Distribution Of Nuclear Pores Research Articles

A single-molecule localization microscopy method for tissues reveals nonrandom nuclear pore distribution in Drosophila.

ABSTRACTSingle-molecule localization microscopy (SMLM) can provide nanoscale resolution in thin samples but has rarely been applied to tissues because of high background from out-of-focus emitters and optical aberrations. Here, we describe a line scanning microscope that provides optical sectioning for SMLM in tissues. Imaging endogenously-tagged nucleoporins and F-actin on this system using DNA- and peptide-point accumulation for imaging in nanoscale topography (PAINT) routinely gives 30 nm resolution or better at depths greater than 20 µm. This revealed that the nuclear pores are nonrandomly distributed in most Drosophila tissues, in contrast to what is seen in cultured cells. Lamin Dm0 shows a complementary localization to the nuclear pores, suggesting that it corrals the pores. Furthermore, ectopic expression of the tissue-specific Lamin C causes the nuclear pores to distribute more randomly, whereas lamin C mutants enhance nuclear pore clustering, particularly in muscle nuclei. Given that nucleoporins interact with specific chromatin domains, nuclear pore clustering could regulate local chromatin organization and contribute to the disease phenotypes caused by human lamin A/C laminopathies.

Nuclear pore protein TPR associates with lamin B1 and affects nuclear lamina organization and nuclear pore distribution.

The organization of the nuclear periphery is crucial for many nuclear functions. Nuclear lamins form dense network at the nuclear periphery and play a substantial role in chromatin organization, transcription regulation and in organization of nuclear pore complexes (NPCs). Here, we show that TPR, the protein located preferentially within the nuclear baskets of NPCs, associates with lamin B1. The depletion of TPR affects the organization of lamin B1 but not lamin A/C within the nuclear lamina as shown by stimulated emission depletion microscopy. Finally, reduction of TPR affects the distribution of NPCs within the nuclear envelope and the effect can be reversed by simultaneous knock-down of lamin A/C or the overexpression of lamin B1. Our work suggests a novel role for the TPR at the nuclear periphery: the TPR contributes to the organization of the nuclear lamina and in cooperation with lamins guards the interphase assembly of nuclear pore complexes.

Stage-dependent remodeling of the nuclear envelope and lamina during rabbit early embryonic development.

Utilizing 3D structured illumination microscopy, we investigated the quality and quantity of nuclear invaginations and the distribution of nuclear pores during rabbit early embryonic development and identified the exact time point of nucleoporin 153 (NUP153) association with chromatin during mitosis. Contrary to bovine early embryonic nuclei, featuring almost exclusively nuclear invaginations containing a small volume of cytoplasm, nuclei in rabbit early embryonic stages show additionally numerous invaginations containing a large volume of cytoplasm. Small-volume invaginations frequently emanated from large-volume nuclear invaginations but not vice versa, indicating a different underlying mechanism. Large- and small-volume nuclear envelope invaginations required the presence of chromatin, as they were restricted to chromatin-positive areas. The chromatin-free contact areas between nucleolar precursor bodies (NPBs) and large-volume invaginations were free of nuclear pores. Small-volume invaginations were not in contact with NPBs. The number of invaginations and isolated intranuclear vesicles per nucleus peaked at the 4-cell stage. At this stage, the nuclear surface showed highly concentrated clusters of nuclear pores surrounded by areas free of nuclear pores. Isolated intranuclear lamina vesicles were usually NUP153 negative. Cytoplasmic, randomly distributed NUP153-positive clusters were highly abundant at the zygote stage and decreased in number until they were almost absent at the 8-cell stage and later. These large NUP153 clusters may represent a maternally provided NUP153 deposit, but they were not visible as clusters during mitosis. Major genome activation at the 8- to 16-cell stage may mark the switch from a necessity for a deposit to on-demand production. NUP153 association with chromatin is initiated during metaphase before the initiation of the regeneration of the lamina. To our knowledge, the present study demonstrates for the first time major remodeling of the nuclear envelope and its underlying lamina during rabbit preimplantation development.

Remodeling of nuclear landscapes during human myelopoietic cell differentiation maintains co-aligned active and inactive nuclear compartments.

