Morphological changes during diapause stages in the embryonic cortex of the annual killifish <Emphasis Type="Italic">Millerichthys robustus</Emphasis> (Cyprinodontiformes: Cynolebiidae) under natural conditions

The annual killifish inhabits in extreme locations with unpredictable rainy season where survives through the massive generation of embryos resistant of drought, capable to remain in a state of metabolic dormancy (three moments of diapause during embryonic development) protected by embryonic cortical structures: perivitelline space, egg envelope and its ornamented structures (trapeze-shaped projections and filaments in Millerichthys robustus). This research describes, for the first time, changes in cortical structures during three diapause stages in embryos of annual fish M. robustus during an annual life cycle. Embryos were collected in three periods through the year in a temporal water body: flood, drought and wet. During flood period all embryos were found in diapause I (during epiboly, dispersion of the blastomeres stage) with maximum thickness in all cortical structures and presence of egg envelope filaments. During drought period all embryos were in diapause II (development during somitogenesis, before the organogenesis) and its structures reduced its thickness significantly and lost the egg envelope filaments. Interestingly, embryos in diapause II and III (embryonic development completed in a pre hatching stage) were found during wet period (an example of bet-hedging strategy) in which all structures presented a recovery tending to its original condition observed during flood period. This research demonstrates that annual fish embryos respond to their exposure to seasonal environmental variations with dynamic structural changes that are fundamental for their survival.     

Survival of water stress in annual fish embryos: dehydration avoidance and egg envelope amyloid fibers

Diapausing embryos of Austrofundulus limnaeus survive desiccating conditions by reducing evaporative water loss. Over 40% of diapause II embryos survive 113 days of exposure to 75.5% relative humidity. An early loss of water from the perivitelline space occurs during days 1-2, but thereafter, rates of water loss are reduced to near zero. No dehydration of the embryonic tissue is indicated based on microscopic observations and the retention of bulk (freezable) water in embryos as judged by differential scanning calorimetry. Such high resistance to desiccation is unprecedented among aquatic vertebrates. Infrared spectroscopy indicates frequent intermolecular contacts via beta-sheet (14%) in hydrated egg envelopes (chorions). These beta-sheet contacts increase to 36% on dehydration of the egg envelope. Interestingly, the egg envelope is composed of protein fibrils with characteristics of amyloid fibrils usually associated with human disease. These features include a high proportion of intermolecular beta-sheet, positive staining and green birefringence with Congo red, and detection of long, unbranched fibrils with a diameter of 4-6 nm. The high resistance of diapause II embryos to water stress is not correlated with ontogenetic changes in the egg envelope.     

Electron microscopic study of the cortical reaction of an ophiuroid echinoderm.

The egg coats of an ophiuroid echinoderm (Ophiopholis aculeata) are described by electron microscopy before and after fertilization. The unfertilized egg is closely invested by a vitelline coat about 40 A thick, and the peripheral cytoplasm is crowded with cortical granules five or six deep. During the cortical reaction, which rapidly follows insemination, exocytosis of cortical granules takes place. Some of the cortical granule material is evidently added to the vitelline coat to form a composite structure, the fertilization envelope, which is made up of a 400 A thick middle layer separating inner and outer dense layers, each about 50 A thick. The elevation of the fertilization envelope from the egg surface creates a perivitelline space in which the hyaline layer soon forms. The hyaline layer is about 2 micron thick, finely granular, and apparently derived from cortical granule material. The extracellular layers of the early developmental stages of ophiuroids and echinoids are quite similar in comparison to those of asteroids; this finding helps support Hyman's argument that the ophiuroids are more closely related to the echinoids than to the asteroids.     

