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International Journal of Molecular... Feb 2022Maturing male germ cells undergo a unique developmental process in spermiogenesis that replaces nucleosomal histones with protamines, the process of which is critical...
Maturing male germ cells undergo a unique developmental process in spermiogenesis that replaces nucleosomal histones with protamines, the process of which is critical for testicular development and male fertility. The progress of this exchange is regulated by complex mechanisms that are not well understood. Now, with mouse genetic models, we show that barrier-to-autointegration factor-like protein (BAF-L) plays an important role in spermiogenesis and spermatozoal function. BAF-L is a male germ cell marker, whose expression is highly associated with the maturation of male germ cells. The genetic deletion of BAF-L in mice impairs the progress of spermiogenesis and thus male fertility. This effect on male fertility is a consequence of the disturbed homeostasis of histones and protamines in maturing male germ cells, in which the interactions between BAF-L and histones/protamines are implicated. Finally, we show that reduced testicular expression of BAF-L represents a risk factor of human male infertility.
Topics: Animals; Biomarkers; Gene Expression Regulation, Developmental; Germ Cells; Histones; Humans; Infertility, Male; Intracellular Signaling Peptides and Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nuclear Proteins; Protamines; Spermatids; Spermatogenesis; Testis
PubMed: 35216101
DOI: 10.3390/ijms23041985 -
Journal of Controlled Release :... Apr 2023Proteins and peptides often require frequent needle-based administrations. Here, we report a non-parenteral delivery method for proteins through physical mixing with...
Proteins and peptides often require frequent needle-based administrations. Here, we report a non-parenteral delivery method for proteins through physical mixing with protamine, an FDA-approved peptide. Protamine was shown to promote tubulation and rearrangement of cellular actin, leading to enhanced intracellular delivery of proteins compared to poly(arginine) (R8). While the R8-mediated delivery resulted in significant lysosomal accumulation of the cargo, protamine directed the proteins to the nuclei with little lysosomal uptake. Intranasal delivery of insulin mixed with protamine effectively reduced blood glucose levels in diabetic mice 0.5 h after administration and the effect lasted for ∼6 h, comparable to subcutaneously injected insulin at the same dose. In mice, protamine was shown to overcome mucosal and epithelial barriers and modulate adherens junctions, promoting insulin penetration to the lamina propria layer for systemic absorption.
Topics: Mice; Animals; Protamines; Diabetes Mellitus, Experimental; Insulin; Cell-Penetrating Peptides
PubMed: 36878318
DOI: 10.1016/j.jconrel.2023.03.002 -
Journal of Cardiac Surgery Nov 2022The objective of this study is to evaluate protamine sulfate effects on graft's blood flow by comparing transit-time flow measurement (TTFM) values before and after... (Observational Study)
Observational Study
OBJECTIVE
The objective of this study is to evaluate protamine sulfate effects on graft's blood flow by comparing transit-time flow measurement (TTFM) values before and after protamine administration.
METHODS
This is an observational study with data collected between years 2018 and 2020. Immediate graft patency was evaluated using TTFM. Only patients with TTFM parameters registered before and after protamine infusion were included. The main three parameters studied were: mean graft flow (MGF), pulsatility index (PI), and diastolic flow (DF). In the first analysis, all conduits were evaluated regardless of the surgical technique used. In a second analysis, on-pump and off-pump groups were compared. Evaluated grafts were left internal thoracic artery, saphenous vein graft (SVG), radial artery, and right internal thoracic artery. Since SVG was numerically the most used graft, an exclusive analysis was created.
RESULTS
Our study included 575 patients, resulting in a total of 1686 grafts, mean 2.93 grafts/patient. Off-pump surgery was performed in 158 patients. Before protamine infusion, inadequate TTFM parameters were observed in 3.8% of grafts. Overall, after protamine administration, MGF decreased in all grafts, but its reduction was not statistically significant. PI values increased in the SVG and DF values reduced in LIMA grafts. SVG group analysis showed that after protamine PI values were higher in OM1 and RCA. DF values increased in RCA. The comparison between off and on-pump surgeries, showed that in off-pump cases TTFM measures did not present statistically significant differences.
