Functional Characterization of Double-knockout Mouse Sperm Lacking SPAM1 and ACR or SPAM1 and PRSS21 in Fertilization

Abstract

Abstract. Mammalian fertilization requires sperm to penetrate the cumulus to reach the oocyte. Although sperm hyaluronidase has long been believed to participate in the penetration process, our previous works revealed that neither of two sperm hyaluronidases, SPAM1 and HYAL5, are essential for fertilization. In this study, we have produced doubleknockout mice lacking SPAM1 and either one of two sperm serine proteases, ACR and PRSS21, and characterized the mutant sperm. The SPAM1/ACR-and SPAM1/PRSS21-deficient males were fertile, whereas epididymal sperm of the mutant mice exhibited a reduced capacity to fertilize the oocytes in vitro. Despite normal motility, the ability of sperm to traverse the cumulus matrix was more severely impaired by the loss of SPAM1 and ACR or SPAM1 and PRSS21 than by the loss of only SPAM1. Moreover, SPAM1/ACR-and SPAM1/PRSS21-deficient sperm accumulated on the surface (outer edge) of the cumulus more abundantly than SPAM1-deficient sperm. These results suggest that ACR or PRSS21 or both may function cooperatively with SPAM1 in sperm/cumulus penetration. Key words: Cumulus, Hyaluronidase, Mouse, Sperm, Serine protease (J. Reprod. Dev. 58: [330][331][332][333][334][335][336][337] 2012) M ammalian fertilization involves a multistep set of physiological events, including sperm interaction with the cumulus matrix and zona pellucida (ZP) in the oocyte-cumulus complex (OCC) [1][2][3][4][5][6]. According to a currently favored model [1,2,7,8], only capacitated sperm enter the OCC, traverse the cumulus matrix and reach the oocyte ZP. Following binding of sperm to the ZP, the sperm undergo the acrosome reaction. As a consequence of the exocytotic event, the acrosomal components, including hydrolytic enzymes, are released from the acrosome to enable the sperm to penetrate the ZP. Sperm hyaluronidases and serine proteases are thought to play important roles in the fertilization events.Mouse epididymal sperm contain at least two hyaluronidases, SPAM1 (PH20) [9] and HYAL5 [10], and two serine proteases, ACR (acrosin) [11] and PRSS21 (testisin/TESP5) [12]. SPAM1 is glycosylphosphatidylinositol (GPI) anchored only on the plasma membrane of sperm, and acrosome-reacted sperm still retain approximately half of total SPAM1 [10,13]. HYAL5, on the other hand, is localized on the plasma and acrosomal membranes of acrosome-intact sperm and is mostly released from the sperm membranes during the acrosome reaction [10]. ACR is present in the acrosome as a matrix protein, whereas the sperm membrane contains PRSS21 as a GPI-anchored protein [11,12]. Contrary to early expectation, single-knockout mice lacking SPAM1 (Spam1 -/-) [13], HYAL5 (Hyal5 -/-) [14], ACR (Acr -/-) [15] or PRSS21 (Prss21 -/-) [16] were all fully fertile. Despite the male fertility, in vitro fertilization (IVF) assays demonstrated delayed penetration of Spam1 -/-and Acr -/-epididymal sperm through the cumulus matrix and ZP, respectively [13][14][15]. The Prss21 -/-sperm also exhibited multiple defects in the sperm functions in vitro [16]. Th...

Effects of latrunculin A on the relocation of sperm IZUMO1 during gamete interaction in mouse

“…Rat anti‐IZUMO1 monoclonal antibody (Mab125) (Ikawa et al, ; Inoue et al, ) was generously provided by Dr. M. Ikawa (Osaka University, Japan). Goat anti‐IZUMO1 polyclonal antibody (item sc‐79539) and rat anti‐ZP2 monoclonal antibody (clone IE‐3; item sc‐32752) (Zhou, Kang, & Baba, ) were purchased from Santa Cruz Biotechnology (Dallas, TX). Alexa Fluor 568‐conjugated PNA (item L32458) or Alexa Fluor 568‐conjugated phalloidin (item A12380) and secondary antibodies were purchased from Molecular Probes (Eugene, OR).…”

“…Blocked samples were incubated with anti‐IZUMO1 antibody (1:200 dilution of Mab125) and Alexa Fluor 488 rabbit anti‐rat IgG plus Hoechst 33342, as described above (section 4.4). Fluorescence images were captured using an Extended Depth of Focus z‐axis motor (Nikon Products), and processed by NIS‐AR software (Nikon Co.), as described previously (Zhou et al, ).…”

Recombinant Human Hyaluronidase-Facilitated Subcutaneous Immunoglobulin Infusion in Primary Immunodeficiency Diseases

“…Theoretically, a neutralizing antibody to rHuPH20 could cross-react with human PH20, inhibiting this activity. However, studies of PH20 knockout mice immunocontraceptive trials with anti-PH20 antibodies in mice and rabbits have shown that it plays a nonessential role in fertility [27][28][29][30]. Large doses of rHuPH20, such as those used to elicit high titers of neutralizing anti-rHuPH20 antibodies in animal studies, did not result in an observable effect on fertility, reproduction, fetal development or postnatal life [31].…”

The Acrosomal Matrix

“…The authors suggest a role in cumulus dispersal and penetration, which is interesting since sperm lacking known hyaluronidases SPAM1 (Baba et al 2002) and HYAL5 (Kimura et al 2009) were able to fertilize oocytes. Together with double knockout experiments of SPAM1 and ACR or SPAM1 and PRSS21 (Zhou et al 2012), these results suggest that ACR and PRSS21 may work in concert with hyaluronidases for cumulus penetration. These results are also consistent with the notion that acrosomal exocytosis occurs before or during interaction with the cumulus (Jin et al 2011).…”