A targeted genetic investigation into teleost reproductive biology has revealed that disabling the bone morphogenetic protein 15 gene does not prevent male Atlantic salmon from undergoing successful testicular development, entering spermiation, or producing motile spermatozoa. The study, conducted by researchers at the Institute of Marine Research in Norway and Utrecht University, generated frameshift and in-frame mutations in Atlantic salmon using CRISPR-Cas9 genome editing to characterize the physiological role of the signalling molecule in the male gonad. While bone morphogenetic protein 15 has long been recognized as an indispensable regulator of ovarian folliculogenesis and oocyte development across diverse mammalian and fish species, its function within the vertebrate testis has remained largely unexplored. By tracking edited male salmon across multiple years and consecutive spawning seasons, the authors demonstrated that complete loss of functional Bmp15 protein does not abolish male fertility, showing that the factor is dispensable for completing spermatogenesis under standard aquaculture conditions.
While the gene is heavily expressed in the ovaries of vertebrates, where its disruption often leads to ovarian arrest or failure of ovulation, research in female Atlantic salmon previously revealed that bmp15 knockout causes ovulatory dysfunction alongside high embryonic mortality. Conversely, in female zebrafish, targeted loss of bmp15 causes complete female-to-male sex reversal resulting from an inability to maintain ovarian estrogen synthesis. Prior to the current trial in Atlantic salmon, the only genetic knockout model evaluating male bmp15 function in vertebrates was established in domestic pigs, where constitutive gene knockdown yielded only mild reductions in sperm viability without altering gonadal morphology, testicular steroidogenesis, or downstream pathway activation. Because bmp15 transcript is naturally expressed at detectable levels within the male gonads of several teleosts, investigators sought to determine whether deleting the gene could disrupt testis development or induce male sterility.
The study was partly driven by aquaculture challenges in commercial salmon farming, where precocious sexual maturation of males and potential genetic introgression of farm escapees into wild stocks represent pressing ecological and welfare issues. Developing gene-edited lines that selectively induce reproductive sterility has emerged as a promising strategy to mitigate unwanted genetic exchange between farmed and wild populations. Because previous investigations had identified germ cell-specific expression of bmp15 within salmon gonads, researchers hypothesized that the factor could serve as an effective target to block male gamete production. To test this hypothesis experimentally, fertilized salmon eggs obtained from AquaGen AS were co-injected in late 2015 with Cas9 messenger RNA and single guide RNAs targeting exon 1 of the bmp15 gene on chromosome 18, together with a guide RNA targeting the slc45a2 pigmentation gene as a visual mutation tracer. The bmp15 target sequence was designed to disrupt the putative propeptide cleavage site located at amino acid positions 310 to 313, preventing the maturation and activation of the encoded protein.
Rearing and sampling procedures were carried out under Norwegian food safety and animal welfare permits at the Matre Aquaculture Research Station in Matredal. Mosaic founder fish were reared to reproductive maturity to establish an experimental first-generation lineage by fertilizing gametes from highly edited broodstock. Non-edited wild-type controls were generated alongside the mutants, and all fry were commingled in shared rearing tanks upon beginning external feeding to ensure uniform environmental conditions across freshwater, seawater post-smolt, and brackish water phases. Following passive integrated transponder tagging and fin-clip DNA isolation, genetic sex was confirmed by quantitative polymerase chain reaction assays targeting the sexually dimorphic on the Y-chromosome locus. High-throughput sequencing of targeted amplicons classified the surviving experimental males into distinct cohort groups: 53 wild-type fish, 18 homozygous in-frame mutants, 26 compound in-frame and frameshift mutants, and 32 homozygous frameshift mutants, alongside two heterozygous individuals that were excluded from subsequent detailed comparisons.
Somatic metrics collected at 18, 22, and 26 months of age revealed that general physical growth did not diverge significantly among juvenile fish during early seawater development. However, by 26 months of age, corresponding to the initial spawning period, homozygous frameshift mutants exhibited significantly reduced body length and body weight when compared directly against wild-type controls. Maturation-stratified analyses revealed that this growth reduction was confined exclusively to the subgroup of males that remained sexually immature, with the smaller somatic profile persisting in non-spermiating frameshift mutants throughout intermediate samplings up to 40 months of age. By 48 months of age, this pattern reversed, and only spermiating frameshift fish displayed statistically lower body mass and length, though small sample sizes among older immature mutants limited formal statistical power. Somatic growth was therefore modulated in an age- and maturation-dependent manner rather than reflecting a uniform suppression of body growth across all life stages.
Histological examinations and testicular gene expression profiling were initiated at the 18-month post-smolt stage to evaluate early cellular dynamics. In this preliminary sampling, homozygous frameshift males presented testes transiting into early puberty, displaying increased mitotic activity among type A spermatogonia and a higher density of interstitial somatic cells. Testicular transcript measurements in these immature frameshift fish showed reduced expression of anti-Müllerian hormone and gonadal soma-derived factor, paired with elevated transcript levels of insulin-like growth factor 3 and the germ cell marker vasa. By contrast, males carrying in-frame mutations showed a germ cell-poor testicular histology alongside lowered vasa and growth differentiation factor 9 transcript abundance. By 22 months of age, quantitative measurements revealed that wild-type salmon experienced a transient peak in testicular bmp15 mRNA expression during early spermiogenesis. Concurrently, 63.6 percent of the mutant males had progressed to the rapid testicular growth phase containing advanced spermatids, compared to only 33.3 percent of the wild-type controls, with the proportion reaching 75 percent within the homozygous frameshift group.
