Annexins expressed on the cell surface serve as receptors for adhesion to immobilized fetuin-A

Fetuin-A is a major constituent of the fetal bovine serum used extensively in cell culture media. We hereby present data demonstrating that breast carcinoma cells can adhere to immobilized fetuin-A in a calcium-dependent fashion. Interestingly, the cells can also divide and attain confluency under these conditions. Using a proteomic approach, we have identified annexin-II and -VI as the putative cell surface receptors for fetuin-A in the presence of Ca2+ ions. Biotinylation of cell surface proteins followed by immunoprecipitation revealed that annexin-VI was expressed on the extracytoplasmic surface of the cell membranes. Finally, to demonstrate that annexin-II and -VI were the adhesive receptors for fetuin-A, siRNA knockdown of expression of the annexins significantly reduced the calcium-mediated adhesion. Interestingly, we demonstrated that the tumor cells could also adhere to immobilized fetuin-A in the presence of magnesium ions, and that this adhesion was most likely mediated by integrins because neutralizing antibodies against beta1 integrins substantially reduced the adhesion. Our studies suggest that the expression of annexin-II and -VI and possibly other members of the family mediate novel adhesion and signaling mechanisms in tumor cells.     

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.     

Characterization of a fucose-binding protein from bull sperm and seminal plasma that may be responsible for formation of the oviductal sperm reservoir

Oviductal sperm reservoirs have been found in cattle, mice, hamsters, pigs, and horses. In cattle (Bos taurus), the reservoir is evidently formed when sperm bind to fucosylated ligands resembling Le(a) trisaccharide on the surface of oviductal epithelium. The aim of this study was to characterize the fucose-binding protein on bull sperm. Fresh ejaculated sperm were extracted with 0.5 M KCl in Hepes-balanced salts. Extracts were subjected to affinity chromatography using immobilized Le(a) trisaccharide (alpha-L-Fuc[1,4]-beta-D-Gal[1,3]-D-GlcNAc). Two-dimensional PAGE of the affinity chromatography eluates revealed a prominent protein of approximately 16.5 kDa and a pI of 5.8. This protein inhibited binding of sperm to oviductal explants. A similar analysis of proteins extracted from capacitated sperm (which do not bind to oviductal epithelium) showed a reduction in the amount of the 16.5-kDa protein. When examined by epifluorescence microscopy, live uncapacitated sperm labeled over the acrosome with a fucose-BSA-fluorescein isothiocyanate (FITC) conjugate, while capacitated sperm did not. When capacitated sperm were treated with 16.5-kDa protein, labeling with fucose-BSA-FITC was partially restored. The comparative ease with which the protein was removed from sperm and its apparent reassociation with sperm suggested that it could be a peripheral protein derived from epididymal or accessory gland fluids. Blots of SDS-PAGE gels of seminal plasma proteins revealed the presence of a Le(a)-binding protein with an apparent mass of 16.5 kDA: Amino acid sequencing of two tryptic fragments of the protein purified from sperm extracts identified it as PDC-109 (BSP-A1/A2), a product of the seminal vesicles.     

Adsorption of oviductal fluid proteins by the bovine sperm membrane during in vitro capacitation.

125I-labeled oviductal fluid (ODF) proteins and antiserum to ODF were used to determine whether ODF proteins associate with the sperm membrane during in vitro capacitation. Luteal and nonluteal pools of ODF were obtained from oviduct catheters during the estrous cycle. Washed sperm (50 x 10(6) sperm/ml) were incubated up to 4 h in a protein-free modified Tyrode's medium (MTM), or MTM supplemented with 40% ODF, or 0.5 ng 125I-labeled ODF proteins. Solubilized sperm membrane proteins and incubation media containing ODF proteins were separated by gel electrophoresis. Membranes isolated from bovine sperm, previously incubated with ODF, adsorbed five 125I-proteins: A doublet at 85-95 kDa, and others at 24, 34, 53, and 66 kDa. The amount of 66 kDA 125I-protein associated with the sperm decreased during the incubation, whereas the amount of 85 to 95-kDa protein did not. Western blot analyses also detected the presence of ODF proteins (53, 66, 85-95, and 116 kDa) in solubilized membranes from sperm incubated in ODF. The 85 to 95-kDa protein in ODF decreased in apparent molecular weight by 5 kDa when associated with the sperm membrane. At 53 kDa, ODF proteins which associated with sperm were transformed from two to three separate proteins. These studies indicate that the surface of sperm is modified by adsorption of ODF proteins to the membrane during in vitro capacitation.     

