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Frontiers in Endocrinology 2022Male fertility throughout life hinges on the successful production of motile sperm, a developmental process that involves three coordinated transitions: mitosis,... (Review)
Review
Male fertility throughout life hinges on the successful production of motile sperm, a developmental process that involves three coordinated transitions: mitosis, meiosis, and spermiogenesis. Germ cells undergo both mitosis and meiosis to generate haploid round spermatids, in which histones bound to the male genome are replaced with small nuclear proteins known as protamines. During this transformation, the chromatin undergoes extensive remodeling to become highly compacted in the sperm head. Despite its central role in spermiogenesis and fertility, we lack a comprehensive understanding of the molecular mechanisms underlying the remodeling process, including which remodelers/chaperones are involved, and whether intermediate chromatin proteins function as discrete steps, or unite simultaneously to drive successful exchange. Furthermore, it remains largely unknown whether more nuanced interactions instructed by protamine post-translational modifications affect chromatin dynamics or gene expression in the early embryo. Here, we bring together past and more recent work to explore these topics and suggest future studies that will elevate our understanding of the molecular basis of the histone-to-protamine exchange and the underlying etiology of idiopathic male infertility.
Topics: Chromatin; Histones; Humans; Male; Protamines; Semen; Spermatozoa
PubMed: 35813619
DOI: 10.3389/fendo.2022.895502 -
PLoS Medicine Jun 2021The dose of protamine required following cardiopulmonary bypass (CPB) is often determined by the dose of heparin required pre-CPB, expressed as a fixed ratio. Dosing... (Randomized Controlled Trial)
Randomized Controlled Trial
BACKGROUND
The dose of protamine required following cardiopulmonary bypass (CPB) is often determined by the dose of heparin required pre-CPB, expressed as a fixed ratio. Dosing based on mathematical models of heparin clearance is postulated to improve protamine dosing precision and coagulation. We hypothesised that protamine dosing based on a 2-compartment model would improve thromboelastography (TEG) parameters and reduce the dose of protamine administered, relative to a fixed ratio.
METHODS AND FINDINGS
We undertook a 2-stage, adaptive randomised controlled trial, allocating 228 participants to receive protamine dosed according to a mathematical model of heparin clearance or a fixed ratio of 1 mg of protamine for every 100 IU of heparin required to establish anticoagulation pre-CPB. A planned, blinded interim analysis was undertaken after the recruitment of 50% of the study cohort. Following this, the randomisation ratio was adapted from 1:1 to 1:1.33 to increase recruitment to the superior arm while maintaining study power. At the conclusion of trial recruitment, we had randomised 121 patients to the intervention arm and 107 patients to the control arm. The primary endpoint was kaolin TEG r-time measured 3 minutes after protamine administration at the end of CPB. Secondary endpoints included ratio of kaolin TEG r-time pre-CPB to the same metric following protamine administration, requirement for allogeneic red cell transfusion, intercostal catheter drainage at 4 hours postoperatively, and the requirement for reoperation due to bleeding. The trial was listed on a clinical trial registry (ClinicalTrials.gov Identifier: NCT03532594). Participants were recruited between April 2018 and August 2019. Those in the intervention/model group had a shorter mean kaolin r-time (6.58 [SD 2.50] vs. 8.08 [SD 3.98] minutes; p = 0.0016) post-CPB. The post-protamine thromboelastogram of the model group was closer to pre-CPB parameters (median pre-CPB to post-protamine kaolin r-time ratio 0.96 [IQR 0.78-1.14] vs. 0.75 [IQR 0.57-0.99]; p < 0.001). We found no evidence of a difference in median mediastinal/pleural drainage at 4 hours postoperatively (140 [IQR 75-245] vs. 135 [IQR 94-222] mL; p = 0.85) or requirement (as a binary outcome) for packed red blood cell transfusion at 24 hours postoperatively (19 [15.8%] vs. 14 [13.1%] p = 0.69). Those in the model group had a lower median protamine dose (180 [IQR 160-210] vs. 280 [IQR 250-300] mg; p < 0.001). Important limitations of this study include an unblinded design and lack of generalisability to certain populations deliberately excluded from the study (specifically children, patients with a total body weight >120 kg, and patients requiring therapeutic hypothermia to <28°C).
