Mutant generation of the testis genes and phenotype analyses in Drosophila

Abstract

Multiple genes work together to maintain the normal functions of the reproductive system. However, for many of these genes, little is known about their specific functions and mechanisms. In the present study, eight Drosophila genes, including CG4161, CG11475, CG2921, CG10541, CG7276, CG3800, CG8117 and CG16779, were selected for detailed studies based on their testis expression, undefined functions, and having highly homologous and conserved genes in humans (Homo sapiens) and mouse (Mus musculus). We analyzed their expression levels in different tissues, and determined their probably functions in male reproduction. The results showed that the first five genes were mainly expressed in testis, while other three showed ubiquitous expression in all tissues examined. Using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) and homology-directed repair (HDR) strategies, we have systematically generated the mutants of these genes and studied their functions in male reproduction. Loss of function of CG7276 and CG3800 resulted in partial sterility and reduction of the offspring number, while other genes showed no significant impact on fertility. CG7276 -/- and CG3800 -/- mutants were partial sterile and showed various types of abnormities, including seminal vesicle atrophy, spermatogonial stem cell reduction and cellular distribution disorders. Results of DNA and F-actin staining also indicated that CG7276 and CG3800 could play important roles in spermiogenesis. The establishments of these mutants have provided means to unravel the functions and mechanisms of these genes in animal models.