Identification of ampullary and isthmic oviductal fluid proteins that associate with the bovine sperm membrane

The objective of this study was to define the complement of ampullary and isthmic oviductal fluid proteins that associate with the sperm membrane during the non-luteal and luteal stages of the oestrous cycle. Oviductal fluid was obtained from three dairy cows via indwelling cannulae in the ampulla and isthmus of the same oviduct. Daily samples of oviductal fluid were combined by region and stage of the cycle to create pools consisting of non-luteal isthmic, luteal isthmic, non-luteal ampullary, and luteal ampullary oviductal fluid. An aliquot from each oviductal fluid pool was biotinylated by incubation in biotin reagent. Spermatozoa from three fertile bulls were pooled, washed and incubated for 4 h in each of the four pools of biotinylated oviductal fluid. After incubation, sperm membrane proteins were solubilized, and proteins subjected to one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. The biotinylated oviductal fluid proteins that associated with the sperm membranes were detected by avidin–horseradish peroxidase and diaminobenzidine substrate. Eight oviductal fluid proteins with apparent molecular weights of 97, 75, 66, 55, 48, 34, 28 and 24 kDa were consistently detected to associate with the sperm membrane. Differences in ampullar and isthmic fluid proteins that associated with sperm during the luteal and non-luteal stage of the oestrous cycle were not detected.     

Spermadhesin AQN1 is a candidate receptor molecule involved in the formation of the oviductal sperm reservoir in the pig

Sperm are stored in the isthmic region of the oviduct under conditions that maintain viability and suppress early capacitation steps until ovulation occurs. The initial contact between sperm and oviductal epithelium is mediated by carbohydrate-protein interactions. In the pig, the carbohydrate recognition system has been shown to involve oligomannosyl structures. The spermadhesins AWN and AQN1 are the dominant porcine carbohydrate-binding sperm proteins. The objective of this study was to demonstrate that AQN1 contributes to sperm binding to the oviductal epithelium. AQN1 showed a broad carbohydrate-binding pattern as it recognizes both alpha- and beta-linked galactose as well as Manalpha1-3(Manalpha1-6)Man structures, whereas AWN bound only the galactose species. Binding of ejaculated sperm to oviductal epithelium was inhibited by addition of AQN1 but not by AWN. Mannose-binding sites were localized over the rostral region of the sperm head. Flow cytometry showed that, under capacitating conditions, the population of live sperm was shifted within 30 min toward an increase in the proportion of cells with low mannose- and high galactose-binding. The loss of mannose-binding sites was accompanied by the loss of AQN1 in sperm extracts and the significant reduction in the sperm-oviduct binding. The oviductal epithelium was shown by GNA-lectin histochemistry and by SDS-PAGE and lectin blotting of the apical membrane fraction to express mannose components that could be recognized by AQN1. These results demonstrate that the sperm lectin AQN1 fulfils the criteria for an oviduct receptor in the pig and may play a role in the formation of the oviductal sperm reservoir.     

Sulfated glycoconjugates are powerful modulators of bovine sperm adhesion and release from the oviductal epithelium in vitro

The mechanisms of sperm adhesion and release within the mammalian oviduct are still poorly understood. In this in vitro study, a previously developed adhesion assay was used to analyze the effects of heparin, N-desulfated heparin, fucoidan, dextran sulfate, and dextran on bovine sperm-oviductal cell adhesion and release. Results showed that 1) all sulfated glycoconjugates were powerful inhibitors of sperm binding to oviductal monolayers in a dose-dependent manner, whereas N-desulfated heparin and dextran had no effect; 2) sperm pretreatment with heparin and fucoidan markedly inhibited adhesion; 3) treatment of oviductal monolayers with heparinase I, II, or sodium chlorate (an inhibitor of sulfation) had no effect on sperm adhesion; 4) sulfated glycoconjugates were also powerful and quick inducers of sperm release from oviductal monolayers; and 5) addition of sulfated glycoconjugates to the cocultures caused a sudden increase of bound-sperm flagellar beat frequencies, followed by a release of highly motile sperm. In conclusion, these data support the hypothesis that sulfated glycoconjugates may act as signals that induce sperm release and migration from the oviductal reservoir.     

