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Expression and localization of five members of the testis-specific serine kinase (Tssk) family in mouse and human sperm and testis.

Molecular human reproduction (2010-08-24)
Yahui Li, Julian Sosnik, Laura Brassard, Michael Reese, Nikolay A Spiridonov, Tonya C Bates, Gibbes R Johnson, Juan Anguita, Pablo E Visconti, Ana M Salicioni
ABSTRACT

Members of the testis-specific serine/threonine kinases (Tssk) family may have a role in sperm differentiation in the testis and/or fertilization. To gain insight into the functional relevance of these kinases, their expression was examined both at the mRNA and protein levels. Quantitative PCR analysis confirmed that all five Tssk mRNAs are almost exclusively expressed postmeiotically in the testis. Recombinant mouse and human Tssks were cloned and used for validation of an array of commercial and custom-made antibodies against Tssks. Immunolocalization in mouse testis, and in mouse and human sperm, showed that Tssk1, Tssk2, Tssk4 and Tssk6, but not Tssk3, were present in mouse sperm and in germ cells from mouse testis. TSSK1, TSSK2 and TSSK6 were also detected in human sperm, while TSSK3 was absent. In both mouse and human sperm, Tssk1 was partially soluble, while Tssk2, Tssk4 and Tssk6 were insoluble in non-ionic detergents. In vitro recombinant TSSK2 activity assays showed maximum enzymatic activity at 5 mM Mg(2+) and a Km for ATP of ∼10 µM. These, observations together with findings that the Tssk1/Tssk2 double knock-out as well as the Tssk6 null mice are sterile without presenting other detectable defects, suggest that these kinases could be used as targets for male contraception.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Protein Kinase A from bovine heart, ≥0.4 units/μg protein, lyophilized powder
Sigma-Aldrich
Protein Kinase CβI isozyme human, ≥95% (SDS-PAGE), recombinant, expressed in baculovirus infected insect cells, buffered aqueous glycerol solution
Sigma-Aldrich
Protein Kinase Cζ isozyme human, ≥75% (SDS-PAGE), recombinant, expressed in baculovirus infected insect cells, buffered aqueous solution
Sigma-Aldrich
Protein kinase CβII isozyme human, >85% (SDS-PAGE), recombinant, expressed in baculovirus infected insect cells, buffered aqueous glycerol solution
Sigma-Aldrich
Protein Kinase Cδ isozyme human, >95% (SDS-PAGE), recombinant, expressed in baculovirus infected insect cells, buffered aqueous glycerol solution
Sigma-Aldrich
Protein kinase Cγ isozyme human, >95% (SDS-PAGE), recombinant, expressed in baculovirus infected insect cells, buffered aqueous glycerol solution

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