Sorting and reorganization of centrosomes during oocyte maturation in the mouse

In animal oocytes, the centrosome exists as an acentriolar aggregate of centrosomal material that is regulated in a dynamic manner throughout the process of meiotic maturation. Recently, it has been demonstrated that in female meiotic systems spindle assembly is likely regulated by chromosomal and microtubule/microtubule-associated influences. The purpose of this study was to analyze the distribution of the integral centrosomal protein, pericentrin, during the course of meiotic maturation. The function of the centrosome during meiotic progression was evaluated by exposing oocytes to pharmacological agents that perturb cytoplasmic homeostasis (cycloheximide, nocodazole, cytochalasin D, taxol, and vanadate). Pericentrin was localized to the spindle poles during metaphase of meiosis-I as O- and C-shaped structures. At anaphase, these structures fragment, become displaced from the spindle poles, and associate with the lateral spindle margin. The metaphase spindle at meiosis-II had incomplete pericentrin rings at both spindle poles. Vanadate treatment, a known inhibitor of dynein-ATPase, resulted in meiotic arrest, constriction of the spindle pole, and an aggregation of pericentrin at the spindle poles. After taxol exposure, pericentrin incorporation into both spindle poles and cytoplasmic centrosomes was increased. Treatment of oocytes with cycloheximide, nocodazole, and cytochalasin D, influenced early events associated with chromosome capture and spindle assembly and altered the number and distribution of cytoplasmic centrosomes. Thus, although pericentrin incorporation is not required for meiotic spindle formation, the dynamic reorganization of pericentrin and changes in centrosome microtubule nucleating capacity are involved in critical cell cycle transitions during meiotic maturation.     

Ultrastructural correlates of meiotic maturation in mammalian oocytes

Immature mammalian oocytes reside in ovarian follicles with junctionally coupled granulosa cells. When released from a currently undefined meiotic arresting influence, these oocytes resume meiosis to progress from late diplotene (germinal vesicle stage) through the first meiotic division to metaphase II. Oocytes remain at metaphase II until fertilization activates them to complete meiosis. This review summarizes ultrastructural events that occur during meiotic maturation in mammals. Developmental correlates that promise a clearer understanding of regulatory mechanisms operating to control maturation are emphasized. By use of TEM of thin sections, freeze-fracture analysis, and replicated oocyte cortical patches, we demonstrate stage-specific changes in the oocyte nucleus, reorganization of cytoplasmic organelles, correlations between oocyte maturational commitment and the junctional integrity of associated granulosa cells, and definition of the components comprising the oocyte cortical cytoplasm.     

Mouse oocyte differentiation during antral follicle development

During antral follicle development mouse oocytes undergo rearrangement of granulosa cell interactions and the oocytes released from follicles at the beginning or at the end of antral development are either devoid of denuded oocytes (DO) or strictly associated with cumulus-intact (CI) cumulus cells. In this study, these two oocyte classes were analyzed before germinal vesicle (GV) and after in vitro maturation (IVM) to evaluate (a) the ultrastructural aspect of oolemma microvilli by scanning electron microscopy analysis and (b) specific morphological markers of differentiation (chromatin organization, mitochondria, cortical granules, microfilaments, and spindle of metaphase II- MII-). At GV-stage, CI oocytes exhibited remarkable differences (a) in the oolemma microvillar ultrastructure and distribution with respect to DO and (b) in the chromatin organization that was typical of meiotically competent germ cells. By contrast, homogeneous patterns of distribution of mitochondria, cortical granules, and microfilaments characterized both the oocyte classes. At the end of culture, CI oocytes, even when matured without cumulus cells, reached more efficiently the MII stage and acquired an ultrastructural microvillous configuration different from DO. In addition, MII-arrested DO had a higher percentage of meiotic spindles with abnormal morphology in comparison with preovulatory oocytes, while cortical granule and microfilament patterns revealed no appreciable differences between the groups. With regard to mitochondria, a polarized distribution of these organelles was found in 82% of DO and in 97% of CI oocytes. These observations suggested that the achievement of the full antral follicle development is a condition for the acquisition of specific qualitative properties that are essential for the production of fertilizable oocytes, both in in vivo and in vitro models as well.     