Carbohydrate-based interactions of oviductal sperm reservoir formation-studies in the pig.

Competitive inhibition of sperm to explants of the oviductal epithelium was used to study the complementary receptor system that may be involved in the establishment of the oviductal sperm reservoir in the pig. Sperm binding to the oviductal explants is expressed as Binding Index (BI = sperm cells/0.01 mm(2)). From a set of glycoproteins with known oligosaccharide structures, only asialofetuin and ovalbumin showed inhibitory activity, indicating that ovalbumin may block high affinity binding sites (IC(50) congruent with 1.3 microM) and asialofetuin low affinity sites (IC(50) congruent with 18 microM) of the complementary receptor systems, whereas fetuin carrying terminal sialic acid has no effect. Ovalbumin glycopeptides were isolated by Con A affinity chromatography and reverse-phase HPLC following tryptic digestion. Glycopeptides and enzymatically released glycans were analyzed by MS, and were shown to represent preferentially the two high mannose type glycans (Man)(5)(GlcNAc)(2) and (Man)(6)(GlcNAc)(2), and as a minor component the hybrid type glycan (Hex)(4)(GlcNAc)(5). Glycopeptides (84% inhibition) and glycans (81% inhibition) significantly reduced sperm-oviduct binding at a concentration of 3 microM, whereas the deglycosylated peptides showed no inhibitory activity. Mannopentaose (IC(50) congruent with 0.8 microM) representing the oligomannose residue of the high mannose glycans of ovalbumin was as effective as ovalbumin. These data indicate that the carbohydrate-based mechanisms underlying the formation of the oviductal sperm reservoir in the pig is the result of the concerted action of at least the high-affinity binding sites for oligomannose or nonreducing terminal mannose residues and low-affinity binding of galactose.     

Beta-defensin 126 on the surface of macaque sperm mediates attachment of sperm to oviductal epithelia

Beta-defensin 126 (DEFB126) coats the entire surface of macaque sperm until sperm become capacitated, and the removal of DEFB126 from over the head of sperm is required for sperm-zona recognition. Viable sperm collected from cervix and the uterine lumen of mated female macaques had DEFB126 coating the entire surface, suggesting that DEFB126 is retained on sperm en route to the oviduct. DEFB126 plays a major role in attachment of sperm to oviductal epithelial cells (OECs). Following treatment to either remove or alter DEFB126, sperm were coincubated with explants of OECs, which were assessed for sperm binding following rinsing to remove superficially attached sperm. Sperm treated with either 1 mM caffeine + 1 mM dibutyryl cyclic adenosine monophosphate (dbcAMP) (induces capacitation and complete release of DEFB126 from sperm), 2 mM caffeine (removes DEFB126 from over the head and midpiece but does not induce capacitation), anti-DEFB126 immunoglobulin, or neuraminidase (cleaves sialic acid from terminal positions on glycosylation sites of DEFB126) resulted in similar and significant levels of inhibition of sperm-OEC binding. Preincubation of OECs with soluble DEFB126 also resulted in significantly reduced sperm-OEC binding. Furthermore, reduced OEC binding capability of sperm lacking DEFB126 could be restored by addition of soluble DEFB126 to the sperm surface prior to incubation with OECs. Finally, purified DEFB126, infused into oviducts in situ, associated primarily with the apical membranes of secretory-type epithelial cells. In summary, treatments of macaque sperm that result in either removal, masking, or alteration of DEFB126 result in loss of sperm-OEC binding that is independent of changes in sperm motility. DEFB126 may be directly involved in the formation of a reservoir of sperm in the oviduct of macaques.     

