Distinct Protrusions Research Articles

Effects of the Biomimetic Microstructure in Electrospun Fiber Sutures and Mechanical Tension on Tissue Repair.

Electrospun microfibers, designed to emulate the extracellular matrix (ECM), play a crucial role in regulating the cellular microenvironment for tissue repair. Understanding their mechanical influence and inherent biological interactions at the ECM interface, however, remains a complex challenge. This study delves into the role of mechanical cues in tissue repair by fabricating Col/PLCL microfibers with varying chemical compositions and alignments that mimic the structure of the ECM. Furthermore, we optimized these microfibers to create the Col/PLCL@PDO aligned suture, with a specific emphasis on mechanical tension in tissue repair. The result reveals that within fibers of identical chemical composition, fibroblast proliferation is more pronounced in aligned fibers than in unaligned ones. Moreover, cells on aligned fibers exhibit an increased aspect ratio. In vivo experiments demonstrated that as the tension increased to a certain level, cell proliferation augmented, cells assumed more elongated morphologies with distinct protrusions, and there was an elevated secretion of collagen III and tension suture, facilitating soft tissue repair. This research illuminates the structural and mechanical dynamics of electrospun fiber scaffolds; it will provide crucial insights for the advancement of precise and controllable tissue engineering materials.

The male stem cell niche of Drosophila melanogaster: Interactions between the germline stem cells and the hub

The Drosophila male stem cell niche is a well characterized structure in which a small cluster of somatic cells send self-renewal signals to neighbouring germ cells. Although the molecular information involved in the stem cell fate have been identified, much less is understand on the mechanisms driving their short-range specific release. Our ultrastructural analysis reveals distinct protrusions of the stem cell plasma membrane that interdigitate with membrane protrusions of the facing hub cells. Some of these protrusions are very elongated and extend into the hub and could correspond to the Mt-Nanotubes. Therefore, the interface between the stem cells and the hub appears more complex than previously reported and the membrane protrusions of the stem cells might represent specialized surface areas involved in the niche-stem cell communication. We also noticed the presence of clathrin-coated vesicles in the germline plasma membrane that might be also involved in delivering information from the hub.

The Septum Inguinalis: A Clue to Hernia Genesis?

Purpose: Double ipsilateral inguinal (“pantaloon”) hernias and also the more advanced “combined” inguinal hernia involve disruption of the inguinal floor. In the case of pantaloon hernias, the medial boundary of the internal ring remains intact but in combined hernias this is fully disrupted, producing a single hernial protrusion. Deepening the pathophysiology of these hernias may be helpful in addressing hernia genesis, thus improving strategies for the treatment of this disease. Materials and Methods: A cohort of 22 patients who underwent inguinal hernia repair showed double ipsilateral (pantaloon) hernia, comprising distinct direct and indirect protrusions separated by a tissue septum. In 19 patients, the septal arrangement dividing the 2 hernias showed macroscopically evident structural damages, then excised and histologically studied. Different tissue markers were used for the identification of the structural damages. Results: Macroscopically, the divisor septum represents the boundary between internal ring and Hesselbach’s triangle. Anteriorly it is composed by fibers of the internal oblique and transverse muscles, which form a complex with the inferior epigastric vessels on the corresponding posterior side of the inguinal floor. In the patient cohort studied, this anatomical structure showed a progressive sufferance characterized by chronic compressive damage. Conclusion: The anatomical structure which separates the indirect and direct components of a pantaloon hernia, herein referred to as the “septum inguinalis”, has been shown progressively alter in both macro- and microscopically until it f undergoes disruption with development of a combined hernia. Understanding of this anatomical concept may help surgeons to perform sound repairs of these complex hernias.

Development of decellularized amniotic membrane as a bioscaffold for bone marrow-derived mesenchymal stem cells: ultrastructural study

