Systematic mapping and functional analysis of a family of human epididymal secretory sperm-located proteins

Abstract

The mammalian spermatozoon has many cellular compartments, such as head and tail, permitting it to interact with the female reproductive tract and fertilize the egg. It acquires this fertilizing potential during transit through the epididymis, which secretes proteins that coat different sperm domains. Optimal levels of these proteins provide the spermatozoon with its ability to move to, bind to, fuse with, and penetrate the egg; otherwise male infertility results. As few human epididymal proteins have been characterized, this work was performed to generate a database of human epididymal sperm-located proteins involved in maturation. Two-dimensional gel electrophoresis of epididymal tissue and luminal fluid proteins, followed by identification using MALDI-TOF/MS or MALDI-TOF/TOF, revealed over a thousand spots in gels comprising 745 abundant nonstructural proteins, 408 in luminal fluids, of which 207 were present on spermatozoa. Antibodies raised to 619 recombinant or synthetic peptides, used in Western blots, histological sections, and washed sperm preparations to confirm antibody quality and protein expression, indicated their regional location in the epididymal epithelium and highly specific locations on washed functional spermatozoa. Sperm function tests suggested the role of some proteins in motility and protection against oxidative attack. A large database of these proteins, characterized by size, pI, chromosomal location, and function, was given a unified terminology reflecting their sperm domain location. These novel, secreted human epididymal proteins are potential targets for a posttesticular contraceptive acting to provide rapid, reversible, functional sterility in men and they are also biomarkers that could be used in noninvasive assessments of male fertility.

Fig. 1.

Separation and identification of human epididymal proteins by 2D-PAGE and MALDI-MS Spectra. Reference maps of (A) the epididymal tissue pellet; (B) the epididymal fluid; example spectra of (C) HEL-S-162eP, (D) HEL-S-128m. The MS map (C, upper panel) and MS/MS map (C, lower panel) marked with b ions and y ions for glyceraldehyde-3-phosphate dehydrogenase identification. The sequence of precursor at m/z 1530.78 was analyzed by MS/MS to be VPTANVSVVDLTCR and the protein identified as glyceraldehyde-3-phosphate dehydrogenase. The MS map (D, upper panel) and MS/MS map (D, lower panel) marked with b ions and y ions for peroxiredoxin 6 identification. The sequence of precursor at m/z 1530.78 was analyzed by MS/MS to be LPFPIIDDR and identified as peroxiredoxin 6.

Fig. 2.

Immunohistochemical location of sperm-located and nonlocated proteins in tissue sections. Representative examples of proteins located in different epididymal regions (left panels, Caput; middle panels, Corpus; right panels, Cauda) in different tissue compartments. 1, negative control, preimmune rabbit serum; 2, secreted, sperm-bound HEL-S-97n, in the epididymal epithelium of all three regions; 3, secreted, HEL-S-153, in the epididymal epithelium and on microvilli of all three regions; 4, nonsecreted HEL-T-60, in the epididymal epithelium and on microvilli of all three regions. Each bar represents 200 μm.

Fig. 3.

Immunocytochemistry of washed ejaculated human spermatozoa incubated with antibodies against epididymal secretory proteins. Merged micrographs of fluorescence staining. Proteins selected for association with specific sperm domains: a, acrosome' HEL-S-25a65307; b, equatorial region'HEL-S-55e65307; c, postequatorial region'HEL-S-69p65307; d, neck'HEL-S-97n65307; e, midpiece'HEL-S-123m65307; f, principal piece'HEL-S-133P65307; g, end piece'HEL-S-150E65307; h, whole sperm'HEL-S-154w (previously HEL-75)65307; i, acrosome and neck'HEL-S-157an65307; j, acrosome and midpiece'HEL-S-158am65307; k, acrosome and principal piece'HEL-S-160aP65307; l, equatorial and principal piece'HEL-S-162eP65307; m, postequatorial and annulus'HEL-S-163pA65307; n, neck and annulus'HEL-S-164nA65307; o, midpiece and principal piece'HEL-S-181mP; p, pre-immune serum as negative control. Red staining (PI) indicates the nuclei, green staining (FITC) indicates the location of epididymal secretory proteins on spermatozoa. Each bar represents 5 μm.

Fig. 4.

Flow chart of identification and categorization of the 207 sperm-related proteins from 745 initially identified tissue and fluid proteins. The number of proteins examined was reduced by ignoring 117 structural proteins (unlikely to be secreted) and antibodies were raised to the remaining 628 proteins with 98% success. With the 619 usable antibodies, Western blots revealed that 408 proteins were present in epididymal fluid and 211 not. Two hundred and seven proteins were sperm-located (174 proteins found in epididymal fluid, 33 not) and the remaining proteins (412) were not associated with spermatozoa, whether found in fluid (234) or not (178). Filled sperm head, sperm-located proteins; clear sperm head, non sperm-located proteins; IHC: immunohistochemistry; WB: western blots of epididymal luminal fluid; +65306 positive staining 65307 −65306 negative staining.

Fig. 5.

Pie diagrams of the proportion of epididymal proteins categorized by function. Functional classification of (A) all 745 epididymal proteins, (B) 408 epididymal secretory proteins, (C) 207 epididymal sperm-located proteins.