Korean J Fertil Steril.  2006 Sep;33(3):189-198.

Identification of Oocyte-Specific Diva-Associated Proteins using Mass Spectrometry

• Yoon SJ
• Kim JW
• Choi KH
• Lee SH
• Lee KA

Abstract

OBJECTIVE
We previously described that Diva is highly expressed in matured metaphase II (MII) oocytes compared to immature germinal vesicle (GV) oocytes in mouse.1 We report here that the expression of Diva transcript as well as protein is oocyte-specific. To elucidate its physiological role in oocyte, the binding partner(s) of Diva has been identified by using immunoprecipitation (IP) followed by Mass Spectrometry.
METHODS
NIH/3T3 cells were transiently transfected for 24 h with either empty vector for control or FLAG-tagged mouse Diva construct, and IP was performed with anti-FLAG antibody. The immuno-isolated complexes were resolved by SDS-PAGE on a 12% gel followed by Coomassie Blue staining. For in-gel digestion, 15 bands of interest were excised manually and digested with trypsin. All mass spectra were acquired at a positive reflector mode by a 4700 Proteomics Analyzer (Applied Biosystems, Framingham, MA). Proteins were identified by searching the NCBI nonredundant database using MASCOT Peptide Mass Fingerprint software (Matrixscience, London).
RESULTS
Diva-associated complexes were formed in FLAG-tagged mouse Diva-overexpressed NIH/3T3 cells via IP using anti-FLAG-conjugated beads. Among the excised 15 bands, actin and actin-binding proteins such as tropomyosin, tropomodulin 3, and alpha-actinin were identified. Binding between Diva and actin or tropomyosin was confirmed by IP followed by Western blot analysis. Both bindings were also detected endogenously in mouse ovaries, indicating that Diva works with actin and tropomyosin.
CONCLUSIONS
This is the first report that immuno-isolated Diva-associated complexes are related to actin filament of the cytoskeletal system. When we consider the association of Diva with actin and tropomyosin, oocyte-specific Diva may play a role in modulating the cytoskeletal system during oocyte maturation.

Keyword

Diva; Oocyte-specific; Immunoprecipitation; Mass spectrometry; Oocyte maturation

MeSH Terms

Actin Cytoskeleton
Actinin
Actins
Animals
Blotting, Western
Dermatoglyphics
Digestion
Electrophoresis, Polyacrylamide Gel
Female
Immunoprecipitation
Mass Spectrometry*
Metaphase
Mice
Microfilament Proteins
Oocytes
Ovary
Proteomics
Tropomodulin
Tropomyosin
Trypsin
Actinin
Actins
Microfilament Proteins
Tropomodulin
Tropomyosin
Trypsin