Synaptic target recognition at Drosophila neuromuscular junctions

Every synaptogenesis begins with "synaptic target recognition," a cell-cell recognition event in which a neuron and its target stably adhere. Despite its importance in developing nervous systems, synaptic target recognition has been difficult to study in complex systems. The relatively simple and genetically accessible Drosophila NMJ model system provides a repertoire of target recognition cues. We describe how these molecules control the targeting of specific growth cones in either a positive (synaptogenic) or negative (anti-synaptogenic) manner. We also propose two alternate signaling paradigms to explain how these initial cell recognition events are coupled to the intracellular signaling pathways that begin the process of synapse maturation.     

Ultrastructural studies on the natural leaf senescence of Cinnamomum camphora

The process of natural leaf senescence of Cinnamomum camphora (C. camphora)—a commercial tree in Asia, was investigated, focusing on changes in cellular ultrastructure, epicuticular wax, and stoma. The changes to mesophyll cells in a senescing leaf predominantly include degradation of the following cellular components: cytoplasm, the central vacuole, small vacuoles, and vesicles with a diameter smaller than 400 nm, which are involved in the degradation of chloroplasts. The sequence of change in epicuticular wax during leaf senescence was different from those in herbaceous plants by atomic force microscope and scanning electron microscopic analysis. Comparing with maturation leaves, senescing leaves develop a wider aperture in their stoma, which would delay the leaf senescence of C. camphora. SCANNING 35:336‐343, 2013. © 2013 Wiley Periodicals, Inc.     

Review : Reticulocyte maturation: mitoptosis and exosome release

During the differentiation of erythroid cells, a vast program of maturation takes place, leading to decay or elimination of organelles, including the nucleus, mitochondria, ribosomes, lysosomes, endoplasmic reticulum and Golgi apparatus. During the last step of red cell maturation, remaining organelles, primarily mitochondria and ribosomes but also vestiges of others are finally cleared from the cell. This cleaning session also affects specific proteins that are partially or entirely removed from the cell surface. The interplay of the various events and their causal relationships are approached here.     

Cryopreparation of mammalian tissue for X-ray microanalysis in STEM

A cryopreparation technique for studies of ultrastructure and distribution of diffusible elements in biological tissue is described. Electron microscopical contrast and characteristic X-ray spectra are found to be poor in completely frozen-hydrated ultrathin cryosections of fresh chemically untreated tissue. Both STEM contrast and detection of characteristic X-rays are enhanced by careful freeze-drying in the microscope. Although the ultrastructure is affected by ice crystals, intracellular compartments can be identified by STEM without staining and studied by X-ray microanalysis.     

Morphodynamics of ovarian follicles during oogenesis in mice

In the mouse, oogonia enter the prophase of the first meiotic division and differentiate into oocyte while developing in the fetal ovary. Shortly after birth, all oocytes are arrested in the dictyate stage of late prophase in the developing follicles; a small number of follicles reach the ovulatory stage; the rest are lost by apoptosis. The resumption of meiotic division and nuclear progression to metaphase II (oocyte maturation) occur in the ovulatory follicles. In this article we review recent morphological data that have clarified how cytokines and glycosaminoglycans (GAGs) are involved in mouse follicular development, atresia, and maturation during oogenesis, as exogenous/endogenous factors. (1) Microvascular networks and angiogenic factors (epidermal growth factor; GAGs) are deeply involved in selective mouse oocyte growth beyond approximately 20-30 microm in diameter. (2) Gonadotropin-inducible neuronal apoptosis inhibitory protein may indirectly affect oocyte survival as a result of the inhibition of apoptotic granulosa-cell death during folliculogenesis. (3) The pattern of oocyte degeneration depends on follicle and oocyte developmental stages, and follicle stimulating hormone accelerates the process of degeneration of oocytes. (4) The process of degeneration of mouse oocytes/eggs is modulated by tumor necrosis factor-alpha that is accumulated in the expanded cumulus during oocyte maturation. (5) A colloidal iron-positive substance was detected in the intercellular spaces of follicular tissue, especially in the cumulus mass. Cells located where the cumulus mass and granulosa cell layer interwound became enlarged during the resumption of oocyte meiosis. Colloidal iron-positive substances accumulated extensively within the intercellular spaces of the enlarged cells.     

