PARENT SESSION
Fertility Regulation
368
POTENTIAL USE OF ARYLSULFATASE-A (AS-A) AS A NON-HORMONAL CONTRACEPTIVE.
Carmona, Euridice1, Weerachatyanukul, Wattana1, Xu, Hongbin1, Shoushtarian, Ali1, Tanphaichitr, Nongnuj1,2, 1 2
ABSTRACT- AS-A (E.C. 3.1.6.8) can desulfate male germ cell specific sulfogalactosylglycerolipid (SGG) in solution, provided that SGG is "solubilized" by a detergent to allow its accessibility to the enzyme's active site pocket. Our results indicate that both SGG and AS-A are localized to the sperm head surface without any GG formation, implying that SGG is not situated next to the AS-A's active site. Both are also involved in sperm-egg binding. In this report, we attempted to desulfate SGG by adding exogenous AS-A to the sperm suspension, with the expectation that GG-containing sperm would be incapable to bind to the eggs. As expected, in vitro sperm-zona pellucida (ZP) binding was inhibited in a concentration dependent manner, following sperm treatment with human liver AS-A, reaching the background level with only 63 nM of AS-A added. Along with this inhibition, we observed an increase in the spontaneous acrosome reaction, i.e., up to 50% within 2 hr of the AS-A treatment (twice higher than the rate observed with control sperm). Sperm viability, as assessed by propidium iodide exclusion, remained high in both AS-A treated and control sperm (95% of total population). In addition, mice transcervically inseminated with unwashed sperm pretreated with 630 nM AS-A showed only 25% eggs fertilized in vivo (scored by the presence of ovulated two-pronuclei eggs in the following day), as compared to 80% in control mice inseminated with untreated sperm. Surprisingly, there was no obvious SGG breakdown, as observed by HPTLC of AS-A-treated sperm lipids. However, fluorescent microscopy revealed that Alexa-430-conjugated AS-A bound to the sperm head, where SGG was. This suggested that exogenous AS-A may bind SGG through sites separate from the enzyme active site pocket, masking SGG to interact with the ZP. Also, this binding may lead to an increase in membrane fluidity and/or SGG aggregation (perhaps through AS-A multimerization ability), both of which could induce the premature acrosome reaction. Regardless of the mechanisms, the contraceptive effects of AS-A are astounding and warrant further investigation/development for its use as a non-hormonal contraceptive. This work was supported by CIHR grant MT 10366.
KEY WORDS: arylsulfatase A, fertilization, contraception, sulfoglycolipid