Mature Spermatozoids Research Articles

Brg1 subunit of chromatin remodelling complex is present during Chara vulgaris (Charophyceae) spermatogenesis

ABSTRACTDuring spermatogenesis, cells developed as a result of numerous mitotic and meiotic divisions transform into mature spermatozoids. In spermatids, remodelling of chromatin structure takes place which is connected with nuclear protein exchange, DNA double strand breaks and epigenetic modifications. Chromatin remodelling complexes, which have mostly been studied in animals, also participate in this process. The Brg1 protein, a functional homologue of the yeast Swi2/Snf2 catalytic subunit of the SWI/SNF complex, is engaged in regulation of cell proliferation and highly expressed in round spermatids in mammals. Immunocytochemical studies with the anti-Brg1 antibody revealed positive reactions in nuclei of the green alga Chara vulgaris at the 64-cell proliferative stage and in spermatid nuclei at the I/II–VII spermiogenesis stages. The most intensive reaction was observed at the early spermiogenesis stages (I/II–III), while at the initial stages of a proliferative phase (4-, 8- and 16-cell) the reaction was not observed, and at 32-cell and VII stages the immunosignals were very weak. Ultrastructural studies with the immunogold technique confirmed the results of the immunocytochemical studies. The highest numbers of gold grains were observed at stages I/II and III of spermiogenesis, and together they constituted above 48% of the total number of gold grains. A much lower, but still substantial, amount of these grains was observed at the 64-cell stage and IV stage (>15% and 17%), respectively. Percentage analysis revealed the lowest number of gold particles at stage VII (3.72%). The significant presence of Brg1 protein at early spermiogenesis stages is correlated with acetylation of the H4K12 histone. It may also be hypothesized that in C. vulgaris the Brg1 subunit participates in processes important for proper chromatin condensation and facilitates maintenance of the correct shape of the spermatid nucleus. On the basis of earlier and current studies it seems that chromatin remodelling in spermatids of this model alga proceeds according to the model presented for mammals.

Ultrastructural, autoradiographic and electrophoretic examinations of Chara tomentosa spermiogenesis

Ultrastructure of a spermatid nucleus changes many times during spermiogenesis. Condensed chromatin forms irregular clusters during phases I-II, a continuous ring adjacent to a nuclear envelope during phases III-V and a network occupying the whole nucleus during phase VI. In advanced spermiogenesis dense chromatin disappears and short randomly positioned fibrils arise, then long parallel ones are found (phase VIII) which during phase IX form a lamellar structure. In mature spermatozoids (phase X) chromatin becomes extremely condensed. <sup>3</sup>H-arginine and <sup>3</sup>H-lysine incorporation into spermatids during 2-min incubation is intensive during phases IN, decreases during phases VI, VII and becomes very low during phases VIII-IX. Capillary electrophoresis has shown that during <em>Chara tomentosa</em> spermiogenesis replacement of histones with basic proteins whose mobility is comparable to that of salmon protamines takes place. At the beginning of spermiogenesis core and linker histones are found in spermatids. During early spermiogenesis protamine-like proteins appear and their amount increases in late spermiogenesis when core histones are still present. In mature spermatozoids only protamine-like proteins represented by 3 fractions: 9.1 kDa, 9.6 kDa, 11.2 kDa are found. Disappearance of linker histones following their modification precedes disappearance of core histones. The results indicate that dynamic rearrangement of chromatin ultrastructure and aminoacid incorporation rate during spermiogenesis are reflected in basic nuclear protein changes.

Failed sperm development as a reproductive isolating barrier between species.

Hybrid male sterility is a common reproductive isolating barrier between species. Yet, little is known about the actual developmental causes of this phenomenon, especially in naturally hybridizing species. We sought to evaluate the developmental causes of hybrid male sterility, using spadefoot toads as our study system. Plains spadefoot toads (Spea bombifrons) and Mexican spadefoot toads (S. multiplicata) hybridize where they co-occur in the southwestern USA. Hybrids are viable, but hybrid males suffer reduced fertility. We compared testes size and developmental stages of sperm cell maturation between hybrid males and males of each species. We found that testes of hybrid males did not differ in mean size from pure-species males. However, hybrids showed a greater range of within-individual variation in testes size than pure-species males. Moreover, although hybrids produced similar numbers of early stage sperm cells, hybrids produced significantly fewer mature spermatozoids than pure-species males. Interestingly, an introgressed individual produced numbers of live sperm comparable to pure-species males, but the majority of these sperm cells were abnormally shaped and non-motile. These results indicate that hybrid incompatibilities in late sperm development serve as a reproductive isolating barrier between species. The nature of this breakdown highlights the possibilities that hybrid males may vary in fertility and that fertility could possibly be recovered in introgressed males.

