Seasonal dependence of cadmium molecular effects on Mytilus galloprovincialis (Lamarck, 1819) protamine‐like protein properties

Mussels have a seasonal reproduction and cadmium is a common stressor in estuarine and coastal environments. In previous studies, we have shown that exposure to subtoxic doses of cadmium produced alterations in the properties of winter Mytilus galloprovincialis sperm protamine‐like (PL) proteins. In this study, it was analyzed the possibility that these cadmium effects may be seasonal. Winter and summer mussels were exposed to CdCl₂, and it was tested the PL‐proteins for cadmium bioaccumulation, electrophoretic pattern, DNA binding, and potentiality to induce DNA oxidative damage. It was found that cadmium exposure did not produce the same effects on PL‐proteins of summer mussels that were produced on PL‐proteins of winter mussels, that is: cadmium bioaccumulation, alterations in the acetic acid‐urea polyacrylamide gels (AU‐PAGE) and sodium dodecyl sulfate‐PAGE pattern, a reduced DNA binding affinity and the ability to induce DNA oxidative damage. PL‐proteins from summer mussels, apart from not being affected by all the abovementioned effects of cadmium, also showed a very low DNA binding affinity, independent of cadmium exposure. This study reveals clock‐associated seasonal responses to cadmium in M. galloprovincialis. Understanding the mechanisms through which environmental signals guide biological rhythms is fundamental to understanding the seasonal sensitivity of this bioindicator, to use M. galloprovincialis in appropriate seasonal periods.     

Molecular effects of copper on the reproductive system of mytilus galloprovincialis

This study aims to assess the effects induced by 24 hr exposure to a subtoxic copper concentration on the reproductive system (gonads, spermatozoa, and protamine‐like [PL] proteins) of Mytilus galloprovincialis. Inductively coupled plasma–mass spectrometry indicated accumulation of this metal in gonads, spermatozoa, and PL proteins of exposed mussels. Further, real‐time polymerase chain reaction analyses showed altered expression levels of mt10 and PL proteins genes in spermatozoa and gonads, respectively, of exposed mussels. Protamine‐like proteins, which represent the main basic component of sperm chromatin of this organism, showed a higher DNA binding affinity and a different DNA binding mode in exposed mussels. Moreover, an increased amount of NaCl was required for the release from sperm nuclei of PL‐III, the main PL protein component. Finally, PL proteins extracted from exposed mussels promoted DNA oxidative damage in the presence of H ₂O _2. These results demonstrate that the tolerable copper amount could also affect the properties of PL proteins and determine the negative effects on the reproductive system of this organism. These analyses could be useful to develop quick and efficient chromatin‐based genotoxicity tests for pollution biomonitoring programs.     

Origin and evolution of chromosomal sperm proteins

In the eukaryotic cell, DNA compaction is achieved through its interaction with histones, constituting a nucleoprotein complex called chromatin. During metazoan evolution, the different structural and functional constraints imposed on the somatic and germinal cell lines led to a unique process of specialization of the sperm nuclear basic proteins (SNBPs) associated with chromatin in male germ cells. SNBPs encompass a heterogeneous group of proteins which, since their discovery in the nineteenth century, have been studied extensively in different organisms. However, the origin and controversial mechanisms driving the evolution of this group of proteins has only recently started to be understood. Here, we analyze in detail the histone hypothesis for the vertical parallel evolution of SNBPs, involving a “vertical” transition from a histone to a protamine‐like and finally protamine types (H → PL → P), the last one of which is present in the sperm of organisms at the uppermost tips of the phylogenetic tree. In particular, the common ancestry shared by the protamine‐like (PL)‐ and protamine (P)‐types with histone H1 is discussed within the context of the diverse structural and functional constraints acting upon these proteins during bilaterian evolution.     

Zinc is sufficiently abundant within mammalian sperm nuclei to bind stoichiometrically with protamine 2

