Guinea pig fertilin exhibits restricted lateral mobility in epididymal sperm and becomes freely diffusing during capacitation.

The guinea pig sperm protein fertilin functions in sperm-egg plasma membrane binding. Fertilin is initially present in the plasma membrane of the whole head in testicular sperm, then becomes concentrated into the posterior head domain during epididymal passage. Fertilin remains localized to the posterior head plasma membrane following the acrosome reaction, when it functions in sperm-egg interaction. Fluorescence redistribution after photobleaching was used to examine the lateral mobility of fertilin in both acrosome-intact and acrosome-reacted sperm. Fertilin exhibited highly restricted lateral mobility in both testicular and epididymal sperm (D < 10(-10) cm(2)/s). However, fertilin in acrosome-reacted sperm was highly mobile within the membrane bilayer (D = 1.8 x 10(-9) cm(2)/s and %R = 84). Measurement of the lateral mobility of fertilin in capacitated, acrosome-intact sperm revealed two populations of cells. In approximately one-half of the cells, lateral mobility of fertilin was similar to sperm freshly isolated from the cauda epididymis; while in the other half fertilin was highly mobile. The release of fertilin from interactions that restrict its lateral mobility may regulate its function in sperm-egg interaction.

Expression and localization of caveolin-1, and the presence of membrane rafts, in mouse and Guinea pig spermatozoa.

In somatic cells, caveolin-1 plays several roles in membrane dynamics, including organization of detergent-insoluble lipid rafts, trafficking of cholesterol, and anchoring of signaling molecules. Events in sperm capacitation and fertilization require similar cellular functions, suggesting a possible role for caveolin-1 in spermatozoa. Immunoblot analysis demonstrated that caveolin-1 was indeed present in developing mouse male germ cells and both mouse and guinea pig spermatozoa. In mature spermatozoa, caveolin-1 was enriched in a Triton X-100-insoluble membrane fraction, as well as in membrane subdomains separable by means of their light buoyant densities through sucrose density gradient centrifugation. These data indicated the presence of membrane rafts enriched in caveolin-1 in spermatozoa. Indirect immunofluorescence analysis revealed caveolin-1 in the regions of the acrosome and flagellum in sperm of both species. Confocal immunofluorescence analysis of developing mouse male germ cells demonstrated partial co-localization with a marker for the acrosome. Furthermore, syntaxin-2, a protein involved in acrosomal exocytosis, was present in both raft and nonraft fractions in mature sperm. Together, these data indicated that sperm membranes possess distinct raft subdomains, and that caveolin-1 localized to regions appropriate for involvement with acrosomal biogenesis and exocytosis, as well as signaling pathways regulating such processes as capacitation and flagellar motility.

Analysis of the process of localization of fertilin to the sperm posterior head plasma membrane domain during sperm maturation in the epididymis.

Fertilin is a heterodimeric (subunits alpha and beta) sperm plasma membrane protein. Both subunits belong to the ADAM protein family of surface proteins that contain a disintegrin and a metalloprotease domain. Fertilin functions in sperm-egg fusion by binding the sperm to the egg plasma membrane via a binding site in the disintegrin domain of fertilin beta. On testicular sperm of guinea pig, fertilin is distributed on the plasma membrane over the entire sperm head, but is found only on the posterior head once sperm have passed through the epididymis. This redistribution of fertilin to the posterior head can be partially mimicked in vitro if testicular sperm are briefly treated with trypsin. In this study we used immunofluorescence and digital image analysis to analyze how fertilin becomes restricted to the posterior head. We found that fertilin became restricted to the posterior head by migration of anterior head fertilin molecules into the posterior head domain. Comparison of immunofluorescence patterns and immunoblots of fertilin from seven regions of the epididymis showed a temporal correlation between the beginning of fertilin's migration to the posterior head and the proteolytic processing of the full-length fertilin beta precursor (the 85-kDa pro-beta form) to a 75-kDa intermediate, pro-beta*. Completion of the migration coincided with the further cleavage of pro-beta* to the 25- to 28-kDa mature form. Our data suggest that the cleavage of fertilin pro-beta to pro-beta* may initiate fertilin's migration into the posterior head domain and, after localization to that membrane domain, pro-beta* is cleaved to mature beta. We also report evidence that a common mechanism may be used to change the localization pattern of other sperm surface molecules. Other surface proteins were shown to become localized to either the posterior or the anterior head membrane domains on sperm at the same time fertilin became localized to the posterior head. These restrictions of surface protein localizations were also shown to immediately precede the development of the sperm's ability to swim and undergo the acrosome reaction, and thus redistribution of surface proteins may be necessary before sperm become functional.

