Batches Of Embryos Research Articles

Design of an automated robotic microinjection system for batch injection of zebrafish embryos and larvae

This paper presents the design of a vision-based automated robotic microinjection system for batch injection of both zebrafish embryos and larvae. A novel visual recognition algorithm based on an automatic threshold and excessive dilatation is introduced to accurately identify the center of zebrafish embryos and larval yolks. A corresponding software system is developed using the producer-consumer model as the framework structure, and a friendly user interface is designed to allow operators to choose from a range of desired functions according to their different needs. In addition, a novel microstructural agarose device is designed and fabricated to simultaneously immobilize mixed batches of embryos and larvae. Moreover, a prototype microinjection system is fabricated by integrating hardware devices with visual algorithms. An experimental study is conducted to verify the performance of the robotic microinjection system. The results show that the reported system can accurately identify zebrafish embryos and larvae and efficiently complete batch microinjection tasks of the mixtures with an injection success rate of 92.05% in 13.88 s per sample. Compared with manual and existing microinjection systems, the proposed system demonstrates the merits of versatility, excellent efficiency, high success rate, high survival rate, and sufficient stability.

Maternal Effects on Embryonic Development and Survival in Walleyes of Lake Nipissing, Ontario

AbstractLife history theory predicts that females may increase reproductive allocation with advancing age as the probability for future reproduction diminishes. In iteroparous fishes, increasing age is usually accompanied by increasing fecundity, but evidence of increasing offspring quality is less consistent. We examined the developmental rate and survival of Walleye Sander vitreus embryos with respect to female age, size, condition, and various ova traits in an exploited stock dominated by young spawners. Embryo batches from individual females were collected in the field on multiple dates and reared to hatch under controlled conditions in both flow‐through and static incubation systems. Survival and thermal inputs to hatch (TU50) were analyzed with respect to spawn date, incubation method, and both maternal and ova traits. Spawning date had a relatively strong effect on embryo survival but only a minor effect on TU50, whereas incubation method had a small effect on survival but a stronger effect on TU50. Embryos reared in static incubators required greater thermal inputs to reach hatch. Using a model selection approach to assess the effects of maternal and ova traits, we found that neither maternal age nor size were strong predictors of survival or TU50. Instead, survival was more strongly related to egg size and fatty acid composition, and TU50 was not strongly related to any maternal or ova trait. Our results suggest that the nature and magnitude of maternal effects on early development and survival may vary among Walleye stocks, modified by physical conditions during incubation.

Temperature–Induced Hatch Failure and Nauplii Malformation in Antarctic Krill

Antarctic krill inhabit areas of the Southern Ocean that can exceed 4.0°C, yet they preferentially inhabit regions with temperatures of −1.5 to ≤1.5°C. Successful embryonic development and hatching are key to their life cycle, but despite the rapid climatic warming seen across their main spawning areas, the effects of elevated temperatures on embryogenesis, hatching success, and nauplii malformations are unknown. We incubated 24,483 krill embryos in two independent experiments to investigate the hypothesis that temperatures exceeding 1.5°C have a negative impact on hatching success and increase the numbers of malformed nauplii. Field experiments were on krill collected from near the northern, warm limit of their range and embryos incubated soon after capture, while laboratory experiments were on embryos from krill acclimated to laboratory conditions. The hatching success of embryo batches varied enormously, from 0 to 98% (mean 27%). Both field and laboratory experiments showed that hatching success decreased markedly above 3.0°C. Our field experiments also showed an approximate doubling of the percentage of malformed nauplii at elevated temperatures, reaching 50% at 5.0°C. At 3.0°C or below, however, temperature was not the main factor driving the large variation in embryo hatching success. Our observations of highly variable and often low success of hatching to healthy nauplii suggest that indices of reproductive potential of female krill relate poorly to the subsequent production of viable krill larvae and may help to explain spatial discrepancies between the distribution of the spawning stock and larval distribution.