Although studies have demonstrated that zinc can bind to sperm nuclear proteins, specifically protamine 2, it has not been shown that the metal is sufficiently abundant inside the sperm nucleus to interact stoichiometrically with these proteins. In this study proton-induced X-ray emission (PIXE) has been used to measure the amount of sulfur and zinc within the nuclei of individual sperm cells to infer the stoichiometry of zinc binding to protamine 2 in six species of mammal: bull, chinchilla, stallion, hamster, human, and mouse (protamine 2 comprises from 0% (bull) to 67% (mouse) of the protamine present in the sperm of these animals). Using the sulfur mass and electrophoretic data on the relative proportion of protamine 1 and protamine 2 in the sperm chromatin of these species, the protamine 1, protamine 2, and total protamine contents within each species sperm nuclei have been determined. The PIXE measurements reveal that the zinc content of the sperm nucleus varies proportionately with the protamine 2 content of sperm chromatin. PIXE analyses of hamster protamines extracted under conditions that appear to at least partially preserve zinc binding also confirm that the majority of the metal is bound to protamine. In five of the species examined, sufficient zinc is present for each protamine 2 molecule to bind one zinc. The results obtained for chinchilla sperm, conversely, indicate the chinchilla protamine 2 molecule may interact differently with zinc. Chinchilla sperm only contain enough zinc for one atom to be bound to two protamine 2 molecules.     

Electrophoretic analysis of nuclear proteins isolated from sperm of the black abalone Haliotis crackeroidii

Nuclear proteins isolated from spermatozoa of the abalone, Haliotis crackeroidii, after sonication and CTAB treatment were characterized electrophoretically. Mature, sonication-resistant sperm nuclei contain only protamine and lack detectable levels of somatic histone. The amino acid composition and size of this protamine suggests that its structure must be intermediate between fish and mammalian protamines.     

Deoxyribonucleoproteins of herring sperm nuclei. I. Chemical composition

The chemical composition of deoxyribonucleoproteins from herring sperm nuclei was analyzed and the results are summarized as follows: 1. Chemical analysis of nuclear proteins and nucleic acids revealed that arginine/P molar ratio in herring sperm nuclei is unity but the ratio of arginine residues in protamine to phosphorus in the total DNA is 0.86. 2. The deoxyribonucleoproteins were isolated and their composition showed that about 14% of the total DNA in herring sperm nuclei is free from protamine and is bound with nonprotamine proteins in the weight ratio of nonprotamine proteins to DNA of 0.25-0.30. The remaining 86% of the total DNA is combined mainly with protamine and a small amount of nonprotamine proteins; the weight ratios of protamine and nonprotamine proteins to DNA are 0.75 and 0.08, respectively. In the latter complex, the molar ratio of arginine residues in protamine to phosphorus in DNA is unity.     

Protamine-like sperm nuclear basic proteins in the primitive frog Ascaphus truei and histone reversions among more advanced frogs

Sperm nuclear basic proteins (SNBPs) that condense chromatin are very diverse. In animals, evolution of SNBPs has proceeded from lysine-rich histone H type in sponges to more arginine-rich protamine-like PL and protamine P types. Yet sporadic PL/P to H reversions are known to occur in both protostomes and deuterostomes. To determine why this is the case, we have examined SNBPs in eleven anuran species. We find that sperm of the primitive, internally fertilizing archeobatrachian frog A. truei (family Ascaphidae) has PL/P type (42 mol % arginine), with an electrophoretic profile similar to SNBPs in another archeobatrachian, externally fertilizing Leiopelma hochstetteri (family Leiopelmatidae). Cytochemistry of sperm nuclei in the advanced, externally fertilizing neobatrachian frogs Crinia signifera and C. deserticola (family Myobatrachidae) indicates that they have reverted to H type SNBPs. This is also known to be the case in externally fertilizing Rana (family Ranidae) and Silurana, a subgenus of Xenopus (family Pipidae). Such a trend, from PL/P type SNBPs in two archeobatrachians to sporadic reversions to H type in more advanced frogs, parallels the ultrastructural simplification from complex A. truei introsperm to neobatrachian aquasperm that Jamieson et al. (1993. Herpetologica 49:52-65) attribute as a secondary reversion to external fertilization.     

Basic Proteins of Plant Nuclei during Normal and Pathological Cell Growth

Histone proteins were studied by microphotometry of plant tissue sections stained with fast green at pH 8.1. For comparative purposes the Feulgen reaction was used for deoxyribose nuclei acid (DNA); the Sakaguchi reaction for arginine; and the Millon reaction for estimates of total protein. Analysis of Tradescantia tissues indicated that amounts of nuclear histone fell into approximate multiples of the gametic (egg or sperm) quantity except in dividing tissues, where amounts intermediate between multiples were found. In differentiated tissues of lily, corn, onion, and broad bean, histones occurred in constant amounts per nucleus, characteristic of the species, as was found also for DNA. Unlike the condition in several animal species, the basic proteins of sperm nuclei in these higher plants were of the histone type; no evidence of protamine was found. In a plant neoplasm, crown gall of broad bean, behavior of the basic nuclear proteins closely paralleled that of DNA. Thus, alterations of DNA levels in tumor tissues were accompanied by quantitatively similar changes in histone levels to maintain the same Feulgen/fast green ratios found in homologous normal tissues.     