Structural change of the endoplasmic reticulum during fertilization: evidence for loss of membrane continuity using the green fluorescent protein.

Green fluorescent protein (GFP) was targeted to the lumen of the endoplasmic reticulum (ER) of starfish eggs by injecting mRNA coding for a chimeric protein containing a signal sequence and the KDEL ER retention sequence. By confocal microscopy, the GFP chimeric protein was localized in intracellular cisternae (membrane sheets) and the nuclear envelope, showing that it had been successfully targeted to the ER. The labeling pattern closely resembled that produced by the fluorescent dicarbocyanine DiI, which has been used previously to label the ER (Jaffe and Terasaki, Dev. Biol. 164, 579-587, 1994). Eggs expressing the GFP chimera were used to examine whether there is a loss of ER continuity at fertilization. The time required for recovery of fluorescence after photobleaching for both the GFP chimera and DiI was much longer in eggs at 1 min postfertilization than in unfertilized eggs or in 20-min-postfertilized eggs. This result provides strong evidence for a transient loss of continuity of the ER associated with Ca release at fertilization.

Sperm surface protein PH-20 is bifunctional: one activity is a hyaluronidase and a second, distinct activity is required in secondary sperm-zona binding.

In previous studies, we have found that the sperm membrane protein PH-20 acts during two different stages of fertilization. On acrosome-intact sperm, PH-20 has a hyaluronidase activity that is required for sperm penetration through the cumulus cell layer that surrounds the oocyte. On acrosome-reacted sperm, PH-20 has a required function in sperm-zona binding (secondary binding). Because hyaluronic acid (HA) has been detected in the zona pellucida, secondary sperm-zona adhesion could depend on repetitive binding and hydrolysis of HA by PH-20 acting as a hyaluronidase. Alternatively, PH-20 may be bifunctional and have a second, different activity required for secondary binding. To distinguish between these two possibilities, in this study we used reagents that inhibit either PH-20's function in sperm-zona binding or its hyaluronidase activity. We found that an anti-PH-20 monoclonal antibody that inhibited sperm-zona binding (approximately 90%) had no effect on hyaluronidase activity. Conversely, apigenin, a hyaluronidase inhibitor, blocked PH-20 hyaluronidase activity 93% without inhibiting sperm-zona binding. Similarly, another anti-PH-20 monoclonal antibody that inhibited hyaluronidase activity 95% only partially inhibited sperm-zona binding (approximately 45%). We also extensively pretreated oocytes with hyaluronidase to remove all accessible HA on or in the zona pellucida and found little or no effect on secondary sperm-zona binding. Our results suggest that PH-20 is bifunctional and has two activities: a hyaluronidase activity and a second, separate activity required for secondary sperm-zona binding.

Structural relationship of sperm soluble hyaluronidase to the sperm membrane protein PH-20.

The sperm plasma membrane protein PH-20 has a hyaluronidase activity that enables acrosome-intact sperm to pass through the cumulus cell layer of the egg. In this study we analyzed the relationship of guinea pig PH-20 and the "classical" soluble hyaluronidase released at the time of the acrosome reaction of guinea pig sperm. PH-20 is a membrane protein, anchored in the plasma and inner acrosomal membranes by a glycosyl phosphatidyl inositol anchor. Several types of experiments indicate a structural relationship of PH-20 and the soluble hyaluronidase released during the acrosome reaction. First, an antiserum raised against purified PH-20 is positive in an immunoblot of the soluble protein fraction released during the acrosome reaction. In the released, soluble protein fraction, the anti-PH-20 antiserum recognizes a protein of approximately 64 kDa, i.e., identical in molecular mass to PH-20 (approximately 64 kDa). Second, the enzymatic activity of the released hyaluronidase is completely inhibited (100%) by the anti-PH-20 antiserum. Third, almost all (97%) of the soluble hyaluronidase is removed from the released protein fraction by a single pass through an affinity column made with an anti-PH-20 monoclonal antibody. These findings suggest that the released, soluble hyaluronidase is a soluble form of PH-20 (sPH-20). During the acrosome reaction, PH-20 undergoes endoproteolytic cleavage into two disulfide-linked fragments whereas the released sPH-20 is not cleaved, suggesting the possible activity of a membrane-bound endoprotease on PH-20. We searched for a cDNA encoding sPH-20 but none was found. This result suggests that sPH-20 may arise from the enzymatic release of PH-20 from its membrane anchor, possibly at the time of acrosome reaction.

