Loss of Prm1 leads to defective chromatin protamination, impaired PRM2 processing, reduced sperm motility and subfertility in male mice

Abstract

One of the key events during spermiogenesis is the hypercondensation of chromatin by substitution of the majority of histones by protamines. In humans and mice, protamine 1 (PRM1/Prm1) and protamine 2 (PRM2/Prm2), are expressed in a species-specific ratio. Using CRISPR-Cas9-mediated gene editing we generated Prm1-deficient mice and demonstrate, that Prm1+/- mice are subfertile while Prm1-/- are infertile. Prm1-deficiency was associated with higher levels of 8-OHdG, an indicator for reactive oxygen mediated DNA-damage. While Prm1+/- males displayed moderate increased levels of 8-OHdG virtually all sperm of Prm1-/- males displayed ROS mediated DNA damage. Consequently, DNA integrity was slightly hampered in Prm1+/-, while DNA was completely fragmented in Prm1-/- animals. Interestingly CMA3 staining which indicates protamine-free DNA revealed, that Prm1+/- sperm displayed high levels (93%), compared to Prm2+/- (29%) and WT (2%) sperm. This is not due to increased histone retention as demonstrated by mass spectrometry (MassSpec) of nuclear proteins in Prm1+/- sperm. Further analysis of the MassSpec data from sperm nuclear proteome revealed, that only one protein (RPL31) is significantly higher abundant in Prm1+/- compared to WT sperm. Comparison of the proteome from Prm1-/- and Prm2-/- to WT suggested, that there are a small number of proteins which differ in abundance. However, their function was not linked mechanistically to primary defects seen in Prm1-/- mice and rather represent a general stress response. Interestingly, using acid urea gels we found that sperm from Prm1+/- and Prm1-/- mice contain a high level of unprocessed, full-length PRM2. Prm2 is transcribed as a precursor protein which, upon binding to DNA is successively processed. Further, the overall ratio of PRM1:PRM2 is skewed from 1:2 in WT to 1:5 in Prm1+/- animals. Our results reveal that Prm1 is required for proper processing of PRM2 to produce the mature PRM2 which, together with Prm1 is able to hypercondense DNA. Hence, the species specific PRM1:PRM2 ratio has to be precisely controlled in order to retain full fertility.

Actl7b-deficiency leads to mislocalization of LC8 type dynein light chains and disruption of murine spermatogenesis

“…Sections of Bouin-fixed testis were processed and stained with Hemalum solution acid (Henricks and Mayer) and Eosin Y solution (Carl Roth, Karlsruhe, Germany) as described (Merges et al, 2022).…”

“…Staining was performed as described (Merges et al, 2022) using 50 μl of sperm swim-out and 50 μl Eosin-Nigrosin stain (0.67 g Eosin Y (color index 45380), 0.9 g sodium chloride, 10 g Nigrosin (color index 50420), 100 ml ddH 2 O). 200 sperm per animal were analyzed.…”

“…Data for proteins detected in all genotypes and replicates were with more than 2 peptides were log2 transformed and median normalized. Abundances were analyzed as described before (Merges et al, 2022). The Bioconductor package proDA (Ahlmann-Eltze and Anders, 2021) was used (peptide spectrum match (PSM)-level data extracted from Protein Discoverer).…”

“…Testis tissue was prepared as described (Merges et al, 2022). In brief, the tissue was fixed in 3% glutaraldehyde at 4°C overnight, washed, post-fixed with 2 % osmium tetroxide at 4°C for 2 h and again washed.…”

“…Samples for sequencing were prepared as described (Merges et al, 2022) and sent to GATC/Eurofins (Cologne, Germany) for sequencing.…”