BmPMFBP1 regulates the development of eupyrene sperm in the silkworm, Bombyx mori

Sperm deliver the male complement of DNA to the ovum, and thus play a key role in sexual reproduction. Accordingly, spermatogenesis has outstanding significance in fields as disparate as infertility treatments and pest-control, making it a broadly interesting and important focus for molecular genetics research in a wide range of species. Here we investigate spermatogenesis in the model lepidopteran insect Bombyx mori (silkworm moth), with particular focus on the gene PMFBP1 (polyamine modulated factor 1 binding protein 1). In humans and mouse, PMFBP1 is essential for spermatogenesis, and mutations of this gene are associated with acephalic spermatozoa, which cause infertility. We identified a B. mori gene labeled as “PMFBP1” in GenBank’s RefSeq database and sought to assess its role in spermatogenesis. Like in mammals, the silkworm version of this gene (BmPMFBP1) is specifically expressed in testes. We subsequently generated BmPMFBP1 mutants using a transgenic CRISPR/Cas9 system. Mutant males were sterile while the fertility of mutant females was comparable to wildtype females. In B. mori, spermatogenesis yields two types of sperm, the nucleated fertile eupyrene sperm, and anucleated unfertile apyrene sperm. Mutant males produced abnormal eupyrene sperm bundles but normal apyrene sperm bundles. For eupyrene sperm, nuclei were mislocated and disordered inside the bundles. We also found the BmPMFBP1 deficiency blocked the release of eupyrene sperm bundles from testes to ejaculatory seminalis. We found no obvious abnormalities in the production of apyrene sperm in mutant males, and double-matings with apyrene-deficient sex-lethal mutants rescued the ΔBmPMFBP1 infertility phenotype. These results indicate BmPMFBP1 functions only in eupyrene spermatogenesis, and highlight that distinct genes underlie the development of the two sperm morphs commonly found in Lepidoptera. Bioinformatic analyses suggest PMFBP1 may have evolved independently in lepidoptera and mammals, and that despite the shared name, are likely not homologous genes.Author summary: The presence of nucleated and anucleated dimorphic sperm produced by a single male is a notable characteristic of lepidopteran insects. Previously we identified the gene BmSxl and BmPnldc1 are required for apyrene and eupyrene sperm development, respectively. However, there remains very little known about the molecular mechanism of eupyrene and apyrene sperm development and function. In human and mouse, the protein PMFBP1 is related to acephalic spermatozoa syndrome. Here, we generate somatic mutants in the silkworm Bombyx mori for this gene. In the silkworm, BmPMFBP1 is essential for male fertility. Loss of BmPMFBP1 function results in defective eupyrene sperm bundles in the early elongation stage of spermatogenesis. The nuclei of the eupyrene sperm bundles were displaced from sperm heads while the apyrene sperm bundles were normal. This deficiency also results in the failure of the release of eupyrene sperm bundles from testes to the ejaculatory seminalis duct. Our study proves the important function of BmPMFBP1 in the development of the eupyrene sperm in the silkworm and identifies a potential target gene for lepidopteran pest control.