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PubMedDate not supplied

The effect of differing rates and injection sites on the amount of protamine delivered before detection of hemodynamic alterations in dogs.

OBJECTIVES: To determine the effect of the route and rate of protamine administration on the amount of protamine that could be delivered before a hemodynamic reaction occurred in dogs. STUDY DESIGN: Prospective randomized experimental study. ANIMALS: Twenty adult mixed-breed dogs weighing 25.1+/-2.5 kg. METHODS: Before vascular surgery, the dogs were heparinized to reach an activated clotting time (ACT) of 300 seconds. After completion of the vascular surgery, protamine was administered intravenously until a hemodynamic reaction was recorded. The 4 groups of dogs were given protamine at 5 mg/min (slow) or 10 mg/min (fast) via the cephalic or the jugular veins. Systemic and pulmonary arterial pressures, central venous pressure (CVP), and pulmonary arterial occlusion pressure (PAOP) were recorded before and after protamine administration. The dose of protamine was recorded when a reaction occurred, which was defined as mean arterial pressure (MAP) <60 mm Hg or mean pulmonary arterial pressure (MPAP) >20 mm Hg or more than double the baseline value. RESULTS: Significant decreases in systolic arterial pressure (SAP), MAP, and diastolic arterial pressure (DAP) and significant increases in systolic (SPAP), mean (MPAP), and diastolic (DPAP) pulmonary arterial pressures were recorded after protamine administration. The cephalic slow group had significantly fewer protamine reactions than other groups (chi-square = 8.57, P = .03, df = 3). Significantly more protamine could be delivered from the cephalic vein (52.5+/-14.5 mg) compared with the jugular vein (37.6+/-16 mg) before a reaction occurred (P = .048). CONCLUSION: The rate of administration did not have an effect on the amount of protamine delivered. Adverse reactions were minimized when protamine was administered via the cephalic vein at a slow rate. CLINICAL RELEVANCE: We would recommend delivering protamine after cardiopulmonary bypass or vascular surgery through a peripheral venous route.

Analysis of Variance↗
PubMed1998

Protein and DNA contents in sperm from an infertile human male possessing protamine defects that vary over time.

Sperm from 2 semen samples collected 6 months apart from an infertile male and 3 semen samples collected over an 18-month period from a fertile human male volunteer have been analyzed for their protamine and DNA content. Hup1M and Hup2b antibodies were used to detect the presence of protamines and protamine precursors in western blots of nuclear proteins isolated from pools of sperm. Phosphorus and sulfur contents, which can be used to estimate the nuclear DNA and protamine contents of sperm from fertile males, were measured within individual sperm heads from each semen sample by particle induced x-ray emission (PIXE). The single-cell data reveal no significant differences in the phosphorus and sulfur contents of sperm heads in the three semen samples obtained from the fertile male. For the initial semen sample produced by the infertile male, Western blot data show a normal complement of protamine 1, small amounts of mature protamine 2, and reveal large amounts of anti-protamine 2 reactive proteins with electrophoretic mobilities similar to protamine 2 precursors. Data from PIXE show elevated levels of sulfur within sperm heads compared with sperm from the fertile male. Western blot data exhibit no evidence of protamines or protamine 2 precursors in the second semen sample produced by the infertile male. Data from PIXE suggest that these sperm are highly deficient in sulfur and protamines. These results show that the degree of maturation of sperm cells present in the semen of some infertile males can vary with time.

DNA↗
PubMed1995

Complexes of a modified low-molecular-weight heparin with protamine are predominantly cleared by a macrophage scavenger receptor-mediated process in rats.