Gametogenesis in the Pacific Oyster Crassostrea gigas: A Microarrays-Based Analysis Identifies Sex and Stage Specific Genes

BackgroundThe Pacific oyster Crassostrea gigas (Mollusca, Lophotrochozoa) is an alternative and irregular protandrous hermaphrodite: most individuals mature first as males and then change sex several times. Little is known about genetic and phenotypic basis of sex differentiation in oysters, and little more about the molecular pathways regulating reproduction. We have recently developed and validated a microarray containing 31,918 oligomers (Dheilly et al., 2011) representing the oyster transcriptome. The application of this microarray to the study of mollusk gametogenesis should provide a better understanding of the key factors involved in sex differentiation and the regulation of oyster reproduction.Methodology/Principal FindingsGene expression was studied in gonads of oysters cultured over a yearly reproductive cycle. Principal component analysis and hierarchical clustering showed a significant divergence in gene expression patterns of males and females coinciding with the start of gonial mitosis. ANOVA analysis of the data revealed 2,482 genes differentially expressed during the course of males and/or females gametogenesis. The expression of 434 genes could be localized in either germ cells or somatic cells of the gonad by comparing the transcriptome of female gonads to the transcriptome of stripped oocytes and somatic tissues. Analysis of the annotated genes revealed conserved molecular mechanisms between mollusks and mammals: genes involved in chromatin condensation, DNA replication and repair, mitosis and meiosis regulation, transcription, translation and apoptosis were expressed in both male and female gonads. Most interestingly, early expressed male-specific genes included bindin and a dpy-30 homolog and female-specific genes included foxL2, nanos homolog 3, a pancreatic lipase related protein, cd63 and vitellogenin. Further functional analyses are now required in order to investigate their role in sex differentiation in oysters.Conclusions/SignificanceThis study allowed us to identify potential markers of early sex differentiation in the oyster C. gigas, an alternative hermaphrodite mollusk. We also provided new highly valuable information on genes specifically expressed by mature spermatozoids and mature oocytes.

Architecture of the mid-stage spermatid of Aneura pinguis (Aneuraceae)

Abstract We investigated the locomotory apparatus of the mid-stage spermatid of Aneura pinguis in order to gain a better understanding of the evolution and development of gamete ultrastructure in liverworts. The locomotory apparatus consists of two staggered but overlapping basal bodies and a multilayered structure made up of a spline and lamellar strip. The basal bodies include a transition region between the basal body and flagellum containing a short stellate pattern and dense matrix. The spline's maximum width is 20 microtubules and reduces in number at the posterior of the multilayered structure; however, this number appears variable at the mid-stage of development and so it is recommended that the mature spermatozoid be investigated for this feature. The anterior basal body is situated over the spline aperture, which measures 2 microtubules in width and 0.85 µm in length. The lamellar strip measures 4.06 µm in length and 0.60 µm in width, and is spatulate in shape with a right lateral offset or keel...

Study of chromatin decondensation factors in human spermatozoids by flow cytometry

To date, the mechanisms responsible for radical change of chromatin structure in male germ cells during fertilization are unclear. Evidence suggesting the existence of proteolytic nuclear enzymes in mature human spermatozoids are presented in this work. The possible role of these previously unknown proteases in decondensation of chromatin of spermatozoids in a fertilized ovum is discussed. Application of the flow cytometry technique has shown that treatment of human spermatozoid nuclei with SH-reagents leads not only to destruction of disulfide bonds between protamine molecules that is necessary for their effective utilization but also induces specific endogenous proteolytic activity that consequently results in rather fast decondensation of chromatin followed by proteolytic cleavage of nuclear proteins. A chromatin decondensation process can be almost totally blocked by serine protease inhibitors and components of seminal fluid. An original cytochemical approach of binding of fluorescently labeled protease inhibitor to the target of investigation has been used in order to visualize the localization of proteases in male germ cell nuclei. The results of our study suggest that one of the factors of chromatin reorganization involved in the formation of male pronucleus is endogenous nuclear protease of spermatozoids, which is activated by glutathione or other SH-components of ovum cytoplasm.

