Analysis of absolute amounts of two meiotic cohesin subunits, RAD21L and REC8, in mouse spermatocytes

RAD2lL and REC8, meiosis-specific paralogs of the canonical cohesin subunit RAD21, are essential for proper formation of axial/lateral elements of the synaptonemal complex, synapsis of homologous chromosomes, and crossover recombination in mammalian meiosis. However, how many meiotic cohesins are present in germ cells has not been investigated because of the lack of an appropriate method of analysis. In the present study, to examine the intracellular amount of meiotic cohesins, we generated two strains of knock-in (KI) mice that expressed a 3xFLAG-tag at the C-terminus of RAD21L or REC8 protein using the CRISPR/Cas9 genome editing system. Both KI mice were fertile. Western blot analyses and immunocytochemical studies revealed that expression levels and localization patterns of both RAD21L-3xFLAG and REC8-3xFLAG in KI mice were similar to those in wild-type mice. After confirming that tagging of endogenous RAD21L and REC8 with 3xFLAG did not affect their expression profiles, we evaluated the levels of RAD21L-3xFLAG and REC8-3xFLAG in the testes of 2-week-old mice in which only RAD21L and REC8 but little RAD21 are expressed in the meiocytes. By comparing the band intensities of testicular RAD21L-3xFLAG and REC8-3xFLAG with 3xFLAG-tagged recombinant proteins of known concentrations in western blot analysis, we found that there were approximately 413,000 RAD21L and 453,000 REC8 molecules per spermatocyte in the early stages of prophase I. These findings provide new insights into the role played by cohesins in the process of meiotic chromosome organization in mammalian germ cells.

Automated summary

This study investigated the expression and localization patterns of RAD21L and REC8 proteins in mouse germ cells using a CRISPR/Cas9 genome editing system. It was found that the expression levels and localization patterns of RAD21L-3xFLAG and REC8-3xFLAG in the knock-in mice were similar to those in wild-type mice. Furthermore, the study revealed that there were approximately 413,000 RAD21L and 453,000 REC8 molecules per spermatocyte in the early stages of prophase I.