A Tale of Two Sperm: Targeting Sperm Developmental Pathways using RNAi Approaches in Bombyx mori

Sexual reproduction is important in the development and continuation of species; however, the topic is not widely understood. Sperm production plays a vital role in reproduction, but sperm does not always develop properly, leading to infertility. Unlike most creatures, butterflies and moths produce two distinct types of sperm; nucleated (eupyrene) and anucleated (apyrene). This phenomenon has raised questions about the role of sperm in reproduction. Bombyx mori, the domesticated silkworm, is the ideal model organism for understanding infertility, as they share several homologous genes with humans and is tractable in a laboratory environment. The BmPMFBP1 gene in B. mori is responsible for the production and regulation of the eupyrene sperm in spermatogenesis. Using RNA interference (RNAi), we targeted the BmPMFBP1 gene to disrupt eupyrene sperm production to observe phenotypic and functional changes in the gene's absence. First, we extracted RNA from caterpillar testes and converted the RNA into cDNA. Then, we used PCR to create a DNA template from the cDNA. Afterwards, we used the DNA template to synthesize double stranded RNA which will be used to disrupt sperm development in B. mori caterpillars. Sperm were isolated from pupal testes and stained for nuclear DNA using DAPI fluorescence. We expect the nuclei sperm bundles to displace from the eupyrene sperm head upon RNAi treatment, meaning the sperm could not be released from the testes, making B. mori sterile. Overall, this study makes strides in understanding male sterility and the process of sperm development.