Developing effective stem cell-based therapies requires the design of complex in vitro culture systems for accurate representation of the physiological stem cell niche. Human amniotic membrane (hAM) has been successfully used in clinical grafting applications due to its unique biological and regenerative properties. Decellularized hAM (d-hAM) has been previously applied to the culture of human bone marrow mesenchymal stem cells (hMSCs), promoting their expansion and differentiation into adipogenic and osteogenic lineages. In the present study, hAM was decellularized by NaOH-treatment, to provide the three-dimensional (3D) bioscaffold for culturing hMSCs. The ultrastructural differences between intact hAM and decellularized hAM were characterized using the transmission electron microscope (TEM), as well as the 3D interaction between d-hAM and hMSCs cultured on the membrane. TEM examination of the intact hAM showed many microvilli on the epithelial layer cells, active Golgi apparatus, smooth endolplasmic reticulum and the characteristic pinocytic vesicles. The epithelial layer with its structures was absent in the d-hAM. However, no observable difference was detected in the ultrastructural characteristics of the compact stromal layer of d-hAM compared to intact hAM. Both contained bundles of extra cellular matrix (ECM) proteins, and scattered elastic fibres. Cultured human mesenchymal stem cells (hMSCs) examined by TEM appeared oval to spherical in shape and had a rough and non-uniform surface with distinct protrusions or irregular fillopodia. Their diameter ranged from 20.49 to 21.6µm. Most of the cellular organelles were also noticed. SEM examination of the prepared samples revealed unique 3D interaction between the hMSC and d-hAM, where the latter seems to envelop the segments of the hMSCs lying on the surrounding membrane. This study shows that the decellularization process affected the epithelial layer only of hAM and had no effect on altering the presence of ECM components present in the stromal layer of the d-hAM. The interaction between hMSCs and d-hAM maybe mediated by hAM components other than human amniotic epithelial cells, such as ECM components or MSCs present in the deeper spongy layer of the membrane or/and the adhesive components of the basement membrane of the removed epithelial layer.

A new phase of rubrene monoclinic crystals on Cd(0001)

The self-assembly 2D thin films of rubrene molecules on a metallic Cd(0001) surface have been investigated using a low-temperature scanning tunneling microscopy. Compared with other precious metals, the divalent hexagonal close packed metal Cd are less reminiscent of a free electron metal because of the strong repulsive interaction between the 4d state and the conduction bands. It manifests some anisotropic behaviors in thermal conductivity, electron mean free path, and superconducting gaps. So in this paper, Cd was used as a substrate to study rubrene. First, isolated rubrene molecules reveal a tilted orientation on the substrate surface and exhibit three distinct protrusions with chiral feature. When the rubrene coverage increased to 0.4 ML, rubrene molecules begin to assemble into monolayer structure on the Cd(0001) substrate. Rubrene molecules adopt a side-lying orientation, and each rubrene molecule takes the form of zigzag with three bright spots. Upon further deposition of rubrene to 1.5 ML, the orientation transition from titled standing-up monolayer to standing-up monoclinic crystals was observed. There are two kinds of structures in the second layer. The first one is chainlike structure, where rubrene molecules are supported by two phenyls and the planes of tetracene are parallel to each other. The plane of tetracene backbone is perpendicular to the substrate plane. Another novel structure in the second layer of rubrene film is a new monoclinic phase. Furthermore, the rubrene monoclinic phase has a slight compressive strain in the a -axis direction, whereas the compressive strain in the c -axis direction becomes more pronounced. In addition, we noticed that one c -axis of a - c plane of monoclinic phase is parallel to the crystal orientation of the substrate. In particular, this monoclinic phase of rubrene was predicated by DFT calculation but has never been confirmed in experiment.

Indications of chemical bond contrast in AFM images of a hydrogen-terminated silicon surface

The origin of bond-resolved atomic force microscope images remains controversial. Moreover, most work to date has involved planar, conjugated hydrocarbon molecules on a metal substrate thereby limiting knowledge of the generality of findings made about the imaging mechanism. Here we report the study of a very different sample; a hydrogen-terminated silicon surface. A procedure to obtain a passivated hydrogen-functionalized tip is defined and evolution of atomic force microscopy images at different tip elevations are shown. At relatively large tip-sample distances, the topmost atoms appear as distinct protrusions. However, on decreasing the tip-sample distance, features consistent with the silicon covalent bonds of the surface emerge. Using a density functional tight-binding-based method to simulate atomic force microscopy images, we reproduce the experimental results. The role of the tip flexibility and the nature of bonds and false bond-like features are discussed.