Electron Microscopic Study of Development in a Sea Cucumber,Stichopus tremulus (Holothuroidea), from Unfertilized Egg through Hatched Blastula

In this, the first fine structural study of sea cucumber embryology, eggs and embryos of Stichopus tremulus developing at 7.5°C are described from spawning through hatched blastulae. Spawned eggs are at about first meiotic metaphase and are surrounded by a jelly layer that remains around the embryos until hatching. No vitelline coat can be demonstrated, but whether it is truly absent or removed by electron microscopic processing is not known. Insemination initiates a rapid cortical reaction, completed within 2 min., which involves a wave of cortical granule exocytosis and fertilization envelope formation. The compactly fibrous fertilization envelope is about 50 nm thick and appears to consist entirely of ejected cortical granule material (if one assumes that there is no vitelline coat). As the fertilization envelope elevates, no hyaline layer appears in the perivitelline space. The first and second polar bodies are emitted, respectively, at about 9 and 15 min. after insemination. The first seven or so cleavages are equal, radial, and occur approximately every 4 hr. The blastocoel opens up at the four-cell stage and, during the earlier cleavages, remains connected with the perivitelline space via numerous gaps between the roughly spherical blastomeres. At the 64-cell stage, these gaps begin to close as the blastomeres start to become cuboidal; in addition, an embryonic cuticle is produced on the apical surface of each blastomere. In embryos of several hundred cells, the blastomeres become associated apicolaterally by junctional complexes, each consisting of a zonula adherens and a septate junction. Several hours before hatching, a single cilium is produced at the apical surface of most blastomeres. At hatching (about 50 hr after insemination), the ciliated blastula leaves behind the fertilization envelope and jelly layer. Swimming blastulae soon begin to elongate in the animal-vegetal axis, and a basal lamina develops on blastomere surfaces facing the blastocoel. The discussion includes a fine structural comparison of egg coats among the five classes of the phylum Echinodermata.     

Breakdown of the cortical alveoli of medaka eggs at the time of fertilization,with a particular reference to the possible role of spherical bodies in the alveoli

Summary The process of cortical change upon fertilization of eggs of the teleostean fish,Oryzias latipes was investigated. A cortical alveolus (CA) contains colloidal material, a spherical body, and often a membranous structure. Upon insemination, breakdown of the cortical alveoli and elevation of the chorion began around the animal pole and ended at the vegetal pole. It was found that the spherical body was extruded with the colloidal material from the CA: the spherical body swelled after the opening of an aperture and was extruded into the perivitelline space through a large aperture. The empty CA shrank and disappeared completely as a result of the transformation of its envelope to numerous microvilli. The spherical body isolated or in the perivitelline space could be digested quickly by proteolytic enzymes. When spherical bodies in the perivitelline space of a fertilized egg were digested enzymatically, the vitellus came into direct contact with the chorion. The present study seems to show that swollen spherical bodies derived from CA play a role in maintaining a certain distance between the chorion and the vitellus after fertilization.     

A cortical granule-specific enzyme,B-1,3-glucanase,in sea urchin eggs

The ultrastructural localization of B-1,3-glucanase in three species of sea urchin eggs was determined using a monospecific antibody in an electronmicroscopic immunogold procedure. In all three species, Lytechinus variegatus, Strongylocentrotus purpuratus, and Arbacia punctulata, B-1,3-glucanase was localized specifically to the cortical granules. No other organelle within the egg contained significant label. During the fertilization reaction, B-1,3-glucanase was released from cortical granules into the perivitelline space and became associated with the hyaline layer. No significant label was found in association with the fertilization envelope.     

An immunocytochemical localization of the cortical granule lectin in fertilized and unfertilized eggs of xenopus laevis

At fertilization, the vitelline envelope surrounding the egg of Xenopus laevis is modified by the addition of an electron-dense component termed the “F layer.” The F layer functions as a block to polyspermy and as a block to the escape of macromolecules from the perivitelline space, thereby causing an osmotically driven envelope elevation. F-layer formation has been hypothesized to result from interaction between a cortical-granule lectin, released in the cortical reaction, and a jelly-coat ligand. Evidence for this hypothesis was sought by determining the location of the cortical-granule lectin both before and after fertilization, using a specific antibody conjugated to horseradish peroxidase. The cortical-granule lectin was localized only in the cortical granules of the unfertilized egg and was located predominantly in the perivitelline space and the F layer of a fertilized egg. These observations support the hypothesis that the F layer is formed by a cortical-granule-Iectin–jelly layer-ligand interaction.     

