Enhancement of Human Sperm Motility by Trophinin Binding Peptide

Cancer Research Center, Burnham Institute for Medical Research, La Jolla, California, USA.
The Journal of urology (Impact Factor: 4.47). 09/2008; 180(2):767-71. DOI: 10.1016/j.juro.2008.03.185
Source: PubMed


Previously we found that the trophinin-binding peptide GWRQ (glycine, tryptophane, arginine, glutamic acid) activated human trophoblastic cells. Although trophinin is expressed in human sperm, to our knowledge the function of this protein is not known. In this study we tested the effect of GWRQ on human sperm to evaluate whether the peptide enhances human sperm motility.
Immunohistochemistry was performed using monoclonal antibodies specific to trophinin, bystin or tastin. GWRQ-MAPS (multivalent 8-branched GWRQ peptide) was chemically synthesized. Human sperm from 4 volunteers with a mean +/- SD age of 35.75 +/- 3.4 years was suspended in buffer with GWRQ or control peptides. In 23 volunteers with a mean age of 25.5 +/- 2.5 years the number of immotile sperm was counted and subtracted from the total number of sperm to determine the number of motile sperm. A Transwell assay was used to measure swim-down motility. Levels of adenosine triphosphate and intracellular calcium in sperm cells incubated with GWRQ or control peptide were measured using a luminescent cell viability assay and a fluo-4 calcium assay, respectively.
The presence of trophinin and the trophinin associated proteins bystin and tastin in human sperm was confirmed by immunohistochemistry. Human sperm incubated with GWRQ-MAPS showed enhanced motility on sperm count assay and swim-down Transwell assay. Sperm cells incubated with GWRQ-MAPS showed decreased adenosine triphosphate and increased intracellular calcium.
Results suggest that GWRQ-MAPS may facilitate optimized in vitro fertilization outcomes and help avert the need for intracytoplasmic sperm injection in cases of severely low sperm motility. Trophinin may have a role in regulating adenosine triphosphatase in human sperm.

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    • "There are as yet no reports of mutant mice completely lacking polyLacs. Previous studies, including our own, suggested that mature human sperm cells are densely covered by carbohydrates [22] [23]. While we attempted to remove glycans from living human spermatozoa using glycosidases, we found human sperm cells treated with endo-b-galactosidase (EBG) moved more rapidly than untreated sperm. "
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    ABSTRACT: Fibroblast growth factors (FGFs) and their receptors are expressed in a variety of mammalian tissues, playing a role in development and cell proliferation. While analyzing human sperm motility, we found that sperm treated with endo-β-galactosidase (EBG), which specifically hydrolyzes poly-N-acetyllactosamine type glycans (polyLacs), enhanced motility. Mass spectrometry analysis revealed that sperm-associated polyLacs are heavily fucosylated, consistent with Lewis Y antigen. Immunohistochemistry of epididymis using an anti-Lewis Y antibody before and after EBG treatment suggested that polyLacs carrying the Lewis Y epitope are synthesized in epididymal epithelia and secreted to seminal fluid. EBG-treated sperm elevated cAMP levels and calcium influx, indicating activation of fibroblast growth factor signaling. Seminal fluid polyLacs bound to FGFs in vitro, and impaired FGF-mediated signaling in HEK293T cells.
    FEBS letters 08/2013; 587(19). DOI:10.1016/j.febslet.2013.07.056 · 3.17 Impact Factor
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    • "Previously we found that monoclonal anti-trophinin antibody or GWRQ-displaying phage particles did not bind to human sperm tails due to heavy glycosylation; however, after mild acid treatment, both antibody and phage bound to sperm tails [16]. In the mouse sperm experiments, mature mouse spermatozoa released from ductus deferens also were not stained by rabbit anti-trophinin antibody described above (data not shown). "
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    ABSTRACT: Background 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. Methods 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). Results 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. Conclusions 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.
    Reproductive Biology and Endocrinology 11/2012; 10(1):101. DOI:10.1186/1477-7827-10-101 · 2.23 Impact Factor
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    ABSTRACT: Trophinin is an intrinsic membrane protein expressed in trophectoderm cells of embryos and in uterine epithelial cells. Trophinin potentially mediates apical cell adhesion at human embryo implantation sites through trophinin-trophinin binding in these two cell types. Trophinin-mediated cell adhesion activates trophectoderm cells for invasion, whereas the effect of adhesion on maternal side is not known. We show that addition of GWRQ peptide, a previously established peptide that mimics trophinin-mediated cell adhesion, to human endometrial epithelial cells expressing trophinin induces their apoptosis. FAS involvement was excluded, as GWRQ did not bind to FAS, and FAS knockdown did not alter GWRQ-induced apoptosis. Immunoblotting analyses of protein kinases revealed an elevation of PKC-d protein in GWRQ-bound endometrial epithelial cells. In the absence of GWRQ, PKC-d associated with trophinin and remained cytoplasmic, but after GWRQ binding to the trophinin extracellular domain, PKC-d became tyrosine phosphorylated, dissociated from trophinin, and entered the nucleus. In PKC-d knockdown endometrial cells, GWRQ did not induce apoptosis.
    Cell cycle (Georgetown, Tex.) 01/2011; 10(1):135-43. DOI:10.4161/cc.10.1.14448 · 4.57 Impact Factor
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