The identity of zona pellucida receptor on spermatozoa: an unresolved issue in developmental biology.

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Contents lists available at ScienceDirect

Seminars in Cell & Developmental Biology journal homepage: www.elsevier.com/locate/semcdb

Review

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The identity of zona pellucida receptor on spermatozoa: An unresolved issue in developmental biology

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Philip C.N. Chiu a,b , Kevin K.W. Lam a , Rachel C.W. Wong a , William S.B. Yeung a,b,∗ a

Department of Obstetrics and Gynaecology, University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, China Centre of Reproduction, Development and Growth, LKS Faculty of Medicine, University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, China b

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Article history: Available online xxx

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Keywords: Spermatozoa Zona pellucida Receptor complex Glycosylation Fertilization

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Mammalian oocytes are surrounded by an acellular zona pellucida (ZP). Fertilization begins when a capacitated spermatozoon binds to the ZP. Defective sperm-ZP interaction is a cause of male infertility and reduced fertilization rates in clinical assisted reproduction treatment. Despite the importance of spermatozoa–ZP binding, the mechanisms and regulation of the interaction are unclear partly due to the failure in the identification of ZP receptor on spermatozoa. Most of the previous studies assumed that the sperm ZP receptor is a single molecular species, and a number of potential candidates had been suggested. Yet none of them can be considered as the sole sperm ZP receptor. Accumulated evidence suggested that the sperm ZP receptor is a dynamic multi-molecular structure requiring coordinated action of different proteins that are assembled into a functional complex during post-testicular maturation and capacitation. The complex components may include carbohydrate-binding, protein-binding and acrosomal matrix proteins which work as a suite to mediate spermatozoa–ZP interaction. This article aims to review the latest insights in the identification of the sperm ZP receptor. Continued investigation of the area will provide considerable understanding of the regulation of fertilization that will be useful for practical application in human contraception and reproductive medicine. © 2014 Published by Elsevier Ltd.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Zona pellucida . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functions of zona pellucida glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Mouse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Human . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glycosylation of zona pellucida protein is important in spermatozoa–zona pellucida interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Mouse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Human . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Humanized mouse zona pellucida . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Identification of zona pellucida receptor on spermatozoa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Carbohydrate-binding candidates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Zona pellucida protein core-binding candidates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . New concepts in zona pellucida receptor on spermatozoa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Multiple molecules are involved in spermatozoa–zona pellucida interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Sperm zona pellucida receptor is a protein complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Dynamic formation of sperm zona pellucida receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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∗ Corresponding author at: Department of Obstetrics and Gynaecology, University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, China. Tel.: +852 22553405; fax: +852 28175374. E-mail addresses: [email protected], [email protected] (W.S.B. Yeung). http://dx.doi.org/10.1016/j.semcdb.2014.04.016 1084-9521/© 2014 Published by Elsevier Ltd.

Please cite this article in press as: Chiu PCN, et al. The identity of zona pellucida receptor on spermatozoa: An unresolved issue in developmental biology. Semin Cell Dev Biol (2014), http://dx.doi.org/10.1016/j.semcdb.2014.04.016

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6.4. Involvement of the acrosomal matrix molecules in spermatozoa–zona pellucida interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lack of standardized assay to evaluate spermatozoa–zona pellucida binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The significance of zona pellucida receptor identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Mammalian oocytes are surrounded by a ∼7–20 ␮m thick porous acellular extracellular matrix termed zona pellucida (ZP), which is a main player in spermatozoa–oocyte interactions and species specific fertilization. Spermatozoa–ZP binding is the first event in fertilization. Traditionally, it is believed that the binding involves interaction of ZP receptor(s) on plasma membrane overlying the acrosome of capacitated spermatozoa with the ZP. Such interaction is referred to as primary binding, and is followed by induction of acrosome reaction of the ZP-bound spermatozoa. Acrosome reaction involves multiple fenestration of the sperm plasma membrane caused by fusion of the outer acrosomal membrane with the plasma membrane, and progressive loss of the plasma membrane resulting in release of the acrosomal contents and exposure of the inner acrosomal membrane [1,2]. After acrosome reaction, the inner acrosomal membrane binds with the ZP in an event known as secondary binding [1,2]. However, this classical model of spermatozoa–ZP interaction has been challenged in recent years that will be described in more detail below. Defective spermatozoa–ZP binding and defective ZP-induced acrosome reaction are major causes of low fertilization rate in clinical in vitro fertilization (IVF) [3–5]. Despite the fundamental importance of spermatozoa–ZP interaction, the mechanisms regulating the process are unclear partly due to failure in the identification of the ZP receptor(s) on spermatozoa [1,2]. In the present article, we discuss the current progress on identification of sperm components involved in the spermatozoa–ZP recognition. Our focus is on studies in mice and humans as they are the most well studied models.

