Egg Envelope Research Articles

Transglutaminase mediates the hardening of fish egg envelope produced by duplication of factor XIIIA gene during the evolution of Teleostei.

During the fertilization of fish eggs, the hardening of the egg envelope is mediated by transglutaminase (hTGase). After fertilization, TGase undergoes processing. We isolated hTGase from extracts of unfertilized and water-activated rainbow trout eggs. Rainbow trout hTGase (Rt-hTGase) appeared as an 80 kDa protein, and its processed form was 55 kDa. Their N-terminal amino acid sequences were nearly identical, suggesting processing in the C-terminal region. The specific activities were not significantly different, indicating that C-terminal processing does not activate the enzyme itself. We cloned the cDNA by reverse transcription polymerase chain reaction (RT-PCR) using degenerate primers followed by RACE-PCR. The deduced amino acid sequence of the cDNA was similar to that of factor XIII subunit A (FXIIIA). Molecular phylogenetic and gene syntenic analyses clearly showed that hTGase was produced by duplication of FXIIIA during the evolution to Teleostei. The 55 kDa processed form of Rt-hTGase is predominantly composed of an enzyme domain predicted from the amino acid sequence of the cDNA. It is hypothesized that the C-terminal domain of Rt-hTGase binds to egg envelope proteins, and that processing allows the enzyme to move freely within the egg envelope, increasing substrate-enzyme interaction and thereby accelerating hardening.

Effects of Light and Water Agitation on Hatching Processes in False Clownfish Amphiprion ocellaris.

False clownfish (Amphiprion ocellaris) employ a hatching strategy regulated by environmental cues, wherein parents provide water flow to encourage embryos to hatch after sunset on the hatching day. Despite previous studies demonstrating the necessity of complete darkness and water agitation for hatching, the regulatory mechanisms underlying these environmental cues remain elusive. This study aimed to investigate how darkness and water agitation affect the secretion of hatching enzymes and the hatching movements of embryos in false clownfish. Assessment of chorion digestion and live imaging of Ca2+ in hatching glands using GCaMP6s, a Ca2+ indicator, revealed that darkness stimulation triggers the secretion of hatching enzymes by increasing Ca2+ levels in hatching gland cells. On the other hand, water agitation primarily stimulated hatching movements in embryos, which led to the rupture of their egg envelopes. These results suggest that changes in light environments following sunset induce embryos to secrete hatching enzymes and that water agitation provided by parents stimulates hatching movements. These responses to environmental cues, light and water agitation, contribute to the rapid and synchronous hatching in false clownfish.

Secondary growth ovarian follicles of the pigmented sterlet sturgeon Acipenser ruthenus L. 1758 (Acipenseriformes, Chondrostei, Actinopterygii, Osteichthyes) – Microscopic study of oocytes, egg envelope and diversification of follicular cells

The individual ovarian follicle of sturgeons (Acipenseriformes, Acipenseridae) contains an oocyte surrounded by follicular cells (FCs), basal lamina, and thecal cells. The late stages of the secondary growth of follicles (mid- and advanced vitellogenic) are not fully explained in Acipenseriformes. To explore and discuss the ultrastructure of oocytes, FCs, an egg envelope, and explain how micropylar cells differentiate and the canals of a multiple micropyle are formed, the samples of ovaries of the mature sterlet sturgeon Acipenser ruthenus were examined. The oocytes are polarized, the nucleus is located in the animal hemisphere, contains lampbrush chromosomes and multiple nucleoli. In the ooplasm three regions are present: a perinuclear (contains the mitochondria), an endoplasm (contains the lipid droplets and yolk platelets), and a periplasm (contains the cortical granules, melanosomes, endocytotic and exocytotic vesicles). The melanosomes in animal hemisphere form two concentric rings separated by a lighter region between them. The FCs are differentiated into bright and dark cells that are both translationally and secretory active. Diversification of FCs involves repeated and cytoskeleton-dependent change of shape. In the advanced follicles the FCs are diversified into micropylar, the animal and vegetal regions cells, and the cells that delaminated from the epithelium in the animal region. The egg envelope is present in the perioocytic space and consists of three layers: (1) an inner layer or vitelline envelope, (2) a middle layer, and (3) an outer layer. The inner layer consists of four sublayers: (a) a filamentous sublayer composed of filaments released from the oocytes, (b) a trabecular 1 sublayer and (c) a trabecular 2 sublayer named due to the sequence of the deposition, and composed of filaments, fibres and trabecules, (d) a homogeneous sublayer located between the trabecular 1 and trabecular 2 sublayers composed of filaments that adhere to each other closely. The middle layer contains two sublayers: a porous 1 and a porous 2 (composed of granular material) which are released by the oocyte and FCs. The outer layer consists of fibrillar material released by the FCs. The egg envelope is pierced by radial canals formed around the microvilli of the oocyte and the microvilli-like processes of FCs. A micropylar field in the egg envelope that covers the animal pole of the oocyte contains 1 – 4 micropylar canals. Micropylar cells are involved in their formation. The shape of these cells is icicle-like and the cytoplasm is differentiated into two regions (a basal and apical bearing a projection) equipped with different sets of organelles.

