Since the successful birth of piglets after the transfer of in vitro-produced embryos in 1986 [1], many researchers have established and enhanced porcine in vitro fertilization (IVF) technologies [2]. However, a major issue of porcine IVF is the abnormally high incidence of polyspermy (≥50 %) [3,4]. Therefore, multiple studies have been conducted to decrease the rate of polyspermy under conditions that support a high penetration rate. For example, the addition of oviductal epithelial cells [5,6], oviductal fluid [7], porcine oviduct-specific glycoproteins [8,9], or Osteopontin, which is present in the oviduct [10], to IVF medium has been performed to reduce the incidence of polyspermy in porcine oocytes. Nevertheless, the fundamental issue of polyspermy during IVF persists.

We recently focused on the behavior of sperm adhesion molecule 1 (SPAM1), which exhibits hyaluronidase activity in sperm, and demonstrated that ellagic acid, which is the strongest hyaluronidase inhibitor, markedly decreased the incidence of polyspermy from 63 to 40 % under conditions maintaining high sperm penetrability (68 %) during IVF of porcine oocytes [11]. These findings suggest that the inhibition of SPAM1 by treatment with ellagic acid effectively prevented polyspermy during porcine IVF. A previous study suggested the ability of SPAM1 and arylsulfatase A (ARSA) to form a stable complex that is dynamically reoriented during sperm capacitation [12]. In addition, extensive research on sperm ARSA revealed that it initiates secondary sperm-zona pellucida (ZP) engagement through its ability to act as a receptor for sulfated carbohydrate chain ligands on ZP glycoproteins [[13], [14], [15], [16], [17]]. Furthermore, our finding showed that the blocking of ZP sulfation by treatment with 50 mM NaClO3 during the in vitro maturation (IVM) of porcine oocytes markedly reduced the incidence of polyspermy with no inhibitory effects on penetration [18]. Accordingly, we inferred that the suppression of polyspermy was associated with the SPAM1-ARSA complex, which promoted us to examine the effects of not only ARSA, but also SPAM1.

Therefore, the present study focused on the prevention of polyspermy during porcine IVF by treatments with sulfatase to desulfate the sulfated carbohydrate chain residues of the ZP and the ARSA-blocking peptide (ARSA-BP) for the specific masking of sperm ARSA. The following experiments were performed to elucidate the involvement of removing the sulfated carbohydrate chain residues of ZP and blocking sperm ARSA in the suppression of polyspermy: 1) an assessment of responses to fertilization parameters, including the penetration rate and incidence of polyspermy, in porcine oocytes matured in vitro; 2) an examination of the number of sperm bound to the ZP, zona solubility, and the induction of the acrosome reaction; 3) an evaluation of developmental potency to the blastocyst stage and the number of apoptotic blastomeres in embryos at the blastocyst stage.