First Hours Of Development Research Articles

EMBRYOGENESIS OF MYLOSSOMA DURIVENTRE (PACU-PEVA)

This paper aimed to test a semi natural reproduction and to study the morphological development of the Mylossoma duriventre embryo until hatching. We selected six females with coelomic cavity bulging and softness and eighteen males that release sperm under light belly compression. We applied two doses of luteinizing hormone (0.0125 mg/kg and 0.125 mg/kg) for females and for males no hormonal induction was made, a semi natural reproduction was performed. After fertilization the embryos were transfer to a incubator and were evaluated every at 15-min intervals during the first hours of development and at one-hour intervals until hatching. The embryos are megalecites with meroblastic division, being visualized six different stages of development: fertilization, cleavage, blastula, gastrula, segmentation and pharyngula. In gastrulation (120-min post fertilization), there is formation of "tail bud" and appearance of somites during segmentation (510-min post fertilization), after the blastopore closure. The hatching occurred with 20 hours of development, at a temperature of 24°C. It was possible to reproduce the pacu peva in captivity, and the morphological development of the embryos showed some specificity when compared to other species and its results regarding embryonic development are essential for a better understanding of biology this species.

Light-sheet fluorescence microscopy for the in vivo study of microtubule dynamics in the zebrafish embryo.

During its first hours of development, the zebrafish embryo presents a large microtubule array in the yolk region, essential for its development. Despite of its size and dynamic behavior, this network has been studied only in limited field of views or in fixed samples. We designed and implemented different strategies in Light Sheet Fluorescence microscopy for imaging the entire yolk microtubule (MT) network in vivo. These have allowed us to develop a novel image analysis from which we clearly observe a cyclical re-arrangement of the entire MT network in synchrony with blastoderm mitotic waves. These dynamics also affect a previously unreported microtubule array deep within the yolk, here described. These findings provide a new vision of the zebrafish yolk microtubules arrangement, and offers novel insights in the interaction between mitotic events and microtubules reorganization.

Quantification reveals early dynamics in Drosophila maternal gradients.

The Bicoid (Bcd) protein is a primary determinant of early anterior-posterior (AP) axis specification in Drosophila embryogenesis. This morphogen is spatially distributed in an anterior-high gradient, and affects particular AP cell fates in a concentration-dependent manner. The early distribution and dynamics of the bicoid (bcd) mRNA, the source for the Bcd protein gradient, is not well understood, leaving a number of open questions for how Bcd positional information develops and is regulated. Confocal microscope images of whole early embryos, stained for bcd mRNA or the Staufen (Stau) protein involved in its transport, were processed to extract quantitative AP intensity profiles at two depths (apical-under the embryo surface but above the nuclear layer; and basal-below the nuclei). Each profile was quantified by a two- (or three-) exponential equation. The parameters of these equations were used to analyze the early developmental dynamics of bcd. Analysis of 1D profiles was compared with 2D intensity surfaces from the same images. This approach reveals strong early changes in bcd and Stau, which appear to be coordinated. We can unambiguously discriminate three stages in early development using the exponential parameters: pre-blastoderm (1-9 cleavage cycle, cc), syncytial blastoderm (10-13 cc) and cellularization (from 14A cc). Key features which differ in this period are how fast the first exponential (anterior component) of the apical profile drops with distance and whether it is higher or lower than the basal first exponential. We can further discriminate early and late embryos within the pre-blastoderm stage, depending on how quickly the anterior exponential drops. This relates to the posterior-wards spread of bcd in the first hour of development. Both bcd and Stau show several redistributions in the head cytoplasm, quite probably related to nuclear activity: first shifting inwards towards the core plasm, forming either protrusions (early pre-blastoderm) or round aggregations (early nuclear cleavage cycles, cc, 13 and 14), then moving to the embryo surface and spreading posteriorly. These movements are seen both with the 2D surface study and the 1D profile analysis. The continued spreading of bcd can be tracked from the time of nuclear layer formation (later pre-blastoderm) to the later syncytial blastoderm stages by the progressive loss of steepness of the apical anterior exponential (for both bcd and Stau). Finally, at the beginning of cc14 (cellularization stage) we see a distinctive flip from the basal anterior gradient being higher to the apical gradient being higher (for both bcd and Stau). Quantitative analysis reveals substantial (and correlated) bcd and Stau redistributions during early development, supporting that the distribution and dynamics of bcd mRNA are key factors in the formation and maintenance of the Bcd protein morphogenetic gradient. This analysis reveals the complex and dynamic nature of bcd redistribution, particularly in the head cytoplasm. These resemble observations in oogenesis; their role and significance have yet to be clarified. The observed co-localization during redistribution of bcd and Stau may indicate the involvement of active transport.

