Direct visualization of pre-protamine 2 detects protamine assembly failures
Objective: Visualisation of the unprocessed pre-PRM2 and evaluation of its abnormal accumulation as a sensor of histone-to-protamine transition disruption in infertile males. Design: Experimental study Setting: Université Grenoble Alpes (UGA France) and Royan Institute for Reproductive Biomedicine (Tehran, Iran) Animals: H2A.L.2-KO, TH2B-tag, Brdt-KO mouse models (UGA). Patients: 49 men undergoing intra cytoplasmic sperm injection and 3 fertile donors (Royan Institute). Interventions: (Non) Main outcome measures: Pre-Prm2 retention rate and its accumulation in mouse models with characterized failure in histone-to-protamine replacement, as well as in human sperm. Proportion of fertilized eggs (2PN) post-ICSI in couples undergoing infertility treatment. Results: Using mouse models with characterized failure in histone-to-protamine replacement, we show that pre-Prm2 retention is tightly linked to nucleosome disassembly. Additionally, in elongating/condensing spermatids, pre-Prm2 and transition protein (TP) are co-expressed spatiotemporally, and their physical interaction suggests that these proteins act simultaneously rather than successively during histone replacement. By using our anti-human pre-PRM2 antibody, we also measured pre-PRM2 retention rates in 49 men from a series of infertile couples undergoing ICSI, which shed new light on the debated relationship between pre-PRM2 retention and sperm parameters. Finally, by monitoring 2-pronuclei (2PN) embryo formation following Intra Cytoplasmic Sperm Injection (ICSI), we evaluated the fertilization ability of the sperm in these 49 patients. Conclusions: Our observations suggest that the extent of pre-PRM2 retention in sperm, rather than pre-PRM2 accumulation per se, is associated with fertilization failure. Furthermore, our data demonstrate that anti-pre-PRM2/pre-Prm2 antibodies are valuable tools which could be used in routine monitoring of sperm parameters in fertility clinics, as well as in experimental research programs to better understand the obscure process of histone-to-protamine transition. Key words: pre-PRM2 accumulation, histone-to-protamine transition, fertilization failure, 2PN formation.