Retinoic acid signaling reduction recapitulates the effects of alcohol on embryo size.

Intrauterine growth restriction (IUGR) is commonly observed in human pregnancies and can result in severe clinical outcomes. IUGR is observed in Fetal Alcohol Syndrome (FAS) fetuses as a result of alcohol (ethanol) exposure during pregnancy. To further understand FAS, the severe form of Fetal Alcohol Spectrum Disorder, we performed an extensive quantitative analysis of the effects of ethanol on embryo size utilizing our Xenopus model. Ethanol-treated embryos exhibited size reduction along the anterior-posterior axis. This effect was evident primarily from the hindbrain caudally, while rostral regions appeared refractive to ethanol-induced size changes, also known as asymmetric IUGR. Interestingly, some embryo batches in addition to shortening from the hindbrain caudally also exhibited an alcohol-dependent reduction of the anterior head domain, known as symmetric IUGR. To study the connection between ethanol exposure and reduced retinoic acid levels we treated embryos with the retinaldehyde dehydrogenase inhibitors, DEAB and citral. Inhibition of retinoic acid biosynthesis recapitulated the growth defects induced by ethanol affecting mainly axial elongation from the hindbrain caudally. To study the competition between ethanol clearance and retinoic acid biosynthesis we demonstrated that, co-exposure to alcohol reduces the teratogenic effects of treatment with retinol (vitamin A), the retinoic acid precursor. These results further support the role of retinoic acid in the regulation of axial elongation.

Risk of Chlamydia abortus transmission via embryo transfer using in vitro produced early bovine embryos

The objectives of this study were to determine (i) whether Chlamydia (C.) abortus would adhere to the intact zona pellucida (ZP-intact) of early in vitro produced bovine embryos; (ii) whether the bacteria would adhere to the embryos (ZP-free) after in vitro infection; and (iii) the efficacy of the International Embryo Transfer Society (IETS) washing protocol.The experimentation was made twice. For each replicate 100 (8–16-cell) bovine embryos produced in vitro were randomly divided into 10 batches. Height batches (4 ZP-intact and 4 ZP-free) of 10 embryos were incubated in a medium containing 4 × 107Chlamydia/ml of AB7 strain. After incubation for 18 h at 37 °C in an atmosphere of 5% CO2, the embryos were washed in accordance with the IETS guidelines. In parallel, two batches (1 ZP-intact and 1 ZP-free) of 10 embryos were subjected to similar procedures but without exposure to C. abortus as a control group. The 10 washing fluids from each batch were collected and centrifuged for 1 h at 13,000×g. Each batch of washed embryos and each wash pellets were tested using PCR.C. abortus DNA was found in all ZP-intact and ZP-free batches of 10 embryos after 10 successive washes. For ZP-intact infected embryos, Chlamydia-DNA was also detected in all 10 wash baths for two batches (2/8) of embryos, whereas for ZP-free infected embryos, Chlamydia-DNA was detected in all 10 wash baths for 6/8 batches of embryos. In contrast, none of the embryos or their washing fluids in the control batches was DNA positive. The bacterial load for batches of 10 embryos after the 10 wash baths was significantly higher for batches of ZP-free embryos (20.7 ± 9 × 103 bacteria/mL) than for batches of ZP-intact embryos (0.47 ± 0.19 × 103 bacteria/mL).These results demonstrate that C. abortus adheres to the ZP as well as the early embryonic cells of in vitro produced bovine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS fails to remove it.

RHOA activity in expanding blastocysts is essential to regulate HIPPO-YAP signaling and to maintain the trophectoderm-specific gene expression program in a ROCK/actin filament-independent manner.