Morphological development of the structures related to annualism in the ovarian follicle of the killifish Millerichthys robustus (Costa,1995) (Teleostei: Cyprinodontiformes)

Ovaries of five females of the annual fish teleost species Millerichthys robustus were processed, and the development of the cortical alveoli, zona pellucida and secondary envelope during oogenesis were described. We also documented the origin of the cortical alveoli in time and space similar to the Balbiani body; the synthesis of three generations of cortical alveoli and an active zona pellucida prior to vitellogenesis, which is implicated in the entry of oils to the interior of the oocyte. We found that in this species, the diameter of the alveoli is greater than in the other teleost fish species reported in the literature, except for Fundulus heteroclitus, in which the diameter is similar. The thickness of the zona pellucida recorded in M. robustus is the greatest reported to date. Likewise, two periods of secondary envelope deposition were documented: filaments during pre‐vitellogenesis and, subsequently, trapeze‐shaped projections during the maturation of the oocytes. We report about development of structures that are considered key for the survival of embryos in annual fish during the long periods of diapause in their extreme habitats. The development of peripheral structures described here probably reflects the changes in the physiology of the oocytes in M. robustus. J. Morphol. 277:1219–1230, 2016. © 2016 Wiley Periodicals, Inc.     

FORMATION OF THE CORTICAL CONCAVITY AT FERTILIZATION IN THE SEA URCHIN EGG

During the initial stages of fertilization envelope elevation in eggs of Strongylocentrotus pur puratus and S. droebachiensis a large concavity of the egg cortex was observed in the light microscope. This concavity corresponded in shape and size with the elevating fertilization envelope. However, after the vitelline layers of eggs were disrupted and the eggs inseminated, the concavity failed to develop although the eggs were fertilized and developed normally. We propose that the concavity is formed owing to increased hydrostatic pressure within the perivitelline space. To further support this hypothesis we measured total egg protein secreted during fertilization, and found that 98% was retained within the perivitelline space. Furthermore, 80% of the total protein was contributed by the hyaline layer. Presumably, colloidal osmotic pressure and/or hydration of fertilization product, trapped beneath the fertilization envelope, is responsible for increased hydrostatic pressure within the perivitelline space, and therefore promotes not only fertilization envelope elevation, but the cortical concavity as well.     

A complex cortical reaction leads to formation of the fertilization envelope in the lobster,Homarus

We have examined the formation of the fertilization envelope in the lobsters Homarus americanus and H gammarus. Oocytes were fixed for electron microscopy either in the ovary or following extrusion from the gonopore. Mature ovarian oocytes are surrounded by a coat (envelope 1), which is comprised of small electron-dense granules and structures resembling “bottlebrushes.” At least part of this coat is synthesized by the follicle cells of the ovary. The cortex of ovarian oocytes contains four types of vesicles that we refer to as high-density vesicles (HDV), low-density vesicles (LDV), moderately dense vesicles (MDV), and ring vesicles (RV). Oocytes that were electrically extruded from the gonopore and fixed immediately had an envelope identical to that of ovarian oocytes. The cortex of gonopore oocytes contained the four types of vesicles found in ovarian oocytes. When unfertilized gonopore oocytes were allowed to incubate in sea water, the oocyte cortex appeared unaltered, but envelope 1 swelled and the bottlebrushes dispersed. When recently fertilized oocytes were fixed during natural spawning or following in-vitro fertilization, each type of vesicle was released in sequence from the cortex of the oocyte. The contents of the HDV and LDV appeared first in the perivitelline space, but their fate could not be determined at later times. The ring-shaped elements of the RV and the moderately electron-dense material of the MDV were released exocytotically somewhat later; these materials coalesced in the perivitelline space to form a new coat (envelope 2). Envelope 1 subsequently condensed to its original thickness and appeared firmly attached to envelope 2. Our results show that the fertilized lobster egg is surrounded by two discrete coats. The outer coat, which is formed in the ovary, undergoes a swelling/condensation cycle at spawning. The inner coat originates from a complex cortical reaction. Together these coats comprise the fertilization envelope of the lobster egg.     