Platelet Release from Infused CD34+-Derived Human Megakaryocytes: Lessons from a Natural-Occurring Platelet Bioreactor

The release of biologically intact platelets from in vitro-grown megakaryocytes remains one of the challenges in an attempt to replace donor-derived platelets with platelets prepared from in vitro-grown megakaryocytes. Thrombopoiesis, the process by which megakaryocytes release platelets, is thought to occur as megakaryocytes exit from the medullar space and transit into the blood stream. Two-photon fluorescence microscopy studies by several groups have documented megakaryocytes transitioning into the blood stream and extending mostly a single protrusion that appears to release large cytoplasmic fragments downstream. These studies have been widely interpreted to support platelet release in the bone marrow environment. A counter proposal made by Dr. William Howell over 75 years ago is that either whole megakaryocytes or large cytoplasmic fragments circulate to the lungs and release platelets in the lungs. Studies by our group infusing in vitro-grown megakaryocytes (both human and mouse) have shown that infused megakaryocytes do indeed become entrapped in the lungs and release platelets. These in vivo-released platelets have a much longer half-life than in vitro-prepared platelets from static cultures and have the same size distribution and nearly the same functionality as infused donor-derived platelets. Using confocal intravital microscopy, we now directly visualized the lungs of recipient NOD-SCID interferon 2 receptor γ-deficient mice during the infusion of calcein-loaded CD34+-derived megakaryocytes. Infused human megakaryocytes were immediately arrested in the pulmonary bed in vessels ~50 µm in diameter. Each cell then extended several distinct protrusions winding down presumed pulmonary capillaries that are presumably wrapped around alveoli. Some of these protrusions reached 200-300 µm in length and assumed the appearance of beads on a string. Consistent with our recently published data, this thrombopoiesis process appears to take 30 minutes to be near-complete, similar to the timeframe we showed for detecting newly released human platelet-like particles after megakaryocyte infusion. At 30 minutes, remaining bodies of the megakaryocytes are still present and we presume these are mostly the remaining nuclei. To better define whether the pulmonary bed is unique for releasing platelets, we also infused megakaryocytes intra-arteriole rather than intravenously to have the megakaryocytes encounter other organ capillary beds before the lung. These studies showed poor platelet release compared to parallel studies in mice receiving the megakaryocytes intravenously. Many of the intra-arterial infused megakaryocytes were entrapped in the spleen and very few were notable in other organs, including the lungs, liver and kidney. Our studies showed that megakaryocytes can shed platelets in the lungs where they may take advantage of the unique three-dimensional organization of the pulmonary vascular bed, flow conditions, vascular surface receptors and glycocalyx as well as a sudden shift from hypoxic to normoxic conditions. Whether these features of the pulmonary vascular bed can be simulated ex vivo and whether this will enhance true platelet release from megakaryocytes in vitro needs to be examined. DisclosuresNo relevant conflicts of interest to declare.

Identification of O-rich structures on platinum(111)-supported ultrathin iron oxide films

Using high-resolution scanning tunneling microscopy (STM) we have studied the oxidation of ultrathin FeO films grown on Pt(111). At the initial stage of the FeO film oxidation by atomic oxygen exposure, we identified three distinct types of line defects, all of which form boundaries between FeO domains of opposite orientation. Two types of line defects appearing bright (type-i) and dark (type-ii) in the STM images at typical scanning parameters are “metallic”, whereas the third line defect exhibits nonmetallic behavior (type-iii). Atomic-scale structure models of these line defects are proposed, with type-i defects exhibiting 4-fold coordinated Fe atoms, type-ii exhibiting 2-fold coordinated O atoms, and type-iii exhibiting tetrahedrally-coordinated Fe atoms. In addition, FeO2 trilayer islands are formed upon oxidation, which appear at FCC-type domains of the moiré structure. At high scanning bias, distinct protrusions on the trilayer islands are observed over surface O ions, which are assigned to H adatoms. The experimental data are supported by density functional theory (DFT) calculations, in which bare and hydroxylated FeO2 trilayer islands are compared. Finally, we compare the formation of O-rich features on continuous FeO films using atomic oxygen with the oxidation of Pt(111)-supported FeO islands accomplished by O2 exposure.

Nanocone formation on ion-bombarded InP surfaces

Surfaces of InP(100) were bombarded by 2.1keV Ar+ ions under normal incidence. The total accumulated ion fluence the samples were exposed to was varied roughly from 1×1017cm−2 to 3×1018cm−2 while ion fluxes in the range of (0.1−2)×1014cm−2s−1 were used. The surface morphology resulting from these ion irradiations was examined by scanning electron microscopy (SEM). For a specific range of ion fluxes and fluences, regular closely-spaced nanocone arrays are found to form, with the cones having typical base diameters of about 100nm. Around their outer peripheries, they commonly exhibit rather distinct protrusions, an observation not reported in previous investigations. The inspection of the initial stages of nanocone formation indicates that the growth of adatom islands, first on the pristine surface and later on previously formed islands, may lead to these 3D nanostructures. This finding would imply that surface diffusion processes of adatoms constitute an important mechanism in the evolution of the observed topography.