What molecular signals are required to maintain the functional trophectoderm (TE) during blastocyst expansion of the late stage of preimplantation development? The activity of ras homology family member A (RHOA) GTPases is necessary to retain the expanded blastocyst cavity and also to sustain the gene expression program specific to TE. At the early stages of preimplantation development, the precursor of the TE lineage is generated through the molecular signals that integrate RHOA, RHO-associated coiled-coil containing protein kinase (ROCK), the apicobasal cell polarity, and the HIPPO-Yes-associated protein (YAP) signaling pathway. By contrast, molecular mechanisms regulating the maintenance of the TE characteristics at the later stage, which is crucial for blastocyst hatching and implantation, are scarcely understood. Expanding mouse blastocysts, obtained from crosses of the F1 (C57BL6 × DBA/2) strain, were exposed to chemical agents that interfere with RHOA, ROCK, or the actin cytoskeleton for up to 8 h, and effects on the blastocyst cavity, HIPPO-YAP signaling, and cell lineage-specific gene expression profiles were examined. Mouse embryos at the embryonic stage E3.5 (expanding blastocysts) and E4.5 (fully expanded blastocysts) were treated with RHOA inhibitor (C3 exoenzyme), ROCK inhibitor (Y27632), or actin filament disruptors (cytochalasin B and latrunculin A). The integrity of the blastocyst cavity was evaluated based on the gross morphology. Effects on HIPPO-YAP signaling were assessed based on the presence of nuclearized YAP protein by immunofluorescence staining and the expression of YAP/TEA domain family member (TEAD) target genes by quantitative RT-PCR (qRT-PCR). The impact of these disruptors on cell lineages was evaluated based on expression of the TE-specific and inner cell mass-specific marker genes by qRT-PCR. The integrity of the apicobasal cell polarity was assessed by localization of protein kinase C zeta (PRKCZ; apical) and scribbled planar cell polarity (SCRIB; basal) proteins by immunofluorescence staining. For comparisons, cultured cell lines, NIH/3T3 (mouse fibroblast) and P19C5 (mouse embryonal carcinoma), were also treated with RHOA inhibitor, ROCK inhibitor, and actin filament disruptors for up to 8 h, and effects on HIPPO-YAP signaling were assessed based on expression of YAP/TEAD target genes by qRT-PCR. Each experiment was repeated using three independent batches of embryos (n = 40-80 per batch) or cell collections. Statistical analyses of data were performed, using one-way ANOVA and two-sample t-test. Inhibition of RHOA deflated the cavity, diminished nuclear YAP (P < 0.01), and down-regulated the YAP/TEAD target and TE-specific marker genes in both E3.5 and E4.5 blastocysts (P < 0.05), indicating that the maintenance of the key TE characteristics is dependent on RHOA activity. However, inhibition of ROCK or disruption of actin filament only deflated the blastocyst cavity, but did not alter HIPPO-YAP signaling or lineage-specific gene expressions, suggesting that the action of RHOA to sustain the TE-specific gene expression program is not mediated by ROCK or the actomyosin cytoskeleton. By contrast, ROCK inhibitor and actin filament disruptors diminished YAP/TEAD target gene expressions in cultured cells to a greater extent than RHOA inhibitor, implicating that the regulation of HIPPO-YAP signaling in expanding blastocysts is distinctly different from that in the cell lines. Furthermore, the apicobasal cell polarity proteins in the expanding blastocyst were mislocalized by ROCK inhibition but not by RHOA inhibition, indicating that cell polarity is not linked to regulation of HIPPO-YAP signaling. Taken together, our study suggests that RHOA activity is essential to maintain the TE lineage in the expanding blastocyst and it regulates HIPPO-YAP signaling and the lineage-specific gene expression program through mechanisms that are independent of ROCK or actomyosin cytoskeleton. Not applicable. This study was conducted using one species, the mouse. Direct translation of the experiments and findings to human fertility preservation and ART requires further investigations. The elucidation of the mechanisms of TE formation is highly pertinent to fertility preservation in women. Our findings may raise awareness among providers of ART that the TE is sensitive to disturbance even in the late stage of blastocyst expansion and that rational approaches should be devised to avoid conditions that may impair the TE and its function. This study was funded by grants from the Ingeborg v.F. McKee Fund of the Hawaii Community Foundation (16ADVC-78882 to V.B.A.), and the National Institutes of Health (P20 GM103457 and R03 HD088839 to V.B.A.). The authors have no conflict of interest to declare.