CONCLUSIONS
Using a mathematical model to guide protamine dosing in patients following CPB improved TEG r-time and reduced the dose administered relative to a fixed ratio. No differences were detected in postoperative mediastinal/pleural drainage or red blood cell transfusion requirement in our cohort of low-risk patients.
TRIAL REGISTRATION
ClinicalTrials.gov Unique identifier NCT03532594.
Topics: Aged; Anticoagulants; Blood Coagulation; Cardiac Surgical Procedures; Cardiopulmonary Bypass; Drug Dosage Calculations; Drug Monitoring; England; Female; Heparin; Heparin Antagonists; Humans; Male; Middle Aged; Models, Biological; Protamines; Thrombelastography; Time Factors; Treatment Outcome; Victoria
PubMed: 34097705
DOI: 10.1371/journal.pmed.1003658 -
The Journal of Thoracic and... Oct 2015
Topics: Antibodies; Anticoagulants; Cardiopulmonary Bypass; Female; Heparin; Heparin Antagonists; Humans; Male; Platelet Activation; Protamines
PubMed: 26320779
DOI: 10.1016/j.jtcvs.2015.08.003 -
European Journal of Vascular and... Sep 2016The aim was to evaluate the safety and efficacy of heparin reversal with protamine after completion of carotid endarterectomy (CEA), summarising the available data from... (Meta-Analysis)
Meta-Analysis Review
OBJECTIVES
The aim was to evaluate the safety and efficacy of heparin reversal with protamine after completion of carotid endarterectomy (CEA), summarising the available data from both randomised and non-randomised studies.
METHODS
The study was a meta-analysis. Pooled odds ratios (ORs) with 95% confidence intervals (95% CIs) were calculated for the outcomes of stroke and wound haematoma among patients receiving or not receiving protamine after CEA. Meta-regression analysis was performed to examine whether the documented differences were modified by potentially meaningful patient related or procedure related predictors, namely publication year, general anesthesia used, number of patients treated, mean age (years), males, neurological symptoms, use of patch, and use of shunt.
RESULTS
Seven studies were included in the meta-analysis reporting on 3,817 patients receiving protamine after CEA and 6,070 patients not receiving protamine for heparin reversal. Only one study was randomised. A statistically significant reduction in wound haematoma requiring re-operation was recorded after heparin reversal with protamine in patients undergoing CEA (OR, 0.42, 95% CI, 0.22-0.80, p = .008). In contrast, no significant difference was observed in stroke rates between groups of patients that received and did not receive protamine (OR, 0.71, 95% CI, 0.49-1.03, p = .07). Meta-regression analysis did not reveal any significant effect mediated by the modifiers examined.
CONCLUSION
On the basis of the available data, heparin reversal with protamine seems to reduce the risk of wound haematoma, without increasing the risk of procedural stroke. However, taking into account the limitations of the analysis, further studies are needed to increase the level of evidence provided by the current meta-analysis.
Topics: Endarterectomy, Carotid; Heparin Antagonists; Humans; Postoperative Hemorrhage; Protamines; Stroke
PubMed: 27389942
DOI: 10.1016/j.ejvs.2016.05.033 -
Cancer Imaging : the Official... Jul 2023Percutaneous hepatic perfusion (PHP) is a palliative intraarterial therapy for unresectable hepatic malignancies. During PHP, high-dose melphalan is infused via the...
PURPOSE
Percutaneous hepatic perfusion (PHP) is a palliative intraarterial therapy for unresectable hepatic malignancies. During PHP, high-dose melphalan is infused via the hepatic artery to saturate tumor in the liver with the chemotherapeutic substance. The venous hepatic blood is filtered by an extracorporeal melphalan specific filtration system. Blood clotting in the extracorporeal filter system is prevented by administering unfractionated heparin (UFH) in high doses, which might be reversed with protamine sulfate after the procedure. Aim of this retrospective two-center-study was to analyze the potential effect of UFH reversal with protamine sulfate on complication rates following PHP.
