Initiation of Parental Genome Reprogramming in Fertilized Oocyte by Splicing Kinase SRPK1-Catalyzed Protamine Phosphorylation.

The paternal genome undergoes a massive exchange of histone with protamine for compaction into sperm during spermiogenesis. Upon fertilization, this process is potently reversed, which is essential for parental genome reprogramming and subsequent activation; however, it remains poorly understood how this fundamental process is initiated and regulated. Here, we report that the previously characterized splicing kinase SRPK1 initiates this life-beginning event by catalyzing site-specific phosphorylation of protamine, thereby triggering protamine-to-histone exchange in the fertilized oocyte. Interestingly, protamine undergoes a DNA-dependent phase transition to gel-like condensates and SRPK1-mediated phosphorylation likely helps open up such structures to enhance protamine dismissal by nucleoplasmin (NPM2) and enable the recruitment of HIRA for H3.3 deposition. Remarkably, genome-wide assay for transposase-accessible chromatin sequencing (ATAC-seq) analysis reveals that selective chromatin accessibility in both sperm and MII oocytes is largely erased in early pronuclei in a protamine phosphorylation-dependent manner, suggesting that SRPK1-catalyzed phosphorylation initiates a highly synchronized reorganization program in both parental genomes.

DOT1L promotes spermatid differentiation by regulating expression of genes required for histone-to-protamine replacement.

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.

Derepression of Y-linked multicopy protamine-like genes interferes with sperm nuclear compaction in D. melanogaster.

Across species, sperm maturation involves the dramatic reconfiguration of chromatin into highly compact nuclei that enhance hydrodynamic ability and ensure paternal genomic integrity. This process is mediated by the replacement of histones by sperm nuclear basic proteins, also referred to as protamines. In humans, a carefully balanced dosage between two known protamine genes is required for optimal fertility. However, it remains unknown how their proper balance is regulated and how defects in balance may lead to compromised fertility. Here, we show that a nucleolar protein, modulo, a homolog of nucleolin, mediates the histone-to-protamine transition during Drosophila spermatogenesis. We find that modulo mutants display nuclear compaction defects during late spermatogenesis due to decreased expression of autosomal protamine genes (including Mst77F) and derepression of Y-linked multicopy Mst77F homologs (Mst77Y), leading to the mutant's known sterility. Overexpression of Mst77Y in a wild-type background is sufficient to cause nuclear compaction defects, similar to modulo mutant, indicating that Mst77Y is a dominant-negative variant interfering with the process of histone-to-protamine transition. Interestingly, ectopic overexpression of Mst77Y caused decompaction of X-bearing spermatids nuclei more frequently than Y-bearing spermatid nuclei, although this did not greatly affect the sex ratio of offspring. We further show that modulo regulates these protamine genes at the step of transcript polyadenylation. We conclude that the regulation of protamines mediated by modulo, ensuring the expression of functional ones while repressing dominant-negative ones, is critical for male fertility.

Widespread displacement of DNA- and RNA-binding factors underlies toxicity of arginine-rich cell-penetrating peptides.

Due to their capability to transport chemicals or proteins into target cells, cell-penetrating peptides (CPPs) are being developed as therapy delivery tools. However, and despite their interesting properties, arginine-rich CPPs often show toxicity for reasons that remain poorly understood. Using a (PR)n dipeptide repeat that has been linked to amyotrophic lateral sclerosis (ALS) as a model of an arginine-rich CPP, we here show that the presence of (PR)n leads to a generalized displacement of RNA- and DNA-binding proteins from chromatin and mRNA. Accordingly, any reaction involving nucleic acids, such as RNA transcription, translation, splicing and degradation, or DNA replication and repair, is impaired by the presence of the CPPs. Interestingly, the effects of (PR)n are fully mimicked by protamine, a small arginine-rich protein that displaces histones from chromatin during spermatogenesis. We propose that widespread coating of nucleic acids and consequent displacement of RNA- and DNA-binding factors from chromatin and mRNA accounts for the toxicity of arginine-rich CPPs, including those that have been recently associated with the onset of ALS.