Malformations and mortality in zebrafish early stages associated with elevated caspase activity after 24 h exposure to MS-222

Tricaine methanesulfonate (MS-222) is a commonly used anaesthetic agent for immobilization of aquatic species. However, delayed development and malformations have been observed in 24 hpf (hours post-fertilization) zebrafish embryos after long-term immobilization. Still, no comprehensive study has been described regarding zebrafish exposure to MS-222 during the first hours of development, which are one of the most sensitive life stages to toxicants. Therefore, this research aimed to assess the toxicity of a 24 h exposure to MS-222 on zebrafish embryonic development. Based on the MS-222 LC50, early blastula stage embryos (~2 hpf) were exposed to 0, 12.5, 25 and 50 mg L−1 for 24 h and then allowed to develop up to 144 hpf. The chromatographic analysis showed that this anaesthetic agent bioaccumulates in 26 hpf zebrafish larvae in a concentration-dependent manner. In addition, increased mortalities and skeletal abnormalities were observed at 144 hpf, namely in the highest tested concentration. Yet, no craniofacial anomalies were observed either by alcian blue or calcein staining methods. Independently of the tested concentration, decreased speed and distance travelled were perceived in 144 hpf larvae. At the biochemical level, decreased in vivo reactive oxygen species (ROS) generation and apoptosis was observed. Additionally, catalase activity was increased at 26 hpf while results of mRNA expression showed a decreased gclc transcript content at the same time-point. Overall, data obtained highlight the toxicological risk of MS-222 and support ROS-mediated cell death signalling changes through the elevation of catalase activity as an adaptative or protective response.

Efficient Early Diagnosis of Sepsis Using Whole-Blood PCR-Reverse Blot Hybridization Assay Depending on Serum Procalcitonin Levels.

Sepsis is one of the medical emergencies, and its early detection, within the first hours of development, and proper management improve outcomes. Molecular diagnostic assays using whole blood collected from patients with suspected sepsis have been developed, but the decision making is difficult because of the possibility of false positives, due to contamination. Here, we evaluated the performance of the reverse blot hybridization assay (REBA) Sepsis-ID test for the detection of sepsis-causing microorganisms using whole-blood samples. In addition, the concentrations of C-reactive protein (CRP) and procalcitonin (PCT) were determined to evaluate whether these biomarkers can provide criteria for performing REBA Sepsis-ID in clinical settings. For this study, EDTA-anticoagulated whole blood was simultaneously collected for REBA Sepsis-ID and blood culture from 440 patients with suspected sepsis, from January to October 2015. In addition, CRP and PCT concentrations were measured in 227 patients. The overall positive rates of REBA Sepsis-ID and blood culture were 16.6% (73/440) and 13.9% (61/440), respectively. The pathogen-positive rates of REBA Sepsis-ID and blood culture were 9.8% (43/440) and 9.5% (42/440), respectively. The areas under the receiver operating characteristic (AUROC) curves of PCT and CRP for predicting pathogen-positive results of REBA Sepsis-ID were 0.72 and 0.69, respectively. The PCT concentrations in the group of patients aged ≥50 years were significantly higher than those in the group aged <50 years. After adjusting for age, the PCT AUROC value was 0.77 for predicting pathogen-positive results of REBA Sepsis-ID. The optimal cutoff values of PCT concentrations for subsequent application of REBA Sepsis-ID were 0.12 ng/mL in all patients and 0.22 ng/mL in patients aged ≥50 years. Our observations showed that REBA Sepsis-ID using whole blood was advantageous for the early detection of sepsis-causing microorganisms, and the PCT concentration could be used to determine the necessity of using REBA Sepsis-ID in clinical settings. The application of REBA Sepsis-ID using whole blood, based on the PCT concentration, may contribute to a highly efficient detection of sepsis-causing microorganisms.