Functionalized magnetic nanoparticles attenuate cancer cells proliferation: Transmission electron microscopy analysis

The penetration and transportation of nanoparticles (NPs) inside the cancer cells is critical to study. In this article, cancer cells (HCT‐116) were treated with functionalized magnetic NPs for the period of 48 hr and studied their ultrastructure by transmission electron microscopy (TEM). The NPs‐treated cells were prepared by chemical fixation and sliced into electron‐transparent arbitrary sections (200 × 200 μm²) by ultramicrotome. Major events of NPs–cell interaction, such as penetration of NPs, encapsulation of NPs into the intracellular compartments, transportation of NPs, and NPs exit, were examined by TEM to understand the mechanism of cell death. The NPs showed the uniform spherical shape with broad size distribution (100–400 nm), while cells displayed irregular morphology with average diameter ~5 μm. Our results showed the successful penetration of NPs deep into the cell, encapsulation, transportation, and exocytosis. Furthermore, we tested the different concentrations (0, 1.5, 12.5, and 50 μg/ml) of NPs on cancer cells and evaluated the cell viability. Laser confocal microscopy and colorimetric analysis together demonstrated that the cell viability is a dose‐dependent phenomenon, where 50 μg/ml specimen showed the highest killing of cancer cells compared to other dosages.     

Preparation of individual electrically and video-recorded eggs for integrated temporal and electron microscopic analyses.

A method for correlative studies of early fertilization events that integrates techniques of intracellular electrophysiological recording, video-imaging, and electron microscopy is described. A key feature of the method is its ability to identify the fertilizing sperm and to record the moment of egg excitation. Since the site of gamete interaction is recognizable throughout all stages of preparation, difficulties associated with locating the site of fertilization and determining specimen orientation for microtomy and electron microscopic examination are eliminated. Virtually all samples yield useful information. An example of interacting gametes fixed 4 sec after initiation of the fertilization potential and serial sectioned is described. The method is applicable to systems other than fertilizing eggs when functional, temporal, and spatial relationships of individual cells need to be correlated with changes in ultrastructure.     

A place for ultrastructural analysis of platelets in cerebral ischemic research

It is well known that estrogen is neuroprotective through various mechanisms which suggest that sex hormone levels, thrombotic mechanisms, and inflammatory processes are strongly interconnected in predicting the outcome and consequences of cerebral ischemia. Because platelet ultrastructure is altered in conditions like thrombosis and associated with stroke, the question arises whether ultrastructural analyses of platelet morphology may provide further insight into the role of estrogen during ischemic insult. In the current study, a hyperglycemic modification to the two‐vessel occlusion model for inducing experimental cerebral ischemia was employed, in order to correlate neural tissue integrity levels between three experimental groups to corresponding platelet ultrastructure so as to determine whether there is an association between cerebral ischemia and the presence of inflammatory or necrotic platelet ultrastructure. It is apparent in the results that under the influence of estrogen in cyclic or intact females, there is lesser neural tissue damage as well as a reduced degree of inflammation evident in platelet activation morphology when compared to males and acyclic or ovariectomized females. It is unmistakable that neural injury is closely shadowed, if not preceded, by inflammatory changes in the coagulation system, particularly manifested in platelet ultrastructure. It is therefore suggested that platelets may indeed be used successfully to follow the progression of events of cerebral ischemia and possibly assist in the assessment of treatment strategies and their effects on hemostasis. Microsc. Res. Tech. 76:795–802, 2013. © 2013 Wiley Periodicals, Inc.     

