Egg envelopes of Armadillidium vulgare (Latreille, 1804) (Crustacea,Isopoda Oniscidea): Ultrastructure and lectins binding

The ultrastructural study carried out on (a) oocytes of Armadillidium vulgare during vitellogenesis, (b) mature eggs taken from the ovaries during the parturial moult of the posterior half of the body, and (c) fertilized eggs collected within a few hours of their release into the brood pouch, has clearly demonstrated that before the fertilization the chorion is the only envelope present in the egg of oniscidean isopods. In the mature eggs, the chorion appears as a uniformly electron‐dense lamina, about 0.4–0.5 µm thick, which does not show any specialized area. A second envelope, described by other authors as vitelline envelope, is formed above the oolemma only right after fertilization and appears separated from the chorion by a space full of liquid. The ways in which the genesis of this envelope is realized are not yet clear; it could be interpreted rather as a fertilization membrane. The investigations carried out with the aid of a battery of FITC‐lectins have highlighted the presence at the chorion surface of unfertilized eggs of various saccharide residues distributed in uniform way. No significant change was observed in the pattern of lectins binding to the chorion of eggs taken from the brood pouch, thus demonstrating how, after the fertilization, no significant rearrangement in the distribution of saccharide residues present on the egg surface occurs in A. vulgare. The ways in which, therefore, the recognition, the binding and the entry of the peculiar sperm of oniscidean isopods into the egg occur, still remain all to be deciphered. Microsc. Res. Tech. 79:792–798, 2016. © 2016 Wiley Periodicals, Inc.     

Structure and function of the extracellular matrix of anuran eggs

The extracellular matrix (ECM) surrounding the anuran egg is composed of jelly coat layers, an envelope, and the perivitelline space, which separates the envelope from the egg plasma membrane. Both the jelly coat layers and egg envelopes are required for fertilization in anurans. This paper reviews the current understanding of the structure-function relations of the ECM, with emphasis on the egg envelope. The fibrous egg envelope exists in four related forms. The envelope forms differ in their ultrastructures, macromolecular compositions, and cellular functions. After the oocyte is released from the ovary, conversion of one envelope form to another is brought about by factors secreted by the oviduct prior to fertilization and by factors released from the egg in the sperm-triggered cortical reaction. An additional extracellular matrix structure, located in the perivitelline space, has recently been identified in Xenopus laevis, as well as a previously undescribed reorganization of envelope fibers occurring at fertilization. The molecular changes in the ECM glycoproteins (limited proteolysis, lectin-ligand binding, and conformational changes) and the oviductal and egg macromolecules responsible for the conversion of envelope forms are discussed. New experimental evidence that supports the lectin-ligand hypothesis for the formation of the fertilization layer is presented. It is proposed that the molecular changes in the ECM are responsible for the ultrastructural alterations of the ECM and for modifications of the fertilization and developmental functions of the anuran egg ECM.     

Gamete interactions and the fate of sperm organelles in fertilized echinoderm eggs

Investigations of gamete fusion, sperm entry and the fate of the sperm nucleus, plasma membrane, mitochondrion, and axonemal complex in fertilized echinoderm eggs are reviewed. The timing of gamete fusion with respect to the onset of electrical activity characteristic of the activated egg and the affects of fixation conditions on the stability of fusing membranes are discussed. Observations from investigations using cationized ferritin labeled gametes and immunogold cytochemistry to demonstrate the mixing of sperm plasma membrane components within the egg plasma membrane, in particular along the surface of the fertilization cone, are compared with results from studies in somatic cells. Transformations of the sperm nucleus into a male pronucleus, consisting of sperm nuclear envelope breakdown, chromatin dispersion, and formation of a pronuclear envelope, are correlated with recent biochemical observation of similar processes in other cellular systems. Fates of the sperm mitochondrion and axonemal complex are examined.     

Comparative ultrastructure of the fertilized egg envelope in Corydoras adolfoi and Corydoras sterbai,Callichthyidae, Teleostei

In teleost, the structural characteristics of fertilized egg and egg envelope are very important for classification of genus or species. The structures of fertilized egg and egg envelope from Corydoras adolfoi and Corydoras sterbai, Callichthyidae, Siluriformes in teleost were examined by scanning and transmission electron microscopes to confirm whether these morphological structures have specificities of species and family or not. The fertilized eggs of C. adolfoi and C. sterbai were non‐transparent, spherical, demersal, and strong adhesive. There were no structural differences between two species through the light microscope. The size of the fertilized eggs of C. adolfoi was 1.95 ± 0.03 mm (n = 20), and that of C. sterbai was 1.92 ± 0.03 mm (n = 20). The perivitelline space was almost not developed in both species. In both species, the adhesive protuberances structures were on the outer surface of egg envelope. And fibrous structures were specially located at attachment part of spawning bed. And the egg envelope consisted of two layers, an inner lamellae layer and an outer strong adhesive layer with high electron dense protuberances structures in cross section. Consequentially, the fertilized eggs, outer surface on the egg envelope and cross section of egg envelope have identical structure. So, these structural characteristics of fertilized eggs and egg envelope show genus Corydoras specificity.     

