Sperm lacking Protamine 2 fail to support normal fertilization and embryogenesis in mouse
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE300618 | GEO | 2026/04/22
REPOSITORIES: GEO
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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:Protamines are the safeguards of the paternal sperm genome. They replace most of the histones during spermiogenesis, resulting in DNA hypercondensation, thereby protecting its genome from environmental noxa. Impaired protamination has been linked to male infertility in mice and humans in many studies. Apart from impaired DNA integrity, protamine-deficient human and murine sperm show multiple secondary effects, including decreased motility and aberrant head morphology. In this study, we use a Prm2-deficient mouse model in combination with label-free quantitative proteomics to decipher the underlying molecular processes of these effects. We show that loss of the sperm`s antioxidant capacity, indicated by downregulation of key proteins like SOD1 and PRDX5, ultimately initiates an oxidative stress-mediated destruction cascade during epididymal sperm maturation. This is confirmed by an increased level of 8-OHdG in epididymal sperm, a biomarker for oxidative stress-mediated DNA damage. Prm2-deficient testicular sperm are not affected and initiate the proper development of blastocyst stage preimplantation embryos in vitro upon intracytoplasmic sperm injection (ICSI) into oocytes. Our results provide new insight into the role of Prm2 and its downstream molecular effects on sperm function and present an important contribution to the investigation of new treatment regimens for infertile men with impaired protamination.
2020-07-29 | GSE149454 | GEO
Project description: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 together with PRM1, mediates chromatin-hypercondensation. The removal of the cP2 domain is believed to be imperative for proper chromatin condensation, yet, the role of cP2 is not yet understood. We generated mice lacking the cP2 domain while the mP2 is still expressed. We show that the cP2 domain is indispensable for complete sperm chromatin protamination and male mouse fertility. cP2 deficient sperm show incomplete PRM2 incorporation, resulting in a severely altered protamine ratio, retention of transition proteins and aberrant retention of the testis specific histone variant H2A.L.2. During epididymal transit, cP2 deficient sperm seem to undergo ROS mediated degradation leading to complete DNA fragmentation. The cP2 domain therefore seems to be a key aspect in the complex crosstalk between histones, transition proteins and protamines during sperm chromatin condensation. 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.
2022-04-06 | GSE200052 | GEO
Project description: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-/- and Prm2-/- sperm show high levels of reactive oxygen species (ROS)-mediated DNA damages and increased histone retention. In contrast, Prm1+/- sperm display only moderate DNA damages. The majority of Prm1+/- sperm were CMA3 positive indicating protamine-free DNA. This is not due to increased histone retention in Prm1+/- sperm. Additionally, we found that sperm from Prm1+/- and Prm1-/- mice contain high levels of incompletely processed PRM2. Further, the PRM1:PRM2 ratio is skewed from 1:2 in WT to 1:5 in Prm1+/- animals. Our results reveal that PRM1 is required for proper PRM2 processing 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.
2022-04-06 | GSE200050 | GEO
Project description:Animal facilities often have surplus unused male mice. To reduce unnecessary mouse sacrifices, we proposed innocuously preselecting sex sperm using CRISPR/Cas9. Specifically, we aimed to significantly decrease the number of copies of the multicopy Ssty2 gene family (repeated over 220 times on the Y chromosome), which plays a pivotal role in regulating X and Y gene expression during sperm differentiation. Transgenic lines were created by microinjecting Cas9 RNA and specific Ssty2 sgRNAs into B6CBAF1 zygotes. Pup genotyping of Ssty2 copies was conducted via qPCR. Epididymal sperm samples were obtained from 2 to 6-month KO and WT males, and after a swim-out, sperm motility was evaluated using the Integrated Semen Analysis System. Motile sperm were categorized by K-means clustering of CASA kinetic parameters. To uncover the impact of Ssty2 depletion on sex-ratio segregation, we selected a line with a substantial decrease in Ssty2 copies, to fewer than 50. Consequently, 70 to 80% of pups were female upon in vivo fertilization, remaining consistent in IVF, but with a normal sex ratio in ICSI. However, we noted a reduction in litter size, declining from an average of 8.5 to 6.4 pups per litter. Testicle size in KO mice exhibited a slight enlargement, with a minor decrease in sperm concentration compared to controls (non-significant). However, over 80% of KO sperm displayed abnormal morphology, showing head, neck, and midpiece defects. Sperm motility comparison revealed a shift, with 60.6% of KO sperm static vs. 35.9% in WT controls, displaying KO sperm as slow and non-progressive motility patterns. In conclusion, by targeting the Ssty2 multicopy gene, we produced CRISPR-mediated KO mice that presented preferential fertilization of sperm carrying the X chromosome via in vivo and IVF.
2025-12-22 | GSE278278 | GEO
Project description:Spermatozoa have a unique genome organization: their chromatin is almost completely devoid of histones and is formed instead of protamines which confer a high level of compaction and preserve paternal genome integrity until fertilization. Histone-to-protamine transition takes place in spermatids and is indispensable for the production of functional sperm. Here we show that the H3K79-methyltransferase DOT1L controls spermatid chromatin remodelling and subsequent reorganization and compaction of spermatozoon genome. Using a mouse model in which Dot1l is knocked-out (KO) in postnatal male germ cells, we found that Dot1l-KO sperm chromatin is less compact and has an abnormal content, characterized by the presence of transition proteins, immature protamine 2 forms and a higher level of histones. Proteomics and transcriptomics analyses performed on spermatids reveal that Dot1l-KO modifies the chromatin prior to histone removal, and leads to the deregulation of genes involved in flagellum formation and apoptosis during spermatid differentiation. As a consequence of these chromatin and gene expression defects, Dot1l-KO spermatozoa have less compact heads and are less motile, which results in impaired fertility.
