Funds are requested to carry out projects on the function of proteins of the Crisp superfamily in the male reproductive tract. Three Crisp genes encode proteins in an organ-specific manner: Crisp-2 is testis- specific, Crisp-3 is submandibular gland-specific and Crisp-1 is expressed primarily in the epididymis. Crisp genes are found in mammals as diverse as rats, guinea pigs, mice and humans and so must have an important function in the organs within which they are expressed. Crisp-1 proteins are synthesized and secreted by the epididymal epithelium and subsequently bind to sperm in a domain-specific manner in rats. Protein D, which binds both loosely and tightly, localizes to the head of the sperm on the plasma membrane overlying the acrosome and has been shown to play a role in fertilization. Protein E binds specifically to plasma membrane of the tail, and also participates in fertilization. Proteins D and E have significant homology to helothermine (a salivary gland toxin that blocks ryanodine receptors) and to a number of insect and snake venom proteins that are capable of regulating ion channels. We hypothesize that proteins D and E function to regulate sperm ion fluxes, particularly calcium. In the first specific aim we explore the roles of Crisp-1 during sperm-egg fusion. In the second aim, we explore their role in capacitation and the acrosome reaction, both processes that require large ion fluxes. Finally, in the last specific aim we address the question of mechanism(s) that mediate binding of protein D and E to sperm in the epididymis. We hypothesize that the signal for domain localization resides in the NH2 terminus of the molecule and the carboxyl terminus, where 14 of the 16 cysteines are found, provides the plasma membrane binding domain. These studies will contribute significant knowledge on the role of a potentially important group of proteins both in the physiology of the epididymis and in fertilization.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD011962-22
Application #
6520746
Study Section
Reproductive Biology Study Section (REB)
Program Officer
Rankin, Tracy L
Project Start
1978-09-29
Project End
2004-03-31
Budget Start
2002-04-01
Budget End
2004-03-31
Support Year
22
Fiscal Year
2002
Total Cost
$208,828
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Liu, Mian; Hamilton, David W; Barany, George (2010) Solid-phase synthesis and evaluation of glycopeptide fragments from rat epididymal cysteine-rich secretory protein-1 (Crisp-1). Molecules 15:6399-410
Roberts, Kenneth P; Ensrud-Bowlin, Kathy M; Piehl, Laura B et al. (2008) Association of the protein D and protein E forms of rat CRISP1 with epididymal sperm. Biol Reprod 79:1046-53
Roberts, Kenneth P; Johnston, Daniel S; Nolan, Michael A et al. (2007) Structure and function of epididymal protein cysteine-rich secretory protein-1. Asian J Androl 9:508-14
Roberts, Kenneth P; Wamstad, Joseph A; Ensrud, Kathy M et al. (2003) Inhibition of capacitation-associated tyrosine phosphorylation signaling in rat sperm by epididymal protein Crisp-1. Biol Reprod 69:572-81
Roberts, Kenneth P; Ensrud, Kathy M; Hamilton, David W (2002) A comparative analysis of expression and processing of the rat epididymal fluid and sperm-bound forms of proteins D and E. Biol Reprod 67:525-33
Roberts, K P; Hoffman, L B; Ensrud, K M et al. (2001) Expression of crisp-1 mRNA splice variants in the rat epididymis, and comparative analysis of the rat and mouse crisp-1 gene regulatory regions. J Androl 22:157-63
Klemme, L M; Roberts, K P; Hoffman, L B et al. (1999) Cloning and characterization of the rat Crisp-1 gene. Gene 240:279-88
Xu, W; Ensrud, K M; Hamilton, D W (1997) The 26 kD protein recognized on rat cauda epididymal sperm by monoclonal antibody 4E9 has internal peptide sequence that is identical to the secreted form of epididymal protein E. Mol Reprod Dev 46:377-82
Xu, W; Hamilton, D W (1996) Identification of the rat epididymis-secreted 4E9 antigen as protein E: further biochemical characterization of the highly homologous epididymal secretory proteins D and E. Mol Reprod Dev 43:347-57
Siiteri, J E; Ensrud, K M; Moore, A et al. (1995) Identification of osteopontin (OPN) mRNA and protein in the rat testis and epididymis, and on sperm. Mol Reprod Dev 40:16-28

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