We have previously described the zonae pellucidae (ZP) binding ability of a pig sperm surface protein, P68. Our recent results on peptide sequencing of 3 P68 tryptic peptides and molecular cloning of pig testis arylsulfatase A (AS-A) revealed the identity of P68 as AS-A. In this report, we demonstrate the presence of AS-A on the mouse sperm surface and its role in ZP binding. Using anti-AS-A antibody, we have shown by immunoblotting that AS-A was present in a Triton X-100 extract of mouse sperm. The presence of AS-A on the sperm plasma membrane was conclusively demonstrated by indirect immunofluorescence, immunogold electron microscopy, and AS-A's desulfation activity on live mouse sperm. The AS-A remained on the head surface of in vivo capacitated sperm, as revealed by positive immunofluorescent staining of oviductal/uterine sperm. Significantly, the role of mouse sperm surface AS-A on ZP binding was demonstrated by dose-dependent decreases of sperm-ZP binding on sperm pretreatment with anti-AS-A IgG/Fab. Furthermore, Alexa-430 conjugated AS-A bound to mouse ZP of unfertilized eggs but not to fertilized ones, and this level of binding increased and approached saturation with increasing Alexa-430 AS-A concentrations. Moreover, in vivo fertilization was markedly decreased when mouse sperm pretreated with anti-AS-A IgG were artificially inseminated into females. All of these results designated a new function for AS-A in mouse gamete interaction.

We have shown previously that male germ cell-specific sulfoglycolipid, sulfogalactosylglycerolipid (SGG), is involved in sperm-zona pellucida binding, and that SGG and its desulfating enzyme, arylsulfatase A (AS-A), coexist in the same sperm head area. However, AS-A exists at a markedly low level in sperm as compared to SGG (i.e., 1/400 of SGG molar concentration). In the present study, we investigated whether perturbation of this molar ratio would interfere with sperm-egg interaction. We demonstrated that purified AS-A bound to the mouse sperm surface through its high affinity with SGG. When capacitated, Percoll gradient-centrifuged mouse sperm were treated for 1 h with various concentrations of AS-A, their binding to zona-intact eggs was inhibited in a dose-dependent manner and reached the background level with 63 nM AS-A. This inhibition could be partially explained by an increase in premature acrosome reaction. The acrosome-reacted sperm population of the 63 nM AS-A-treated sperm sample was twice that of the control sample (treated with 63 nM ovalbumin) at 1 h (i.e., 32% vs. 15%) and rose to 53% at 2 h. This induction was presumably due to SGG aggregation attributed to AS-A, existing as a dimer at neutral pH, and could be mimicked by anti-SGG immunoglobulin (Ig) G/IgM + secondary IgG antibody. Drastic inhibition (75%) of in vivo fertilization was also observed in females inseminated with sperm suspension containing 630 nM AS-A as compared to the rate in females inseminated with sperm suspension included with 630 nM ovalbumin. Our results demonstrate a promising potential for AS-A as a nonhormonal, vaginal contraceptive.

Cumulus cell layers of expanded cumulus oocyte complexes (COCs) are interlinked with networks of hyaluronic acid, chondroitin sulfate B proteoglycans and link proteins, and they can be dispersed by sperm surface hyaluronidases. In this report, we showed that arylsulfatase A (AS-A), existing on the sperm head surface, also had this dispersion action. Purified AS-A free of protease, hyaluronidase and chondroitinase activities could disperse the cumulus matrix of expanded COCs. However, this COC dispersion action was not associated with AS-A desulfation activity, assayed by using p-nitrocatecholsulfate (artificial substrate). COCs incubated for 1 h with sperm pretreated with anti-AS-A IgG in the presence of apigenin (a hyaluronidase inhibitor) did not exhibit matrix dispersion, whereas several cumulus layers were already dispersed in COCs incubated with sperm pretreated with preimmune IgG. Furthermore, sperm from AS-A null mice showed a significant delay in COC dispersion, compared with wild-type sperm. Within 1 h of sperm-COC co-incubation, the size of COCs incubated with AS-A null sperm was 65% of the original dimension, whereas that of COCs inseminated with wild-type sperm was only 17%. A further delay in COC dispersion by AS-A(-/-) mouse sperm was observed when apigenin was present in the co-incubation. We also showed for the first time that AS-A had a specific affinity for chondroitin sulfate B, a component of cumulus matrix proteoglycan networks; this might provide a mechanism of cumulus matrix destabilization induced by sperm surface AS-A.

Arylsulfatase A (AS-A) is a lysosomal enzyme, which catalyzes the desulfation of certain sulfogalactolipids, including sulfogalactosylglycerolipid (SGG), a molecule implicated in cell adhesion. In this report, immunocytochemistry revealed the selective presence of AS-A in the corpus luteum of mouse ovaries. Immunoblotting indicated that mouse corpus luteum AS-A had a molecular mass of 66 kDa, similar to AS-A of other tissues. Corpus luteum AS-A was active, capable of desulfating the artificial substrate, p-nitrocatechol sulfate, at the optimum pH of five. To understand further the role of AS-A in female reproduction, levels of AS-A were determined during corpus luteum development in pseudopregnant mice and during luteolysis after cessation of pseudopregnancy. Immunocytochemistry, immunoblotting and desulfation activity showed that AS-A expression was evident at the onset of pseudopregnancy in the newly formed corpora lutea, and its level increased steadily during gland development. The increase in the expression and activity of AS-A continued throughout luteolysis after the decrease in serum progesterone levels. We also observed the selective presence of SGG on the luteal cell surface in developed corpora lutea, as shown by immunofluorescence of mouse ovary sections as well as high-performance thin-layer chromatography of lipids isolated from mouse and pig corpora lutea. The identity of the "SGG" band on the thin layer silica plate was further validated by electrospray ionization mass spectrometry. Significantly, SGG disappeared in regressing corpora lutea. Therefore, lysosomal AS-A may be involved in cell-surface remodeling during luteolysis by desulfating SGG after its endocytosis and targeting to the lysosome.