When the remaining salmon cohort reached 26 months of age during the December spawning window, individual spermiation was evaluated via gentle abdominal stripping. Initial unadjusted univariate logistic regression indicated an apparent allele-dosage effect, with the estimated odds of spermiation increasing progressively across genotypes to reach a three-fold higher odd in homozygous frameshift mutants relative to wild-type controls. However, because experimental fish originated from diverse parental backgrounds, the researchers genotyped the vestigial-like family member 3a locus, an established genomic determinant accounting for 33 to 39 percent of the phenotypic variation in salmon maturation timing. The analysis revealed that wild-type controls predominantly possessed the homozygous late-maturing LL genotype, whereas mutant fish carried higher frequencies of early-maturing E alleles. Accounting for this underlying genetic architecture using Firth’s penalized logistic regression demonstrated that the vgll3a locus exerted a dominant effect on spermiation, with early-allele carriers exhibiting significantly elevated odds of maturing compared to LL individuals.
After adjusting for the confounding distribution of vgll3a variants, the statistical association between bmp15 frameshift status and spermiation odds was attenuated, showing an odds ratio of 0.8 among early-allele carriers for wild-type relative to mutant fish and an interaction odds ratio of 0.84. Consequently, the study concluded that the heightened maturation rates observed in edited salmon were largely driven by background vgll3a variation rather than a direct causative acceleration induced by bmp15 deficiency alone. Terminal tissue sampling at 26 months confirmed that spermiating fish across all genetic groups exhibited dark skin coloration, soft abdomens, and fully developed testes containing free spermatozoa within the tubule lumens. Measurements of plasma 11-ketotestosterone, the principal circulating androgen in teleost fish, revealed no significant differences between mutant groups and wild-type controls across either immature or spermiating stages, indicating that testicular steroid hormone synthesis remained functionally intact regardless of bmp15 mutational status.
Detailed assessment of non-spermiating males sacrificed at 26 months revealed notable shifts in local testicular gene regulation. Furthermore, real-time quantitative PCR documented significantly reduced transcript levels for the Sertoli cell-derived regulatory factors amh and gsdf in immature frameshift testes, while levels of the germ cell-specific factors vasa and gdf9 remained unaltered. These molecular alterations suggest that the absence of germ cell-expressed Bmp15 alters communication between germ cells and adjacent somatic Sertoli cells. The down-regulation of inhibitory factors such as Amh, which normally suppresses spermatogonial proliferation and differentiation, aligns with the accelerated transition of spermatogonia into active mitotic division observed during earlier developmental windows.
To evaluate whether mutating the bmp15 gene impaired functional gamete quality, milt was harvested from spermiating males during two consecutive reproductive seasons at 36 and 48 months of age. Computer-assisted sperm analysis conducted at approximately 8.1 degrees Celsius examined motility parameters between 8 and 9 seconds following hypoosmotic activation in distilled water. The kinematic evaluations measured the proportion of motile spermatozoa, curvilinear velocity, straight-line velocity, average path velocity, linearity, wobble, and straightness. Across both sampling years, spermatozoa from bmp15 mutant males demonstrated vigorous movement and trajectory parameters that were statistically indistinguishable from wild-type controls. While high individual variability and occasional low-density milt samples were recorded in all cohorts, the absence of functional Bmp15 did not impede flagellar propulsion or kinetic trajectory. These findings contrast with observations in transgenic boars, where constitutive Bmp15 knockdown induced modest declines in sperm viability.
Beyond reproductive performance, full-body radiographic screenings of 26-month-old post-smolts identified an unexpected skeletal phenotype. The proportion of fish exhibiting at least one deformed vertebra or malformations in neural and haemal spines or ribs was significantly elevated in compound and homozygous frameshift mutants compared to wild-type controls. While several other transforming growth factor beta and bone morphogenetic protein family members possess well-documented osteogenic and skeletal remodeling functions, Bmp15 has not previously been linked directly to vertebrate bone biology. The authors noted that crosses using bmp15-deficient female gametes had previously resulted in severe embryonic mortality and structural malformations, suggesting that the observed spinal duplications and vertebral deformities may stem from early embryonic developmental disruptions or maternal effects rather than an ongoing defect in post-embryonic bone mineral homeostasis.
Collectively, the experimental results demonstrate that bone morphogenetic protein 15 is dispensable for male fertility and testicular maturation in Atlantic salmon. These outcomes indicate that bmp15 is not a viable solitary target for engineering male sterility in commercial salmon aquaculture facilities. Furthermore, the findings establish an evolutionary parallel with mammalian models such as the pig, demonstrating that while Bmp15 is essential for female fecundity, its biological role within the male gonad is remarkably limited across divergent vertebrate lineages.
Subject of Research: Investigation of bmp15 gene loss on testicular development and male fertility in Atlantic salmon
Article Title: Loss of bmp15 gene function does not abolish fertility in male Atlantic salmon
Article References: Crespo, D., Fjelldal, P. G., Hansen, T. J., Kjærner-Semb, E., Ayllon, F., Skaftnesmo, K. O., Thorsen, A., Norberg, B., Edvardsen, R. B., Andersson, E., Schulz, R. W., Wargelius, A., & Kleppe, L. (2026). Loss of bmp15 gene function does not abolish fertility in male Atlantic salmon. Frontiers in Genetics, 17, Article 1945517. https://doi.org/10.3389/fgene.2026.1945517
Image Credits: AI Generated
DOI: 10.3389/fgene.2026.1945517
Keywords: Atlantic salmon, CRISPR-Cas9, bmp15, spermatogenesis, spermiation, vgll3a, teleost reproduction, fish genetics, Loss, gene, function, does
