Pulse treatment of sea urchin embryos with A23187 blocks their hatching out

Summary Pulse treatment of sea urchin embryos with 3 µM A23187 for 2 h at 20° C, starting from 3 to 6 h of development, prevented the embryos from hatching. Many embryos thus treated with A23187 produced mesenchyme cells and underwent gastrulation while still enclosed within the fertilization membrane. The pulse treatment in this pre-hatching period exerts markedly stronger inhibitory effects on hatching than on other events in early development. Treatment beginning at times earlier than 2 h and later than 8 h of development caused only a slight delay of hatching. The activity of hatching enzyme, known to increase between 6 and 8 h after fertilization, was quite low, if present at all, in embryos in which hatching was blocked by A23187. Hatching enzyme synthesis is probably blocked by the preceding pulse treatment. However, overall protein synthesis, estimated with methionine S 35 incorporation, was somewhat augmented in embryos by the pulse treatment. The blockage of hatching and the augmentation of overall protein synthesis by A23187 were appreciably reversed by procaine, tetracaine, ruthenium red or verapamil. Probably, an artificial Ca²⁺ signal induced by A23187 activates protein synthesis but blocks the induction of hatching enzyme synthesis.     

Development of chicken embryos following exposure to 60-Hz magnetic fields with differing waveforms.

Previous studies in my laboratory have revealed a reproducible and statistically significant increase in the number of malformations in live chicken embryos that had been exposed during the first 48 h of incubation to a pulsed magnetic field (unipolar pulses, 100-pps, 1-microT peak density). In marked contrast, no adverse effect was seen following similar exposure to 60-Hz, bipolar, unipolar, or split-sine waves at 3-microT peak-to-peak. In the four experiments comprising the present study, differences in the numbers of malformations between control and experimental groups were not statistically significant. Field-free incubation for an additional 72 h after exposure to a bipolar sine wave for 48 h resulted in an increase in normal live embryos in both control and treated groups.     

Induction of developmental abnormalities in larval goldfish, Carassius auratus L., under cool incubation conditions

Compared with incubation at a constant 22° C, exposure of goldfish embryos and larvae to 13° C, under a variety of thermal protocols, caused increased frequencies of abnormal development and, in some cases, reduced survival to hatching. The low-temperature incubation conditions were particularly deleterious when eggs were incubated at 13° C from the outset, regardless of the temperature at which the donor female ovulated and the eggs were fertilized. Significantly higher frequencies of developmental abnormalities were also noted when embryos were transferred from 22°C to 13°C at 6, 24, 128 and, in one case, 175 h after fertilization. In three of five experiments, subjecting embryos and larvae to diel fluctuations between 22 and 13° C, with a 5-h hold at the lower temperature, caused an increase in development abnormalities. These results demonstrate that the thermal requirements of goldfish embryos and larvae necessitate a delay in ovulation and spawning until water is sufficiently warm. Developmental abnormalities can be induced by exposure to cool (13° C) conditions, at least up to the time that swimbladder inflation occurs.     

Preimplantation growth delay and micronucleus formation after in vivo exposure of mouse zygotes to fast neutrons.

Mouse zygotes were irradiated with fast neutrons (0.06 to 1.00 Gy) 1 h after conception and examined at various intervals (24 to 100 h after conception) for embryonic development and micronucleus formation. The frequency of micronuclei per cell increased linearly with dose in 2-cell embryos observed at 24 h after conception and in 4-cell and 8-cell embryos at 48 h after conception. Compared with X rays, the relative biological effectiveness of neutrons for the induction of micronuclei per embryo was 2.5 at 24 h after conception and 3.5 at 48 h after conception. Neutron-induced micronucleus formation was accompanied by morphological growth delay and a significant decrease in the number of cells in the embryos. An inverse relationship was found between the number of cells in embryos and the number of micronuclei when observed at 48 h after conception following irradiation with 0.12 to 1.00 Gy and at 78 h after conception following exposure to 0.50 Gy. The effect of neutron irradiation on embryonic development was likely to be mediated by cell death, as suggested by a significantly increased dead cell index in blastocysts following irradiation of zygotes.     

