PH‐20 but not acrosin is involved in sperm penetration of the macaque zona pellucida

Abstract

In this study, we investigated the functions of PH-20 and acrosin during the interaction of macaque sperm with the zona pellucida. Both of these sperm enzymes have been reported to be present on the inner acrosomal membrane of acrosome reacted sperm, and have been suggested to play a role during secondary sperm-zona binding in other species. Anti-macaque PH-20 IgG, anti-pig acrosin IgG and soybean trypsin inhibitor (SBTI) were used as probes for immunolocalization of the two proteins at the ultrastructural level, and as reagents for blocking sperm penetration of the macaque zona pellucida in vitro. As a control, we performed similar studies with antibodies to CD-46, which is also located on the inner acrosomal membrane, but has no known function in sperm-zona pellucida interaction. After labeling with anti-acrosin IgG, gold label was not present on the sperm surface before the acrosome reaction, but was detected over the entire head of sperm that were induced to acrosome react with calcium ionophore A23187. In contrast, when sperm were induced to acrosome react by binding to intact zona pellucida, acrosin was present in the acrosomal shroud but not on the inner acrosomal membrane. Similar results were obtained when SBTI was used as a probe for enzyme localization. PH-20 and CD-46 were demonstrated on the inner acrosomal membrane of sperm induced to acrosome react by ionophore treatment and by zona binding. Neither anti-acrosin IgG nor anti-CD-46 IgG affected sperm penetration of the zona at concentrations up to 300 microg/ml, but zona penetration was blocked completely when anti-PH-20 IgG (100 microg/ml) was present during sperm-oocyte interaction. Ultrastructural observations of oocytes incubated with anti-PH-20 IgG showed that acrosomal shrouds were present on the zona surface but no sperm had begun to penetrate into the zona substance. We conclude that anti-PH-20 IgG prevented sperm penetration of the macaque zona pellucida by interference with secondary sperm-zona binding, rather than primary sperm-zona binding or the zona-induced acrosome reaction. Acrosin was not detected on the inner acrosomal membrane of sperm that are induced to acrosome react after zona binding, and acrosin does not appear to be critical for sperm penetration of the macaque zona pellucida.

Mechanisms of sperm-egg interactions emerging from gene-manipulated animals

“…These structural data seemed to support the long-held view that hyaluronidase plays a role in fertilization. In fact, in macaque monkeys, zona penetration was completely blocked by anti-PH-20 IgG (100 mg/mL) when present during sperm-oocyte interaction [43]. However -again paradoxically -when PH-20 genedisrupted mice were produced and examined, the mice were found to have an almost normal ability to sire pups [44].…”

Assessment of contraceptive vaccines based on recombinant mouse sperm protein PH20

“…Secondly, PH20 is able to bind directly to the zona pellucida during the early steps of fertilization (Primakoff et al 1985). This is thought to be due to the ability of PH20 to bind hyaluronic acid (HA) in the zona pellucida, a process that is independent of its hyaluronidase activity (Hunnicutt et al 1996, Yudin et al 1999. Finally, the binding of PH20 to HA indirectly triggers an increase in the intracellular calcium of sperm that leads to an increase in the responsiveness of sperm to induction of the acrosome reaction .…”

Cytochrome c Upregulation during Capacitation and Spontaneous Acrosome Reaction Determines the Fate of Pig Sperm Cells: Linking Proteome Analysis

“…The cells were excited at 488 nm using an argon laser; FITC-fluorescence was detected using a 520 nm band pass filter (FL-1). In addition, capacitated sperm cells were identified using fluorescein-labeled anti-CD46, as described previously [20], and/or were determined by counting 200-300 spermatozoa/sample at ×1,000 magnification after double vital staining with trypan blue Giemsa [21]. …”