Bindin Protein: A Potential Target for Male Contraception Or Infertility Treatments

At present, men lack access to non-hormonal methods of contraception that are safe and reversible; with global population numbers steadily rising it is paramount that new solutions for male contraceptives be created as soon as possible.

Bindin is a protein that allows sperm to recognize eggs. Its expression in the reproductive tract and male infertility phenotype when knocked out can be seen when mice lack it.

Sperm Binding to Eggs

Sperm must travel a arduous path through both male and female reproductive tracts to reach an egg, with tight zona pellucida binding being one of many obstacles they must surmount en route. A variety of factors can hinder this goal - for instance morphology and motility correlate with ability of sperm to adhere to zona pellucida and penetrate perivitelline space; but even those sperm that exhibit abnormal characteristics still achieve fertilization (Mortimer & Menkveld 1988).

Over the past two decades, numerous models of sperm-egg recognition have been proposed. At first, researchers attempted to identify an individual zona protein or carbohydrate side chain as the "sperm receptor", however recent genetic data from mice does not support single protein models; replacement of endogenous mouse zona proteins with human ZP2 or ZP3 does not alter taxon specificity or fertility when introduced into null mice with missing HuZP1. Furthermore, interaction of two cell embryos with zona pellucida persists even in animals lacking HuZP1 although with uncleaved HuZP2 interaction persists between endogenous mouse zona proteins or ZP3 replacement (Figure). Finally correlation exists between persistence of interaction of two cell embryos and ZP2 uncleaved on null mice for testing purposes (Figure).

Thus, the three-dimensional structure of zona pellucida rather than any individual protein or carbohydrate likely determines sperm binding to and entry to zona pellucida, leading to models where ZP2, ZP3 and other proteins form an egg-shaped matrix that recognizes sperm (Figure). Furthermore, some researchers have speculated that certain O- and N-linked glycans act as receptors, with glycosidases produced by cortical granules then cutting them off after fertilization has taken place (Figure).

Sperm Motility

Men with normal semen analyses might still struggle to fertilize eggs if their motility percentage of their sperm drops below 40%, according to semen analysis studies. Motility plays an integral part of healthy sperm analysis along with other variables like count, shape and concentration.

Since decades ago, it has been known that protein bindin found on sea urchin sperm heads acts as a species-specific adhesion factor during fertilization to glycoprotein receptors in vitelline envelope of an egg's vitelline envelope - this allows sperm to penetrate plasma membrane of eggs and fuse with them successfully.

CRISPR/Cas9 was used to replace Bindin in sperm cells with a mutant form that displayed diminished ability to attach to egg glycoprotein receptors, according to our TEM analyses. Our studies demonstrated that these mutant sperm could still undergo the acrosome reaction; however, they never formed the fusion complex with an egg; hence their infertility was solely attributable to losing Bindin function.

Bindin may have evolved more quickly due to its sole function of egg binding; proteins with multiple functions tend to change more gradually. But this theory remains just a theory at this stage; we plan on carrying out further investigation in the near future in order to test this theory.

Sperm-Egg Fusion

At the final step of fertilization, egg and sperm membranes fuse to form fertilized eggs. This process is initiated when IZUMO1 from sperm surface proteins binds with JUNO from egg membrane proteins; their interaction is essential to egg fusion; yet how they collaborate remains unknown.

Studies with mouse models have demonstrated that lack of Bindin impairs sperm-egg binding and ZP fusion by interfering with the IZUMO-JUNO interaction. Other candidates such as p-cadherin and CD9 also play roles in this stage, so its mechanism could involve multiple proteins and pathways.

Bindin is replaced by a synthetic peptide mimicking its function (p-Cad), the resultant protein retains its ability to bind ZP and egg membranes but is unable to trigger the acrosome reaction or join zygote and oocyte. This suggests that p-Cad acts as a decoy, drawing sperm toward an acrosome reaction.

SEM and motility analysis demonstrated that Bindin-null sperm were capable of reaching eggs. Furthermore, TEM imaging demonstrated that both p-Cad and IZUMO-JUNO binding sites are located on the acrosome's equatorial regions where egg binding occurs; however, those lacking these proteins were unable to form fertilisation envelopes with any egg they encountered and failed to fertilize an oocyte successfully.

Sperm-Egg Recognition

In 1977, Vacquier and Moy isolated an insoluble granular protein produced during sperm's acrosome reaction that interacts with egg surfaces: this is called "bindin". They observed that this species-specific binding took place with dejellied eggs of Strongylocentrotus purpuratus species (Figure 7.14A), while it also bound iodinated recombinant egg bindin receptor 1 (EBR1) eggs from this same species (Figure 7.14B).

Bindin may play an essential part in the attachment process that enables sperm to penetrate and enter an egg, whereupon they must recognize fertile eggs by means of carbohydrate receptors on its vitelline layer and recognize only one for fertilization. The binding action of bindin may serve as the gateway for penetration by the sperm into fertile eggs that await fertilization, before limiting themselves to just one as part of fertilization.

Scientists have conducted experiments iodinating recombinant Sf-EBR1 and found it bound specifically to species-specific bindin (Figure 2.15). This suggests that repeated domains within EBR1 molecule play an integral part in binding with bindin molecules.

Researchers created a mouse strain wherein their CRISPR/Ca9 gene-targeting eliminated the bindin gene, leading to sperm that neither bound to nor activated eggs (Figures 7.14A and B). When exposed to EBR1 iodinated with EBR1, these sperm exhibited robust acrosomal reactions but failed to form fertilization envelopes (Figure 7.14C). By comparison, wildtype mice that retained their original copy of this gene could agglutinate and activate eggs (Figure 7.14C).