27. During fertilization in mammals proteins lzumo and Juno are required for recognition of sperm and egg. lzumo and Juno are found specifically in sperm and egg, respectively. Which
    one of the following in vivo experiments will demonstrate that lzumo and Juno interact with each other?
    (1) If sperms from a male mouse where lzumo has been knocked out is used to fertilize eggs from a normal female and no fertilization occurs.
    (2) Whole mount immunostaining for lzumo and Juno shows its presence on the sperm and egg
    respectively
    (3) If a CFP fused lzumo protein is mixed with YFP fused Juno protein in a tube, FRET occurs, i.e., when CFP is excited, emission of YFP is observed.
    (4) Two independent kidney cell lines are developed, one expressing lzumo and the other Juno. If the two cells are mixed, they tend to aggregate with each other.

Explanation:

• Experiment (1) shows that sperm from an Izumo knockout male fail to fertilize a normal egg, demonstrating Izumo’s importance but not directly proving its interaction with Juno.

• Experiment (2) confirms the presence/location of Izumo and Juno but does not demonstrate their physical interaction.

• Experiment (3), FRET between CFP-Izumo and YFP-Juno fusion proteins, would indicate direct molecular interaction in vitro but is an artificial system.

• Experiment (4) shows physical interaction between Izumo and Juno by inducing aggregation when cells expressing these proteins are mixed. This cell-cell adhesion model provides strong, functional evidence of direct binding between Izumo and Juno in vivo.

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Introduction

Fertilization in mammals requires precise recognition and binding events between sperm and egg. The sperm protein Izumo and the egg protein Juno form a critical receptor-ligand pair that mediates sperm-egg adhesion, a prerequisite for fusion. Experimental approaches have elucidated this interaction, with in vivo cell adhesion assays providing conclusive evidence.

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Izumo and Juno: Sperm-Egg Recognition Proteins

Izumo is a sperm surface protein essential for fertilization, named after a Japanese shrine related to marriage. Juno (formerly Folr4) is its receptor on the egg membrane. Both proteins are indispensable; knockout of either results in infertility due to failure of sperm-egg fusion.

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Experimental Evidence for Izumo-Juno Interaction

• Knockout studies establish the necessity but are indirect.

• Immunostaining confirms localization but not binding.

• Fluorescence resonance energy transfer (FRET) can demonstrate close molecular proximity in vitro.

• Cell-cell aggregation assays with cells expressing Izumo and Juno produce visible cell clumping, directly demonstrating protein-protein binding in a living system.

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Importance in Fertilization

This receptor-ligand interaction is the first identified essential adhesive step between sperm and egg. It functions to bring the gamete membranes into contact, enabling subsequent membrane fusion and fertilization.

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Conclusion

Of the experimental approaches, mixing kidney cell lines expressing Izumo and Juno to observe cell aggregation provides the clearest physiological demonstration of their interaction, confirming their crucial roles in sperm-egg recognition during mammalian fertilization.

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This article reviews critical experimental evidence supporting the interaction of Izumo and Juno, essential players in mammalian reproductive biology and fertility research.

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Final Answer:
(4) Two independent kidney cell lines are developed, one expressing Izumo and the other Juno. If the two cells are mixed, they tend to aggregate with each other.