Human sperm CRISP2 is released from the acrosome during the acrosome reaction and re-associates at the equatorial segment.

Sperm CRISP2 has been proposed to be involved in sperm-egg fusion. After the acrosome reaction, it appears at the equatorial segment (EqS) of human sperm; the mechanism underlying the appearance of CRISP2 at the EqS remains unknown, though. Here, we provide evidence showing the re-association of sperm acrosomal CRISP2 at the EqS during the acrosome reaction. Results showed that F-actin is not involved in the relocalization of CRISP2. We found that basic, but not acidic, conditions can solubilize CRISP2 from sperm cells, suggesting that CRISP2 is a component of the acrosome and that it is released from the acrosome during the acrosome reaction. Purified, biotinylated human sperm acrosomal CRISP2 binds to the EqS of acrosome-reacted sperm in a dose-dependent manner, revealing that CRISP2 detected at the EqS of acrosome-reacted sperm comes from the population stored in the acrosome. The association of CRISP2 at the EqS is very strong, and does not depend on ionic interactions or intermolecular disulfide bonds. Interestingly, the restriction of CRISP2 at the EqS was diminished when EGTA was present in the media, indicating that Ca(2+) is required for maintaining CRISP2 at the EqS. This study supports the possibility that CRISP2 may help modify the EqS membrane to make this domain fusion-competent.

Roles of mouse sperm-associated alpha-L-fucosidases in fertilization.

Sperm-associated α-L-fucosidases have been implicated in fertilization in many species. Previously, we documented the existence of α-L-fucosidase in mouse cauda epididymal contents, and showed that sperm-associated α-L-fucosidase is cryptically stored within the acrosome and reappears within the sperm equatorial segment after the acrosome reaction. The enrichment of sperm membrane-associated α-L-fucosidase within the equatorial segment of acrosome-reacted cells implicates its roles during fertilization. Here, we document the absence of α-L-fucosidase in mouse oocytes and early embryos, and define roles of sperm associated α-L-fucosidase in fertilization using specific inhibitors and competitors. Mouse sperm were pretreated with deoxyfuconojirimycin (DFJ, an inhibitor of α-L-fucosidase) or with anti-fucosidase antibody; alternatively, mouse oocytes were pretreated with purified human liver α-L-fucosidase. Five-millimolar DFJ did not inhibit sperm-zona pellucida (ZP) binding, membrane binding, or fusion and penetration, but anti-fucosidase antibody and purified human liver α-L-fucosidase significantly decreased the frequency of these events. To evaluate sperm-associated α-L-fucosidase enzyme activity in post-fusion events, DFJ-pretreated sperm were microinjected into oocytes, and 2-pronuclear (2-PN) embryos were treated with 5 mM DFJ with no significant effects, suggesting that α-L-fucosidase enzyme activity does not play a role in post-fusion events and/or early embryo development in mice. The recognition and binding of mouse sperm to the ZP and oolemma involves the glycoprotein structure of α-L-fucosidase, but not its catalytic action. These observations suggest that deficits in fucosidase protein and/or the presence of anti-fucosidase antibody may be responsible for some types of infertility.

Distribution, crypticity, stability, and localization of α-L-fucosidase of mouse cauda epididymal sperm.

Sperm-associated and semen-specific isoforms of α-L-fucosidase are thought to function in fertilization in numerous organisms. Here, we report the localization, distribution, crypticity, and stability of this enzyme in mouse cauda epididymal sperm and cauda fluid. Western analysis revealed that the sperm-associated α-L-fucosidase is present as two isoforms (Mr ∼49 and 56 kDa), whereas the cauda fluid α-L-fucosidase shows a single band at 50 kDa. α-L-Fucosidase activity was detected using the fluorogenic substrate 4-MU-FUC. Of the total α-L-fucosidase activity recovered in the cauda epididymal contents, 74% was found in the cell-free cauda fluid and about 7% was found in sperm cells. During capacitation or permeabilization, cryptic intracellular stores of soluble enzyme were released to the supernatant, while leaving bound enzyme concentrated within the small volume of sperm. Moreover, membrane-associated enzyme activity was still detectable in acrosome-reacted cells. Immunofluorescence studies support the presence of α-L-fucosidase (originally localizing at the acrosomal area) at the equatorial segment after the acrosome reaction. α-L-Fucosidase activity of both cauda fluid and sperm at 37°C, 5% CO(2) was relatively stable and detectable up to 72 hr. The stability and appearance of mouse sperm-associated α-L-fucosidase in the equatorial segment after the acrosome reaction suggest that α-L-fucosidase may be involved in sperm-egg interaction.

Hamster sperm-associated alpha-L-fucosidase functions during fertilization.

Sperm-associated alpha-L-fucosidases have been identified in diverse organisms. Their wide phylogenetic distribution and known properties support the likelihood that L-fucose and alpha-L-fucosidase have fundamental function(s) during gamete interaction. This is consistent with the substantial evidence in the literature documenting the importance of carbohydrate moieties during fertilization. Direct enzyme assays were employed to evaluate the functional distribution of alpha-L-fucosidase in preparations of hamster sperm. In vitro fertilization was performed using Syrian hamster sperm and eggs to identify the functional role of hamster sperm-associated alpha-L-fucosidase during zona pellucida binding/penetration, sperm-egg membrane fusion, and postfusion events. Results reported here document the presence of hamster sperm-associated alpha-L-fucosidase and demonstrate that it functions during fertilization at the stage of sperm-oocyte membrane interaction and/or postfusion events within the zygote. Understanding the role of alpha-L-fucosidase during human fertilization could lead to development of improved infertility treatments.

Stabilization of membrane-associated alpha-L-fucosidase by the human sperm equatorial segment.