Characterization of chromatin-condensing proteins during spermiogenesis in a neogastropod mollusc (Murex brandaris)

During the process of chromatin cndensation in the spermiogenesis of the neogastropod mollusc Murex brandaris, the nuclear protein complement undergoes a complex series of changes. These changes lead to the appearance of three small protamines in the ripe sperm nuclei. We have characterized this system electrophoretically and at the compositions with antibodies elicited against a specific spermatozoan protamine. Our results indicate that the complex pattern of chromatin condensation during spermiogenesis in this species (M. brandaris) may be modulated by a series of post-translational (and intranuclear) modifications of DNA-interacting proteins, such as precursors to the sperm protamines. The amino acid composition of each sperm protamine is remarkably simple (lys + arg + gly ≥96 mol%). This system of spermiogenic/spermatozoal proteins in the neogastropod M. brandaris clearly differs from that in patellogastropods and archaeogastropods, and it may be helpful in understanding evolutionary changes in the chromatin condensation pattern during the spermiogenesis of gastropod molluscs. © 1994 Wiley-Liss, Inc.     

Changes in basic chromosomal proteins during spermatogenesis in the mature rat.

Nuclei of the seminiferous epithelial cells of rat testis were filtered through glass wool to remove sperm heads, flagellae and late-stage spermatids and then centrifuged through sucrose gradients to yield three fractions. The cellular origins of the predominant nuclei in these fractions were identified through the kinetics of labeling with [³H]thymidine. The relative amounts of the different histone fractions changed during the various stages of spermatogenesis in an interesting and systematic manner. For example, the ratio of the trailing (acetylated) to the leading member of the histone F2a1 doublet was greater in spermatid nuclei than in nuclei of a fraction enriched in primary spermatocytes. Similarly, the ratio $\text{X}{}_1\text{F}\text{1}$ was greatest in spermatid nuclei. On the other hand, the ratio $\text{X}{}_3\text{F}\text{2b}$ was greater in the nuclei of pachytene-diplotene primary spermatocytes than in the fraction enriched in nuclei of spermatogonia and preloptotene primary spermatocytes.A basic protein fraction with some of the properties of a protamine was extracted from rat sperm heads and from the nuclei of spermatids. This protein fraction has high contents of arginine and cysteine (after reduction), and it appears to be identical with the protamine described by Kistler et al. In addition, a new protamine was isolated from rat sperm heads which has high arginine content but appears to be devoid of lysine and cyst(e)ine. Two other basic protein fractions with high electrophoretic mobilities were extracted with acid from the nuclei of testicular seminiferous epithetial cells without prior reduction. One of these proteins may be identical with the testis-specific protein of Kistler et al.     

In vitro decondensation of the sperm chromatin in Holothuria tubulosa (sea cucumber) not affecting proteolysis of basic nuclear proteins

Sea urchin and sea star oocyte extracts contain proteolytic activities that are active against sperm basic nuclear proteins (SNBP). This SNBP degradation has been related to the decondensation of sperm chromatin as a possible model to male pronuclei formation. We have studied the presence of this proteolytic activity in Holothuria tubulosa (sea cucumber) and its possible relationship with sperm nuclei decondensation. The mature oocyte extracts from H. tubulosa contain a proteolytic activity to SNBP located in the macromolecular fraction of the egg‐jelly layer. SNBP degradation occurred both on sperm nuclei and on purified SNBP, histones being more easily degraded than protein Ø_o (sperm‐specific protein). SNBP degradation was found to be dependent on concentration, incubation time, presence of Ca²⁺, pH, and this activity could be a serine‐proteinase. Thermal denaturalization of the oocyte extracts (80°C, 10–15 min) inactivates its proteolytic activity on SNBP but does not affect sperm nuclei decondensation. These results would suggest that sperm nuclei decondensation occurs by a mechanism different from SNBP degradation. Thus, the sperm nuclei decondensation occurs by a thermostable factor(s) and the removal of linker SNBP (H1 and protein Ø_o) will be a first condition in the process of sperm chromatin remodeling.     

