Identification of germ cells in large yellow croaker (Larimichthys crocea) and yellow drum (Nibea albiflora) using RT-PCR and in situ hybridization analyses.

Ocean-caught large yellow croaker (Larimichthys crocea) represents an important germplasm resource for the breeding of this species; however, these fish tend to show poor survival in captivity and would be unsuitable breeding purposes. As an alternative to the use of wild-caught croakers, germ cell transplantation has been proposed using the L. crocea specimens as donors and yellow drum (Nibea albiflora) as recipients. In this regard, the identification of L. crocea and N. albiflora germ cells is an essential prerequisite for establishing a germ cell transplantation protocol for these fish. In this study, we cloned the 3' untranslated regions (UTR) of the vasa, dnd, and nanos2 genes in N. albiflora using the rapid amplification of cDNA ends (RACE) method and then aligned and analyzed the sequences of the corresponding genes in L. crocea and N. albiflora. On the basis of gene sequence differences, we designed species-specific primers and probes for RT-PCR analysis and in situ hybridization. RT-PCR analysis revealed that these species-specific primers exclusively amplified DNA from gonads of the respective species, thus confirming that we had six specific primer pairs that could be used to distinguish the germ cells in L. crocea and N. albiflora. Using in situ hybridization analysis, we established that whereas Lcvasa and Nadnd probes showed high species specificity, the probes for Navasa and Lcdnd showed a less specificity. In situ hybridization using Lcvasa and Nadnd thus enabled us to visualize the germ cells in these two species. Using these species-specific primers and probes, we can reliably distinguish the germ cells of L. crocea and N. albiflora, thereby establishing an effective approach for the post-transplantation identification of germ cells when using L. crocea and N. albiflora as donors and recipients, respectively.