Possible mechanisms for early and intermediate stages of sperm chromatin condensation patterning involving phase separation dynamics

During spermiogenesis in some internally fertilizing molluscs and insects, the post-meiotic spermatid nucleus develops via a sequence of complex patterns of the nuclear contents (chromatin and nucleoplasm) on the way to final chromatin condensation. We have examined the TEM data on these sequences for three species: Philaenus spumarius(a homopteran insect), Murex brandaris (a gastropod mollusc), and Eledone cirrhosa(a cephalopod mollusc). For each of these, spatially quantitative study reveals a constant spacing between pattern repeats through changes from granular to fibrillar to lamellar pattern, followed finally by a shrinkage of the spacing. Therefore we distinguish a "patterning" stage followed by a "condensation" stage. The former appears to demand a dynamic explanation, because there is no sign of structural connections to establish the part of the spacing that crosses the nucleoplasm. We consider types of dynamic mechanism, and show that for "nanostructural" dimensions (tens of nanometers as pattern spacing) reaction-diffusion dynamics are quite inappropriate, but that separation of two fluid phases by a mechanism similar to what is known as "spinodal decomposition" is a very attractive possibility.     

Chromatin condensation and acrosome development during spermiogenesis of Ensis ensis (Mollusca,Bivalvia)

We describe chromatin condensation and acrosome development during spermiogenesis of Ensis ensis. The overall shape of the mature spermatozoon corresponds to the primitive type. The nucleus is oval and on its superior pole there is an elongated acrosome; the middle piece contains four mitochondria around the centriolar complex. The condensation of the nuclei seems to occur in three steps: first the diameter of chromatin fibers increases slightly from 17 to 20 nm; second, in midspermatids fiber pairs coalesce; and third, the coalescence continues by addition of other fibers until the nuclei become highly compacted. Chromatin changes are related with nuclear protein composition. Small proacrosomal vesicles show two regions of different electron density. At a later stage they fuse to give a single, spherical vesicle in round spermatids, which migrates to the upper pole and transforms into a tapered acrosome (18 μm long) with a central channel filled with finely fibrous material. © 1994 Wiley-Liss, Inc.     

Changes in density of chromatin in the meristematic cells ofSinapis alba during transition to flowering

Summary Changes in the density of nuclear chromatin in the shoot apical meristem ofSinapis alba L. during floral transition (floral evocation) are described using Feulgen-stained 2 m thick semi-thin sections and scanning cytophotometric techniques. In both G1 and G2 nuclei the chromatin becomes less heterogeneous and less dense in evoked meristems compared to vegetative meristems. When chromatin is resolved into two fractions the dispersed fraction increases relative to the condensed fraction at evocation. This decondensation process occurs earlier in G1 than in G 2 nuclei. These chromatin changes are presumably closely related to the dramatic stimulation of biosynthetic activity and cell division during floral transition.     

Role of follitropin receptor signaling in nuclear protein transitions and chromatin condensation during spermatogenesis

Follitropin receptor (FSHR) in testicular Sertoli cells mediates signaling by pituitary follitropin (FSH) promoting intercellular communication with germ cells for normal spermatogenesis. Using receptor knockout mice we examined changes in sperm nucleoproteins and chromatin architecture. The expressions of transition proteins 1/2 (TP1/2) and protamine-2 (PRM-2) were greatly diminished at 21 days, but returned to normal at 35 days and 3 months after birth. However, protein components in chromatin were quite different. Western blots detected a reduction in PRM1/2 and prolonged retention of mono-ubiquitinated histone 2A (uH2A) in the epididymal sperm from adult mutants. Two forms of mono- and poly-uH2A were present in sonication-resistant testicular spermatids in normal mice, whereas only an elevated mono-uH2A was detectable in mutants. Decrease in PRM1/2 and retention of mono-uH2A was coincident with reduction in TP1/2 in premature spermatids. Thus lack of FSHR signaling impairs expression of TP1/2 and PRM-2 at an early stage of post-natal development causing delayed spermatogenesis. In the adult, absence of FSHR signaling prolongs retention of mono-uH2A, leading to impair transition of basic nucleoproteins and chromatin remodeling during mouse spermatogenesis.     