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

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 demonstrated that Prm1+/- mice were subfertile, whereas Prm1-/- mice were infertile. Prm1-/- and Prm2-/- sperm showed high levels of reactive oxygen species-mediated DNA damage and increased histone retention. In contrast, Prm1+/- sperm displayed only moderate DNA damage. The majority of Prm1+/- sperm were CMA3 positive, indicating protamine-deficient chromatin, although this was not the result of increased histone retention in Prm1+/- sperm. However, sperm from Prm1+/- and Prm1-/- mice contained high levels of incompletely processed PRM2. Furthermore, the PRM1:PRM2 ratio was skewed from 1:2 in wild type to 1:5 in Prm1+/- animals. Our results reveal that PRM1 is required for proper PRM2 processing to produce mature PRM2, which, together with PRM1, is able to hypercondense DNA. Thus, the species-specific PRM1:PRM2 ratio has to be precisely controlled in order to retain full fertility.

DOT1L regulates chromatin reorganization and gene expression during sperm differentiation.

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 remodeling and subsequent reorganization and compaction of the 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. Proteomic and transcriptomic 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.

Protamine dosing and its impact in cardiac surgery transfusion practice-A retrospective bi-institutional analysis.

BACKGROUND: Bleeding after cardiac surgery is common and continues to require 10-20% of the national blood supply. Transfusion of allogeneic blood is associated with increased morbidity and mortality. Excessive protamine in the absence of circulating heparin after weaning off CPB can cause anticoagulation and precipitate bleeding. Hence, adequate dose calculation of protamine is crucial yet under evaluated. STUDY DESIGN: Retrospective cohort study. METHODS: We conducted a retrospective bi-institutional analysis of cardiac surgical patients who underwent cardiopulmonary bypass (CPB)-assisted cardiac surgery to assess the impact of protamine dosing in transfusion practice. Total 762 patients were identified from two institutions using electronic medical records and the Society of Thoracic Surgery (STS) database who underwent cardiac surgery using CPB. Patients were similar in demographics and other baseline characteristics. We divided patients into two groups based on mg of protamine administered to neutralize each 100 U of unfractionated heparin (UFH)-low-ratio group (Protamine: UFH ≤ 0.8) and high-ratio group (Protamine: UFH > 0.8). RESULTS: We observed a higher rate of blood transfusion required in high-ratio group (ratio >0.8) compared with low-ratio group (ratio ≤0.8) (p < .001). The increased requirement was consistently demonstrated for intraoperative transfusions of red blood cells, plasma, platelets, and cryoprecipitate. CONCLUSION: High protamine to heparin ratio may cause increased bleeding and transfusion in cardiac surgical patients. Protamine to heparin ratio of 0.8 or lower is sufficient to neutralize circulating heparin after weaning off cardiopulmonary bypass.

Anticoagulation reversal (vitamin K, prothrombin complex concentrates, idarucizumab, andexanet-α, protamine).

Bleeding events are common in patients prescribed anticoagulants and can have devastating consequences. Several specific and nonspecific agents have been developed to reverse the effects of anticoagulant drugs or toxins. Vitamin K, as the oldest of these antidotes, specifically counteracts the effects of pharmaceuticals and rodenticides designed to deplete stores of vitamin K-dependent factors. In cases of life-threatening bleeding, the addition of prothrombin complex concentrates (PCCs) allows for the immediate replacement of coagulation factors. While the use of PCCs has been extended to the non-specific reversal of the effects of newer direct oral anticoagulants, the specific agents idarucizumab, targeting dabigatran and andexanet-α, binding factor Xa inhibitors, have recently been developed and are being preferentially recommended by most guidelines. However, despite having rapid effects on correcting coagulopathy, there is to date a lack of robust evidence establishing the clear superiority of direct oral anticoagulant-specific reversal agents over PCCs in terms of haemostatic efficacy, safety or mortality. For andexanet-α, a potential signal of increased thromboembolic risks, comparatively high costs and low availability might also limit its use, even though emerging evidence appears to bolster its role in intracranial haemorrhage. Protamine is the specific agent for the reversal of unfractionated heparin anticoagulation used mainly in cardiovascular surgery. It is much less effective for low molecular weight heparin fragments and is usually reserved for cases with life-threatening bleeding.