Stage specific effects of soluble copper and copper oxide nanoparticles during sea urchin embryo development and their relation to intracellular copper uptake

The effects of exposure to either soluble copper (copper sulfate) or copper oxide nanoparticles (nano-CuO) during specific early developmental stages of sea urchin embryos were analyzed. Soluble copper caused significant malformations in embryos (skeletal malformations, delayed development or gut malformations) when present at any given stage, while cleavage stage was the most sensitive to nano-CuO exposure causing skeletal malformations and decreased total antioxidant capacity. The stage specificity was linked to higher endocytic activity during the first hours of development that leads to higher accumulation of copper in specific cells critical for development. Results indicate that nano-CuO results in higher accumulation of copper inside of embryos and this intracellular copper is more persistent as compared to soluble copper. The possible implications later in development are discussed.

Physical features and chitin content of eggs from the mosquito vectors Aedes aegypti, Anopheles aquasalis and Culex quinquefasciatus: Connection with distinct levels of resistance to desiccation

Mosquito eggs are laid in water but freshly laid eggs are susceptible to dehydration, if their surroundings dry out at the first hours of development. During embryogenesis of different mosquito vectors the serosal cuticle, an extracellular matrix, is produced; it wraps the whole embryo and becomes part of the eggshell. This cuticle is an essential component of the egg resistance to desiccation (ERD). However, ERD is variable among species, sustaining egg viability for different periods of time. While Aedes aegypti eggs can survive for months in a dry environment (high ERD), those of Anopheles aquasalis and Culex quinquefasciatus in the same condition last, respectively, for one day (medium ERD) or a few hours (low ERD). Resistance to desiccation is determined by the rate of water loss, dehydration tolerance and total amount of water of a given organism. The ERD variability observed among mosquitoes probably derives from diverse traits. We quantified several attributes of whole eggs, potentially correlated with the rate of water loss: length, width, area, volume, area/volume ratio and weight. In addition, some eggshell aspects were also evaluated, such as absolute and relative weight, weight/area relationship (herein called surface density) and chitin content. Presence of chitin specifically in the serosal cuticle as well as aspects of endochorion external surface were also investigated. Three features could be related to differences on ERD levels: chitin content, directly related to ERD, the increase in the egg volume during embryogenesis and the eggshell surface density, which were both inversely related to ERD. Although data suggest that the amount of chitin in the eggshell is relevant for egg impermeability, the participation of other yet unidentified eggshell attributes must be considered in order to account for the differences in the ERD levels observed among Ae. aegypti, An. aquasalis and Cx. quinquefasciatus.

The Drosophila Gap Gene Network Is Composed of Two Parallel Toggle Switches

Drosophila “gap” genes provide the first response to maternal gradients in the early fly embryo. Gap genes are expressed in a series of broad bands across the embryo during first hours of development. The gene network controlling the gap gene expression patterns includes inputs from maternal gradients and mutual repression between the gap genes themselves. In this study we propose a modular design for the gap gene network, involving two relatively independent network domains. The core of each network domain includes a toggle switch corresponding to a pair of mutually repressive gap genes, operated in space by maternal inputs. The toggle switches present in the gap network are evocative of the phage lambda switch, but they are operated positionally (in space) by the maternal gradients, so the synthesis rates for the competing components change along the embryo anterior-posterior axis. Dynamic model, constructed based on the proposed principle, with elements of fractional site occupancy, required 5–7 parameters to fit quantitative spatial expression data for gap gradients. The identified model solutions (parameter combinations) reproduced major dynamic features of the gap gradient system and explained gap expression in a variety of segmentation mutants.

Differential Regulation of the Catalytic and Accessory Subunit Genes of Drosophila Mitochondrial DNA Polymerase

The developmental pattern of expression of the genes encoding the catalytic (alpha) and accessory (beta) subunits of mitochondrial DNA polymerase (pol gamma) has been examined in Drosophila melanogaster. The steady-state level of pol gamma-beta mRNA increases during the first hours of development, reaching its maximum value at the start of mtDNA replication in Drosophila embryos. In contrast, the steady-state level of pol gamma-alpha mRNA decreases as development proceeds and is low in stages of active mtDNA replication. This difference in mRNA abundance results at least in part from differences in the rates of mRNA synthesis. The pol gamma genes are located in a compact cluster of five genes that contains three promoter regions (P1-P3). The P1 region directs divergent transcription of the pol gamma-beta gene and the adjacent rpII33 gene. P1 contains a DNA replication-related element (DRE) that is essential for pol gamma-beta promoter activity, but not for rpII33 promoter activity in Schneider's cells. A second divergent promoter region (P2) controls the expression of the orc5 and sop2 genes. The P2 region contains two DREs that are essential for orc5 promoter activity, but not for sop2 promoter activity. The expression of the pol gamma-alpha gene is directed by P3, a weak promoter that does not contain DREs. Electrophoretic mobility shift experiments demonstrate that the DRE-binding factor (DREF) regulatory protein binds to the DREs in P1 and P2. DREF regulates the expression of several genes encoding key factors involved in nuclear DNA replication. Its role in controlling the expression of the pol gamma-beta and orc5 genes establishes a common regulatory mechanism linking nuclear and mitochondrial DNA replication. Overall, our results suggest that the accessory subunit of mtDNA polymerase plays an important role in the control of mtDNA replication in Drosophila.