Transmission of modified nucleosomes from the mouse male germline to the zygote and subsequent remodeling of paternal chromatin

Rapidly after gamete fusion, the sperm nucleus loses its specific chromatin conformation and the DNA is repopulated with maternally derived nucleosomes. We evaluated the nature of paternally derived nucleosomes and the dynamics of sperm chromatin remodeling in the zygote directly after gamete fusion. We observed histone H4 acetylated at K8 or K12 already prior to full decondensation of the sperm nucleus, suggesting that these marks are transmitted by the spermatozoon. Tracking down the origin of H4K8ac and H4K12ac during spermiogenesis revealed the retention of nucleosomes with these modifications in the chromocenter of elongating spermatids. We show that sperm constitutive heterochromatin is enriched for nucleosomes carrying specific histone modifications which are transmitted to the zygote. Our results suggest an epigenetic mechanism for inheritance of chromosomal architecture. Furthermore, up to pronucleus formation, histone acetylation and phosphorylation build up in a cascade-like fashion in the paternal chromatin. After formation of the pronucleus, a subset of these marks is removed from the heterochromatin, which suggests a reestablishment of the euchromatin-heterochromatin partition.     

Spermiogenesis and chromatin condensation in the common tree shrew, <Emphasis Type="Italic">Tupaia glis</Emphasis>

We have investigated the cellular characteristics, especially chromatin condensation and the basic nuclear protein profile, during spermiogenesis in the common tree shrew, Tupaia glis. Spermatids could be classified into Golgi phase, cap phase, acrosome phase, and maturation phase. During the Golgi phase, chromatin was composed of 10-nm and 30-nm fibers with few 50-nm to 60-nm knobby fibers. The latter were then transformed into 70-nm knobby fibers during the cap phase. In the acrosome phase, all fibers were packed into the highest-order knobby fibers, each about 80–100 nm in width. These chromatin fibers became tightly packed in the maturation phase. In a mature spermatozoon, the discoid-shaped head was occupied by the acrosome and completely condensed chromatin. H3, the core histone, was detected by immunostaining in all nuclei of germ cell stages, except in spermatid steps 15–16 and spermatozoa. Protamine, the basic nuclear protein causing the tight packing of sperm chromatin, was detected by immunofluorescence in the nuclei of spermatids at steps 12–16 and spermatozoa. Cross-immunoreactivity of T. glis H3 and protamine to those of primates suggests the evolutionary resemblance of these nuclear basic proteins in primate germ cells. This work was supported by the Thailand Research Fund (Senior Research Fellowship to Prof. Prasert Sobhon).     

苔藓植物对环境变化的影响及适应性研究进展

苔藓植物由于其结构相对简单,对环境变化的反应较为敏感,是一类良好的生物指示植物,本文综述了水分、光照、温度等方面的环境因子变化对苔藓植物的影响以及苔藓植物对环境污染的响应及适应的最近研究进展,以期促进国内深入开展苔藓植物对环境污染和全球变化的响应、适应及其生态指示作用等研究。     

Denaturation and condensation of DNA in situ induced by acridine orange in relation to chromatin changes during growth and differentiation of Friend erythroleukemia cells

