SLeX: potential implications for fertility and contraception

Keywords::

• contraception
• fertilization
• sialyl-Lewis(x)
• spermatozoa
• zona pellucida

SLeX is abundant on human zona pellucida

Human oocytes are enveloped by an extracellular matrix known as zona pellucida. The binding of spermatozoa to the zona pellucida marks the first step in fertilization and is an area of intensive research. To understand how this important event in fertilization is regulated, much effort has been invested in searching for specific molecules on the zona pellucida and the sperm surface responsible for the binding. However, the success of the search has been limited.

Human zona pellucida is composed of only four glycoproteins termed zona pellucida glycoprotein (ZP)-1, -2, -3 and -4 [1,2]. Despite the relatively simple molecular composition of the zona pellucida, we do not know much about how its glycoproteins interact with the spermatozoa for two reasons. First, we do not yet have appropriate techniques for studying carbohydrate–protein interaction, which mediate spermatozoa–zona pellucida binding [3,4]. The interaction of sperm zona pellucida binding protein(s) (sperm receptor hereafter) with the carbohydrate moieties on the zona pellucida is evidenced by the observations that chemical or enzymatic removal of ZP3 oligosaccharides abolishes the sperm-binding activity of the glycoprotein [2–5]. Second, the availability of human zona pellucida is limited. Although genes encoding the human zona pellucida glycoproteins have been cloned and their recombinant forms produced, their use in studying spermatozoa–zona pellucida interaction may not be appropriate because they do not have the correct glycosylation. Native human zona pellucida glycoproteins can be purified but the purification requires a huge number of donated oocytes [2].

With the use of an ultrasensitive mass spectrometric method, the sialyl-Lewis(x) (SLeX) sequence (NeuAcα2–3Galβ1–4[Fucα1–3]GlcNAc) was recently found to be the most abundant terminal sequence on the glycans of human zona pellucida [6]. SLeX is a tetrasaccharide structure found at the terminus of glycolipid or glycoprotein. It is a well-characterized ligand of the selectin family and plays crucial roles in cell–cell adhesion [7].

The presence of a high density of SLeX on the glycans of zona pellucida is rare among normal human cells [6]. This glycosylation pattern differs greatly from that of the mouse. No SLeX can be detected in the mouse zona pellucida [8]. The majority of its glycans are of the high mannose or complex type, with polylactosamine chains having terminal sulfate groups and sialic acid [8]. The differences in the zona pellucida glycosylation of the two species indicate that the mouse may not be a suitable model for studying human spermatozoa–zona pellucida binding. It would be interesting to determine whether such differences would lead to species specificity in spermatozoa–zona pellucida binding. Related to this, transgenic mice carrying the human ZP3 gene instead of their own ZP3 gene bound to mouse spermatozoa but not to human spermatozoa [9,10], suggesting that the mouse oocytes can process the glycosylation of human ZP3 in a ‘mouse’ manner, enabling the binding of mouse spermatozoa onto human ZP3.

SLeX on human zona pellucida is important for sperm binding

SLeX neoglycoprotein binds to the human sperm head [6], which is responsible for interacting with the zona pellucida during fertilization. SLeX oligosaccharide, its neoglycoprotein and anti-SLeX antibody inhibit approximately 70% of the spermatozoa–zona pellucida binding [6]. These observations indicate that SLeX is a major ligand for sperm binding. However, the epitope(s) responsible for the other 30% of SLeX-independent binding remains to be determined.

A direct question arising from the finding is whether reduced expression of SLeX on zona pellucida is a cause of fertilization problems in clinical assisted reproduction treatment. Although low spermatozoa–zona pellucida binding usually results from sperm defects [11], oocyte defects cannot be excluded. Our preliminary data show that the expression levels of SLeX are different among oocytes. Study on how these levels are related to the fertilization rate in clinical assisted reproduction is ongoing in our laboratory.

Use of SLeX in identification of sperm receptors

Several carbohydrate-binding proteins have been proposed to be the sperm receptors in humans. These include fucosyltransferase-5 [12], galactosyltransferase [13], sperm agglutination antigen-1 [14] and α-D-mannosidase [15]. However, they are unlikely to be solely responsible for mediating spermatozoa–zona pellucida binding [3] as antibodies against and competitors/substrates of these proteins cannot completely inhibit spermatozoa–zona pellucida binding and/or zona pellucida-induced acrosome reaction. The identity of human sperm receptors remains obscure.