A Comparative Morphological Study on the Characteristics of Egg Envelopes of Three Cultrinae Fishes (Cyprinidae, Teleostei) in Korea.

Three species of subfamily Cultrinae currently live in Korea, but Erythroculter erythropterus has been introduced into the Nakdonggang River and has taken over the habitat, reducing the habitat of Culter brevicauda. Only the endangered species C. brevicauda still lives in the Yeongsangang River, and it is necessary to be careful not to introduce E. erythropterus in the future. Hemiculter eigenmanni is also found throughout the country. In order to effectively manage and conserve the species in its various habitats and against invasions, this study was initiated. The ultrastructure of the egg envelopes of three species of Cultrinae inhabiting the Geumgang and Yeongsangang Rivers-E. erythropterus, C. brevicauda, and H. eigenmanni-were observed. It was found that the zona radiata of the egg envelopes of all three species were divided into two layers, an outer and inner layer, with the outer surface having a non-structural form. This form is characteristic of fishes with muddy, stagnant habitats or spawning grounds. The number of pore canals on the surface of the egg envelopes was 83 for E. erythropterus, 75 for C. brevicauda, and 58 for H. eigenmanni per 10 μm2, and the thickness was 7.89 ± 0.34 μm, 12.27 ± 0.46 μm, and 7.42 ± 0.24 μm, respectively. The shape of the micropyle demonstrated a funnel shape narrowing toward the inner diameter in all three species, and the size of the inner diameter was 6.62 ± 0.29 μm in E. erythropterus, 4.19 ± 0.39 μm in C. brevicauda, and 3.98 ± 0.46 μm in H. eigenmanni. The differences between species were identified in the number of pore canals, thickness, and micropyle inner diameter of egg envelopes, which were species-specific. Our study reveals a morphological mechanism in the egg envelope that prevents the formation of interspecific hybrids, and these features can be taxonomic traits that clarify species names. It also provides useful data for the production (breeding) of the second generation in aquaculture.

Fluctuating and Stable High Temperatures Differentially Affect Reproductive Endocrinology of Female Pupfish.

For many fishes, reproductive function is thermally constrained such that exposure to temperatures above some upper threshold has detrimental effects on gametic development and maturation, spawning frequency, and mating behavior. Such impairment of reproductive performance at elevated temperatures involves changes to hypothalamic-pituitary-gonadal (HPG) axis signaling and diminished gonadal steroidogenesis. However, how HPG pathways respond to consistently high versus temporally elevated temperatures is not clear. Here, sexually mature Amargosa River Pupfish (Cyprinodon nevadensis amargosae) were maintained under thermal regimes of either stable ∼25°C (low temperature), diurnal cycling temperatures between ∼27 and 35°C (fluctuating temperature), or stable ∼35°C (high temperature) conditions for 50 days to examine effects of these conditions on HPG endocrine signaling components in the pituitary gland and gonad, ovarian and testicular gametogenesis status, and liver gene expression relating to oogenesis. Female pupfish maintained under stable high and fluctuating temperature treatments showed reduced gonadosomatic index values as well as a lower proportion of oocytes in the lipid droplet and vitellogenic stages. Females in both fluctuating and stable 35°C conditions exhibited reduced ovarian mRNAs for steroid acute regulatory protein (star), cholesterol side chain-cleavage enzyme, P450scc (cyp11a1), and 3β-hydroxysteroid dehydrogenase (3bhsd), while ovarian transcripts encoding 11β-hydroxysteroid dehydrogenase (11bhsd) and sex hormone-binding globulin (shbg) were elevated in females at constant 35°C only. Ovarian aromatase (cyp19a1a) mRNA levels were unaffected, but circulating 17β-estradiol (E2) was lower in females at 35°C compared to the fluctuating temperature condition. In the liver, mRNA levels for choriogenins and vitellogenin were downregulated in both the fluctuating and 35°C conditions, while hepatic estrogen receptor 2a (esr2a) and shbg mRNAs were elevated in 35°C females. Taken together, these results demonstrate the potential for elevated temperatures to impair ovarian steroidogenesis and reduce egg envelope and vitellogenin protein production in female C. n. amargosae pupfish, while also shedding light on how thermal regimes that only intermittently reach the upper thermal range for reproduction have differential impacts on reproductive endocrine pathways than constantly warm conditions.