DNA in situ is progressively denatured when the cells or nuclei are treated with increasing concentration of acridine orange (AO). This transition can be monitored by flow cytometry as a decrease in green fluorescence. The complexes of denatured DNA and AO undergo immediate condensation and aggregation; this step is manifested by appearance of red luminescence and formation of precipitates that can be detected by electron microscopy. The precipitates form preferentially in heterochromatin as well as in ribosomes and polysomes. Their formation and further aggregation affects cellular light scatter properties in both the forward and right-angle direction. The AO-induced DNA denaturation and condensation was studied in nuclei of Friend erythroleukemia cells from exponentially growing, differentiated or quiescent cells. The DNA in nuclei of quiescent cells, from plateau-phase cultures, was the most sensitive to denaturation; it denatured (measured by changes in luminescence) at an AO concentration between 50 and 80 microM with the midpoint of the transition (Cd) at 70 microM. DNA in nuclei of differentiated cells (dimethyl-sulfoxide-induced erythroid differentiation) was more resistant (Cd = 77-83 microM), whereas DNA in exponentially growing cells was the most resistant (Cd = 86 microM). Extraction of proteins with 0.1 M HCl at 0 degree C abolished the differences between the cells and shifted the transition to a lower AO concentration (Cd = 46 microM). For comparison, the midpoint transitions representing condensation of free, nucleic acids measured as light scatter changes occurred at 13, 22, 31 and 53 microM of AO, for rRNA, tRNA, and denatured and native-calf thymus DNA, respectively. Denaturation and condensation of DNA, which can be induced by AO either in isolated nuclei or viable permeabilized or fixed cells provides a new approach to discriminate cell subpopulations with different chromatin structure by flow cytometry. The molecular mechanisms of this phenomenon are discussed.     

Changes in distribution of basic nuclear proteins and chromatin organization during spermiogenesis in the greater bandicoot rat, <Emphasis Type="Italic">Bandicota indica</Emphasis>

Male germ cells of the greater bandicoot rat, Bandicota indica, have recently been categorized into 12 spermiogenic steps based upon the morphological appearance of the acrosome and nucleus and the cell shape. In the present study, we have found that, in the Golgi and cap phases, round spermatid nuclei contain 10-nm to 30-nm chromatin fibers, and that the acrosomal granule forms a huge cap over the anterior pole of nucleus. In the acrosomal phase, many chromatin fibers are approximately 50 nm thick; these then thickened to 70-nm fibers and eventually became 90-nm chromatin cords that are tightly packed together into highly condensed chromatin, except where nuclear vacuoles occur. Immunocytochemistry and immunogold localization with anti-histones, anti-transition protein2, and anti-protamine antibodies suggest that histones remain throughout spermiogenesis, that transition proteins are present from step 7 spermatids and remain until the end of spermiogenesis, and that protamines appear at step 8. Spermatozoa from the cauda epididymidis have been analyzed by acid urea Triton X-100 polyacrylamide gel electrophoresis for basic nuclear proteins. The histones, H2A, H3, H2B, and H4, transitional protein2, and protamine are all present in sperm extracts. These findings suggest that, in these sperm of unusual morphology, both transition proteins and some histones are retained, a finding possibly related to the unusual nuclear form of sperm in this species.     

Spermatogenesis and chromatin condensation in male germ cells of sea cucumber Holothuria leucospilota (Clark, 1920)

Male germ cells in the testis of Holothuria leucospilota can be divided into 12 stages based on ultrastructure and patterns of chromatin condensation. The spermatogonium (Sg) is a spherical-shaped cell with a diameter of about 6.5-7microm. Its nucleus mostly contains euchromatin and small blocks of heterochromatin scattered throughout the nucleus. The nucleolus is prominent. Primary spermatocytes are divided into six stages, i.e., leptotene (LSc), zygotene (ZSc), pachytene (PSc), diplotene (DSc), diakinesis (DiSc) and metaphase (MSc). The early cells are round while in DiSc and in MSc cells are oval in shape. From LSc to MSc, the sizes of cells range from 3.5 to 4microm. LSc contains large blocks of heterochromatin as a result of increasingly condensed 17nm fibers. In ZSc, the nucleus contains prominent synaptonemal complexes but a nucleolus is absent. In PSc, heterochromatin blocks are tightly packed together by 26nm fibers and appeared as large patches in DSc. Heterochromatin patches were enlarged to form chromosomes in DiSc and MSc and then the chromosome are moved to be aligned along equatorial region. The secondary spermatocyte (SSc) is an oval cell about 4.5-5.5microm. Their nuclei contain large clumps of heterochromatin along the nuclear envelope and in the center nuclear region. Spermatids are divided into two stages, i.e., early spermatid (ESt) and late spermatid (LSt). The nuclei decrease in size by a half and become spherical; thus the chromatin fibers condensed into 20nm and are closely packed together leaving only small spaces in LSt. The spermatozoa (Sz), with chromatin tightly packed in the spherical nucleus with a diameter of 2microm and a small acrosome situated at the anterior of the nucleus. The tail consists of a pair of centrioles lying perpendicular to each other and surrounded by a mitochondrial ring, and an axonemal complex, surrounded by a plasma membrane.     