BackgroundPrevious studies of higher order chromatin organization in nuclei of mammalian species revealed both structural consistency and species-specific differences between cell lines and during early embryonic development. Here, we extended our studies to nuclear landscapes in the human myelopoietic lineage representing a somatic cell differentiation system. Our longterm goal is a search for structural features of nuclei, which are restricted to certain cell types/species, as compared to features, which are evolutionary highly conserved, arguing for their basic functional roles in nuclear organization.ResultsCommon human hematopoietic progenitors, myeloid precursor cells, differentiated monocytes and granulocytes analyzed by super-resolution fluorescence microscopy and electron microscopy revealed profound differences with respect to global chromatin arrangements, the nuclear space occupied by the interchromatin compartment and the distribution of nuclear pores. In contrast, we noted a consistent organization in all cell types with regard to two co-aligned networks, an active (ANC) and an inactive (INC) nuclear compartment delineated by functionally relevant hallmarks. The ANC is enriched in active RNA polymerase II, splicing speckles and histone signatures for transcriptionally competent chromatin (H3K4me3), whereas the INC carries marks for repressed chromatin (H3K9me3).ConclusionsOur findings substantiate the conservation of the recently published ANC-INC network model of mammalian nuclear organization during human myelopoiesis irrespective of profound changes of the global nuclear architecture observed during this differentiation process. According to this model, two spatially co-aligned and functionally interacting active and inactive nuclear compartments (ANC and INC) pervade the nuclear space.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0038-0) contains supplementary material, which is available to authorized users.

Peptide Ordering within Nuclear Pores in Living Cells

It is a profound belief of biologists, based upon decades of structural biochemistry, that understanding the three-dimensional structure of individual proteins and protein complexes illuminates the basis of protein function. X-ray crystallography and other structural techniques have revealed remarkable high-resolution maps of even large, multimolecular structures. However, the approaches which give such spectacular information in vitro are unable to probe structures of proteins that are functioning in their natural milieu in living cells. This frontier between structural biology and cellular biology is only beginning to be addressed. An example of the progress is work from the laboratory of Sanford Simon (Rockefeller University, New York). Earlier works by Mattheyses et al. (1) and Kampman et al. (2), and a new article by Atkinson et al. (3) (this issue) describe the elegant application of fluorescence polarization to probe the orientation of specific protein components in functioning nuclear pore complexes in living cells. Nuclear pores are an essential component of eukaryotic cells that are responsible for the highly regulated, bidirectional transport of proteins and RNA between the cytoplasm and nucleus. They are very large structures that contain a large aqueous channel that spans the double membrane of the nuclear envelope. Pores are composed of ∼30 different proteins (nucleoporins) present in multiple copies, with several thousand proteins comprising a single nuclear pore. Crystallography has provided structural information about isolated nucleoporins and their interaction sites (4). Electron microscopy of intact nuclear pore complexes has revealed a structure with octagonal symmetry (5,6). Simon and colleagues have taken advantage of the random distribution of nuclear pores around the approximately circular cross section of the nuclear envelope. Nuclear pore proteins labeled with green fluorescent protein were introduced into cells by molecular biology techniques. By illumination of the sample with polarized light and collection of the emission in two orthogonally polarized channels, the fluorescence anisotropies of nuclear pores with their varying orientations around the nuclear envelope cross section were determined. Thus, the intrinsic geometry of the positions of the nuclear pores in the nuclear envelope enabled investigation of the anisotropy at various angles. Channel nucleoporins have a predicted α-helical region and an unfolded domain containing phenylalaine-glycine (FG) repeats. The ensemble of FG domains from multiple nucleoporins in a channel is thought to function as a diffusion barrier to regulate transport of large macromolecules (Fig. 1 (7)). This article addresses the question of whether FG regions in different nucleoporins are oriented relative to the central axis of functioning nuclear pores in living cells. The findings were significant: some of the FG domains of specific nucleoporins were ordered. Ordering was more likely for nucleoporins lining the center of the nuclear pore complex than for those at the cytoplasmic or nuclear entrances. Ordering was not altered by active transport or attachment of cargo. Although one might have predicted (wished?) a change with function, the time resolution of anisotropy measurements (2 s) was likely insufficient to capture fluctuations due to individual transport events. Figure 1 Schematic of nuclear pore. Adapted from Fig. 12-10 in Alberts et al. (7). These studies are a hopeful beginning, demonstrating the possibility of asking structural questions within living cells. We can look forward to improved techniques, including faster time resolution approaches and the application of single-molecule fluorescence imaging, that will enable the detection of function-related structural changes in proteins in their normal, intracellular milieu. These types of biophysical techniques should be applicable to numerous other dynamic protein structures within cells.

