ESP13.2, a Member of the  -Defensin Family, Is a Macaque Sperm Surface-Coating Protein Involved in the Capacitation Process

Abstract

Female macaques produced isoantibodies to a limited number of sperm surface proteins following immunization with sperm components released by phosphatidylinositol-specific phospholipase C (PI-PLC). Washed, acrosome-intact, fixed sperm injected into rabbits elicited a major immune response to one of the same PI-PLC-released proteins, which was shown to be a sperm surface-coating protein. After purification and digestion of the glycoprotein, four peptides were analyzed for amino acid sequence, and all had 100% homology with an epididymal secretory protein, ESP13.2, reported previously to be a small, cationic-rich peptide and a member of the beta-defensin family. Antibodies to purified ESP13.2 recognized a number of protein bands on Western blots of nonreduced PI-PLC-released sperm components and nonreduced whole-sperm extracts. After chemical disulfide reduction, only a single, broad band from 31 to 35 kDa was recognized by anti-ESP13.2 antibodies. Indirect immunofluorescence showed ESP13.2 over the entire surface of ejaculated macaque sperm. Fluorescence was only slightly reduced after sperm were washed through 80% Percoll. A 24-h incubation in capacitating medium significantly reduced the amount of ESP13.2 over the head and midpiece, whereas exposure of the incubated sperm to dbcAMP and caffeine (capacitation activators) resulted in almost complete loss of ESP13.2 from the sperm surface. After activation, ESP13.2 was the primary component released into the medium as judged electrophoretically. Lignosulfonic acid, a potent inhibitor of macaque fertilization in vitro, completely blocked release of ESP13.2 from the sperm surface, even following treatment with activators. These findings suggest that the beta-defensin, ESP13.2, has a function in the capacitation of macaque spermatozoa and may modulate sperm surface-receptor presentation at the time of fertilization.

Identification, characterization, and evolution of a primate β-defensin gene cluster

“…40 Figure 3 Expression of novel human b-defensin genes in different tissues. Total RNAs isolated from human (1) parotid glands, (2) thyroid, (3) breast, (4) lungs, (5) liver, (6) spleen, (7) kidney, (8) colon, (9) adrenal, (10) ovary, (11) seminal vesicle, (12) cauda, (13) corpus, (14) caput, (15) prostate, and (16) testis were used for RT-PCR analysis. PPIA was amplified as a control to check the quality and quantity of the template.…”

“…We demonstrate that, similar to other bdefensins, 3,4,11 the expression of these novel 20q defensins is restricted to the testis and epididymis. DEFB118 location on sperm, 25 DEFB126 involvement in the capacitation process, 13 the Bin1b role in sperm motility and maturation, 14 taken together with the male tract specific expression and differential regional expression of the 20q defensins provide evidence that these bdefensins have evolved to perform reproduction-specific tasks, combining immunity and fertility in their functional repertoires. In addition, the male organs may require more elaborate protection against pathogens than other organ systems.…”

“…3,4,11 In addition, the b-defensin-like epididymal protein (EP2)/sperm-associated antigen 11 (SPAG11) isoforms are also predominantly expressed in the male reproductive tract. 12 Recent studies specifically implicate b-defensins in pivotal roles in reproductive biology including ESP13.2/DEFB126 in sperm capacitation 13 and Bin1b/EP2E in sperm maturation, 14 thereby redefining our concepts concerning the multifunctional roles of b-defensins.…”

Comparative genomic analysis of a mammalian β-defensin gene cluster

“…The modified duplicates may perform a new function as hypothesized by neofunctionalization, may improve on the ancestral function as explained by subfunctionalization, or may lose all function as in nonfunctionalization. Most defensins tested appear to have retained the ancestral function of host defense (36,79), and in addition, some have gained reproductive functions, thereby corresponding to the subneofunctionalization hypothesis, i.e., Bin1b (86), DEFB126 (83,84).…”

“…␤-Defensins are increasingly recognized as mediators of reproductive functions, a model predicted by their expression primarily in the male reproductive tract. DEFB126, a sperm-bound ␤-defensin, blocks spermzona recognition and binding (67) and, before its release during capacitation (83), protects spermatozoa from immunorecognition and attachment of anti-sperm antibodies (84). The rat ␤-defensin-like SPAG11E is involved in motility acquisition during sperm maturation (86).…”