Survival of largemouth bass, bluegill and channel catfish embryos after electroshocking

Survival to hatching was determined after electroshocking embryos of largemouth bass Micropterus salmoides , bluegill Lepomis macrochirus and channel catfish Ictalurus punctatus . Embryos at different developmental stages were exposed for 20 s to homogeneous electric fields (4–16 V cm⁻¹) of direct current (DC) or 60 or 120 Hz pulsed direct current (PDC) in water of 100 μS cm⁻¹ ambient conductivity. For all species, DC reduced survival of embryos at developmental stages before, during, or soon after epiboly; but survival did not differ from controls during later developmental stages. Survival of largemouth bass and bluegill was not reduced by 60 or 120 Hz PDC except for bluegill exposed at 12 h post‐fertilization. Channel catfish embryo survival was <5% when exposed to 60 or 120 Hz PDC at 8 h post‐fertilization, survival improved for embryos exposed at 67 h to 60 Hz but not to 120 Hz, and all embryos survived exposure to PDC at 150 h post‐fertilization. Exposure durations as short as 5 s resulted in <10% survival of largemouth bass during sensitive stages. All bluegill embryos aged 22 h post‐fertilization hatched prematurely after exposure to 16 V cm⁻¹ DC, but survival was not affected. The use of PDC for electroshocking near largemouth bass and bluegill nests could reduce the negative effects on survival of these species; however, PDC can reduce survival of channel catfish embryos.     

Hypergravity susceptibility of ventral root activity during fictive swimming in tadpoles (Xenopus laevis)

1. Fictive swimming is an experimental model to study early motor development. As vestibular activity also affects the development of spinal motor projections, the present study focused on the question whether in Xenopus laevis tadpoles, the rhythmic activity of spinal ventral roots (VR) during fictive swimming revealed age-dependent modifications after hypergravity exposure. In addition, developmental characteristics for various features of fictive swimming between stages 37/38 and 47 were determined. Parameters of interest were duration of fictive swimming episodes, burst duration, burst frequency (i.e., cycle length), and rostrocaudal delay. 2. Ventral root recordings were performed between developmental stage 37/38, which is directly after hatching and stage 47 when the hind limb buds appear. The location of recording electrodes extended from myotome 4 to 17. 3. Hypergravity exposure by 3 g-centrifugation lasted 9 to 11 days. It started when embryos had just terminated gastrulation (stage 11/19-group), when first rhythmical activity in the ventral roots appeared (stage 24/27-group), and immediately after hatching (stage 37/41-group). Ventral root recordings were taken for 8 days after termination of 3 g-exposure. 4. Between stage 37/38 (hatching) and stage 47 (hind limb bud stage) burst duration, cycle length and rostrocaudal delay recorded between the 10th and 14th postotic myotome increased while episode duration decreased significantly. In tadpoles between stage 37 and 43, the rostrocaudal delay in the proximal tail part was as long as in older tadpoles while in caudal tail parts, it was shorter. During this period of development, there was also an age-dependent progression of burst extension in the proximal tail area that could not be observed between the 10th and 14th myotome. 6. After termination of the 3 g-exposure, the mean burst duration of VR activity increased significantly (p < 0.01) when 3 g-exposure started shortly after gastrulation but not when it started thereafter. Other parameters for VR activity such as cycle length, rostrocaudal delay and episode duration were not affected by this level of hypergravity. 7. It is postulated that (i) functional separation of subunits responsible for intersegmental motor coordination starts shortly after hatching of young tadpoles; and that (ii) gravity exerts a trophic influence on the development of the vestibulospinal system during different periods of embryonic development leading to the formation of more rigid neuronal networks earlier in the spinal than in the ocular projections.     