The structure and ultrastructure of the egg capsule of Brachyptera risi (Plecoptera,Nemouroidea, Taeniopterygidae) with some remarks concerning choriogenesis

The organization of the egg capsule of the euholognathan stonefly, which represents the family Taeniopterygidae (Nemuroidea) was investigated using light and electron microscopy techniques. The presence of a complex, multilayered egg capsule, composed of a vitelline envelope, multilayered chorion, and extrachorion is described. The morphology of the eggshell of Brachyptera risi was compared with that of euholognthan and systellognathan egg coverings and the ground plan of the egg capsule in Plecoptera was discussed. Microsc. Res. Tech. 78:180–186, 2015. © 2014 Wiley Periodicals, Inc.     

Polyspermy prevention in marine invertebrates

In marine invertebrates, as in most other organisms, normal development requires that only one sperm nucleus joins with the egg nucleus at fertilization. The principal mechanisms employed are (1) prevention of sperm-egg plasma membrane fusion and (2) modifications of the egg extracellular coat to prevent sperm binding and/or penetration. In a third strategy, fertilization is polyspermic, but only one sperm nucleus fuses with the egg nucleus. Other factors such as gamete density during spawning, chemotaxis, and localized sites for sperm entry may also affect the numbers of sperm reaching the egg.     

Ubiquitin-dependent proteolysis in mammalian spermatogenesis,fertilization, and sperm quality control: killing three birds with one stone

Ubiquitin and ubiquitin-like proteins control the degradation of substrates as diverse as cyclins, viral envelope proteins, plasma membrane receptors, and mRNAs. The ubiquitinated substrates are targeted towards the lysosomal or proteasomal degradation sites. The number and position of ubiquitin molecules bound to substrates' lysine residues and the number and position of ubiquitin molecules in polyubiquitin chains determine the astonishing substrate specificity of ubiquitin-mediated proteolysis. Ubiquitin is likely to be expressed in mammalian gametes and embryos at any given developmental step, but the information on ubiquitin dependence of gametogenesis and fertilization is sketchy. Ubiquitin ligases E1, E2, E3, and UBC4 are active in the testis. Ubiquitin and proteasomal subunits can be detected in the human sperm centrosome that undergoes dramatic reduction during spermatid elongation. Spermatid histones are ubiquitinated as they are being transiently replaced by transitional proteins and permanently by protamines. Ubiquitin tagging of the sperm mitochondrial membranes may serve as a death sentence for paternal mitochondria at fertilization, thus promoting the maternal inheritance of mitochondrial DNA (mtDNA) in mammals. The defective spermatozoa become surface-ubiquitinated during sperm descent down the epididymis. Finally, new evidence suggests the involvement of ubiquitin-proteasome pathway in the zona penetration by the acrosome-reacted spermatozoon. Such differential patterns of ubiquitination in the testis and epididymis, and inside the egg, may be necessary for reproductive success in humans and animals. Deciphering and eventually manipulating the ubiquitin-dependent proteolysis in the reproductive system could allow us to redirect the mode of mtDNA inheritance after cloning and ooplasmic transplantation, provide germ line therapy in some cases of male infertility, develop new contraceptives, manage polyspermia during in vitro fertilization, and establish objective markers for infertility diagnostics, semen evaluation, and prediction of future fertility.     

The structure and formation of the egg membranes in Nasonia vitripennis (Walker) (Hymenoptera, Pteromalidae)

A histochemical and fine structural study of the vitelline membrane and chorion was carried out. Following the uptake of protein at the oöcyte plasma membrane during yolk synthesis, the egg membranes are laid down in sequence in the intercellular space between the oöcyte and the follicle cells. The follicular epithelial cells alone contribute towards their formation. The structure of the chorion is much simpler than that of Bombus, the only other hymenopteran previously investigated.     

