The online version of this article (doi:10.1186/1477-7827-10-101) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
SKP carried out sperm motility assays and analyzed the data. JY carried out the sperm motility assay and immunohistochemistry. LW prepared sperm cells from wild type and mutant mice. TKS participated sperm motility assay. KM analyzed GWRQ-MAPS peptide by mass spectrometry. KJS purified GWRQ-MAPDS and genotyped trophinin gene knockout mouse. MSC and SHL analyzed the data. NT participated sperm motility assay. SH participated the data analysis. DN generated trophinin gene knockout mouse line. KS and MNF design the study, analyzed the data and drafted the manuscript. All authors read and approved the final manuscript.
Trophinin is an intrinsic membrane protein that forms a complex in the cytoplasm with bystin and tastin, linking it microtubule-associated motor dynein (ATPase) in some cell types. Previously, we found that human sperm tails contain trophinin, bystin and tastin proteins, and that trophinin-binding GWRQ (glycine, tryptophan, arginine, glutamine) peptide enhanced motility of human sperm.
Immunohistochemistry was employed to determine trophinin protein in mouse spermatozoa from wild type mouse, by using spermatozoa from trophinin null mutant mice as a negative control. Multivalent 8-branched GWRQ (glycine, tryptophan, arginine, glutamine) peptide or GWRQ-MAPS, was chemically synthesized, purified by HPLC and its structure was confirmed by MALDI-TOF mass spectrometry. Effect of GWRQ-MAPS on mouse spermatozoa from wild type and trophinin null mutant was assessed by a computer-assisted semen analyzer (CASA).
Anti-trophinin antibody stained the principal (central) piece of the tail of wild type mouse sperm, whereas the antibody showed no staining on trophinin null sperm. Phage particles displaying GWRQ bound to the principal piece of sperm tail from wild type but not trophinin null mice. GWRQ-MAPS enhanced motility of spermatozoa from wild type but not trophinin null mice. CASA showed that GWRQ-MAPS enhanced both progressive motility and rapid motility in wild type mouse sperm.
Present study established the expression of trophinin in the mouse sperm tail and trophinin-dependent effect of GWRQ-MAPS on sperm motility. GWRQ causes a significant increase in sperm motility.
Additional file 1: Figure S1. Purification and validation of synthetic GWRQ-MAPS peptide. Gel filtration of crude GWRQ-MAPS (A), HPLC purification (B), and mass spectrometry analysis of purified GWRQ-MAPS (C). HPLC showed purity of GWRQ-MAPS more than 99%. Signal at m/z 5120.835 (major) is theoretical mass for GWRQ-MAPS, 5135.886, minus 15. Apparent dimer (m/z 10244.897) and trimer (m/z 15335.252) were formed during storage after HPLC purification. (TIFF 2 MB)12958_2012_1037_MOESM1_ESM.tiff
Additional file 2: Figure S2. Dose-dependency of GWRQ-MAPS on motility of wild type mouse sperm. Fresh spermatozoa from three C57BL/ 6 mice were analyzed by CASA. Each bar represents the result obtained by ten randomly selected fields each containing >100 spermatozoa. Asterisks show statistical significance (p<0.05). (TIFF 814 KB)12958_2012_1037_MOESM2_ESM.tiff
Additional file 3: Figure S3. CASA parameters. Data were collected by analyzing fresh sperm frosignificant (pm wild type (n=10) and trophinin null (n=3) mice. Asterisks represent statistical < 0.05) by unpaired two-tailed t-test. N. S., not significant. (TIFF 1 MB)12958_2012_1037_MOESM3_ESM.tiff
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- Enhancement of mouse sperm motility by trophinin-binding peptide
Seong Kyu Park
Toshiaki K Shibata
Kyung Jun Shim
Mun Seog Chang
Seung Ho Lee
Michiko N Fukuda
- BioMed Central
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