Phosphorylation of H2AX histone as indirect evidence for double-stranded DNA breaks related to the exchange of nuclear proteins and chromatin remodeling in Chara vulgaris spermiogenesis

Phosphorylation of H2AX histone results not only from DNA damage (caused by ionizing radiation, UV or chemical substances, e.g. hydroxyurea), but also regularly takes place during spermiogenesis, enabling correct chromatin remodeling. Immunocytochemical analysis using antibodies against H2AX histone phosphorylated at serine 139 indirectly revealed endogenous double-stranded DNA breaks in Chara vulgaris spermatids in mid-spermiogenesis (stages V, VI and VII), when protamine-type proteins appear in the nucleus. Fluorescent foci were not observed in early (stages I-IV) and late (VIII-X) spermiogenesis, after replacement of histones by protamine-type proteins was finished. A similar phenomenon exists in animals. Determination of the localization of fluorescent foci and the ultrastructure of nuclei led to the hypothesis that DNA breaks at stage V, when condensed chromatin adheres to the nuclear envelope. This is transformed into a net-like structure during stage VI, probably allowing chromosome repositioning to specific regions in the mature spermatozoid. However, at stages VI and VII, DNA breaks are necessary for transformation of the nucleosomal structure into a fibrillar and finally the extremely condensed status of sleeping genes at stage X.

Morphological changes of sperm nuclei during spermatogenesis in the brown alga Cystoseira hakodatensis (Fucales, Phaeophyceae)

SUMMARY Morphological changes and chromatin condensation of sperm nuclei were observed during spermatogenesis in the fucalean brown alga Cystoseira hakodatensis (Yendo) Fensholt. Ultrastructural studies have shown that the mature spermatozoid has an elongated and concave nucleus with condensed chromatin. The morphological changes and the chromatin condensation process during spermatogenesis was observed. Nuclear size decreased in two stages during spermatogenesis. During the first stage, spherical nuclei decreased in size as they were undergoing meiotic divisions and the subsequent mitoses within the antheridium. During the second stage, the morphological transformation from a spherical into an elongated nucleus occurred. Afterwards, chromatin condensed at the periphery in each nucleus, and chromatin-free regions were observed in the center of the nucleus. These chromatin-free regions in the center of nucleus were compressed by the peripheral chromatin-condensed region. As the result, the elongated and concave nucleus of the mature sperm consisted of uniformly well-condensed chromatin.

Novel features of Equisetum arvense spermatozoids: insights into pteridophyte evolution

Summary • To characterize structural diversity within Equisetum and among pteridophytes, architectural features of the sperm cell are described here in a second subgenus of Equisetum, a divergent basal group in the fern clade. • Transmission electron microscopy observations of prereleased spermatozoids of Equisetum arvense were correlated with three-dimensional scanning electron microscopy images of swimming cells. • The mature spermatozoid completes a helix of approximately 2.5 revolutions. At the cell anterior is a complex multilayered locomotory apparatus with staggered flagella. Mitochondria (elongated–rounded) are aggregated near the locomotory apparatus and organelles extend along the cell length. The spline contains up to 300 microtubules and wraps in part around the long cylindrical nucleus. In swimming sperm cells, the anterior of the cell remains tightly coiled while the posterior relaxes and extends in a trailing fashion. • Spermatozoids of Equisetum arvense are smaller than those of Equisetum hyemale but structurally similar, except for nuclear shape. Conservation of cellular features suggests recent radiation of the genus. Equisetum spermatozoids share several critical features with ferns, including Psilotum, and support monophyly of a fern–Equisetum assemblage. Entry of the male gametes of Equisetum in their entirety into the archegonial venters indicates possible biparental inheritance of chloroplast and mitochondrial genomes.