MATERIALS AND METHODS
All patients receiving PHP treatment between 10/2014 and 04/2021 were classified according to their intraprocedural coagulation management: 92 patients/192 PHP received full UFH reversal with protamine (group); 13 patients/21 PHP in group received a reduced amount of protamine, and 28 patients/43 PHP did not receive UFH reversal with protamine (group). Periinterventional clinical reports, findings and laboratory values were retrospectively evaluated. Complications and adverse events were classified according to Common Terminology Criteria for Adverse Events (CTCAEv5.0).
RESULTS
Thromboembolic events were recorded after 10 PHP procedures (5%) in group, six of which (3%) were major events (CTCAE grade 3-5). No (0%) thromboembolic events were recorded in group and group. Hemorrhagic events were registered after 24 PHP (13%) in group two of which (1%) were major (CTCAE grade 3-4). In group, only minor bleeding events were recorded, and one major hemorrhagic event was documented in group (2%). There was a significant difference between the percentage of post-interventional thrombopenia in group (39%) and group (14%) versus group (23%) (p=.00024). In group one patient suffered from a severe anaphylactic shock after the administration of protamine.
CONCLUSION
Our retrospective study implies that there might be a link between the practice of protamine sulfate administration to reverse the full hemodilutive effect of UFH after PHP and the post-interventional risk of thromboembolic events as well as clinically significant thrombopenia. Our data suggest that the standard use of protamine sulfate after PHP in low-risk patients without clinical signs of active bleeding should be critically re-evaluated.
Topics: Humans; Heparin; Melphalan; Retrospective Studies; Protamines; Thrombocytopenia; Perfusion
PubMed: 37452405
DOI: 10.1186/s40644-023-00590-7 -
PLoS Genetics Jun 2022Protamines are unique sperm-specific proteins that package and protect paternal chromatin until fertilization. A subset of mammalian species expresses two protamines...
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 protamine incorporation and 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.
Topics: Animals; Chromatin; Histones; Humans; Infertility, Male; Male; Mammals; Mice; Protamines; Semen; Spermatozoa
PubMed: 35763544
DOI: 10.1371/journal.pgen.1010272 -
Human Gene Therapy Dec 2022Lentiviral transduction of human mesenchymal stem cells (MSCs) induces long-term transgene expression and holds great promise for multiple gene therapy applications....
Lentiviral transduction of human mesenchymal stem cells (MSCs) induces long-term transgene expression and holds great promise for multiple gene therapy applications. Polybrene is the most commonly used reagent to improve viral gene transfer efficiency in laboratory research; however, it is not approved for human use and has also been shown to impair MSC proliferation and differentiation. Therefore, there is a need for optimized transduction protocols that can also be adapted to clinical settings. LentiBOOST (LB) and protamine sulfate are alternative transduction enhancers (TEs) that can be manufactured to current Good Manufacturing Practice standards, are easily applied to existing protocols, and have been previously studied for the transduction of human CD34 hematopoietic stem cells. In this study, we investigated these reagents for the enhancement of lentiviral transduction of adipose-derived MSCs. We found that the combination of LB and protamine sulfate could yield comparable or even superior transduction efficiency to polybrene, with no dose-dependent adverse effects on cell viability or stem cell characteristics. This combination of TEs represents a valuable clinically compatible alternative to polybrene with the potential to significantly improve the efficiency of lentiviral transduction of MSCs for gene therapy applications.
Topics: Humans; Lentivirus; Transduction, Genetic; Hexadimethrine Bromide; Genetic Vectors; Mesenchymal Stem Cells; Cell Differentiation; Protamines
PubMed: 35859364
DOI: 10.1089/hum.2022.117 -
Drug Delivery Dec 2020Protamine is a natural cationic peptide mixture used as a drug for the neutralization of heparin and in formulations of slow-release insulin. In addition, Protamine can...
