A possible function for P21 CDKN1A in a mid-pachytene/XY body checkpoint in mouse spermatocytes – short report ISSN 2753-8176 (online), DOI:10.13140/RG.2.2.10079.94880

A possible function for P21 CDKN1A in a mid-pachytene/XY body checkpoint in mouse spermatocytes – short report ISSN 2753-8176 (online), DOI:10.13140/RG.2.2.10079.94880 Ana Pedro1 1Gwyntwr1386 Healthcare CIC, Regus Chester Business Park, Heronsway, Chester, CH49QR, UK Keywords: P21 CDKN1A, mid-pachytene checkpoint, XY body 1. Corresponding author: anapedrolaboratories@gmail.com. It was suggested the existence of a mid-pachytene checkpoint where this checkpoint could be activated at the earliest at stage IV (1). On a previous publication (2), we found that the cyclin dependent kinase inhibitor P21 CDKN1A might be involved in a mid-pachytene checkpoint among other checkpoints in the mouse seminiferous epithelium. Mice deficient for Atm are male sterile with arrest and apoptosis occurring at testis epithelial stage IV, which in normal spermatocytes corresponds to mid-pachynema (3). In Atm/p53 or Atm/p21 double mutants, assembly of Rad51 foci on axial elements is defective, and gametogenesis proceeds only to pachytene of prophase I (4). Here we provide more evidence for P21 CDKN1A involvement on a mid-pachytene checkpoint by analysing mouse spermatocyte spreads. As we previously found (2), during zygotene and the zygotene-pachytene transition (Z-P), γ-H2AX starts decreasing while P21 raises to a peak of expression at Z-P, when γ-H2AX only appears covering the forming X-Y body. Moreover, during pachytene as γ-H2AX continues to fade, P21 shows another peak of expression where it is supposed it works a mid-pachytene checkpoint monitoring those pachytene spermatocytes with meiotic recombination defects (3,4). γ-H2AX totally disappears during diplotene (2). Rad51 is a conserved eukaryotic protein that catalyses the HR repair of DNA DSBs that occur during mitosis and meiosis (5). Rad51 as an essential role in the maintenance of spermatogonia as well as meiotic progression in mice (6). We found that accordingly to our previous descriptions (2) (fig.1), Rad51 might be located during early pachytene along the axes of meiotic chromosomes including the XY chromosomes, repairing DNA DSBs which occurred during meiotic recombination. Moreover, as we previously described (2), while γ-H2AX in early pachytene is fading from the meiotic chromosome axes, P21 at this stage appears fade too and showing some correlation with the distribution of γ-H2AX at meiotic chromosome axes (fig.1). However, at mid-pachytene while γ-H2AX concentrates at the XY body, P21 expression achieves a peak (fig.1). This suggests P21 might be monitoring the repair of DSBs during meiotic recombination and gets to a low level in early pachytene when all DSBs are repaired, to achieve another peak at mid-pachytene to monitor DSB repair and synapsis completion in the XY body, letting the meiotic cell cycle progress as XY body DSBs are also repaired (7). Further investigation into this will be needed. Materials and methods This study was performed within the scope of Ana Pedro’s PhD Thesis approved on the 07/07/2009 by the Dean of University of Valladolid, Spain. Spermatocyte spreadings Spermatocyte spreadings were prepared as described in 8. P21 CDKN1A, γ-H2AX and Rad51 staining We used for double immunofluorescence we used mouse anti-γ-H2AX, 1:2000 (Upstate, #05-636), rabbit anti-γ-H2AX (1:100, Trevigen, #4411-PC-020), P21 CDKN1A Neomarkers, ab-9, #RB-032-P0, 1:150), Santa Cruz Biotechnology, Inc., mouse anti-Rad51 (1:30, Neomarkers, # MS-988-P0), anti-mouse Alexa Fluor ® 488, 1:1500 (Molecular Probes, # A-21202), anti-rabbit Alexa Fluor ® 594, 1:1000 (Molecular Probes # A-21207), antimouse Alexa Fluor ® 594 (Molecular Probes, # A-21203) References 1. Ashley et al (2004). Correlation of meiotic events in testis sections and microspreads of mouse spermatocytes relative to the mid-pachytene checkpoint. Chromosoma, 113, 126-136 2. Pedro A (2021). Possible functions of P21 CDKN1A within the germ cells of the mouse seminiferous epithelium. Preprints. doi: 10.20944/preprints202104.0558.v1 3. Ashley et al (2004).The mammalian mid-pachytene checkpoint: meiotic arrest in spermatocytes with a mutation in Atm alone or in combination with a Trp53 (p53) or Cdkn1a (p21/cip1) mutation. Cytogenet Genome Res; 107 (3-4):256-62 4. Barlow et a.(1997). Partial rescue of the prophase I defects of Atm-deficient mice by p53 and p21 null alleles. Nat. Genet.17(4):462-6. 5. Cloud et al. (2012). Rad51 is an accessory factor for Dmc1-mediated joint molecule formation during meiosis. Science;337:1222–5 6. Qin et al (2022).RAD51 is essential for spermatogenesis and male fertility in mice. Cell Death Discovery (2022) 8:118 7. Abe et al. 2020. The Initiation of Meiotic Sex Chromosome Inactivation Sequesters DNA Damage Signaling from Autosomes in Mouse Spermatogenesis. Current Biology.Volume 30, Issue 3,3 February 2020, Pages 408-420.e5 8. Barchi et al (2005). Surveillance of different recombination defects in mouse spermatocytes yields distinct responses despite elimination at an identical developmental stage.Mol Cell Biol;25:7203–7215.