Meiotic chromatin diminution in a vertebrate, the holocephalan fish Hydrolagus collie (Chondrichthyes, Holocephali).

A histochemical, microdensitometric, and electron microscopic study of testes of the ratfish Hydrolagus colliei shows that an instance of the rare phenomenon of germ line chromatin diminution occurs in this vertebrate species. In primary spermatocytes at metaphase I a spherical mass of heterochromatin accumulates at one side of the metaphase plate. At anaphase I the heterochromatic mass is left in the equatorial cytoplasm and is passed into one of the two secondary spermatocytes formed during cytokinesis. As nuclear membranes are being restored, a double membrane envelope is also formed around the heterochromatic mass, which is then termed the 'chromatin diminution body' (CDB). At second meiotic division the CDB is included in the cytoplasm of one of the four spermatids and retained there, apparently unchanged, until mid-spermiogenesis. At that time the CDB becomes adherent to the spermatid plasma membrane and is pinched off from the spermatid by a process of apocrine exocytosis, taking a layer of spermatid plasma membrane along with it. Simultaneously this tri-membrane CDB is taken into the adjacent Sertoli cell by endocytosis, thereby acquiring a fourth membrane layer, a part of the Sertoli cell plasma membrane. The CDBs are subsequently phagocytized, possibly first fusing with dense, multilaminate bodies in the Sertoli cell cytoplasm. The CDB chromatin mass is strongly positive with the Feulgen method for DNA and the alkaline fast green method for histones. Microdensitometric analysis shows that the discarded chromatin amounts to about 10% of the diploid nuclear content and that it appears to be part of the normal diploid complement rather than DNA amplified during meiosis.

Dynamic aspects of spermiogenic chromatin condensation patterning by phase separation during the histone-to-protamine transition in charalean algae and relation to bryophytes.

During early-to-middle spermiogenesis in multicellular, internally fertilizing charalean green algae (Chara fibrosa, Chara vulgaris, Chara tomentosa, Nitella missouriensis), patterning of chromatin/nucleoplasm in developing spermatid nuclei changes from granules → fibers → contorted lamellae → condensed chromatin. Cytochemical, immunocytochemical, electrophoretic studies on C. vulgaris and C. tomentosa spermatids (Kwiatkowska, Poplonska) and amino acid analysis of protamines in Chara corallina sperm (Reynolds, Wolfe), indicate that more positively charged protamines replace histones directly during spermiogenesis, not indirectly through other intermediate transitional proteins as in internally fertilizing neogastropods and sharks with more ordered spermatid lamellae. We hypothesize that such lamellar-mediated patterning is due to liquid-liquid phase separation by spinodal decomposition. This is a spontaneous thermodynamic process that involves diffusive instability of a lamellar chromatin network, a dominant pattern repeat distance and bicontinuity of chromatin/nucleoplasm phases. C. vulgaris sperm show contorted lamellae in the posterior region, whereas C. corallina sperm display contorted peripheral lamellae and interior fibrils. Among internally fertilizing liverworts, which may have evolved from Zygnematales, mid-spermatid nuclei lack lamellae. Instead they display self-coiled chromatin rods in Blasia pusilla, contain short chromatin tubules in Haplomitrium hookeri resembling those in internally fertilizing mosses and a hornwort and indirectly replace histones with protamines in Marchantia polymorpha.

On the diversity of sperm histones in the vertebrates: II. A cytochemical study of the basic protein transitions during spermiogenesis in the cartilaginous fish Hydrolagus colliei.

Spermiogenesis in the ratfish (Hydrolagus colliei) is characterized by unusual changes in the basic proteins of the nucleus. Cytochemical analysis indicates that early spermatids in this cartilaginous fish contain the somatic type of histones. However, late spermatids contain the salmon type of sperm histone whereas spermatozoa display the mouse/grasshopper type designated by Bloch ('69). Such an unusual cytochemical shift from protamine in the spermatid to "stable protamine" in the sperm has not been documented heretofore.

Detection of sperm histone diversity among vertebrates by alkaline fast green staining.

