Mouse sperm protamine mass spectrometry
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Mus Musculus (mouse)
TISSUE(S): Sperm
SUBMITTER: 
Lindsay Moritz
LAB HEAD: Sue Hammoud
PROVIDER: PXD028917 | Pride | 2023-05-17
REPOSITORIES: Pride
Similar Datasets
Project description:Conventional dogma presumes that protamine-mediated DNA compaction in sperm is achieved by electrostatic interactions between DNA and the arginine-rich core of protamines. However, phylogenetic analysis reveals several non-arginine residues conserved within, but not across species. The significance of these residues or post-translational modifications are poorly understood. Here, we investigated the functional role of K49, a rodent-specific lysine residue in mouse protamine 1 (P1) that is acetylated early in spermiogenesis and retained in sperm. In vivo, alanine substitution (P1 K49A) results in loss of programmatic histone retention, decreased sperm motility, decreased male fertility, and in zygotes, premature P1 removal from paternal chromatin, altered DNA replication, and embryonic arrest. In vitro, P1 K49A decreases protamine-DNA binding and alters DNA compaction/decompaction kinetics. Hence, a single amino acid substitution outside the P1 arginine core is sufficient to profoundly alter protein function and developmental outcomes, suggesting that protamine non-arginine residues are essential for reproductive fitness.
2023-05-09 | GSE225270 | GEO
Project description:Conventional dogma presumes that protamine-mediated DNA compaction in sperm is achieved by electrostatic interactions between DNA and the arginine-rich core of protamines. However, phylogenetic analysis reveals several non-arginine residues conserved within, but not across species. The significance of these residues or post-translational modifications are poorly understood. Here, we investigated the functional role of K49, a rodent-specific lysine residue in mouse protamine 1 (P1) that is acetylated early in spermiogenesis and retained in sperm. In vivo, alanine substitution (P1 K49A) results in loss of programmatic histone retention, decreased sperm motility, decreased male fertility, and in zygotes, premature P1 removal from paternal chromatin, altered DNA replication, and embryonic arrest. In vitro, P1 K49A decreases protamine-DNA binding and alters DNA compaction/decompaction kinetics. Hence, a single amino acid substitution outside the P1 arginine core is sufficient to profoundly alter protein function and developmental outcomes, suggesting that protamine non-arginine residues are essential for reproductive fitness.
2023-05-09 | GSE225271 | GEO
Project description:Conventional dogma presumes that protamine-mediated DNA compaction in sperm is achieved by electrostatic interactions between DNA and the arginine-rich core of protamines. However, phylogenetic analysis reveals several non-arginine residues conserved within, but not across species. The significance of these residues or post-translational modifications are poorly understood. Here, we investigated the functional role of K49, a rodent-specific lysine residue in mouse protamine 1 (P1) that is acetylated early in spermiogenesis and retained in sperm. In vivo, alanine substitution (P1 K49A) results in loss of programmatic histone retention, decreased sperm motility, decreased male fertility, and in zygotes, premature P1 removal from paternal chromatin, altered DNA replication, and embryonic arrest. In vitro, P1 K49A decreases protamine-DNA binding and alters DNA compaction/decompaction kinetics. Hence, a single amino acid substitution outside the P1 arginine core is sufficient to profoundly alter protein function and developmental outcomes, suggesting that protamine non-arginine residues are essential for reproductive fitness.
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Project description:Protamines replace histones as the main nuclear protein in sperm cells having a crucial role in compacting the paternal genome. Human spermatozoa contain the protamine 1 (P1) and the family of protamine 2 (P2) proteins. Alterations in protamine PTMs or on the P1/P2 ratio could be associated with male infertility. Top-down proteomics enables large-scale analysis of intact proteforms derived from alternative splicing, missense or nonsense genetic variants or PTMs. In contrast to current gold standard techniques, top-down allows a more in-depth analysis of protamine PTMs and proteoforms, opening up new perspectives to unravel their impact on male fertility. We analyzed two normozoospermic semen samples by top-down and discussed in detail the difficulties we found in the data analysis and the suggested solutions, as this step is one of the current bottlenecks in top-down proteomics with currently available bioinformatic tools. Our strategy for the data analysis combines two different software, ProSight PD (PS) and TopPIC suite (TP), with a clustering algorithm to decipher protamine proteoforms. We identified up to 32 protamine proteoforms at different levels of characterization. This in-depth analysis of the protamine proteoform landscape of an individual boosts personalized diagnosis of male infertility.