Structure and Regulated Expression of the δ-Aminolevulinate Synthase Gene from Drosophila melanogaster

The structure of the single copy gene encoding the putative housekeeping isoform of Drosophila melanogaster delta-aminolevulinate synthase (ALAS) has been determined. Southern and immunoblot analyses suggest that only the housekeeping isoform of the enzyme exists in Drosophila. We have localized a critical region for promoter activity to a sequence of 121 base pairs that contains a motif that is potentially recognized by factors of the nuclear respiratory factor-1 (NRF-1)/P3A2 family, flanked by two AP4 sites. Heme inhibits the expression of the gene by blocking the interaction of putative regulatory proteins to its 5' proximal region, a mechanism different from those proposed for other hemin-regulated promoters. Northern and in situ RNA hybridization experiments show that maternal alas mRNA is stored in the egg; its steady-state level decreases rapidly during the first hours of development and increases again after gastrulation in a period where the synthesis of several mRNAs encoding metabolic enzymes is activated. In the syncytial blastoderm, the alas mRNA is ubiquitously distributed and decreases in abundance substantially through cellular blastoderm. Late in embryonic development alas shows a specific pattern of expression, with an elevated mRNA level in oenocytes, suggesting an important role of these cells in the biosynthesis of hemoproteins in Drosophila.

Cloning and transcriptional regulation of the gene encoding the vacuolar/H + ATPase B subunit of Dictyostelium discoideum

The main function of vacuolar H + ATPases in eukaryotic cells is to generate proton and electrochemical gradients across the membrane of inner compartments. We have isolated the gene encoding the B subunit of Dictyostelium discoideum vacuolar H + ATPase ( vatB) and analyzed its transcriptional regulation. The deduced protein comprises 493 amino acids with a calculated molecular mass of 54 874 Da. The predicted protein sequence is highly homologous to previously determined V/H + ATPase B subunit sequences. The protein is encoded by a single gene in the Dictyostelium genome. The gene is maximally expressed during growth and it decreases during the first hours of development. Gene expression is rapidly enhanced by phagocytosis, but not by fluid-phase endocytosis. Acidic and alkaline conditions affect vatB gene expression differently.

Transcriptional regulation during the activation and development of Artemia franciscana encysted embryos.

Early embryos of the crustacean Artemia franciscana (brine shrimp) can get into cryptobiosis under adverse environmental conditions. Under these circumstances, the embryo arrests all metabolic activities, gets dehydrated and is surrounded by a hard shell. These cysts are viable for long periods and can be activated when environmental conditions are favorable. Once the cyst is activated, the embryo resumes development and gives rise in a few hours to a swimming nauplii that continues the development, through several molts, to the adult animal. The activation of the cyst can be reproduced in the laboratory which makes of the Artemia cyst an useful model system to study the mechanisms that regulate the exit of a latent state, as well as embryonic development Activation of the cyst requires resumption of all metabolic activities, including energetic metabolism, protein synthesis, gene transcription, etc. While the mechanisms involved in the activation of the metabolism and protein synthesis have been studied with some detaii in the last years (1), little is know about the process of transcriptional activation, The only well established data is the presence of active RNApolymerases in the cyst which implies that the cysts have the enzymatic activity required for transcription (2), These data suggest that the lack of transcriptional activity in the cyst must be regulated through the accessibility of the RNA polymerases to the gene promoters. Our laboratory is trying to test this hypothesis and to get more information on the mechanisms that regulate gene transcription during the activation of the cyst In the last years we have isolated cDNAand genomic clones coding for several genes whose expression is induced during the embryonic deveiopment that immediately follows cyst activation, Tohese genes encode the SarcolEndoplasmic reticulum CaATPase (3), the ex1 subunit of the Nail<ATPase (4) and three actin isoforms (5). The mRNAs encoded by all these genes, except for on actin isoform, are expressed in the cyst but their levels are markedly increased during the first hours ot embryonic development (6). The mRNA encoded by the Actin 302 gene is not expressed in the cyst and is induced after 10 hours of development (6), These studies show that gene transcription is activated in the first hours of development, after the activation of the cyst Once the expression pattern of these genes had been characterized, we have approached the study of the mechanism invoived in the regulation of their expression through the isolation and characterization of their promoter regions. The exon~ntron structure of these genes was estabiished and their transcription initiation sites determined (3-5). The sarco/endoplasmic reticulum CaATPase gene was exceptional since this gene can be transcribe from two different promoters to give two mRNAs of different size that code for two protein isoforms differingat their C-terminal amino acids and that are expressed in different tissues (7,8).