Observation of the embryonic development in Pseudoplatystoma coruscans (Siluriformes: Pimelodidae) under light and scanning electron microscopy

Pseudoplatystoma coruscans is a very popular species for tropical fish culture as it has boneless meat of delicate taste and firm texture. Few studies on fish reproductive biology refer to the morphological features of eggs. The goal, therefore, of this present work was to perform a structural and ultrastructural analysis of fertilization and embryonic development in P. coruscans. The incubation period, from fertilization to hatching, lasts 13 h at 28/29 degrees C and 18 h at 27 degrees C. The oocytes had a mean diameter of 0.95 mm and hatched larvae were 2.55 mm in diameter. Analysing their development, we observed round, yellow oocytes that bore a double chorion membrane and a single micropyle. At 10 s after fertilization, several spermatozoa were detected attached to the oocyte surface. After 1 min of development, a fertilization cone that obstructed the micropyle could be observed. Segmentation started between 20 and 30 min after fertilization, when the egg cell was then formed. The first cleavage occurred between 30 and 45 min after fertilization, prior to reaching the morula stage (75 and 90 min after fertilization). The epiboly movement started at 120 and 180 min after fertilization and ended at 360 and 480 min after fertilization. Differentiation between cephalic and caudal region was detected after 420 and 600 min after fertilization and larvae hatched between 780 and 1080 min after fertilization. Seven main embryonic development stages were identified: egg cell, cleavage, morula, blastula, gastrula, segmentation with differentiation between cephalic and caudal regions, and hatching.     

Effects of co-culture and embryo number on the in vitro development of bovine embryos

It is generally accepted that culturing embryos in groups or with somatic cells improves both the yield and quality of the blastocysts obtained. The aims of this study were 1) to compare the yield and quality of the embryos obtained after culture in several number conditions and in several culture systems and 2) to assess the effect of co-culture started at various stages of embryo development. Under cell-free culture conditions (modified synthetic oviduct fluid [mSOF] supplemented with 10% fetal calf serum [FCS] 48 h post insemination, the rate of Day 10 blastocysts was lower when embryos were cultured in small groups (1 to 6 per drop) than in large groups (4 versus 23% ; P < 0.01). There was no group effect when embryos were co-cultured either with Buffalo rat liver (BRL) cells in TCM 199, or in a culture system allowing the progressive development of cumulus cells in mSOF, even if co-culture started at 66 or 114 h post insemination. However, embryos cultured singly had lower cell numbers than embryos cultured in large groups when co-culture started at 114 h post insemination. This suggests that 1) somatic cells improve the development of singly cultured bovine embryos up to the blastocyst stage after the 9-16 cell stage; 2) co-culture affects blastocyst cell number of singly cultured embryos by acting roughly between the 5-8 and the 9-16 cell stage; and 3) cooperation between embryos could replace the effect of co-culture either on the yield of blastocysts or on blastocyst cell number. Blastocysts appeared significantly earlier in co-culture with cumulus cells in mSOF than in co-culture with BRL cells in TCM 199 (detection of the blastocysts: 7.3 +/- 0.1 d post insemination with cumulus cells versus 8.1 +/- 0.1 d with BRL cells; P < 0.001) and had a significant higher number of cells (143 +/- 9 versus 85 +/- 11; P < 0.001). This system thus seems suitable for the culture of small numbers of embryos resulting from in vitro maturation and fertilization of oocytes from individual donor cows.     

Ultraviolet light‐induced impairment of goldfish embryo development and evidence for photorepair mechanisms

Goldfish Carassius auratus embryos were subjected to artificial ultraviolet‐B (UVB) radiation (280–320 nm) at various times during development to evaluate the effects on production of anatomically normal larvae. The UVB radiation used in these experiments included a higher proportion of shorter wavelengths compared to the natural spectrum. The development of embryos exposed to UVB for 2 or 4 h at 26 h post‐fertilization was severely impaired whereas similar exposures at 50 or 74 h post‐fertilization had no effect. A 2 h exposure to UVB commencing at 2 h post‐fertilization did not adversely affect embryonic development whereas a 4 h exposure to a lower dose did. At 50 h post‐fertilization, when embryos were normally resistant to UVB, denial of access to visible light and UVA before, during and after exposure to UVB caused impairment of development. Analysis of DNA fragment length after incubation with an endonuclease suggested that UVB damage at 50 h was caused by formation of pyrimidine dimers. This study demonstrated that the sensitivity of goldfish embryos to UVB varied during development and that resistance to UVB in later developmental stages included a photorepair mechanism.     