Identification of the sperm receptors has been difficult because of the scarcity of human zona the pellucida glycoproteins for use in the determination of zona pellucida binding capacity of potential candidates. Unlike the zona pellucida, SLeX is readily available commercially. Thus a sufficient quantity of SLeX can be obtained easily for isolation of sperm receptors by techniques such as co-precipitation, and for use as probe to confirm the zona pellucida-binding ability of the identified proteins.

Identification of men with spermatozoa–zona pellucida binding defects

Infertility affects approximately 15% of couples of reproductive age. The condition has a major impact on public health. Defective zona pellucida binding in men is a cause of infertility. It occurs in 78 and 64% of oligospermic and teratozoospermic men, respectively [11]. In clinical assisted reproduction, the defect manifests as a reduced fertilization rate [11]. Although assisted reproduction with intracytoplasmic sperm injection can improve the fertilization rate, the micromanipulation procedure is associated with increased risks of sex chromosome abnormalities, genomic imprinting defects and transmission of genetic abnormalities to the offspring [16,17]. Thus, the procedure should be performed only on those who are in need. The identification of men with a spermatozoa–zona pellucida binding defect is a challenge to reproductive medicine specialists.

The common tests used for spermatozoa–zona pellucida binding are the hemizona binding assay and the competitive intact zona pellucida binding assay [11]. Both assays require donated human oocytes, which are difficult to obtain in sufficient numbers for routine clinical use. Moreover, these tests require special skills, such as microdissection of the oocyte to obtain the hemizonae. Standardization of the protocol for these assays among laboratories is also difficult. To circumvent these difficulties, researchers have tried to use recombinant human zona pellucida proteins instead. Although these recombinant proteins have the same polypeptide sequence, they do not have glycosylation identical to their native counterparts. They bind to spermatozoa with low affinity and are poor agents for induction of acrosome reaction [2,5].

The identification of SLeX as the major carbohydrate ligand for human spermatozoa–zona pellucida binding prompts the development of a convenient and readily standardized assay for the identification of men with potential dysfunctional spermatozoa–zona pellucida binding. Commercially available multivalent SLeX ligands may serve as surrogates for the zona pellucida in such assays. The extent of their binding to sperm samples is expected to be proportional to the zona pellucida binding capacity of the samples. While reduced binding of SLeX in the assay suggests potential binding problems, one has to be cautious in interpreting the results, as the assay assesses only the SLeX-dependent binding, which accounts for approximately 70% of the zona pellucida binding capacity of the samples.

Implication on contraception

Immunocontraception or contraceptive vaccine targeting molecules, including those on gametes, required for the establishment are methods, some of which, such as steroid contraceptives are associated with systematic side effects [18]. It is unlikely that vaccine targeting at SLeX can be used as a contraceptive for two reasons. First, SLeX is also present and has important physiological functions in other cell types. For instance, SLeX–selectin interaction is crucial to the trafficking of immune cells through the vascular system and within different tissues [7]. Thus, the induction of antibodies against SLeX by vaccination is likely to produce side effects. This drawback may be solved by targeting the sperm receptor for SLeX instead. Second, an in vitro experiment demonstrates that anti-SLeX antibody can only partially inhibit spermatozoa–zona pellucida binding [6]. Therefore, vaccination against SLeX alone against or its sperm receptor will not give full protection from pregnancy. The incomplete blockage of spermatozoa–zona pellucida binding by anti-SLeX antibody is consistent with the accumulated evidence that the sperm receptor is a multi-molecular structure involving coordinated actions of different proteins that are assembled during capacitation [19,20]. A possible solution to this latter problem is to use a multi-epitope approach [18] – that is, the establishment of a vaccine against sperm receptors for both the SLeX-dependent and -independent binding.

Conclusion

The identification of SLeX as a major ligand for spermatozoa–zona pellucida binding has opened a new research avenue for the regulation of fertilization in humans. The finding is useful in the development of assays for identifying men with a spermatozoa–zona pelluida binding defect and the establishment of new contraceptives. To make these developments a success, identification of the ligand(s) involved in the SLeX-independent binding of spermatozoa to the zona pellucida is needed.

Financial & competing interests disclosure

This work is supported by a research grant from University Research Committee, University of Hong Kong. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.