Safety and Efficacy of Insulins in Critically Ill Patients Receiving Continuous Enteral Nutrition.

OBJECTIVE: There is a relative lack of consensus regarding the optimal management of hyperglycemia in patients receiving continuous enteral nutrition (EN), with or without a diagnosis of diabetes. METHODS: This retrospective study examined 475 patients (303 with known diabetes) hospitalized in critical care setting units in 2019 in a single center who received continuous EN. Rates of hypoglycemia, hyperglycemia, and glucose levels within the target range (70-180 mg/dL) were compared between patients with and without diabetes, and among patients treated with intermediate-acting (IA) biphasic neutral protamine Hagedorn 70/30, long-acting (LA) insulin, or rapid-acting insulin only. RESULTS: Among those with type 2 diabetes mellitus, IA and LA insulin regimens were associated with a significantly higher proportion of patient-days in the target glucose range and fewer hyperglycemic days. Level 1 (<70 mg/dL) and level 2 (<54 mg/dL) hypoglycemia occurred rarely, and there were no significant differences in level 2 hypoglycemia frequency across the different insulin regimens. CONCLUSION: Administration of IA and LA insulin can be safe and effective for those receiving insulin doses for EN-related hyperglycemia.

Effect of naringenin on post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo (Bubalus bubalis) bull sperm.

Our objectives were to explore the effect of naringenin addition in the semen extender on the post-thaw 1) sperm quality, 2) fertility-associated gene expression, and 3) fertilization potential of buffalo bull sperm. In experiment 1, semen samples (n = 32) from four Nili-Ravi buffalo bulls were pooled (n = 8) and diluted with the tris-citric acid (TCF-EY) extender containing different concentrations of naringenin, i.e., placebo (DMSO), 0 (control), 50, 100, 150 and 200 µM naringenin. After dilution, semen samples were packed in 0.5 mL French straws, cryopreserved and analyzed for post-thawed sperm quality and gene expression. Computer-assisted Semen Analysis, Hypo-osmotic Swelling test, Normal Apical Ridge assay, Rhodamine 123, Acridine orange, Propidium iodide staining and Thiobarbituric Acid Reactive Substances assay were performed to assess sperm motility parameters, plasma membrane functionality, acrosome integrity, mitochondrial membrane potential, DNA integrity, viability and lipid peroxidation, respectively. Expression levels of sperm acrosome-associated SPACA3, DNA condensation-related PRM1, anti-apoptotic BCL2, pro-apoptotic BAX, and oxidative stress-associated ROMO1 genes were evaluated through qPCR. Results revealed that total and progressive motility, plasma membrane functionality, acrosome integrity, mitochondrial membrane potential, DNA integrity and viability were higher (P < 0.05) with 50, 100 and 150 µM naringenin compared to 200 µM naringenin, placebo and control groups. Moreover, all naringenin-treated groups improved catalase activity, and reduced lipid peroxidation compared to placebo and control groups (P < 0.05). Relative expression levels of SPACA3 and PRM1 genes were higher (P < 0.05) with 150 µM naringenin compared to all groups except 100 µM (P > 0.05). No difference (P > 0.05) in the expression level of BCL2 gene was observed among all groups. Furthermore, BAX gene was expressed higher (P < 0.05) in the 200 µM naringenin group, whereas no difference (P > 0.05) in expression was noticed among the remaining groups. In addition, ROMO1 gene was expressed lower (P < 0.05) in all naringenin-treated groups compared to the control. In experiment 2, the in vivo fertility of semen doses (n = 400; 200/group) containing optimum concentration of naringenin (150 µM; depicted better in vitro sperm quality in experiment 1) was compared with control during the breeding season. Buffaloes were inseminated 24 h after the onset of natural estrus and palpated transrectal for pregnancy at least 60 days post-insemination. The fertility rate of 150 µM naringenin group was higher (P = 0.0366) compared to the control [57.00 ± 0.03 % (114/200) vs. 46.50 ± 0.04 % (93/200), respectively]. Taken together, it is concluded that naringenin supplementation in semen extender improves post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo bull sperm, more apparently at 150 µM concentration.

Assessment of Perioperative Protamine Reactions in Patients With Fish Allergies: A Retrospective Observational Study.

