Immunofluorescent microscopic study of meiosis in zebrafish.

In recent years, zebrafish (Danio rerio) has been used as a model vertebrate organism for studies of human disease, development, and genetics. This chapter describes detailed methods for the preparation of whole-mount meiotic oocytes and spermatocytes as well as cryostat sectioning of ovaries and testes for immunofluorescence microscopy studies of zebrafish.

Initiation and resolution of interhomolog connections: crossover and non-crossover sites along mouse synaptonemal complexes.

Programmed double-strand breaks at prophase of meiosis acquire immunologically detectable RAD51-DMC1 foci or early nodules (ENs) that are associated with developing chromosome core segments; each focus is surrounded by a gammaH2AX-modified chromosome domain. The 250-300 ENs per nucleus decline in numbers during the development of full-length cores and the remaining foci are relatively evenly distributed along the mature cores (gamma distribution of nu=2.97). The ENs become transformed nodules (TNs) by the acquisition of RPA, BLM, MSH4 and topoisomerases that function in repair and Holliday junction resolution. At the leptotene-zygotene transition, TNs orient to positions between the aligned cores where they initiate structural interhomolog contacts prior to synaptonemal complex (SC) formation, possibly future crossover sites. Subsequently, TNs are associated with SC extension at the synaptic forks. Dephosphorylation of TN-associated histone gammaH2AX chromatin suggests annealing of single strands or repair of double-strand breaks DSBs at this time. Some 200 TNs per pachytene nucleus are distributed proportional to SC length and are evenly distributed along the SCs (nu= approximately 4). At this stage, gammaH2AX-modified chromatin domains are associated with transcriptionally silenced sex chromosomes and autosomal sites. Immunogold electron microscope evidence shows that one or two TNs of the 10-15 TNs per SC acquire MLH1 protein, the hallmark of reciprocal recombination, whereas the TNs that do not acquire MLH1 protein relocate from their positions along the midline of the SCs to the periphery of the SCs. Relocation of TNs may be associated with the conversion of potential crossovers into non-crossovers.