3D nuclear architecture reveals coupled cell cycle dynamics of chromatin and nuclear pores in the malaria parasite Plasmodium falciparum

The deadliest form of human malaria is caused by the protozoan parasite Plasmodium falciparum. The complex life cycle of this parasite is associated with tight transcriptional regulation of gene expression. Nuclear positioning and chromatin dynamics may play an important role in regulating P. falciparum virulence genes. We have applied an emerging technique of electron microscopy to construct a 3D model of the parasite nucleus at distinct stages of development within the infected red blood cell. We have followed the distribution of nuclear pores and chromatin throughout the intra-erythrocytic cycle, and have found a striking coupling between the distributions of nuclear pores and chromatin organization. Pore dynamics involve clustering, biogenesis, and division among daughter cells, while chromatin undergoes stage-dependent changes in packaging. Dramatic changes in heterochromatin distribution coincide with a previously identified transition in gene expression and nucleosome positioning during the mid-to-late schizont phase. We also found a correlation between euchromatin positioning at the nuclear envelope and the local distribution of nuclear pores, as well as a dynamic nuclear polarity during schizogony. These results suggest that cyclic patterns in gene expression during parasite development correlate with gross changes in cellular and nuclear architecture.

Cell-cycle-dependent dynamics of nuclear pores: pore-free islands and lamins

Nuclear pores are sophisticated gateways on the nuclear envelope that control macromolecular transport between the cytoplasm and nucleoplasm. So far the structural and functional aspects of nuclear pores have been extensively studied, but their distribution and density, which might reflect nuclear organization and function, remain unknown. Here, we report the cell-cycle-dependent dynamics of nuclear pores. Large distinct subdomains lacking nuclear pores are present on the nuclear surface of HeLaS3 cells in early cell-cycle stages. Such ;pore-free islands' gradually become dispersed in G1-S phase. Surprisingly, the islands are enriched with inner nuclear membrane proteins lamin A/C and emerin, but exclude lamin B. Lamin-A/C-enriched pore-free islands were also observed in human normal diploid fibroblasts and several cell lines, showing the generality of this phenomenon. Knockdown and ectopic expression analyses demonstrated that lamin A/C, but not emerin, plays an essential structural and regulatory role in the nuclear pore distribution and the formation of pore-free islands. These data thus provide strong evidence that the dynamics of nuclear pores are regulated by the reorganization of inner nuclear structures.

Nuclear deformation characterizes Werner syndrome cells

Mutations in the lamin A gene have been shown, among other defects, to give rise to Hutchinson-Gilford progeria syndrome (HGPS) and to atypical Werner syndrome (WS), both of which are progeroid disorders. Here, we have investigated well-characterized WS patient cell strains that are compound heterozygous for mutations in the WRN gene. As in HGPS and in atypical WS, we found nuclear deformations to be characteristic of all cell strains studied. In WS cells centrosome number, assembly of the nuclear lamina and nuclear pore distribution occurred normally. Furthermore, nuclear deformations were not associated with a defect in lamin A expression. We propose that nuclear deformation is a universal characteristic of progeroid cells and may result from slow cell cycle progression.

Genetic network interactions among replication, repair and nuclear pore deficiencies in yeast

The yeast RAD27 gene encodes a functional homolog of the mammalian FEN1 protein, a structure-specific endo/exonuclease which plays an important role in DNA replication and repair. Previous genetic interaction studies, including a synthetic genetic array (SGA) analysis, showed that the survival of rad27Δ cells requires several DNA metabolic processes, in particular those mediated by all members of the Rad52-dependent recombinational repair pathway. Here, we report the results of our SGA analysis of the collection of non-essential yeast genes against the rad27Δ mutation, which resulted in the identification of a novel synthetic lethal interaction conferred by mutations affecting the Nup84 nuclear pore subcomplex ( nup133Δ, nup120Δ and nup84Δ). Additional screens showed that all Rad52 group genes are required for the survival of the nup133Δ and nup120Δ mutants, which are defective in nuclear pore distribution and mRNA export, but not of the nup133Δ N mutant, which is solely defective in pore distribution. This requirement for the DNA double-strand break (DSB) repair pathway is consistent with the observation that, like rad27Δ, the nup133Δ, nup120Δ and nup84Δ mutants are sensitive to methyl methanesulfonate (MMS). Furthermore, nup133Δ cells exhibit an increased number of spontaneous DNA repair foci containing Rad52. Altogether, these data suggest that the pathological interactions between the rad27Δ and specific nupΔ mutations result from the accumulation of unrepaired DNA damages.