Ultrastructural characterization of the locomotory cytoskeletal system of the spermatozoid inGinkgo biloba

The development of the locomotory cytoskeletal system of sperm is carefully coordinated with the development of the sperm inGinkgo biloba. Here we report further ultrastructural characterization of the locomotory cytoskeletal system in the developing spermatid and mature spermatozoid, particularly with respect to the initiation and early development of the flagellar apparatus. A multilayered structure (MLS) assembles from an electron-dense matrix that self-organizes after blepharoplast breakup and then further elongates. At the tail of the assembling MLS, the spline microtubules connect to an anterior beak of the nuclear envelope. Nuclear-pore complexes are found on the nuclear envelope close to this beak. The mitochondria which elongate and line up one behind the other are tightly associated with the MLS. The MLS ofG. biloba is composed of an upper layer of parallel spline microtubules and a lower layer consisting of a fibrous lamellar strip composed of paralled fibers about 9 nm in diameter. Higher-magnification images show that the fully assembled fibers of the lamellar strip consist of subunits which suggest that protofilaments are involved in the assembly processes. A unique cytoskeletal system of the spermatozoid inG. biloba is given by the anterior bundle of microtubules. This bundle, in which microtubules are arranged parallel to each other, forms between the plasmalemma and the MLS and is about 214–392 nm in cross section. These microtubules expand spirally along the MLS band. Other details of cellular fine structure of the mature spermatozoid are described.

Correlative Sem And Tem Of Plant Spermatozoids: Equisetum arvensel.

The phylogenetic position of mosses, ferns and fern allies is enhanced by detailed knowlegde of gametophyte reproductive structures, in particular the spermatozoid (freeswimming sperm). The number and placement of flagella in conjunction with their ultrastructure and that of the multilayered structure (MLS) has been used to establish related groups. Herein, ultrastructural details of pre-release spermatozoids obtained via TEM are compared with the morphological appearance of fully mature (free-swimming) spermatozoids obtained via SEM. This represents the first intensive study of any plant sperm cell that correlates 3-dimensional SEM images with TEM ultrastructural features.Equisetum arvense L. is a fern ally commonly refered to as ‘horsetail’. The mature spermatozoid (Fig. 1) is twisted into approximately 3.5 revolutions, or gyres. A complex locomotory apparatus (Fig. 2), involving approximately 55 flagella, dominates the anterior region of the cell. The basal bodies subtending these flagella feature a well-defined stellate pattern and occur in 4 irregular rows staggered over approximately 1.75 gyres.

Architecture of the Spermatozoid of Selaginella australiensis

Architecture of the Spermatozoid of Selaginella australiensis

Monoclonal antibodies to surface and cytoskeletal components of the spermatozoid ofPteridium aquilinum

Detergent extracted spermatozoids of the fernPteridium aquilinum were used as mixed antigen preparations for raising monoclonal antibodies in order to obtain reagents for detecting as yet uncharacterized components of the plant cytoskeleton. Selected antibodies were studied by immunofluorescence microscopy of developing spermatids and mature spermatozoids. Some reacted directly with fixed cells, others required permeabilization treatments with cold methanol or Triton X-100. AntibodiesPas2D9 andPas6D7 bind to glycoprotein antigenic determinants that are exposed on the surface of the plasma membrane. Several antibodies interact with cytoskeletal components.Pas1D3,Pas5D8 andPas5F4 bind to the cytoskeleton of permeabilized cells including the flagella. Three react specifically with the flagellar band or associated components:Pas2G6 reacts with the whole flagellar band but shows a prominent binding to basal bodies,Pas5E2 binds exclusively to basal bodies, andPas5E7 detects mitochondria associated with the flagellar band. Cross-reactions to wheat root tip cells at different stages of the cell cycle are described inMarc andGunning (1988).

Immunofluorescent localization of cytoskeletal tubulin and actin during spermatogenesis inPteridium aquilinum (L.) Kuhn