Biology of spermatozoa maturation: an overview with an introduction to this issue

Mammalian spermatozoa undergo morphological, biochemical, and physiological modifications initially in the testis (testicular maturation) and later in the epididymis (epididymal maturation). These maturational changes are commensurate with the functional events that occur in developing germ cells and maturing spermatozoa. This special issue reviews the recent, relevant topics dealing with spermatozoa maturation and focuses on the events that occur in internal components such as the nucleus, the acrosome, the perinuclear theca, the fibrous sheath, and the cytoplasmic droplet as well as the plasma membrane. These structures/elements and the constituent proteins of which they are comprised undergo a variety of sequential modifications starting from their origination in developing germ cells up to epididymal maturation. Several steps of the maturation processes on the sperm plasma membrane are mediated by external enzymes and secretions derived from the epithelium lining of the genital tract. Degradation of some of the constituent proteins and the elimination of defective spermatozoa are controlled by the degradation/recycling system, the ubiquitin system. These maturational modifications are necessary for spermatozoa to become fertilization-competent cells and to be stored safely in the male.     

The grid-cell-culture technique: the direct examination of virus-infected cells and progeny viruses

We describe a method for the structural analysis and identification of viruses, without purification or concentration steps which could alter virus morphology. Virus-infected cells grown on carbon-Parlodion-coated electron microscope grids release large numbers of progeny viruses which adsorb to the surface of the grid and are revealed by negative staining. The technique is rapid, sensitive and can be used at three levels. (1) Negative staining of whole cell preparations revealed both extracellular and intracellular viruses or nucleocapsids beneath the plasma membrane; (2) non-ionic detergent extraction of cells infected with certain viruses reveals cytoskeleton-associated, virus-specific structures normally only observed after thin sectioning; (3) cultures prepared by either procedure are suitable for colloidal gold immunological studies. Extracellular and cytoskeletal-associated viruses were heavily and specifically labelled with gold. The results indicate that the technique may be used to rapidly identify unknown viruses on the basis of size, topography, morphology and mode of maturation from the infected cell, as well as the presence of characteristic intracellular cytoskeletal-associated structures. The technique also has potential use in the sero-grouping and sero-typing of viruses with specific monoclonal antibodies.     

Organisation of Xenopus oocyte and egg cortices

The division of the Xenopus oocyte cortex into structurally and functionally distinct "animal" and "vegetal" regions during oogenesis provides the basis of the organisation of the early embryo. The vegetal region of the cortex accumulates specific maternal mRNAs that specify the development of the endoderm and mesoderm, as well as functionally-defined "determinants" of dorso-anterior development, and recognisable "germ plasm" determinants that segregate into primary germ cells. These localised elements on the vegetal cortex underlie both the primary animal-vegetal polarity of the egg and the organisation of the developing embryo. The animal cortex meanwhile becomes specialised for the events associated with fertilisation: sperm entry, calcium release into the cytoplasm, cortical granule exocytosis, and polarised cortical contraction. Cortical and subcortical reorganisations associated with meiotic maturation, fertilisation, cortical rotation, and the first mitotic cleavage divisions redistribute the vegetal cortical determinants, contributing to the specification of dorso-anterior axis and segregation of the germ line. In this article we consider what is known about the changing organisation of the oocyte and egg cortex in relation to the mechanisms of determinant localisation, anchorage, and redistribution, and show novel ultrastructural views of cortices isolated at different stages and processed by the rapid-freeze deep-etch method. Cortical organisation involves interactions between the different cytoskeletal filament systems and internal membranes. Associated proteins and cytoplasmic signals probably modulate these interactions in stage-specific ways, leaving much to be understood.     

