The major focus of the Walker laboratory is to define how hormonal and environmental signals are transduced into changes in gene expression. 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 to support the production of spermatozoa.

One area of concentration is our new finding that the CREB transcription factor is rapidly phosphorylated (within 1 min) and activated after addition of testosterone to Sertoli cells isolated from rat testes. This novel action of androgen does not appear to be mediated through classical pathways involving the androgen receptor directly activating gene expression. Instead, testosterone induces CREB phosphorylation through signaling pathways initiated through the androgen receptor that require calcium influx and/or the MAP kinase cascade. This finding is significant as it not only allows for the characterization of a unique signaling cascade for androgen but it also may provide a mechanism by which testosterone can support spermatogenesis. The goals of this major focus of the laboratory include 1) characterization of the signaling pathways activated by androgen and 2) identification CREB and MAP kinase-regulated genes induced by androgen in Sertoli cells 3) identifying the physiological importance of the novel testosterone actions in vivo. Thus far, studies suggest that testosterone-bound androgen receptor recruits Src kinase that then activates the MAP kinase cascade through intermediary proteins. Microarray data has identified a number of potential gene targets for testosterone-mediated activation of the MAP kinase pathway and has opened a new area of interest - adhesion proteins responsible for maintaining contact between Sertoli cells and spermatid germ cells. Once the testosterone and MAP kinase regulation of this category of genes has been confirmed, we hope to initiate in vivo studies in which MAP kinase actions are blocked in Sertoli cells to determine whether germ cells will be prematurely released from Sertoli cells. These findings would then explain at least one mechanism by which androgen acts to maintain fertility.
In another study that is underway, a repressor of helix-loop-helix transcription factors named Id2 was found to be transiently induced by FSH in Sertoli cells and the promoter region required for stimulation was identified. Preliminary studies show that the Id2 protein represses transcription from the androgen receptor promoter suggesting that transient induction of Id2 may be responsible for the reported delayed induction of androgen receptor gene expression by FSH and cAMP. Because Id2 activity is also required for the proliferation of many cell types, studies are in progress to determine the molecular mechanisms and signaling pathways by which Id2 regulates the FSH-induced expansion of Sertoli cells prior to puberty. Some of the goals of this project are to determine the relative expression levels of proliferation-promoting Id proteins versus differentiation-inducing helix loop-helix transcription factors throughout post natal Sertoli cell development. The mechanisms by which thyroid hormone and helix-loop-helix transcription factors promote differentiation and the cessation of Sertoli cell proliferation are also being investigated. Finally, the regulation of cell cycle controlling genes by Id and helix-loop-helix transcription factors will be addressed.

Ongoing studies in the lab seek to characterize the factors that modulate expression of the CREB gene in the testis of rodents and monkeys and identify genes critical for progression of spermatogenesis that are regulated by cAMP and the CREB family of transcription factors. The results of this work may then lead to information needed to provide therapies for infertility and solutions for male contraception.

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 the retention and release of spermatozoa in the testis.
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 thyroid hormone and E-box transcription factors cause the Sertoli cell to differentiate.
3) The use of tissue grafts onto mice as bio-incubators to develop testes and produce sperm. In collaboration with Stefan Schlatt, we are studying the development of Sertoli cells grafted onto the backs of nude mice. Our studies will focus on determining whether Sertoli cells mature naturally in the grafts or can be induced to differentiate in the grafts to support germ cell production.