Ability of sulfated glycoconjugates and disulfide-reductants to release bovine epididymal sperm bound to the oviductal epithelium in vitro

In Bos taurus, at ejaculation, epididymal sperm acquire a number of proteins secreted in the seminal plasma that increase their ability to interact with the female reproductive tract. Sperm-oviduct interaction comprises a transient sperm adhesion to the isthmus, the lower portion of the oviduct, followed by sperm release around ovulation. Oviductal fluid molecules, such as sulfated glycoconjugates and disulfide-reductants, are able to release bovine ejaculated sperm bound to the oviductal epithelium in vitro through the reduction of sperm surface protein disulfides to sulfhydryls. To understand whether the sperm molecules sensitive to releasing signals are already exposed on the surface of epididymal sperm, we studied the ability of cauda epididymal sperm to adhere to the oviductal epithelium and to be released by sulfated glycoconjugates and the disulfide-reductant penicillamine. Surface protein sulfhydryls in cauda epididymal sperm were analyzed in the initial suspension, in sperm bound to the in vitro-cultured oviductal epithelium, and in released sperm. Results showed that epididymal sperm are able to bind the oviductal epithelium in vitro, although at a lower extent than frozen-thawed ejaculated sperm; the interaction is mediated by oviductal cell microvilli that closely bind to the plasma membrane of the sperm head rostral region, as previously shown for ejaculated sperm. The sulfated glycoconjugates heparin, fucoidan, and dextran sulfate, as well as the disulfide-reductant penicillamine, are all powerful inducers of sperm release. The level of sulfhydryls in sperm surface proteins was (1) high in the initial sperm suspension; (2) low in bound sperm; (3) markedly increased in sperm released by heparin or by penicillamine. In conclusion, epididymal sperm are already able to bind the oviductal epithelium and to respond to the inducers of release through the reduction of sperm surface protein disulfides to sulfhydryls.     

Importance of calcium for the binding of oviductal fluid proteins to the membranes of bovine spermatozoa

Our laboratory has recently shown that in vitro-cultured oviductal cells secrete sperm motility maintaining factor(s). Since the binding of oviductal proteins to spermatozoa (SPZ) has been demonstrated in many species, the motility factor was postulated to bind the membranes of SPZ. Therefore, the current study was performed to evaluate which proteins from in vivo oviductal secretions bind to sperm membranes, to characterize binding conditions, and to evaluate the effect of this binding on sperm survival. Bovine oviducts were dissected, and oviductal cells and fluid were collected by pressing the oviductal tube with a glass slide. This mixture was incubated in Tris-EDTA buffer at 37°C for 30 min, and the cells were washed twice by centrifugation. The supernatant containing oviductal fluid proteins (OFP) was reserved, filtered, frozen (for later motility tests), or lyophilized and labeled with ¹²⁵|. Frozen-thawed SPZ were incubated either immediately, following capacitation, ionophore-induced acrosome reaction, death by heating, or flagellar removal with labeled OFP for 30 min. The resulting pellet after three washes was dissolved in SDS and submitted to 10% SDS-PAGE. An autoradiogram showed that 72, 66, 39, 38, and 36 kDa proteins bind strongly to the five types of SPZ used, and that this binding is very specific, since unlabeled OFP inhibited binding while serum proteins did not. Furthermore, for 39, 38, and 36 kDa proteins, the presence of calcium in the incubation medium was essential for dose-dependent binding, whereas magnesium was not. Preincubation of SPZ for 30 min at 37°C with oviductal fluid, followed by one wash and 6 hr of incubation in control media, showed that the percentage of motile SPZ is significantly higher (52 ± 6%) compared with SPZ not preincubated with oviductal fluid (24 ± 6%: P < 0.01). In summary, a limited number of proteins from oviductal secretions bind to the surface of bovine SPZ only in the presence of calcium, and this binding appears to be important for subsequent sperm viability. © 1996 Wiley-Liss, Inc.     