Effects of collision shock on semi-buoyant fish egg hatchings in high-speed streams

During downstream drifting in high-speed steams, semi-buoyant fish eggs can run onto rocks, boats, and other surfaces. This will result in the abrupt and severe collision shock to the fish eggs, causing damage to fish eggs and potentially leading to their death. For protection of fish stocks and river management, it is crucial to understand the impact of collision shock on their hatching and to clarify the upper threshold of collision velocity. Through an indoor free-fall experiment, we quantified the influences of collision shock on semi-buoyant fish eggs. We discovered that, when fish eggs collided with a rock at a speed of 4.4 m/s, about 70% of the eggs did not successfully hatch. Forty-five percent of the egg envelopes were broken when the collision velocity was 4.4 m/s and collided with water. Development stage, particularly when the eggs were at the cell cleavage stage, was the most susceptible factor to collision shock. Our results offer new insights for preserving fish stocks in high-speed streams and can be used to inform river management strategies.

Біологічні особливості штучного відтворення осетроподібних (Acipenseriformes) видів риб (огляд)

Purpose. To analyze the scientific data of domestic and foreign authors regarding the peculiarities of artificial reproduction of sturgeonss (Acipenseriformes). To investigate the state of study of the phenomenon of polyspermic fertilization in sturgeons and the factors that induce it in the conditions of artificial reproduction and cultivation. Findings. A review analysis of the results of scientific studies by domestic and foreign authors was carried out on the pecularities of artificial reproduction of sturgeons and the phenomenon of polysperm fertilization of their eggs in controlled conditions of aquaculture. It is shown that information on the problems of sturgeon polyspermy and its induction by various factors was practically absent in the available professional literature in recent years. Since during the artificial reproduction of sturgeons, a significant number of embryos have atypical development, which leads to their death even before hatching, it has been suggested that atypical forms of cell division were caused by polyspermy. The conducted studies added to the knowledge about the peculiarities of the structure of the sturgeon egg envelope and the processes that occur during fertilization. Despite the fact that there is a natural mechanism to prevent the possibility of polyspermic fertilization, this phenomenon occurs quite often, causing an impairment of zygote fragmentation at the initial stages of embryogenesis, and therefore a decrease in the yield of larvae. Understaning of the mechanism of blocking polyspermy in sturgeons and the factors that induce it requires further studies. The list of scientific articles includes 61 sources, 58 in English. The published publications mainly cover the period of the last twenty years. Practical value. A review of data from scientific and professional sources on the specified topic will allow a comprehensive approach to the understanding of the peculiarities of artificial reproduction of sturgeons, the phenomenon of polyspermy and increasing the yield of larvae in sturgeon farming. Review material on this topic can be interesting and useful for scientists, practitioners, students whose interests are related to aquaculture and used in the educational process of training specialists in specialty 207 “Aquatic bioresources and aquaculture”. Keywords: aquaculture, sturgeons (Acipen­seriformes), eggs, egg envelope, micropyle, fertilization, cortical reaction, polyspermy, embryos.