Testis sections from fifteen species from six classes of vertebrates were stained with alkaline fast green (AFG) to correlate staining differences with the known biochemical diversity of histones in the spermatozoa. After trichloroacetic acid (TCA) hydrolysis the sperm of some species known to contain sperm-specific histones did not stain. This correlation held if fixation in neutral buffered formalin was limited to 3 to 6 hr and hydrolysis was done at 90 C. The species whose sperm did stain after TCA hydrolysis could be divided into three groups. In some species the sperm no longer stained if, after TCA, the sections were treated with thioglycollic acid. These sperm contained basic proteins that were rich in cysteine. In turn, the group of species whose sperm continued to stain after TCA and thioglycollic acid treatments could be subdivided. The sperm of some were stained specifically without DNA hydrolysis if the AFG was made up with sodium chloride. These sperm contained sperm-specific histones. In other species the sperm did not stain under these conditions, and these sperm had a basic protein complement similar to that found in somatic cell nuclei. These correlations suggest that AFG staining can be used to detect sperm histone diversity in a wide range of organisms.

On the diversity of sperm histones in the vertebrates. I. Changes in basic proteins during spermiogenesis in the newt Notophthalmus viridescens.

Sperm histones display great variability in contrast to the conservation of most classes of somatic histones. To study this paradox, this series of papers examines the variation of histone patterns in the testis and sperm of vertebrates, particularly amphibians and reptiles, and attempts to relate such variation to genetically based sex determination as hypothesized by Bloch [Genetics Supplement 61, 93 (1969)]. In the present study we have investigated spermiogenesis in the newt Notophthalmus viridescens viridescens. Cytochemical experiments indicate that the basic nuclear proteins undergo progressive shifts from somatic type histone leads to very arginine-rich "stable protamine" in the later spermatids leads to protamine in the mature sperm. Electrophoresis of Notophthalmus histones extracted from chromatin reveals that the pattern of testes specific basic proteins in the urodele is distinct from the pattern of testicular proteins in the anurans Bufo americanus and Xenopus laevis. Species within the class Amphibia therefore exhibit considerable diversification in their type of basic sperm proteins.

Particulate cytochromes of mung bean seedlings.

Efforts have been made to solubilize cytochrome components from particulate fractions of etiolated mung bean seedlings. Low temperature spectrophotometry reveals that the cytochrome composition of mitochondria isolated from whole seedlings is the same as that reported by Bonner for mung bean hypocotyls. On the basis of the identity in position of the alpha-bands in low temperature difference spectra for mitochondria, for a partially purified haemoprotein from mitochondria, and for purified cytochrome b-555, it is suggested that cytochrome b-555 is an intrinsic component of mung bean mitochondria. Difference spectra show that both the mitochondrial and microsomal fractions contain at least 2 b-type cytochromes. Cytochrome b-555 is almost certainly present in the microsomes, since the low temperature difference spectrum for the cytochrome is identical with the spectrum for this particulate fraction.By freezing and thawing mung bean mitochondria in 4% cholate and centrifuging, cytochrome oxidase activity can be concentrated in the supernatant fraction, although it is not completely solubilized. The oxidase is inhibited by high concentrations of cytochrome c. A particle-bound cytochrome c can be obtained from mitochondria by digestion with snake venom. However, the autoxidizability of the preparation indicates that the cytochrome has been solubilized in a modified form. A CO-binding pigment can be obtained from mung bean microsomes by digestion with snake venom.

On the diversity of sperm histones in the vertebrates: IV. Cytochemical and amino acid analysis in Anura.