Neutralization of heparin with its antidote protamine is associated with side effects such as pulmonary hypertension. The pharmacodynamic effects of protamine treatment are well documented. However, little is known about metabolic fate of heparin-protamine complexes. Twenty Wistar rats received a 131I-labeled low-molecular-weight heparin tracer intravenously. Four groups of animals were formed: a control group receiving the tracer, a second group receiving protamine after tracer application, a third group receiving maleylated bovine serum albumin (mal-BSA) prior to tracer and protamine injections to inhibit scavenger receptors of the reticuloendothelial system, and the last group receiving preformed heparin-protamine aggregates. All animals were examined by scintigraphy. Blood and tissue samples were analyzed for radioactivity. Protamine injection 2 min after heparin tracer application lead to a rapid decline in blood radioactivity. The radioactivity in the liver increased from 17% for the control to 43% after protamine application. Injection of mal-BSA prior to protamine prevented tracer accumulation in the liver and increased urine excretion (34% versus 20%). In vitro preformed heparin-protamine precipitates were rapidly trapped in the liver. We present evidence that, like the polyanionic heparin, polyanionic heparin-protamine complexes are phagocytosed by a scavenger receptor-mediated mechanism by macrophages, predominantly in the liver. The amount, the size, and the charge density of the complexes might trigger a mediator release from macrophages leading to phenomena such as pulmonary hypertension.

Animals↗
PubMed1990

Vertebrate protamine gene evolution I. Sequence alignments and gene structure.

The availability of the amino acid sequence for nine different mammalian P1 family protamines and the revised amino acid sequence of the chicken protamine galline (Oliva and Dixon 1989) reveals a much close relationship between mammalian and avian protamines than was previously thought (Nakano et al. 1976). Dot matrix analysis of all protamine genes for which genomic DNA or cDNA sequence is available reveals both marked sequence similarities in the mammalian protamine gene family and internal repeated sequences in the chicken protamine gene. The detailed alignments of the cis-acting regulatory DNA sequences shows several consensus sequence patterns, particularly the conservation of a cAMP response element (CRE) in all the protamine genes and of the regions flanking the TATA box, CAP site, N-terminal coding region, and polyadenylation signal. In addition we have found a high frequency of the CA dinucleotide immediately adjacent to the CRE element of both the protamine genes and the testis transition proteins, a feature not present in other genes, which suggests the existence of an extended CRE motif involved in the coordinate expression of protamine and transition protein genes during spermatogenesis. Overall these findings suggest the existence of an avian-mammalian P1 protamine gene line and are discussed in the context of different hypotheses for protamine gene evolution and regulation.

Amino Acid Sequence↗
PubMed1984

Protamine-DNA association in mammalian spermatozoa.

We have previously identified two subsets of basic nuclear proteins of mouse sperm: the protamines and a group of less basic proteins and, with the aid of a polyvalent antiserum, we have demonstrated their differential extractibility by NaCl in reducing solution (Rodman et al., J cell sci 53 (1982) 227) [9]. By affinity purification with isolated mouse sperm protamines we have obtained a protamine-specific fraction of that antiserum and a fraction that contains antibodies to the subset of less basic proteins. With those immunochemical probes we have shown the following The antigenic sites recognized by the protamine-specific antibodies are accessible, intranuclearly, only after the DNA has been removed by DNase I. The antibodies and DNA compete for binding sites on the protamines. DNA removal and consequent availability of the antigenic sites of the protamine molecules to the antibodies are possible only after displacement of the less basic proteins and chromatin decondensation have been induced. Immunoreactivity by the less basic proteins takes place without intervention of DNase. Those data indicate that the protamines are DNA-bound but that the less basic proteins are not or, alternatively, their putative DNA-binding sites do not coincide with their immuno-reactive sites. Those data also suggest that a function of the subset of less basic proteins may be to provide a shield for the protamine-DNA complex. The mouse protamine-affinity-bound antibodies are highly cross-reactive with protamines of other mammalian sperm suggesting that, despite considerable molecular diversity among mammalian protamines, the DNA-binding sites are conserved.

Animals↗
PubMed1988

Anticoagulant effects of protamine sulfate in a canine model.

