Immunocontraceptive target repertoire defined by systematic identification of sperm membrane alloantigens in a single species

Abstract

Sperm competence in animal fertilization requires the collective activities of numerous sperm-specific proteins that are typically alloimmunogenic in females. Consequently, sperm membrane alloantigens are potential targets for contraceptives that act by blocking the proteins' functions in gamete interactions. Here we used a targeted proteomics approach to identify the major alloantigens in swine sperm membranes and lipid rafts, and thereby systematically defined the repertoire of these sperm-specific proteins in a single species. Gilts with high alloantibody reactivity to proteins in sperm membranes or lipid rafts produced fewer offspring (73% decrease) than adjuvant-only or nonimmune control animals. Alloantisera recognized more than 20 potentially unique sperm membrane proteins and five sperm lipid raft proteins resolved on two-dimensional immunoblots with or without prior enrichment by anion exchange chromatography. Dominant sperm membrane alloantigens identified by mass spectrometry included the ADAMs fertilin α, fertilin ß, and cyritestin. Less abundant alloantigens included ATP synthase F1 β subunit, myo-inositol monophosphatase-1, and zymogen granule membrane glycoprotein-2. Immunodominant sperm lipid raft alloantigens included SAMP14, lymphocyte antigen 6K, and the epididymal sperm protein E12. Of the fifteen unique membrane alloantigens identified, eleven were known sperm-specific proteins with uncertain functions in fertilization, and four were not previously suspected to exist as sperm-specific isoforms. De novo sequences of tryptic peptides from sperm membrane alloantigen "M6" displayed no evident homology to known proteins, so is a newly discovered sperm-specific gene product in swine. We conclude that alloimmunizing gilts with sperm membranes or lipid rafts evokes formation of antibodies to a relatively small number of dominant alloantigens that include known and novel sperm-specific proteins with possible functions in fertilization and potential utility as targets for immunocontraception.

Fig 1. Fertility of gilts immunized with sperm membranes or lipid rafts.

A) Detection of SMA with anti-TWM alloantisera from four different immunized gilts. Blots of TWM (triple-washed membranes, 50 μg protein/gel) resolved by 2-D gel electrophoresis were each probed with individual alloantisera (1/5000 dilution, sera collected 4 wk after boost), and immunoreactivity visualized with identical exposure times. Note the variability of the immune response evident as the high immunoreactivity of sera from two gilts (“High Ab,” #466 & 474), and lower immunoreactivity of two others (“Low Ab,” #465 & 473). B) Fertility of immunized gilts (TWM n = 4; lipid rafts n = 2) compared to gilts injected with adjuvant only ("FCA", n = 4). Gilts were bred on second estrus after collection of alloantisera used for the blots in Panel A. Data are expressed as mean litter size ± SD, with results for TWM-immunized high responder ("High Ab" blots in panel A) and low responder animals shown separately. For reference, shown also is our swine farm’s average litter size for non-immunized sows ("None") over a preceding 7-month period (2528 offspring/247 litters = 10.3 ± 0.6). C) Effect of alloimmunization on fertility. We compared litter sizes of high responder gilts immunized with TWM or lipid rafts to litter sizes of gilts injected with Freund's Complete Adjuvant only ("FCA"). Shown are mean litter sizes and standard error of the means with n = 4 in each group. High immunity to SMA or SLRA produced a 3.8-fold decrease in litter size (from 10.5 to 3.3 piglets/litter, p = 0.0119).

Fig 2. Detection of acidic SMA in membranes isolated by Method 1 on western blots probed with sperm membrane alloantisera pool anti-TWM #1.