Laminin expression in the mouse lung increases with development and stimulates spontaneous organotypic rearrangement of mixed lung cells.

The recent establishment of a role for laminin in mouse lung organogenesis (Schuger et al. 1990a,b, 1991) prompted us to study its expression in the developing lung. Laminin A and B chains were detected in the murine lung from the first hours of development onward. In situ hybridization of mRNA as well as SDS-PAGE studies of lung cells in monoculture indicated that both epithelium and mesenchyme produce complete laminin molecules. Quantitative analysis of the in situ hybridization studies showed a gradual increase in laminin expression during development which was further supported by immunohistochemistry and ELISA. The overall pattern of expression suggested that the effects of laminin in morphogenesis were not restricted to a particular stage of development. Furthermore, the increase in expression during late development supported a role for the molecule in the fetal lung, which was not previously established. We next determined whether the increase in laminin production modulated the behavior of fetal lung cells as compared with their embryonic counterparts. We previously showed that organotypic pattern formation does not occur in cultures of mixed embryonic lung cells unless exogenous laminin is added (Schuger et al., 1990b). Organotypic pattern formation is the result of cell sorting into epithelial and mesenchymal compartments and further rearrangement in a pattern resembling the tissue of origin. In the present study, we demonstrated that organotypic pattern formation occurs spontaneously in cultures of mixed fetal lung cells, which express high laminin levels. Pattern formation was abolished by antibodies to laminin. These studies suggest a correlation between laminin expression and the ability of lung cells in culture to reproduce normal tissue patterns. We conclude that laminin is critical for epithelial-mesenchymal recognition and further morphogenic interaction during both the embryonic and fetal stages of lung development.

Triploidy induced by cold shock in the Asian catfish, Clarias batrachus (L.)

Artificially fertilized eggs of diploid females of Clarias batrachus were subjected to cold shock during the first hour of development. Eggs cold shocked at 5°C for 1.0 h, starting 5 min after fertilization, yielded 100% triploid fry as shown by chromosome counts. Hatching success of eggs cold-shocked for 1.0 h was not significantly different from the control but longer durations resulted in 100% mortality.

Cell differentiation in the absence of intracellular and extracellular cyclic AMP pulses in Dictyostelium discoideum

The mutant HSB1 was obtained by colony immunoblotting of mutagenized Dictyostelium discoideum cells with a monoclonal antibody against contact site A, a membrane glycoprotein. HSB1 cells undergo early development in the absence of intracellular and extracellular cAMP pulses. The receptor-mediated activation of adenylate cyclase is defective, but the basal activity increases normally during the first hours of development. Other preaggregative gene products, such as the inhibitor of phosphodiesterase, the contact site A glycoprotein, EDTA-stable contacts, and four developmentally regulated gene transcripts are also expressed in the mutant. Postaggregative gene transcripts fail to accumulate, even after pulsatile addition of cAMP. However, complete development of mutant cells is obtained by complementation with cells of the parent strain AX2.

Effects of 100-Hz magnetic fields with various waveforms on the development of chick embryos.

Chick embryos were exposed during their 52 first hours of development to 100-Hz magnetic fields. Sinusoidal, square and pulsed waveforms were used at average field strengths from 0.1 A/m to 80 A/m. After exposure, the embryos were examined for abnormalities and classified by the developmental stages. When bipolar oscillations (oscillating at both sides of the zero-level) were used, the percentage of abnormal embryos was significantly increased above 1 A/m. In exposure to unipolar square waves, no significant effect on the percentage of abnormalities could be demonstrated. The developmental stage was possibly affected by unipolar square waves at 0.1 A/m, all other field strengths and waveforms being apparently ineffective.