Ultrastructure of the female pedal gonad in Phoxichilidium femoratum (Chelicerata, Pycnogonida)

Phoxichilidium femoratum is a common species of sea spiders – a small and unique group of chelicerates with unusual adult anatomy. In particular, substantial parts of the reproductive system in pycnogonids (unlike euchelicerates) are located in the appendages. Existing studies of pycnogonid gonads are often limited to light-microscopic level, cover a small range of species, and focus on the contents of the gonad diverticula. Ultrastructural data are rare and contradictory, and the organisation of the gonad wall and the gonoducts is unknown. Here we present a detailed light and transmission electron microscopy-based examination of the pedal portion of the adult female reproductive system in Phoxichilidium femoratum Rathke, 1799. We describe its gross anatomy and the ultrastructure of the gonad diverticulum, oviduct and gonopore, as well as development of the oocytes. Each gonad diverticulum is enclosed in the extracellular matrix of the horizontal septum and bears some internal cellular lining. However, neither the gonad lining, nor the septum sheath cells, ever form a continuous epithelial layer. Oocytes, which undergo maturation in the diverticulum, remain, until very late in the process, attached to the gonad wall though specialised stalk cells. Interestingly, stalk cells do not participate in egg envelope or yolk formation: both are synthesized endogenously in the oocytes. The oviduct is supplied with musculature, which assists in egg transport to the gonopore, whereas the gonopore itself is surrounded by specialised glands.

Asymmetry in previtellogenic and early vitellogenic oocytes, ultrastructure of follicular cells and egg envelope in the pigmented sterlet, Acipenser ruthenus L. 1758 (Chondrostei, Acipenseriformes).

Ovarian follicles of sterlets (Acipenser ruthenus) are composed of a single oocyte surrounded by follicular cells (FCs), basal lamina, and thecal cells. Previtellogenic oocytes are polarized. Homogeneous ooplasm (contains ribosomes) and granular ooplasm (comprises nuage aggregations of nuclear origin, rough endoplasmic reticulum (RER), Golgi complexes, ribosomes, and mitochondria) are distinguished. Granular ooplasm is initially located near the nucleus, contacts the plasma membrane of the oocyte (oolemma) and forms a thin layer underneath its entire perimeter. Next, a ring that surrounds the nucleus is formed and sends strands directed toward the oolemma. The lipid body composed of lipid droplets forms adjacent to this ring. Later, the granular ooplasm and strands enlarge toward the oolemma, lipid body disperses, and homogeneous ooplasm is no longer present. A thin cortical ooplasm is formed underneath the oolemma and does not contain any organelles. The oocyte nucleus moves to the center. The nucleoplasm contains lampbrush chromosomes, nuclear bodies, and multiple nucleoli. Early vitellogenic oocytes are polarized, too. Three regions in the ooplasm are distinguished: the perinuclear (contains lipid droplets near the nuclear envelope), the endoplasm (contains yolk platelets and lipid droplets), and the periplasm (contains yolk spheres, pigment granules, and microtubules). In all these regions the RER, Golgi complexes, nuage, and mitochondria are present. Micropinocytotic vesicles, Golgi vesicles and precursors of the internal layer of the egg envelope are in the cortical ooplasm. Some FCs delaminate from the follicular epithelium, degenerate and vesicles are released into the perioocytic space. They may contain precursors of egg envelope and may be involved in "cell-cell" communication. The egg envelope (zona radiata, zona pellucida) is made up of three layers: the vitelline envelope (inner layer), the middle layer, and the outer layer. In its deposition, both the oocyte and FCs are engaged.

Adverse effects of knocking down chitin synthase A on female reproduction in Culex pipiens pallens (Diptera: Culicidae).

Current mosquito-borne disease vector control strategies, largely based on chemical insecticides, are seriously threatened by increasing resistance worldwide. There is also growing concerned about the adverse effects of insecticides on nontarget organisms and the environment, therefore effective and ecologically friendly alternative approaches are urgently needed. Targeting critical steps of reproduction is considered a potential way to control mosquito populations. Herein, we focused on the roles of chitin synthase A (encoded by chsa) in the reproduction of female mosquitoes. The injection of small interfering RNA targeting Cpchsa in female Culex pipiens pallens (Diptera: Culicidae) had antireproductive effects, including decreased follicle numbers, egg-laying, and hatching rate. Scanning electron microscopy observations showed that Cpchsa silencing caused a defective egg envelope, including absence of the vitelline membrane and cracked chorion layers, which resulted in abnormal permeability. Widely distributed nurse cell apoptosis and follicular epithelial cell autophagy were observed in Cpchsa-silenced ovaries during the vitellogenesis phase. Consistent with the detective egg envelope formation during oogenesis, the exochorionic eggshell structures were also affected in eggs deposited by Cpchsa-silenced mosquitoes. This study provided fundamental evidence for the role of chitin synthase A in the female reproductive process of mosquitoes and might result in a novel alternative strategy for mosquito control. © 2023 Society of Chemical Industry.