A novel approach to identify bovine sperm membrane proteins that interact with receptors on the vitelline membrane of bovine oocytes

At fertilization, the sperm triggers intracellular calcium oscillations, which are pivotal to oocyte activation and development. A working hypothesis for the interaction between the sperm and the oocyte is that disintegrin ligands on the inner acrosomal membrane of the sperm bind to integrin receptors on the oocyte vitelline membrane. The aim of these experiments was to find and identify the sperm protein ligands involved in bovine sperm-oocyte interactions. In situ fluorescent labeling of proteins and 2-D gel electrophoresis were used to identify specific sperm membrane proteins that interact with proteins in the oocyte vitelline membrane. Sperm were labeled with a fluorescent dye and used to fertilize zona-free oocytes. Sperm-oocyte complexes were either lysed immediately, or following covalent cross-linking of proteins with dibromobimane. The cross-linking reagent serves the critical function of covalently linking proteins together so that they will remain as a unit through lysis of the cells and 2-D gel analysis, and which can be subsequently identified by mass spectrometry. Lysates were electrophoretically run on the same 2-D gel. The comparison of uncross-linked and cross-linked protein spots revealed that some proteins shifted position based on binding. These spots were picked and proteins identified by mass spectrometry. These results provide a list of specific sperm proteins that interact with oocyte membrane proteins and establish a group of candidate ligands, one or more of which may be responsible for induction of outside-in signaling resulting in oocyte activation and fusion of the gametes.     

Prostasome-like particles are involved in the transfer of P25b from the bovine epididymal fluid to the sperm surface

In bulls, P25b is a sperm protein associated with the plasma membrane covering the acrosome. The amount of P25b bound to a constant number of spermatozoa varies from one individual to the other, low levels being associated with bull subfertility. In this study, we describe the epididymal origin of P25b using Western blot analysis. Whereas P25b was undetectable on caput spermatozoa, the amount of P25b associated to a constant number of spermatozoa increases from the corpus to the cauda. Prostasome-like particles were prepared by ultracentrifugation of epididymal fluid. P25b appears to be also associated with those membranous vesicles in increasing amounts along the epididymis. P25b is anchored to the plasma membrane of spermatozoa through glycosylphosphatidyl-inositol as shown by the ability of phospholipase C. but not of high salt treatment, to release P25b. Coincubation experiments revealed that prostasome-like particles are able to transfer P25b to spermatozoa, this process being more efficient at slightly acidic pH. P25b thus appears to be a marker of sperm epididymal maturation in bulls.     

Effect of oviductal proteins on sperm functions and lipid peroxidation levels during cryopreservation in buffaloes

A study was undertaken to find out the effect of addition of oviductal proteins on sperm functions and lipid peroxidation (LPO) levels in buffaloes. Oviductal flushings were collected from apparently healthy buffalo genital tracts (nonluteal and luteal stage of estrous cycle), centrifuged (3000 rpm; 30 min), filtered (0.2 microm) and frozen at -20 degrees C. The proteins in pooled nonluteal and luteal oviductal fluid were precipitated overnight using ammonium sulphate, centrifuged (10,000 rpm; 30 min) and dialyzed (>10 kDa). After protein estimation, aliquots of samples containing 10 mg proteins were lyophilized in cryovials and stored frozen at -20 degrees C. Six pooled good quality ejaculates collected by artificial vagina method from two Murrah buffalo bulls were utilized for the study. After fresh semen analysis, each pooled ejaculate was split into three parts and extended in Tris-Egg yolk-Citrate extender (20% egg yolk: 7% glycerol), so that final dilution yielded approximately 60 million sperm cells/ml and cryopreserved in 0.5 ml French straws (30 million sperm cells per straw) in LN2 (-196 degrees C). Before freezing, the nonluteal and luteal oviductal proteins (NLOP &LOP) were incorporated at the concentration of 1mg/ml of extended semen. The equilibrated and frozen thawed (37 degrees C for 30s) semen was evaluated for motility, viability and acrosomal integrity, bovine cervical mucus penetration test and hypo-osmotic sperm swelling test. Besides these tests, LPO level was assessed in sperm and seminal plasma in equilibrated and frozen thawed semen. Results revealed that addition of oviductal proteins to semen before freezing convey beneficial effect in terms of spermatozoan motility, viability and acrosomal integrity. Nonluteal oviductal proteins favored significantly (P < 0.05) higher sperm penetration distance in cervical mucus (23.00+/-1.15 mm) than the control group (15.00+/-3.46 mm) in frozen thawed semen. Similarly, swollen sperm percentage was also significantly (P < 0.05) higher in NLOP treated group than the LOP included and control groups. In frozen thawed spermatozoa, the LPO level was significantly (P < 0.05) lower in NLOP added group than the LOP added and control group. It was inferred that incorporation of oviductal proteins in extender before freezing reduced the lipid peroxidation levels in buffalo spermatozoa during cryopreservation and thereby improved the post-thaw semen quality.     