The variability of sperm histones in frogs has been studied by cytochemical and amino acid analyses. Cytochemically, Rana sperm proteins fall into Bloch's ('69, '76) type 4 somatic-like histone category, while Xenopus and Bufo have type 3 intermediate sperm histones. Extractability in 5% trichloroacetic acid (TCA) at different temperatures splits this type 3 category into two groups: type 3B intermediate sperm histones of Bufo are extractable at 85-90 degrees C, while Xenopus intermediate type 3A sperm histones require temperatures of 95-100 degrees C for extraction. Amino acid analysis confirms that Rana sperm histones are of the nucleosomal type, with a testis-specific, very lysine-rich H1 histone. The sperm protein in Bufo is richer in arginine than the proteins in Xenopus. Both of these genera contain lysine and histidine as well as arginine in their sperm proteins. These results confirm earlier electrophoretic data (Kasinsky et al., '78) and indicate that sperm histones in the order Anura can vary markedly between different genera.

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 from Scyliorhinus canicula (a chondrichthyan) and typical protamines from bony fish generates an apparent contradiction: Two relatively close species (H. colliei and S. 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.

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.

Origin of H1 linker histones.

In which taxa did H1 linker histones appear in the course of evolution? Detailed comparative analysis of the histone H1 and histone H1-related sequences available to date suggests that the origin of histone H1 can be traced to bacteria. The data also reveal that the sequence corresponding to the 'winged helix' motif of the globular structural domain, a domain characteristic of all metazoan histone H1 molecules, is evolutionarily conserved and appears separately in several divergent lines of protists. Some protists, however, appear to have only a lysine-rich basic protein, which has compositional similarity to some of the histone H1-like proteins from eubacteria and to the carboxy-terminal domain of the H1 linker histones from animals and plants. No lysine-rich basic proteins have been described in archaebacteria. The data presented in this review provide the surprising conclusion that whereas DNA-condensing H1-related histones may have arisen early in evolution in eubacteria, the appearance of the sequence motif corresponding to the globular domain of metazoan H1s occurred much later in the protists, after and independently of the appearance of the chromosomal core histones in archaebacteria.

Sperm-Specific Basic Proteins in the Holocephalan Fish Hydrolagus colliei (Chondrichthyes, Chimaeriformes) and Comparison with Protamines from an Elasmobranch.

Seven basic proteins can be isolated from sperm nuclei of the holocephalan ratfish Hydrolagus colliei. Two of these proteins (R3 and m0) are devoid of cysteine, whereas five of them (R1, R2, m1, m2, and m3) contain low levels of this amino acid residue. The proteins R1, R2, and R3 are major ones in the sperm nuclei of H. colliei, and they are analogous to basic proteins Z1, Z2, and Z3 (scylliorhinines) from the sperm of the elasmobranch Scyliorhinus canicula. However, taking into account the partial sequence of R3 protein and the number of cysteines in R1 and R2, these proteins do not seem to be homologous to the scylliorhinines. A comparison of sperm basic proteins between H. colliei (a holocephalan) and S. canicula (an elasmobranch) suggests a remarkable divergence of these proteins from a common ancestral pattern during the evolution of Chondrichthyes.

Characterization of spermatid/sperm basic chromosomal proteins in the genus Xenopus (Anura, Pipidae).

Spermatid/sperm basic chromosomal proteins from 17 species and subspecies of the genus Xenopus (Anura, Pipidae) were compared. Electrophoresis on acetic acid/urea/Triton X-100 polyacrylamide gels revealed that each Xenopus species with a diploid chromosome number of 36, 72, or 108 showed multiple, diverse spermatid/sperm-specific basic chromatin proteins with mobilities greater than the somatic histones. The numbers and mobilities of these proteins were characteristic of each Xenopus species and each subspecies of Xenopus laevis. Cytochemical tests revealed that the sperm basic nuclear proteins of these Xenopus species and subspecies were rich in arginine and lysine and contained more arginine than the nuclear proteins of somatic cells. X. tropicalis (2n = 20) and X. sp. n. (Zaire) displayed spermatid/sperm-specific basic chromatin proteins which migrated within the histone H1 region of acetic acid/urea/Triton X-100 polyacrylamide gels. Cytochemically the sperm nuclei of these species resembled those of somatic cells. These observations suggest that spermatid/sperm basic nuclear proteins can be used as molecular markers for individual species and subspecies of the genus Xenopus.

Determinants of sperm nuclear shaping in the genus Xenopus.

