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Sperm Exocytosis Increases the Amount of PH-20 Antigen on the Surface of Guinea Pig Sperm Ann E

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Sperm Exocytosis Increases the Amount of PH-20 Antigen on the Surface of Guinea Pig Sperm Ann E Sperm Exocytosis Increases the Amount of PH-20 Antigen on the Surface of Guinea Pig Sperm Ann E. Cowan, Paul Primakoff, and Diana G. Myles Department of Physiology, University of Connecticut Health Center, Farmington, Connecticut 06032 Abstract. Evidence has been presented that the PH-20 antigenic sites on the sperm surface. These new anti- protein functions in sperm adhesion to the egg zona genic sites are revealed on the surface by insertion of pellucida (Primakoff, P., H. Hyatt, and D. G. Myles, the IAM into the plasma membrane. Our evidence in- 1985, J. Cell Biol., 101:2239-2244). The PH-20 pro- dicates that before the acrosome reaction an intracellu- tein migrates from its original surface domain to a lar population of PH-20 antigen is localized to the new surface domain after the acrosome reaction IAM. When migration of the surface population of the (Myles, D. G., and P. Primakoff, 1984, J. Cell Biol., PH-20 protein is prevented, PH-20 protein can still be 99:1634-1641). The acrosome reaction is an exocytotic detected on the IAM of acrosome-reacted sperm. Also, event that results in insertion of a region of the secre- PH-20 protein can be detected on the IAM of permea- tory granule membrane, the inner acrosomal mem- bilized acrosome-intact sperm by indirect immunofluo- brane (IAM), into the plasma membrane. After the rescence. Thus, the sperm cell regulates the amount of acrosome reaction, PH-20 protein migrates to the IAM PH-20 protein on its surface by sequestering about from its initial domain on the posterior head surface. two-thirds of the protein on an intracellular membrane We have now found a new dynamic feature of the and subsequently exposing this population on the cell regulation of PH-20 protein on the sperm surface; exo- surface by an exocytotic event. This may be a general cytosis increases the surface expression of PH-20 pro- mechanism for regulating cell surface composition tein. After the acrosome reaction there is an approxi- where a rapid increase in the amount of a cell surface mately threefold increase in the number of PH-20 protein is required. ELL surface function can be regulated by controlling In this paper we report a second mechanism for the control the level of expression of surface molecules, their of the surface expression of the PH-20 antigen that acts to C time of appearance, and their localization in the regulate the amount of PH-20 on the cell surface. Initially, membrane. Our laboratory has been studying the function the PH-20 protein is found on both an intracellulax mem- and localization of a guinea pig-sperm surface antigen, the brane, the acrosomal membrane, and on the plasma mem- PH-20 protein. We have obtained evidence that this protein brane. We term the acrosomal membrane population PH- has a role in sperm binding to the egg zona pellucida 20~ and the plasma membrane population PH-20pM. (Primakoff et al., 1985). Furthermore, we have found that When the acrosome reaction occurs, PH-20~ is added to the PH-20 protein is localized to the posterior head surface the cell surface and serves to increase surface expression of of acrosome-intact sperm, but after the acrosome reaction its the PH-20 protein. Thus, sperm exocytosis, which is re- pattern of localization is altered (Myles and Primakoff, quired for fertilization (Noda and Yanagimachi, 1976) results 1984). The acrosome reaction is an exocytotic event that in both an "upregulation" of the PH-20 antigen and a dra- results in insertion of a region of the secretory granule mem- matic alteration in its surface localization. We speculate that brane, the inner acrosomal membrane (IAM), ~ into the these changes in the surface expression of PH-20 protein may plasma membrane. After the acrosome reaction, the PH-20 be important in regulating sperm-zona interaction. antigen migrates rapidly and completely to the IAM. This is an example of a dynamic regulation of the localization of a Materials and Methods cell surface molecule. The PH-20 migration is a specific pro- cess, since other antigens localized to the posterior head Antibodies surface remain on the posterior head after the acrosome reaction. The three monoclonal antibodies (MAbs) used in this study (PH-20, PH-21, and PH-22) are described in Primakoff and Myles (1983) and Primakotf et 1. Abbreviations used in this paper: FITC, fluorescein isothiocyanate; IAM, al. (1985). All three MAbs recognize the PH-20 protein and belong to the inner acrosomal membrane; MAb, monoclonal antibody; OAM, outer subclass IgG1 (Primakoff et al., 1985). Competition experiments