Enhancement of radiation effects by mercury in early postimplantation mouse embryos in vitro

Summary Two-cell mouse embryos were X-irradiated (1 Gy) and immediately thereafter exposed to mercuric chloride (3 µM) up to the blastocyst stage. When combined treatment started shortly (about 1 to 2 h) before mitosis to the four-cell stage, blastocyst formation, hatching of blastocysts, trophoblast outgrowth and ICM formation were impaired stronger than expected from the addition of the single effects. The enhancement of risk was maximal for hatching of blastocysts and no further increase was observed for trophoblast outgrowth and ICM formation. When exposure of embryos to X-rays and mercury began about 5 to 6 h before mitosis to the four-cell stage, only additive effects were obtained for the endpoints mentioned above.     

Valproate-induced teratogenesis in Japanese rice fish (Oryzias latipes) embryogenesis

Fertilized eggs of Japanese rice fish (medaka) at three developmental stages (Iwamatsu stages 4-30) were exposed to waterborne valproic acid (VPA) (0-80 mM) in hatching solution for 48 h. The amount of valproate to cause 50% mortality (IC(50)) is found to be developmental stage-specific. The embryos were more sensitive to valproate at early stages of development (Iwamatsu stages 4-10) than in the embryos in late stages (Iwamatsu stages 17-30). Valproate exposed embryos have microcephaly and disrupted cardiovasculature with delayed vessel circulation, thrombus formation, and slow heart rate. The hatching efficiency is also reduced by valproate exposure due to developmental delay. The mRNA analysis of nine genes belong to oxidative stress (catalase, gsr, gst), neurogenesis (iro3, wnt1, shh, otx2, nlgn3b) and cell cycle regulation (ccna2) have been done. It was observed that the genes belong to oxidative stress remained unaltered after valproate exposure. However, some of the genes belong to neurogenesis (wnt1,shh, otx2 and nlgn3b) and cell cycle (ccna2) showed developmental stage-specific alteration after valproate exposure. This study indicates that valproate is able to induce some of the phenotypic features which are analogous to human fetal valproate syndrome (FVS). Modulation of genes expressed in neural tissues indicates that this fish can be used to analyze the mechanisms of many neurobehavioral disorders like Autism spectrum disorder (ASD) in human.     

Hatching plasticity in the tropical gastropod Nerita scabricosta

Hatching plasticity has been documented in diverse terrestrial and freshwater taxa, but in few marine invertebrates. Anecdotal observations over the last 80 years have suggested that intertidal neritid snails may produce encapsulated embryos able to significantly delay hatching. The cause for delays and the cues that trigger hatching are unknown, but temperature, salinity, and wave action have been suggested to play a role. We followed individual egg capsules of Nerita scabricosta in 16 tide pools to document the variation in natural time to hatching and to determine if large delays in hatching occur in the field. Hatching occurred after about 30 d and varied significantly among tide pools in the field. Average time to hatching in each pool was not correlated with presence of potential predators, temperature, salinity, or pool size. We also compared hatching time between egg capsules in the field to those kept in the laboratory at a constant temperature in motionless water, and to those kept in the laboratory with sudden daily water motion and temperature changes. There was no significant difference in the hatching rate between the two laboratory treatments, but capsules took, on average, twice as long to hatch in the laboratory as in the field. Observations of developing embryos showed that embryos in the field develop slowly and continuously until hatching, but embryos in the laboratory reach the hatching stage during the first month of development and remain in stasis after that. Instances of hatching plasticity in benthic marine invertebrates, like the one in N. scabricosta, could greatly enhance our ability to investigate the costs and benefits of benthic versus planktonic development, a long‐standing area of interest for invertebrate larval biologists.     

