Zebrafish mutagenesis: The isolation and characterization of reproductive mutations in the adult zebrafish

Experimental approaches using random mutagenesis have been classically used for studying development, and have been employed for several large screens on zebrafish. However, mutagenesis has not generally been used for studying physiological processes, especially in adults. We have used a chemical mutagenesis scheme to recover mutations affecting ovarian and testicular maturation and gametogenesis in the adult zebrafish. Point mutations were induced in adult male AB zebrafish using N-ethyl-N-nitrosourea (ENU). A three-generation (F2) crossing scheme was used to recover recessive mutations in the F3 generation. At sexual maturity (3 months), whole body cross-sections from families of the F3 generation were mass embedded in paraffin blocks, sectioned at 7?m, and stained with hematoxylin and eosin. A screen employing light microscopy was used to visualize alterations in gametogenesis or gonadal structure. A total of 125 mutagenized genomes in 81 families were analyzed, and 9 gonadal mutations resulting in distinct phenotypes were confirmed to be germline mutations. Male mutations included testes with greatly reduced sperm and large numbers of spermatogonia, as well as testes with spermatogenesis arrested at the spermatocyte stage of development. Female mutations included ovaries containing degenerating oocytes surrounded by hypertrophied follicle walls and/or stromal proliferation, ovaries with large numbers of resorbing oocytes (pre- and post-ovulatory oocytes), and ovaries containing abnormal post-ovulatory follicles. Mutations were further characterized using different histological and cytochemical methods to understand the types of tissues and cells present in the mutant gonads, as well as how those tissues differed when compared to wild-type fish. A number of reproductively related candidate genes were isolated from zebrafish. These candidate genes were selected based on their published roles in various cells and tissues, and were isolated using several techniques, including library screening and reverse-transcriptase polymerase chain reaction (RT PCR). Candidate genes were then characterized in wild-type fish by examining tissue expression patterns with RT PCR, as well as Northern analysis in some instances. Finally, one of the original goals was to map individual mutations to linkage groups. Although mapping experiments were never successful due to the low numbers of mutant individuals, attempts were made to map several of the reproductive mutations.