Fertilization in Oikopleura dioica (Tunicata,Appendicularia): Acrosome reaction,cortical reaction and sperm-egg fusion

Summary Fine structural changes in the egg and sperm are described during gamete interaction in Oikopleura dioica, an appendicularian tunicate. The unfertilized egg has a vitelline layer 80 nm thick and a perivitelline space about 5 m wide. In the peripheral cytoplasm are a few cortical granules 0.6×0.7 m in diameter and areas rich in parallel cisternae of rough endoplasmic reticulum alternating with areas rich in long mitochondria. In the deeper cytoplasm the predominant organelles are multivesicular bodies. From 25 s to 60 s after insemination, the egg transiently elongates, although with no obvious cytoplasmic rearrangement, and the egg surface becomes bumpy. During this interval sperm enter the egg, and the cortical granules undergo exocytosis. After expulsion into the perivitelline space, the cortical granule contents do not appear to change their shape or blend with the vitelline layer, which neither elevates further nor loses its ability to bind sperm. On encountering the egg, the sperm undergoes an acrosome reaction involving exocytosis of the acrosome and production of an acrosomal tubule. The acrosomal contents bind the sperm to the vitelline layer, and the posterior portion of the acrosomal membrane and the anterior portion of the nuclear envelope evaginate together to form an acrosomal tubule, which fuses with the egg plasma membrane to form a fertilization cone. By 45 s after insemination, the sperm nucleus, centriole, mitochondrion and at least the anterior portion of the axoneme are within the fertilization cone. By 60 s sperm entry is complete. In having eggs with a cortical reaction and sperm with an acrosome reaction, O. dioica resembles echinoderms and enteropneusts and differs markedly from ascidian tunicates, which lack both these features. The relatively unmodified pattern of gamete interaction in O. dioica in comparison with the highly modified pattern in ascidians is difficult to reconcile with the neoteny theory that appendicularians have evolved via ascidian ancestors. The present results are more consistent with the idea that an appendicularian-like ancestor gave rise to ascidians.     

Staging and the time course of embryonic development in kurosoi,Sebastes schlegeli

Synopsis Intraovarian development of embryos was observed in 31 female rockfish kurosoi,Sebastes schlegeli, which had been raised in the laboratory. Three of the females had newly fertilized eggs; the others' embryos were at various stages. The developmental series (33 stages) was not very different from that in oviparous fishes. However, several characteristic features were noted: thin egg envelope, narrow perivitelline space, elliptical encapsulated embryo in the late stages and depletion of yolk at parturition. The time course of development up to birth at different temperatures was adjusted to that at 9.8°C using the Arrhenius equation and processed for the relationship between the stage number (X) and stage-to-birth time in days (Y). An equation, was obtained, and the gestation period was calculated to be 48 days at 9.8°C.     

Embryo of an annual fish (Austrolebias charrua) in the last dormancy stage,diapause III

Embryo of an annual fish (Austrolebias charrua) in the last dormancy stage, diapause III. The embryo, surrounded by a transparent vitelline envelope, is in the pre‐hatching stage. A prominent eye and part of the pigmented body and tail are apparent. Why annual fishes? Annual fishes (Order Cyprinodontiformes) are a special kind of teleost, found in Africa and South America, with developmental strategies closely related to their life cycle. These fishes inhabit temporary pools that undergo drying during summer, when all adults die. The embryos remain buried in the bottom mud and are resistant to desiccation. In the subsequent rainy season they hatch a few hours after the pool is flooded and a new reproductive cycle begins. This developmental pattern is characterized by the presence of a unique stage between cleavage and embryogenesis, dispersion‐aggregation of blastomeres and because the embryos show reversible developmental arrests (diapauses) at different stages. Annual fish embryos are transparent, large, hardy and easy to maintain in the laboratory. Adults show continuous production of eggs and juveniles reach sexual maturity a few weeks after hatching (an unusual condition in fishes). Their particular developmental features confer unique opportunities for research on cell behavior during early development, the effect of environmental factors on development, the regulation of diapauses and the mechanisms involved in sex determination, among others topics. Image provided by Nibia Berois, Universidad de la República, Montevideo, Uruguay.     

p62/p56 are cortical granule proteins that contribute to formation of the cortical granule envelope and play a role in mammalian preimplantation development