A) Pre-enrichment of acidic SMA by anion exchange chromatography. B) 2-D electrophoretic profiles of acidic immunoreactive SMA eluted with 220–260 mM NaCl (fractions 3–5 pool), left panels, and 280–310 mM NaCl (fractions 6–8 pool), right panels. Proteins from sperm particulate fraction containing released plasma membranes were isolated by Method 1 as described in “Materials and Methods”. Following pre-enrichment, acidic SMA eluted with a continuous NaCl gradient were solubilized in solution containing 9.8 M urea (disulfides not reduced), loaded onto 18 cm IPG strip (pH 4–7), and resolved by IEF followed by SDS-PAGE. Immunoreactive SMA were detected by western blotting (top panels) using anti-TWM #1 (1/5000 dilution of pooled sera from [3]), and all SMA were visualized with Coomassie blue (bottom panels). Immunodominant SMA were cored from duplicate preparative gels and analyzed by MS. Two high-molecular weight SMA with M_r 100,000 and 150,000 (dotted arrows) not detected on the corresponding 2-D gel (right panels) were not analyzed by MS. Likewise, the analysis of SMA with a M_r ≥ 250,000 (circled in dashed line) was not pursued. SMA #8–12 (boxed in dashed line) were previously detected in fractions 3–5 pool (left panel) and were not reanalyzed. C) Confirmation of immunoreactivity of individual spots cored from 2-D gel as shown in B).

Fig 3. Detection of acidic SMA in membranes isolated by Method 2 on western blots probed with sperm membrane alloantisera pool anti-TWM #1.

A) Pre-enrichment of acidic SMA by anion exchange chromatography. B) Profile of acidic immunoreactive SMA eluted with 240–280 mM NaCl. Acidic SMA eluted with a continuous NaCl gradient were solubilized (disulfides not reduced) and resolved by IEF(pH 4–7)/SDS-PAGE as for Fig 2. Immunoreactive SMA were detected by western blotting (top panel) using anti-TWM #1 (1/5000 dilution of pooled sera from [3]), and all SMA visualized with Coomassie blue (bottom panel). Immunodominant SMA were cored from duplicate preparative gels and analyzed by MS. Five SMA (#8–12) previously immunodetected in fractions 3–5 pool (Fig 2B) were not labeled on the blot. Immunoreactive SMA with M_r 100,000–250,000 (circled in dashed line) not visualized on the corresponding 2-D gel were not analyzed further. C) Confirmation of individual spot’s immunoreactivity previously resolved on 2-D gel as shown in B).

Fig 4. Detection of SMA by 2-D western blotting with new alloantisera to sperm membranes (anti-TWM #2).

A) Immunoreactive SMA detected by western blotting (top panel) using anti-TWM #2 (1/5000 dilution of pooled high responder sera) and visualization of all SMA with silver stain (bottom panel). Particulate fractions enriched in plasma membranes of boar spermatozoa were isolated by Method 2 as described in “Materials and Methods”. TWM proteins were solubilized (disulfides not reduced) and resolved by IEF (pH4-7)/SDS-PAGE as for Figs 2 and 3, and SMA detected by Western blotting with anti-TWM #2. Immunodominant SMA (except M5) were cored from duplicate gel, and analyzed by MS. Black arrows indicate acidic SMA previously identified as ADAMs (Fig 2B). B) A longer exposure of the blot shown in A) allowed the immunodetection of SMA M3-M7.

Fig 5. Detection of SLRA by 2-D western blotting with alloantisera to raft proteins (anti-rafts).

A) Profile of immunoreactive SLRA detected by western blotting using anti-rafts alloantisera (pool from gilts 470 and 471). B) Visualization of all raft proteins with silver stain. Lipid rafts in detergent extracts of boar spermatozoa were isolated by sucrose gradient ultracentrifugation. Raft proteins, fraction 4 as determined by light-scattering at 620 nm and the presence of flotillin-2, were precipitated with acetone (A and B, left panels). Alternatively, fractions 4 and 5 were pooled and ultracentrifuged (A and B, right panels). Raft proteins were solubilized in rehydration solution containing 9.8 M urea (disulfides not reduced), resolved by IEF/SDS-PAGE, and SLRA were detected by Western blotting (1/5000 dilution of pooled sera from gilts #470 and #471). Immunodominant SLRA were cored from a duplicate gel for analysis by MS.