Annexin A2 is involved in pig (Sus scrofa)sperm-oviduct interaction.

The oviduct is a dynamic organ which modulates gamete physiology. Sperm-oviduct interaction provides the formation of a sperm storage reservoir and allows the selection of sperm with certain qualities in eutherian mammals. In sows, the oviductal sperm binding glycoprotein (SBG) has been proposed to be involved in sperm selection. In this work, based on its affinity to sperm periacrosomal membrane proteins, we isolate another pig oviductal cell protein that interacts with sperm. Peptide identification by LC/MS-MS allowed the identification of this protein as annexin A2. The presence of this annexin, as well as annexin A1 and annexin A5 in sow oviductal cells was confirmed by Western blot with specific antibodies. The three proteins were localized in sow oviduct by immunohistochemistry, showing the presence of annexin A2 at the apical surface of the oviductal epithelial cells. Based on our data and the fact that annexins have been stated as candidate receptors of bovine sperm for sperm reservoir formation, we propose that this family of proteins is involved in sperm-oviduct interaction, annexin A2 being the main sperm binding isoform in pig.

Annexins are candidate oviductal receptors for bovine sperm surface proteins and thus may serve to hold bovine sperm in the oviductal reservoir.

The sperm of eutherian mammals are held in a storage reservoir in the caudal segment of the oviduct by binding to the mucosal epithelium. The reservoir serves to maintain the fertility of sperm during storage and to reduce the incidence of polyspermic fertilization. Bovine sperm bind to the epithelium via seminal vesicle secretory proteins in the bovine seminal plasma protein (BSP) family, namely, PDC109 (BSPA1/A2), BSPA3, and BSP30K, which coat the sperm head. Our objective was to identify the receptors for bull sperm on the oviductal epithelium. Proteins extracted from apical plasma membrane preparations of bovine oviductal epithelium were subjected to affinity purification using purified BSPs bound to corresponding antibodies conjugated to Protein A agarose beads. Oviductal protein bands of approximately 34 and 36 kDa were eluted by EGTA from the beads and identified by tandem mass spectrometry as annexins (ANXAs) 1, 2, 4, and 5. Subsequently, antibodies to each of the ANXAs were found to inhibit sperm binding to explants of oviductal epithelium. Anti-ANXA antibodies labeled the apical surfaces and cilia of the mucosal epithelium in sections of bovine oviduct. Western blots confirmed the presence of ANXAs in apical plasma membranes. Because fucose had been determined to be a critical component of the oviductal receptor, the ANXAs were immunoprecipitated from solubilized apical plasma membranes and were probed with Lotus tetragonolobus lectin to verify the presence of fucose. Thus, these ANXAs are strong candidates for the sperm receptors on bovine oviductal epithelium.

Bovine seminal plasma proteins PDC-109, BSP-A3, and BSP-30-kDa share functional roles in storing sperm in the oviduct.

On ejaculation, sperm become coated with proteins secreted by the male accessory sex glands. In the bull, these proteins consist predominantly of the bovine seminal plasma family of proteins (BSPs): PDC-109 (BSP-A1/-A2), BSP-A3, and BSP-30-kDa. PDC-109 plays a role in forming an oviductal sperm reservoir by enabling sperm to bind to oviductal epithelium. Because PDC-109 has high sequence identity with the other BSPs, we tested BSP-A3 and BSP-30-kDa for the capacity to bind sperm to oviductal epithelium. BSP-A3 and BSP-30-kDa each increased binding of epididymal sperm to epithelium and were as effective as PDC-109 in competitively inhibiting binding of ejaculated sperm. Because binding extends the motile life of sperm, BSPs were tested for the ability to maintain sperm motility. BSP-treated epididymal sperm incubated with plasma membrane vesicles from bovine oviductal epithelium maintained progressive motility longer than untreated sperm. To our knowledge, this is the first report of this protective effect of BSPs. Similarities in function among the BSPs were reflected in their three-dimensional structure, whereas surface maps of electrostatic potential indicated differences in binding affinities and kinetics. Such differences may provide sperm with greater adaptability to variations among females. Altogether, these results indicate that BSPs play a crucial role in fertilization by maintaining sperm motility during storage.

Calcium/calmodulin and calmodulin kinase II stimulate hyperactivation in demembranated bovine sperm.

Hyperactivated motility is observed among sperm in the mammalian oviduct near the time of ovulation. It is characterized by high-amplitude, asymmetrical flagellar beating and assists sperm in penetrating the cumulus oophorus and zona pellucida. Elevated intracellular Ca2+ is required for the initiation of hyperactivated motility, suggesting that calmodulin (CALM) and Ca2+/CALM-stimulated pathways are involved. A demembranated sperm model was used to investigate the role of CALM in promoting hyperactivation. Ejaculated bovine sperm were demembranated and immobilized by brief exposure to Triton X-100. Motility was restored by addition of reactivation medium containing MgATP and Ca2+, and hyperactivation was observed as free Ca2+ was increased from 50 nM to 1 microM. However, when 2.5 mM Ca2+ was added to the demembranation medium to extract flagellar CALM, motility was not reactivated unless exogenous CALM was readded. The inclusion of anti-CALM IgG in the reactivation medium reduced the proportion hyperactivated in 1 microM Ca2+ to 5%. Neither control IgG, the CALM antagonist W-7, nor a peptide directed against the CALM-binding domain of myosin light chain kinase (MYLK2) inhibited hyperactivation. However, when sperm were reactivated in the presence of CALM kinase II (CAMK2) inhibiting peptides, hyperactivation was reduced by 75%. Furthermore, an inhibitor of CAMK2, KN-93, inhibited hyperactivation without impairing normal motility of intact sperm. CALM and CAMK2 were immunolocalized to the acrosomal region and flagellum. These results indicate that hyperactivation is stimulated by a Ca2+/CALM pathway involving CAMK2.

PDC-109 (BSP-A1/A2) promotes bull sperm binding to oviductal epithelium in vitro and may be involved in forming the oviductal sperm reservoir.

Sperm reservoirs have been found in the oviducts of several species of mammals. In cattle, the reservoir is formed by the binding of sperm to fucose-containing glycoconjugates on the surface of oviductal epithelial cells. A fucose-binding molecule was purified from sperm extracts and identified as PDC-109 (BSP-A1/A2), a protein that is secreted by the seminal vesicles and associates with the plasma membrane of sperm upon ejaculation. The objective of this study was to demonstrate that PDC-109 promotes bull sperm binding to oviductal epithelium. PDC-109 was purified from bovine seminal plasma, and polyclonal antibodies were produced in rabbits. The antibodies detected PDC-109 on ejaculated sperm by indirect immunofluorescence and Western blots of extracts, but PDC-109 was not detected on epididymal sperm. When added to epididymal sperm, purified PDC-109 was absorbed onto the plasma membrane overlying the acrosome, as demonstrated by indirect immunofluorescence and by labeling sperm directly with fluorescein-conjugated PDC-109. When added to explants of oviductal epithelium, significantly fewer epididymal sperm than ejaculated sperm became bound. Addition of PDC-109 to epididymal sperm increased epithelial binding to the level observed for ejaculated sperm. In addition, binding of ejaculated sperm to oviductal epithelium was inhibited by addition of excess soluble PDC-109. Ejaculated sperm lost the ability to bind to oviductal epithelium after heparin-induced capacitation, but treatment with PDC-109 restored binding. These results demonstrate that PDC-109 enables sperm to bind to oviductal epithelium and plays a major role in formation of the bovine oviductal sperm reservoir.