2. Zona pellucida The ZP of mouse and human oocytes is made up of a delicate network of thin interconnected filaments forming a regular alternating pattern of wide and tight meshes [6]. The wide meshes correspond to pores of the ZP, whereas the tight meshes represent the compact parts of the ZP surrounding the pores. The pores are larger at the outer surface than at the inner surface of the ZP. The porous region of the human ZP occupies about 25% of the total area of the ZP [7]. The amorphous spongy outer surface of the ZP with larger pores may facilitate sperm penetration, as human ZP with a more compact and smoother outer surface is less penetrable [8,9]. The mammalian ZP is composed of either three or four glycoproteins designated as ZP glycoprotein-1 (ZP1), ZP2, ZP3 and ZP4. There are four genetic loci encoding the mouse ZP genes. The mouse ZP is composed of only three ZP glycoproteins, ZP1, ZP2 and ZP3 [10]. Zp4 is a pseudogene in mouse and does not express the cognate protein [12]. The ZP of pig [13], cow [14] and dog [12] are also made up of 3 glycoproteins but instead of ZP1, ZP4 is present. Among studied mammals, only human, other primates and rat express four ZP glycoproteins [2,15–17]. Spermatozoa–ZP interaction is largely species-specific, especially for human spermatozoa, which bind only to the ZP from humans and hominoid species (gibbon, gorillas), but not from other sub-hominoids (baboons, rhesus monkeys, squirrel monkeys) or lower mammalian species [1,18,19]. It is interesting to note that different mammals produce the ZP with various

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combinations of the ZP glycoproteins, which may have implications in species specific fertilization. The ZP glycoproteins are conserved during evolution. For instance, human ZP2 shows 57, 64 and 94% amino acid sequence identity with mouse, porcine and bonnet monkey ZP2, whereas human ZP3 has 67, 74 and 94% identity with mouse, porcine and bonnet monkey ZP3, respectively. In addition, there is an overall conserved backbone structure of the ZP glycoproteins [20]. The ZP glycoproteins genes are located on different chromosomes, and the high homology between the ZP genes suggests that they arose by gene duplication [17].

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Most of our knowledge on the roles of ZP glycoproteins in fertilization comes from studies in mice. In mice, it is generally believed that ZP3 is the primary sperm receptor that binds to the anterior head of capacitated spermatozoa and induces acrosome reaction [1,2]. After acrosome reaction, ZP2 binds only to acrosome-reacted spermatozoa and acts as a secondary sperm receptor [1,2,21]. Mouse ZP1 is postulated to cross-link the filaments formed by ZP2–ZP3 heterodimers and provides structural integrity and stability to the ZP matrix [1,22]. The crucial role of ZP glycoproteins in fertilization is substantiated in mice with knockout of individual ZP glycoproteins. The ZP of Zp1 null mice is loose and its filaments are not interconnected, consistent with the proposed function of ZP1 in maintaining the integrity of ZP. The mutant females are fertile, but have reduced fecundity and their embryos hatch precociously [23]. Zp2 null mice have a thin ZP [24]. No two-cell embryos can be recovered from the mice after mating with normal males, suggesting a role of the mouse ZP2 in fertilization and early embryo development [24]. Zp3 null female mice are sterile; they have ZP-free oocytes with compromised developmental potential and disorganized corona radiata [25,26]. 3.2. Human A critical appraisal of the biochemical properties and biological activities of the human ZP glycoproteins is difficult due to paucity of human oocytes for research. To circumvent this difficulty, investigators have studied the biological activities of recombinant human ZP glycoproteins on spermatozoa. Human ZP1 has been expressed in the prokaryotic (Escherichia coli) and eukaryotic (baculovirus) systems [27,28]. These recombinant ZP1 bind to the anterior head of the capacitated acrosome-intact but not the acrosome-reacted spermatozoa. In contrast to the mouse ZP1, the glycosylated baculovirus-expressed ZP1 exhibits a dose-dependent induction of acrosome reaction of the capacitated spermatozoa, though the unglycosylated E. coliexpressed ZP1 does not, indicating a critical role of glycosylation of ZP1 in acrosome reaction. The ‘ZP domain’ (amino acid residues 273–551) of the human ZP1 is sufficient to induce acrosome reaction [28]. Prokaryotic and eukaryotic-derived recombinant human ZP2 bind to the equatorial segment of the acrosome-reacted


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spermatozoa [29,30]. They fail to induce acrosome reaction of the capacitated spermatozoa [30], consistent with the role of ZP2 as a secondary sperm receptor as in the mice [20]. Baculovirus-expressed [30,31] and mammalian cell-expressed recombinant ZP3 [32,33] bind to the acrosomal region of capacitated human spermatozoa and induce acrosomal exocytosis dose-dependently. Acrosome reacted spermatozoa do not bind these ZP3. The baculovirus-expressed recombinant human ZP4 has similar binding properties and acrosome reaction inducing activity [30,31]. These results are consistent with the function of ZP3 and ZP4 as the primary sperm receptor in humans. The data obtained from the recombinant ZP proteins have to be interpreted with caution because their glycosylation are different from that of the native counterparts [34,35]. Glycosylation is cell type- and species-specific. Culture conditions can affect the glycosylation of the recombinant proteins [36,37]. The importance of glycosylation in spermatozoa–ZP interaction is exemplified by the observation that porcine ZP proteins expressed in Sf9 cells bind to bovine but not to porcine spermatozoa [38]. To solve the concern, we purified and studied native human ZP glycoproteins [15,39]. The native human ZP3 binds to the acrosomal region of acrosome-intact spermatozoa. The binding are lost after acrosome reaction. Native ZP3 but not recombinant ZP3 binds to the midpiece of human spermatozoa as well. Recombinant ZP2 binds mainly to the equatorial segment of the acrosome reacted spermatozoa [29,30], whereas native ZP2 binds to the acrosome, post-acrosome and midpiece [15] of human spermatozoa. Similar to E. coli-expressed recombinant ZP4, the binding sites of native human ZP4 are in the acrosomal region, and are lost after acrosome reaction [30]. Glycosylation affects the activities of the ZP glycoproteins. Although both the native and recombinant human ZP3 and ZP4 induce acrosome reaction, the potency of native glycoproteins is much higher than that of the recombinant counterparts. Native human ZP3 and ZP4 at a concentration of 25 pmol/ml can induce acrosome reaction in 24–31% of capacitated human spermatozoa within 15 min [39]. The potency of induction of acrosome reaction is comparable to that of intact human ZP [40] and is higher than that of the recombinant human ZP [39]. 4. Glycosylation of zona pellucida protein is important in spermatozoa–zona pellucida interaction