Impact of ACTH administration on the oviductal sperm reservoir in sows: the local endocrine environment and distribution of spermatozoa

The objective of the study was to investigate if short-term stress in sows (simulated by injections of synthetic adrenocorticotrophic hormone (ACTH)) during standing oestrus had a negative effect on the local environment in the utero-tubal junction (UTJ) and isthmus and the distribution of spermatozoa in these segments. Fourteen sows were monitored for ovulation using ultrasonography in two consecutive oestruses. The sows were fitted with jugular catheters and, from onset of the second oestrus, blood samples were collected every second hour. In the 2nd oestrus, seven sows were given ACTH every second hour, from the onset of standing oestrus until the sow ovulated (ACTH-group), whereas the other seven sows remained as controls (C-group) and were given NaCl solution. The sows were artificially inseminated 16-18 h before expected ovulation. Six hours after ovulation the sows were anaesthetised, and blood samples were repeatedly taken from veins draining the uterus and the UTJ-isthmus, respectively. This oviduct was thereafter removed and divided in four adjacent sections consisting of: (i) the UTJ, (ii) the first, and (iii) the second isthmus segment prior to (iv), the ampullary-isthmic junction (AIJ) and the ampulla. The three first-mentioned segments were flushed to retrieve spermatozoa, whereas the last one was flushed to collect oocytes/ova. The number of spermatozoa attached to the zona pellucida was counted. The concentrations of cortisol in jugular blood of the ACTH-group sows during the time of ACTH-injections were significantly higher than of the C-group sows (p<0.05), as were the levels of progesterone (p<0.001). Progesterone and cortisol concentrations measured in the blood samples draining the UTJ-isthmic region 6 h after ovulation did not significantly differ between the groups, but the C-group displayed significantly higher concentrations of progesterone in the UTJ-isthmic region compared with the levels measured in parallel samples taken of jugular blood (p<0.01). The C-group, but not the ACTH-group, also displayed a significant elevation in progesterone concentration 6h after ovulation compared with the basal levels before ovulation (p<0.01). Numbers of retrieved spermatozoa were not significantly different between the C-group and the ACTH-group. However, there was a tendency for a larger number of spermatozoa among sows in the ACTH-group, especially in the isthmic segment adjacent to the AIJ. In conclusion, simulated stress induced by injections of ACTH during standing oestrus results in elevated concentrations of progesterone before ovulation and may interfere with the rise of progesterone after ovulation. However, ACTH-injections appeared to augment transport of spermatozoa through the female genital tract of pigs.     

Arabinogalactan proteins at the cell surface ofBrassica sperm andLilium sperm and generative cells

Antibodies to arabinogalactan proteins were tested for binding to sperm cells ofBrassica campestris and to generative cells and sperm ofLilium longiflorum. Two monoclonal antibodies, JIM8 and JIM13, bound toBrassica sperm in pollen grains and pollen tubes and to isolated sperm. Sperm pairs retained within the vegetative cell inner plasma membrane fluoresced more brightly than single sperm, indicating that the vegetative cell inner plasma membrane that surrounds sperm pairs also contains arabinogalactan proteins. Isolated sperm pairs exhibited a uniform fluorescence while single sperm had patches of fluorescence. InLilium, isolated generative cells and single sperm cells bound antibodies in a patchy pattern. Antibodies to arabinogalactan proteins may be useful in describing the overall shape of sperm cells and for identifying sperm among other cell types.     

