Sterilization of aquaculture species has been recognized as beneficial for various reasons. First, it can promote growth by conserving energy that would otherwise be used for gonadal development. Second, it can prevent genetic disturbance to wild populations caused by escaped farmed individuals reproducing in natural environments. Third, it helps farmers protect their aquaculture strains from being illegally produced outside their premises. Furthermore, it could serve as a means to address regulatory challenges associated with implementing genome editing technologies for the aquaculture industry in some countries.
Recent studies using zebrafish and medaka have revealed that certain microRNAs influence fertility by regulating gene expression in their reproduction. This study investigates the effect of microRNA202 (mir202) knockout(KO) on sterility in Nile tilapia (Oreochromis niloticus), a globally important aquaculture species. We also study the method of large-scale production of mir202 KO fish.
Using the CRISPR/Cas9 genome editing tool, the mir202 gene was knocked out. Homozygous KO individuals were produced in the F2 generation, and the gonad development of each sex in KO individuals was physiologically and genetically analyzed. In addition to mir202, to create a monosex population, genes essential for male differentiation (gsdf and dmrt1) and female differentiation (cyp17 and cyp19a1a) were simultaneously knocked out. As a result of the mir202KO, female individuals became sterile. To apply this technology in an aquaculture setting, we studied the germ cell transplantation (GCT) method into dndKO hosts to evaluate the possibility of the sterility phenotype in the next generation.
In mir202KO females, arrested ovarian development was observed. The ovaries from 8-month-old were tiny, string-like structures, observed with only stage I-III oocytes inside. In males, while testes developed, a significant delay in development and reduction in sperm count were noted, resulting in an extremely low natural fertilization rate of 1.27 ± 2.61%. Based on these findings, we produced an all-female sterile population with a double KO with the masculinization-related gene (mir202 and gsdf). Furthermore, by transplanting oogonia obtained from these sterile females into dndKO hosts, we achieved the large-scale production of sterile individuals in the next generation.
These results demonstrate that the integration of sterilization through mir202 knockout and germ cell transplantation offers an effective method for large-scale production of all-female sterile populations of Nile tilapia.