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Oligosaccharides chains are found on the asparagine (N-linked) and serine/threonine (O-linked) residues of the ZP glycoproteins [41,42]. Since Oikawa and coworkers first demonstrated that wheat germ agglutinin blocked fertilization [43], there is great interest on the role of carbohydrates in spermatozoa–ZP interaction.

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The glycan of mouse ZP has diverse N- and O-linked carbohydrate sequences [44,45]. Several evidence indicate the involvement of ZP glycans during spermatozoa–ZP binding in mice. First, chemical or enzymatic removal of all the glycans of the mouse ZP3 abolishes its sperm binding ability [34,46,41]. Second, elective removal of the O-linked glycans of the mouse ZP3 by alkaline hydrolysis abrogates its ability in induction of acrosome reaction [41]. Third, O-linked oligosaccharides recovered from the mouse ZP3 by mild alkaline hydrolysis under reducing conditions [41,47] and certain O-linked-related oligosaccharides [48] inhibit the binding of spermatozoa to the ZP. Ser332 and Ser334 residues of the mouse ZP3 were once thought to be the sperm binding O-glycosylation sites because mutation at these sites abolished the binding of recombinant mouse ZP3 to the

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spermatozoa [49]. However, when Zp3 transgene with mutation at these sites were introduced into Zp3 null mice, the resulting mice were fertile [50]. Glycoproteomic analyses confirm that the mouse ZP3 is not O-glycosylated at these sites in vivo [50,51]. Recently, two other conserved O-glycosylation sites on the on the surface of mouse ZP3 glycoprotein are proposed to mediate spermatozoa–ZP binding [52]. The involvement of O-linked glycans in spermatozoa–ZP interaction is challenged by the increased fertility of transgenic mice producing oocytes with specific deletion of ␤1–3 galactosyltransferase, an enzyme required for biosynthesis of core 1/core 2 O-linked glycans [53]. These oocytes produce minimal amount of O-linked glycans. Yet they can be fertilized normally. N-linked glycans contribute to nearly half of the molecular mass of mouse ZP3 and over 40% of the mouse ZP2 [54]. Early study did not support the involvement of the N-linked glycans in mouse spermatozoa–ZP interaction as their elective removal by endo-␤-N-acetyl-d-glucosamine treatment did affect the action of mouse ZP3 on acrosome reaction [41]. More recent studies on transgenic mice showed the contrary. Inactivation of N-acetylglucosaminyltransferase V, an enzyme essential for the synthesis of triantennary and tetraantennary N-linked glycans, yields female mutant exhibiting about 50% fecundity of the control mice [55]. In addition, oocyte with specific deletion of Nacetylglucosaminyltransferase I, an enzyme essential for complex type N-linked glycan synthesis in oocytes, leads to a 81% decrease in gamete binding and a 50% loss in fertility [56,57].

4.2. Human In humans, several oligosaccharide moieties such as Nacetyl glucosamine, fucose, mannose and glycoconjugates bearing selectin ligands are involved in spermatozoa–ZP binding [58–61]. The importance of glycosylation in human spermatozoa–ZP binding is also demonstrated with a glycoprotein known as glycodelin. Glycodelin isoforms obtained from amniotic fluid and follicular fluid are potent inhibitors of spermatozoa–ZP in humans [62,63]. In contrast, another glycodelin isoform in the cumulus matrix with the same protein core but different glycosylation promotes the binding [63,64]. The biological relevance of glycans of human ZP is reinforced by reduction in the ZP-binding and acrosome reaction induction ability of native human ZP3 and ZP4 after removal of their N-linked glycans [15,39]. Recently, sialyl lewis x (SLeX) [NeuAc␣2-3Gal␤14(Fuc␣1-3) GlcNAc] was identified as the most abundant terminal sequence on the glycans of human ZP [42]. SLeX is responsible for initiating human spermatozoa–ZP binding, as the binding is largely inhibited by glycoconjugates terminated with SLeX sequences or by antibodies directed against the sequence [42]. Studies using recombinant ZP proteins further support an important role of N-linked glycosylation in spermatozoa–ZP interaction, consistent with the data obtained from native ZP glycoproteins and a predominance of N-linked glycosylation in the human ZP [15,39,42]. Unglycosylated recombinant human ZP3 and ZP4 cannot induce acrosome reaction [30]. Insect cells-derived glycosylated recombinant human ZP3 and ZP4 expressed in the presence of tunicamycin, which inhibits N-linked glycosylation, have reduced acrosome reaction inducing capability when compared to those expressed in the absence of tunicamycin [30]. On the other hand, removal of O-linked glycans does not change the acrosome reaction inducing ability of the ZP molecules, suggesting that O-linked glycosylation may not have a significant contribution in the process [30].