OBJECTIVES: To retrospectively assess the incidence and severity of perioperative protamine reactions in adult patients with documented history of fish allergy. DESIGN: Retrospective observational study. SETTING: Large academic tertiary referral center. PARTICIPANTS: Adults with fish allergies undergoing surgeries involving protamine, between January 1, 2008, and March 1, 2018. INTERVENTIONS: Perioperative protamine administration in patients with documented fish allergy. MEASUREMENTS AND MAIN RESULTS: Perioperative protamine and anaphylactic reactions were reviewed. A diagnosis of anaphylaxis or protamine reaction was based on clinical suspicion, perioperative events, and postoperative evaluations. Among 214 patients, 2 cases (<1%) of anaphylaxis or protamine reactions occurred. Cardiac procedures were most common (67%). The median intraoperative heparin dosage was 46,000 IU, and the median protamine dosage was 310 mg. Nearly all patients (99%) were admitted to the intensive care unit postoperatively, with a median hospital stay of 6.5 days (interquartile range, 5.2-14.6 days). There were 3 deaths (1%) within 30 days, and 15 (7%) within 1 year. CONCLUSIONS: The study findings suggest that in patients with a history of fish allergy, cross-reactivity with protamine is unlikely, as anaphylaxis and/or protamine reactions were rare in this patient population in the perioperative environment. Based on these findings, this study does not recommend avoiding protamine solely based on a history of fish allergy when heparin reversal is required during surgery.

Ameliorative effect of crocin on post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo (Bubalus bubalis) bull sperm.

Buffalo bull sperm suffer more cryoinjuries due to lipid peroxidation of high structural polyunsaturated fatty acid contents than cattle sperm. Consequently, the post-thaw fertilization potential of buffalo bull sperm is compromised. Crocin is a carotenoid known for its antioxidant potential through scavenging reactive oxygen species. Objectives of the current study were to investigate the effect of crocin addition in the semen extender on post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo bull sperm. Semen samples (n = 32) from four Nili-Ravi buffalo bulls were extended with tris-citric acid extender containing different concentrations of crocin (0 mM; control, 0.5, 1, 1.5 and 2 mM). The extended semen was packed in 0.5 mL French straws (25 × 106 sperm/straw) and cryopreserved in liquid nitrogen. Computer-assisted semen analysis, hypo-osmotic swelling test, normal apical ridge assay, Rhodamine 123, acridine orange, propidium iodide staining, and thiobarbituric acid reactive substances assay were performed to assess sperm motility parameters, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential, DNA integrity, viability, and lipid peroxidation, respectively. Expression levels of sperm acrosome-associated SPACA3, DNA condensation-related PRM1, anti-apoptotic BCL2, pro-apoptotic BAX, and oxidative stress-associated ROMO1 genes were evaluated through qPCR. The fertility of semen doses containing the most potent concentration of crocin (based on optimum post-thaw semen quality) was compared with control during the breeding season. Buffaloes (n = 400; 200/group) were inseminated 24 h after the onset of oestrus and transrectally palpated for pregnancy at least 60 days post-insemination. Results revealed that 0.5 and 1 mM crocin improved sperm post-thaw total motility, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential and viability, and 1 and 1.5 mM crocin enhanced catalase activity and reduced lipid peroxidation compared to control (p < .05). Moreover, 1 mM crocin improved sperm post-thaw progressive motility, kinematics, and DNA integrity, and 1.5 mM crocin enhanced plasma membrane integrity than control (p < .05). Expression levels of SPACA3, PRM1 and BCL2 genes were higher (p < .05) with 1 mM crocin compared to other groups. In contrast, no difference (p > .05) was noticed in expressions of BAX and ROMO1 genes among all groups. The fertility rate of semen doses containing the most potent concentration (1 mM) of crocin was higher (p = .0465) compared to control (56 ± 0.03% vs. 46 ± 0.04%, respectively). In conclusion, 1 mM crocin in the semen extender improves post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo bull sperm.

Clues of incomplete reversal of heparin in cardiac surgery.