The fission yeast Nup107-120 complex functionally interacts with the small GTPase Ran/Spi1 and is required for mRNA export, nuclear pore distribution, and proper cell division.

We have characterized Schizosaccharomyces pombe open reading frames encoding potential orthologues of constituents of the evolutionarily conserved Saccharomyces cerevisiae Nup84 vertebrate Nup107-160 nuclear pore subcomplex, namely Nup133a, Nup133b, Nup120, Nup107, Nup85, and Seh1. In spite of rather weak sequence conservation, in vivo analyses demonstrated that these S. pombe proteins are localized at the nuclear envelope. Biochemical data confirmed the organization of these nucleoporins within conserved complexes. Although examination of the S. cerevisiae and S. pombe deletion mutants revealed different viability phenotypes, functional studies indicated that the involvement of this complex in nuclear pore distribution and mRNA export has been conserved between these highly divergent yeasts. Unexpectedly, microscopic analyses of some of the S. pombe mutants revealed cell division defects at the restrictive temperature (abnormal septa and mitotic spindles and chromosome missegregation) that were reminiscent of defects occurring in several S. pombe GTPase Ran (Ran(Sp))/Spi1 cycle mutants. Furthermore, deletion of nup120 moderately altered the nuclear location of Ran(Sp)/Spi1, whereas overexpression of a nonfunctional Ran(Sp)/Spi1-GFP allele was specifically toxic in the Deltanup120 and Deltanup133b mutant strains, indicating a functional and genetic link between constituents of the S. pombe Nup107-120 complex and of the Ran(Sp)/Spi1 pathway.

Sertoli cell nuclear pore number changes in some stages of the spermatogenic cycle of the rat seminiferous epithelium

In an earlier study we described changes in the number and distribution of nuclear pores during maturation of germ cells at given stages of the spermatogenic cycle; these changes were related to the activity of nucleus-cytoplasm transport. Similarly, the present work was performed by combining freeze-fracture techniques with Sertoli nuclei identification criteria, using fragments of tubules isolated by transillumination under stereo-microscopy. We studied the density of nuclear pores in freeze-fracture replicas of the Sertoli nuclear envelope at stages XIII-XIV-I compared with stages IX-XII. Pore counts were carried out on photographs of the platinum replicas using a digitalized morphometric board. The results were statistically analyzed using Student's t test. The difference in density (pore number/μm2 ± SEM) was significant between stages IX-XII (8.25 ± 0.63) and XIII-XIV-I (10.80 ± 0.60). We postulate that this density appears to be increased at the time of increased metabolic requirements of the Sertoli cell.

Dynamics of nuclear pore distribution in nucleoporin mutant yeast cells.

To follow the dynamics of nuclear pore distribution in living yeast cells, we have generated fusion proteins between the green fluorescent protein (GFP) and the yeast nucleoporins Nup49p and Nup133p. In nup133- dividing cells that display a constitutive nuclear pore clustering, in vivo analysis of GFP-Nup49p localization revealed changes in the distribution of nuclear pore complex (NPC) clusters. Furthermore, upon induction of Nup133p expression in a GAL-nup133 strain, a progressive fragmentation of the NPC aggregates was observed that in turn led to a wild-type nuclear pore distribution. To try to uncouple Nup133p-induced NPC redistribution from successive nuclear divisions and nuclear pore biogenesis, we devised an assay based on the formation of heterokaryons between nup133- mutants and cells either expressing or overexpressing Nup133p. Under these conditions, the use of GFP-Nup133p and GFP-Nup49p fusion proteins revealed that Nup133p can be rapidly targeted to the clustered nuclear pores, where its amino-terminal domain is required to promote the redistribution of preexisting NPCs.

Formation of Nuclear Bodies in Cells Overexpressing the Nuclear Pore Protein POM121

POM121 is an integral membrane protein specifically localized in the pore membrane domain of the nuclear envelope. We have investigated the intracellular distribution of rat POM121 heterologously overexpressed in monkey COS cells by immunofluorescence and fluorescence digital imaging microscopy. At low levels of expression overexpressed POM121 was distributed in the nuclear envelope in a punctate fashion, partially overlapping with the distribution of nuclear pores. At high levels of expression, however, the overexpressed protein accumulated in intranuclear bodies. These bodies represent a novel subnuclear structure, displaying a defined cylindrical structure and a distinct localization at or adjacent to the inner nuclear membrane. The C-terminal portion of POM121, which contains a pentapeptide repeat domain common to a subfamily of related nucleoporins, was sufficient to mediate targeting to the nuclear envelope as well as formation of intranuclear bodies.

