Acrosomal protein NEP4 is required for sperm retention in, but not entry into, the female seminal receptacle in Drosophila melanogaster.

Sperm storage in the female reproductive tract is essential for efficient progeny production in animals with internal fertilization. This is particularly so for insects, especially females that have specialized organs for long-term storage. Nep4 mutant males of Drosophila melanogaster produce an average number of morphologically normal sperm, but they cannot fertilize eggs because Nep4 sperm nuclei fail to decondense after entry into the egg. Furthermore, although Nep4 mutant sperm enter the sperm storage organs in a typical manner, they are largely lost from the seminal receptacle within 24 h. It is potentially advantageous for females to discard such defective sperm. However, whether females are actively involved in this rapid sperm loss is not known. First, this study characterized NEP4 expression in wild-type testes. Between the 2 isoforms, the soluble isoform (Nep4-PB) but not the membrane-bound isoform (Nep4-PA) was essential for male fertility. NEP4 protein was localized to the acrosome of mature sperm and remained so in female storage organs. Second, we performed rescue experiments; the rapid sperm-loss phenotype of Nep4 sperm could not be rescued by mating to an eggless female nor by insemination by a wild-type male beforehand. This indicates that Nep4 sperm are likely unable to stay in and autonomously exit the seminal receptacle. This study provides evidence that sperm retention and maintenance in female sperm storage organs involve different mechanisms from those for sperm entry into the storage organs and that the acrosome of Drosophila sperm plays an important role in this process.