Introduction
Early puberty is a major problem in farmed Atlantic salmon males as it stunts growth and entails welfare problems due to the maturation-associated loss of osmoregulation capacity in seawater. A better understanding of the regulation of puberty is the basis for developing improved rearing protocols to avoid these problems. As puberty onset is controlled by activation of the brain-pituitary-gonad (BPG) axis, our aim here was to study if puberty is initiated when the gene encoding the follicle-stimulating hormone receptor (fshr) is rendered non functional. It is known that lack of fshr in male mice is associated with smaller testis and a reduced Sertoli cell number, but the mutants are still fertile. On the contrary, both medaka and zebrafish fshr KO males show no clear phenotype regarding testis size and fertility. Apparently normal spermatogenesis and fertility in these model fish may be related to Lh receptor-activation and the ensuing androgen production stimulating testis maturation. In salmon, this may be different since in other salmonids Fsh, like in mammals, cannot activate the Lh receptor and since Lh is usually not secreted until the spawning period. We therefore expected a phenotype different from fshr KO zebrafish and medaka males.
Material and methods
We made fshr mutants using CRISPR-Cas9 technology. In view of the long generation time, we first studied highly mutated F0 generation fshr crispants. fshr crispants and control males were reared in a common garden, and precocious maturation was induced by exposing the one-year old postsmolts to continuous light and 16º C water temperature for a period of three months. Samplings (testis, pituitary and plasma) took place 1, 2, 5 and 9 months post induction. For the F1 generation, the sampling times were 2, 7 and 11 months post induction.
Results
At the first sampling (1 month), all males displayed low GSI values and no effect of the fshr KO could be detected on plasma androgen levels or stage of spermatogenesis. However, in the samples collected during the last two samplings, we observed slightly (5 months) and clearly (9 months) lower GSI values in fshr KO mutants compared to control. The results in F0 crispants were variable, but we observed initially a delay in the start of maturation. The males then went through maturation but displayed a shorter cycle compared to control males. Later analysis suggested that the weak and variable phenotype in F0 crispants may be related to presence of in-frame mutations, which may leave some of the mutated protein at least partially functional. Most mutants reached maturity and we crossed four highly mutated fish (2 of each sex), to create an F1 generation. At one-year of age, we pit-tagged and genotyped 200 F1 fish for sex and fshr genotyping and were able to identify sibling fish having either wild-type, fshr-/- or fshr +/- genotypes with known mutations. When stimulating pubertal maturation in F1 fish, clear effects were observed. None of the fshr-/- (N=10) males entered puberty while 22% of the fshr+/- males (N=72) and 85% of the wild-type males (N=24) entered maturation.
Conclusion
We show that Atlantic salmon males do not enter puberty when lacking fshr, which is dissimilar to findings in zebrafish and medaka, but similar to findings in mouse.
Keywords: Atlantic salmon, follicle-stimulating hormone receptor, CRISPR-Cas9 technology, testis maturation