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4.3. Humanized mouse zona pellucida The introduction of human ZP3 gene into ZP3-deficient mice lead to the production of chimeric ZP composing of mouse ZP1, mouse ZP2 and human ZP3 [65]. Despite the presence of human ZP3, mouse but not human spermatozoa bind to the chimeric ZP [66]. On the other hand, capacitated human spermatozoa bind to the human–mouse chimeric ZP made up of human ZP2 either alone or coexpressed with other human ZP proteins, and undergo acrosome reaction [67,68]. Using recombinant peptides, the site of gamete recognition is localized to a defined domain in the Nterminus of ZP2 [68]. These observations suggest ZP2 is the primary ligand for sperm binding, independent of ZP3 and ZP-derived glycans. However, the spermatozoa-chimeric ZP binding can only be observed after incubating the spermatozoa with the ZP for 4 h [68], which is much longer than the reported duration (1–2 h) required for substantial binding of the human spermatozoa to the human ZP in vitro [69,70]. Spontaneous acrosome reaction occurs in about 22% of human spermatozoa after 4 h incubation [71]. Thus, it is possible that the human spermatozoa could have undergone spontaneous acrosomal exocytosis after prolonged culture, leading to the binding of acrosome reacted spermatozoa to the human ZP2 of the chimeric ZP [1,2,21,34]. Even if ZP2 is the real ligand for the primary binding, the slower binding kinetics and lower binding capacity of the chimeric ZP may indicate the involvement of species-specific glycosylation on the proper functioning of the molecule. This possibility is supported by the observation that recombinant human ZP2 glycoprotein with different glycosylation from that of the native counterparts has different binding pattern on human spermatozoa [15,30]. The ZP glycans may take part in direct interaction with the sperm ZP receptors or may provide the proper tertiary structure that increases the availability of the ZP glycoproteins to their binding proteins on spermatozoa. 5. Identification of zona pellucida receptor on spermatozoa Based on the contact mechanics theory, Kolzlovsky and Gefen developed a biomechanical spermatozoa–oocyte contact model [72]. In order to allow an efficient spermatozoa–ZP interaction, the model shows that a high density of sperm ZP receptors on the sperm head is required to provide sufficient biochemical binding forces for counteracting the propulsive forces generated by the swimming spermatozoa. Despite the theoretical presence of a large number of ZP receptors on spermatozoa, their identities are controversial. Multiple approaches have been used to identify sperm proteins involved in spermatozoa–ZP interaction. Some groups studied the immunoproteome of spermatozoa revealed by anti-sperm antibodies in serum, seminal plasma or on sperm surface [73–75]. Other researchers identified the candidate receptor by one- or twodimensional gel electrophoresis of the sperm proteins followed by probing with solubilized ZP or ZP glycoproteins [76,77]. Immunoprecipitation with recombinant ZP protein or known ZP-binding inhibitory factor as a bait on sperm lysate is another widely used strategy. Sp56 [78] and sperm fucosyltransferase-5 (sFUT5) [64] were identified by this approach. Other methods such as screening of ZP-binding inhibitory antibodies and the corresponding antigens after animal immunization [79] and yeast two-hybrid system have also been used [80]. 5.1. Carbohydrate-binding candidates The importance of glycosylation of the ZP glycoproteins on spermatozoa–ZP interaction leads researchers to look for sperm lectin-like proteins as ZP receptors. A number of molecules had been proposed. The most well studied molecule in mice is

␤1,4-galactosyltransferase (GalTase). It binds to the ␤-linked Nacetylglucosamine residues at the terminals of O-glycans on mouse ZP3 [47,81]. Inhibition of GalTase activity or blocking the recognition site of the enzyme greatly diminished sperm binding [81]. Both anti-GalTase antibodies and affinity-purified GalTase inhibited spermatozoa–ZP interaction dose-dependently [82]. While these observations are highly suggestive of GalTase as a ZP receptor, knockout mouse model fails to demonstrate that it is the sole ZP binding protein. The mutant male mice are fertile, though their spermatozoa are defective in mouse ZP3-induced acrosome reaction in vitro [83]. Murine eggs from wild type mice bind 3–4 times more spermatozoa from male mice deficient in GalTase than spermatozoa from wild type mice [83]. The role of GalTase in primary binding is also challenged by glycomic analysis showing that mouse ZP glycans are rarely terminated with ␤-linked GlcNAc sequences [44]. Lectin binding studies of freshly ovulated mouse oocytes are consistent with the structural analysis [84]. A second carbohydrate-binding candidate is Sp56. The molecule was initially localized to the plasma membrane of acrosome-intact mouse spermatozoa [85]. It was hypothesized that Sp56 binds to the terminal galactose residues of O-glycans at Ser-332 and Ser-334 of the mouse ZP3 [49,85]. Native or recombinant Sp56 inhibited sperm binding to the mouse ZP, but not to the embryos in vitro [86,87]. However, subsequent studies revealed that Sp56 was in the acrosomal matrix [88], a location incompatible with mediation of primary ZP binding. Similar to GalTase, ablation of the Sp56 gene does not affect male fertility, spermatozoa–ZP binding and acrosome reaction [89]. These contradictory data was resolved recently by the demonstration that Sp56 was progressively released to the sperm surface during capacitation and formed a high order aggregates that mediated initial gamete binding in mice [87,90]. SED1 (secreted protein that contains notch-like epidermal growth factor (EGF) repeats and discoidin/F5/8 type C domains) is the mouse homolog of a boar sperm plasma membrane protein isolated by affinity chromatography with porcine ZP proteins [91]. SED1 is secreted by the epididymal epithelium and binds to the mouse sperm plasma membrane overlying the acrosome during passage of the spermatozoa through the epididymis. Recombinant SED1, anti-SED1 antibodies and truncated SED1 proteins containing a discoidin/C domain competitively inhibit sperm-ZP binding [92]. As SED1 binds to ZP3 on immunoblots, SED1 is believed docking to sialylated and/or sulfated complex carbohydrate residues of ZP3 [93]. The protein has been genetically ablated by different groups of investigators. In each case, the male mice are fertile, with either decreased [92,94] or normal [95,96] fecundity. On the other hand, in vitro assays demonstrate a nearly complete failure of binding of SED1-null spermatozoa to ZP of ovulated eggs [92]. With the combined use of chemical cross-linker, immunoprecipitation and mass spectrometry analysis, we showed that sFUT5 was a candidate ZP receptor on human spermatozoa [62]. It is an intrinsic component of the sperm plasma membrane located on the acrosomal region of human spermatozoa and has its catalytic domain (C-terminal) oriented toward the sperm surface. Purified sFUT5 binds strongly to intact and solubilized human ZP. The mouse spermatozoa also express FUT activities [97]. The ability of antiFUT5 antibody and substrates of FUT5 including oligosaccharides or artificial carbohydrate chains with N-acetyllactosamines terminals, in inhibiting spermatozoa–ZP binding in vitro is consistent with a role for sFUT5 in gamete interaction [48,62]. Subsequent studies demonstrate the presence of sFUT5 in the lipid raft of capacitated mouse [98] and human spermatozoa [99], which plays an important role in spermatozoa–ZP binding [100]. Other carbohydrate-binding proteins, such as sperm agglutination antigen-1 [101], fertilization antigen-1 [102] and zonadhesin [103], have been proposed to be the candidate ZP receptor on spermatozoa. However, the failure of genetic ablation of these