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Project description:Protamine 1 (P1) and protamine 2 family (P2) are the most abundant nuclear basic spermspecific proteins, packing 85-95% of the paternal DNA. P1 is synthesized as a mature form, whereas P2 components (HP2, HP3 and HP4) arise from the proteolysis of the precursor (pre-P2). Due to the particular protamine physical-chemical properties, their identification by standardized bottom-up mass spectrometry (MS) strategies are not straightforward. Therefore, the aim of this study was to identify the sperm protamine proteoforms profile including P1 and P2 members and their post-translational modifications in normozoospermic individuals using two complementary strategies, a top-down MS approach and a proteinase K-digestion based bottom-up MS approach. By top-down MS approach, both described and new truncated P1 and pre-P2 proteoforms were identified. Likewise, intact P1, pre-P2, and P2 mature forms and their phosphorylation pattern were detected, as well as a +61 Da modification in different proteoforms. Additionally, the bottom-up MS approach revealed phosphorylated residues for pre-P2 and the new P2 isoform 2, which is not annotated at UniProtKB database. Implementing these strategies in comparative studies of different infertile phenotypes would permit to determine alterations in the protamine proteoforms pattern and elucidate the role of phosphorylation/dephosphorylation dynamics in male fertility.
2019-11-11 | PXD014618 | Pride
Project description:MTD project_description Protamines are testis-specific proteins, which replace the majority of histones during spermiogenesis. This results in a hypercondensation of the sperm DNA, finally causing a conformational change from a nucleosomal chromatin structure into a mainly toroid like structure. This is assumed to protect the paternal genome and to contribute to the formation of a hydrodynamic sperm head shape. In mice and humans, two protamines, Protamine 1 (Prm1) and Protamine 2 (Prm2), are expressed. Impaired sperm protamination has been repeatedly correlated with male subfertility in mice and humans. Apart from defects in DNA condensation and impaired DNA integrity, protamine-deficient human and murine sperm show multiple secondary defects, which cannot be explained by the chromosomal function of protamines. These include impaired sperm motility, decreased viability as well as acrsosomal malformations. In this study, we utilized a Prm2-deficient mouse model to investigate the underlying molecular mechanisms of these defects. Since trancription is silenced upon incorporation of protamines, we used LC-MS in combination with label-free quantification to compare the sperm proteome of Prm2-deficient (Prm2-/-) males with the proteome of wildtype (Prm2+/+) and Prm2 heterozygous (Prm2+/-) males. We show that Prm2 deficient mice display a strongly altered proteomic profile compared to controls. Of note, a significant downregulation of proteins SOD1 and PRDX5, which have a well-known function for the detoxification of reactive oxygen species was observed. Thus, together with a series of additional experiments, we could for the first time show that Prm2-deficiency triggers a reactive oxygen species (ROS) mediated sperm destruction cascade during epididymal sperm maturation, finally causing described secondary sperm defects.
2020-07-29 | PXD018843 | Pride
Project description:Spermatogenesis is a complex process of sperm generation, including mitosis, meiosis, and spermiogenesis. During spermiogenesis, histones in post-meiotic spermatids are removed from chromatin and replaced by protamines. Although histone-to-protamine exchange is important for sperm nuclear condensation, the underlying regulatory mechanism is still poorly understood. Here, we identify PHD finger protein 7 (PHF7) as an E3 ubiquitin ligase for histone H3K14 in post-meiotic spermatids. Generation of Phf7-deficient mice and Phf7 C160A knockin mice with impaired E3 ubiquitin ligase activity reveals defects in histone-to-protamine exchange caused by dysregulation of histone removal factor Bromodomain, testis-specific (BRDT) in early condensing spermatids. Surprisingly, E3 ubiquitin ligase activity of PHF7 on histone ubiquitination leads to stabilization of BRDT by attenuating ubiquitination of BRDT. Collectively, our findings identify PHF7 as a critical factor for sperm chromatin condensation and contribute to mechanistic understanding of fundamental phenomenon of histone-to-protamine exchange and potential for drug development for the male reproduction system.
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