KCTD19 and its associated protein ZFP541 are independently essential for meiosis in male mice

Abstract

Meiosis is a cell division process with complex chromosome events where various molecules must work in tandem. To find meiosis-related genes, we screened evolutionarily conserved and reproductive tract-enriched genes using the CRISPR/Cas9 system and identified potassium channel tetramerization domain containing 19 (Kctd19) as an essential factor for meiosis. In prophase I, Kctd19 deficiency did not affect synapsis or the DNA damage response, and chiasma structures were also observed in metaphase I spermatocytes of Kctd19 KO mice. However, spermatocytes underwent apoptotic elimination during the metaphase-anaphase transition. We were able to rescue the Kctd19 KO phenotype with an epitope-tagged Kctd19 transgene. By immunoprecipitation-mass spectrometry, we confirmed the association of KCTD19 with zinc finger protein 541 (ZFP541) and histone deacetylase 1 (HDAC1). Phenotyping of Zfp541 KO spermatocytes demonstrated XY chromosome asynapsis and recurrent DNA damage in the late pachytene stage, leading to apoptosis. In summary, our study reveals that KCTD19 associates with ZFP541 and HDAC1, and that both KCTD19 and ZFP541 are essential for meiosis in male mice.

Trim41 is required to regulate chromosome axis protein dynamics and meiosis in male mice

“…A monoclonal antibody against TRIM41 was produced as previously described [ 16 ]. The DNA encoding mouse TRIM41 (residue 35–85 aa, NP_663352.2) was inserted into pGEX6p-1 (GE healthcare), and the expression vector was transformed into E .…”

“…Spread nuclei from spermatocytes were prepared as previously described [ 15 , 16 ]. Seminiferous tubules were unraveled using forceps in ice-cold DMEM (11995065, Thermo Fisher Scientific) and incubated in 1 mg/mL collagenase (C5138, Sigma-Aldrich) in DMEM (20 mL) at 37°C for 15 min.…”

“…For cytological analysis of metaphase/anaphase I cells, seminiferous tubule squashes were performed as previously described [ 16 , 57 ]. Briefly, seminiferous tubules were incubated in fix/lysis solution [0.1% Triton X-100, 0.8% PFA in PBS] at room temperature for 5 min.…”

“…The molecules involved in these complex events are not fully characterized yet, especially in mammals, due to difficulties in culturing and genetically manipulating spermatogenic cells in vitro . Thus, physiological screening with KO mice has been a powerful strategy to identify meiosis-related factors [ 14 – 16 ].…”

Mysteries and unsolved problems of mammalian fertilization and related topics

“…Of course, erasure and re-establishment of proper sex-specific genomic imprinting in these cells must take place simultaneously. Recently, Hirosawa-Takeda et al [ 368 ] and Oura et al [ 369 ] found that the zinc-finger protein (ZFP541) gene is involved in the initiation of meiosis of male germ cells. Whether activation of such genes in somatic cells induces meiosis is of academic interest.…”