Alternative signal pathways underly fertilization and egg activation in a fish with contrasting modes of spawning

BackgroundThe processes of fertilization and egg activation are vital for early embryogenesis. However, while the mechanisms associated with key events during these processes differ among species and modes of spawning, the signal pathways underlying these processes are opaque for many fishes, including economically important species.ResultsWe investigated phenotypic traits, ultrastructure and protein expression levels in the eggs of the topmouth culter (Culter alburnus), a protected and economically important freshwater fish that exhibits two spawning modes, producing semi-buoyant eggs and adhesive eggs. Unfertilized eggs of C. alburnus were examined, as well as eggs at fertilization and 30 min post fertilization. Our results showed that in semi-buoyant eggs, energy metabolism was activated at fertilization, followed by elevated protein expression of cytoskeleton and extracellular matrix (ECM)-receptor interactions that resulted in rapid egg swelling; a recognized adaptation for lotic habitats. In contrast, in adhesive eggs fertilization initiated the process of sperm-egg fusion and blocking of polyspermy, followed by enhanced protein expression of lipid metabolism and the formation of egg envelope adhesion and hardening, which are adaptive in lentic habitats.ConclusionOur findings indicate that alternative signal pathways differ between modes of spawning and timing during the key processes of fertilization and egg activation, providing new insights into the molecular mechanisms involved in adaptive early embryonic development in teleost fishes.

Egg envelope formation of medaka Oryzias latipes requires ZP proteins originating from both the liver and ovary

Teleost oocytes are surrounded by a structure called chorion or egg envelopes, which is composed of zona pellucida (ZP) proteins. As a result of the gene duplication in teleost, the expression site of the zp genes, coding the major component protein of egg envelopes, changed from the ovary to the maternal liver. In Euteleostei, there are three liver-expressed zp genes, named choriogenin (chg) h, chg hm, and chg l, and the composition of the egg envelope is mostly made up of these Chgs. In addition, ovary-expressed zp genes are also conserved in the medaka genomes, and their proteins have also been found to be minor components of the egg envelopes. However, the specific role of liver-expressed versus ovary-expressed zp genes was unclear. In the present study, we showed that ovary-synthesized ZP proteins first form the base layer of the egg envelope and then Chgs polymerize inwardly to thicken the egg envelope. To analyze the effects of dysfunction of the chg gene, we generated some chg knockout medaka. All knockout females failed to produce normally fertilized eggs by the natural spawning. The egg envelopes lacking Chgs were significantly thinner, but layers formed by ZP proteins synthesized in the ovary were found in the thin egg envelope of knockout as well as wildtype eggs. These results suggest that the ovary-expressed zp gene is well conserved in all teleosts, including those species in which liver-derived ZP proteins are the major component, because it is essential for the initiation of egg envelope formation.

DISTRIBUTION OF INVADER <i>MNEMIOPSIS LEIDYI</i> (CTENOPHORA) IN EARLY STAGES OF DEVELOPMENT IN THE SOUTH-EASTERN BALTIC SEA IN AUTUMN 2021

The embryos within the egg envelope (eggs) and cydippid stage larvae of alien species of ctenophore Mnemiopsis leidyi were recorded in the South-Eastern Baltic Sea (within the economic zone of the Russian Federation) in the autumn of 2021. The adults of Mnemiopsis leidyi were not found. The data on the occurrence and distribution of ctenophore in the early stages of development in the SEB were given. The ctenophore eggs were recorded in the open part of the SEB at the stations with the depths of 65-115 m in the intermediate layer and below the upper boundary of the halocline. A large number of eggs (51-57 ind./m3) were observed on the north-eastern slope of the Gdansk Deep. The area of occurrence of ctenophore eggs in 2021 increased compared to the data of 2010-2015. The significant correlation between the eggs abundance and the water salinity and temperature was not revealed. Using the NEMO 4.0 numerical model, the path of advective water penetration into the study area from the western basins of the Baltic Sea, which probably introduced the ctenophore eggs, was traced.

