Species Diversity in the Structure of Zonadhesin, a Sperm-specific Membrane Protein Containing Multiple Cell Adhesion Molecule-like Domains

Abstract

A hallmark of gamete interactions at fertilization is relative or absolute species specificity. A pig sperm protein that binds to the extracellular matrix of the egg in a species-specific manner was recently identified and named zonadhesin (Hardy, Prior to fertilization, the sperm cell interacts with multiple cells including fellow sperm cells, Sertoli cells, epithelial cells within the male reproductive tract or female reproductive tract, cells associated with the egg, and the egg itself. In some cases, the interactions are attractive in nature and in others repulsive. In an attempt to identify sperm proteins that bind to the egg extracellular matrix, Hardy and Garbers (1) solubilized sperm membranes from the pig and determined which proteins bound to the pig zona pellucida. Subsequently, the cDNA encoding a protein that bound to the zona pellucida in a relatively species-specific manner was cloned and named zonadhesin (2). Pig zonadhesin is a transmembrane protein with a very short intracellular region. The putative extracellular region contains a mucin-like domain and five tandem repeats homologous to the D-domains of prepro-von Willebrand factor (ppvWF) 1 (3, 4). The cloning of the pig sperm zonadhesin cDNA raised the questions of whether other species would contain homologs of zonadhesin and, if so, whether these would be variable within the above domain structure. Here, the mouse homolog of zonadhesin is cloned and shown to contain additional repetitive sequences within the D-domain region. Furthermore, three tandem repeats of a MAM domain were identified at the N terminus. The MAM domain is a module composed of about 160 amino acids including four conserved Cys; it is found in a number of functionally diverse transmembrane proteins such as the meprins and receptor protein-tyrosine phosphatases (5). Thus, zonadhesin displays a large species variation between pig and mouse and also contains multiple domains previously suggested as involved in cell adhesion processes.D MATERIALS AND METHODSRNA Isolation-Total RNA was isolated from 40 mouse testes or similar amounts of other mouse tissues (including brain, heart, kidney, liver, lung, small intestine, and spleen) in guanidinium thiocyanate and N-lauroyl sarcosine, followed by extraction with acidic phenol/CHCl 3 and precipitation with isopropyl alcohol (6). Poly(A) ϩ RNA was purified from total RNA by oligo(dT)-cellulose chromatography (7).Northern Blots-30 g of total RNA from various mouse tissues was loaded on each lane of a formaldehyde-agarose gel (8) and blotted overnight using 10 ϫ SSC as the transfer buffer. The blot was then fixed by UV light and hybridized with a random primer-labeled (Amersham Life Science, Inc.), 1.4-kb probe from the 3Ј-end of the mouse zonadhesin cDNA as described previously (1). After hybridization, the blot was washed with 0.2 ϫ SSC and 0.2% SDS at 65°C.Screening of cDNA Libraries-The first mouse cDNA clone (#18) was obtained by a low stringency screening of a mouse testis cDNA library (Stratagene) using a cDNA fragment correspo...

Comparisons of pollen coat genes across Brassicaceae species reveal rapid evolution by repeat expansion and diversification

“…Homologous genes that function in reproduction have been sequenced in abalone, sea urchins, fruit flies, and mammals (1). Positive selection has been detected in at least 17 cases (33), including abalone sperm lysin (42) and mammalian egg proteins (43), and rapid evolution has been found in many others (1,2,(44)(45)(46)(47)(48). Numerous genes involved in pathogenesis and pathogen defense are also under positive selection because of the advantages of change for both the host and the pathogen (33).…”

The role of the acrosomal matrix in fertilization

“…Subsequently, cDNAs or genes have been partially or completely sequenced from mouse, human, rabbit, hamster, and non-human primates (Bi et al, 2003, Gao and Garbers, 1998, Gao et al, 1997, Hardy and Garbers, 1995, Herlyn and Zischler, 2007, Lea et al, 2001, Olson et al, 2004) Testicular porcine zonadhesin is extractable with non-ionic detergents and would not be considered a component of the acrosomal matrix by the operational definition listed above (Bi et al, 2003). In round and elongating spermatids, zonadhesin localizes to the developing outer acrosomal membrane.…”

Heterogeneous Processing and Zona Pellucida Binding Activity of Pig Zonadhesin

“…Among these molecules zonadhesin is unique in its ability to bind directly and in a species-specific manner to native, particulate ZP (4,5). Zonadhesin from pig (5), mouse (6), rabbit, 2 and human (7) 3 spermatozoa is a mosaic protein with a predicted Type I integral membrane topology. In each of these species, the large extracellular region of the protein comprises primarily three domain types (meprin/A5 antigen/mu receptor tyrosine phosphatase, mucin, and von Willebrand D (VWD)) that are present in other adhesion molecules (8 -10).…”