A novel nuclear pore protein Nup133p with distinct roles in poly(A)+ RNA transport and nuclear pore distribution.

Temperature-sensitive nucleoporin nup49-316 mutant cells accumulate poly(A)+ RNA inside the nucleus when shifted to restrictive temperature. We performed a synthetic lethal screen with this mutant allele to identify further components of the mRNA export machinery. A synthetic lethal mutant slv21 was isolated, which exhibited a ts phenotype and showed nuclear accumulation of poly(A)+ RNA at 37 degrees C. The wild-type gene complementing slv21 was cloned and sequenced. It encodes a novel protein Nup133p which is located at the nuclear pore complex. NUP133 is not an essential gene, but cells in which NUP133 is disrupted grow slowly at permissive temperatures and stop growing at 37 degrees C. Concomitant with the growth inhibition, nup133- cells accumulate poly(A)+ RNA inside the nucleus whereas nuclear import of a karyophilic reporter protein is not altered. Strikingly, nup133- cells display extensive clustering of nuclear pore complexes at a few sites on the nuclear envelope. However, the nuclear pore clustering phenotype and intranuclear accumulation of poly(A)+ RNA are not obligatorily linked, since an amino-terminally truncated Nup133p allows normal poly(A)+ RNA export, but does not complement the clustering phenotype of nup133- cells.

Characterization of nuclear pore distribution in freeze-fracture replicas of seminiferous tubules isolated by transillumination

Transilluminated seminiferous tubules were staged and utilized to determine the distribution of nuclear pore complexes in seminiferous tubules of the rat. Segments of seminiferous tubules of adult albino rats were separated and identified (in stages VII–VIII, IX–XI, XII–XIV, and V–VI), and then processed by freeze-fracture. Type A spermatogonia, the only spermatogonia located in seminiferous segments possessing stages IX–XI and XII–XIV, are oval cells in contact with the basal lamina. They either exhibit a random distribution of nuclear pores or a slight degree of clumping. Type B spermatogonia, found in segments possessing stages V–VI, exhibit, instead, a noticeable pore clustering. The identification of intermediate spermatogonia was not undertaken in this study. Preleptotene spermatocytes are easily identified in freeze-fracture by their location in segments with stages VII–VIII, by their arrangement in numerous groups between the basal lamina and the pachytene spermatocytes, and by their comparatively small size. They exhibit noticeable pore clustering. Leptotene (segments containing stages IX–XI) and zygotene (XII–XIV) spermatocytes show a more homogeneous distribution of nuclear pores. Pachytene spermatocytes are identified by their large size, by consistent detachment from the basal lamina and by being rather numerous and found in all the stages explored. Diplotene spermatocytes have the largest nuclei of all germ cells. They are always detached from the basal lamina and found only in seminiferous segments containing stage XIII. Pachytenes display a regular geometric array of pore aggregation with striking clustering, whereas diplotene nuclear pores take on a random distribution. Secondary spermatocytes, only present in stage XIV intermingled with metaphaseanaphase profiles, are characterized in replicas by a paucity of evenly distributed nuclear pores.

Freeze-fracture organization of chromatin and cytoplasm in neurons and astroglia of rat cerebellar cortex

The cytology of the cell nucleus and cytoplasm of neurons and astroglia of the rat cerebellar cortex has been investigated by freeze-fracture electron microscopy. The main differential characteristics in the cytoplasm of the several cell types of the cerebellar cortex were: (1) the organization of endoplasmic reticulum elements, including special configurations of lamellar bodies and hypolemmal complexes, (2) the polarity, extension and arrangement of Golgi cisterns and associated tubulovesicular elements; (3) the connection pattern among different membrane-bounded cellular compartments; and (4) the architecture of endomembranes (i.e. presence of pits and fenestrations). In the nucleus, the main differential features were the the three-dimensional view of the nuclear envelope, the distribution of nuclear pores and the aggregation pattern of chromatin, visualized as clusters of nuclear particles in cross-fractures. The quantitative analysis of chromatin revealed four peaks of nuclear particle sizes (8, 12, 17 and 21 nm) that may correspond to variable degrees of coiling of the polynucleosomal chain in the chromatin fibre. Significant differences were observed in the proportion, numerical density and size distribution of aggregated nuclear particles in heterochromatin domains among the different cell types of the cerebellar cortex. The percentage of nuclear particles in aggregates varied from 10% in Purkinje cells to 64% in granule cells. Astrocytes and Bergmann glia showed intermediate values (about 40%). The percentage of nuclear particles in aggregates showed a significant (P less than 0.05) negative linear correlation with the nuclear volume, the number of pores per unit nuclear volume and the total number of pores per nucleus. In granule cells and astroglia, heterochromatin domains had a greater percentage of large nuclear particles (greater than 10 nm) than did euchromatin domains, whereas in interneurons, Purkinje and Golgi cells heterochromatin and euchromatin showed a similar proportion of large particles. Nuclear particles in euchromatin exhibited a similar pattern of distribution in all cerebellar cells.