Ultrastructure of the seminiferous epithelium and intertubular tissue of the human testis

The ultrastructural features of the human testis are reviewed with emphasis upon the process of spermatogenesis and the cytology of the Leydig cells. The seminiferous epithelium is structurally partitioned by the Sertoli cells into basal and adluminal compartments via the specialized tight junctions between the Sertoli cells. Spermatogonia reside in the basal compartment, and, via a series of cell divisions, produce the primary spermatocytes, which at the commencement of their development move into the adluminal compartment, and thus the lengthy process of meiotic maturation is initiated. The fine structure of primary spermatocytes is described together with the complex transformation of the spermatids into spermatozoa during the process of spermiogenesis. Earlier studies of the organization of the human seminiferous epithelium showed that germ cells at different developmental stages formed identifiable collections termed cell associations or stages, but since several stages were seen in a single tubule cross-section, this gave the impression of an extremely irregular pattern of spermatogenic development. When the topographic arrangement of germ cells was re-examined with the aid of computer modelling, a highly ordered distribution was revealed, conforming to a helical pattern based on the geometry of spirals. Thus spermatogenesis in the human testis is subjected to a precise regulation in keeping with the ordered arrangement of the germ cells seen in other mammalian species. The intertubular tissue of the human testis is composed of loose connective tissue containing blood vessels, occasional lymph capillaries, macro-phages, mast cells, and the Leydig cells which occur either as single cells or form small clusters. The Leydig cell cytoplasm contains an abundant supply of smooth endoplasmic reticulum and mitochondria with tubular cristae, both features being characteristic of steroidogenic cells. Human Leydig cells contain large Reinke crystalloids of variable size and number, but their function remains obscure. The frequent occurrence of paracrystalline inclusions within the cytoplasm of the human Leydig cell suggests that these elements are precursors of the Reinke crystalloids.     

Method of platinum-carbon coating of ultrathin sections for transmission and scanning electron microscopy: an application for study of biological composites

Many biological materials are composites containing two or more components with different mechanical properties. This study is concerned with the application of a method of platinum-carbon coating (Pt/C) of ultrathin sections for TEM and SEM studies of the design of natural composite materials. The changes in profile of the ultrathin resin-embedded sections during different stages of the preparation reflect the material properties of the various components: stiffer regions deform less than softer ones. Such changes in the section profile can be visualized by the Pt/C method and used as evidence of specific material properties in particular regions of composite materials. The method increases the relief contrast, improves the 3D-view of structures, and in combination with standard TEM and SEM procedures can provide clear demonstrations of material design. The distribution of chitin crystallites in the insect cuticle and the ultrastructure of the pore canal system specialized for the transport of epidermal secretions to the cuticle surface were studied here as examples.     

Multiple intracellular signals coordinate structural dynamics in the sea urchin egg cortex at fertilization

Fertilization of the sea urchin egg is accompanied by a sequence of structural changes in the egg cortex that include exocytosis, endocytosis, and microvillar growth. This architectural reorganization is coordinated by two intracellular signals: a rapid, transient rise in cyto-solic free calcium and a slower, longer lasting increase in cytoplasmic pH. In this report we provide ultrastructural views of these events in quick-frozen eggs and discuss their relationship to the calcium and pH signals.     

Single-channel response of hamster oocytes to fertilization with homologous spermatozoa.

Electrophysiological events occur early after fertilization, along with changes in intracellular Ca2+ concentration. Passive electrical parameters were determined in golden hamster oocytes by whole cell patch-clamp method. In separate experiments the effect of 4-aminopyridine on resting oocytes was tested. The single-channel patch clamp configuration was employed to assess the electrical response to fertilization with homologous sperm. Structure of oocytes submitted to patch clamp was evaluated with scanning electron microscopy and found to be preserved. Oocyte diameter was 70.2 +/- 2.2 microm; their resting parameters were: membrane potential 23.8 +/- 0.8 mV; total membrane specific resistance 519.1 +/- 94.6 ohms.cm2, and specific capacity 0.99 +/- 0.03 microF.cm(-2). Total membrane current was decreased by 42 % by 4-aminopyridine. Control oocytes and oocytes exposed to sperm differed in their membrane currents in response to a voltage ramp clamping membrane potential from - 100 mV to + 100 mV. In both cases, currents were largest at the most negative potentials, but sperm-exposed oocytes had larger currents. Additionally, while in control oocytes the current was inward at negative potentials but outward at positive potentials, in the presence of spermatozoa oocytes was inward within the whole voltage range tested. This latter current may represent Ca2+ entry.