The actin regulators Enabled and Diaphanous direct distinct protrusive behaviors in different tissues during Drosophila development.

Actin-based protrusions are important for signaling and migration during development and homeostasis. Defining how different tissues in vivo craft diverse protrusive behaviors using the same genomic toolkit of actin regulators is a current challenge. The actin elongation factors Diaphanous and Enabled both promote barbed-end actin polymerization and can stimulate filopodia in cultured cells. However, redundancy in mammals and Diaphanous' role in cytokinesis limited analysis of whether and how they regulate protrusions during development. We used two tissues driving Drosophila dorsal closure--migratory leading-edge (LE) and nonmigratory amnioserosal (AS) cells--as models to define how cells shape distinct protrusions during morphogenesis. We found that nonmigratory AS cells produce filopodia that are morphologically and dynamically distinct from those of LE cells. We hypothesized that differing Enabled and/or Diaphanous activity drives these differences. Combining gain- and loss-of-function with quantitative approaches revealed that Diaphanous and Enabled each regulate filopodial behavior in vivo and defined a quantitative "fingerprint"--the protrusive profile--which our data suggest is characteristic of each actin regulator. Our data suggest that LE protrusiveness is primarily Enabled driven, whereas Diaphanous plays the primary role in the AS, and reveal each has roles in dorsal closure, but its robustness ensures timely completion in their absence.

Surface grafted glycopolymer brushes to enhance selective adhesion of HepG2 cells

This work demonstrates the application of carbohydrate based methacrylate polymer brush, poly(2-lactobionamidoethyl methacrylate), for the purpose of cell adhesion studies. The first part of the work illustrates the effects of the structure of the aminosilane based ATRP initiator layer on the polymerization kinetics of 2-lactobionamidoethyl methacrylate) (LAMA) monomer on thermally oxidized silicon wafer. Both monolayer and multilayered aminosilane precursor layers have been prepared followed by reaction with 2-bromoisobutyrylbromide to form the ATRP initiator layer. It is inferred from the kinetic studies that the rate of termination is low on a multilayered initiator layer compared to a disordered monolayer structure. However both initiator types results in similar graft densities. Furthermore, it is shown that thick comb-like poly(LAMA) brushes can be constructed by initiating a second ATRP process on a previously formed poly(LAMA) brushes. The morphology of human hepatocellular carcinoma cancer cells (HepG2) on the comb-like poly(LAMA) brush layer has been studied. The fluorescent images of the HepG2 cells on the glycopolymer brush surface display distinct protrusions that extend outside of the cell periphery. On the other hand the cells on bare glass substrate display spheroid morphology. Further analysis using ToF-SIMS imaging shows that the HepG2 cells on glycopolymer surfaces is enriched with protein fragment along the cell periphery which is absent in the case of cells on bare glass substrate. It is suggested that the interaction of the galactose units of the polymer brush with the asialoglycoprotein receptor (ASGPR) of HepG2 cells has resulted in the protein enrichment along the cell periphery.

Three dimensional structure of the anthrax toxin translocon–lethal factor complex by cryo‐electron microscopy

We have visualized by cryo-electron microscopy (cryo-EM) the complex of the anthrax protective antigen (PA) translocon and the N-terminal domain of anthrax lethal factor (LF(N) inserted into a nanodisc model lipid bilayer. We have determined the structure of this complex at a nominal resolution of 16 Å by single-particle analysis and three-dimensional reconstruction. Consistent with our previous analysis of negatively stained unliganded PA, the translocon comprises a globular structure (cap) separated from the nanodisc bilayer by a narrow stalk that terminates in a transmembrane channel (incompletely distinguished in this reconstruction). The globular cap is larger than the unliganded PA pore, probably due to distortions introduced in the previous negatively stained structures. The cap exhibits larger, more distinct radial protrusions, previously identified with PA domain three, fitted by elements of the NMFF PA prepore crystal structure. The presence of LF(N), though not distinguished due to the seven-fold averaging used in the reconstruction, contributes to the distinct protrusions on the cap rim volume distal to the membrane. Furthermore, the lumen of the cap region is less resolved than the unliganded negatively stained PA, due to the low contrast obtained in our images of this specimen. Presence of the LF(N) extended helix and N terminal unstructured regions may also contribute to this additional internal density within the interior of the cap. Initial NMFF fitting of the cryoEM-defined PA pore cap region positions the Phe clamp region of the PA pore translocon directly above an internal vestibule, consistent with its role in toxin translocation.

