Oral Presentation ESA-SRB 2023 in conjunction with ENSA

Super resolution analysis of meiotic recombination in murine spermatocytes (#153)

Lynette Mirembe 1 , Sarah Hilet 1 , Ashley Rozario 1 , Donna Whelan 1 , Cathryn A Hogarth 2
  1. Department of Rural Clinical Sciences, La Trobe Rural Health School, La Trobe University, Bendigo, Victoria, Australia
  2. Department of Rural Clinical Sciences, La Trobe Rural Health School, La Trobe University, Wodonga, Victoria, Australia

Meiosis is the key step in sexual reproduction driving genetic variability. Genetically unique haploid gametes arise because of meiotic recombination, the pairing of homologous chromosomes and switching of their genetic material. Errors in recombination result in chromosomal abnormalities that can lead to germ cell loss or unhealthy gametes.  Advances in microscopy and immunofluorescence imaging have been key in unravelling the molecular events that take place during recombination but molecular-level understanding of the precise distribution of key meiotic proteins during recombination remains poor. This study employed dSTORM super-resolution microscopy (SRM), which uses the single molecule localisation approach, to map key proteins at 10 to 20 nm spatial resolution, a 10-fold resolution increase in comparison to conventional fluorescence imaging. To capture high resolution snapshots of meiosis, SYCP3, γH2AX, MLH1 and RAD51 were indirectly immunolabeled in primary spermatocytes and SRM was performed on a customised inverted microscope equipped with high power continuous wave lasers and a sensitive sCMOS camera. SRM of SYCP3 revealed the double-helix structure of the lateral elements of the synaptonemal complex. Imaging of γH2AX and RAD51 identified significant cluster-like colocalization with the XY chromosomes during the pachytene stage, which are often unable to be detected using conventional fluorescence imaging. Single foci of MLH1 signal were also detected unilaterally within sister chromatids. This study is the first description of SRM mapping of meiotic recombination. Ultimately, the ability to visualise meiosis and recombination in high resolution will allow for an improved understanding of the effects of mutations and toxins on this essential process in germ cell development.