Applied DC magnetic fields cause alterations in the time of cell divisions and developmental abnormalities in early sea urchin embryos

Most work on magnetic field effects focuses on AC fields. The present study demonstrates that exposure to medium-strength (10 mT-0.1 T) static magnetic fields can alter the early embryonic development of two species of sea urchin embryos. Batches of fertilized eggs from two species of urchin were exposed to fields produced by permanent magnets. Samples of the continuous cultures were scored for the timing of the first two cell divisions, time of hatching, and incidence of exogastrulation. It was found that static fields delay the onset of mitosis in both species by an amount dependent on the exposure timing relative to fertilization. The exposure time that caused the maximum effect differed between the two species. Thirty millitesla fields, but not 15 mT fields, caused an eightfold increase in the incidence of exogastrulation in Lytechinus pictus, whereas neither of these fields produced exogastrulation in Strongylocentrotus purpuratus. Bioelectromagnetics 18:255–263, 1997. © 1997 Wiley-Liss, Inc.     

Effects of male accessory sex gland secretions on early embryonic development in the golden hamster

The ventral prostates, dorsolateral prostates, coagulating glands, seminal vesicles and/or ampullary glands were bilaterally removed from adult male hamsters. Removal of these glands did not affect the fertilization rate and cleavage of the embryos at 48 h post coitum (p.c.). Air-dried preparations of the embryos showed a delay in cleavage at 72 h p.c. and a significant number of degenerated embryos was also found in females mated with males from which all the male accessory sex glands had been removed. A significant implantation loss was also observed at 122 h p.c. The results suggest that, in the golden hamster, removal of the male accessory sex gland causes a slower cleavage rate in embryonic development and a significant embryonic loss during pregnancy.     

Sensitivity of Embryos and Larvae of the Sea Urchin Strongylocentrotus intermedius to Effects of Copper Ions

The effect of copper ions in seawater (0.02 mg/l) on the early stages of development of the sea urchin Strongylocentrotus intermedius was studied. Copper exposure from fertilization or the prism stage retarded development and growth and led to abnormalities in the morphology of the embryos and larvae. However, if development to the pluteus stage proceeded in clean seawater, an increased copper concentration did not inhibit the growth of larvae. If sea urchin embryos at fertilization and the prism stage were maintained for 1–2 days in seawater containing 0.02 mg Cu/l and then transferred to clean seawater, the adverse consequences of this exposure remained present after 48 h.     

Effect of cysteamine supplementation of in vitro matured bovine oocytes on chilling sensitivity and development of embryos

In vitro techniques for production of bovine embryos including in vitro oocyte maturation (IVM), fertilization (IVF) and culture (IVC) are becoming increasingly employed for a variety of research purposes. However, decreased viability following cryopreservation by conventional methods has limited commercial applications of these technologies. A practical alternative to facilitate transport would be to arrest development by chilling without freezing. The present research was undertaken to evaluate chilling sensitivity of IVM-IVF embryos at different stages of development, and to determine possible beneficial effects of cysteamine treatment during IVM, previously shown to enhance embryo development in culture, on survival following chilling at different stages. Embryos produced by standard IVM-IVF-IVC methods were chilled to 0 degrees C for 30 min at 2-cell (30-34 h post-insemination, hpi), 8-cell (48-52 hpi) or blastocyst (166-170 hpi) stages. Viability after chilling was assessed by IVC with development to expanded blastocyst stage determined on days 7 and 8 post-insemination (pi) and hatching blastocyst stage determined on days 9 and 10 pi. Control embryos at the same stages were handled similarly, but without chilling, and development during culture similarly assessed. The effect of cysteamine supplementation (100 microM) of the IVM medium was determined for both chilled and non-chilled (control) embryos. Cysteamine supplementation during IVM had no significant effect on oocyte maturation or fertilization, but increased the proportions of oocytes developing to blastocyst stage by day 7 (13.7+/-0.9% versus 7.2+/-0.9%; P<0.05), total blastocysts (20.5+/-0.9% versus 15.3+/-1.3%; P<0.05), and hatching blastocysts (16.8+/-1.6% versus 12.0+/-1.5%; P<0.05). The greater survival in terms of hatching (78.6+/-7.0) following chilling of blastocysts produced by IVM-IVF of oocytes matured in media supplemented with cysteamine offers promise for applications requiring short-term storage to facilitate transport of in vitro produced bovine embryos.     

