Coexistence of two chromatin structures in sperm nuclei of the bivalve molluscProtothaca thaca

The chromatin of the spermatozoa from the bivalve molluscProtothaca thaca, has a peculiar composition in which coexist core histones with sperm-specific proteins H1 and Pt1, the latter being a protein exhibiting features intermediate between histones and protamines. In this paper, we report an analysis of chromatin organization using micrococcal nuclease digestion, salt fractionation of soluble chromatin derived from nuclease digestion and crosslinking experiments. The results obtained indicate that it is possible to obtain two types of chromatin, one which is soluble, more accessible to micrococcal nuclease action and which does not contain Pt1, and another insoluble type, more resistant to micrococcal nuclease and enriched in protein Pt1. The crosslinking experiments show that the protein Pt1 interacts with itself and with core histones but not with sperm-specific H1. These results have led us to propose a special structural arrangement for this chromatin. Based in the data reported here we propose the coexstence in the genome ofP. thaca of two interspersed chromatin domains, one nucleosomal and the other nonnucleosomal containing protein Pt1.     

Nucleosomal organization of a part of chromatin in mollusc sperm nuclei with a mixed basic protein composition

The structural organization of mature sperm chromatin from three representatives of theMytilidae family has been studied. The acid-soluble proteins in these species nuclei are primarily sperm-specific (approximately 80%) with the remainder being core histones. Previously, we have shown that the mature sperm nuclei of these molluscs are compact, dense structures formed by interaction of the spermspecific proteins with DNA (1). Here we show that: a) although the histones are minor chromatin protein fraction, they still organize a part (20–25%) of the total DNA into nucleosomes; b) one of the sperm-specific proteins, different from somatic H1 or H5 histones participates in the formation of the beaded structures.     

A dual chromatin organization in the sperm of the bivalve mollusc Spisula solidissima

Most of the DNA in the sperm of the bivalve mollusc. Spisula solidissima, is found to be associated with a specific high-molecular-mass, protamine-like component, sharing features common both to protamines and to histones. We have found that this component coexists, in the mature sperm nucleus, with a complete set of histones, including an H1-like histone. Such histones account for approximately 20% of the whole protein content in the sperm chromatin, the overall protein/DNA ratio (w/w) being 0.87. These data, together with micrococcal nuclease digestions in combination with salt fractionation, have allowed us to propose a structural model for this chromatin in which short nucleosomal domains are interspersed in a highly saturated protamine-DNA complex.     

The structural organization of sperm chromatin

The structure of rabbit, fowl, and Xenopus laevis sperm chromatin was explored by study of the reaction of their decondensed nuclei with DNase 1 and micrococcal nuclease. Those of rabbit and fowl were readily digested by DNase 1, and the polyacrylamide gel electrophoresis profiles of DNAs extracted from the digests were similar, each being polydisperse with a single discrete band of DNA smaller than 72 base pairs. There were differences, however, between the sperm chromatins in the course of their digestion by micrococcal nuclease. A limit digest at about 45% acid solubility was obtained with Xenopus sperm chromatin, while 90% of fowl sperm DNA was rendered acidsoluble by the enzyme. The gel profiles of the limit digests were polydisperse, but only those of rabbit and fowl sperm chromatins possessed a discrete band of DNA smaller than 72 base pairs. Bleomycin did not react with DNA of rabbit, fowl, or Xenopus spermatozoa. Since bleomycin reacts with somatic cell chromatin, and the course of DNase 1 or micrococcal nuclease digestion of sperm chromatin was different from that found for somatic cell chromatin, it would appear that sperm chromatin does not have the repeating nucleosometype structure of somatic cell chromatin. The nuclease digestion studies further suggest that the organization of rabbit and fowl sperm chromatins is similar, and is different from that of Xenopus sperm chromatin. The dependence of the structure of sperm chromatin on the composition of its basic proteins, and a possible structure for a protamine-type sperm chromatin, are discussed.     

