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Barry T. Hinton |
Professor
Department of Cell Biology, University of Virginia Health Sciences Center, P.O. Box 800732, Charlottesville, Virginia 22908, USA.
Tel: +1 434-924-2174 Fax: +1 434-982-3912
Email: bth7c@virginia.edu |
Research Description
The goal of this laboratory is to understand the mechanisms by which the epididymal epithelium is able to maintain a specialized luminal fluid microenvironment that is critically important for the maturation and survival of spermatozoa. This laboratory utilizes in vivo micropuncture and microperfusion techniques, immunochemical, microanalytical and modern molecular biological techniques to achieve this goal. There are four major projects currently ongoing:
(1) To test the hypothesis that growth factors of Sertoli cell origin enter the epididymal duct and bind to their receptors on the apical surface of the epididymal cells. Second messengers are then activated which in turn activate transcription factors that transactivate specific epididymal genes. The gene of interest in this laboratory is gamma-glutamyl transpeptidase (GGT) which plays a role in the metabolism of glutathione, an important antioxidant. Multiple GGTs are expressed in the epididymis and each are under the control of specific promoters. GGT mRNA IV is of special interest because it is regulated by testicular factors. Our published and unpublished data has led us to hypothesize that fibroblast growth factors (FGF) from Sertoli cells interact with specific FGF receptors (FGFR-1) localized on the apical surface of epididymal cells, activate the MAP kinase pathway which in turn activate members of Ets transcription factor family, in particular PEA3, ERM and ER81. These transcription factors then transactivate GGT mRNA IV. We are using a novel in vivo electroporation technique to examine the transactivation of GGT IV promoter and laser capture techniques to identify the FGFR types in specific epididymal cells. We are currently investigating those proteins, e.g. Grb2 and FRS2, that bind to FGFR-1 and regulate its activity.
(2) Fluid homeostasis is critical for normal development and function of the male reproductive system particularly the epididymis. Alterations in fluid balance can have adverse effects and may be the source of certain forms of male infertility. Our laboratory is interested in the expression and regulation of transport proteins responsible for regulating the epididymal fluid microenvironment. We are currently studying the expression and regulation of a transport protein that may be responsible for transporting L-carnitine into the lumen of the epididymis. L-carnitine is present in high concentrations in the epididymal tissues of many species and studies suggest that L-carnitine may be involved in water homeostasis. L-carnitine is transported against a concentration gradient greater than 2000-fold, with intraluminal concentrations reaching as high as 50mM in the cauda epididymides of the rat. Studies in our laboratory have demsontrated that OCTN2, a member of the organic cation transporter family, is expressed in the rat epididymis in a region-dependent manner with highest expression observed in the regions previously shown to be involved in L-carnitine uptake. Moreover, we localized OCTN2 protein to the basolateral region of epididymal cells suggesting that this protein is likely responsible for the transport of L-carnitine across the epididymal epithelium. We are currently using gene-silencing technologies to test the hypothesis that OCTN2 is responsible for transporting L carnitine into the lumen of the epididymis. Further, we have been performing experiments testing the hypothesis that OCTN2 and the apical organic solute transporters, FLIPT1 and 2 are regulated by changes in osmolarity.
(3) We are investigating the role of the orphan receptor protein tyrosine kinase, c-Ros, in the post-meiotic, epididymal maturation of sperm. This gene is involved in the development of the initial segment (IS) of the epididymis. Male mice with a knock-out mutation in the gene for c-Ros are infertile, and have IS's that are small and poorly developed; however, these mice appear to be normal in all other aspects. As expected, c-Ros is expressed in the developing IS, as well as several other tissues during embryogenesis. However, c Ros may play a role in the sperm maturation process since this gene is expressed at high levels in the IS of adult male mice. We are attempting to experimentally separate these two roles of c-Ros to determine the function of c-Ros in the adult male. Since the ligand for this receptor is unknown and there are no specific inhibitors for the protein kinase, we are generating conditional knock-out mice to regulate the expression of the c-Ros gene in a temporal fashion. We will allow the IS to develop normally through puberty, and then turn off the c-Ros gene in the adult. We will then analyze the resulting phenotype. We are also interested in the signalling pathways and downstream genes controlled by this receptor protein tyrosine kinase, as well as interacting adaptor proteins and phosphatases.
(4) We have begun experiments to understand the regulation of tubular morphogenesis of the Wolffian/Mesonephric duct. At E14 the mouse developing Wolffian duct is approximately 1 mm in length and grows to approximately 1 m. The mechanisms by which this tube undergoes proliferation and differentiation is unknown. Preliminary evidence would suggest that growth factors and the hedeghog signaling pathway are required for normal growth, folding and differentiation.
Publications (.doc)
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