In recent years, a strong demand for new breeding technologies in aquaculture has emerged. In addition to chromosome set manipulation techniques, micromanipulation technologies have been used and are being developed to improve strains. The technique of microinjection is one of the most powerful tools. It is used to analyze the function of a gene by overexpression (e.g. transgenic and ectopic mRNA overexpression) or gene silencing1 in basic studies, and to generate transgenic fish,2,3 nuclear transplanted fish4 and germline chimeric fish5 in practical studies.
The microinjection method is very simple and efficient, however, there is a serious problem in that the eggs of teleosts have a chorion that hardens with time after fertilization. In many teleosts, the hardened chorion is too hard and elastic to be penetrated by a glass needle for injection. It is necessary in the microinjection method for DNA or RNA to be injected during early embryogenesis, in many cases before the first cleavage. Therefore, dechorionation and prevention of chorion hardening have been studied in many teleosts. It has been reported that some digestive enzymes are effective for dechorionation of some cyprinid fish,6–8 and an alkaline solution containing glutathione prevents the hardening of the chorion in medaka and salmon.9,10 Other methods for marine fish such as melting the surface of the chorion using sodium hypo-chlorite solution (NaOCl)11 are known. For these methods to be carried out, the eggs have to be fertilized artificially, or naturally fertilized eggs have to be collected immediately after being spawned. However, it is still difficult to control spawning in many industrial marine fish such as the Pacific bluefin tuna Thunnus orientalis, which is one of the most popular and an economically important fish eaten as a luxury food in Japan. The bluefin tuna eggs rise to the sea surface after they are spawned, and the chorion of the eggs becomes too hard to insert a needle for microinjection by the first cleavage. Neither chorion removal with enzymes nor the digestion of the surface of the chorion with NaOCl occurred satisfactorily in the authors preliminary experiments (data not shown). Therefore, other methods for dechorionation or preventing the hardening of the chorion are necessary for industrial marine fish.
In this study, the authors used naturally spawned eggs of Pacific bluefin tuna, cultured in floating net cages at the Amami Experiment Station, Fisheries Laboratory of Kinki University. The cages were located near the Keten coast in the Strait of Oshima; therefore, it was possible to transport the eggs to the laboratory within 15 min after they were collected. Spawned eggs were captured as soon as possible after rising to the sea surface, and 50% of the eggs were put in Mg2+/Ca2+-free Ringer's solution for marine fish (1.2% NaCl, 0.06% KCl) containing 0.2% urea (MCU solution), while the remaining 50% of the eggs were kept in sea water (control). The eggs were incubated at atmospheric temperature during transportation, and at 26°C after they reached the laboratory. Some of the eggs in the MCU solution were transferred to sea water at the 2-, 4-, 8- and 16-cell stages, and the survival rate was estimated at 2 h post spawning (hps: 64- to 128-cell stages), 5 hps (early gastrula period) and 12 hps (8–10 somite period).
Under the same conditions, the authors attempted to inject green fluorescent protein (GFP)-nos1-3′UTR mRNA into the blastodisc during the 1-cell stage (before approx. 50 min post spawning [mps]). The mRNA includes the open reading frame of GFP fused to the 3′ untranslated region (UTR) of nos1, constructed for the visualization of primordial germ cell (PGC).12 It is known in several teleosts that this mRNA is localized to the area where some determinants of germ cells are also localized, during early embryogenesis, and maintains the potential to transcribe to a fluorescent protein only in PGC. Therefore, it is expected that it is the index whether this mRNA fragment accurately injected a blastodisc during early cleavage stages.
As a result, the chorion in MCU solution did not harden, and the glass needle penetrated easily until the 8-cell stage (approx. 1.25 hps), though that of untreated eggs (control) were already too hard to be penetrated by the needle at the 2-cell stage (approx. 35 mps).
All embryos incubated in MCU solution showed abnormal development or were dead before the blastula stage (Fig. 1). Of the eggs transferred to sea water at the 2-cell stage, 84% (31/37) developed normally until 12 hps; results showed that later the time of transfer, the lower the survival rate. None of the eggs transferred at the 16-cell stage (1.5 hps) survived to 5 hps (Fig. 2a). The survival rates of injected eggs were only slightly lower than those of uninjected eggs under each condition (Fig. 2b). Of the embryos that were injected with GFP-nos1-3′UTR mRNA and transferred at the 2-cell stage, 66% (25/38) developed normally until 12 hps, and 32% (8/25) of the embryos showed clear GFP fluorescence, Also, strong fluorescent cells were observed, located near both sides of the embryonic body, where the position of PGC had been reported in other teleosts13–17(Fig. 3b, arrowhead). The injected embryos transferred at the 4-cell stage did not survive up to 12 hps (0/66). Therefore, it was considered that the method in this study only enabled the use of microinjection if procedures and transfer to sea water were accomplished by the 2-cell stage.
The results in this study suggest that the MCU solution prevents hardening of the chorion, but may cause damage to the embryo simultaneously. It is considered that the effect of injection on the embryos in the MCU solution was small (Fig. 2b), therefore it is difficult to believe that MCU solution can cause embryos direct damage. For these reasons, it is thought that the abnormal development of embryos was caused by chorion damaged. Therefore, it is necessary to observe a structure of a chorion following treatment of MCU solution, moreover, observation of the influence of the MCU solution it is required to the later stages, at least to the hatching stage.
It was possible for gene introduction to eggs of naturally- spawned fish, as some embryos injected with GFP-nos1-3′UTR mRNA showed the GFP fluorescence. However, it has not been made clear in this study whether the bright cells were PGC, but the possibility that injected mRNA in this method was localized to the same position as is shown in other animals. The results suggest that this method can be applied to other marine fish that spawn floating eggs, considering the results of experiments not shown here using the floating eggs of other fish.
In conclusion, it has become possible to prevent the hardening of the chorion of naturally spawned eggs of marine fish and to culture them. Hereafter, it will be important to examine detailed conditions in other fish, and to develop a method for obtaining a high yield even by microinjection.