Protamine sulfate is considered a weak anticoagulant, yet little is known concerning the mechanism of this effect or its relation to prior heparin exposure. This investigation defined the influence of increasing doses of protamine, with and without prior heparin anticoagulation, on the activated clotting time (ACT), thrombin clotting time (TCT), prothrombin time (PT), partial thromboplastin time (PTT), fibrinogen level, platelet count, and platelet aggregation to ADP in dogs (n = 8). Four doses of intravenous protamine sulfate (1.5, 3.0, 6.0, and 15.0 mg/kg) were studied in each animal, with at least 5 days between individual studies. Four dogs received heparin, 150 IU/kg 10 min prior to protamine sulfate administration, and four dogs received protamine sulfate alone. Protamine sulfate caused anticoagulation, both in the presence and absence of heparin, with significant changes occurring in the ACT, PTT, platelet count, and platelet aggregation. Relevant changes did not occur in the TCT, PT, or fibrinogen levels. Platelet effects were capable of causing bleeding with standard or excess use of protamine sulfate, especially if platelet numbers were already decreased, as might occur in surgical procedures where thrombocytopenia commonly accompanies major blood loss and replacement. The ACT, reflecting both the coagulation cascade and platelet function, was the test most profoundly affected by protamine overdosage, and therefore may be misleading as a measure of protamine reversal of heparin. The TCT, which is sensitive to heparin anticoagulation but not protamine-induced anticoagulation, should be more accurate in differentiating inadequate heparin reversal from the effects of excess protamine.

Animals↗
PubMed1999

Administration of protamine after coronary stent deployment.

BACKGROUND: Prompt reversal of anticoagulation by protamine administration could be an important therapeutic option to treat serious procedural complications such as vessel rupture or major bleeding from the puncture site during coronary stent implantation. However, this therapeutic option is rarely used because of the possible risk of stent thrombosis. METHODS: We retrospectively analyzed the incidence of acute and subacute stent thrombosis and vascular complications in 90 patients who received protamine (protamine group) and 1763 patients who did not receive protamine (control group) after successful coronary stent implantation. The 2 groups were matched for clinical, angiographic, and procedure characteristics. RESULTS: No patients in the protamine group had adverse effects such as hypotension or vascular collapse during protamine administration. Acute stent thrombosis did not occur in any protamine group patient but did occur in 12 patients in the control group (0.7%) (P =.47). Subacute stent thrombosis occurred in 2 patients in the protamine group (2.1%) and in 15 in the control group (0.8%) (P =.20). By logistic regression analysis, protamine was not a determinant of stent thrombosis. CONCLUSIONS: Reversal of anticoagulation by protamine after stent implantation does not predispose to stent thrombosis. This result has important clinical consequences because it allows the use of protamine in the treatment of coronary perforation and serious bleeding complications that may occur during coronary stent deployment.

Aged↗
PubMed1997

Differential effects of novel protamine variants on myocyte contractile function with left ventricular failure.

BACKGROUND: Protamine administration can cause left ventricular (LV) dysfunction, which may have clinical significance in the setting of congestive heart failure (CHF). Protamine variants have recently been constructed with heparin reversal capacity similar to protamine. The purpose of this study was to examine the potential differential effects of these protamine variants on isolated myocyte contractile function in normal myocytes and in myocytes after the development of CHF. METHODS: Contractile function was measured by means of computer-aided videomicroscopy in myocytes from five normal pigs and five pigs with CHF induced by rapid pacing (240 beats/min for 3 weeks). Myocyte contractility was examined in the presence of 40 micrograms/ml native protamine or one of three protamine variants: (1) reduced charge (+18) and lysine substituted for arginine; (2) lysine-substituted variant with glutamic acid substituted for the initial proline; or (3) arginine-rich peptide with a terminal arginine-glycine-aspartic acid (RGD) amino acid sequence. RESULTS: In the presence of native protamine, myocyte percent shortening fell from baseline in both the normal (2.86 +/- 0.15 versus 4.58 +/- 0.08, p < 0.05) and the CHF groups (1.01 +/- 0.06 versus 2.07 +/- 0.05, p < 0.05). With both of the lysine-substituted protamine variants, percent shortening fell from baseline in the normal group (3.42 +/- 0.20 for arginine and 3.74 +/- 0.20 for glutamic acid versus 4.58 +/- 0.08, p < 0.05), and was unchanged in the CHF group (1.94 +/- 0.13 versus 2.07 +/- 0.05, p = 0.34 for arginine; and 1.96 +/- 0.10 versus 2.07 +/- 0.05, p = 0.31, for glutamic acid). However, with the arginine/RGD variant, percent shortening fell from baseline in both the normal (2.86 +/- 0.23 versus 4.58 +/- 0.08, p < 0.05) and the CHF groups (1.32 +/- 0.10 versus 2.07 +/- 0.05, p < 0.05). CONCLUSIONS: Specific changes in the primary and secondary structures of protamine had different effects on myocyte contractile function. Furthermore, the negative effects of lysine-substituted protamine variants on myocyte contractility were less pronounced in both CHF and normal myocytes. Thus protamine variants may be of clinical use, particularly in the setting of preexisting LV dysfunction.