Atomic-Scale Characterization of Graphene Grown on Copper (100) Single Crystals

Growth of graphene on copper (100) single crystals by chemical vapor deposition has been accomplished. The atomic structure of the graphene overlayer was studied using scanning tunneling microscopy. A detailed analysis of moiré superstructures present in the graphene topography reveals that growth occurs in a variety of orientations over the square atomic lattice of the copper surface. Transmission electron microscopy was used to elucidate the crystallinity of the grown graphene. Pristine, defect-free graphene was observed over copper steps, corners, and screw dislocations. Distinct protrusions, known as "flower" structures, were observed on flat terraces, which are attributed to carbon structures that depart from the characteristic honeycomb lattice. Continuous graphene growth also occurs over copper adatoms and atomic vacancies present at the single-crystal surface. The copper atom mobility within vacancy islands covered with suspended graphene sheets reveals a weak graphene-substrate interaction. The observed continuity and room-temperature vacancy motion indicates that copper mobility likely plays a significant role in the mechanism of sheet extension on copper substrates. Lastly, these results suggest that the quality of graphene grown on copper substrates is ultimately limited by nucleation at the surface of the metal catalyst.

Chemical and structural components of work function changes in the process of palladium hydride formation within thin Pd film

Surface phenomena which occur in the process of palladium hydride PdH x formation during H 2 interaction “in situ” with thin palladium films were studied by means of two methods: (i) measurements of work function changes Δ Φ , and (ii) determination of topographical images using atomic force microscopy (AFM). A very strong decrease of the work function up to ∼ 2 V was registered in the course of the reaction at 78 K when thin Pd films deposited on Pyrex glass were applied. However a small Δ Φ was noticed when using Pd films deposited on glass precovered with a thick platinum film. AFM topography exhibited a net of distinct protrusions in the first case, but the topography was almost unchanged in the second. We suggest that the above unusually high decrease of the work function in the process of PdH x formation within a Pd/glass system is caused by the change of thin Pd film topography, and not by very strong polarization of hydrogen adspecies on PdH x surface.

A Three-dimensional Working Model of the Multienzyme Complex of Aminoacyl-tRNA Synthetases Based on Electron Microscopic Placements of tRNA and Proteins

It has become evident that the process of protein synthesis is performed by many cellular polypeptides acting in concert within the structural confines of protein complexes. In multicellular eukaryotes, one of these assemblies is a multienzyme complex composed of eight proteins that have aminoacyl-tRNA synthetase activities as well as three non-synthetase proteins (p43, p38, and p18) with diverse functions. This study uses electron microscopy and three-dimensional reconstruction to explore the arrangement of proteins and tRNA substrates within this "core" multisynthetase complex. Binding of unfractionated tRNA establishes that these molecules are widely distributed on the exterior of the structure. Binding of gold-labeled tRNA(Leu) places leucyl-tRNA synthetase and the bifunctional glutamyl-/prolyl-tRNA synthetase at the base of this asymmetric "V"-shaped particle. A stable cell line has been produced that incorporates hexahistidine-labeled p43 into the multisynthetase complex. Using a gold-labeled nickel-nitrilotriacetic acid probe, the polypeptides of the p43 dimer have been located along one face of the particle. The results of this and previous studies are combined into an initial three-dimensional working model of the multisynthetase complex. This is the first conceptualization of how the protein constituents and tRNA substrates are arrayed within the structural confines of this multiprotein assembly.

Comparison of the Symmetry Breaking in the Surface Molecular Structures of One- and Two-Dimensional Bis(ethylenedithio)tetrathiafulvalene Compounds

Scanning tunneling microscopy (STM) and atomic force microscopy (AFM) were performed on the cationic molecular surfaces of bis(ethylenedithio)tetrathiafulvalene (BEDT-TTF)-based organic conductors with one-dimensional (β-(BEDT-TTF)2AsF6) and two-dimensional (κ-(BEDT-TTF)2Cu(NCS)2) conductivity. For the β-(BEDT-TTF)2AsF6 surface, highly similar STM/AFM images to those of β-(BEDT-TTF)2PF6 were observed. Two types of alternately aligned molecular rows existed in the direction of the crystal a-axis; one is characterized by bright and distinct protrusions, and the other is a little darker with less distinct rows forming a zigzag structure. On the other hand, in the case of κ-(BEDT-TTF)2Cu(NCS)2, similar one-dimensional modulation was confirmed by AFM for the first time in addition to the STM observation. However, the observed difference in the molecular images was only in their brightness, and their shapes were quite similar. Since the surface structures were confirmed by both STM and AFM, the obtained results indicate that the observed modulations are associated with the structural changes in the surface molecular arrangements. The modulations were discussed in relation to the crystal structures.