The infection of murine gammaherpesvirus-68 delays the early embryonic development of zebrafish

Zebrafish is a model animal for aquaculture as well as bio-medicine. Murine gamma-herpesvirus 68 (MHV68) is widely regarded as a model for the study of human gamma herpesviruses such as Kaposi's sarcoma-associated herpesvirus (KSHV). In this study, the infection of MHV68 on zebrafish was investigated through incubating fertilized zebrafish eggs with MHV68. The development of zebrafish delayed or stopped at early stage separately when the zebrafish eggs were incubated with MHV68 at different dose for 2 h. The lytic gene of MHV68, including ORF65, RTA and ORF57, were detected in the genome of larvae hatched from the infected eggs. The transcription of the latent gene LANA but not of these lytic genes was detected by RT-PCR. The mRNA level of growth factor TGF-β1, but not of FGF3, was obviously decreased in the larvae from the MHV68 treated eggs. The mRNA levels of cytokines, such as IFNφ3, ISG15 and TNFα, varied post MHV68 infection; however, the transcription of the cytosolic DNA sensors, including DDX41, DHX9 and cGAS, did not change significantly. Moreover, the data generated in zebrafish cell line demonstrated that MHV68 could infect ZF4 cells. All the data support the conclusion that MHV68 could infect zebrafish embryo in the egg envelope and go into latent infection, which will delay the development of the embryos through some unknown mechanism.

Medaka, Oryzias latipes, egg envelopes are created by ovarian-expressed ZP proteins and liver-expressed choriogenins

The medaka (Oryzias latipes) egg envelope (chorion) is composed of three major glycoproteins, Zona Interna (ZI)-1, -2, and -3, that originate in the spawning female liver as the precursor proteins Choriogenin (Chg.)H, Chg.Hm, and Chg.L, respectively. These ZI and Chg. proteins contain a structural ZP protein domain that is conserved among the egg envelope proteins of all animals. While ovarian expression of ZP proteins (e.g., ZPCs and ZPB) has been reported in medakas, the functions of these proteins remain unknown. Thus, the present study aimed to determine whether the ovary-expressed medaka ZP protein, mZPC5, is involved in forming the chorion matrix.The mZPC5 gene (mzpc5) was expressed in the ovaries but not the livers of mature female medakas, as shown by reverse transcription-polymerase chain reaction assays with mzpc5-specific primers. In situ hybridization analysis revealed that ovarian mzpc5 expression was restricted to the ooplasm of early (stage I–III) previtellogenic oocytes, and its expression signal weakened with oocyte growth. Following sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis with anti-mZPC5 antibodies, two immunoreactive proteins were detected in the ovary and chorion extracts. These proteins were approximately 50 and 74 kDa in size, like ZI-3 and ZI-2, respectively.Immunohistochemical assays using anti-mZPC5 and anti-Chg.H antibodies localized the mZPC5 protein in the ooplasm of early previtellogenic oocytes. With oocyte growth, mZPC5 tended to accumulate in the chorion, co-localizing with Chg.H.We previously showed that ovary-expressed ZP proteins could not compensate for Chg.L function loss in gene knock-out (chg.l -/-) medakas. As in our previous study, the chg.l-/- females produced oocytes with thin chorions, resulting in infertile soft eggs. However, in the present study, mZPC5 and Chg.H were co-localized in the chg.l-/- chorions. These results suggested that in the medaka previtellogenic oocyte, 1) mZPC5 is secreted from the ooplasm and deposited on the outer surface of its plasma membrane, creating the thin chorion layer; and 2) following the accumulation of liver-derived Chgs., the 3D structure of the chorion matrix is formed cooperatively with mZPC5 and Chgs. during oogenesis. More research is needed to confirm the functions of mZPC5 in chorion structure and physiology.

Decapod egg membranes: powerful barriers or regulatory structures?