The purpose of this study was to identify specific cortical granule protein(s) that form the cortical granule envelope and examine their role(s) in fertilization and preimplantation development. The polyclonal antibody A-BL2 was used to show that the cortical granules of mice, rats, hamsters, cows, and pigs contain a pair of proteins designated p62/p56. These proteins are released from hamster cortical granules at fertilization and contribute to formation of the cortical granule envelope, an extracellular matrix present in the perivitelline space of fertilized mammalian oocytes. P62/p56 were present in the cortical granule envelope throughout preimplantation development and were found in blastomere cortices of 4-cell to blastocyst stage embryos. Hamster oocytes fertilized in vivo in the presence of A-BL2 were all monospermic, suggesting that p62/p56 do not function in blocking polyspermy. Likewise treatment of morula to blastocyst stage hamster embryos with A-BL2 had no effect on the implantation of blastocysts. However, cleavage divisions were inhibited in vivo in a dose-dependent manner when fertilized oocytes or 2-cell embryos were treated with A-BL2. Inhibition of cell division was more pronounced in 2-cell embryos than in fertilized oocytes. This study identifies p62/p56 as cortical granule proteins that contribute to the formation of the cortical granule envelope and further supports the idea that after their release at fertilization, p62/p56 function in regulating preimplantation development at the level of oocyte and blastomere cleavage.     

Life stage dependent responses to desiccation risk in the annual killifish Nothobranchius wattersi

To assess whether the annual killifish Nothobranchius wattersi responds plastically to a desiccation risk and whether this response is life stage dependent, life‐history traits such as maturation time, fecundity and life span were experimentally measured in N. wattersi that were subjected to a drop in water level either as juveniles, as adults or both as juveniles and adults. Fish that were exposed to simulated pool drying as juveniles did not show changes in reproductive output or life span. Adults reacted by doubling short term egg deposition at the cost of a shorter lifespan. Overall, these results suggest that annual fish species can use phenotypic plasticity to maximize their reproductive output when faced with early pond drying, but this response appears to be life‐stage specific. In addition to frogs and aquatic insects, phenotypic plasticity induced by forthcoming drought is now also confirmed in annual fishes and could well be a common feature of the limited number of fish taxa that manage to survive in this extreme environment.     

Fine structure of reproductive glands in two primitive marine pulmonates (Basommatophora: Siphonariidae)

Within the clade Euthyneura the marine basommatophorans are particularly neglected. More morphological and molecular studies are needed because their phylogenetic relationships with other pulmonates remain unresolved. The present study examines the most conspicuous reproductive gland, the glandular complex in two marine limpets, Siphonaria capensis and S. serrata (Pulmonata: Basommatophora) at both gross and fine structural levels. These two sympatric species with different developmental modes were selected to compare the structure and function of this enormous glandular structure. In both S. capensis and S. serrata, the glandular complex shows an undifferentiated state composed of an acidophilic albumen gland and a basophilic mucous gland. The glands contain secretory cells and supporting cells (= ciliated cells) that are highly ciliated. When the histochemical properties of the glandular complex were compared with those of siphonariid egg masses (of each species) it could be established that the albumen gland was responsible for the production of perivitelline fluid whereas the mucous gland secreted substances that help in the assembly of mucous layers surrounding the egg capsules. We suggest that the presence of a single glandular complex comprised of two glands is the most primitive organization of reproductive glands in pulmonates. Furthermore, the histology, fine structure and histochemistry of these glands are very similar to those of the reproductive glands of opisthobranchs.     

Fate of the sea urchin egg receptor for sperm following fertilization

The sea urchin egg receptor for sperm is a 350 kDa glycoprotein containing a large extracellular domain that contains the sperm binding site, a transmembrane domain and a short COOH- terminal intracellular domain. During oogenesis, the receptor protein is first detected in Golgi-associated vesicles and cortical granules. Not until the egg is mature does the receptor appear on the cell surface; at this stage the intact receptor is found in approximately equal quantities on the egg cell surface and in cortical granules. As a potentially unique type of receptor, we were interested in its fate following fertilization. Several techniques have revealed that, following sperm binding, the amount of receptor markedly decreases. Using western blot analysis as well as direct measurement of the receptor protein, it was found that the membrane-bound form of the receptor rapidly disappeared following sperm binding to the egg, with only 3% of the receptor remaining after 30 s. Analysis by immupoelectron microscopy revealed that 30 s after sperm binding, 30% of the initial level of receptor was present. This remaining 30% was found mostly within the perivitelline space formed by the raised fertilization envelope. The disparity between these two sets of results (i.e. 3 vs 30%) is most likely accounted for by the exocytosis of receptor molecules from cortical granules; this fraction of the receptor would have been lost during isolation of the membrane-bound form of the receptor. Thus, unlike other cell surface receptors, the sea urchin egg receptor for sperm is not endocytosed and recycled following ligand binding. Rather, it disappears, presumably as a result of proteolysis. Transiently, the cortical granule form of the receptor is found released into the perivitelline space where it may bind to sperm and thereby prevent polyspermy. Despite the apparent secretion of this form of the receptor, experiments with antibodies to the extracellular and intracellular domains indicate that the receptors in cortical granules and in the plasmic membrane are similar, if not identical.     