Dependence of removal of sperm-specific proteins from Xenopus sperm nuclei on the phosphorylation state of nucleoplasmin

In Xenopus laevis , nucleoplasmin from fully grown oocytes is not highly phosphorylated, but is more extensively phosphorylated during oocyte maturation to retain this state until mid-blastula transition. Incubation of demembranated sperm with nucleoplasmin from oocytes or mature eggs revealed that egg nucleoplasmin is twice as potent as oocyte nucleoplasmin in removing sperm-specific basic proteins from chromatin (protamine-removing activity: PRA). Dephosphorylation of egg nucleoplasmin by alkaline phosphatase induced a remarkable decline of PRA in nucleoplasmin. Treatment of oocyte nucleoplasmin with cdc2 protein kinase induced an increase of the extent of phosphorylation, but to a level lower than that exhibited by egg nucleoplasmin, suggesting the involvement of other unspecified kinase(s) in phosphorylating nucleoplasmin during oocyte maturation. Incubation of sperm with cdc2 kinase induced selective phosphorylation of sperm-specific basic proteins, accompanied by their enhanced removal from sperm chromatin upon exposure to high-salt solutions. These results suggest that removal of sperm-specific basic proteins from sperm chromatin in fertilized eggs is facilitated by phosphorylation of both nucleoplasmin and sperm-specific basic proteins.     

Presence of a highly specific histone H1-like protein in the chromatin of the sperm of the bivalve mollusks

Chromatin organization in the sperm of the bivalve mollusks results from the interaction between a discrete number of protamine-like proteins (PL) and DNA. A small variable amount of histones is also present. An extensive study carried out on a relatively large number of species, within the class Bivalvia, has shown that it is possible to arrange these mollusks into five major categories on the basis of their PL composition (Ausio, J. Comp. Biochem. Physiol. 85, 439–449, (1986) [1]). In the present work, we have extended this analysis to a larger number of species and found that in spite of the inter- and intra-specific similarity of all PL proteins in their chemical composition, they exhibit different degrees of structural variability. Moreover one of these PL proteins is present in all the species analyzed, and bears an enormous resemblance to histones of the H1 family. The evolutionary significance of this finding is discussed.     

Spermatogenesis in the endosymbiont-bearing bivalve Loripes lucinalis (Veneroida: Lucinidae)

In the endosymbiont-bearing bivalve Loripes lucinalis, spermatogenesis is similar to that described for numerous bivalve species and leads to the formation of an aquasperm. The head and midpiece measure 10.5 ± 1.5 μm in length. The head is made up of a cylindrical nucleus slightly tapered apically and capped by a short conical acrosome. The nucleus lacks both an anterior and posterior nuclear invagination. The acrosome is 1.0 ± 0.1 μm long and consists of an acrosomal cone containing a diffuse subacrosomal material and an apical electron-lucent vacuole. There is no axial rod. The midpiece is made up of four mitochondrial spheres that surround the distal and proximal centrioles. The base of the distal centriole is joined to the plasmic membrane by the pericentriolar complex made up of nine radial arms. A cytoplasmic collar is observed that sheaths the flagellum as it emerges from the distal centriole. The spermatozoa present in mature acini are grouped into characteristic rings, which may have a nutritive function, with the acrosome oriented toward the centre of these ring formations. Also present within the gonad are somatic cells that seem to play a nutritive role in relation to the germinal cells. These nutritive cells undergo a cycle of development and lysis that corresponds to the spermatogenic cycle of the bivalve. These cells are large and rich in glycogen and lipid inclusions. In-depth examination of nutritive cells and gametes reveals that the male gonad is devoid of microorganisms in either a vegetative or cryptic form, suggesting that a vertical transmission through paternal gonadal inheritance is a very unlikely means of symbiont transmission in L. lucinalis. © 1996 Wiley-Liss, Inc.     

Nucleosomal organization of macronuclear chromatin in oxytricha fallax

The spacing of nucleosomes in chromatin from the macronucleus of the protozoan, Oxytricha fallax, was examined by staphylococcal nuclease digestion and gel electrophoresis. Macronuclear chromatin is ofmparticular interest because macronuclear DNA is not organized into chromosomes but instead exists as small molecules a few thousand base pairs long. When macronuclear chromatin is partially digested with staphylococcal nuclease the resulting fragments form a series of sizes which are multiples of 198 base pairs. Further digestion produces a ‘core’ DNA fragment 139 base pairs long. When chromatin from metabolically inactive cysts was examined it also showed a nucleosomal organization, with spacing identical to that in active vegetative cell chromatin. The spacing of nucleosomes in chromatin containing rRNA genes was examined also. DNA from staphylococcal nuclease digested macronuclear chromatin was electrophoresed in agarose gels, transferred to nitrocellulose and hybridized to I¹²⁵ labeled rRNA.The hybridization showed a series of rDNA fragments whose sizes were identical to the sizes of total DNA fragments. In the macronucleus of O. fallax, differences in chromatin function or activity do not seem to be reflected in differences in nucleosome spacing.     