Polarity of the surface and cortex of the amphibian egg from fertilization to first cleavage

The anuran egg is polarized along its animal–vegetal axis and becomes bilaterally symmetrical before first cleavage. Functional sperm entry is regionally restricted to the animal hemisphere of the egg, and functional sperm entry does not occur after egg activation. This regional and functional restriction in sperm entry correlates with the presence of long, slender microvilli and with the presence of the filamentous component of the glycocalyx. After sperm fusion, the egg undergoes activation, including a depolarization of the membrane potential and exocytosis of granules in the cortex. Both of these activation responses are the result of a propagated increase in intracellular calcium. The egg's ability to undergo a propagated activation response develops after germinal vesicle breakdown and depends on the development of the cortical endoplasmic reticulum. Once activated, the radial symmetric egg acquires bilateral symmetry due to a rotation of the egg cortex relative to the inner cytoplasm. A transient array of parallel microtubules forms near the vegetal cortex and may be part of the motor driving the cortical rotation.     

Ultrastructure of oocytes of the Urostreptus atrobrunneus (Diplopoda,Spirostreptida, Spirostreptidae): A potential urban centers plague

The knowledge of the process of egg formation is indispensable for understanding the mechanisms involved in the reproduction of different species. In this context, the objective of this work was to describe the ultrastructure of the oocytes of Urostreptus atrobrunneus (Spirostreptida), a potential plague of urban centers in different locations of São Paulo State. The lack of knowledge about the morphology, physiology, and the reproductive behavior of the species have hindered an effective control of it. The oocytes of U. atrobrunneus presented three development stages: young oocyte or type I; intermediary oocyte or type II; and mature oocyte or type III. During the oocyte development, the cytoplasm become filled with several globules of protein, drops of lipids, and sphaerocrystals, and it was not observed in many organelles in the oocytes with exception of mitochondria, abundant, principally in young oocytes. The vitelline membrane is also deposited in a discontinuous form and the chorion does not present differentiation of layers. The follicular epithelium alters its shape according to the development phase of the oocyte. Part of the vitellus is from exogenous origin and part is endogenous. Before this, only two studies about the ultrastructural analysis of the female germ cells of diplopods were published. Microsc. Res. Tech. © 2012 Wiley Periodicals, Inc.     

From fertilization to cancer: the role of centrosomes in the union and separation of genomic material

Centrosomes play crucial roles in the union of sperm and egg nuclei during fertilization and in the equal separation of genomic material during cell division. While many studies in recent years have focused on the molecular composition of centrosomes, this article focuses on the structural behavior of centrosomes and on factors that play a role in centrosome functions under normal, artificially altered, and abnormal conditions. We review here how studies in the classic sea urchin egg model have contributed to our knowledge on the centrosome cycle within the cell cycle, on compaction and decompaction of centrosomal material, and on the contributions of maternal and paternal centrosomes during fertilization. Centrosome material is activated in unfertilized eggs by increasing pH with ammonium and by increasing calcium with the ionophore A23187, which are conditions that are normally induced by sperm. D(2)O and taxol also induce centrosome aggregation in the unfertilized egg. Maternal and paternal centrosome material both contribute to the formation of a functional centrosome but the formation of a bipolar centrosome requires material from the paternal centrosome. Fertilization of taxol-treated eggs reveals that the male centrosome possesses the capability to attract maternal centrosome material. When pronuclear fusion of the male and female pronuclei is inhibited with agents such as the disulfide reducing agent dithiothreitol (DTT) a bipolar mitotic apparatus is formed from the paternal centrosome. Furthermore, one centrosome of the bipolar mitotic apparatus is capable of organizing an additional half spindle that attaches to the female pronucleus indicating a functional and perhaps structural connection between centrosomes and chromatin. Sea urchin eggs are also useful to study centrosome abnormalities and consequences for the cell cycle. While classic studies by Theodor Boveri have shown that dispermic fertilization will result in abnormal cell division because of multiple centrosomes contributed by sperm, abnormal cell division can also be induced by chemical alterations of centrosomes. Compaction and decompaction of centrosome structure is studied using chloral hydrate or the chaotropic agent formamide, which reveals that centrosomes can be chemically altered to produce mono- or multipolar abnormal mitosis and unequal distribution of genomic material upon release from formamide. The patterns of abnormal centrosome reformations after recovery from formamide treatment resemble those seen in cancer cells which argues that structural defects of centrosomes can account for the formation of abnormal mitosis and multipolar cells frequently observed in cancer. In summary, the sea urchin model has been most useful to gain information on the role of centrosomes during fertilization and cell division as well as on adverse conditions that play a role in centrosome dysfunctions and in disease.     