Rapid and slow mechanisms for loss of cell adhesiveness during fertilization in Chlamydomonas.

Although vegetative cells, gametes, and zygotes of the biflagellated alga Chlamydomonas bear flagella, only the flagella of mt+ and mt- gametes are adhesive. The molecules responsible for adhesiveness, mt+ and mt- agglutinins, are long rod-shaped glycoproteins displayed on the flagellar membrane. These flagellar agglutinins, which gametes use both as adhesion and signaling molecules during the early events of fertilization, are lost from the flagella during adhesion. Flagellar adhesiveness can be maintained, however, by recruitment and activation of preexisting, inactive agglutinins from the plasma membrane of the cell body (Hunnicutt et al, 1990, J. Cell Biol. 111, 1605-1616) unless the gametes of opposite mating types fuse to form zygotes. Upon cell fusion, flagellar adhesiveness is lost. In the studies presented here, we have employed an in vitro bioassay to measure agglutinins in both cell bodies and flagella at various times during gametogenesis, during fertilization, and after zygote-formation. By use of the bioassay, which can detect agglutinins that are functionally inactive in vivo, we found that vegetative cells are devoid of agglutinins. These adhesion molecules appear only after gametogenesis is underway with the cell body agglutinins appearing first and then the flagellar agglutinins. Surprisingly, 30 min after zygote formation, when the zygotes' flagella are no longer adhesive, the flagellar agglutinin activity detectable with the bioassay remains high. One interpretation of these results is that zygotes continue to recruit agglutinins from the cell body to the flagella, but cell fusion abrogates activation of the agglutinins. Within 45-90 min after fusion both the cell body and flagellar agglutinins are lost and can be detected in the medium. These mechanisms, which render the zygotes nonadhesive to other zygotes and unmated gametes, contribute to the Chlamydomonas equivalent of a block to polyspermy.

Cell body and flagellar agglutinins in Chlamydomonas reinhardtii: the cell body plasma membrane is a reservoir for agglutinins whose migration to the flagella is regulated by a functional barrier.

Fertilization in Chlamydomonas reinhardtii is initiated when gametes of opposite mating types adhere to each other via adhesion molecules (agglutinins) on their flagella. Adhesion leads to loss of active agglutinins from the flagella and recruitment of new agglutinins from a pool associated with the cell body. We have been interested in determining the precise cellular location of the pool and learning more about the relationship between agglutinins in the two domains. In the studies reported here we describe methods for purification of mt+ cell body agglutinins by use of ammonium sulfate precipitation, chromatography (molecular sieve, ion exchange, and hydrophobic interaction), and sucrose gradient centrifugation. About 90% of the total agglutinins were associated with the cell body and the remainder were on the flagella. Cell body agglutinins were indistinguishable from mt+ flagellar agglutinins by SDS-PAGE, elution properties on a hydrophobic interaction column, and in sedimentation properties on sucrose gradients. The nonadhesiveness of cell bodies suggested that the cell body agglutinins would be intracellular, but our results are not consistent with this interpretation. We have demonstrated that brief trypsin treatment of deflagellated gametes destroyed all of the cell body agglutinins and, in addition, we showed that the cell body agglutinins were accessible to surface iodination. These results indicated that C. reinhardtii agglutinins have a novel cellular disposition: active agglutinins, representing approximately 10% of the total cellular agglutinins, are found only on the flagella, whereas the remaining 90% of these molecules are on the external surface of the cell body plasma membrane in a nonfunctional form. This segregation of cell adhesion molecules into distinct membrane domains before gametic interactions has been demonstrated in sperm of multicellular organisms and may be a common mechanism for sequestering these critical molecules until gametes are activated for fusion. In experiments in which surface-iodinated cell bodies were permitted to regenerate new flagella, we found that the agglutinins (as well as the 350,000 Mr, major flagellar membrane protein) on the newly regenerated flagella were iodinated. These results indicate that proteins destined for the flagella can reside on the external surface of the cell body plasma membrane and are recruited onto newly forming flagella as well as onto preexisting flagella during fertilization.