Details concerning the appearance and behaviour of blepharoplasts during spermatogenesis, and the assembly of the cytoskeletal motile apparatus of spermatids were elucidated by immunofluorescence microscopy using antibodies to tubulin and actin, applied to material prepared from antheridia of the fernPteridium aquilinum (L.) Kuhn. Blepharoplast immunofluorescence with antitubulin first appears as spheres at the future spindle poles prior to the last spermatogenous division. Developing spermatids each have one blepharoplast, which gives rise to a triangular layer corresponding to the incipient microtubule ribbon. Compared to the ribbon, immunoreactivity of the multilayered structure is relatively weak. Intensely fluorescing basal bodies appear, increase in number, and become arranged in rows along two edges of the microtubule ribbon as it widens and elongates. Along the dorsal edge is a dense file of basal bodies spaced at about 0.3 μm intervals, parallel to each other and oriented at 145° to the multilayered structure. This spacing and orientation is maintained throughout spermatid development. Basal bodies at the opposite edge are initially oriented at 115° to the multilayered structure but become rearranged into small groups that rotate so that the angle is reduced to 55–70° by the time the assembly of flagella commences on both sets of basal bodies. By this stage the microtubule ribbon has encircled about 2/3 of the nuclear circumference and the nucleus is assuming a crescent shape. In fully developed spermatozoids the groups of basal bodies are oriented at 25° to the multilayered structure, parallel to the long body of the now helical nucleus. Immunofluorescence using antiactin showed that towards the completion of nuclear shaping, actin forms a strip along the helical multilayered structure. Detergent-extraction of mature spermatozoids revealed that actin is associated also with the flagellar band, particularly with basal bodies.

The Blepharoplast of Zamia pumila L.

Blepharoplasts of Zamia pumila L. are paired 26-μm-diameter multicentriolar bodies in the spermatogenous cell of the pollen tube. Each blepharoplast has ca. 46,000-49,000 centrioles surrounding an electron-dense core. During division of the spermatogenous cell, the centrioles migrate outwardly, collectively forming a sphere of somewhat greater diameter than the blepharoplast, which then collapses to form a somewhat irregular mass of evenly distributed centriolar clusters adjacent to each of the two spermatid nuclei. A multilayered structure appears in the midst of these clusters and organizes the spiral band to which the spline apparatus (an array of cytoskeletal microtubules) and numerous flagellar basal bodies are attached. The mature spermatozoid has ca. 40,000-50,000 flagella.

Immunofluorescence microscopy of the flagella and multilayered structure in two mosses, Sphagnum palustre L. and Polytrichum juniperinum Hedw.

Antibody against tubulin from porcine brain was used to examine the distribution of tubulin in developing spermatids of Polytrichum and mature spermatozoids of Sphagnum. Cells were prepared for indirect immunofluorescence microscopy after fixation in buffered paraformaldehyde and brief incubation in cellulase. Pretreatment with cold methanol resulted in considerably enhanced immunofluorescence but exposure to Triton X-100, with or without sonication, had no effect. The antibody showed similar immunological cross-reactivity with the flagella (both basal bodies and axonemes) and the spline microtubules of the multilayered structure. This is the first direct evidence that this rigid array of stable cytoskeletal microtubules consists of tubulin. Particularly intense fluorescence from the lamellar strata of the MLS in developing spermatids provides strong support for the notion that the lamellae comprise a highly structured microtubule organizing centre (MTOC), responsible for the ordered assembly of the overlying spline tubules. The demonstration of immunological cross-reactivity with antitubulin from porcine brain tubulin, within a plant structure other than fully formed microtubules, suggests that immunocytochemistry may have considerable potential for the detection of other MTOCs. By contrast, no detectable fluorescence emanated from the granular matrix cementing the flagellar basal bodies to the spline or the spindle-shaped sheath of fibres present in the spermatozoids of Sphagnum. Disruption of the mature gametes by sonication and treatment with Triton X-100 reveals the presence of particularly strong links between the spline and subjacent nuclear envelope.

Etude ultrastructurale comparée de la spermiogenènes des monogènes: I. Megalocotyle (Monopisthocotylea Capsalidae)

In Megalocotyle the zones of differentiation (ZD) are short and incomplete; there is no median cytoplasmic process or free flagella. Some peripheral microtubules are present in early ZD, but they disappear. Each nucleus of the common cytoplasmic mass inserts one extremity into a ZD. The mitochondria fuse around this extremity and form a beadlike perforated sphere. Later this sphere slides along the nucleus away from the plasmic membrane like a bead on a string. Each mitochondrial bead (MB), as it moves, trails part of its bulk in the form of a solid streamer; two parallel centrioles follow the MB, each generating a trailing 9+“1” flagellum. The result is a four-part ensemble extending from each MB to each ZD: the two axonemes, the streamer, and the nucleus, with no membrane separating it from the surrounding cytoplasm. Subsequently a cytoplasmic canal (CC) is formed. The arching membranes, originally located at the base of the ZD, migrate along the four-part ensembles as far as the MB, leaving behind two membranes which define the CC. This formation of the CC is remarkable in that it occurs after migration of the centrioles. At the end of spermiogenesis, the cytoplasmic mass reveals 64 parallel CC each containing an immature spermatozoid whose free rear extremity lengthens out. Each spermatozoid is then detached at its anterior extremity set in the cytoplasmic mass. As in most monopisthocotylean monogeneans, the mature spermatozoid of Megalocotyle lacks peripheral microtubules, although its early ZD has microtubules which subsequently disappear. This feature distinguishes it from trematodes and polyopisthocotylean monogeneans, whose mature spermatozoid keep their ZD microtubules.