Osmoregulatory abilities and mechanisms of adults and larvae of decapod crustaceans have been extensively investigated. However, how embryos carried by their mothers can deal with changing or extreme salinities is less understood. The egg membranes are believed to isolate embryos from a challenging environment, although osmoregulatory ability has been demonstrated in early developing embryos (naupliar stage) of two crabs. To establish whether embryos are isolated by their membranes and/or are able to osmoregulate, we measured the survival and volume change over 48 h of oocytes and embryos in different stages of three carideans (Betaeus lilianae, Palaemon macrodactylus and P. argentinus) and the brachyuran Neohelice granulata, subjected to different salinities. In addition, we recorded osmolality changes in homogenates of the same stages in P. argentinus and N. granulata after 2 h of exposure and mapped the presence of putative sites of ions exchange in the membrane of all species. High mortality, when it occurred, was associated with low salinity and mortality variation with the stage of development depended on the species. All species precipitated silver salts in or under the egg envelope, with a different pattern between carideans and the brachyuran. Changes in osmolality and egg volume after hypo- or hyper-osmotic salinity challenges indicate that eggs are not fully isolated by their membranes, and that some osmoregulatory mechanisms are in play to maintain developmental homeostasis. We suggest that egg membranes can participate in osmoregulation by selectively transporting ions to an intramembrane space, with differences between carideans and brachyurans.

Light and electron microscopic morphology of the fertilized egg and fertilized egg envelope of Poropanchax normani, Poeciliidae, Teleostei

We examined the morphology of the fertilized egg and the fine structure of fertilized egg envelopes of Poropanchax normani belonging to the family Poeciliidae, also known as Norman’s lampeye using light and electron microscopes. The fertilized eggs with narrow perivitelline space were found to be spherical and demersal, additionally containing small oil droplets in the vitelline membrane. Further, a bundle of adhesive filaments was observed to be present on one side of the fertilized egg. These filaments possessed remarkably high elasticity and were approximately 1-3 mm in length. The size of the fertilized egg was determined to be about 1.49 ± 0.07 mm (n = 30). The outer surface appeared smooth, and adhesive filaments originating at different location of the surface of the envelope were found to be distributed around the egg envelope and were joined together to form a single long bundle in scanning electron microscopic observation. A peak-like structure formed of several straight wrinkles was observed around the micropyle. However, the complete structure of the micropyle could not be studied due to the depth at which it was located. Additionally, the total thickness of the egg envelope was ascertained to be approximately12.5–14.5 μm. The egg envelope consisted of two distinct layers, an outer electron dense layer and an inner lamellar layer, further consisting of 10 sublayers of varying thicknesses. Collectively, it was observed that the morphological characteristics of the fertilized egg, fine structures surrounding the micropyle, outer surface, adhesive structure consisting adhesive filaments, and sections of fertilized egg envelope displayed species specificity.

Microscopic study of the primary growth ovarian follicles of the pike-perch Sander lucioperca (Linnaeus 1758) (Actinopterygii, Perciformes)

The ovaries of Sander lucioperca (Actinopterygii, Perciformes) are made up of the germinal epithelium and ovarian follicles, in which primary oocytes grow. Each follicle is composed of an oocyte surrounded by flattened follicular cells, the basal lamina, and thecal cells. The early stages of oocyte development (primary growth = previtellogenesis) are not fully explained in this species. The results of research with the use of stereoscopic, light, fluorescence, and transmission electron microscopes on ovarian follicles containing developing primary oocytes of S. lucioperca are presented. The polarization and ultrastructure of oocytes are described and discussed. The deposition of egg envelopes during the primary growth and the ultrastructure of the eggshell in maturing follicles of S. lucioperca are also presented. Nuclei in primary oocytes comprise lampbrush chromosomes, nuclear bodies, and nucleoli. Numerous additional nucleoli arise in the nucleoplasm during primary growth and locate close to the nuclear envelope. The Balbiani body in the cytoplasm of oocytes (ooplasm) is composed of nuage aggregations of nuclear origin and mitochondria, endoplasmic reticulum (ER), and Golgi apparatus. The presence of the Balbiani body was reported in oocytes of numerous species of Actinopterygii; however, its ultrastructure was investigated in a limited number of species. In primary oocytes of S. lucioperca, the Balbiani body is initially located in the perinuclear ooplasm on one side of the nucleus. Next, it surrounds the nucleus, expands toward the plasma membrane of oocytes (oolemma), and becomes fragmented. Within the Balbiani body, the granular nuage condenses in the form of threads, locates near the oolemma, at the vegetal oocyte pole, and then dissolves. Mitochondria and cisternae of the rough endoplasmic reticulum (RER) are present between the threads. During primary growth micropylar cells differentiate in the follicular epithelium. They contain cisternae and vesicles of the RER and Golgi apparatus as well as numerous dense vesicles suggesting high synthetic and secretory activity. During the final step of primary growth several follicular cells delaminate from the follicular epithelium, migrate toward the oocyte and submerge in the most external egg envelope. In the ooplasm, three regions are distinguished: perinuclear, endoplasm, and periplasm. Cortical alveoli arise in the perinuclear ooplasm and in the endoplasm as a result of the fusion of RER vesicles with Golgi ones. They are evenly distributed. Lamellar bodies in the periplasm store the plasma membrane and release it into a space between the follicular cells and the oocyte. The developing eggshell in this space is made up of two egg envelopes (the internal one and the external) that are pierced by canals formed around the microvilli of oocytes and the processes of follicular cells. In the deposition of egg envelopes the oocyte itself and follicular cells are engaged. In maturing ovarian follicles the eggshell is solid and the internal egg envelope is covered with protuberances.

