description): Spermiogenesis is a sequential assembly process based on carefully timed translation of stored mRNAs in transcriptionally silent cells; however, the molecular mechanisms that determine when individual mRNAs will be translated are not well understood. A large part of the problem results from our having no reliable method of purifying sub-populations of spermatids at different stages of maturation for molecular analysis. The proposed study aims to resolve this deficiency. The applicant proposes to develop a method that uses transgenic mice that expresses the jellyfish green fluorescent protein (GFP) in post- meiotic male germ cells, in combination with fluorescence-activated cell sorting (FACS), to purify homogeneous sub-populations of staged spermatids. The goals of this project are: 1) Isolate at least 20 spermatid sub- populations by FACS. 2) Evaluate the purity and stage of spermatids represented in each sub-population based on morphological, ultrastructural, and molecular criteria; select 10 homogeneous sub-populations representing different stages spanning all of spermiogenesis. 3) Purify mRNP particle- associated mRNA (stored information) and polysome-associated mRNA (active information) from each of these homogeneous sub-populations, prepare a cDNA library from each mRNA sample, and evaluate each by RT-PCR. This work will establish a reliable protocol for sorting spermatids and will yield ordered libraries of most or all of the active and stored spermiogenic information. Our long-term goals are to use this technology to catalog and characterize most or all spermiogenic mRNAs and to determine when each one is used during sperm maturation. In the current submission, the applicant is requesting two years of R03 funding to rigorously establish the technology on which this research, as well as countless other molecular investigations on spermiogenesis, can be founded.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Small Research Grants (R03)
Project #
1R03HD039242-01
Application #
6163562
Study Section
Pediatrics Subcommittee (CHHD)
Program Officer
Rankin, Tracy L
Project Start
2000-08-01
Project End
2002-07-31
Budget Start
2000-08-01
Budget End
2001-07-31
Support Year
1
Fiscal Year
2000
Total Cost
$68,280
Indirect Cost
Name
Montana State University - Bozeman
Department
Veterinary Sciences
Type
Schools of Earth Sciences/Natur
DUNS #
625447982
City
Bozeman
State
MT
Country
United States
Zip Code
59717
Tucker, Tammy A; Kundert, Jean A; Bondareva, Alla A et al. (2005) Reproductive and neurological Quaking(viable) phenotypes in a severe combined immune deficient mouse background. Immunogenetics 57:226-31
Schmidt, Edward E; Bondareva, Alla A; Radke, Jay R et al. (2003) Fundamental cellular processes do not require vertebrate-specific sequences within the TATA-binding protein. J Biol Chem 278:6168-74
Sealey, Amy L; Hobbs, Nicole K; Schmidt, Edward E (2002) Molecular genotyping of the mouse scid allele. J Immunol Methods 260:303-4
Hobbs, Nicole K; Bondareva, Alla A; Barnett, Sheila et al. (2002) Removing the vertebrate-specific TBP N terminus disrupts placental beta2m-dependent interactions with the maternal immune system. Cell 110:43-54