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.     

Common phylogenetic origin of protamine-like (PL) proteins and histone H1: Evidence from bivalve PL genes

Sperm nuclear basic proteins (SNBPs) can be grouped into three main categories: histone (H) type, protamine (P) type, and protamine-like (PL) type. Protamine-like SNBPs represent the most structurally heterogeneous group, consisting of basic proteins which are rich in both lysine and arginine amino acids. The PL proteins replace most of the histones during spermiogenesis but to a lesser extent than the proteins of the P type. In most instances, PLs coexist in the mature sperm with a full histone complement. The replacement of histones by protamines in the mature sperm is a characteristic feature presented by those taxa located at the uppermost evolutionary branches of protostome and deuterostome evolution, while the histone type of SNBPs is predominantly found in the sperm of taxa which arose early in metazoan evolution; giving rise to the hypothesis that protamines may have evolved through a PL type intermediate from a primitive histone ancestor. The structural similarities observed between PL and H1 proteins, which were first described in bivalve molluscs, provide a unique insight into the evolutionary mechanisms underlying SNBP evolution. Although the evolution of SNBPs has been exhaustively analyzed in the last 10 years, the origin of PLs in relation to the evolution of the histone H1 family still remains obscure. In this work, we present the first complete gene sequence for two of these genes (PL-III and PL-II/PL-IV) in the mussel Mytilus and analyze the protein evolution of histone H1 and SNBPs, and we provide evidence that indicates that H1 histones and PLs are the direct descendants of an ancient group of "orphon" H1 replication-dependent histones which were excluded to solitary genomic regions as early in metazoan evolution as before the differentiation of bilaterians. While the replication-independent H1 lineage evolved following a birth-and-death process, the SNBP lineage has been subject to a purifying process that shifted toward adaptive selection at the time of the differentiation of arginine-rich Ps.     

The sperm proteins from amphioxus mirror its basal position among chordates and redefine the origin of vertebrate protamines

The sperm nuclear basic proteins (SNBPs) that participate in chromatin condensation in spermatozoa belong to 3 groups: histone (H), protamine-like (PL), and protamine (P) type. They share a common origin with histone H1 resulting from the segregation of PL components, corresponding to different regions of an H1 precursor molecule (N-terminal, winged-helix, C-terminal domains), becoming independent and following a subsequent process of parallel vertical evolution (H <--> PL <--> P). In the present work, we describe the sequence and primary structure of the main SNBP component in the sperm of the cephalochordate Branchiostoma floridae (amphioxus), revealing that it represents the deuterostome counterpart of the PL-III SNBP component from molluscs corresponding to the H1 N-terminal region. Until now, this has been a missing piece needed to complete the evolutionary history of SNBPs in metazoan genomes. The discovery of this PL lineage in deuterostomes definitively validates the parallel vertical evolution of SNBPs across metazoans, giving further support to the "basal" position of amphioxus among chordates, with respect to tunicates. Sequence analyses suggest that later on in evolution, the appearance of positively selected arginine-rich protamines, derived from the H1 C-terminal region, led to the extinction of this PL lineage in the genomes of early protostomes and deuterostomes. Given that tunicates are now viewed as a sister group of vertebrates, the lysine to arginine transition responsible for the origin of vertebrate protamines must be set a step back from tunicates.     

The Sperm Nuclear Basic Proteins (SNBPs) of the Sponge Neofibularia nolitangere: Implications for the Molecular Evolution of SNBPs

We have isolated and characterized for the first time, the SNBPs from an organism (Neofibularia nolitangere) of the phylum Porifera (Sponges). We have shown that these proteins consist of histones which, as expected, exhibit an amino acid composition very similar to that of other eukaryotic histones. The finding of histones in the sperm of these primitive organisms provides support to the notion that histones (SNBPs of the histone, H, type) were the proteins present at the onset of SNBP evolution. In contrast, a discrete number of alternative SNBP types (protamine-like, PL; protamine, P, types) seem to have appeared later on in the course of evolution and are found in both protostomes and deuterostomes, most likely as a result of processes of parallel evolution. Received: 5 March 1997 / Accepted: 6 March 1997     

A sperm nuclear basic protein from the sperm of the marine worm Chaetopterus variopedatus with sequence similarity to the arginine-rich C-termini of chordate protamine-likes

The sperm nuclear basic proteins (SNBPs) of the marine annelid worm Chaetopterus variopedatus have been shown previously to consist of a mixture of two SNBPs: histone H1-like (CvH1) and C.variopedatus protamine-like (CvPL). Here, we report the structural characterization of CvPL. The protein has a molecular weight of 8370.5 Da, a K/R ratio of 0.34, and a secondary structure, which are intermediate between those of protamine (P) and protamine-like (PL) SNBPs. The N-terminal sequence of CvPL shows a high extent of similarity with the arginine-rich C-terminal domain of chordate PL-type SNBPs. Furthermore, the protein binds to DNA in a similar fashion as vertebrate PLs and their own CvH1, but in a way that is different from that of the lysine-rich somatic H1 histones. We have experimentally determined the molar ratio CvH1:CvPL to be ～1:6 in C. variopedatus sperm. Based on all of these, a model is proposed for the organization of the sperm chromatin by CvH1 and CvPL.     