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potential ZP receptors in affecting male fertility, and the inability of antibodies against and competitors/substrates of these molecules to completely block spermatozoa–ZP binding and/or ZP-induced acrosome reaction, suggest that none of them is the solely mediator of spermatozoa–ZP binding [1,2,104].

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The binding of unglycosylated E. coli-expressed recombinant ZP proteins to the capacitated spermatozoa [30,105] suggests that the ZP receptors on spermatozoa also interact with the protein core of ZP molecules. However, when compared to the carbohydratedependent interaction, our knowledge on the molecular basis underlying ZP protein core-dependent interaction is limited. One ZP protein core-binding candidate is zona receptor kinase (ZRK). It was first found in a dot blot assay during screening of a testicular expression library, and was identified as a 95-kDa phosphotyrosine-containing sperm protein receptor for ZP3 [106]. It is present in the spermatozoa of mice and humans [106]. Exposure of the mouse spermatozoa to a monoclonal anti-ZRK antibody mimics the inhibitory activity of ZP3 on spermatozoa–ZP binding and induction of acrosome reaction [107]. However, the subsequent identification of human ZRK as the proto-oncogene c-mer [108] raised doubts on the role of ZRK in spermatozoa–ZP binding. Another ZP protein-core-binding protein on spermatozoa is ADAM3 (a disintegrin and metalloprotease 3). Mice lacking ADAM3 are infertile [109]. In addition, disruption of genes including Ace, Adam2, Adam1a, Calr3, Clgn, Pdilt, Pmis2, Rnasse10 and Tpst2 reduces ZP-binding to cumulus-free oocytes and impairs sperm migration into the oviduct similar to that observed in Adam3 null males [110–117]. All these null mutants lack ADAM3, suggesting a common denominator for the observed phenotype. However, ADAM3-deficient spermatozoa fertilize eggs in vivo successfully when being injected into the oviduct, and in vitro in the presence, but not in the absence, of the cumulus oophorus [116,117]. Thus, rather than ZP recognition, ADAM3 is more likely to be involved in passage of the spermatozoa through the utero-tubal junction or cumulus oophorus. Of note, the orthologous gene of ADAM3 in humans is a pseudogene [118] and men are fertile in the absence of the ADAM3 protein. Acrosin is localized to the acrosomal matrix as an inactive zymogen, proacrosin [119]. Both proacrosin and acrosin have been purified from the cauda epididymal and ejaculated spermatozoa of various species, including mouse and human [120]. The binding of acrosin/proacrosin to recombinant ZP2 proteins [120], indicating its possible role in secondary binding. The mechanism of the interaction involves a non-enzymatic recognition of polysulphate groups on ZP2 by the basic residues on the surface of acrosin/proacrosin [120]. Although acrosin/proacrosin deficient mouse spermatozoa display a delay of almost 30 min in both ZP penetration and fertilization when compared with the wild-type and the heterozygous counterparts, the male mice lacking them remain fertile [121,122]. Sperm adhesion molecule 1 (SPAM1, PH-20) is a glycosyl phosphatidylinositol (GPI)-anchored protein on the plasma membrane and the inner acrosomal membrane of spermatozoa. SPAM1 contains a ZP binding domain near the C-terminus and a hyaluronidase domain on the N-terminus. Functional-blocking antibodies against SPAM1 inhibited the binding of acrosomereacted, but not acrosome intact, spermatozoa to the ZP by about 90% [123]. Consistently, treatment of acrosome-reacted spermatozoa with a phosphatidylinositol-specific phospholipase C that cleaves SPAM1 from its GPI anchor, reduces the surface PH-20 contents as well as the ZP-binding capabilities of the treated spermatozoa, suggesting an essential role of the molecule in secondary binding [123]. Again, male mice lacking SPAM1 are totally fertile,