Estradiol increases the number of nuclear pores in the arcuate neurons of the rat hypothalamus

Freeze-fracture replicas of hypothalamic arcuate neurons and of Purkinje and granule cells of the cerebellar cortex from adult female rats were assessed in order to test the possible influence of estradiol on nuclear pores. Rats were ovariectomized and injected either with estradiol or with vehicle. An additional group of rats in proestrus was also studied. Pore diameter was not affected by ovariectomy or estrogen treatment. In arcuate neurons, the number of nuclear pores per nuclear membrane area, the total number of pores per nucleus, and the percentage of nuclear pores arranged in clusters were decreased by ovariectomy and increased within 30 minutes after estradiol administration to ovariectomized rats. The effect of estradiol on nuclear pores was sustained for several days; the number of pores and the percentage of pores in clusters reverted to control values by 1 month after the hormonal treatment. None of the above mentioned changes was observed in Purkinje and granule cells of the cerebellar cortex. These results indicate that estradiol may modulate the number and distribution of nuclear pores in arcuate neurons and suggest that the modification of the ultrastructure of the nuclear envelope may be one of the first effects of gonadal steroids on target cells.

Unequal distribution of nuclear pores in oryzalin-induced mini-nuclei in protonema cells of the mossFunaria hygrometrica: influence of the nucleolus

Mini-nuclei, formed in tip cells ofFunaria caulonemata after oryzalin treatment, have unequally distributed nuclear pores. The region of the nuclear envelope near the nucleolus, in a distance of up to 3 μm, is devoid of pores. In other areas pores occur with a high frequency.

High-voltage cytochemistry for three-dimensional observation of nuclei and calculation of total numbers of nuclear pores in retinal Mueller glia

Introduction: We have demonstrated that Glucose-6-phosphatase (G6Pase) activity is localized to the endoplasmic reticulum and nuclear envelope of Mueller glia in the normal and pathological guinea pig retina. Using a combination of this cytochemical technique and high voltage electron microscopy, the distribution of nuclear pores could be clearly observed on the nuclear envelope of Mueller glia because of their anatomical lack of reaction products. This technique was developed to study the three-dimensional structure of nuclei and to calculate total numbers of nuclear pores utilizing a computer graphic analysis system in the normal and pathological retina.Materials and methods: Normal and photocoagulated retina of pigmented adult guinea pigs were perfused with a cold mixture of 0.25% glutaraldehyde and 2% paraformaldehyde in 0.1M cacodylate buffer, and the enucleated globes were hemisected and immersed in the same fixative for 30 min. After sectioning and incubation in the reaction medium for the detection of G6Pase activity by the method of Wachstein-Meisel, the sections were postfixed, dehydrated and embedded in Spurr’s epoxy resin. Serial thick sections (1.0um) were prepared for the observation by a Hitachi high voltage electron microscope (H 1250-M) with an accelerating voltage of 1000 Kv. and pictures were analyzed and three-dimensionally reconstructed by TRI (RATOC Co., Ltd.).

The polar development of membrane structure during spermiogenesis by freeze fracture and double replica technique

With the developing process from the spermatid to the sperm, the great changes of membrane structure are token place both in plasma membrane and nuclear membrane. This note will report the development of polarization in membrane and the distribution of nuclear pores and protein particles during spermiogenesis, and also compare the number of protein in P-face with that in E-face.Mice, Rats, Rabbits and Dogs were operated by taking out the seminiferous tube. It was fixed in 2.5% glutaradehyde for 2 hrs 5 washed in cacodylated buffer, and then the sample was put in to 30% glycerol solution for 6-12hrs. Samples were put into liquid nitrogen rapidly and afterwards moved to BALZAR BAF-400D instrument to get Freeze-Fracture replica and double replica for observation with TEM.The expression and the development of the polarization in membrane structure of the spermatid. The spermatid in different stepshas different characteristics and there is more polarization for membrane structure following spermiogenesis.