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PubMed2001

Round spermatids from infertile men exhibit decreased protamine-1 and -2 mRNA.

During spermiogenesis, histone-to-protamine exchange causes chromatin condensation. Spermatozoa from infertile men are known to exhibit an increased protamine-1 (PRM1) to protamine-2 (PRM2) protein ratio. Since patients undergoing testicular sperm extraction (TESE) followed by intracytoplasmic sperm injection (ICSI) reveal low fertilization rates, whether the outcome of ICSI could be related to the percentage of round spermatids expressing PRM1-mRNA and PRM2-mRNA was investigated. Applying in-situ hybridization, 55 testicular biopsies from men undergoing TESE/ICSI were investigated. The percentage of PRM1-mRNA and PRM2-mRNA positive spermatids was significantly (P < 0.0001) decreased in men with at least qualitatively normal spermatogenesis (PRM1-mRNA: 58.4 +/- 13.8%; PRM2-mRNA: 56.4 +/- 11.3%) and impaired spermatogenesis (PRM1-mRNA: 32.6 +/- 10.8%; PRM2-mRNA: 31.7 +/- 11.1%) compared with men with obstructive azoospermia and quantitatively normal spermatogenesis (PRM1-mRNA: 79.9 +/- 4.6%; PRM2-mRNA: 78.1 +/- 5.7%). A positive correlation (r(PRM1) = 0.733; r(PRM2) = 0.784; P < 0.001) was demonstrated between the score and the percentage of PRM1-mRNA and PRM2-mRNA positive spermatids. While successful fertilization was neither related to the score, nor to the percentage of PRM1-mRNA and PRM2-mRNA positive spermatids, a significant (P < 0.05) relationship was demonstrated between successful fertilization and the PRM1-mRNA to PRM2-mRNA ratio. Therefore, the PRM1-mRNA to PRM2-mRNA ratio in round spermatids may serve as a possible predictive factor for the outcome of ICSI.

Adult↗
PubMed1996

Immunoelectron microscopical distribution of histones H2B and H3 and protamines during human spermiogenesis.

The fine structural distribution of histones H2B and H3, and protamines were localized by means of specific antibodies and ultrastructural immunocytochemistry in nuclei of human spermatids and spermatozoa. The antibodies were used to detect the nuclear basic proteins on section of testis and ejaculated spermatozoa by immunoelectron microscopy. A quantitative analysis of labelling density was performed on micrographs using an interactive image analysis system. The labelling density of somatic-type histones H2B and H3 and of their testis-specific variants was constant in the nuclei of young spermatids with round nuclei (stages 1-2), and then increased in intermediate spermatids (stages 3-4). Histone H3 labelling decreased at the end of the elongation phase (stage 5) while histone H2B labelling decreased in mature spermatids (stage 6) only. Spermatozoa were found to be weakly labelled by the anti-histone antibodies. The first signs of labelling of protamines and basic intermediate proteins appeared in spermatid nuclei at stage 4, increased further in stage 6 spermatids and persisted in all sperm nuclei. The present work shows that histone-to-protamine replacement occurs at the beginning of the spermatid maturation phase in human. However, histones are partially retained in mature spermatids and sperm nuclei.

Adult↗
PubMed1977

Phosphorylated protamines. I. Binding stoichiometry and thermal stability of complexes in DNA.

To decipher on a molecular level the role of protamine phosphorylation in spermiogenesis, clupeine Z species containing one, two or three serine phosphates were prepared utilizing a recently developed chemical procedure. The melting of complexes with calf thymus DNA showed that thermal stability decreases with increasing degree of phosphorylation. The stoichiometry of the nucleoprotamine complexes was investigated analyzing the melting curves and using the fluorescamine assay recently described. Phosphorylation significantly reduces binding stoichiometry defined as DNA-nucleotides covered by a protamine molecule. Thus, phosphorylated protamines are more densely packed along DNA; the implications on processes occurring in spermiogenesis as i. e. histone replacement, are discussed. A general discussion on the variability in protein-DNA stoichiometry values obtained by different procedures is included.