Animals↗
PubMed1995

Effective and less toxic reversal of low-molecular weight heparin anticoagulation by a designer variant of protamine.

PURPOSE: This investigation assessed protamine reversal of heparin anticoagulation by formation of a protamine-heparin alpha-helix by use of a new designer-variant protamine [+18BE] that was made from an existing protamine variant [+18B] whose non-alpha-helix-forming amino acid proline (P) was replaced by an alpha-helix-forming glutamic acid (E). The rate of administration of the new [+18BE] variant protamine on efficacy and toxicity in comparison to that of [+21] standard protamine and [+18B] was also studied. METHODS: Acetyl-EAA(K2A2K2A)4K2-Amide [+18BE] was administered intravenously in a 1:1 dose to low-molecular-weight heparin (LMWH)-anticoagulated (intravenous 150 IU antifactor Xa/kg) dogs over 10 seconds or 3 minutes (n = 7, each group). Reversal efficacy was documented by measuring activated clotting time, thrombin clotting time, antifactor Xa, and antifactor IIa. Toxicity was defined by measuring systemic blood pressure, heart rate, cardiac output, pulmonary artery pressure, and oxygen consumption. Measurements were made at baseline, after administration of LMWH, before its reversal, and for 30 minutes thereafter. Results were compared with those after LMWH reversal with [+21] standard protamine and the [+18B] variant. A total toxicity score (TTS) was calculated for each compound from maximal declines in blood pressure, heart rate, cardiac output, and oxygen consumption. RESULTS: LMWH anticoagulation reversal was significantly (p < 0.01) less toxic over 10 seconds and 3 minutes with the [+18BE] designer variant (TTS -2.3, -2.2) compared with the [+21] standard protamine (TTS -6.4, -7.2). Percent LMWH reversal at 3 minutes revealed [+18BE] to have antifactor Xa activity as high as 91%, compared with 68% for protamine [+21], when given over 3 minutes (p < 0.05). CONCLUSIONS: This investigation documents that a new designer variant of protamine [+18BE] has superior efficacy compared with [+21] standard protamine for reversal of LMWH anticoagulation and that this occurs with a highly favorable toxicity profile.

Amino Acid Sequence↗
PubMed1997

A novel protamine variant reversal of heparin anticoagulation in human blood in vitro.

PURPOSE: Protamine reversal of heparin anticoagulation during cardiovascular surgery may cause severe hypotension and pulmonary hypertension. A novel protamine variant, [+18RGD], has been developed that effectively reverses heparin anticoagulation without toxicity in canine experiments. Heretofore, human studies have not been undertaken. This investigation hypothesized that [+18RGD] would effectively reverse heparin anticoagulation of human blood in vitro. METHODS: Fifty patients who underwent anticoagulation therapy during vascular surgery had blood sampled at baseline and 30 minutes after receiving heparin (150 IU/kg). Activated clotting times were used to define specific quantities of [+18RGD] or protamine necessary to completely reverse heparin anticoagulation in the blood sample of each patient. These defined amounts of [+18RGD] or protamine were then administered to the heparinized blood samples, and percent reversals of activated partial thromboplastin time, thrombin clotting time, and antifactor Xa/IIa levels were determined. In addition, platelet aggregation assays, as well as platelet and white blood cell counts were performed. RESULTS: [+18RGD] and protamine were equivalent in reversing heparin as assessed by thrombin clotting time, antifactor Xa, antifactor IIa levels, and white blood cell changes. [+18RGD], when compared with protamine, was superior in this regard, as assessed by activated partial thromboplastin time (94.5 +/- 1.0 vs 86.5 +/- 1.3% delta, respectively; p < 0.001) and platelet declines (-3.9 +/- 2.9 vs -12.8 +/- 3.4 per mm3, respectively; p = 0.048). Platelet aggregation was also decreased for [+18RGD] compared with protamine (23.6 +/- 1.5 vs 28.5 +/- 1.9%, respectively; p = 0.048). CONCLUSIONS: [+18RGD] was as effective as protamine for in vitro reversal of heparin anticoagulation by most coagulation assays, was statistically more effective at reversal than protamine by aPTT assay, and was associated with lesser platelet reductions than protamine. [+18RGD], if less toxic than protamine in human beings, would allow for effective clinical reversal of heparin anticoagulation.