OBJECTIVES: In our center, an unusual rate of patients had abnormalities of hemostasis in immediate postoperative period of cardiac surgery. Our objectives were to identify the cause of these sudden hemostasis abnormalities and to evaluate the performances of point of care coagulation testing. METHODS: In this prospective and descriptive study, we included 33 consecutive patients undergoing elective cardiac surgery for 1 month. Heparin-induced anticoagulation and calculation of the protamine dose were tested by the Hemostasis Management System Plus device (Medtronic, Minneapolis, MN, USA). Fifteen minutes after the end of the protamine infusion, activated clotting time (ACT), activated partial thromboplastin time and anti Xa activity were measured. In case of unusual clinical bleeding, a Quantra analysis (Stago, HemoSonics LLC, Charlottesville, VA) was added. RESULTS: Residual antiXa activity >0.2 IU/mL after neutralization was present in 44% of patients. Our investigation concluded incomplete heparin reversal. There was no association between cellular reinfusate and the presence of heparin. The unusual rate of hemostasis abnormalities was explained by a less efficient protamine reversal of heparin. ACT and Clot Time Ratio (CTR, Quantra system) correlated with AntiXa with Spearman's coefficients of 0.85 (p < .0001) and 0.95 (p = .0012), respectively. About ACT, a threshold of 150 seconds had a sensitivity of 85% [58-97] and a specificity of 85% [58-97%] for detection of AntiXa>0.2. For CTR, a threshold of 1.4 had a sensitivity of 67% [30-94] and a specificity of 100% [18-100]. CONCLUSION: The use of point of care coagulation testing is effective in detecting incomplete reversal of heparin.

Thioredoxin-dependent disulfide bond reduction is required for protamine eviction from sperm chromatin.

Cysteine oxidation in protamines leads to their oligomerization and contributes to sperm chromatin compaction. Here we identify the Drosophila thioredoxin Deadhead (DHD) as the factor responsible for the reduction of intermolecular disulfide bonds in protamines and their eviction from sperm during fertilization. Protamine chaperone TAP/p32 dissociates DNA-protamine complexes in vitro only when protamine oligomers are first converted to monomers by DHD. dhd-null embryos cannot decondense sperm chromatin and terminate development after the first pronuclear division. Therefore, the thioredoxin DHD plays a critical role in early development to facilitate the switch from protamine-based sperm chromatin structures to the somatic nucleosomal chromatin.

Isolation and Purification of Protamine from the Cultured Takifugu flavidus and Its Physicochemical Properties.

Protamine is a cationic peptide derived from fish sperm and has several important functional properties: antibacterial properties, acting as a carrier for injectable insulin and as a heparin antagonist, combatting fatigue, etc. Thus, it has been widely used in medicinal applications and food products. Cultured Takifugu flavidus is a type of pufferfish with a delicious taste that is popular in China, and its production is increasing significantly. Therefore, protamine was extracted via acid extraction from the sperm of Takifugu flavidus and further isolated and purified via sephadex gel chromatography, ion exchange chromatography, and desalination chromatography. Furthermore, the physicochemical properties of protamine were investigated. The results showed that the sperm of the cultured T. flavidus were non-toxic, and the extracted and purified protamine had high contents of arginine (36.90%) and lysine (27.02%), respectively. The secondary structure of protamine was mainly ß-folded and irregularly curled. Additionally, protamine exhibited high thermal stability with a denaturation temperature of 176 °C. This study would provide a theoretical basis for the structural analysis, bioactivity, and resource development of pufferfish protamine and help to promote the development of the pufferfish industry.

Importin-9 regulates chromosome segregation and packaging in Drosophila germ cells.

Germ cells undergo distinct nuclear processes as they differentiate into gametes. Although these events must be coordinated to ensure proper maturation, the stage-specific transport of proteins in and out of germ cell nuclei remains incompletely understood. Our efforts to genetically characterize Drosophila genes that exhibit enriched expression in germ cells led to the finding that loss of the highly conserved Importin ß/karyopherin family member Importin-9 (Ipo9, herein referring to Ranbp9) results in female and male sterility. Immunofluorescence and fluorescent in situ hybridization revealed that Ipo9KO mutants display chromosome condensation and segregation defects during meiosis. In addition, Ipo9KO mutant males form abnormally structured sperm and fail to properly exchange histones for protamines. Ipo9 physically interacts with proteasome proteins, and Ipo9 mutant males exhibit disruption of the nuclear localization of several proteasome components. Thus, Ipo9 coordinates the nuclear import of functionally related factors necessary for the completion of gametogenesis. This article has an associated First Person interview with the first author of the paper.

