Correlation of epididymal protease inhibitor and fibronectin in human semen

PLoS One. 2013 Dec 16;8(12):e82600. doi: 10.1371/journal.pone.0082600. eCollection 2013.

Abstract

Objective: Epididymal protease inhibitor (Eppin) was located on the surface of spermatozoa and modulates the liquefaction of human semen. Here, we identify the correlative protein partner of Eppin to explore the molecular mechanism of liquefaction of human semen.

Methods: (1) Human seminal vesicle proteins were transferred on the membrane by Western blotting and separated by 2-D electrophoresis and incubated in recombinant Eppin. The correlative protein was identified by Mass Spectrometry (MS) (2). Western blotting was used to determine the relation of rEppin and rFibronectin(Fn); (3) Co-localization in spermatozoa were detected using immunofluorescence; (4) Correalation of Eppin and Fn was proved by co-immunoprecipitation.

Results: Fn was identified as the binding partner of recombinant Eppin by MS. Recombinant of Eppin was made and demonstrated that the Eppin fragment binds the fn 607-1265 fragment. The Eppin-Fn complex presents on the sperm tail and particularly in the midpiece region of human ejaculated spermatozoa. Immunoprecipitation indicated that Eppin in the spermatozoa lysates was complexed with Fn.

Conclusions: Our study demonstrates that Eppin and Fn bind to each other in human semen and on human ejaculated spermatozoa. Eppin-Fn complex may involve in semen coagulation, liquefaction and the survival and preparation of spermatozoa for fertility in the female reproductive tract.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Blotting, Western
  • Electrophoresis, Polyacrylamide Gel
  • Epididymis / metabolism
  • Fibronectins / chemistry
  • Fibronectins / metabolism*
  • Humans
  • Male
  • Molecular Sequence Data
  • Protein Binding
  • Proteinase Inhibitory Proteins, Secretory / chemistry
  • Proteinase Inhibitory Proteins, Secretory / isolation & purification
  • Proteinase Inhibitory Proteins, Secretory / metabolism*
  • Semen / chemistry
  • Semen / metabolism*
  • Semen Analysis
  • Seminal Vesicle Secretory Proteins / chemistry
  • Seminal Vesicle Secretory Proteins / metabolism

Substances

  • Eppin protein, human
  • Fibronectins
  • Proteinase Inhibitory Proteins, Secretory
  • Seminal Vesicle Secretory Proteins

Grants and funding

This work was supported by grants from National Natural Science Foundation of China (NO.81270685) (http://isisn.nsfc.gov.cn/egrantweb/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.