Perivitelline space: does it play a role in blocking polyspermy in mammals?

The perivitelline space of mammalian oocytes changes in size and composition during preimplantation development. Often overlooked in the past, this space contains a hyaluronan-rich extracellular matrix prior to fertilization and a cortical granule envelope following release of the cortical granules at fertilization. The hyaluronan-containing matrix of unfertilized oocytes is well developed in some species such as opossums and humans but is scant in rodents including the hamster and mouse. The significance of the hyaluronan-rich matrix, which attaches to the plasma membrane of the oocytes, is not fully understood. However, hyaluronan, which can inhibit membrane fusion, is present in the perivitelline space (PVS) of unfertilized oocytes and must be negotiated by the fertilizing sperm. Following fertilization, the cortical granule envelope forms as the cortical granules disperse, thereby causing the PVS to increase significantly in size. Calcium is important in the dispersion of the cortical granules following exocytosis. Once formed, the cortical granule envelope in some species is about the same thickness as the zona pellucida, but it is not readily visualized unless it is stained with fluorescent probes or examined ultrastructurally after following stabilization with ruthenium red. The envelope contains proteins that remain in the PVS until the time of blastocyst hatching. Although little work has been done on the functions of the cortical granule envelope, several studies are consistent with the idea that it plays a role in blocking polyspermy. While nicotine increases polyspermy in sea urchins, its effects on polyspermy in human smokers have not been characterized, but could be addressed in human in vitro fertilization labs.     

Morphology of mammalian sperm membranes during differentiation,maturation, and capacitation

The mammalian spermatozoon is a highly polarized cell whose surface membrane can be divided into five functionally, structurally, and biochemically distinct domains. These domains are formed during spermatogenesis, continue to be modified during passage through the epididymis, and are further refined in the female reproductive tract. The integrity of these domains appears to be necessary for the sperm to perform its function—fusion with the egg and subsequent fertilization. The domains can be identified morphologically by their surface contours and texture, the content, distribution, and organization of intramembranous particles after freeze-fracture, and by the density of surface and cytoplasmic electron-dense coatings in thin sections. By using a variety of labels that stain carbohydrates (lectins), lipids (filipin and polymyxin B), and monoclonal antibodies to specific membrane constituents, the biochemical composition of these contiguous membrane regions has also been partly elucidated. We review here what is known about the structure, composition, and behavior of each membrane domain in the mature sperm and include some information regarding domain formation during spermatogenesis. The sperm is an excellent model system to study the creation and maintenance of cell polarity, granule exocytosis, and fertilization. Hopefully this review will provide impetus for future studies aimed more directly at addressing the relationship of its morphology to its functions.     

Electron microscopic observation of the sagittal structure of Drosophila mature sperm

Observation of sperm development and determination of their morphological characteristics are very important to the understanding of phylogenetic relationships and the study of sperm function during fertilization. Although ultrastructural studies of sperm development in the testes of the fruit fly Drosophila have been performed, there are few reports describing electron microscopic morphology of mature sperm, that is, those released from the testes to the seminal vesicles. Here, we present the first report of the sagittal organization of Drosophila sperm head and neck regions by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The head and tail structures of a mature sperm, for example, the acrosome, nucleus, and flagellum, were easy to distinguish by the morphological characteristics of the sperm surface by SEM. The morphological relationships between the surface and internal structures of mature sperm were confirmed by observing longitudinal sections with TEM. Our approach overcame the technical difficulties involved in sample preparation for electron microscopic observation of the Drosophila mature sperm head, and therefore, this study serves as an important foundation for future genetic dissection of sperm ultrastructure and function in male sterile mutants. Microsc. Res. Tech. 77:661–666, 2014. © 2014 Wiley Periodicals, Inc.     

Fine structure and formation of the eggshell in scorpionfly Panorpa liui Hua (Mecoptera: Panorpidae)

The morphology and formation of the eggshell in the scorpionfly Panorpa liui Hua were examined with light microscopy and scanning and transmission electron microscopy. During oogenesis of the scorpionfly, the follicle cells multiply by mitotic divisions and diversify into four morphologically distinct subpopulations, three of which are engaged in the eggshell formation and mold various parts of chorion. The eggshell consists of three layers: An inner vitelline membrane, an outer chorion, and a precursor extrachorion. The chorion constitutes a very compact endochorion, a rough fibrillar exochorion, and a polygonal meshwork of elevated ridges. At proximal end of ovarioles the chorion of matured oocyte is covered with a loose membrane, which might be the remnant of follicle cells. The jelly substance, which acts as lubricant to protect the oviducts and ovulated eggs during ovulation, might add to the top of polygonal ridges as the outermost extrachorion after oviposition. The eggshell formation process in Panorpa is tentatively proposed. Microsc. Res. Tech. 2009. © 2009 Wiley-Liss, Inc.     