CONCLUSION
Significant variations were observed in TTFM values before and after protamine administration. Although different, those values remained within the normal reference ranges. We recommend that flow measurement should be performed before protamine infusion.
Topics: Blood Flow Velocity; Coronary Artery Bypass; Coronary Circulation; Humans; Mammary Arteries; Protamines; Vascular Patency
PubMed: 36116058
DOI: 10.1111/jocs.16948 -
EJHaem Feb 2022In this prospective, single-centre observational study of 30 patients undergoing cardiopulmonary bypass (CPB), the effect of unfractionated heparin (UFH), CPB surgery...
In this prospective, single-centre observational study of 30 patients undergoing cardiopulmonary bypass (CPB), the effect of unfractionated heparin (UFH), CPB surgery and protamine sulphate on complement and on post-operative blood loss were assessed. Although C3 and C4 levels decreased significantly immediately following the administration of UFH, C3a, C5a, Bb fragment and SC5b-9 remained unchanged. During CPB, C3 and C4 continued to fall whilst both alternative and classical pathways activation markers, Bb, C3a, C5a and SC5b-9 increased significantly. Protamine sulphate had no effect on classical pathway components or activation markers but decreased alternative pathway activation marker Bb. Over the 12-24 h post-surgery, both classical and alternative pathway activation markers returned to baseline, whilst C3 and C4 levels increased significantly but not to baseline values. Total drain volume 24 h after the surgery showed a moderate inverse correlation with post-protamine C3 ( = -0.46, = 0.01) and C4 ( = -0.57, = 0.0009) levels, whilst a moderate positive correlation was observed with post-protamine C3a ( = 0.46, = 0.009), C5a ( = 0.37, = 0.04) and SC5b-9 ( = 0.56, = 0.001) levels but not with Bb fragment ( = 0.25, = 0.17). Thus, inhibition of complement activation may be a therapeutic intervention to reduce post-operative blood in patients undergoing CPB.
PubMed: 35846208
DOI: 10.1002/jha2.371 -
Gene Jul 2019The functional sperm is the key factor for species continuation. The process spermatogenesis, to produce mature sperm is quite complex. It begins with the proliferation... (Review)
Review
The functional sperm is the key factor for species continuation. The process spermatogenesis, to produce mature sperm is quite complex. It begins with the proliferation and differentiation of spermatogonia, which develop from primary spermatocytes to secondary spermatocytes and round spermatids, which eventually develop into fertile mature sperm. Spermiogenesis is the latest stage of spermatogenesis, where the round spermatids undergo a series of dramatic morphological changes and extreme condensation of chromatin to construct mature sperm with species-specific shape. During spermiogenesis, chromatin remodeling is a unique progress. It leads the nucleosome from a histone-based structure to a mostly protamine-based configuration. The main events of chromatin remodeling are the replacement of histone by histone variants, hyperacetylation, transient DNA strand breaks and repair, variants by transition proteins and finally by protamines. In this review, we synthesize and summarize the current knowledge on the progress of chromatin remodeling during spermiogenesis. We straighten out the chronological order of chromatin remodeling and illustrate the possible regulation mechanisms of each step.
Topics: Animals; Chromatin; Chromatin Assembly and Disassembly; DNA; Histones; Humans; Male; Sperm Maturation; Spermatids; Spermatocytes; Spermatogenesis; Spermatozoa
PubMed: 31085275
DOI: 10.1016/j.gene.2019.05.027 -
Development (Cambridge, England) May 2023Unique chromatin remodeling factors orchestrate dramatic changes in nuclear morphology during differentiation of the mature sperm head. A crucial step in this process is...