The effect of temporal variability in salinity on the invasive red alga Gracilaria vermiculophylla

Non-native, invasive species are often characterized by being tolerant to environmental stressors, leaving them more fit relative to native species. The red alga Gracilaria vermiculophylla originates from the NW Pacific but has recently spread along the coastlines of Western Europe, where it has become abundant in many shallow, soft-bottom estuaries. Salinity is important for the local and regional distribution of algae. The distribution of G. vermiculophylla in Europe suggests that it thrives well in hyposaline environments and that it may be more fit than some native algae under such conditions. Little, however, is known about the ecophysiology of G. vermiculophylla and it is therefore difficult to predict its spread and future distribution. Laboratory experiments with G. vermiculophylla showed that steady-state salinity above 15?psu was optimal for growth and that the growth rate was reduced at salinities below 15?psu. Variable salinity reduced the growth rate and larger oscillations were more stressful than small ones. Exposure to very low salinity (0–5?psu) was stressful for the alga and algae exposed to these low levels for 2–4 days were unable to recover fully. Gracilaria vermiculophylla did not seem to perform better in hyposaline conditions than many native, estuarine species. The present distribution of G. vermiculophylla in Scandinavia can be explained well by its response to salinity, but this may not explain its present success relative to many naturally occurring algal species.     

Immunological evidence for a P2 protamine precursor in mature rat sperm.

High molecular weight proteins in Rattus norvegicus that are immunoreactive with an anti-protamine 2 specific antibody but not with an anti-protamine 1 specific antibody are described. These proteins were detected by coupling high-performance liquid chromatography (HPLC) with an enzyme-linked immunosorbent assay (ELISA). Briefly, following HPLC separation of rat sperm nuclear proteins, the HPLC fractions were probed with the antibodies. We estimate that the antibody probes are 100-1000 times more sensitive than UV absorbance measurements. Immunoblot analysis following acid-urea electrophoretic separation of rat sperm nuclear proteins, and of the HPLC fractions, also detected putative protamine 2 precursor proteins. The proteins reactive with the anti-protamine 2 antibody are most likely not mature protamine 2, since they were detected in a region of the chromatogram where we would not expect protamine 2 to migrate based on the chromatographic locations of human and mouse protamine 2. Likewise, the immunoblotting experiments demonstrated that the anti-protamine 2 antibody recognized proteins with slower electrophoretic mobilities than would be expected for a mature protamine 2. An anti-protamine 1 monoclonal antibody, Hup1N, that binds rat protamine 1 is also described. Hup1N allowed for identification of the HPLC fractions that contained rat protamine 1. Finally, we demonstrated that Hup1N binds protamine 1 from a large number of species, suggesting a conserved epitope for Hup1N.     

Heat shock proteins mediate trade‐offs between early‐life reproduction and late survival in Drosophila melanogaster

Ageing and the resulting increased likelihood mortality are the inescapable fate of organisms because selection pressures on genes that exert their function late in life is weak, promoting the evolution of genes that enhance early‐life reproductive performance at the same time as sacrificing late survival. Heat shock proteins (HSP) are known to buffer various environmental stresses and are also involved in protein homeostasis and longevity. The characteristics of genes for HSPs (hsp) imply that they affect various life‐history traits, which in turn affect longevity; however, little is known about the effects of hsp genes on life‐history traits and their interaction with longevity. In the present study, the effects of hsp genes on multiple fitness traits, such as locomotor activity, total fecundity, early fecundity and survival time, are investigated in Drosophila melanogaster Meigen using RNA interference (RNAi). In egg‐laying females, RNAi knockdown of six hsp genes (hsp22, hsp23, hsp67Ba, hsp67Bb, hsp67Bc and hsp27‐like) does not shorten survival but rather increases it. Knockdown of five of those genes on an individual basis reduces early‐life reproduction, suggesting that several hsp genes mediate the trade‐off between early reproduction and late survival. The data indicate a positive effect of hsp genes on early reproduction and also negative effects on survival time, supporting the antagonistic pleiotropic effects predicted by the optimality theory of ageing.     

Synthesis and processing of mammalian protamines and transition proteins

Mouse and rat seminiferous tubule fragment cultures were used to examine synthesis and processing of mammalian protamines and transition proteins. The tubule fragments were incubated with [³H]-arginine, [³H]-histidine, [³⁵S]-cysteine, or [³²P]-PO₄, and radiolabeled proteins were analyzed by acid/urea polyacrylamide gel electrophoresis and fluorography or autoradiography. Newly synthesized protamines were recovered from sonication-resistant nuclei (SRN) and could not be detected in cytoplasmic fractions, indicating that protamines are deposited into nuclei immediately after synthesis. Newly synthesized mouse protamine 1 (mP1) and the precursor to mouse protamine 2 (pre-mP2) migrated more slowly during electrophoresis than their predominant testicular forms, identified by staining with Coomassie blue R-250. Within 1 hour of synthesis, the electrophoretic mobilities of mP1 and pre-mP2 increased to match those of their predominant forms. These changes are consistent with initial charge-neutralizing modifications of the newly synthesized protamines, followed by removal of at least some of the modifying ligands, to unmask protamine basicity. Steady-state phosphorylation rates were high for rat protamine 1 (rP1) and were independent of phosphate content; both rP1 molecules of low and high phosphate content were rapidly phosphorylated. Pre-mP2-3, a major processing intermediate derived by proteolysis of pre-mP2, was also rapidly phosphorylated. Like the protamines, transition protein 2 (TP2) was rapidly phosphorylated and increased in electrophoretic mobility soon after synthesis. In contrast, transition protein 1 (TP1) was not phosphorylated and did not exhibit multiple electrophoretic forms. © 1994 Wiley-Liss, Inc.     