95 kd sperm proteins bind ZP3 and serve as tyrosine kinase substrates in response to zona binding

In the mouse, the zona pellucida (ZP) glycoprotein ZP3 both binds intact sperm and induces acrosomal exocytosis. The subsequent signaling pathway(s) is still uncertain, but Gi-like proteins have been implicated. By analogy with other signal transduction mechanisms, we examined anti-phosphotyrosine antibody reactivity in mouse sperm. Antibodies reacted with three proteins of 52, 75, and 95 kd. Indirect immunofluorescence localized reactivity to the acrosomal region of the sperm head. The 52 kd and 75 kd phosphoproteins are detected only in capacitated sperm, whereas the 95 kd protein is detected in both fresh and capacitated sperm. For the 95 kd protein, the level of immunoreactivity is not related to sperm motility but is enhanced by both capacitation and sperm interaction with solubilized ZP proteins. In addition, binding of radiolabeled whole ZP or purified ZP3 to blots of separated sperm proteins identified two ZP binding proteins of 95 kd and 42 kd. 95 kd sperm proteins that bind to ZP3 also react with anti-phosphotyrosine antibodies (in a ZP concentration-dependent manner), supporting the idea that the same 95 kd sperm protein serves as a ZP3 receptor and as a tyrosine kinase substrate. These findings and our evidence on acrosome reaction triggering via sperm receptor aggregation suggest that a 95 kd protein in the sperm plasma membrane is aggregated by ZP3, which stimulates tyrosine kinase activity leading to acrosomal exocytosis.     

Sperm binding to oviductal epithelial cells in the rat: role of sialic acid residues on the epithelial surface and sialic acid-binding sites on the sperm surface

The aim of this study was to assess the participation of carbohydrate residues in the adhesion of spermatozoa to the oviductal epithelium in the rat. We first examined, by lectin labeling, the distribution of glycoconjugates in rat oviducts obtained under various hormonal environments. Several classes of glycoconjugates were abundant in the epithelium, and the expression of some of these molecules varied differentially in ampulla and isthmus, along the estrous cycle and with estradiol and progesterone treatment. Proestrous rats were intraoviductally injected with lectins Dolichos biflorus, Erythrina cristagalli, Helix pomatia, Arachis hypogea, Ulex europaeus I, Triticum vulgaris, or Tritrichomonas mobilensis and were inseminated with 10-20 million epididymal spermatozoa in each uterine horn. Three hours later, the total number of spermatozoa present in the oviduct and the proportion adhering to the epithelium were determined. Intraoviductal administration of lectins did not affect the total number of spermatozoa recovered from the oviduct and only the sialic acid-binding lectin TML decreased the percentage of sperm cells adhering to the epithelium. The involvement of sialic acid in sperm-oviduct adhesion was further explored, inseminating spermatozoa preincubated with mannose, galactose, sialic acid, fucose, fetuin, or asialofetuin. Sialic acid and fetuin inhibited sperm-oviduct binding while other carbohydrates had no effect. Using TML lectin immunohistochemistry, we found that sialic acid-rich glycoconjugates are equally localized in the epithelium of ampulla and isthmus of proestrous rats. The electrophoretic pattern of sialic acid-rich glycoproteins of the epithelium showed 15 major protein bands, for which molecular mass ranged from 200 to 50 kDa with no difference between ampulla and isthmus or between estrous cycle stages. Binding sites for sialic acid-fluorescein isothiocyanate were demonstrated on the surface of rat spermatozoa, and biotinylated sialic acid recognized 11 plasma membrane proteins of sperm cells. These groups of sialic acid-rich glycoproteins in the oviductal epithelium and of sialic acid-binding proteins in the plasma membrane of sperm cells are good candidates for further studies to characterize the molecules responsible for sperm binding. We conclude that there are segment-specific changes of sugar residues present in the oviductal epithelium associated with different endocrine environments. Sperm-oviduct adhesion in the rat occurs by interaction of sialoglycoconjugates present in the epithelial cells with sialic acid-binding proteins on the sperm surface. This replicates the situation previously found in hamsters, disclosing for the first time that species-specificity in the sugar involved in sperm binding is not absolute.