A Unique Seminal Plasma Protein, Zona Pellucida 3-Like Protein, has Ca²⁺ -Dependent Sperm Agglutination Activity

Identification of seminal proteins provides a means of investigating their roles. Despite their importance in the study of protein function, such as regulation of sperm motility, it is difficult to select candidates from the large number of proteins. Analyzing the rate of molecular evolution is a useful strategy for selecting candidates, and expressing the protein allows the examination of its function. In the present study, we investigated seminal plasma proteins of the cichlid Oreochromis mossambicus, which exhibits a unique mode of fertilization and a rapidly evolving gene that encodes a seminal plasma protein, zona-pellucida 3-like (ZP3-like), which does not belong to the same molecular family as other ZPs. Seminal plasma proteins of O. mossambicus were separated by two-dimensional electrophoresis, and 19 major proteins were identified by mass spectrometry (MALDI-Tof Mass). Because proteins that are under positive selection often impact sperm function, the rates of molecular evolution of these proteins were analyzed in terms of non-synonymous/synonymous substitutions (ω). Among the 19 proteins, positive selection was supported for five genes; functional assays were carried out on four of the proteins encoded by these genes. Of the four positively selected proteins, only ZP3-like protein agglutinated sperm in a dose- and Ca²⁺ -dependent manner. The other three proteins did not affect sperm motility. Because of the unique fertilization type, in which fertilization occurs in the buccal cavity, the need to retain sperm within the cavity during spawning, and the agglutination of sperm, which may be partly assisted by ZP3-like protein, may contribute to fertilization success. Fertilization in the buccal cavity may be related to its rapid molecular evolution.