Previous reports from this laboratory documented the existence of two novel isoforms of alpha-L-fucosidase in human semen and showed that membrane-associated alpha-L-fucosidase is cryptically held within the acrosomal compartment and enriched within the sperm equatorial segment. The occurrence of these novel isoforms is provocative. Sperm proteins potentially involved in sperm-egg interactions must maintain their functional integrity as they travel through the female reproductive tract. The goal of this project was to investigate the stability of membrane-associated alpha-L-fucosidase in human sperm. Whole seminal plasma and Percoll purified sperm cell populations were incubated for 72 h at 37 degrees C, with 5% CO(2) or ambient air. At various times during prolonged incubation, sperm cells were permeabilized with 0.1% Triton X-100 and enzyme assays using the fluorogenic substrate 4-MU-fuc were performed to evaluate the stability of both the seminal plasma and membrane-associated alpha-L-fucosidase. Here, we report seminal plasma alpha-L-fucosidase activity rapidly decreased within 24 h. Conversely, alpha-L-fucosidase activity from Percoll purified sperm cell populations persisted up to 72 h. Data from these experiments support the notions that (i) membrane-associated alpha-L-fucosidase is stable for extended periods of time, consistent with a possible role in sperm-egg interaction and (ii) membrane domains and compartmentalization within the human sperm are key to preserving protein integrity.

Crypticity and functional distribution of the membrane associated alpha-L-fucosidase of human sperm.

Two distinctive isoforms of the enzyme alpha-L-fucosidase are found within human semen in substantial amounts, suggesting specialized functions during reproduction. The membrane-associated isozyme of human sperm cells was previously characterized biochemically, and here we report on its subcellular localization. Intact, detergent permeabilized, capacitated, and acrosome-reacted sperm were investigated using antifucosidase immunofluorescence, binding of the fluorescent fucosylated glycoconjugate RITC-BSA-fucose (RBF), and enzyme activity in the presence and absence of selected inhibitors. Both immunolocalization and RBF binding show that fucosidase is broadly distributed over the membrane systems of human sperm, but is relatively enriched within the equatorial segment. Upon detergent treatment or induction of acrosome reaction (AR), a portion of enzyme activity is recoverable in the supernatant, presumably associated with released remnants of the outer acrosomal membrane. Surprisingly, cell-bound enzyme activity increases sharply following permeabilization of intact sperm, representing cryptic fucosidase that is relatively stable and corresponds with strong fluorescence in the equatorial segment and other sperm membranes. These observations support the notion that the fucosidase has a role in the intimate species signature interactions between sperm and oocyte.

Purification and characterization of human seminal plasma alpha-L-fucosidase.

Human seminal plasma alpha-L-fucosidase (EC 3.2.1.51) has been purified 7100-fold to very high purity and specific activity (83,000 nmol/min/mg protein) by affinity chromatography on agarose-epsilon-aminocaproyl-fucopyranosylamine. The purified alpha-L-fucosidase appeared to contain a single subunit of 56-57 kDa (as determined by SDS-PAGE and Western analysis). Lectin blotting and N-glycanase treatment studies indicated that this subunit is N-glycosylated and contains sialic acid residues. Human seminal plasma alpha-L-fucosidase was shown to contain three multimeric forms of 110, 236 and 314 kDa respectively (as determined by Sephadex G-200 chromatography) and therefore probably exists in dimeric, tetrameric and hexameric forms. Kinetic analysis with the 4-methylumbelliferyl-alpha-L-fucopyranoside (4MU-Fuc) substrate indicated a broad acidic optimum (pH 4.0-4.5) with a second neutral optimum (pH 6.4-7.4) with 60-80% of maximal activity. Apparent K(M) and V(max) values for the 4MU-Fuc substrate were determined to be 0.06 mmol/l and 92 micromol/min/mg protein respectively, using Lineweaver-Burk double reciprocal plots. Isoelectric focusing and neuraminidase treatment studies provided further evidence that the purified seminal plasma alpha-L-fucosidase is a sialoglycoprotein with several isoforms between pI values 5-7. The acidic isoforms between pI values 5-6 appear to be related chemically to the more neutral isoforms by sialic acid residues since neuraminidase treatment converted the former into the latter isoforms.

Purification and characterization of plasma membrane-associated human sperm alpha-L-fucosidase.

Detergent and salt extraction studies, as well as cytochemical localization with fluorescein isothiocyanate-bovine serum albumin-L-fucose, have provided further evidence for the plasma membrane association of a novel human sperm, alpha-L-fucosidase. This alpha-L-fucosidase has been solubilized and purified 8600-fold to high specific activity (35 000 U/mg protein) by affinity chromatography on agarose-C(24)-fucosylamine. To our knowledge, this is the first report concerning the purification and characterization of a mammalian plasma membrane-associated alpha-L-fucosidase. Both SDS-PAGE and Western blot analysis indicated the alpha-L-fucosidase is highly purified and contains a single subunit with a molecular mass of 51 kDa. N-glycanase studies indicated the subunit contains N-glycans, and lectin blot analysis detected the presence of mannose, but no terminal galactose or sialic acid residues. Isoelectric focusing indicated the presence of two major alpha-L-fucosidase isoforms (pIs 6.5 and 6.7) and a possible minor isoform (pI 6.3). Treatment of alpha-L-fucosidase with neuraminidase did not change its isoform profile, providing further evidence for the enzyme's lack of sialic acid residues. Kinetic analysis with 4-methylumbelliferyl alpha-L-fucopyranoside indicated that sperm alpha-L-fucosidase has a pH optimum near 7, an apparent K(m) of 0.08 mM, and a V(max) of 6.8 micro mol/min/mg protein. The unusual properties of human sperm alpha-L-fucosidase argue in support of a potentially important, but presently unknown, role for this enzyme in human reproduction.