Effects of continuous and repeated dehydration on carbon fixation by bryophytes from the maritime Antarctic

 The effects of dehydration and rehydration on carbon exchange in 14 bryophytes from the maritime Antarctic were investigated using an infra-red gas analysis system. Continuous long-term (1–12 months) and repeated (1–6 one-month cycles) desiccation responses were investigated under controlled conditions. Loss of photosynthetic rate increased with length of dehydration period in all species, although some desiccation tolerance was observed even in those bryophytes from the most hydric habitats. Percentage retention of photosynthetic rate increased from hydric to xeric species, but this pattern was not repeated in terms of absolute rates of carbon fixation due to the high initial rates in the hydric species. Repeated cycles caused a greater loss of photosynthetic rate than continuous dehydration in hydric species, but the opposite situation occurred in mesic and xeric mosses. The latter groups were possibly better able to utilise the short periods of rehydration during cycles. In most bryophytes an increase in the percentage loss of photosynthetic rate following dehydration-rehydration occurred from spring to summer to autumn samples. This pattern was clearest in the hydric species and reduced in the xeric species. These variations were largely due to changes in the initial rates of photosynthesis during the growing season. It is suggested that this increased photosynthetic capacity is stress-sensitive, and is lost during either desiccation or winter freezing; the base photosynthetic capacity, being stress-tolerant, survives either of these events. The results obtained support the hypothesis that water availability is of importance in determining the distribution of bryophytes in the Antarctic. However, only the broad scale of variation in plant communities could be explained by these observations; other factors must be important in determining the finer scale of species distribution and community composition. The results are applicable to attempts to model the productivity of Antarctic bryophytes from known or predicted environmental data. Received: 15 April 1996 / Accepted: 28 September 1996     

Analysis of chromatin and DNA during chromosome endoreduplication in the endosperm ofTriticum durum Desf.

Summary Chromatin structure was studied in nuclei of the endosperm of durum wheat (Triticum durum Desf., cv. Creso), where a large number of cells undergo chromosome endoreduplication during caryopsis development. Optical density profiles of interphase nuclei at different ploidy levels after Feulgen staining were determined cytophotometrically. It was observed that, within each development stage, polyploid nuclei (6–12C and 12–24C) show more condensed chromatin than euploid nuclei (3–6C): this should indicate that endoreduplication is accompanied by some reduction of nuclear activity. Within the same ploidy level, 3–6C and 6–12C nuclei become increasingly condensed with development (except for the last stage), while 12-24C nuclei are identical at all stages. DNA methylation at different stages of caryopsis development was then analyzed in genomic DNA, highly repeated sequences and ribosomal DNA, by digestion with cytosine-methylation-sensitive restriction enzymes. We observed that (i), depending on the enzyme, DNA from caryopses may show higher mean length than DNA from shoot apices and variations occur during endosperm development; (ii) highly repeated DNA sequences also show some variation in base methylation between apices and endosperms and among endosperm development stages, even though to a lesser extent than genomic DNA; (iii) rDNA shows variations only between endosperm and apices while no variation was observed among endosperm development stages in relation to chromosome endoreduplication. Our data may be explained by assuming the occurrence, during endosperm development, of processes of chromatin condensation possibly involved in silencing the activity of extra copies of DNA resulting from chromosome endoreduplication. At least in part, DNA methylation is involved in the process of chromatin condensation. rDNA shows no variation during endosperm development: this suggests that rDNA copies are actively transcribed in both triploid and endoreduplicated nuclei.     