Detection and developmental regulation of the mRNA for the regulatory subunit of the cAMP-dependent protein kinase of D. discoideum by cell-free translation.

cAMP is an important effector of the development of Dictyostelium discoideum amoebae and could exert its effects on gene expression through the cytosolic cAMP-dependent protein kinase (cAK). Antibodies, specific for the regulatory subunit (R) of the cAK, were used to investigate the developmental regulation of the corresponding mRNA (R-mRNA) by in vitro translation and immunoprecipitation. Under such conditions, a single polypeptide of the same mol. wt. as R (42 kd) is detected, showing that the protein is not synthesized as a large precursor. The level of the R-mRNA, which is low in vegetative cells, increases 10- to 20-fold during the first hours of development. Its expression is stimulated by the treatment of AX3 cells with cAMP either added to a concentration of 1 mM or given as 0.1 microM pulses every 5 min, whereas such treatments have little or no effect in cells of strain AX2. The R-mRNA remains highly expressed (0.01-0.03% of translatable mRNA) throughout post-aggregative development; it is not affected by mechanical disaggregation of the multicellular organism. The parallel developmental time courses of the translatable R-mRNA and the R protein produced in vivo suggest that the expression of this polypeptide is regulated at the level of mRNA synthesis.

Separation of Dictyostelium discoideum cells into density classes throughout their development and their relationship to the later cell types

This paper describes a fast, non-destructive method for the separation of large quantities of Dictyostelium discoideum cells into density classes at all stages of development. The cells were separated by low-speed centrifugation on preformed, linear Percoll density gradients. On these gradients, cells at all developmental stages showed a unimodal variation in density and this variation in density rapidly increased during the first hours of development. The density was affected by the amount of salt present in the gradient medium, which suggests that it is regulated by a permeability property of the cells. Slug cells showed a unimodal variation in density and did not form bands corresponding to the cell types. However, were able to isolate density fractions which showed a good enrichment of prespore and prestalk cells: 95% and 90%, respectively. Preaggregation cells separated on density gradients yielded fractions which contained different amounts of three developmentally regulated enzymes. Hence, cells at this stage are already heterogeneous in their enzymatic content. Sorting experiments showed a strong correlation between density and developmental fate; the least dense (light) cells preferentially became prestalk cells, and the dense (heavy) cells became prespore cells. This was found for cells at all developmental stages; even vegetative-stage cells showed considerable heterogeneity with regard to density, which was related to their developmental fate. The light cells become prestalk cells, and the heavy cells become prespore cells. Vegetative cells from the various density fractions differed in their DNA content and temporal onset of mitotic activity when resuspended in medium. Therefore, we suggest that the separation of vegetative cells on density gradients results in a separation of cells into cell-cycle phases. Hence, there appear to be cell-cycle-linked differences among vegetative cells, which bias their differentiation towards either the spore or stalk pathway.

Thermal induction of diploid gynogenesis and triploidy in the eggs of the rainbow trout (Salmo gairdneri Richardson).

Sporadic diploid gynogenetic fry resulted from fertilization with gamma-irradiated sperm. Their frequency increased appreciably when thermal shock treatments, beginning during the first hour of development (--0.4 degrees C lasting 6 hrs 45 min; 26-30 degrees C lasting 10 min), were used. When the eggs were fertilized with normal sperm, a good triploidization rate was induced by means of heat shocks (27-30 degrees C lasting 10 min). It is supposed that the retention of the second polar body caused the gynogenetic diploidy observed.

Induction of triploidy in the newt Pleurodeles waltlii by heat shock or hydrostatic pressure: interpretation of the different types of ploidy using a chromosomal marker.

Eggs of diploid females of Pleurodeles waltii, fertilized artificially by waltlii sperm, have been submitted to increased temperature of hydrostatic pressure during the first hour of development. Of the resulting viable individuals about 95% were triploid, the remainder diploid or tetraploid. Other types of ploidy have been observed in abnormal embryos (n, 2n/4n, 5n, n/5n, 6n). Some of the treated eggs came from females with a marker chromosome (pericentric inversion). The karyotypes of the animals developing from such eggs confirm that triploidy results from retention of the second polar body and make it possible to interpret the origin of the other types of ploidy. From the results it can be envisaged how gynogenetic animals can be obtained from eggs inseminated by inactivated sperm and shock-treated to restore diploidy.