5

though epididymal spermatozoa of SPAM1 deficient mice exhibit a delayed dispersal of the cumulus cells in vitro [124]. In addition to sperm proteins, several epididymal sperm proteins have been reported to be involved in fertilization. CRISP1 belongs to the highly conserved cysteine-rich secretory protein (CRISP) family, which are abundantly expressed in the male reproductive tract of mammals. CRISP1 is an androgen-regulated glycoprotein from the epididymis and is acquired by spermatozoa during their passage through the epididymis [125]. Three observations suggest the involvement of CRISP1 in spermatozoa–ZP interaction. First, anti-CRISP1 antibody or exogenous CRISP1 protein inhibit the binding of the spermatozoa to the ZP of mouse [126] and human [127]. Second, CRISP1 protein binds to the mouse [126] or human ZP [127]. Third, in vitro fertilization assays show that Crisp1−/− spermatozoa exhibit reduced penetration through the ZP-intact oocytes. However, Crisp1−/− mice are fertile [128]. The mechanism of action of CRISP1 on spermatozoa–ZP remains to be clarified. Another example is human carbonyl reductase P34H, a member of the short chain dehydrogenase/reductase superfamily. P34H is predominantly expressed in the human corpus epididymis [129]. During epididymal transit of spermatozoa, P34H progressively accumulates on the sperm surface covering the acrosome [130]. Anti-P34H antibody specifically inhibits spermatozoa–ZP binding [131] and human spermatozoa lacking P34H cannot bind to the ZP [132]. Furthermore, the loss of P34H from the sperm surface has been associated with idiopathic infertility in men [132,133]. 6. New concepts in zona pellucida receptor on spermatozoa Our understanding of the sperm-ZP interaction has progressed substantially over the past decade due to advances in technology including biophysical, biochemical, molecular biology and transgenic techniques. In contrast to the classical model of spermatozoa–ZP interaction which involves a relatively simple, single receptor–ligand primary binding and ZP glycoprotein induced acrosome reaction, followed by the secondary binding, more recent studies reveal a more complicated and at times contradictory story. 6.1. Multiple molecules are involved in spermatozoa–zona pellucida interaction Most early studies assumed that the sperm ZP receptor was a single molecular species. However, a surprisingly large number of sperm molecules bind to the ZP and potentially involved in spermatozoa–ZP interaction. These together with the failure in producing an infertile phenotype in the respective knockout animals prompt a revision of the traditional concept of having a single, constitutively expressed ZP receptor on spermatozoa. From an evolution perspective for continuation of species, it is logical for nature to develop a system with more than one ZP receptors on spermatozoa; they have redundant function, such that the defect associated with mutation or loss of only one of them can be ready replaced by others. This provides an explanation why 100% inhibition of fertilization is rarely achieved in vivo and in vitro as elimination of one ZP-binding protein can be partly compensated by the others. In addition, since the threshold of residual sperm binding that must be achieved to ensure fertility has not been established, therefore, 20–30% residual binding mediated by another candidate ZP receptor may be sufficient to completely recover fertility in knockout mice. The identification of sperm proteins that bind either to the glycans or the protein core of ZP molecules are in line with the existence of multiple sperm ZP receptors, i.e. some receptors interact with the glycans while the other with the accessible protein regions


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of the ZP glycoproteins. This is exemplified in studies of SLeX as the major carbohydrate ligand for initiating human spermatozoa–ZP binding. Glycoconjugates terminated with SLeX suppresses ZP binding but do not induce acrosome reaction, a hallmark of primary ZP binding [42]. Similarly, spermatozoa from GalTasedeficient mouse are able to bind to the ZP, but do not respond to ZP3-induced acrosome reaction [83]. These results indicate that spermatozoa–ZP interaction involves at least two receptors, one for binding and the other for induction of acrosome reaction. Consistently, results obtained in both the mouse and human models indicate that about 80% of the binding sites on spermatozoa are carbohydrate dependent, whereas the remaining 20% are for protein–protein interaction [55]. This concept is further supported by the observations that ZP glycoproteins bind to more than one membrane fractions of mouse spermatozoa [134]. Binding kinetic study also suggests that spermatozoa–ZP binding involves a series of binding events, each presumably mediated by a specific receptorligand interaction [135]. 6.2. Sperm zona pellucida receptor is a protein complex