THE OCCURRENCE AND PHYLOGENETIC SIGNIFICANCE OF A MULTILAYERED STRUCTURE IN COLEOCHAETE SPERMATOZOIDS

Spermatozoid‐forming cells of Coleochaete scutata were found in packets of four arranged in concentric internal bands. Spermatozoids, which occur singly in antheridial cells, are spherical to ovoid, approximately 7 μm long by about 3.9 μm wide. As compared to relatively unspecialized zoospores, male gametes undergo a number of specialized cellular changes during development. The spherical nuclei and cytoplasm of mature spermatozoids are increased in density. Posterior plastids are reduced and contain large starch grains. Many small mitochondria are clustered near the cell anterior. The plasmalemma is covered with a layer of flattened, diamond‐shaped scales, while body scales of zoospores are pyramidal. The two flagella of both zoospores and spermatozoids are covered with flattened, diamond‐shaped scales and hairs. The spermatozoids contain an anterior multilayered structure (MLS) structurally similar to, though smaller than, the MLS observed in zoospores. An asymmetrical cytoskeleton consisting of a band of 30–45 microtubules extends from the MLS down one side of the spermatozoid close to the plasmalemma. An immature MLS was observed in an early stage of spermatozoid development. The finding of an MLS and asymmetrical cytoskeleton in specialized male gametes as well as relatively unspecialized zoospores of Coleochaete strengthens assumptions of homology between MLSs of green algal reproductive cells and those found in flagellated spermatozoids of archegoniate plants. The structure of the spermatozoid of Coleochaete supports the hypothesis that this alga may be relatively close to the phylogenetic line which led directly to archegoniates.

SPERMATOGENESIS IN LYCOPODIUM: THE MATURE SPERMATOZOID

The mature spermatozoid of Lycopodium cernuum is a blunt ended, fusiform cell, 8–10 μm long by 4–5 μm wide. A multilayered structure (MLS) and a subtending anterior mitochondrion are located at the anterior of the cell. The MLS is coiled through 1–1.5 gyres in a shallow sinistral helix around the periphery of the cell. The MLS would be triangular in outline if unwound and laid flat, about 1.4 μm wide, 7.5–8 μm long, and 80 nm thick. The MLS comprises four layers, S1–S4. The S1 forms the spline, a supportive sheet of microtubules; the S2, lamellate in younger stages, is an homogeneous, darkly staining layer in the mature sperm; the S3 and S4 retain their lamellate appearance and are delimited by lateral connections. Approximately 200 S1 microtubules extend posteriorly from the MLS at about 45° to the MLS long axis and form a partial sheath around the nucleus. The two basal bodies are located on opposite sides of the cell external to the MLS. Each is tangential to the curve of the MLS and surrounded by a globular matrix. At their attachment, the axonemes are oriented laterally and are antiparallel to each other. Distally, the flagella, each about 38 μm long, trail behind the cell as it swims. The nucleus is roughly ovoid, about 4 μm diam, and centrally or sometimes laterally located. The greater volume of the nucleus is occupied by condensed, amorphic chromatin. Cavities within the chromatin are often seen to contain spheroidal inclusions that have two differently staining regions. The inclusions are also located at the periphery of the chromatin. The posterior of the cell is occupied by several small mitochondria and an amyloplast, about 2 μm diam containing numerous starch grains.

A microtubule--nuclear envelope complex in the spermatozoid of Pteridium.

The microtubules on the outside of the nucleus of the mature spermatozoid of Pteridium form a complex with the envelope, and the perinuclear space is eliminated. These dissimilar components are held firmly together, possibly by hydrophobic bonding. The outer part of the chromatin is also attached to the inner boundary of the envelope, and pulls away when the envelope is detached.