The morphogenesis of sperm nuclei was investigated in six different species or subspecies of the genus Xenopus (Pipidae, Anura). The sequence of nuclear morphogenesis was similar in each species used in this study. Electrophoretic comparison of the basic chromatin proteins from late spermatids and sperm of each species demonstrated that the complements of histones and spermatid-sperm-specific basic proteins were extremely diverse suggesting that shape was not determined by specific basic proteins or mechanisms of histone removal. This conclusion was reinforced by the observation that Xenopus sperm DNA decondensed by 2.0 M NaCl remained contained in residual structures which resembled intact sperm nuclei. These observations suggested that morphogenesis of sperm nuclei is directed by proteins or RNA molecules which are not directly responsible for chromatin condensation.

Nuclear basic protein changes during spermiogenesis in the longnose skate and the spiny dogfish.

Nuclear shape and the organization of nuclei within the seminiferous follicles have been used to divide spermiogenesis in the longnose skate, Raja rhina, into eight stages and in the spiny dogfish, Squalus acanthias, into seven stages. Cytochemical techniques for basic proteins reveal that as spermatid nuclei begin to elongate they have the somatic histone complement, and as they complete elongation they contain a very arginine-rich, TCA-extractable complement, or the salmon sperm histones type. After the nuclei have developed their ultimate corkscrew shape the final transition takes place to a very arginine-rich, TCA-stable complement, or the mammalian sperm histone type. These nuclei were not rich in sulfhydryl groups, but they were extractable with sodium thioglycolate. In addition, a number of variables affecting TCA extraction and subsequent alkaline fast green staining are described.

Chromosomal Proteins of the Sperm of a Cephalochordate (Branchiostoma floridae) and an Agnathan (Petromyzon marinus): Compositional Variability of the Nuclear Sperm Proteins of Deuterostomes.

We have isolated and characterized for the first time the chromosomal proteins from the nucleus of the sperm of a lancelet (amphioxus) Branchiostoma floridae (Hubbs, 1922) (Phylum Chordata: Subphylum Cephalochordata) and of a lamprey Petromyzon marinus (Linnaeus, 1758) (Phylum Chordata: Subphylum Vertebrata: Class Agnatha). In the first case, the major protein component of the sperm-chromatin of a lancelet is a highly specialized protamine-like (PL) protein that has structural and compositional features similar to those of PL-III from bivalve mollusks. In contrast, the chromatin of the sperm of the lamprey has a structural arrangement and protein composition (histones) very similar to that found in the somatic cells of all eukaryotic organisms. Among the deuterostomes, chromosomal protein variability is considerably greater in representatives of the Phylum Chordata than in echinoderms. The possible evolutionary significance of these findings is discussed.

Chromatin reorganization during spermiogenesis of the mollusc Thais hemostoma (Muricidae): implications for sperm nuclear morphogenesis in cenogastropods.

Thais is a cenogastropod mollusc belonging to the Muricidae family. The sperm nuclear morphogenesis of Thais develops in two well-defined and peculiar steps. In the first one, the round early spermatidyl nucleus is penetrated by an endonuclear channel, which arranges as a helix at the inner nuclear surface and organizes the condensing chromatin all around. In the second step, the spiral channel stretches, dragging along the associated chromatin and leading to a definitive cylinder-shaped sperm nucleus. Simultaneously with these changes in nuclear shape, the chromatin is sequentially organized in granules, fibres, lamellae, and, finally, in a very condensed structure, whereas the spermiogenic DNA-associated proteins become more basic and simple. The sperm nucleus contains a small group of protamines consisting of only four types of amino acid (lysine, arginine, glycine, and serine). The most remarkable fact on nuclear spermiogenesis in Thais is that, whereas the chromatin condensation process, the nuclear proteins, and the final shape of sperm nucleus are very similar to those in other muricidae studied, the pathway of nuclear morphogenesis is completely different. We propose an independent genetic control for those two spermiogenic events (chromatin condensation and nucleomorphogenesis). Finally we discuss briefly the main traits of nucleomorphogenesis of muricid molluscs.