Organization and modifications of sperm acrosomal molecules during spermatogenesis and epididymal maturation

The mammalian acrosome is a highly specialized organelle overlying the anterior part of the sperm nucleus and contains a variety of proteins, including hydrolytic enzymes and matrix molecules. Functionally, the anterior acrosome is involved in the acrosome reaction or sperm-zona pellucida interaction, while the equatorial segment (posterior acrosome) is involved in sperm-egg fusion. The acrosome is formed during spermiogenesis, during which associated molecules are transported from the Golgi apparatus and organized. Many of the molecules thus arranged gradually become compartmentalized during sperm passage through the epididymis. Some of them are further modified during the fertilization process. The findings indicate that acrosomal molecules are not only restricted to a specific region (domain) of the acrosome but also undergo ongoing relocation in a stage-specific manner during sperm maturation in the testis and epididymis. Such maturation-associated modifications are considered essential for sperm molecules to reach the correct or final site before fertilization. This review focuses on the organization and modifications of the acrosomal molecules as well as their compartmentalization within the acrosome.     

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.     

Interactions of sperm perinuclear theca with the oocyte: implications for oocyte activation,anti-polyspermy defense,and assisted reproduction

Perinuclear theca (PT) is the cytoskeletal coat of mammalian sperm nucleus that is removed from the sperm head at fertilization. PT harbors the sperm borne, oocyte-activating factor (SOAF), a yet-to-be-characterized substance responsible for triggering the signaling cascade of oocyte activation, thought to be dependent on intra-oocyte calcium release. The present article reviews the current knowledge on the biogenesis and molecular composition of sperm PT. Possible functions of sperm PT during natural and assisted fertilization, and in the initiation of embryonic development are discussed. Furthermore, evidence is provided that SOAF is transferred from the sperm PT to oocyte cytoplasm through the internalization and rapid solubilization of the post-acrosomal PT. It is shown that during natural fertilization the sperm PT dissolves in the oocyte cytoplasm concomitantly with sperm nuclear decondensation and the initiation of pronuclear development. SOAF activity is preserved in the differentially extracted sperm heads only if the integrity of PT is maintained. After intracytoplasmic sperm injection (ICSI), activation occurs only in those oocytes in which the injected spermatozoon displays complete or partial dissolution of PT. In the latter case, the residual PT of the sub-acrosomal and/or post-acrosomal sperm region may persist on the apical surface of the sperm nucleus/male pronucleus and may cause a delay or arrest of zygotic development. We propose that the sperm PT harbors SOAF in the post-acrosomal sheath, as this is the first part of the sperm cytosol to enter the oocyte cytoplasm and its disassembly appears sufficient to initiate the early events of oocyte activation. Dissolution of the sub-acrosomal part of the PT, on the other hand, appears necessary to insure complete DNA decondensation in the internalized sperm nucleus and initiate DNA synthesis of both pronuclei. The release of the SOAF from the sperm head into oocyte cytoplasm at fertilization ultimately leads to the activation of oocyte mechanism including the completion of the meiotic cell cycle, pronuclear development and anti-polyspermy defense.     

Cellular and molecular mechanisms leading to cortical reaction and polyspermy block in mammalian eggs

Following fusion of sperm and egg, the contents of cortical granules (CG), a kind of special organelle in the egg, release into the perivitelline space (cortical reaction), causing the zona pellucida to become refractory to sperm binding and penetration (zona reaction). Accumulating evidence demonstrates that mammalian cortical reaction is probably mediated by activation of the inositol phosphate (PIP(2)) cascade. The sperm-egg fusion, mediated by GTP-binding protein (G-protein), may elicit the generation of two second messengers, inositol 1,4,5 triphosphate (IP(3)) and diacylglycerol (DAG). The former induces Ca(2+) release from intracellular stores and the latter activates protein kinase C (PKC), leading to CG exocytosis. Calmodulin-dependent kinase II (CaMKII) may act as a switch in the transduction of the calcium signal. The CG exudates cause zona sperm receptor modification and zona hardening, and thus block polyspermic penetration. Oolemma modification after sperm-egg fusion and formation of CG envelope following cortical reaction also contribute to polyspermy block.