Sperm nuclear basic proteins of two closely related species of Scorpaeniform fish (Sebastes maliger, Sebastolobus sp.) with different sexual reproduction and the evolution of fish protamines

In this paper, we present a review of sperm nuclear basic proteins (SNBPs) in teleost fish. The distribution of the three basic groups of SNBPs [histone (H)-type, protamine-like (PL)-type and protamine (P)-type], their evolution and possible relation to the mode of fertilization are described. In this regard, we have characterized the SNBPs from two closely related species of Scorpaeniform fish: internally fertilizing Sebastes maliger and externally fertilizing Sebastolobus sp., both in the family Scorpaenidae. Despite the different reproductive behavior of these two closely related rockfish species, in both instances the SNBP consists of protamines. However, there is a significant increase in the arginine content of the protamine in the internally fertilizing rockfish. The relevance of this observation is discussed within the context of the P-type SNBP in teleosts. The rapid evolution of teleost protamines, including those in rockfish, has also allowed us to obtain a molecular phylogeny for this group of bony fish that is almost indistinguishable from that currently available from the use of conventional anatomical/paleontological markers.     

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     

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.     

Replacement of nucleosomal histones by histone H1-like proteins during spermiogenesis in Cnidaria: Evolutionary implications

We have analyzed the chromosomal protein composition of the sperm from several species belonging to three different classes (Hydrozoa, Scyphozoa, Anthozoa) of the phylum Cnidaria. In every instance, the sperm nuclear basic proteins (SNBPs) were found to consist of one to two major protein fractions that belong to the histone H1 family, as can be deduced from their amino acid composition and solubility in dilute perchloric acid, and the presence of a trypsin-resistant core. In those species where mature spawned sperm could be obtained, we were able to show that these proteins completely replace the somatic histones from the stem cells that are present at the onset of spermatogenesis. The presence of a highly specialized histone H1 molecule in the sperm of this phylum provides support for the idea that the protamine-like proteins (PL) from higher groups in the phylogenetic tree (and possibly protamines as well) may all have evolved from a primitive histone H1 ancestor.     

H2A.Bbd: an X-chromosome-encoded histone involved in mammalian spermiogenesis

Despite the identification of H2A.Bbd as a new vertebrate-specific replacement histone variant several years ago, and despite the many in vitro structural characterizations using reconstituted chromatin complexes consisting of this variant, the existence of H2A.Bbd in the cell and its location has remained elusive. Here, we report that the native form of this variant is present in highly advanced spermiogenic fractions of mammalian testis at the time when histones are highly acetylated and being replaced by protamines. It is also present in the nucleosomal chromatin fraction of mature human sperm. The ectopically expressed non-tagged version of the protein is associated with micrococcal nuclease-refractory insoluble fractions of chromatin and in mouse (20T1/2) cell line, H2A.Bbd is enriched at the periphery of chromocenters. The exceedingly rapid evolution of this unique X-chromosome-linked histone variant is shared with other reproductive proteins including those associated with chromatin in the mature sperm (protamines) of many vertebrates. This common rate of evolution provides further support for the functional and structural involvement of this protein in male gametogenesis in mammals.     

Characterization of the PL-I-related SP2 protein from Xenopus

The complete cDNA sequence of Xenopus laevis sperm specific proteins SP1 and SP2 has been determined. This information when taken together with N-terminal sequencing and mass spectroscopy data indicates that these two proteins share a product precursor relationship in which SP2 results from cleavage of a short N-terminal peptide of SP1. The secondary and tertiary structures of SP2 have been characterized using circular dichroism and three dimension structure prediction. These structural analyses have conclusively shown that SP1/SP2 proteins are related to proteins of the histone H1 family, particularly to vertebrate histone H1x. Hence, they can be considered bona fide members of the protamine-like- I (PL-I) group of sperm nuclear basic proteins (SNBPs) that have been described in other vertebrate and invertebrate groups. SP2 binds to nucleosomal DNA in a way that is very similar to that of histone H1. However, its interaction with circular DNA does not exhibit an enhanced preference for the supercoiled conformation, and it appears to be mainly driven by ionic interactions.     

Protamine-like proteins: evidence for a novel chromatin structure.

