lines. Dmrt1's role as an important regulator of testis function suggests that identification of factors acting upstream and downstream of it will provide insight into the genetic processes that regulate gene expression in the testis and enhance our understanding of the molecular events necessary for testis differentiation and fertility.

DESCRIPTION (provided by applicant): Fshr is a critical component of the endocrine axis that controls gonad function, gametogenesis, and fertility. Characterization of the mechanisms regulating Fshr transcription provides the opportunity to reveal proteins involved in selective gene expression in Sertoli and granulosa cells and expand our understanding of factors regulating reproductive health. To date, transcriptional studies of Fshr have focused only on promoter and 5'flanking sequences. While these analyses are critical to our understanding of Fshr, it appears that regulatory sequences outside this region are important for Fshr transcriptional regulation. Fortunately, our ability to identify such regulatory sequences has advanced significantly due to the extensive progress in genome research, which provides a wealth of new information and resources. The proposed studies combine computation analysis, transcriptional assays, and transgenic approaches to further our understanding of the processes controlling Fshr transcription. In Aim I, studies are proposed to use mouse knockout models and chromatin immunoprecipitation to evaluate the in vivo roles of transcription factors implicated in Fshr regulation and expand our understanding of the mechanisms regulating transcription through the proximal promoter. In Aim II, comparative genomics, DNase I hypersensitivity mapping and transient transfection analysis will be used to identify distal regulatory regions within the Fshr locus. DNA/protein binding studies will be used to identify proteins associated with important regulatory elements. Aim III proposes to use transgenic mice for in vivo characterization of Fshr regulatory elements. An Fshr-containing yeast artificial chromosome (YAC) will be used to generate transgenic mice that will be characterized for expression in various tissues. Mutations within proposed regulatory elements will be generated in the YAC by homologous recombination and tested for correct expression in transgenic mice, to evaluate their role in vivo. The proposed studies will reveal regulatory elements and proteins needed for Fshr expression, leading not only to a better understanding of its transcriptional control but to the basic principles governing gonadal function, fertility, and generalized gene transcription. By advancing our understanding in these areas, we enhance our ability to evaluate matters of human reproductive health and to develop strategies to assist fertility problems and management.

21+ years old; Adult; Animals; Antibodies; Assay; Band Shift Mobility Assay; Bandshift Mobility Assay; Binding; Binding (Molecular Function); Binding Sites; Bioassay; Biologic Assays; Biological; Biological Assay; Bromide, Ethidium; CHIP assay; Cell Survival; Cell Viability; Cells; ChIP (chromatin immunoprecipitation); Combining Site; Common Rat Strains; Computer Simulation; Computerized Models; Contraceptive Agents; Contraceptive Agents, Male; Contraceptives; Contraceptives, Male; Cultured Cells; Development; Drug Delivery; Drug Delivery Systems; Drug Targeting; Drug Targetings; Electrophoretic Mobility Shift Assay; Elements; Expression Profiling; Expression Signature; Fecundability; Fecundity; Fertility; Fluorescence; Fluorescence Polarization; Future; Gametes; Gene Targeting; Genes; Genital System, Male, Testis; Germ Cells; Germ-Line Cells; Goals; Gonadal structure; Gonads; High Throughput Assay; Homidium Bromide; Human, Adult; In Vitro; Kansas; Laboratories; Lead; Male Contraceptive Agents; Mammals, Rats; Mathematical Model Simulation; Mathematical Models and Simulations; Mobility Shift Assay; Models, Computer; Molecular; Molecular Fingerprinting; Molecular Interaction; Molecular Profiling; Monitor; Pb element; Physiologic; Physiological; Production; Proteins; Rat; Rattus; Reactive Site; Reproductive Cells; Response Elements; Sertoli Cells; Sex Cell; Simulation, Computer based; Spermatogenesis; Structure of sertoli cell; Sustentacular Cell of Testis; Targetings, Gene; Testicles; Testing; Testis; Transfection; Universities; adult human (21+); base; chromatin immunoprecipitation; computational modeling; computational models; computational simulation; computer based models; computerized modeling; computerized simulation; contraceptive target; design; designing; drug development; drug discovery; gel shift assay; gene product; heavy metal Pb; heavy metal lead; high throughput screening; in silico; in vivo; inhibitor; inhibitor/antagonist; initial cell; interest; male; male antifertility drug; molecuar profile; molecular signature; novel; sertoli cell; sexual cell; small molecule; transcription factor; virtual simulation

Project Summary/Abstract.! This R13 application requests funds to support the XXIVth North American Testis Workshop, to be held April 19 - April 22, 2017 in Miami, Florida. The requested funds are intended to help defray travel costs for 10 young investigators, selected for short talks from submitted abstracts, and housing costs for 20 invited speakers. Since 1972, the North American Testis Workshop has been the premiere international forum for basic and clinician-scientists to present and discuss their recent findings on testis regulation and function. The main goals for the Workshop are to: 1) facilitate solutions for the high incidence of male infertility and the need for a safe, reversible male contraceptive and 2) foster development of the field's future leaders, with emphasis on trainees, women and underrepresented minorities.! The Workshop's theme is ?From Testis Differentiation to Sperm Production?. The program includes four major lectures (the keynote address and three benchmark lectures) and 16 invited talks that are grouped into six sessions as follows: Germline Establishment & Homeostasis (Session I), Germ cell Differentiation and Maintenance: Role of RNA & RBPs (Session II), Testis Development & Differentiation (Session III), Transcriptional & Endocrine Regulation in the Testis (Session IV), Sperm Development & Maturation (Session V), and Genetics & Epigenetics of Male Reproduction (Session VI). After invited talks, sessions will end with short talks selected from abstracts submitted by young investigators (trainees and Assistant Professors in rank for no more than 2 years). Two poster sessions, with all posters displayed throughout the meeting, are also planned. The meeting will begin with the keynote address by Dr. Dirk de Rooij, from Utrecht University, The Netherlands. Dr. de Rooij is a longstanding leader in the field of testis biology who has contributed extensively to our understanding of the testis, by documenting fundamental characteristics of different testicular cell types and their relation to spermatogenesis and male fertility. His presentation will offer new insights on spermatogonial renewal and differentiation in humans and non-primates and provide a strong foundation for many subsequent presentations. Dr. Haifan Lin, from Yale University, will give the first benchmark lecture. Dr. Lin is internationally recognized for his research on germline self-renewal and development and the Piwi-piRNA pathway and its role in epigenetic programming and posttranscriptional regulation. His presentation will provide a strong foundation for Sessions I & II. Dr. John Aitken, from the University of Newcastle, Australia, will present the second benchmark lecture. Dr. Aitken is a leading authority on male infertility and his lecture is relevant to presentations throughout the meeting but mostly to those in Sessions V and VI. Dr. Oliver Rando, from the University of Massachusetts, will give the third benchmark lecturer. Dr. Rando is well known for his research on chromatin and epigenetic inheritance and his talk on the sperm epigenome, paternal inheritance and influences from diet and the environment merges nicely with topics in Sessions V and VI.