Effect of elevated temperature and of hypoxia on NO activity in the central nervous system of bivalve molluscs

By light and electron microscopy methods the effect of changes of environmental conditions on the state of the nitroxidergic system has been studied in molluscs on the background of action of elevated temperature and hypoxia. Analysis is performed of biological effect of isolated and combined effects of the studied factors on dynamics of NO synthesis. A higher resistance of CNS neurons to the combined action of hyperthermia and hypoxia is revealed in molluscs with the initially high level of nitrogen oxide production. In molluscs with the initially low level of development of the nitroxidergic system, induction of NO formation in stress has been found to be accompanied by a change of morphology of nervous structures. It is suggested that nitrogen oxide participates in evolutionary established mechanisms of protection of mollusc nerve cells from hypoxia, while the initial high level of NO production reflects larger adaptational possibilities characteristic of these organisms.     

Changes in ribonucleoprotein particle and chromatin organization induced by liposomes in isolated nuclei

Nuclei isolated from rat liver, incubated in the presence of liposomes of different phospholipids, undergo typical modifications: chromatin dispersion and reduction of the interchromatin granules in nuclei incubated with negatively charged liposomes and increase of the chromatin density and of the number and size of the interchromatin granules in nuclei incubated with neutral liposomes. The possibility that the observed modifications are caused by an impairment of the transport and translocation of ribonucleoproteins belonging to the inner nuclear matrix, is suggested by the results obtained by radiotracer techniques on the release of RNA from liposome-incubated nuclei.     

Fragmentation of condensed chromatin in nuclei of a plant sperm,pteridium aquilinum (L.) Kuhn

Nuclei of pteridium sperm have been dispersed by turbulence in natural or slightly alkaline buffer after stripping off the cytoplasm with nonionic detergent. The nuclei tended to break up into fragments arranged in a linear order. These fragments fluoresced brightly with acridine orange as did intact nuclei. Grounds are given for identifying the smaller fragments with chromosomes. It is proposed that the sperm nucleus of British Pteridium, possibly an autotetrapolid, consists of a sequence of paired homologues.     

Chromatin organization in nuclei of sea urchin embryos. Comparison with the chromatin organization of the sperm.

The chromatin in sea urchin embryo nuclei and that in sperm heads are both organized in nucleosomes but show marked differences when analyzed by endonuclease digestion. Sperm chromatin DNA appears to be totally organized in nucleosomes that are highly resistant to nuclease hydrolysis. The kinetics of formation of acid-soluble oligonucleotides is slow and concerns only about 50% of the total DNA. In contrast, the DNA of embryo chromatin does not appear to be totally organized in nucleosomes since 5 to 10% is rapidly and preferentially hydrolysed into acid-soluble oligonucleotides without any appreciable fragmentation of the remaining parts. Futher digestion causes the formation of the usual pattern of DNA bands, as detected by gel electrophoresis. The length of the DNA segment associated with the embryo nucleosomes appears to be shorter than that of the DNA segment associated with the sperm nucleosomes. The kinetics of formation of acid-soluble oligonucleotides upon digestion of embryo chromatin is much faster than that of sperm chromatin and concerns almost all the chromatin DNA.     

Nuclear matrix involvement in sperm head structural organization

Summary— In the sperm nuclei the DNA is packaged into a highly condensed form and is not organized into nucleosome and solenoid but is bound and stabilized mainly by the protamines that arrange the DNA in an almost crystalline state. As demonstrated for somatic cells, the sperm DNA has been reported to be organized in loop domains attached to the nuclear matrix structures. However, the possible role of the sperm head matrix in maintaining the loop organization in absence of a typical nucleosomal structures has not been fully elucidated. By using in situ nick translation at confocal and electron microscope level, we analyzed the organization of the DNAprotamine complex and its association with the sperm nuclear matrix. The data obtained indicate that the chromatin organization in sperm nuclei is maintained during the sperm condensation by means of interactions with the nuclear matrix at fixed sites. The fine stucture of sperm nucleus and of sperm nuclear matrix, investigated on sections and replicas of freeze-fractured specimens, suggests that the lamellar array, observed by freeze-fracturing in the sperm nuclei, could depend on the inner matrix which presents a regular organization of globular structures possibly involved in the maintenance of chromatin domains in highly condensed sperm nuclei also.     