Protamine-like (PL) proteins are DNA-condensing proteins that replace somatic-type histones during spermatogenesis. Their composition suggests a function intermediate to that of histones and protamines. Although these proteins have been well characterized at the chemical level in a large number of species, particularly in marine invertebrates, little is known about the specific structures arising from their interaction with DNA. Speculation concerning chromatin structure is complicated by the high degree of heterogeneity in both the number and size of these proteins, which can vary considerably even between closely related species. After careful examination and comparison of the protein sequences available to date for the PL proteins, we propose a model for a novel chromatin structure in the sperm of these organisms that is mediated by somatic-type histones, which are frequently found associated with these proteins. This structure supports the concept that the PL proteins may represent various evolutionary steps between a sperm-specific histone H1 precursor and true protamines. Potential post-translational modifications and the control of PL protein expression and deposition are also discussed.     

Molecular effects on spermatozoa of Mytilus galloprovincialis exposed to hyposaline conditions

Salinity represents a critical environmental and an ecological factor in the reproduction of marine species. As global climate changes and anthropogenic factors affect salinity, in this study, we have analyzed the responses of Mytilus galloprovincialis spermatozoa to hyposaline stress. We exposed mussels, in laboratory tanks, for 24 hr at 18°C to control (35.9 psu) and three hyposaline (17.1, 22.6, and 26.2 psu) conditions, and evaluated the expression of sperm hsp70 and protamine‐like proteins genes. Further we analyzed the electrophoretic pattern, the DNA binding and the release from sperm nuclei of protamine‐like proteins. For all experimental approaches used, the results obtained at 17.1 psu condition were very similar to those obtained in the control condition, while alterations were always recorded at 22.6 and 26.2 psu conditions. Particularly, at 22.6 and 26.2 psu, was observed: 42.5‐ and 17.1‐fold increase in hsp70 expression, respectively, and hypoexpression of PL‐II/PLIV protamine‐like proteins genes. Further, electrophoretic mobility shift assays and salt‐induced release of nuclear proteins from sperm nuclei, revealed alterations in the PL proteins/DNA binding, in these two hyposaline conditions. The similarity between the results obtained in control and in the more severe hyposaline condition (17.1 psu) could indicate a phenomenon of fertility preservation strategy due to gamete plasticity.     

A unique vertebrate histone H1-related protamine-like protein results in an unusual sperm chromatin organization

Protamine-like proteins constitute a group of sperm nuclear basic proteins that have been shown to be related to somatic linker histones (histone H1 family). Like protamines, they usually replace the chromatin somatic histone complement during spermiogenesis; hence their name. Several of these proteins have been characterized to date in invertebrate organisms, but information about their occurrence and characterization in vertebrates is still lacking. In this sense, the genus Mullus is unique, as it is the only known vertebrate that has its sperm chromatin organized by virtually only protamine-like proteins. We show that the sperm chromatin of this organism is organized by two type I protamine-like proteins (PL-I), and we characterize the major protamine-like component of the fish Mullus surmuletus (striped red mullet). The native chromatin structure resulting from the association of these proteins with DNA was studied by micrococcal nuclease digestion as well as electron microscopy and X-ray diffraction. It is shown that the PL-I proteins organize chromatin in parallel DNA bundles of different thickness in a quite distinct arrangement that is reminiscent of the chromatin organization of those organisms that contain protamines (but not histones) in their sperm.     

The primary structure of a chondrichthyan protamine: A new apparent contradiction in protamine evolution

We have determined the primary structure of protamine R3 from ratfish (Hydrolagus colliei), a species belonging to the order Chimaeriformes (an old phylogenetic line among the chondrichthyes). Protamine R3 contains 48 residues organized as follows: ARRRH SMKKK RKSVR RRKTR KNQRK RKNSL GRSFK (Q/A)HGFL KQPPR FRP. Comparison of this sequence with both protamine Z3 fromScyliorhinus canicula (a chondrichthyan) and typical protamines from bony fish generates an apparent contradiction: Two relatively close species (H. colhei andS. canicula, both chondichthyes) display different protamines, whereas species more distant in evolution (S. canicula and bony fish) contain very similar protamine molecules. We note that this is not an isolated case in the evolution of sperm nuclear basic proteins (SNBPs) and discuss the possible significance of this fact. Correspondence to: M. Chiva     

Protamines in the internally fertilizing neobatrachian frog Eleutherodactylus coqui

