Myogenic progenitor cell (MPC) is responsible for postembryonic muscle growth and regeneration. Progranulin (PGRN) is a pluripotent growth factor that is correlated with neuromuscular disease, which is characterised by denervation, leading to muscle atrophy with an abnormal quantity and functional ability of MPC. However, the role of PGRN in MPC biology has yet to be elucidated. Here, we show that knockdown of zebrafish progranulin A (GrnA) resulted in a reduced number of MPC and impaired muscle growth. The decreased number of Pax7-positive MPCs could be restored by the ectopic expression of GrnA or MET. We further confirmed the requirement of GrnA in MPC activation during muscle regeneration by knockdown and transgenic line with muscle-specific overexpression of GrnA. In conclusion, we demonstrate a critical role for PGRN in the maintenance of MPC and suggest that muscle atrophy under PGRN loss may begin with MPC during postembryonic myogenesis.

Genetically modified (GM) fish with desirable features such as rapid growth, disease resistance, and cold tolerance, among other traits, have been established in aquaculture. However, commercially available GM fish are restricted because of global concerns over the incomplete assessments of food safety and ecological impact. The ecological impact concerns include gene flow and escape of the GM fish, which may cause extinction of wild natural fish stocks. Infertility control is a core technology for overcoming this obstacle. Although polyploidy technology, GnRH-specific antisense RNA, and RNAi against GnRH gene expression have been used to cause infertility in fish, these approaches are not 100% reliable and are not heritable. In the present study, zebrafish was used as a model to establish an inducible platform of infertility control in GM fish. Nitroreductase, which converts metronidazole substrate into cytotoxin, was fused with EGFP and expressed specifically by oocytes in the Tg(ZP:NTR-EGFP) by a zona pellucida promoter. Through consecutive immersion of metronidazole from 28 to 42 days posthatching, oocyte-specific EGFP expression was eliminated, and atrophy of the gonads was detected by anatomical analysis. These findings reveal that oocyte-specific nitroreductase-mediated catalysis of metronidazole blocks oogenesis and leads to an undeveloped oocyte. Furthermore, oocyte cell death via apoptosis was detected by a TUNEL assay. We found that the gonadal dysgenesis induced by metronidazole resulted in activation of the ovarian killer gene bok, which is a proapoptotic gene member of the Bcl-2 family and led to infertility. These results show that oocyte-specific nitroreductase-mediated catalysis of metronidazole can cause reliable infertility in zebrafish and could potentially be used as a model for other aquaculture fish species.

Multiple advantages-including the short generation time, large numbers of fertilized eggs, low cost of cultivation and easy maintenance favor the use of fish as bioreactors for the production of pharmaceutical proteins. In the present study, zebrafish eggs were used as bioreactors to produce mature tilapia insulin-like growth factors (IGFs) proteins using the oocyte-specific zona pellucida (zp3) promoter. The chimeric expression plasmids, pT2-ZP-tIGFs-IRES-hrGFP, in which hrGFP was used as reporter of tilapia IGFs expression, were designed to established Tg (ZP:tIGFs:hrGFP) transgenic lines for the expression of tilapia IGF-1 and IGF-2. Recombinant tilapia IGF-1 and IGF-2 were expressed as soluble forms in cytoplasm of fertilized eggs. The content level of tilapia IGF-1 and IGF-2 were 6.5 and 5.0% of the soluble protein, respectively. Using a simple Ni-NTA affinity chromatography purification process, 0.58 and 0.49 mg of purified tilapia IGF-1 and IGF-2 were obtained, respectively, from 650 fertilized eggs. The biological activity of the purified tilapia IGF-1 and IGF-2 was confirmed via a colorimetric bioassay to monitor the growth stimulation of zebrafish embryonic cells (ZF4), tilapia ovary cells (TO-2) and human osteosarcoma epithelial cells (U2OS). These results demonstrate that the use of zebrafish eggs as bioreactors is a promising approach for the production of biological recombinant proteins.