Aged↗
PubMed1989

Activated partial thromboplastin time-protamine dose relation in the presence and absence of heparin.

A protamine titration is one of the methods to determine the protamine dose necessary to neutralize heparin. The protamine dose response was studied with the activated partial thromboplastin time (APTT) in the presence of a known amount of heparin and in the absence of heparin, using freshly prepared human plasma. When heparin was present in the plasma, APTT values plateaued between a minimal neutralizing dose of protamine and a protamine dose five times greater. At doses above the APTT plateau, protamine exerted its own anticoagulant action as evidenced by an increase in APTT values. In the absence of heparin, APTT did not change until the protamine concentration reached 50 micrograms/mL. Then the APTT began to increase above this critical concentration. The increases in APTT values caused by an increase of 50 micrograms/mL of protamine were significantly greater without heparin than they were in the presence of heparin. These results suggest that protamine has a wide safety range when neutralizing heparin without exerting its own anticoagulant action. Although the mechanisms are under speculation, the heparin-protamine complex may inhibit the anticoagulant action of protamine in vitro.

Blood Coagulation↗
PubMed1998

Protamine inhibits angiogenesis and growth of C6 rat glioma; a synergistic effect when combined with carmustine.

Protamine inhibits angiogenesis and blocks endothelial, fibroblast and platelet growth factors. Human and experimental gliomas spread and grow in response to both paracrine and autocrine release of these factors. Our objective was to study the effect of protamine administration on cell proliferation, angiogenesis and tumoral growth of C6 glioma. Additionally, we compared the antitumoral effect of protamine with that of another inhibitor of angiogenesis, suramin, and investigated a potential synergistic antitumoral action of low doses of protamine combined with the antineoplastic carmustine. C6 glioma cells were implanted subcutaneously in Wistar rats. A highly malignant glioma developed in 80% of animals; when the tumour reached a diameter of 1.5 cm, either protamine, suramin, carmustine or protamine plus carmustine were administered in various doses. Tumour parameters were measured and compared between groups. In a dose-dependent manner, protamine reduced tumour volume (P < 0.001), mitotic index (P < 0.05), vascular density (P < 0.05) and cell viability (P < 0.005) of C6 glioma. An ultrastructural study demonstrated membranous inclusions in the cytoplasm of 28% of tumoral and endothelial cells of tumours from animals treated with protamine. The inhibition of tumoral growth produced by moderate doses of protamine was similar to that produced by toxic doses of suramin. The combination of protamine and carmustine had a synergistic curtailing effect on tumoral growth (P < 0.001). Our results indicate that protamine is an effective agent against glioblastoma; in non-toxic doses it could potentiate the antineoplastic effect of nitrosoureas for the treatment of glial tumours.

Animals↗
PubMed1997

Measurement of fluorescent-labeled LMM-heparin in biological fluids using protamine-linked microbeads.