The internally fertilizing primitive frog Ascaphus truei (family Ascaphidae) from the Pacific Northwest is the only frog with an intromittent organ. The more advanced neobatrachian frog Eleutherodactylus coqui (family Leptodactylidae) from Puerto Rico has secondarily acquired internal fertilization but mates by cloacal apposition. Nonetheless, both frogs have introsperm with an elongated head containing highly condensed chromatin. Characterization of sperm nuclear basic proteins (SNBPs) in E. coqui by acid-urea polyacrylamide gel electrophoresis indicates that, as in A. truei, testes from a single animal contain several protamines. Amino acid analysis indicates a composition for the most rapidly moving protamine of each species as follows: in E. coqui, ARG (35.6 mol %) + LYS (3.8 mol %) + HIS (7.6 mol %) = 47 mol % total basic residues and in A. truei, ARG (42.1 mol %) + LYS (11.1 mol %) = 53.2 mol % total basic residues. Transmission electron microscopy shows that E. coqui introsperm, like those in A. truei, are elongate with highly condensed chromatin. However, E. coqui introsperm lacks an axial perforatorium that extends into an endonuclear canal. These morphological features are plesiomorphic (primitive) and shared by A. truei with urodeles and basal amniotes (Jamieson et al. (1993) Herpetologica 49:52-65). In E. coqui introsperm, the nucleoprotein complex has a cross-sectional axis of 420 + 20 angstroms and shows a knobby chromatin structural organization in TEM. The presence of arginine-enriched protamines in both a basal anuran like the ascaphid A. truei and a more advanced neobatrachian like the leptodactylid E. coqui supports the hypothesis that internal fertilization acts as a constraint on the range of SNBP diversity in animals.     

Evidence for a shared structural role for HMG1 and linker histones B4 and H1 in organizing chromatin.

The high mobility group proteins 1 and 2 (HMG1/2) and histone B4 are major components of chromatin within the nuclei assembled during the incubation of Xenopus sperm chromatin in Xenopus egg extract. To investigate their potential structural and functional roles, we have cloned and expressed Xenopus HMG1 and histone B4. Purified histone B4 and HMG1 form stable complexes with nucleosomes including Xenopus 5S DNA. Both proteins associate with linker DNA and stabilize it against digestion with micrococcal nuclease, in a similar manner to histone H1. However, neither histone B4 nor HMG1 influence the DNase I or hydroxyl radical digestion of DNA within the nucleosome core. We suggest that HMG1/2 and histone B4 have a shared structural role in organizing linker DNA in the nucleosome.     

The essential role of Drosophila HIRA for de novo assembly of paternal chromatin at fertilization

In many animal species, the sperm DNA is packaged with male germ line–specific chromosomal proteins, including protamines. At fertilization, these non-histone proteins are removed from the decondensing sperm nucleus and replaced with maternally provided histones to form the DNA replication competent male pronucleus. By studying a point mutant allele of the Drosophila Hira gene, we previously showed that HIRA, a conserved replication-independent chromatin assembly factor, was essential for the assembly of paternal chromatin at fertilization. HIRA permits the specific assembly of nucleosomes containing the histone H3.3 variant on the decondensing male pronucleus. We report here the analysis of a new mutant allele of Drosophila Hira that was generated by homologous recombination. Surprisingly, phenotypic analysis of this loss of function allele revealed that the only essential function of HIRA is the assembly of paternal chromatin during male pronucleus formation. This HIRA-dependent assembly of H3.3 nucleosomes on paternal DNA does not require the histone chaperone ASF1. Moreover, analysis of this mutant established that protamines are correctly removed at fertilization in the absence of HIRA, thus demonstrating that protamine removal and histone deposition are two functionally distinct processes. Finally, we showed that H3.3 deposition is apparently not affected in Hira mutant embryos and adults, suggesting that different chromatin assembly machineries could deposit this histone variant.     

Loss of the cleaved-protamine 2 domain leads to incomplete histone-to-protamine exchange and infertility in mice

Protamines are unique sperm-specific proteins that package and protect paternal chromatin until fertilization. A subset of mammalian species expresses two protamines (PRM1 and PRM2), while in others PRM1 is sufficient for sperm chromatin packaging. Alterations of the species-specific ratio between PRM1 and PRM2 are associated with infertility. Unlike PRM1, PRM2 is generated as a precursor protein consisting of a highly conserved N-terminal domain, termed cleaved PRM2 (cP2), which is consecutively trimmed off during chromatin condensation. The carboxyterminal part, called mature PRM2 (mP2), interacts with DNA and together with PRM1, mediates chromatin-hypercondensation. The removal of the cP2 domain is believed to be imperative for proper chromatin condensation, yet, the role of cP2 is not yet understood. We generated mice lacking the cP2 domain while the mP2 is still expressed. We show that the cP2 domain is indispensable for complete sperm chromatin protamination and male mouse fertility. cP2 deficient sperm show incomplete protamine incorporation and a severely altered protamine ratio, retention of transition proteins and aberrant retention of the testis specific histone variant H2A.L.2. During epididymal transit, cP2 deficient sperm seem to undergo ROS mediated degradation leading to complete DNA fragmentation. The cP2 domain therefore seems to be a key aspect in the complex crosstalk between histones, transition proteins and protamines during sperm chromatin condensation. Overall, we present the first step towards understanding the role of the cP2 domain in paternal chromatin packaging and open up avenues for further research.