The effects of leucine treatment on zebrafish sperm motility the activation of PI3K/Akt signaling pathway. (A) Representative immunoblots showed p-Akt and Akt proteins after leucine treatment. Actin was used as the control. Relative protein level was shown from 3 independent experiments. (B) Quantification of the ratio of pAkt/Akt from 3 independent experiments. (C) The mRNA expression of PI3KR1 and PI3KCA were checked by qRT-PCR. (D) Comparisons of semen kinematic parameters between control group and leucine-treated group. Two kinematics parameters including progressive sperm and slow sperm were assessed with the computer-assisted sperm analysis (CASA) system. (E) Representative immunofluorescence pictures to detect mTOR and p-mTOR signals in control and leucine-treated zebrafish testes. Scale bar: 50 μm. Bars represent mean ± SEM of more than three separate experiments. ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001.

The effects of leucine treatment on autophagy in zebrafish testis. (A) The mRNA expression of autophagy related genes atg5, atg7, atg12 and p62 were evaluated by qRT-PCR. (B) Representative immunoblots showed P62 and Lc3 proteins after leucine and LY294002 administration. Actin was used as the control. Relative protein levels of P62 and Lc3-II were shown from 3 independent experiments. (C) Representative immunofluorescence pictures to detect Lc3 levels in control and leucine-treated zebrafish testes. Scale bar: 50 μm. Bars represent mean ± SEM of more than three separate experiments. ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001.