Formation and structure of the fertilization envelope in Xenopus laevis

This paper reports the morphological events that occur when the vitelline envelope (VE) of an unfertilized egg of Xenopus laevis is transformed into the fertilization envelope (FE) surrounding the zygote. The VE is about 1 μm thick and is composed of an interlacing network of small filaments. The FE is constructed from the VE plus an electron-dense layer (fertilization layer), about 2–6 μm thick, on the outer surface of the VE, i.e., at the interface between the VE and the innermost jelly-coat layer. The fertilization layer is a stable component of the FE and is not removed by mercaptan solutions used to dejelly eggs. The events of FE formation were observed in the light and electron microscopes after dejellied eggs were activated by pricking. The FE is established when material from the cortical granules is extruded into the perivitelline space. The cortical granule material passes through the VE as the envelope lifts away from the egg surface. Some cortical granule material deposits in the interstices of the VE, but most of it forms the fertilization layer on the outer surface of the envelope. The cortical reaction is completed about 8–9 min after addition of sperm when eggs are fertilized in vitro.     

Cloning and characterization of a candidate nutritive glycoprotein from the albumen gland of the freshwater snail, Helisoma duryi (Mollusca: Pulmonata)

Abstract. The albumen gland is a female accessory sex gland that synthesizes and secretes perivitelline fluid around pulmonate eggs. The perivitelline fluid is composed of mainly galactogen and proteins, and is thought to provide nourishment to the embryos during development. We have previously identified the major secretory protein of the albumen gland of the freshwater snail Helisoma duryi as a native glycoprotein of ∼288 kDa, consisting of four 66-kDa subunits. In this study, the major albumen gland protein in H. duryi was purified, cloned, and the full-length cDNA sequence determined. Nucleotide sequence analysis revealed that the albumen gland protein (HdAGP) shared 83% identity with a partial cDNA sequence from a developmentally regulated albumen gland protein in Biomphalaria glabrata . The HdAGP mRNA was detected by RT-PCR in the albumen gland, ovotestis, mantle and digestive gland. SDS-PAGE analysis of the albumen gland protein in egg masses at different stages of development showed that the amount of HdAGP steadily decreased during embryogenesis, suggesting its possible catabolism by the developing embryos. Protein domain searches suggested that the HdAGP shared limited sequence identity, and adopted a similar three-dimensional conformation to the bactericidal, permeability increasing, protein family, raising the possibility of a potential bactericidal function for this important reproductive/developmental protein.     

Physiological control of water flux in conifers

Summary Pre-hatching developmental times for prosobranch gastropods are greatly influenced by temperature and taxonomic affinity. If the data used here (including all available data from the Muricacea) are a representative sample, then reasonably accurate estimates of developmental time can be obtained for most prosobranchs knowing only temperature and taxon. Times are also significantly affected by egg or hatching size. Correlations between developmental time and hatching form are probably accounted for by egg size. Prehatching periods are little, if at all, longer for metamorphosed hatchlings than for swimming hatchlings; in any event, differences are small relative to typical free swimming periods. Therefore, the planktonic period is a substantial addition to the total pre-juvenile period. Many embryos die before hatching. More would survive if development were faster; development is, therefore, prolonged at a measurable selective cost. Factors promoting extended developmental periods should be evaluated with these costs in mind. For example, providing much of the yolk as nurse-eggs may allow a species to have a large hatching size and at the same time a relatively brief developmental time.