and tests acrosomai membrane. for inhibition of sperm-zona binding show that at least two and probably © The Rockefeller University Press, 0021-9525/86/10/1289/09 $1.00 The Journal of Cell Biology, Volume 103, October 1986 1289-1297 1289 three different epitupes are recognized by the three MAbs (Primakoff ct al., NaCI, 4 mM KC1, 4 mM Hepes (pH 7.4), 10 mM glucose, and 2 mM 1985). CaC12) at 37"C (Green, 1978). Previous results have shown that migration The PH-22 MAb was purified from culture supernatants by protein of PH-20 protein from the posterior head to the IAM occurs rapidly (within A-affinity chromatography using the BioRad Monoclonal Antibody Puri- 5-10 rain after A23187 addition) when sperm are acrosome-reacted by this fication System as described in the suppliers instructions (Bio-Rad Labora- method (Myles and Pfimakoff, 1984). Alternatively, sperm were incubated tories, Richmond, CA). The Fab' fragment of the PH-22 MAb was produced for 1 h in modified Tyrode's medium lacking calcium (109 mM NaCi, 2.8 by pepsin digestion to produce a F(ah02 fragment that was subsequently re- mM KC1, 0.5 mM MgCI2, 25 mM NaI-ICO3, 5.6 mM glucose, 10 mM lac- duced to produce the Fall (Parham, 1983). Purified PH-22 IgG (5 mg) was tic acid, 1 mM Na-pyruvate, pH 7.6, and 0.3% BSA [fraction V, Calbio- incubated for 8 h at 37°C with 125 ~l of pepsin-coated agarose beads (Bio- chem-Behring Corp.]) supplemented with 75 ktg/mi lysophosphatidyl cho- Rad Laboratories) in 0.1 M citrate buffer (pH 3.5) in a total volume of 0.5 line (Sigma Chemical Co.) at 37"C, and the acrosome reaction induced by mi to completely digest the IgG into P(ab')2. The pH was neutralized by the addition of an equal volume of modified Tyrode's medium containing addition of 1/10th volume 0.3 M Tris (pH 8.6), and the pepsin-coated beads 4 mM Ca 2+ (Fleming and Yanagimachi, 1981). In contrast with sperm removed by centrifugation. The F(at02 was reduced with 20 mM cysteine acrosome-reacted using A23187, sperm acrosome-reacted using lysophos- for 1 h at 37°C, and alkylated with 30 mM iodoacetamide for 15 min at room phatidyl choline are hypermotile and capable of fertilizing eggs. This temperature. Fab' was purified from residual F(aht)2 by Sephadex G-100 method for inducing the acrosome reaction resulted in a much slower rate chromatography. The FalY was completely free of contaminating material as of PH-20 migration (see Fig. 2). Sperm were scored as acrosome-intact or determined by nonreducing SDS-polyacrylamide gels stained with Coomas- acrosome-reacted using phase-contrast optics. sic Blue. The PH=22 Fab' was iodinated using the Bolton-Hunter reagent (New En- Protease Treatment gland Nuclear, Boston, MA), as detailed in the suppliers instructions, t2sI- Labeled Fal~ was separated from hydrolysis products and unreaeted reagent Sperm at 5 x 107/mi in Mg2+ Hepes were digested with 2 mg/mlPrunase by Sephadex G-50 chromatography. (Calbioehem-Behring Corp.) at 37°C for 30 rain. The sperm were washed Rhodamine labeling of the purified PH-22 MAb was done using once, resuspended at 2 x 107/ml for Ca 2+ Hepes medium at 37°C, and tetraethyl rhodamine isothiocyanate (RITC; Sigma Chemical Co., St. acrosome-reacted by the addition of 1 Ig/ml A23187. The sperm were Louis, MO) in 0.1 M sodium carbonate buffer (pH 9.5) at a ratio of 25 I~g washed, fixed in formaldehyde, and stained for immunefluorescence. RITC/mg protein (Goding, 1976). The rhodamine conjugate was separated from free dye by chromatography on a Sephadex G-25 column. Assay of Antigenic Activity Second antibodies used were fluorescein isothiocyanate (FITC)- or RITC F(ab)2 goat anti-mouse IgG and Fah goat-anti-mouse IgG (Cooper Bio- The amount of antigenic activity in cell fractions was measured by determin- medical, Malvern, PA). The latter antibody was conjugated with rhodamine ing the ability of the fraction to inhibit an indirect solid-phase radioactive as described above for purified PH-22 MAb. binding assay. The indirect binding assay has been described previously (Myles et al., 1981). Briefly, an octylghicoside extract of acrosome-intact lmmunofluorescence sperm was bound to the wells of a 96-well plate. The plates were washed, unbound sites blocked with 3 % ovalbumin in PBS, and MAb to be tested Immunotluorescence staining was carried out on live sperm or sperm fixed was incubated in the wells. After washing, MAb bound to the wells was de- in 1.5% formaldehyde (made fresh from paraformaldehyde) as previously tected using an l~I-rabhit anti-mouse IgG (New England Nuclear). For described (Myles ct al., 1981). For immunefluorescence of permeabilized the inhibition experiments, an appropriate dilution of MAb was chosen that sperm, sperm were isolated from the cauda epididymis in Mg 2+ Hepes was not saturating in the solid-phase assay.
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