Involvement of sperm sulphatases in early sperm-zona interactions in the hamster

0 IVF Laboratory, Cromwell Hospital , London SWS OTU, England 1 MRC Experimental Embryology and Teratology Unit , Woodmansteme Road, Carshalton, Surrey, SM5 4EF, England Sulphatase preparations iromAbalone entrails, the limpet Patella vulgata and ox liver, as well as artificial substrates for these enzymes, were used in the hamster in vitro fertilization system to study the possible roles of sperm sulphatases in sperm-zona pellucida interactions. />-nitrophenyl sulphate, />-nitrocatechol sulphate, ascorbate 2-sulphate, as well as D-glucopyranose and Dgalactopyranose, both sulphated at the 3, 4 or 6 position but not the 2 position, inhibited fertilization in a dose-dependent manner. Sperm-egg fusion was not inhibited by the substrates used and eggs pre-treated with sulphates could readily be fertilized. Sperm motility and therefore viability was unaffected by inhibitory concentrations of substrates as determined by rhodamine 123 labelling of motile spermatozoa. Acrosomal integrity of rhodamine-labelled spermatozoa was assessed and found to be unaffected by inhibitory levels of substrates. Fertilization was inhibited by high concentrations of the two molluscan sulphatases but not by purified ox liver sulphatase. Pre-treatment of eggs with these enzymes did not prevent fertilization. Long-term exposure of hamster oocytes to iV-acetylhexosaminidase or limpet sulphatase caused thinning and distension of the zona pellucida but these changes were not observed with the ox liver sulphatase. The results suggest that a glycosulphatase is probably released from hamster spermatozoa during sperm-egg adhesion and, or, penetration. If sperm-egg adhesion molecules are sulphated, the commercially available sulphatases would be unsuitable for their characterization. - Freshly ovulated mammalian oocytes are normally surrounded by three investments, namely the cumulus oophorous, the corona radiata and the zona pellucida. During fertilization, the acellular and translucent zona pellucida presents the major obstacle to capacitated spermatozoa, as they must attach to and pass through this layer in a species-specific manner before fusing with the oocyte. Spermzona pellucida have been suggested to be regulated by interactions leading to fertilization glycoproteins, which are exposed on the surfaces of interacting gametes in a timedependent manner (Ahuja, 1984). The contents of the sperm acrosome are rich in many hydrolases, including acrosin, hyaluronidase, /3-./V-acetylhexosaminidase and arylsulphatase. The localization of individual hydrolases, particulary the sulphatase and /3-iV-acetylhexosaminidase, within the acrosome varies considerably between species and is poorly understood (Harrison, 1982). It has been suggested that large quantities of these enzymes released during the acrosome reaction assist sperm passage through the various barriers surrounding the egg, but an understanding of how these enzymes might interact with surface glycoproteins in regulating sperm-zona interactions is totally lacking. Surface glycoproteins responsible for adhesion between cells in different systems are often sulphated in nature and are regulated jointly by the enzymes responsible for their sulphation and desulphation (Rapraieger & Epel, 1981). The components of the zona pellucida (Dunbar, Wardrip & Hedrick, 1980), as well as the sperm surface (Murray, Narsimhan, Levine & Pinteric, 1980), are also highly sulphated. Sulphatases have been demonstrated in the uterus (Legault, Bleau, Chapdelaine & Roberts, 1980), the testis (Farooqui & Srivastra, 1979) and spermatozoa (Akruk, Farooqui, Williams & Srivastra, 1979), and a sulphotransferase activity has been found in the epididymis (Bauthillier, Bleau, Chapdelaine & Roberts, 1984; Langlais et al. 1981). The position and number of sulphate groups on sugars are believed to exert a pronounced influence on the conformation of sulphated polysaccharides. It is therefore natural to ask whether sperm sulphatases released during fertilization have a role in early sperm-zona interactions and whether the surface macromolecules regulating these interactions are sulphated in nature. This paper describes preliminary attempts to answer these questions by studying the effects of exogenous sulphatases and artificial substrates for these enzymes on the fertilization of hamster oocytes in vitro. All the components of the culture medium, sulphatase preparations from Abalone entrails and the limpet Patella vulgata, and jack bean iV-acetylhexosaminidase, were purchased from Sigma (London). /3-nitrophenyl sulphate, /3-nitrocatechol sulphate and ascorbate 2-sulphate were also obtained from Sigma. Sulphated monosaccharides and a purified preparation of liver sulphatase were kindly given by Dr A. B. Roy of the Australian National University. Collection and culture of gametes The culture medium used for sperm capacitation as well as regular handling of gametes was a modified Tyrode solution containing 15 % (v/v) heat-inactivated human serum (Ahuja & Tzartos, 1981). The medium was buffered at pH 7-2-7-4 and gassed with 5 % CO2 in air. The osmolarity of the medium was 300-310 mosmol/kg. Heat-inactivated serum was always added to the medium immediately before starting the experiment. Female hamsters (5-6 weeks old) were induced to superovulate by injections of 30 I.U. of pregnant mare's serum gonadotrophin (PMSG) (Intervet, Cambridge) followed 72 h later by 30 I.U. of human chorionic gonadotrophin (HCG) (Intervet); 15-17 h after the injection of HCG, the hamsters were killed and their oviducts were dissected out. Cumulus masses containing the eggs were recovered by tearing open the oviducts under oil, using two pairs of forceps. Cumulus masses obtained from different oviducts were pooled and used for subsequent experiments. Mature spermatozoa were obtained by making three small incisions at the distal (caudal) end of the epididymis of a mature male hamster; the small sample of contents thus released was immediately transferred to 1 ml of culture medium. Motile spermatozoa swimming away from the central mass were collected with the help of a mouth pipette and stereomicroscope and were deposited in 50-jul culture drops under oil. The whole operations was carried out on a bench incubator at 37 C. As determined by haemocytometer counts, the sperm concentration in thefinalsuspension was 4 X 106 to 5 X 106 spermatozoa per ml. Freshly obtained epididymal sperm were capacitated by incubating together with cumulus cells in culture medium at 37 C in an atmosphere of 5 % CO2 in air. Samples (10 /A) of sperm suspension were pipetted under oil and a sample of fresh cumulus cells (roughly 0"2 vol. of the culture drop) was added to each sperm droplet. After about 30-60 min incubation, the eggs were withdrawn and the dishes were transferred to the incubator for a total of S h. At the end of this incubation period, the majority of spermatozoa had gained the characteristic 'whiplash' motility and (...truncated)