Chemical Phenomena↗
PubMed1977

Phosphorylated protamines. II. Circular dichroism of complexes with DNA, dependency on ionic strength.

The influence of protamine phosphorylation upon the conformation of nucleoprotamine complexes was studied at different ionic strengths using circular dichroism. The sharp onset of CD spectral changes upon decreasing the NaC1 concentrationwas correlated with the beginning of complex formation and can be used to determine apparent binding affinities in terms of a critical ionic strength. It is show that phosphorylation strongly reduces the binding strength of protamines towards DNA. Directly mixed and reconstituted complexes reveal differences in their CD spectra, which decrease with increasing ionic strength. Spectra of complexes between threefold phosphorylated clupeine Z and DNA obtained by reconstitution or direct mixing at higher ionic strength resemble the phi-type spectra of DNA and are unique for the phosphorylated species. The implications of protamine phosphorylation for chromatin or DNA condensation havebeen discussed.

Circular Dichroism↗
PubMed1979

Heterogeneity of biologically active deadenylated protamine mRNA components isolated from rainbow trout testes.

Poly(A)+ protamine mRNA's were isolated from rainbow trout testes and deadenylated by treatment with calf thymus RNase H. Four subcomponents of deadenylated PmRNA (PmRNA1-4) were purified by electrophoresis on a 6% polyacrylamide gel in 8 M urea. Translation of each PmRNA subcomponent in the wheat germ S-30 cell-free system showed that all subcomponents are biologically active but each codes for two or more protamine polypeptides suggesting molecular heterogeneity. However, the deadenylated mRNA's can be categorized into two groups based on the spectrum of protamines whose synthesis they stimulate.

Animals↗
PubMed1989

Conformation of the fowl protamine, galline, and its binding properties to DNA.

1. CD spectra showed that the fowl protamine, galline, has an unordered structure rich in reverse turns in neutral solution. Eight reverse turns were predicted to be present in the galline molecule on the basis of its amino acid sequence. Spectrophotometric analyses revealed that galline efficiently bound to DNA in 0.25 mM EDTA/10 mM Tricine-HCl, pH 7.4, but hardly so in 30 mM NaCl/3 mM sodium citrate, pH 7.0. Citrate ions bound specifically to the galline molecule, causing a conformational change in it. As a result, galline could not interact with DNA. 2. The concentration of unbound galline in a mixture of DNA and galline in 100 mM NaCl/50 mM Tricine-HCl, pH 7.4, at 37 C was determined by measurement of the intrinsic fluorescence of tyrosine residues of galline in the supernatant after ultracentrifugation of the mixture. The Scatchard plot showed positive co-operativity in the binding of galline to DNA and the binding parameters were determined: the co-operative binding constant (Kc) = 3.3 X 10(7)M-1, the co-operativity factor (q) = 800, and the number of nucleotides of DNA occupied by one galline molecule (n) = 28. The Kc and q values were intermediate between those for clupeine Z from herring sperm and S-methyl protamine from boar sperm. That is, the binding constants of protamine as to DNA decrease in the order of herring, fowl, and boar, while the co-operativities in binding increase in that order.

Amino Acid Sequence↗
PubMed1981

Closely related mRNA sequences of protamines in rainbow trout testis.

Recombinant plasmids containing DNA complementary to protamine messenger RNA of rainbow trout (Salmo gairdnerii) have been isolated and sequenced. One of the clones contained the entire coding sequence of a protamine gene together with the complete 3' non-coding region. Another clone had an identical nucleotide sequence in the coding region but four base substitutions in the 3' non-coding region. On the basis of a comparison of the nucleotide sequences available at present, the structure and divergence of the protamine gene family are discussed.

Animals↗
PubMed2003

Protamine 2 deficiency leads to sperm DNA damage and embryo death in mice.