Gonad morphology and reproductive cycle of the mangrove oyster Crassostrea brasiliana (Lamarck, 1819) in the baía de Guaratuba,Paraná, Brazil

This study aimed to describe the gonadal histology and the reproductive cycle of Crassostrea brasiliana in the mangroves of Guaratuba Bay in southern Brazil. Adults were collected monthly from January 2010 to April 2011 from three sampling sites in intertidal oyster beds. The animals were evaluated using biometric and histological analyses of the gonads. The gonadal tissue samples were processed according to the standard histological procedures, and permanent slides were prepared using Harris' haematoxylin and eosin. The oysters were identified at the species level using a molecular protocol. Females (69%) predominated over males (26%), with 4% indeterminate and 1% hermaphroditic. Mature females were more prevalent in February, March and December 2010 and in March 2011. Mature males were more prevalent in February and April 2010 and in March 2011. The presence of hermaphroditic individuals was sporadic, and oysters in immature stages or sexual repose were observed in only a few collections between the months of May and October 2010. The reproduction of C. brasiliana in Guaratuba Bay occurs intermittently, but with greater intensity during the summer, with a larger number of females produced.     

Genetic differentiation in relation to marine landscape in a broadcast-spawning bivalve mollusc (Placopecten magellanicus)

Marine bivalves are sessile or sedentary as adults but have planktonic larvae which can potentially disperse over large distances. Consequently larval transport is expected to play a prominent role in facilitating gene flow and determining population structure. The sea scallop (Placopecten magellanicus) is a dioecious species with high fecundity, broadcast spawning and a c. 30-day planktonic larval stage, yet it forms discrete populations or 'beds' which have significantly different dynamics and characteristics. We analysed variation at six microsatellite loci in 12 locations throughout the geographic range of the species from Newfoundland, Canada, to New Jersey, USA. Significant differentiation was present and the maximum pairwise theta value, between one of the Newfoundland samples in the north and a sample from the southern portion of the range, was high at 0.061. Other proximate pairs of samples had no detectable genetic differentiation. Mantel tests indicated a significant isolation by distance, but only when one of the populations was excluded. A landscape genetic approach was used to detect areas of low gene flow using a joint analysis of spatial and genetic information. The two major putative barriers inferred by Monmonier's algorithm were then used to define regions for an analysis of molecular variance (amova). That analysis showed a significant but low percentage (1.2%) of the variation to be partitioned among regions, negligible variation among populations within regions, and the majority of the variance distributed between individuals within populations. Prominent currents were concordant with the demarcation of the regions, while a novel approach of using particle tracking software to mimic scallop larval dispersal was employed to interpret within-region genetic patterns.     

Developmental pattern of telomerase expression in the sand scallop, Euvola ziczac

Abstract. Telomerase is a ribonucleoprotein that can maintain telomeres, the repetitive sequences of DNA found at the end of eukaryotic chromosomes, and confer long-term proliferative capacity on cells. Telomerase expression is essential during periods of intense cell division such as the early developmental process. In later development, some species retain telomerase activity while others repress telomerase activity in what is thought to be a tumor-protective mechanism. Despite the importance of telomerase expression in both development and neoplastic disease, no studies to date have characterized its expression in bivalves. We present the first report of telomerase expression in a bivalve species, the sand scallop, Euvola ziczac. Telomerase activity was detected throughout the early stages of development and in all adult tissues examined. Analysis of DNA isolated from adult tissues indicated long telomeres, with terminal restriction fragment lengths >20 kb in both somatic and germ tissues. Ubiquitous telomerase expression throughout development and into adulthood would suggest a lack of telomere-related senescence and suggests that these scallops do not use telomerase repression as a mechanism to suppress the formation of neoplasm.     

Molecular crowding affects diffusion and binding of nuclear proteins in heterochromatin and reveals the fractal organization of chromatin

The nucleus of eukaryotes is organized into functional compartments, the two most prominent being heterochromatin and nucleoli. These structures are highly enriched in DNA, proteins or RNA, and thus thought to be crowded. In vitro, molecular crowding induces volume exclusion, hinders diffusion and enhances association, but whether these effects are relevant in vivo remains unclear. Here, we establish that volume exclusion and diffusive hindrance occur in dense nuclear compartments by probing the diffusive behaviour of inert fluorescent tracers in living cells. We also demonstrate that chromatin‐interacting proteins remain transiently trapped in heterochromatin due to crowding induced enhanced affinity. The kinetic signatures of these crowding consequences allow us to derive a fractal model of chromatin organization, which explains why the dynamics of soluble nuclear proteins are affected independently of their size. This model further shows that the fractal architecture differs between heterochromatin and euchromatin, and predicts that chromatin proteins use different target‐search strategies in the two compartments. We propose that fractal crowding is a fundamental principle of nuclear organization, particularly of heterochromatin maintenance.