On the diversity of sperm basic proteins in the vertebrates: V. Cytochemical and amino acid analysis in Squamata, Testudines, and Crocodylia.

The variability of sperm basic proteins in representatives of three reptilian orders, Squamata, Testudines, and Crocodylia, has been examined by cytochemistry, acid-urea polyacrylamide gel electrophoresis, and amino acid analysis of amidoblack-stained bands. Snakes contain type 3B intermediate sperm basic proteins by cytochemical criteria. Electrophoresis of basic proteins from epididymis chromatin as well as from testis and ductus deferens cell suspensions shows two fast-moving bands in the vicinity of herring protamine. These proteins are triprotamines containing about 27 mol % arginine, along with lysine and histidine. Lizards have type 1 protamines in their sperm nuclei cytochemically and also show a two-banded electrophoretic pattern similar to that of snakes. However, these proteins are triprotamines, similar to those in snakes with 25 mol % arginine. It may be that these are testis-specific proteins of the spermatid stage in lizards since a cytochemical transition can be observed from type 3A intermediate proteins in spermatids of testis to type 1 protamine in mature sperm of ductus deferens. Turtles contain type 3A intermediate sperm basic proteins cytochemically and basic proteins from epididymis chromatin display both a prominent band and a minor band close to, but slightly slower than, the two bands for snakes and lizards. Amino acid analysis of these bands shows that these basic proteins are also triprotamines but with a higher level of arginine, about 48 mol %, than that in snake and lizard sperm proteins. Basic proteins from epididymis chromatin of a single Mississippi alligator show three main bands moving close to the bands of snakes, lizards, and turtles. These proteins have amino acid compositions typical for triprotamines, with 28-39 mol % arginine. The data indicate that the sperm basic proteins of representatives of 25 species in three reptilian orders are very similar, in contrast to the diversity of sperm protein types found in frogs (Kasinsky, Huang, Kwauk, Mann, Sweeney, and Yee: J. Exp. Zool., 203:109-126, '78; Kasinsky, Huang, Mann, Roca, and Subirana: J. Exp. Zool., 234:33-46, '85a). This appears to be part of a macroevolutionary trend from diversity of sperm basic proteins in frogs to relative constancy in reptiles (Kasinsky, Mann, Pickerill, Gutovich, and Byrd, Jr.:J. Cell Biol., 91:1879, '81; Kasinsky, Mann, Lemke, and Huang: In: Chromosomal Proteins and Gene Expression, Plenum Press, New York, pp. 333-352, '85b). We present the hypothesis that one factor for such a trend resides in the fact that fertilization is internal in reptiles but external in anurans.

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.

Characterization and evolutionary relevance of the sperm nuclear basic proteins from stickleback fish.

We have characterized the sperm nuclear basic proteins (SNBPs) of the sticklebacks in the suborder Gasterosteoidei. The complete amino acid sequence of the protamines from Aulorhynchus flavidus, Pungitius pungitius, Gasterosteus aculeatus, (anadromous) and G. wheatlandi, as well as the sequences of the protamines of several species pairs of freshwater G. aculeatus, have been determined. Analysis of the primary structure of these proteins has shown that: a) despite the relatively low amino acid complexity and small molecular mass of these basic proteins, they are very good molecular markers at the generic level. The bootstrap parsimony analysis using their sequences provides a phylogenetic relationship for the old anadromous species of Gasterosteoidei which is identical to that obtained from morphological and behavioral analysis; b) the comparison of the sequences also suggests that protamines from the suborder Gasterosteoidei have most likely evolved from a common gene in the early Acanthopterygii by an extension of the carboxy terminal portion of the molecule; c) protamines are not good markers for recent postglacial freshwater isolates of G. aculeatus. However, in the unique case of Enos Lake (British Columbia), we have been able to detect an additional minor protamine component in the benthic forms of G. aculeatus that is not present in the limnetic forms. Thus, this new protamine must have appeared during the past 12,000 years concomitantly with the speciation of benthics and limnetics in this lake.