Cytokinesis is incomplete in spermatogenic cells, and the descendants of each stem cell form a clonal syncytium. As a result, a heterozygous mutation in a gene expressed postmeiotically affects all of the haploid spermatids within a syncytium. Previously, we have found that disruption of one copy of the gene for either protamine 1 (PRM1) or protamine 2 (PRM2) in the mouse results in a reduction in the amount of the respective protein, abnormal processing of PRM2, and inability of male chimeras to transmit either the mutant or wild-type allele derived from the 129-genotype embryonic stem cells to the next generation. Although it is believed that protamines are essential for compaction of the sperm nucleus and to protect the DNA from damage, this has not been proven experimentally. To test the hypothesis that failure of chimeras to transmit the 129 genotype to offspring was due to alterations in the organization and integrity of sperm DNA, we used the single-cell DNA electrophoresis (comet) assay, ultrastructural analysis, and the intracytoplasmic sperm injection (ICSI) procedure. Comet assay demonstrated a direct correlation between the fraction of sperm with haploinsufficiency of PRM2 and the frequency of sperm with damaged DNA. Ultrastructural analysis revealed reduced compaction of the chromatin. ICSI with PRM2-deficient sperm resulted in activation of most metaphase II-arrested mouse eggs, but few were able to develop to the blastocyst stage. These findings suggest that development fails because of damage to paternal DNA and that PRM2 is crucial for maintaining the integrity of sperm chromatin.

Animals↗
PubMed2001

A highly conserved sequence essential for translational repression of the protamine 1 messenger rna in murine spermatids.

Translational regulation of the protamine 1 mRNA is mediated by sequences in its 3' untranslated region. In this study, we demonstrate that a highly conserved sequence, the translational control element, is solely responsible for protamine 1 translational regulation. Mutation of the conserved sequence causes premature translation of a transgene containing a fusion between the human growth hormone coding sequence and the protamine 1 3' untranslated region. Temporal expression of the transgene was monitored in prepubertal animals by Northern and Western blotting and in adult animals by immunocytochemistry. Messenger RNAs lacking the translational control element sediment in the messenger ribonucleoprotein particle and ribosomal fractions of polysome gradients, suggesting that the translational control element is required for translational repression but not for incorporation of mRNAs into ribonucleoprotein particles.

Acrosome↗
PubMed1987

C5a and thromboxane generation associated with pulmonary vaso- and broncho-constriction during protamine reversal of heparin.

The authors conducted a study in humans to determine the mediators associated with acute pulmonary vaso- and broncho-constriction occurring episodically with protamine reversal of heparin anticoagulation. Of 48 adult patients investigated prospectively after termination of cardiopulmonary bypass, two presented a sudden increase of airway pressure, acute pulmonary hypertension, and systemic hypotension 1-3 min after right atrial protamine injection. In these two subjects, plasma levels of C5a increased from 0.7 and 2.2 to 9.8 and 9.9 ng/ml, respectively, and thromboxane B2 increased from 0.26 and 0.34 to 7.5 and 16.2 ng/ml 1 minute after drug injection. A third subject not identified prospectively had an identical reaction and mediator profile (C5a, 10.2 ng/ml; TxB2, 18.6 ng/ml at 1 min). The plasma levels of these mediators were unchanged in the remaining patients (C5a, 0.7 +/- 1.1 [x +/- S.D.] to 0.6 +/- 0.9 ng/ml; TxB2, 0.16 +/- 0.12 to 0.15 +/- 0.07 ng/ml). Plasma histamine was not involved in this type of reaction, but increased from 0.7-10.4 ng/ml in a fourth patient who became hypotensive without acute pulmonary hypertension, bronchoconstriction, or elevation of C5a or TxB2. The authors' data indicate that the generation of high plasma levels of C5a anaphylatoxins and thromboxane is associated with pulmonary vaso- and broncho-constriction induced by protamine reversal of heparin in humans.

Adult↗
PubMed1975

Coagulation dynamics following thermal injury: effect of heparin and protamine sulfate.

A burned rat model was developed to examine post-burn alterations in coagulation. Fibrin split product concentration, as measured by the staphylococcal clumping test, was noted to rise significantly within the first 24 hours following injury. Prophylactic in vivo systemic anticoagulation with heparin was ineffective in modifying this response. However, systemic administration of protamine sulfate prevented post-burn elevation of fibrin split products. In vitro fibrin split product concentration in burn sera following the addition of heparin and protamine sulfate, was also analyzed. The results of these experiments elucidated the biochemical effects of protamine sulfate on circulating fibrin degradation products in the rat burn model.