identified different sub-types of membrane rafts, which may have distinct functional roles in spermatozoa–ZP interaction [144]. 6.4. Involvement of the acrosomal matrix molecules in spermatozoa–zona pellucida interaction It was previously presumed that an intact outer acrosomal membrane and plasma membrane are required for the spermatozoa to recognize and to bind to the ZP. However, recent observation showed that the acrosomal matrix could be exposed to the sperm surface during capacitation [145]. The mechanism for the progressive exposure of intra-acrosomal contents during capacitation is not yet clear. It has been suggested that as capacitation proceeds, the outer acrosomal membrane evaginates to form vesicles that enlarge and become tethered to the plasma membrane through complementary vesicle-associated soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) and target membrane SNARE proteins [146–149]. Thus, the ZP-binding proteins can be carried to the sperm surface from the acrosomal vesicle via an exocytotic process. One good example is Sp56 which is an acrosomal matrix molecule. It is partially exposed/released to the sperm surface during capacitation and mediates initial gamete binding [87]. The observation explains why many previously identified candidate ZP receptors on spermatozoa, such as proacrosin and ZP-binding protein 2, turn out to be components of the acrosomal matrix. The role of acrosomal matrix molecules in spermatozoa–ZP interaction is further complicated by a recent study demonstrating that most mouse spermatozoa undergo acrosome reaction before they encounter the ZP during their interactions with the cumulus cells in vivo [150]. The study also presents evidence that acrosome-reacted spermatozoa readily bound to the ZP [150]. This is supported by the observation that acrosome-reacted mouse spermatozoa collected from the perivitelline space can fertilize fresh, cumulus-enclosed, ZP-intact oocytes and produce live offsprings [151]. These observations challenge two traditional concepts. First, acrosome intact spermatozoa are required for fertilization and they lose their fertilizing capacity soon after acrosome reaction [1,2,152]. Second, the molecules on the sperm plasma membrane are required for spermatozoa–ZP binding.

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Recent evidence indicate that the sperm ZP receptor is a composite structure involving coordinated actions of multiple recognition molecules that are assembled into a functional complex during post-testicular maturation and capacitation [100,136–138]. The involvement of a suite of multi-molecular protein complexes on spermatozoa in mediating their interactions with the ZP is analogous to the dynamic, tightly regulated processes that drive many cell–matrix and cell–cell interaction [139,140]. One of the human sperm ZP receptor complexes has been characterized. The complex contains arylsulfatase A (ARSA), SPAM1 and heat shock 70 kDa protein 2 (HSPA2) [141]. The complex is translocated to the apical region of the sperm head during capacitation as a result of changes in membrane fluidity and activation of a capacitation-associated protein kinase A signal transduction pathway. In this complex, ARSA mediates spermatozoa–ZP interaction, while SPAM1 is involved in cumulus matrix dispersal. HSPA2 is unlikely to be directly involved in the binding of human spermatozoa to the ZP surface, but may act as an accessory protein within the complex that facilitates the organization and expression of the ZP receptor complex on the surface of spermatozoa after capacitation-associated tyrosine phosphorylation [141–143].

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7. Lack of standardized assay to evaluate spermatozoa–zona pellucida binding

One of the means for organizing the multimeric molecular complexes on sperm plasma membrane is to cluster the constituent proteins in the lipid raft. Lipid rafts are heterogeneous, dynamic, sterol- and sphingolipid-enriched membrane microdomains that serve as platforms through protein–protein and protein–lipid interactions for cell adhesion and signaling. Protein complexes in lipid rafts exhibiting ZP binding affinity are present in the mouse [98] and human spermatozoa [99]. Changes in the sperm lipid rafts are associated with capacitation [100,136,137]. Specifically, there is a uniform distribution of the rafts in the uncapacitated mouse and human spermatozoa. After capacitation, the rafts are confined within the peri-acrosomal region of the sperm head [98,99]. Proteomic analyses of the mouse and human spermatozoa confirms that many of the putative ZP binding proteins, such as ADAM, sFUT5, GalTase, SED-1, sperm surface protein Sp17, zona pellucidabinding protein 1 and Sp56, are relocated to the lipid rafts after capacitation [98,99]. These data suggest that the lipid rafts serve as macromolecular scaffolds that selectively concentrate ZP binding molecules to the appropriate site on the sperm surface [100]. Recent biochemical analyses of the sterol, GM1, phospholipid, and protein composition of the mouse sperm membrane fractions have further

Many of the difficulties for getting conclusive results on molecules involved in spermatozoa–ZP binding are related to the lack of a reliable assay for evaluation of the process. In humans, spermatozoa–ZP binding assays involve incubation of the spermatozoa with the whole human oocytes [153,154] or half of the ZP [155]. However, sperm binding to the ZP in these assays varies depending on the techniques used and the conditions defined by the researchers, which may not be appropriate for physiological binding in fertilization. For example, the number of ZP bound spermatozoa is affected by the diameter of the pipette using to strip off the loose bound spermatozoa [156], the source of the oocyte used [157–159], sperm preparation protocols [156,160], the base medium used [156] and inseminated sperm concentration [161]. Therefore, future development of a synthetic biomaterial that could provide a standard matrix to assess spermatozoa–ZP binding in a reproducible manner would be helpful in the identification of the sperm ZP receptor. The increasing evidence that the sperm ZP receptors are molecular complexes also require new methods for evaluation of their functions. Conventional studies on spermatozoa–ZP binding involved blocking the molecule of interest by antibodies and