Production of an aurone by bryophytes in the reproductive phase

The aurone, aureusidin 6-O-glucuronide, has been isolated from the antheridiophores of two liverworts, Marchantia berteroana, M. polymorpha and from Conocephalum supradecompositum. It occurs only in these organs in Marchantia. The appearance of this aurone in bryophyte reproductive structures suggests parallel evolution within the angiosperms and the bryophytes.     

On the role of inter-nucleosomal interactions and intrinsic nucleosome dynamics in chromatin function

Evidence is emerging that many diseases result from defects in gene functions, which, in turn, depend on the local chromatin environment of a gene. However, it still remains not fully clear how chromatin activity code is ‘translated’ to the particular ‘activating’ or ‘repressing’ chromatin structural transition. Commonly, chromatin remodeling in vitro was studied using mononucleosomes as a model. However, recent data suggest that structural reorganization of a single mononucleosome is not equal to remodeling of a nucleosome particle under multinucleosomal content – such as, interaction of nucleosomes via flexible histone termini could significantly alter the mode (and the resulting products) of nucleosome structural transitions. It is becoming evident that a nucleosome array does not constitute just a ‘polymer’ of individual ‘canonical’ nucleosomes due to multiple inter-nucleosomal interactions which affect nucleosome dynamics and structure. It could be hypothesized, that inter-nucleosomal interactions could act in cooperation with nucleosome inherent dynamics to orchestrate DNA-based processes and promote formation and stabilization of highly-dynamic, accessible structure of a nucleosome array. In the proposed paper we would like to discuss the nucleosome dynamics within the chromatin fiber mainly as it pertains to the roles of the structural changes mediated by inter-nucleosomal interactions.     

Effect of timing of cutting on patterning and proportion regulation during regeneration of the planarian Dugesia tigrina

The time interval between cuts that are made to obtain a tissue fragment from a planarian was found to be important to the process of its regeneration. Short fragments made by two transverse cuts across the body were more likely to regenerate abnormally when the interval between the two cuts was 5 or 12 min than when it was 1.5 min. The longer intervals specifically altered the regression line in the correlation between the length:width ratio of fragments and frequency of abnormal regenerates. This effect occured regardless of which region of the body the fragment was taken from. The time interval also affected body proportioning in regenerates and to the greatest degree in fragments derived from the region located immediately behind the head. These results indicate that events occuring shortly after a cut is made in a planarian significantly affect structure patterning and proportioning of the regenerate.     

Effects of cryopreservation on head morphometry and its relation with chromatin status in brown bear (Ursus arctos) spermatozoa

The Cantabrian brown bear (Ursus arctos) is a highly endangered species in Spain and basic studies are necessary in order to bank its germplasm. Sperm heads are mainly made up of chromatin, thus their shape depends partly on chromatin structure. Thawed semen from 10 bears was used to analyze chromatin status by sperm chromatin structure assay (SCSA) and head morphometry by the computer-assisted sperm morphology assessment (CASMA) system. Morphometry was analyzed before and after freezing–thawing in order to evaluate the effects of cryopreservation on sperm heads. Each spermatozoon was measured for four primary parameters (length, L; width, W; area, A; perimeter, P) and derived parameters (ellipticity: L/W, circularity: 4πA/P², elongation: (L − W)/(L + W), regularity: πLW/4A). All the derived parameters significantly differed between bears. Likewise, cryopreservation affected head morphometry by reducing its size. Clustering based on morphometric parameters separated three subpopulations, one of them being significantly more influenced by the cryopreservation process. We obtained high correlations between head morphometry and SCSA parameters: standard deviation of DNA fragmentation index (SD-DFI) was correlated with perimeter and area (r = 0.75 and r = 0.62, respectively) and DFIm and DFIt (moderate and total DNA fragmentation index) were correlated with perimeter (r = 0.65 and r = 0.67, respectively). Nevertheless, classification of males according to SCSA or head morphometry did not completely agree so the two assays might explain male variability differently. We conclude that cryopreservation affected morphometry at least in a subset of spermatozoa. These results might improve future application of sperm banking techniques in this species.