Animals↗
PubMed2004

Role of endothelin-1 and thromboxane A2 in the pulmonary hypertension induced by heparin-protamine interaction in anesthetized dogs.

This study aimed to study the role of thromboxane A2 (TXA2) and endothelin-1 (ET-1) in the pulmonary hypertension induced by interaction of heparin-protamine in anesthetized dogs. The effect of inhaled nitric oxide (NO) was also investigated in this model. Dogs were anesthetized and instrumented for acquisition of mean arterial blood pressure, mean arterial pulmonary pressure (MPAP), and pulmonary pressure gradient (PPG). Cardiac index (CI), heart rate, and index of systemic vascular resistance were also obtained. Intravenous administration of heparin (500 IU/kg) 3 minutes before protamine (10 mg/kg) caused marked pulmonary hypertension, as evaluated by the increase in MPAP and PPG. This was accompanied by systemic hypotension, CI decrease, and tachycardia. Indomethacin (10 mg/kg), dazoxiben (10 mg/kg), or tezosentan (10-mg/kg bolus plus 10-mg/kg/h infusion) significantly reduced the increase in MPAP and PPG, but had no effect on the systemic hypotension. Similar results were obtained with inhaled NO (3 ppm). Plasma TXB2 levels were markedly elevated during the pulmonary hypertension, and this was abolished in indomethacin-treated dogs. Our study shows that interaction of heparin-protamine in anesthetized dogs lead to TXA2- and ET-1-mediated pulmonary hypertension. Drugs that interfere with the synthesis of these mediators as well as inhaled NO may be of beneficial value to control this disorder.

Administration, Inhalation↗
PubMed2002

Use of heparin and protamine sulfate during laparoscopic donor nephrectomy.

BACKGROUND: Our aim was to evaluate the necessity of heparin and protamine administration during laparoscopic donor nephrectomy. METHODS: Data from 52 consecutive living-related laparoscopic donor nephrectomies performed at University of California Los Angeles between August 1999 and August 2001 were used for this analysis. For the purpose of this analysis, the patients were divided into three cohorts: group A received both heparin and protamine; group B received heparin only; and group C received neither. Intraoperative blood loss, length of admission, recipient creatinine at follow-up, and intraoperative and postoperative complications were compared between the groups. Statistical analysis was performed using a two-tailed test. RESULTS: There were no significant differences between the groups with regard to patient age and gender. Intraoperative blood loss did not differ between group B (99+/-73 mL) and group C (82+/-54 mL) ( =0.4). None of the patients required blood transfusion. No graft loss occurred in any group. Length of hospital stay, excluding any preoperative days, was similar (2.8+/-0.7, 2.9+/-1.6, and 2.5+/-0.8 days, for groups A, B, and C, respectively, ( >0.05). No systemic thromboembolic complications were noted in any of the groups. One patient in group B was converted to an open procedure because of a difficult dissection unrelated to heparin administration. The mean recipient creatinine levels at follow-up in the recipients of kidneys from groups A, B, and C were not significantly different (1.1, 1.3, and 1.3; >0.05) through the extended follow-up period of 691, 286, and 97 days, respectively. CONCLUSIONS: According to our experience, there is no apparent benefit in the administration of heparin alone or in the administration of protamine sulfate to reverse heparin anticoagulation during laparoscopic donor nephrectomy if heparin is given. This is not only in terms of bleeding complications but is also true in regard to recipient renal function through the follow-up period. It is important to note that our warm ischemic times were less than 2 minutes, because longer warm ischemic times may make the use of heparin a more important consideration. This is the first time that these questions have been studied in the laparoscopic donor nephrectomy population.

Adult↗
PubMed2003

Evaluation of the effect of protamine on human prostate carcinoma PC-3m using contrast enhanced Doppler ultrasound.

