Recombination study by MLH1 immunostaining of mouse spermatocytes after dietary treatments

Meiotic recombination is a critical process for sexually reproducing organisms. This exchange of genetic information between homologous chromosomes during meiosis is important not only because it generates genetic diversity, but also because it is often required for proper chromosome segregation. Consequently, the frequency and distribution of crossovers are tightly controlled to ensure fertility and offspring viability. However, in many systems it has been shown that environmental factors can alter the frequency of crossover events. We have explored for the first time the effect of dietary changes on crossover frequency per nucleus. Our study was performed in spermatocytes of 3 mouse inbred strains by analyzing the number and position of crossovers along the synaptonemal complexes, as well as the length of such synaptonemal complexes, by immunostaining with antibodies against MLH1 (which allows the identification of the crossover sites) and SYCP3 (a component of the synaptonemal complex). Our results show that male recombination rate is sensitive to dietary changes, and this sensitivity depends on the genetic background in mice. This is first to report a nutrition effect on genome-wide levels of recombination.

Methods
We performed two studies: in the initial one, adult males from the three strains were analyzed for the effect of two diets on recombination (undernourishment (reduction to 50% daily intake) and breeding diets (Teklad Global 18% Protein Rodent Diet)) provided during 24 days relative to a control group kept ad libitum with maintenance diet (Teklad Global 14% Protein Rodent Maintenance Diet). After the 24-day diet period, adult male mice were euthanized by cervical dislocation and weighed. After removing and weighing the testes, chromosome spreads for immunostaining as previously described (Anderson et al. 1999; de Boer et al. 2009; Milano et al. 2019). MLH1 immunostaining allows for identification of about 90% of mammalian crossover sites (Anderson et al. 1999; Cole et al. 2012). All slides were imaged on a Zeiss LSM 710 confocal microscope and analyzed using Zeiss Zen lite software. Only mid and mid-late pachytene stage spermatocytes were scored. For each spermatocyte, we counted the number of foci localizing to the SC of the 19 autosomes (Anderson et al. 1999); total SC length and interfocus distances were also measured in autosomes only.