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. Mol. Reprod. Dev. 50:345–353, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

大鼠精核蛋白的纯化

用大鼠睾丸制备长形精子细胞核及用附睾制备的附睾精子核,用盐酸胍法提取总碱性蛋白（Ｔｏｔａｌ　ｂａｓｉｃ　ｐｒｏｔｅｉｎ,ＴＢＰ）。通过Ｓｅｐｈａｄｅｘ　Ｇ－１００分子筛层析,其第三峰用５％ＴＣＡ沉淀,得到纯大鼠精核蛋白（Ｒａｔ　ｐｒｏｔａｍｉｎｅ,ＲＰ）。ＲＰ也可用ＴＢＰ通过ＨＰＬＣ反相柱得到纯化,ＲＰ位于２６％－２８％乙腈梯度。     

Use of chromomycin A3 staining in bovine sperm cells for detection of protamine deficiency

Sperm chromatin integrity is essential for accurate transmission of male genetic information, and normal sperm chromatin structure is important for fertilization. Protamine is a nuclear protein that plays a key role in sperm DNA integrity, because it is responsible for sperm DNA stability and packing until the paternal genome is delivered into the oocyte during fertilization. Our aim was to investigate protamine deficiency in sperm cells of Bos indicus bulls (Nelore) using chromomycin A₃ (CMA₃) staining. Frozen semen from 14 bulls were thawed, then fixed in Carnoy's solution. Smears were prepared and analyzed by microscopy. As a positive control of CMA₃ staining, sperm from one bull was subjected to deprotamination of nuclei. The percentage of CMA₃-positive bovine sperm did not vary among batches. Only two bulls showed a higher percentage of CMA₃-positive sperm cells compared to the others. CMA₃ is a simple and useful tool for detecting sperm protamine deficiency in bulls.     

Sperm Nuclear Basic Proteins (SNBPs) of Agnathans and Chondrichthyans: Variability and Evolution of Sperm Proteins in Fish

We have characterized for the first time SNBPs from the hagfish Eptatratus stouti (Myxini) and the lamprey Lampetra tridentatus (Cephalaspidomorphi) and have found that histones are the major protein components of the sperm of these agnathans. We have also conducted a systematic analysis of SNBPs from different groups of chondrichthyan fishes, including the skate Raja rhina and seven species of sharks. Together with our previous data showing the sporadic nature of SNBP evolution in bony fish (Saperas, N., Ausio, J., Lloris, D. and Chiva, M. [1994] J. Mol. Evol. 39: 282–295), the present study provides a unique insight into the overall evolutionary complexity and variability of the nuclear sperm proteins of fishes. It would appear that despite the discontinuous evolution of these proteins, the macroevolutionary pattern of histone (H type) → protamine-like (PL type) → protamine (P type) has been conserved in fish evolution, as it has in the evolution of other Deuterostomes. Received: 11 June 1996 / Accepted: 6 August 1996     

Protamine precursors in human spermatozoa

Basic proteins isolated from human sperm nuclei are highly heterogeneous. Three groups of nuclear basic proteins have been characterized: somatic-type as well as testis-specific histones, protamines and basic proteins with an electrophoretic mobility which is intermediate between that of histones and that of protamines. Human protamines can be separated into 2 protein families with different amino acid composition and amino-acid sequence. Protamines HP1 differ in their degree of phosphorylation. Protamines HP2, 3 and 4 differ by their amino-terminal sequence. Intermediate basic proteins (HPI1, HPI2, HPS1, HPS2) share a common C-terminal sequence of 54 residues identical to the amino-acid sequence of protamine HP3; only their N-terminal regions are different. Taking into account these structural homologies, the intermediate basic protein HPI1 appears as a precursor of protamines HP2 and HP3.