The Ly6/uPAR Protein Bouncer is Necessary and Sufficient for Species-Specific Fertilization

S. Herberg, K. R. Gert, A. Schleiffer, & A. Pauli (2018)

The Ly6/uPAR Protein, referred to as Bouncer, may be an important player in the molecular mechanisms leading to fertilization. Discovered through the analysis of protein-coding genes Bouncer was shown to be highly expressed in the oocytes of zebrafish. Through genetic analysis it was concluded that Bouncer was the homolog of SPACA4, a protein expressed in the sperm of mammals, amphibians and reptiles. Species that utilize internal fertilization employed testis-specific expression of SPACA4 while species that use external fertilization were oocyte-specific through the use of Bouncer.

The function of Bouncer was further analyzed through the use of CRISPR/Cas9 to create lines of Zebrafish that were not able to produce a mature Bouncer protein. Crosses between heterozygous parental lines showed that Bouncer was not necessary for the development of embryos. However in vivo mating experiments showed that when eggs from females who lacked the Bouncer protein were fertilized less than 1 in 1000 developed into a cleavage stage embryo. In comparison, nearly all eggs that carried the Bouncer gene developed into or past the cleavage stage. It was also shown that female sterility was directly linked to a lack of the Bouncer protein.

The absence of Bouncer results in a failure of an embryo to develop alongside no obvious morphological defect. Instead, the lack of the protein prevents the sperm from entering the egg at all. This was proven both by a lack of sperm entry in Bouncer deficient eggs during In vitro fertilization as well as the successful fertilization of Bouncer deficient eggs through the use of intracytoplasmic sperm injection, a process that directly injects the sperm cell into the egg.

Further study showed that the protein produced by bouncer was concentrated within the egg membrane and vesicles within the egg, further strengthening the argument that it controls the entry and attachment of sperm into the egg during fertilization. When sperm cells were introduced to egg cells treated to have a completely exposed surface significantly more sperm cells were found to attach to the Bouncer containing eggs than to those without. This same imaging also supported that Bouncer does not recruit sperm to the egg itself. 

Researchers noted that the Bouncer proteins produced by different species had high degrees of difference in their amino acid makeup. Because of this it was considered that the Bouncer protein may play a significant role in preventing different species from producing viable offspring. To test this zebrafish were altered to produce the Bouncer protein of medaka, a species of fish that is not closely related to and cannot normally breed with the zebrafish. Further experiments with IVF showed that the zebrafish eggs that produced only medaka Bouncer could only be successfully fertilized by medaka sperm while zebrafish eggs that produced both forms of Bouncer could be fertilized by both species’ sperm. However, the fertilization rates between medaka sperm and zebrafish eggs with medaka Bouncer were still lower than same species egg and sperm, suggesting that Bouncer interacts with a compound in the sperm as well. 

Many other species employ an interaction between a sperm specific protein as well as an egg protein during species-specific binding. It is important to note however that all other recorded interactions are with the sperm and egg coat, not the sperm and egg membrane such has been found with the zebrafish. Zebrafish are externally fertilizing organisms who utilize Bouncer while internally fertilizing organisms showcase opposing expression through the usage of SPACA4. There is thought to be a link between this and post copulatory mate choice, though this has not been proven. Due to its similar nature to Bouncer, SPACA4 is thought to also play a significant role in fertilization in mammals. Observations from the Bouncer data collection support the idea that SPACA4 interacts within the membrane of the sperm to support binding to the egg cell during fertilization. Further experiments would need to be conducted to cement the link between fish Bouncer and mammalian SPACA4.