Synchronization of meiosis in male mice

The first wave of spermatogonia entry into meiosis initiates at 8 days postpartum (8 dpp). Then, spermatocytes progress to meiotic prophase and reach the leptotene, zygotene and pachytene stages at approximately 11, 13 and 15 dpp, respectively. Hence, the proportion of cells at leptotene/zygotene is 55%, 41% and 26% at these three ages, respectively (Goetz et al., 1984). To obtain a more enriched proportion of leptotene/zygotene spermatocytes, germ cell development was synchronized in vivo by manipulating the retinoic acid metabolism, as described in Romer et al., 2018. Briefly, at day two post-partum, mice were treated daily (by pipette feeding) with WIN 18,446 (100µg/gram of body weight), an inhibitor of retinoic acid synthesis that blocks the differentiation of spermatogonia and thus meiosis entry (Hogarth et al., 2013). After 8 to 10 days of treatment, meiosis was initiated synchronously by a single intraperitoneal injection of 100 µg of retinoic acid in 10 µL of DMSO. Between 8 and 9 days after the injection, mice were sacrificed and testes were harvested. At this time point, about 80–85% of spermatocytes were at leptotene/zygotene stage, as assessed by SYCP3, SYCP1 and γH2AFX staining on spermatocyte spreads performed using a small proportion of testis tissue. The remaining testis tissue was processed for nuclei purification and FACS sorting.