Unique chromatin remodeling factors orchestrate dramatic changes in nuclear morphology during differentiation of the mature sperm head. A crucial step in this process is histone-to-protamine exchange, which must be executed correctly to avoid sperm DNA damage, embryonic lethality and male sterility. Here, we define an essential role for the histone methyltransferase DOT1L in the histone-to-protamine transition. We show that DOT1L is abundantly expressed in mouse meiotic and postmeiotic germ cells, and that methylation of histone H3 lysine 79 (H3K79), the modification catalyzed by DOT1L, is enriched in developing spermatids in the initial stages of histone replacement. Elongating spermatids lacking DOT1L fail to fully replace histones and exhibit aberrant protamine recruitment, resulting in deformed sperm heads and male sterility. Loss of DOT1L results in transcriptional dysregulation coinciding with the onset of histone replacement and affecting genes required for histone-to-protamine exchange. DOT1L also deposits H3K79me2 and promotes accumulation of elongating RNA Polymerase II at the testis-specific bromodomain gene Brdt. Together, our results indicate that DOT1L is an important mediator of transcription during spermatid differentiation and an indispensable regulator of male fertility.
Topics: Animals; Male; Mice; Cell Differentiation; Chromatin Assembly and Disassembly; Histone-Lysine N-Methyltransferase; Histones; Protamines; Semen; Spermatids
PubMed: 37082969
DOI: 10.1242/dev.201497 -
Frontiers in Cell and Developmental... 2023Protamines (PRM1 and PRM2) are small, arginine-rich, nuclear proteins that replace histones in the final stages of spermiogenesis, ensuring chromatin compaction and...
Protamines (PRM1 and PRM2) are small, arginine-rich, nuclear proteins that replace histones in the final stages of spermiogenesis, ensuring chromatin compaction and nuclear remodeling. Defects in protamination lead to increased DNA fragmentation and reduced male fertility. Since efficient sperm production requires the translocation of protamines from the cytoplasm to the nucleus, we investigated whether SPAG17, a protein crucial for intracellular protein trafficking during spermiogenesis, participates in protamine transport. Initially, we assessed the protein-protein interaction between SPAG17 and protamines using proximity ligation assays, revealing a significant interaction originating in the cytoplasm and persisting within the nucleus. Subsequently, immunoprecipitation and mass spectrometry (IP/MS) assays validated this initial observation. Sperm and spermatids from knockout mice exhibited abnormal protamination, as revealed by chromomycin A3 staining, suggesting defects in protamine content. However, no differences were observed in the expression of and mRNA or in protein levels between testes of wild-type and knockout mice. Conversely, immunofluorescence studies conducted on isolated mouse spermatids unveiled reduced nuclear/cytoplasm ratios of protamines in knockout spermatids compared to wild-type controls, implying transport defects of protamines into the spermatid nucleus. In alignment with these findings, experiments involving somatic cells, including mouse embryonic fibroblasts, exhibited compromised nuclear translocation of PRM1 and PRM2 in the absence of SPAG17. Collectively, our results present compelling evidence that SPAG17 facilitates the transport of protamines from the cytoplasm to the nucleus.
PubMed: 37766963
DOI: 10.3389/fcell.2023.1125096 -
Supportive Care in Cancer : Official... Oct 2022Cancer patients have an increased risk of bleeding compared to non-cancer patients with anticoagulant therapy. A bleeding risk assessment before initiation of...
Cancer patients have an increased risk of bleeding compared to non-cancer patients with anticoagulant therapy. A bleeding risk assessment before initiation of anticoagulation is recommended. Currently low molecular weight heparin (LMWH) and direct oral anticoagulants (DOACs) are the mainstays of treatment for cancer-associated venous thromboembolism (VTE). Since DOACs are administered orally, they offer some convenience and ease of administration; however, LMWH may be preferred in certain cancers. Given the prevalence of anticoagulant therapies in cancer patients, clinical providers must be able to recognize potentially critical bleeding sites and modalities to reverse major hemorrhage. Reversal agents or antidotes to bleeding may be required when bleeding is persistent or life-threatening. These include vitamin K, fresh frozen plasma (FFP), protamine, prothrombin complex concentrate (PCC) or andexanet alfa, and idarucizumab. Inferior vena cava (IVC) filter insertion can be also considered in those with major bleeding. Evidence for timing and need for re-initiation of anticoagulant therapy after a major bleeding remains sparse, but a multi-disciplinary approach and shared decision-making can be implemented in the interim.