A quantitative assay for fluorescent heparin in a purified system and in plasma was developed (Piazolo et al: Semin Thromb Hemostas 20:227-235, 1994). The protamine microbeads (1.6 microns) showed a broad size distribution and a large standard variation in low concentrations. Our aim was to optimize these protamine microbeads for the measurement of fluorescent heparin. The following results were obtained: Paramagnetic protamine microbeads of different average diameters (0.8, 1.6, 2.8, and 4.5 microns) were synthesized by cyclocarbodiimide and tosyl activation. These microbeads bind heparin and are assayed using flow cytometry. The protamine concentration on the surface of the beads ranged between 2.0 and 61 mg/mL. The protamine microbeads bound fluorescent heparin and were analyzed by flow cytometry. The protamine microbeads bound LMM-heparin-tyramine-FITC dose dependently in saline solution, plasma, and blood. There are substantial differences between the microbeads of different origins with regard to the amount of protamine bound, the sensitivity of the detection, and the reliability for the determination of heparins in plasma and blood. The minimal sensitivity of the final method was 0.001 U/mL LMMH-tyramine-FITC in saline solution and in plasma. Human blood cells were not bound to protamine microbeads. The half-maximal binding of LMMH-tyramine-FITC of the different protamine-coated microbeads ranged from 1.7 to 8.0 micrograms/mL in saline solution, 2.3 to 8.7 micrograms/mL in plasma, and 3.1 to 6.4 micrograms/mL in blood. We conclude that all protamine microbeads can be used to quantify the concentration of LMMH-tyramine. Protamine Dynabeads M-450 (diameter 4.5 microns) have advantages over other microbeads because of their more homogeneous size distribution, a higher selectivity, and they can be measured together with leukocytes. They are currently used to develop a competitive binding assay for heparin in plasma.

Binding, Competitive↗
PubMed2004

Formation of native-like mammalian sperm cell chromatin with folded bull protamine.

The DNA of most vertebrate sperm cells is packaged by protamines. The primary structure of mammalian protamine I can be divided into three domains, a central DNA binding domain that is arginine-rich and amino- and carboxyl-terminal domains that are rich in cysteine residues. In native bull sperm chromatin, intramolecular disulfide bonds hold the terminal domains of bull protamine folded back onto the central DNA binding domain, whereas intermolecular disulfide bonds between DNA-bound protamines help stabilize the chromatin of mature mammalian sperm cells. Folded bull protamine was used to condense DNA in vitro under various solution conditions. Using transmission electron microscopy and light scattering, we show that bull protamine forms particles with DNA that are morphologically similar to the subunits of native bull sperm chromatin. In addition, the stability provided by intermolecular disulfide bonds formed between bull protamine molecules within in vitro DNA condensates is comparable with that observed for native bull sperm chromatin. The importance of the bull protamine terminal domains in controlling the bull sperm chromatin morphology is indicated by our observation that DNA condensates formed under identical conditions with a fish protamine, which lacks cysteine-rich terminal domains, do not produce as uniform structures as bull protamine. A model is also presented for the bull protamine.DNA complex in native sperm cell chromatin that provides an explanation for the positions of the cysteine residues in bull protamine that form intermolecular disulfide bonds.

Amino Acid Sequence↗
PubMed2005

Effects of protamine and heparin can be detected and easily differentiated by modified thrombelastography (Rotem): an in vitro study.

BACKGROUND: Precise coagulation monitoring might help prevent heparin-protamine mismatch and thus decrease postoperative blood loss. We therefore measured coagulation time (CT) by modified thrombelastography (Rotem) as a possible differential monitor of the effects of heparin and protamine. METHODS: Undiluted and diluted blood samples from 26 healthy volunteers were spiked with increasing concentrations of heparin (0.1, 0.2, 0.4, 0.8 and 1 U ml(-1)). In addition, undiluted blood was spiked with protamine hydrochloride (0.1, 0.2, 0.4, 0.8 and 1.6 U ml(-1)), and we tested the effect of protamine on the reversal of heparin 0.4 U ml(-1). Heparin-containing samples were analysed using the heparin-sensitive INTEM test and the heparinase-containing HEPTEM test; protamine series were also analysed with the EXTEM test (tissue factor activation). RESULTS: CT by the INTEM test [CT-INTEM; median (min/max)] increased significantly and dose-dependently with increasing concentrations of heparin [control, 175 s (146/226); heparin, 1.0 U ml(-1) 1320 s (559/2100); P<0.001] and protamine [control, 172 s (150/255); protamine, 1.6 U ml(-1) 527 s (300/1345); P<0.0001]. Up to heparin concentrations of 0.4 U ml(-1), results were similar in undiluted and diluted blood samples. As expected, CT-HEPTEM remained within the normal range for all tested heparin concentrations (median 180-183 s), but increased similarly to CT-INTEM for increasing protamine concentrations. CONCLUSION: CT measurement using the Rotem technique appears to be a valuable tool for heparin-protamine management. For detection of heparin alone, protamine alone and the two combined, the ratio of CT-INTEM:CT-HEPTEM can be used to distinguish the effects of heparin excess (CT-INTEM:CT-HEPTEM>1) from those of protamine excess (CT-INTEM:CT-HEPTEM=1).