PURPOSE: We evaluated the ability of contrast enhanced Doppler ultrasound (CEDU) to demonstrate changes in tumor vascularity during angiogenesis inhibitive therapy in PC-3m human prostate carcinoma. MATERIALS AND METHODS: Pieces of human prostate carcinoma PC-3m tumor (Institute of Urology, Beijing University, Beijing, People's Republic of China) were transplanted subcutaneously into 48 male BALB/C nude mice (Center of Animal Quarantine, Beijing, People's Republic of China). Protamine (Shanghai Biochemistry Pharmaceutical Co., People's Republic of China) was injected subcutaneously as an angiogenesis inhibitor. The animals were randomly divided into 3 groups according to protamine dose. The color flow signal-pixel rate (SPR) of the images was calculated using the number of pixels showing color Doppler signals as a ratio of the total number of pixels covering the lesion. RESULTS: The SPR of the high and low dose groups were significantly lower than that of the control group (p <0.01). Mean SPR +/- SD in the 3 groups was 0.09 +/- 0.05, 0.11 +/- 0.05 and 0.22 +/- 0.10, respectively. SPR correlated significantly with microvessel density (r = 0.86 to 0.94, p <0.01). CONCLUSIONS: CEDU can effectively reveal the change in vascularity in a tumor that was treated with protamine. In addition to microvessel density, CEDU may become one of several independent prognostic indexes of angiogenesis inhibitor therapy.

Angiogenesis Inhibitors↗
PubMed1978

Effects of protamine sulfate on dengue type 1 viral activities.

Protamine treatment of type 1 dengue-infected mouse brain suspension resulted in precipitation of several viral specific activities. Complement-fixation activity was almost completely precipitated by protamine. The complement-fixation components recovered in the precipitate were comparable to a non-precipitated reference dengue 1 antigen in their homologous and heterologous reactions. Dengue hemagglutinin was also precipitated by the same treatment. The precipitated hemagglutinin was resolved into three components by buoyant density centrifugation, whose densities were 1.236, 1.215, and 1.178 g/ml, respectively. Three similar HA components were detected in non-protamine treated virus preparations. In both instances the highest-titered HA fraction possessed a buoyant density of 1.21--1.22 g/ml. These HA components were tested in the hemagglutination-inhibition reaction and were proved to be virus-specific. Cesium chloride density gradient centrifugation was shown to be useful for removing possible inhibitor(s) of viral specific hemagglutinin.

Animals↗
PubMed1983

Protamine titration after cardiopulmonary bypass.

A method of protamine titration, with the use of the 'Haemochron 400' system for reversal of heparinisation after cardiopulmonary bypass is described. Twenty-three patients, average age 47 years, undergoing this procedure for either valve replacement or coronary artery bypass grafting were studied. Accurate reversal of heparinisation was achieved using comparatively small doses of protamine. A linear relationship between the dose ratio of protamine and heparin and the time interval between their respective administrations was defined where only a single dose of heparin had been administered.

Adult↗
PubMed1992

Granulomatous hypersensitivity to protamine as a complication of insulin therapy.

A patient is described who developed a granulomatous skin reaction to injections of protamine-insulin for the treatment of her diabetes mellitus. By means of skin testing it was shown that this reaction was caused by protamine. No adverse effects were seen when she was injected with insulin which did not contain protamine.

Aged↗
PubMed1977

On the competition between protamines and histones: studies directed towards the understanding of spermiogenesis.

Specific contacts between the crossinteracting histones effectively prevent their protamine-mediated displacement from nucleoproteins revealing a complex competition pattern not expected from their relative affinities to DNA. Histone H1 is the only species freed under a variety of conditions if native chromatin is used; the results found with chromatins obtained by conventional methods reflect the extent to which histone redistribution has occurred. It is concluded that the events occurring during spermiogenesis must be apt to disrupt histone interactions found in the nucleosomes. The possible role of a protamine phosphorylation/dephosphorylation mechanism is discussed on the basis of comparative experiments with protamines phosphorylated to different extents.

Animals↗