Loss of bmp15 gene function does not abolish fertility in male Atlantic salmon

Introduction Bone morphogenetic protein 15 (BMP15), a transforming growth factor β (TGF-β) superfamily member, is well known for its crucial involvement in regulating female reproduction in mammals, and recently also in fishes. However, the biological function(s) of Bmp15 in male reproduction are largely unknown in vertebrates in general. Methods In this study, we used CRISPR/Cas9 technology to generate Atlantic salmon bmp15 mutants to investigate its potential role in testis development, spermiation rates and sperm motility. Results No apparent phenotypic differences were observed at either of the two sampling points (18 and 22 months of age) carried out prior to the spawning season, in any of the mutant groups. However, at 22 months of age, mutant males showed a higher frequency of individuals in the rapid testicular growth phase than wild-type (WT) controls (63.6% versus 33.3%). Then, males that remained sexually immature and homozygous for the frameshift mutations ( bmp15 fs/fs ) were significantly shorter and lighter than WT controls from 26 months of age (at spawning) onwards; they also exhibited a higher incidence of deformed vertebrae and significantly lower testicular transcript levels for two growth factors ( amh, gsdf ) when examined at the age of 26 months. Regarding testis maturation, 26 month-old fs/fs mutant males showed a higher frequency of spermiating fish than WTs. However, further analyses revealed variation in vgll3a genotype - linked to age at maturity - among the fish investigated, as well as a vgll3a genotype-dependent association with spermiation probability. No significant differences were found regarding the plasma levels of 11-ketotestosterone, the main androgen in fish, irrespective of the maturation stage. Also regarding sperm motility and trajectory, no significant differences were detected between mutants and WTs in the two following reproductive seasons, under the conditions of this study. Conclusion This is the first mutant model in fish investigating Bmp15-mediated action in the male gonad and, similarly to the only other genetic model available in vertebrates to date–the pig –, no remarkable testicular phenotypes were revealed. Future studies will be required to determine whether these effects are evolutionary conserved across vertebrates, or whether distinct or additional phenotipic consequences emerge in other fishes or tetrapods.