Heterogeneous Processing and Zona Pellucida Binding Activity of Pig Zonadhesin

Abstract

Zonadhesin is a mosaic protein in sperm membrane fractions that binds directly and in a species-specific manner to the extracellular matrix (zona pellucida) of the oocyte. The active form of pig zonadhesin from capacitated, epididymal spermatozoa comprises two covalently associated polypeptide chains of M r 105,000 (p105) and M r 45,000 (p45). Here we report detection and characterization of multiple zonadhesin isoforms in freshly ejaculated cells. Antibodies to the predicted von Willebrand D0-D1, D1, and D3 domains of pig zonadhesin recognized p105, p45, and additional M r 60,000 -90,000 polypeptides in particulate fractions of uncapacitated cells. Although the p105/45 form constituted a minority of all zonadhesin forms in sperm membrane fractions, it was the predominant form capable of binding to the pig zona pellucida. Zonadhesin-binding sites were distributed over the entire zona pellucida. Anion exchange chromatography resolved active, p105/45 zonadhesin from the p60 -90 inactive forms. Without disulfide bond reduction some zonadhesin was M r >300,000, including M r 300,000 and 900,000 proteins comprising in part multimers of p105/45. The multimeric forms did not bind the zona pellucida as avidly as did the p105/45 monomer. Expressed D1 and D3 domain fragments containing the CG(L/V)CG sequence motif spontaneously formed multimers at ؊246 mV E h in vitro. Double Cys 3 Ser mutants of the D1 fragment formed multimers with the same apparent kinetics as the wild type protein. Zonadhesin localized to the apical head of pig spermatozoa. We conclude that a heterogeneous combination of specific proteolysis and intermolecular disulfide bond formation in the sperm head generates multiple forms of zonadhesin with differing avidities for the zona pellucida.Adhesion of mammalian spermatozoa to the zona pellucida (ZP) 1 is a complex process mediated by binding of sperm proteins to complementary ligands in the ZP (1, 2). The complexity of this process derives partly from cellular changes that occur during gamete interactions. Spermatozoa undergo physiological changes in the female reproductive tract that are required for fertilization and are collectively called capacitation (1, 3). Although the molecular basis of capacitation is only partly understood, in some if not all species avidity of sperm-ZP adhesion increases as capacitation progresses. After capacitation is completed, the membranes involved in initial adhesion events are lost from the sperm surface during the acrosome reaction, but adhesion is sustained by interaction of newly exposed structures with the ZP (1, 2). Unique adhesion molecule pairs likely function at different times during fertilization, and the activities of these molecules may change as fertilization progresses (2). It is therefore important to assess the biochemical and functional properties of sperm adhesion molecules at each stage in the fertilization process. Several sperm proteins that may mediate adhesion to the ZP have been identified and characterized (2). Among these molecules zonadhesi...

The molecular evolution of sperm zonadhesin

“…Direct evidence for the binding partner of ZAN came from its aforementioned isolation through a ZP affinity matrix. Subsequent Western blotting and partial aa sequencing of the isolated peptides revealed that the ZP avidity of oligomers containing D1-3 is particularly strong Garbers 1994, 1995; see also Hickox et al, 2001). Meanwhile, ZP binding activity has also been demonstrated for a rabbit ZAN D4 construct (Lea et al, 2001) so that ZP apparently represents the common binding partner of ZAN D1-4 domains.…”

“…Immunofluorescence and immunoelectron microscopy of spermatozoa from different boreoeutherian species such as horse, pig, mouse, rat, rabbit, cattle, mole, hamster, and humans repeatedly localized zonadhesin at the apical head of spermatozoa (Gao and Garbers 1998;Hickox et al, 2001;Lea et al, 2001;Breazeale et al, 2002;Bi et al, 2003, Olson et al, 2004. Additionally, ZAN might be expressed in Sertoli-cells as inferred from immunofluoresence data of rabbit testis (Lea et al, 2001).…”

“…A great leap forward in this field is represented by the work of Hardy and Garbers (1994) who concentrated proteins from large quantities of pig (Sus scrofa) sperm membranes through an affinity matrix made of porcine ZP. One of the isolated proteins that turned out to bind speciesspecifically to ZP (Hardy and Garbers 1994) was termed colleagues used immunoprecipitation and other methods to unravel the activation of pig ZAN by processing, glycosylation and oligomerization (Hickox et al, 2001;Bi et al, 2003). Beyond this, Hardy and collaborators were first to document that transcripts of human ZAN undergo alternative splicing into six variants (see AY046055 at NCBI; see also Gasper and Swanson 2006).…”

Causes and consequences of the evolution of reproductive proteins

“…Nevertheless, many new molecular tools are available to help the evolutionary biologist study the actual effects of protein variation on fertilization potential. For example, the effects of amino acid changes at positively selected sites could be tested using site-directed mutagenesis and in vitro binding assays (e.g., Kinloch et al, 1995;Chen et al, 1998;Hickox et al, 2001;Williams et al, 2003Williams et al, , 2006. Alternatively, manipulation of genes could be performed in vivo using transgenics or targeted gene transfer (Flotte, 2007;Gao et al, 2007;Waehler et al, 2007).…”

Identification of sex hormone binding globulin-interacting proteins in the brain using phage display screening

“…Posttranslational processing leads to multiple forms of zonadhesin with differing apparent avidities for the ZP (45). In the pig, the p105/45 form binds preferentially to conspecific ZP (45). It is interesting that with the discovery of membrane receptors for progesterone in oocytes (46)(47)(48), that rapid steroid effects in oocytes are receiving renewed interest.…”