Loss of the cleaved-protamine 2 domain leads to incomplete histone-to-protamine exchange and infertility in mice

Abstract

Protamines are unique sperm-specific proteins that package and protect paternal chromatin until fertilization. A subset of mammalian species expresses two protamines (PRM1 and PRM2), while in others PRM1 is sufficient for sperm chromatin packaging. Alterations of the species-specific ratio between PRM1 and PRM2 are associated with infertility. Unlike PRM1, PRM2 is generated as a precursor protein consisting of a highly conserved N-terminal domain, termed cleaved PRM2 (cP2), which is consecutively trimmed off during chromatin condensation. The carboxyterminal part, called mature PRM2 (mP2), interacts with DNA and mediates chromatin hyper-condensation, together with PRM1. The removal of the cP2 domain is believed to be imperative for proper chromatin condensation and the prevention of DNA damage. Yet, the role of cP2 is not yet understood. Using CRISPR-Cas9 mediated gene editing, we generated mice lacking the cP2 domain while the mP2 is still expressed. We show that deletion of one allele of the cP2 domain is sufficient to render male mice infertile. cP2 deficient sperm show incomplete PRM2 incorporation, retention of transition proteins and a severely altered protamine ratio. During epididymal transit, cP2 deficient sperm seem to undergo ROS mediated degradation leading to complete DNA fragmentation, inviability and immotility of mature sperm. The cP2 domain therefore seems to be necessary for the complex crosstalk leading to the successive and complete removal of transition proteins and complete protamination of sperm chromatin. Overall, we present the first step towards understanding the role of the cP2 domain in paternal chromatin packaging and open up avenues for further research.

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

“…Defects in PRM2 processing were also described for histone variant H2A.L.2-KO, transition protein (TPs)-1 and -2 (TP1/TP2)-double KO, TP2-KO and cleaved PRM2 cP2-KO mouse models [40,[56][57][58], all of which display fertility problems. Interestingly, a recent study showed that mutation of a single non-arginine residue in PRM1 (P1 K49A/K49A ) leads to impaired PRM2 processing in mice [59].…”

“…Arévalo et al have shown that mice lacking the highly conserved N-terminal part of PRM2, called cleaved PRM2 (cP2), display defective PRM2 processing and show a PRM1:PRM2 ratio of approx. 5:1 [40]. Mice lacking cP2 on one allele are infertile.…”

“…Peptide preparation: Protein solutions (5.5 M urea, 20% 2-mercaptoethanol, 5% acetic acid) were dried in a vacuum concentrator and subjected to in solution preparation of peptides as described previously [40]. Briefly, cysteines were alkylated with acrylamide and digested with trypsin, followed by desalting.…”

“…Briefly, cysteines were alkylated with acrylamide and digested with trypsin, followed by desalting. LC-MS measurements were performed according to Arévalo et al [40]. Briefly, peptides were separated on a self-packed reversed-phase column within a 90 min gradient.…”

The Art of Packaging the Sperm Genome: Molecular and Structural Basis of the Histone-To-Protamine Exchange

“…P1 is expressed in its mature form, while P2 is initially expressed as a longer precursor (pro-P2) and undergoes selective proteolytic processing to produce its mature form (P2) once bound to DNA ( 88 , 89 ). Truncation of the amino terminus of P2, the portion of the protein that is typically cleaved (cP2) in the nucleus, causes infertility due to inefficient import of the protein into the nucleus, resulting in altered protamine ratios and immotile sperm; suggesting that the longer isoform may be required for protamine-chaperone interactions ( 90 ).…”