Hormonal regulation of Sertoli cell signaling pathways, gene expression and differentiation

Description of projects available to graduate students:
Research Interest: Hormonal regulation of Sertoli cell signaling pathways, gene expression and differentiation

The major focus of the Walker laboratory is to define how hormonal and environmental signals are transduced into changes in the activity of signaling pathways, gene expression and the differentiation of Sertoli cells in the testis. The mammalian testis is used as a model system to determine how follicle-stimulating hormone, testosterone and other hormones alter cell signaling pathways to regulate the genes that are required for male fertility. Research in the Walker lab focuses on two major topics: 1) The molecular mechanisms of testosterone in the Sertoli cells that are required to support of male fertility and 2) The regulation of Sertoli cell differentiation

1) Testosterone is essential for male fertility. This steroid hormone acts in the testis through the androgen receptor (AR) in the Sertoli cell to support male germ cell development and survival as well as the release of mature spermatozoa. However, there is a lack of information regarding the molecular mechanisms by which testosterone acts to support spermatogenesis and fertility.
Our lab recently characterized a rapid (<1 min) and sustained mechanism of testosterone action (the non-classical pathway) that causes the phosphorylation and activation of the Src and ERK kinases, as well as the CREB transcription factor. This pathway of testosterone action was also found to be required for germ cells to attach to Sertoli cellls and for the relase of mature sperm. Thus, the non-classical pathway is required for at least two processes that are critical for male fertility.
We are developing transgenic mice in which endogenous AR is replaced only on Sertoli cells with AR mutants that can activate only the non-classical or the classical pathway of testosterone signaling. We will identify the spermatogeneic processes and factors that are regulated by each pathway in vivo. We will also translate our findings from the mouse models to studies of monkey and human testis ex vivo models. The result of these studies will provide 1) information needed to treat specific male infertility conditions and 2) long-needed new targets for male contraceptive research.

2) The number of Sertoli cells present in the testis determines the number of sperm that can be produced and thus the limit of male fertility. During puberty Sertoli cells differentiate and stop proliferating. Because Sertoli cells rarely die, the number of Sertolic ells present aafter puberty remains constant. We are studying the mechanism that determine the final number of Sertoli cells. Specifically, we are investigating the factors that cause Sertoli cells to differentiate. We have found that the expression and activity of USF transcription factors increases dramatically during Sertoli cell differentiation and that USF is an important regulator of differentiation-associated genes. In the future, we will investigate whether USF is required for Sertoli cell differentiation and determine what factors cause the increase in USF expression observed during Sertoli cell differentiation.

Opportunities for graduate training include:
1. The molecular mechanisms of testosterone action in the Sertoli cell. The aim of this project is to determine how testosterone acts via non-classical (non-genomic) mechanisms to rapidly (within 5 min) activate kinases and transcription factors in Sertoli cells. Studies are underway to determine whether cell signaling pathways initiated by testosterone are required for male fertility. New inhibitors of non-classical testosterone signaling and targets for male contraceptives are being investigated.
2) Regulation of Sertoli cell proliferation and differentiation. The aim of this project is to determine the molecular mechanisms that cause Sertoli cells to stop dividing and initiate differentiation. Studies focus on the factors that support Sertoli cell proliferation as well as the mechanisms by which E-box transcription factors cause the Sertoli cell to differentiate.

Recent publications
1: Shupe J, Cheng J, Puri P, Kostereva N, Walker WH. Regulation of sertoli-germ cell adhesion and sperm release by FSH and nonclassical testosterone signaling. Mol Endocrinol. 2011 Feb;25(2):238-52. Epub 2010 Dec 22. PubMed PMID: 21177760.
2: Walker WH. Non-classical actions of testosterone and spermatogenesis. Philos Trans R Soc Lond B Biol Sci. 2010 May 27;365(1546):1557-69. Review. PubMed PMID: 20403869; PubMed Central PMCID: PMC2871922.
3: Gassei K, Ehmcke J, Wood MA, Walker WH, Schlatt S. Immature rat seminiferous tubules reconstructed in vitro express markers of Sertoli cell maturation after xenografting into nude mouse hosts. Mol Hum Reprod. 2010 Feb;16(2):97-110. Epub 2009 Sep 21. PubMed PMID: 19770206; PubMed Central PMCID: PMC2803008.
4: Viswanathan P, Wood MA, Walker WH. Follicle-stimulating hormone (FSH) transiently blocks FSH receptor transcription by increasing inhibitor of deoxyribonucleic acid binding/differentiation-2 and decreasing upstream stimulatory factor expression in rat Sertoli cells. Endocrinology. 2009 Aug;150(8):3783-91. Epub 2009 May 7. PubMed PMID: 19423764; PubMed Central PMCID: PMC2717885.
5: Walker WH. Molecular mechanisms of testosterone action in spermatogenesis. Steroids. 2009 Jul;74(7):602-7. Epub 2008 Nov 27. Review. PubMed PMID: 19095000.
6: Wood MA, Walker WH. USF1/2 transcription factor DNA-binding activity is induced during rat Sertoli cell differentiation. Biol Reprod. 2009 Jan;80(1):24-33. Epub 2008 Sep 3. PubMed PMID: 18768914; PubMed Central PMCID: PMC2804807.

Techniques graduate student will learn:
1) Analyses of in vivo gene expression (RT-PCR, RNAse protection and Northern assays)
2) Transfection of mammalian cells to assay gene expression
3) Identification of regulatory sequences in gene promoters (mutagenesis, subcloning, computer-assisted DNA sequence analysis)
4) Assays of DNA-protein interactions (EMSA, footprinting)
5) Analysis of cellular protein levels (SDS-PAGE, Western Blotting)
6) Localization of proteins to specific cells in tissue (immunohistochemistry)
7) Culturing of primary cell cultures from tissues
8) Construction of plasmid and viral vectors
9) Histological analysis of gene and protein expression in testis tissue by light and fluorescence microscopy
10) Analysis of cell signaling pathways
11) Assays of hormoal stimulation of target tissues and cells