Motility enables sperm to reach the egg for fertilization, and normal sperm motility is largely dependent on formation and function of the flagella. An elongating spermatid-specific, the manchette, has been proposed to play a central role in sperm flagella formation. Meiosis expressed protein 1 (MEIG1) is a key protein that regulates manchette stability and sperm flagella formation. The protein was identified as a binding partner of Sperm Associated-Antigen 16L (SPAG16L), a sperm tail axoneme protein;it also binds the protein product (PACRG) of the Parkin co-regulated gene. The reproductive phenotype of Pacrg-deficient mice mirrors that of the Meig1 mutant mice. PACRG is expressed post-meiotically and is localized to the manchette. MEIG1 and SPAG16L are present in the cytoplasm of spermatocytes. However, both proteins migrate to the manchette in elongating spermatids. MEIG1 loses its manchette localization in the Pacrg-deficient elongating spermatids. In Meig1-deficient mice, SPAG16L loses its manchette localization. These observations suggest a role for MEIG1 in protein escort/targeting. MEIG1 consists of only 88 amino acids, and no functional domains were identified with bioinformatic tools. It is phosphorylated in vivo, and phosphorylation might modulate MEIG1 function, although this has not been established. Our preliminary studies using nuclear magnetic resonance (NMR) revealed that MEIG1 forms an unique structure that provides a large surface area for interaction with other proteins, and several amino acids, including several potential sites for phosphorylation in the aromatic and charged regions, may form protein-protein interaction surfaces. The long-term objective of this research is to investigate the role of MEIG1 complexes in mammalian sperm flagellogenesis. We propose three aims: 1) To dissect the SPAG16L/MEIG1/PACRG complex in vivo and in vitro;2) To identify domains of MEIG1 that mediate interactions with PACRG and SPAG16L;3). To determine the role of post-translational modification in MEIG1 function. We hypothesize that MEIG1 functions as a chaperone that associates with multiple proteins, maintains the integrity of the manchette, and plays a role in assembly of the sperm flagella. PACRG recruits MEIG1 to the manchette through binding to a specific domain of MEIG1, and MEIG1 binds other proteins such as SPAG16L through a different domain. Mutations/deletions of the domains will reduce or abolish MEIG1 interaction with these proteins. MEIG1 binds to PACRG and SPAG16L with differential binding affinities to facilitate docking of MEIG1 to PACRG associated with the manchette and off-loading of its cargo (e.g., SPAG16L). We anticipate that MEIG1 has several phosphorylated amino acids in vivo, and phosphorylation controls its affinity to cargo proteins. Little is known about the mechanisms that lead to the proper targeting and assembly of these molecules into the sperm flagellum. The research proposed in this application will, for the first time, reveal the molecular basis of the escort of proteins to the site of flagellum assembly, and the structure/function relationships of a unique chaperone that is essential for normal spermiogenesis.

Public Health Relevance

The proposed studies will reveal the molecular basis of the escort of proteins to the site of flagellum assembly, and the structure/function relationships of a unique chaperone that is essential for normal spermiogenesis. Because of the dramatic restructuring of spermatids that occurs during spermiogenesis, the process affords a unique opportunity to understand general principles of intracellular trafficking and the assembly of complex organelles. The proposed studies will not only fill a gap in our knowledge of the process of spermiogenesis, but also hold the promise of elucidating new mechanisms for protein escort and maintenance of protein stability.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD076257-02
Application #
8675898
Study Section
Cellular, Molecular and Integrative Reproduction Study Section (CMIR)
Program Officer
Moss, Stuart B
Project Start
2013-06-15
Project End
2018-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
2
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Virginia Commonwealth University
Department
Obstetrics & Gynecology
Type
Schools of Medicine
DUNS #
City
Richmond
State
VA
Country
United States
Zip Code
23298
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