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antagonists followed by determination of the change in binding. The approach is not suitable for studying complexes. Although the use of double and triple transgenic animals can potentially serve the purpose, the procedure is labor intensive, expensive and inefficient. Novel technique is required for studying multi-protein complexes in spermatozoa. One such technique is inducible degradation of protein [162], which allows real time monitoring of the consequence of removal of a specific complex component in a live cell. 8. The significance of zona pellucida receptor identification The world population has exceeded 7.22 × 109 and increasing at a rate of around 1.14% per year (World POPClock Projection, 2014). Alarmingly, 95% of this population growth is occurring in developing countries, where it is being driven partly by insufficient resources for family-planning [163]. In USA, half of all pregnancies are unintended, 43% of which end in abortion [164]. These statistics show the inadequacy of the current contraceptive measures and highlight the need for new approaches to contraception. Research on the ZP receptor is therefore warranted as a means of providing novel targets for contraceptive intervention. The identification of the ZP receptor also has implication in assisted reproduction. Subfertility affects about 15% of couples of reproductive age and has a major impact on public health. Treatment of human infertility with IVF and intracytoplasmic sperm injection (ICSI) has been successful. The main difference between standard IVF and ICSI is that sperm functions such as ZP-binding and acrosome reaction, while essential for normal fertilization with IVF, are not necessary with ICSI. IVF is preferred where fertilization is likely to occur as it has a lower cost. In addition, ICSI may have detrimental effects on the resulting embryos [165–168]. Currently, semen analysis is an important consideration for the choice between IVF and ICSI. However, standard semen analysis provides only limited information on the sperm fertilizing ability and defective ZP interaction can occur in 13% men with normal semen parameters [169]. While ICSI is very successful in treating patients with defective spermatozoa–ZP interaction [170], there is no easy method of identifying these patients. Very often, these patients will receive IVF in their first treatment cycle based on their normal semen parameters. They are at risk of having reduced or failure of fertilization in IVF, wasting one cycle and the associated financial cost, and suffering emotionally. To improve the clinical management of these patients, it is important to diagnose defective ZP interaction by reliable test before commencing assisted reproduction treatment. The identification of ZP receptor can be used as simple test for prediction of the fertilization potential of semen samples. 9. Conclusion This review provides an update on our recent knowledge on spermatozoa–ZP interactions. Our understanding of the process has progressed substantially over the past decade. The failure of identifying the responsible molecules for process prompts a revision of the classical concept of having a single sperm ZP receptor. Several new concepts on the process emerge. First, the plasma membrane of spermatozoa is a dynamic structure with diverse microdomains that undergo dramatic changes/reorganization during sperm maturation and capacitation. Since the fundamental mechanisms that regulate these dynamic changes of sperm surface proteins are poorly understood, it is difficult to identify the specific molecule(s) that take part in the spermatozoa–ZP interaction. Second, spermatozoa–ZP interaction is a complex binding event includes multiple receptor–ligand interactions. Third, since the spermatozoa–ZP binding is a relatively species-specific event,

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it is reasonable to expect that different mammalian species may involve different molecules for the process. Therefore, results of gene-targeting strategies that examine the role of candidate sperm receptors and ZP glycoproteins in one species may not be applicable to another species. Fourth, acrosomal matrix molecules can be exposed to the sperm surface during capacitation and take part in the spermatozoa–ZP interaction. The findings signify further increase in the difficulties in the identification of sperm ZP receptor as routine assays do not enable monitoring of the translocation of the matrix molecules. To conclude, while significant progress has been made in our understanding of the molecular basis of spermatozoa–ZP interaction, the identity of the sperm ZP receptor remains an unresolved issue. Many research challenges remain. Further investigation of the area will provide considerable understanding of fundamental aspects of fertilization that will be useful for practical application in human reproductive medicine and infertility. Uncited reference

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[11].

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Acknowledgements

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This work was supported in part by grants from the Committee Q3 on Research and Conference Grant, The University of Hong Kong, Hong Kong Research Grant Council (RGC grants HKU 764512M, HKU 764611M and 201109176154). References

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G Model YSCDB 1569 1–10 8 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864


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G Model YSCDB 1569 1–10 10 1123 1124 1125 1126 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 1172 1173


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Biology of mammalian fertilization: role of the zona pellucida.

The influence of solubilized porcine zona pellucida protein on the binding capacity of human spermatozoa.

Fertilizing capacity of epididymal and testicular spermatozoa microinjected under the zona pellucida of the mouse oocyte.

Developmental biology. The germ of the issue.

Informed consent: an unresolved issue.

Calcium-dependent binding of mouse epididymal spermatozoa to the zona pellucida.

Preterm birth, an unresolved issue.

Isolation and biochemical characterization of a zona pellucida-binding glycoprotein of boar spermatozoa.

Characterization of low Mr zona pellucida binding proteins from boar spermatozoa and seminal plasma.

Pharmacological treatment of atherosclerosis: an unresolved issue.

Oocyte-specific expression of mouse Zp-2: developmental regulation of the zona pellucida genes.

Correcting tricuspid regurgitation: an unresolved issue.

Paraphilic coercive disorder: an unresolved issue.

The complete primary structure of the spermadhesin AWN, a zona pellucida-binding protein isolated from boar spermatozoa.

The hemizona assay (HZA) as an experimental model to evaluate the inhibition of sperm binding to the murine zona pellucida by isolated zona pellucida protein.

Aldosterone and the heart: still an unresolved issue?

Maturation-associated changes in the rat zona pellucida.

Immunocytochemical localization of an oviductal zona pellucida glycoprotein in the oviductal epithelium of the golden hamster.

Structure of the mouse egg extracellular coat, the zona pellucida.

Genomic organization and polypeptide primary structure of zona pellucida glycoprotein hzp3, the hamster sperm receptor.

Sexual Dysfunction in Females after Cancer Treatment: an Unresolved Issue

Sperm selection capacity of the human zona pellucida.

Amyloid properties of the mouse egg zona pellucida.

Binding of a 15 kDa glycoprotein from spermatozoa of boars to surface of zona pellucida and cumulus oophorus cells.