Topics: Administration, Oral; Anticoagulants; Antidotes; Hemorrhage; Heparin, Low-Molecular-Weight; Humans; Neoplasms; Protamines; Vitamin K
PubMed: 35579752
DOI: 10.1007/s00520-022-07136-w -
Biophysical Journal Jun 2021DNA looping plays an important role in cells in both regulating and protecting the genome. Often, studies of looping focus on looping by prokaryotic transcription...
DNA looping plays an important role in cells in both regulating and protecting the genome. Often, studies of looping focus on looping by prokaryotic transcription factors like lac repressor or by structural maintenance of chromosomes proteins such as condensin. Here, however, we are interested in a different looping method whereby condensing agents (charge ≥+3) such as protamine proteins neutralize the DNA, causing it to form loops and toroids. We considered two previously proposed mechanisms for DNA looping by protamine. In the first mechanism, protamine stabilizes spontaneous DNA fluctuations, forming randomly distributed loops along the DNA. In the second mechanism, protamine binds and bends the DNA to form a loop, creating a distribution of loops that is biased by protamine binding. To differentiate between these mechanisms, we imaged both spontaneous and protamine-induced loops on short-length (≤1 μm) DNA fragments using atomic force microscopy. We then compared the spatial distribution of the loops to several model distributions. A random looping model, which describes the mechanism of spontaneous DNA folding, fit the distribution of spontaneous loops, but it did not fit the distribution of protamine-induced loops. Specifically, it failed to predict a peak in the spatial distribution of loops at an intermediate location along the DNA. An electrostatic multibinding model, which was created to mimic the bind-and-bend mechanism of protamine, was a better fit of the distribution of protamine-induced loops. In this model, multiple protamines bind to the DNA electrostatically within a particular region along the DNA to coordinate the formation of a loop. We speculate that these findings will impact our understanding of protamine's in vivo role for looping DNA into toroids and the mechanism of DNA condensation by condensing agents more broadly.
Topics: Chromosomes; DNA; Lac Repressors; Nucleic Acid Conformation; Protamines
PubMed: 34023297
DOI: 10.1016/j.bpj.2021.04.022 -
BioRxiv : the Preprint Server For... May 2024There is a well-established link between abnormal sperm chromatin states and poor motility, however, how these two processes are interdependent is unknown. Here, we...
There is a well-established link between abnormal sperm chromatin states and poor motility, however, how these two processes are interdependent is unknown. Here, we identified a possible mechanistic insight by showing that Protamine 2, a nuclear DNA packaging protein in sperm, directly interacts with cytoskeletal protein Septin 12, which is associated with sperm motility. Septin 12 has several isoforms, and we show, that in the sperm, the short one (Mw 36 kDa) is mislocalized, while two long isoforms (Mw 40 and 41 kDa) are unexpectedly lost in sperm chromatin-bound protein fractions. Septin 12 co-immunoprecipitated with Protamine 2 in the testicular cell lysate of WT mice and with Lamin B1/B2/B3 in co-transfected HEK cells despite we did not observe changes in Lamin B2/B3 protein or SUN4 expression in testes. Furthermore, the sperm have on average a smaller sperm nucleus and aberrant acrosome biogenesis. In humans, patients with low sperm motility (asthenozoospermia) have imbalanced histone- protamine 1/2 ratio and modified levels of cytoskeletal proteins. We detected retained Septin 12 isoforms (Mw 40 and 41 kDa) in the sperm membrane, chromatin-bound and tubulin/mitochondria protein fractions, which was not true for healthy normozoospermic men. In conclusion, our findings expand the current knowledge regarding the connection between Protamine 2 and Septin 12 expression and localization, resulting in low sperm motility and morphological abnormalities.
PubMed: 38854089
DOI: 10.1101/2024.05.28.596175