Correlation of epididymal protease inhibitor and fibronectin in human semen

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.

Figure 1. Far-western immunoblot analysis demonstrating that only the N-terminal of recombinant epididymal protease inhibitor (N-rEppin) and rEppin can binds to reducing seminal plasma proteins.

A The monomer and multimers of rEppin, N-rEppin, C-rEppin in Western-blotting; N-Ep: N-rEppin (Eppin_18-76Mr 10000); Ep: rEppin (Mr 18000); C-Ep: C-rEppin (Eppin_75-133Mr 12000); B: Amido Black Staining of seminal plasma proteins(Stain the membrane for 5 minites with Amido Black, rinse the stained membrane for 30 to 45 sec with TBST three times),; lane 1: Non-reducing seminal plasma proteins, lane 2-4: Reducing seminal plasma proteins; M: marker; C: M: marker; lane 5-6: lane 1-2 (B) incubated with rEppin and probed with anti-his tag; lane 7: lane3 (B) incubated with C-rEppin and probed with anti-his tag; lane 8: lane 4(B) incubated with N-rEppin and probed with anti-his tag.

Figure 2. SDS-PAGE in two dimensions and Western blot analysis of seminal vesicle fluid shows a molecular weight 55-72KDa spot (arrowhead) binded to Eppin.

A. Seminal vesicle fluid was separated by 2-D electrophoresis\ and was stained overnight with 0.01% Bio-Rad R-250 Coomassie in 10% acetic acid. B. Far Western blot: Incubate the membrane with 6-His-Eppin protein and probed with anti-His. We can see the positive spot molecular weight 55-72KDa on the blot (arrowhead).

Figure 3. Mass spectrometric identification of Fn.

A. Seminal vesicle fluid was separated by SDS gels (two-dimensional), stained Coomassie blue stained, and the protein spot trypsin digested for identification. B. Matched peptides were shown in Bold Red.

Figure 4. Immunoblot and Far-Western immunoblot analysis demonstrate that Eppin and Fibronectin can combine with each other.

A. Immunoblot analysis demonstrating that Eppin binds Fibronectin. Lanes 1: protein of 18-kDa recombinant Eppin (arrow) and multimer forms (*) and were recognized by the rabbit Eppin antibody. Lanes 2: rEppin blot incubated in rFn overnight, washed, and probed with anti-Fn mAbs; lane 3: rEppin blot incubated without rFn overnight, washed, and probed with anti-Fn mAbs. The position of molecular weight standards (kDa) is indicated on the left. B. Far-Western immunoblot analysis demonstrating that rFn on a blot will bind Eppin. Lane 1: protein of 73.2-kDa recombinant rFn (*) blot probed with anti-Fn mAbs; lane 2: rFn blot incubated in r-Eppin, washed, and probed with rabbit Eppin antibody; lane3: rFn blot probed with rabbit Eppin antibody.

Figure 5. Immunolocalization of Fn and eppin in the sperm.

A Phase contrast image of spermatozoon. B. Immunolocalization of Eppin on the head and tail of ejaculate human spermatozoa with anti-Eppin antibodies and detected with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit antibodies (green). C. Immunolocalization of Fn on the head and tail of ejaculate human spermatozoa with anti-Fn antibodies and detected with Cy3 (cyanine-3)-conjugated goat anti-mouse antibodies (red). D. Dual-immunofluorescence staining of Fn (red) and Eppin (green) in human sperm showing that the strong colocalization (→) is seen in the postacrosomal and midpiece region of the head (yellow areas). E. Control exposure with fluorescein isothiocyanate (FITC) conjugated goat anti-rabbit IgG and Cy3 (cyanine-3)-conjugated anti-mouse IgG, with isotype mouse antibody and rabbit antibody instead of primary antibody.

Figure 6. Fn and Eppin interact in the spermatozoa.

Anti-Fn mAbs antibody was linked to Plus-agarose beads followed by incubation with 500 µg spermatozoa lysate. The antibody used for immunoprecipitation indicated that Eppin in the spermatozoa lysates was complexed with Fn. Unbound (Sn), or bound (IP) proteins were separated by PAGE. C indicates proteins bound to Plus-agarose linked to normal rabbit IgG as a negative control.