Protamine is a natural cationic peptide mixture used as a drug for the neutralization of heparin and in formulations of slow-release insulin. In addition, Protamine can be used for the stabilization and delivery of nucleic acids (antisense, small interfering RNA (siRNA), immunostimulatory nucleic acids, plasmid DNA, or messenger RNA) and is therefore included in several compositions that are in clinical development. Notably, when mixed with RNA, protamine spontaneously generates particles in the size range of 20-1000 nm depending on the formulation conditions (concentration of the reagents, ratio, and presence of salts). These particles are being used for vaccination and immuno-stimulation. Several grades of protamine are available, and we compared them in the context of complex formation with messenger RNA (mRNA). We found that the different available protamine preparations largely vary in their composition and capacity to transfect mRNA. Our data point to the source of protamine as an important parameter for the production of therapeutic protamine-based complexes.
Topics: Cells, Cultured; Drug Compounding; HEK293 Cells; Humans; Leukocytes, Mononuclear; Particle Size; Protamines; RNA, Messenger; Transfection
PubMed: 32804028
DOI: 10.1080/10717544.2020.1790692 -
Oncogene Apr 2022The small arginine-rich protein protamine condenses complete genomic DNA into the sperm head. Here, we applied its high RNA binding capacity for spontaneous...
The small arginine-rich protein protamine condenses complete genomic DNA into the sperm head. Here, we applied its high RNA binding capacity for spontaneous electrostatic assembly of therapeutic nanoparticles decorated with tumour-cell-specific antibodies for efficiently targeting siRNA. Fluorescence microscopy and DLS measurements of these nanocarriers revealed the formation of a vesicular architecture that requires presence of antibody-protamine, defined excess of free SMCC-protamine, and anionic siRNA to form. Only these complex nanoparticles were efficient in the treatment of non-small-cell lung cancer (NSCLC) xenograft models, when the oncogene KRAS was targeted via EGFR-mediated delivery. To show general applicability, we used the modular platform for IGF1R-positive Ewing sarcomas. Anti-IGR1R-antibodies were integrated into an antibody-protamine nanoparticle with an siRNA specifically against the oncogenic translocation product EWS/FLI1. Using these nanoparticles, EWS/FLI1 knockdown blocked in vitro and in vivo growth of Ewing sarcoma cells. We conclude that these antibody-protamine-siRNA nanocarriers provide a novel platform technology to specifically target different cell types and yet undruggable targets in cancer therapy by RNAi.
Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Oncogene Proteins, Fusion; Protamines; Proto-Oncogene Protein c-fli-1; RNA, Small Interfering; RNA-Binding Protein EWS; Technology; Xenograft Model Antitumor Assays
PubMed: 35220407
DOI: 10.1038/s41388-022-02241-w -
Nucleic Acids Research Jun 2020Protamine proteins dramatically condense DNA in sperm to almost crystalline packing levels. Here, we measure the first step in the in vitro pathway, the folding of DNA...
Protamine proteins dramatically condense DNA in sperm to almost crystalline packing levels. Here, we measure the first step in the in vitro pathway, the folding of DNA into a single loop. Current models for DNA loop formation are one-step, all-or-nothing models with a looped state and an unlooped state. However, when we use a Tethered Particle Motion (TPM) assay to measure the dynamic, real-time looping of DNA by protamine, we observe the presence of multiple folded states that are long-lived (∼100 s) and reversible. In addition, we measure folding on DNA molecules that are too short to form loops. This suggests that protamine is using a multi-step process to loop the DNA rather than a one-step process. To visualize the DNA structures, we used an Atomic Force Microscopy (AFM) assay. We see that some folded DNA molecules are loops with a ∼10-nm radius and some of the folded molecules are partial loops-c-shapes or s-shapes-that have a radius of curvature of ∼10 nm. Further analysis of these structures suggest that protamine is bending the DNA to achieve this curvature rather than increasing the flexibility of the DNA. We therefore conclude that protamine loops DNA in multiple steps, bending it into a loop.
Topics: DNA; Microscopy, Atomic Force; Nucleic Acid Conformation; Pliability; Protamines
PubMed: 32392345
DOI: 10.1093/nar/gkaa365