Ultrastructure of Egg Envelopes and Early Embryos of Rohdella amazonica (Trematoda: Aspidogastrea) Parasitic in Banded Puffer Fish, Colomesus psittacus

Egg structure and early embryonic development of the aspidogastrean, Rohdella amazonica, a basal trematode, were studied by transmission electron microscopy (TEM) to gain insight into functional, developmental, and phylogenetic characteristics. Gravid worms were removed from the intestine of naturally infected banded puffer fish Colomesus psittacus, collected from the Bay of Marajó, Paracauari River (Pará, Brazil) and processed by standard TEM methods. By the time of pronuclear fusion, the fertilized zygote was already enclosed in a thick, electron-dense pre-operculate eggshell and an underlying layer of vitellocytes that fused into a vitelline syncytium as they were still secreting their shell granules. When cleavage commenced, a small number of macromeres moved to the area just underneath the eggshell, where they fused to form a single syncytial embryonic envelope. Simultaneously, the smaller blastomeres continued to divide as they maintained contact with each other, but remained separate from the vitelline syncytium. Concurrent with these cellular changes, a thickened knob expanded at one pole of the eggshell and began to form an opercular suture. By the time the operculum was fully formed, the vitelline syncytium had mostly degenerated, while the smaller blastomeres had become cohesive as a single mass that preceded the differentiation and morphogenesis of the cotylocidium larva. The general pattern of cleavage and eggshell formation resembles that of other trematodes and polylecithal cestodes, but the single embryonic envelope has been reported only in a few basal taxa. The only other aspidogastrean studied in detail to date is very similar, indicating close phylogenetic affinity and conservatism within this basal neodermatan and neoophoran group.

The light-dependent daily cycle of ovulation in the oviparous medaka fish, Oryzias latipes (Atherinomorpha: Beloniformes: Adrianichthyidae) artificially pregnant with developing embryos.

In the oviparous medaka fish, Oryzias latipes, mature spermatozoa that were artificially introduced into the ovarian cavity retaining ovulated eggs could internally fertilize these eggs. This enabled us to examine the effect of ovarian gestation on the ovulation cycle. Most freshly ovulated eggs could be internally fertilized in the ovarian cavity. Yet eggs ovulated 24 h after single insemination remained unfertilized in the ovarian cavity. Artificially pregnant females persisted in a daily cycle of ovulation, which occurred shortly before the onset of light under the present reproductive conditions. Females continuously ovulated a certain number of eggs despite ovarian gestation, that is, the presence of embryos within the ovarian cavity. Repeated cycles of ovulation led to crowding in the ovarian cavity because the group of fertilized eggs, with their hardened egg envelope (chorion or zona radiata), plugged the genital orifice. The development of fertilized eggs was retarded and ceased around the initiation stage of blood circulation, but when they were transferred from the ovarian cavity into regular saline, they regained their ability to develop normally up to hatching. These results show that in oviparous female medaka, ovarian gestation exerted little effect on the time of ovulation and the number of ovulated eggs.