2023-03-30 | PXD030734 | Pride
Project description:<p>The epididymis is the primary organ responsible for sperm maturation, with its caudal region serving as the primary storage site for mature sperm. The unique small molecule metabolites present in the cauda epididymal fluid not only facilitate the long-term storage of sperm but also ensure their structural and functional integrity prior to fertilization. Consequently, an in-depth investigation into the composition and functions of these metabolites in the cauda epididymal fluid is essential for elucidating the regulatory mechanisms governing sperm maturation and fertilization. Yaks, as livestock unique to the Qinghai-Tibet Plateau, hold significant scientific and economic value. Compared to cattle, male yaks exhibit poorer reproductive performance, characterized by reduced sperm motility, elevated sperm abnormality rate, and lower artificial insemination conception rates. To investigate the relationship between sperm motility defects in yaks and the microenvironment during their maturation, this study collected cauda epididymal fluid from yaks and cattle for untargeted metabolomic sequencing. The results indicated that 1,098 and 1,297 kinds of metabolites annotated by the Human Metabolome Database (HMDB) database were identified in yak and cattle cauda epididymal fluid, respectively, using positive and negative ion modes. In both modes, the three categories containing the most types of metabolites were organic acids and derivatives, organoheterocyclic compounds, and lipids and lipid-like molecules. Within the lipid metabolites, fatty acids and conjugates were the most prevalent in both modes. In the positive ion mode, compared to cattle cauda epididymal fluid, 79 metabolites were significantly upregulated and 212 were significantly downregulated in yak cauda epididymal fluid. In the negative ion mode, compared to cattle cauda epididymal fluid, 110 metabolites were significantly upregulated and 230 metabolites were significantly downregulated in yak cauda epididymal fluid. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment results showed that the most significantly enriched pathway in the positive ion mode was the biosynthesis of amino acids, while in the negative ion mode, it was ABC transporters. Among the significant differentially expressed metabolites, 18 have been associated with sperm mobility, maturation or function. The functions of these metabolites involve regulation of oxidative stress, sperm capacitation, spontaneous acrosome reaction, mitochondrial energy metabolism, mRNA methylation, and flagellar motility. Taken together, these findings establish a foundational dataset and offer novel perspectives for elucidating the molecular mechanisms underlying yak sperm maturation, enhancing sperm motility, optimizing reproductive techniques, and ultimately improving reproductive performance.</p>
2025-09-29 | MTBLS13067 | MetaboLights
Project description:Protamines play a crucial role in nuclear condensation during spermiogenesis, a process associated with significant chromatin remodeling and replacement of histones by protamines. While much research has focused on the function of protamines in sperm development and fertility, their effects in non-sperm cells remain largely unexplored. In this study, we investigated the impact of overexpressing murine and human protamine 1 and 2 (PRM1 and PRM2) on nuclear architecture, histone eviction, DNA methylation, and transcription in HEK293T cells and mesenchymal stromal cells (MSCs). Overexpression of protamines resulted in nuclear condensation and particularly PRM1 showed notable enrichment in nucleoli and cycle abnormalities. Immunofluorescence staining indicated a significant reduction in specific histone modifications (H3K9me3, H3K4me1, and H3K27Ac) in response to protamine expression, especially in MSCs. Interestingly, despite these changes in nuclear organization, the methylome remained largely stable upon protamine expression. However, particularly expression of protamines significantly diminished transcription, especially of the ribosomal genes upon PRM1 expression. Our studies indicate that PRM1 and PRM2 condense distinct genomic regions in somatic cells, resulting in widespread silencing of gene expression.
2025-12-02 | GSE297024 | GEO
Project description:Protamines play a crucial role in nuclear condensation during spermiogenesis, a process associated with significant chromatin remodeling and replacement of histones by protamines. While much research has focused on the function of protamines in sperm development and fertility, their effects in non-sperm cells remain largely unexplored. In this study, we investigated the impact of overexpressing murine and human protamine 1 and 2 (PRM1 and PRM2) on nuclear architecture, histone eviction, DNA methylation, and transcription in HEK293T cells and mesenchymal stromal cells (MSCs). Overexpression of protamines resulted in nuclear condensation and particularly PRM1 showed notable enrichment in nucleoli and cycle abnormalities. Immunofluorescence staining indicated a significant reduction in specific histone modifications (H3K9me3, H3K4me1, and H3K27Ac) in response to protamine expression, especially in MSCs. Interestingly, despite these changes in nuclear organization, the methylome remained largely stable upon protamine expression. However, particularly expression of protamines significantly diminished transcription, especially of the ribosomal genes upon PRM1 expression. Our studies indicate that PRM1 and PRM2 condense distinct genomic regions in somatic cells, resulting in widespread silencing of gene expression.
2025-12-02 | GSE296730 | GEO
Project description:Various behavioural abnormalities and congenital anomalies were observed in the F2 offspring of ICSI (Kanatsu-Shinohara et al., 2023, J. Clin.). Invest. 133(22): e170140). The data presented here aim to clarify the cause of these abnormalities. Gene expression in the ICSI-F1/F2 placenta was investigated using RNA sequencing (RNA-seq). ICSI was performed using C57BL/6 epididymal sperm and oocytes. The ICSI-F2 generation was produced using the sperm of the ICSI-F1 generation via in vitro fertilisation. Placentae recovered by caesarean section at E19.5 were examined for gene expression. Each group contains four biological replicates.
2026-04-21 | GSE300654 | GEO