Adult↗
PubMed2006

Protamines and male infertility.

Protamines are the major nuclear sperm proteins. The human sperm nucleus contains two types of protamine: protamine 1 (P1) encoded by a single-copy gene and the family of protamine 2 (P2) proteins (P2, P3 and P4), all also encoded by a single gene that is transcribed and translated into a precursor protein. The protamines were discovered more than a century ago, but their function is not yet fully understood. In fact, different hypotheses have been proposed: condensation of the sperm nucleus into a compact hydrodynamic shape, protection of the genetic message delivered by the spermatozoa, involvement in the processes maintaining the integrity and repair of DNA during or after the nucleohistone-nucleoprotamine transition and involvement in the epigenetic imprinting of the spermatozoa. Protamines are also one of the most variable proteins found in nature, with data supporting a positive Darwinian selection. Changes in the expression of P1 and P2 protamines have been found to be associated with infertility in man. Mutations in the protamine genes have also been found in some infertile patients. Transgenic mice defective in the expression of protamines also present several structural defects in the sperm nucleus and have variable degrees of infertility. There is also evidence that altered levels of protamines may result in an increased susceptibility to injury in the spermatozoan DNA causing infertility or poor outcomes in assisted reproduction. The present work reviews the articles published to date on the relationship between protamines and infertility.

Amino Acid Sequence↗
PubMed2007

Altered protamine expression and diminished spermatogenesis: what is the link?

During the elongating spermatid stage of spermiogenesis, human sperm chromatin undergoes a complex transition in which histones are extensively replaced by protamines in a carefully regulated transition including histone modifications and intermediate and temporary replacement of the histones by sperm-specific transition proteins. The replacement of most histones by protamines 1 and 2 facilitates a high order of chromatin packaging necessary for normal sperm function and may also be necessary for DNA silencing and imprinting changes within the sperm cell. Protamines 1 and 2 are usually expressed in nearly equal quantities, but elevated or diminished protamine 1/protamine 2 ratios are observed in some infertile men and is often associated with severe spermatogenesis defects. Human and animal studies demonstrate that expression of the protamine proteins is uniquely regulated by transcription/translation factors, including storage of the mRNA in ribonucleoprotein (RNP) particles composed of the mRNA, transcription factors and a kinesin molecule necessary for transport of the RNP to the cytoplasm and removal of transcriptional activators from the nucleus. Recent studies indicate that most patients with abnormal protamine protein levels have elevated levels of protamine transcript in the mature sperm cell, indicating a possible defect in transcription or translation. The regulation of protamine expression is unique and includes several possible mechanisms which may be responsible for dysregulation of protamine expression and concurrent broad spectrum defects in spermatogenesis. We suggest two hypotheses: (i) that abnormal protamine expression is indicative of a generalized defect in mRNA storage and/or translation which affects other mRNA transcripts or (ii) that protamines may act as a checkpoint of spermatogenesis.

Animals↗
PubMed1994

Identification of phosphoseryl residues in protamines from mature mammalian spermatozoa.

Protamines isolated from ejaculated human, stallion, bull, boar, and ram spermatozoa were subjected to phosphoserine conversion reaction and protein sequencing. Phosphoserines were detected as S-ethylcysteines. Endogenously phosphorylated protamines have previously been found only in ejaculated human sperm. In this study, we demonstrate that ejaculated sperm from other species also contain protamines phosphorylated at serine residues. In P1-protamines, the endogenously phosphorylated serines were located at the N-terminal region in all species studied, whereas in major forms of human and stallion P2-protamines, the serine residues located in the middle region of the molecule were predominantly phosphorylated. These results support the current DNA binding model in the case of the P1-protamines. The internal location of the phosphorylated serines in the P2-protamines indicates, however, that the binding of these proteins to DNA or their interaction with other protamine molecules may differ from that of P1-protamines. This also suggests that, during sperm maturation, P2-protamines may have a role different from that of P1-protamines.

Amino Acid Sequence↗