Superovulation in the cow using estradiol 17beta or GnRH in conjunction with FSH-P

A study was designed to evaluate the superovulatory response in the cow when either estradiol 17beta or gonadotrophin releasing hormone (GnRH) was used in a superovulatory regimen with follicle stimulating hormone (FSH-P). Fifty-four cyclic crossbred females were superovulated in replicates between Days 8 and 12 of their cycle. All animals were treated with 28 mg of FSH-P in twice-daily decreasing doses, each receiving 500 mug cloprostenol (PGF) 48 h after initiation of treatment. Group 1 served as FSH-P controls, Group 2 received FSH-P and 400 mug of estradiol 17beta 36 h after PGF, and Group 3 received FSH-P and 250 mug GnRH 48 h after PGF. Inseminations with one vial of frozen semen were done at 12, 24 and 36 h after the onset of estrus. Ova/embryos were collected nonsurgically at Day 7 postestrus. Numbers of corpora lutea (CL) were recorded after palpation per rectum and the recovered ova and embryos were evaluated. All females were bled for endocrine examination. There were no differences in ovarian response among these treatments. Mean total ova/embryos collected in Group 3 was significantly higher than in Groups 1 or 2 (P < 0.05); however, no significant difference existed between groups in the mean numbers of fertilized or transferable embryos. Similarly, no significant differences existed between groups for recovery rate, fertilization rate, or percentage of transferable embryos. Serum estradiol levels were significantly higher at the expected end of ovulation in Group 2, and this tended to be associated with higher fertilization and transferable embryo rates. Furthermore, a significant positive correlation was found to exist between CL numbers and each of the ova/embryo parameters and the estradiol levels at estrus.     

Exogastrulation and interference with the expression of major yolk protein by estrogens administered to sea urchins

Although estrogens have been detected in some echinoderm species, their role is not clearly understood; so we examined the effects of estrogens administered to sea urchin embryos and larvae. A typical malformation was exogastrulation, induced by the exposure to ethynylestradiol (EER) in a defined period of 12 h from 12 h after fertilization (HAF). Morphogenesis for gastrulation was delayed in the treated embryos: protrusion of the archenteron started at 30 HAF when gastrulation had already finished in normal embryos. Exogastrulation induced by EER was cancelled by the antiestrogen chemical, ICI182,780. Feeding larvae were less sensitive to estrogens than those in early embryogenesis and, at certain concentrations, developed without abnormal morphology. The effect of estrogens was examined at the level of gene expression of the major yolk protein (MYP). MYP expression started during the larval stage and was suppressed by estrone at the six-armed stage, but not by β-estradiol, and in later stage larvae, the expression was not affected by treatment with either estrogen. Estrogens affect sea urchins in the early stage of embryogenesis, leading to abnormal morphogenesis and interference with gene expression.     

Assessment of female gamete quality in the Pacific oyster Crassostrea gigas

Summary

The possible relationship between certain oocyte and embryo characteristics and larvae viability was investigated with reference to the following aspects: (1) morphological—oocyte diameter and shape; (2) cytological—overall ultrastructure and membrane integrity; (3) biochemical—content of lipids, proteins and carbohydrates; and (4) physiological—respiration. The rate of survival and incidence of abnormality were estimated 24 h after fertilization. The first results showed that 80–90% of oocytes were cytologically viable before fertilization. Eighty to 90% of oocytes are apparently viable before fertilization on the basis of staining with Trypan blue, but this parameter shows little correlation with larval viability. However, Trypan blue staining is of value in allowing the recognition of oocytes with damaged membranes. Respiration was measured for unfertilized oocytes 5 min after stripping, after 6 h, and for 3-h embryos. Positive correlations were found between the O₂-consumption of embryos and both the rate of fertilization and the hatching rate of 24-h larvae. In contrast, no correlation was found between hatching parameters and the O₂-consumption of unfertilized oocytes. These results suggest that embryos possess quality indicators, relating to metabolic characteristics, which can be quantified more easily than those of oocytes.