Hypoglycemic and hypolipidemic effect of pentaamino acid fullerene C60 derivative in rats with metabolic disorder.

Pentaamino acid fullerene C60 derivative is a promising nanomaterial, which exhibited antihyperglycemic activity in high-fat diet and streptozotocin-induced diabetic rats. This study investigates the effect of pentaaminoacid C60 derivative (PFD) in rats with metabolic disorders. Rats were assigned to 3 groups (of 10 rats each) as follows: Group 1 (normal control), group 2 included the protamine-sulfate-treated rats (the untreated group of animals with the model metabolic disorder); group 3 (Protamine sulfate + PFD) included the protamine-sulfate-treated model rats that received an intraperitoneal injection of PFD. Metabolic disorder in rats was initiated by protamine sulfate (PS) administration. The PS + PFD group was injected intraperitoneally with PFD solution (3 mg/kg). Protamine sulfate induces biochemical changes (hyperglycemia, hypercholesterolemia, and hypertriglyceridemia) in the blood and morphological lesions in rat liver and pancreas. The potassium salt of fullerenylpenta-N-dihydroxytyrosine in protamine sulfate-induced rats normalized blood glucose level and the serum lipid profile and improved hepatic function markers. Treatment with PFD restored pancreas islets and liver structure of protamine sulfate-induced rats compared to the untreated group. PFD is a promising compound for further study as a drug against metabolic disorders.

Sperm mRNA screening by reverse transcription-recombinase polymerase amplification (RT-RPA) assay: Decision-making for differential extraction.

The presence of sperm cells is an indicator for differential extraction on sexual assault samples. In general, sperm cells are identified by microscopic analysis; however, this conventional method takes time and effort, even for trained personnel. Here, we present a reverse transcription-recombinase polymerase amplification (RT-RPA) assay targeting sperm mRNA marker (PRM1). The RT-RPA assay requires only 40 min for PRM1 detection and demonstrates a sensitivity of 0.1 µL of semen. Our results indicate that the RT-RPA assay may be a rapid, simple, and specific strategy for screening sperm cells in sexual assault samples.

The contemporary role of protamine in the cardiac catheterization laboratory.

Access to the arterial circulation and full anticoagulation carries a risk of serious bleeding during and after percutaneous coronary intervention. Important sources of bleeding include the arterial access site and coronary artery perforation. Prompt and effective management of hemorrhagic complications is an essential interventional skill. Protamine sulfate is well-known as a heparin reversal agent. Despite this, there is heterogeneity in the use of protamine during interventional procedures. While protamine is generally well-tolerated, it is associated with a risk of hypersensitivity reaction, including anaphylaxis, among others. The purpose of this review is to summarize the existing evidence about and experience with the use of protamine sulfate in the setting of percutaneous coronary and structural interventional procedures.

Citrylglutamate synthase deficient male mice are subfertile with impaired histone and transition protein 2 removal in late spermatids.

Chromatin remodelling in spermatids is an essential step in spermiogenesis and involves the exchange of most histones by protamines, which drives chromatin condensation in late spermatids. The gene Rimklb encodes a citrylglutamate synthase highly expressed in testes of vertebrates and the increase of its reaction product, ß-citrylglutamate, correlates in time with the appearance of spermatids. Here we show that deficiency in a functional Rimklb gene leads to male subfertility, which could be partially rescued by in vitro fertilization. Rimklb-deficient mice are impaired in a late step of spermiogenesis and produce spermatozoa with abnormally shaped heads and nuclei. Sperm chromatin in Rimklb-deficient mice was less condensed and showed impaired histone to protamine exchange and retained transition protein 2. These observations suggest that citrylglutamate synthase, probably via its reaction product ß-citrylglutamate, is essential for efficient chromatin remodelling during spermiogenesis and may be a possible candidate gene for male subfertility or infertility in humans.