Intervascular Smooth Muscle Fibers and Muscular Bolsters in Nasal Swell Bodies of Humans

There is strong clinical evidence that the cavernous tissue (swell bodies) of nasal mucosa plays an important role in congestion of the nose. Still, the complex mechanisms responsible for the unique behavior of these vessels have not yet been identified, and even the morphology of these structures is still a matter of controversy. The present study was performed on nasal mucosal specimens from inferior turbinates of humans by means of histology and transmission electron microscopy. Besides the evaluation of the vascular wall structure of the nasal swell bodies, special attention was given to two morphological peculiarities: intervascular smooth muscle fibers and muscular bolsters. Intervascular smooth muscle fibers are composed of bundles of smooth muscle cells varying in diameter between 14 and 35 microns; they are attached to the muscular coat of the vessels of the cavernous tissue. Muscular bolsters are distinct protrusions of the vascular wall in nasal swell bodies; they are found not only at the transition between different vessels, but also irregularly within the course of veins of the cavernous tissue. The authors report on their understanding of the functional significance of intervascular smooth muscle fibers and muscular bolsters within the cavernous tissue for swelling mechanisms in the nose and discuss their results in light of the literature.

Perforated axospinous synapses with multiple, completely partitioned transmission zones: probable structural intermediates in synaptic plasticity.

Analysis of axospinous synapses in the rat dentate gyrus, using three-dimensional reconstructions from electron micrographs of serial sections, revealed a novel synaptic subtype. Synapses of this subtype exhibit partitions that emanate from the postsynaptic spine head and invaginate the presynaptic axon terminal, dividing its portion contracted by the spine into distinct protrusions. Such complete spine partitions provide barriers between two to four discrete transmission zones, each one being formed by a separate axon terminal protrusion and delineated by a separate segment of the postsynaptic density (PSD). Spine partitions that differ from the complete ones were found in two other synaptic subtypes. One of these is characterized by a sectional partition the base of which is placed between the arms of a horseshoe-shaped PSD. Synapses of another subtype exhibit a focal partition the base of which is restricted to a perforation in a fenestrated PSD. Although both sectional and focal partitions invaginate a presynaptic axon terminal, they do not divide into separate protrusions and do not split a single transmission zone into disjointed entities. All three subtypes of partitioned synapses have nonpartitioned counterparts exhibiting segmented, horseshoe-shaped, or fenestrated PSDs. These observations suggest a model of structural modifications underlying synaptic plasticity. According to this model, synapses with multiple, completely partitioned transmission zones that appear to be designed as elements of an unusually high strength, represent pivotal structural intermediates in synaptic plasticity. The formation of such synapses from those that belong to other subtypes is postulated to result in a sustained increase in the efficacy of synaptic transmission. Conversely, a disassembly of complete partitions with the transformation of multiple transmission zones into a single one is proposed to lead to a persistent depression of synaptic responses.

Extrusion of nucleoli from pronuclei of the rat.

Electron microscope observations of osmium tetroxide-fixed rat eggs indicate that small nucleoli are extruded from pronuclei in a sharply demarcated time period after sperm penetration. Approximately 4(1/2) hours after sperm penetration, fine fibrous material aggregated in distinct loci along the inner surface of the nuclear envelope and condensed into small, dense bodies. The term tertiary nucleolus or extrusion body is used to designate the forming bodies. The small tertiary nucleoli form distinct protrusions from the pronuclei during the following developmental period and finally bud off into the cytoplasm, carrying with them a small portion of the double nuclear envelope. The extrusion bodies can be observed only in the vicinity of the pronuclei and have not been seen near the cell membrane. The fate of the tertiary nucleoli is not known; apparently they transform or disappear after they have passed into the cytoplasm. Eleven hours after sperm penetration, tertiary nucleoli are not present near the nuclear membrane and the extrusion activity has apparently ceased. Large and small nucleoli react similarly to cytochemical reagents: they are Feulgen negative; they are positive to the Millon, Sakaguchi, brom-phenol blue, and PAS reactions. Azure B stain combined with nuclease extraction indicates the presence of small amounts of RNA in the nucleoli.