Effect of pesticides mixture of dimethoate and methidathion on acetylcholinestrase during embryo development using chick embryo model

Pesticides mixture (dimethoate 30% and methidathion 40%) prepared by the farmers in Yemen and engaged in the cultivation of Qat, which is chewed by people every day including pregnant women. Therefore, the developing embryos in the society are more vulnerable than adults to the chronic cholinergic intoxication. This study aimed to examine the chronic effect of pesticides mixture on the AChE of developmental embryo in an avian model, which does not share the maternal potential confounds. For this, fresh fertile chicken eggs (Gallus gallus domesticus) were used. The lethal concentration of pesticides mixture for 50% killing (LD50) values was computed on the basis of probit analysis and was found to be 40 ppm. 1/5 th LD50 and 1/10 th LD50 (8 and 4 ppm) were chosen to be the tested doses. Eggs weighing 54±1 gm were separated in to 3 batches of 10 eggs each batch. One batch of embryos was injected with normal saline and the other batches of embryos injected with pesticides mixture of 4 and 8 ppm each alternative day starting from incubation day 7 for 2 weeks. On day 21 after 12 hours of the last dose an amount of 200 µl blood was collected from the blood vessels surrounding the embryonic membranes and the heamolyzate was used for the assessment of the AChE activity calorimetrically. Result of this study indicated that 1/10 th of the LD50 had only marginal effect on the AChE activity (40.6%). Whereas 1/5 th of the LD50 of pesticides mixture caused significant inhibition of AChE activity (69%) which could not be reversible. So neuro-developmental consequences such as behavioral changes and memory impairment may prolong throughout the life span of the embryo.

90 Risk of Coxiella burnetii Transmission via Embryo Transfer Using In Vitro Early Caprine Embryos

Previous experiments using in vitro infection have shown that Coxiella burnetii has a strong tendency to adhere to the zona pellucida (ZP) of in vivo-derived goat embryos, and the washing procedure recommended by the International Embryo Technology Society (IETS) for bovine embryos failed to remove it (Alsaleh et al. 2013 Theriogenology 80, 571-575). The aim of this study was, for in vitro-produced caprine embryos infected in vitro, to (1) evaluate the ability of C. burnetii to adhere to intact ZP, (2) test the efficacy of IETS recommended rules for the washing of bovine embryos to eliminate C. burnetii, and (3) determine, by confocal microscopy, the bacteria location. One hundred ZP-intact caprine embryos, produced in vitro, at the 8- to 16-cell stage were randomly allocated into 11 batches of 8 to 9 embryos. Nine batches were incubated for 18 h with 109 Coxiella/mL of CbB1 strain (ISP, INRA, Val de Loire, France). The embryos were then recovered and washed in batches in 10 successive baths following the IETS guidelines. In parallel, 2 batches of embryos were subjected to similar procedures but without exposure to C. burnetii to serve as the control group. One of the 9 batches of infected embryos and 1 of the 2 non-infected control batches were used to perform immunolabelling to localise the bacteria. Coxiella burnetii DNA was detected by conventional PCR in all 8 batches of infected embryos after 10 successive washings. However, bacterial DNA was not detected in the embryos of the control group. The first 5 washing media of the infected groups were consistently positive and Coxiella DNA was detected up to the tenth wash in 2 batches. After immunolabelling, the observation of embryos under confocal microscopy allowed us to localise C. burnetti on the external part of the ZP without deep penetration. The presence of C. burnetii was seen on the surface of the ZP, with bacterial loads differing from one embryo to another in the same batch. This study clearly demonstrates that C. burnetii, after in vitro infection at 109 Coxiella mL−1, stick strongly to the external part of the ZP of in vitro-produced early caprine embryos without profound penetration. The 10-washings protocol recommended by IETS to eliminate the pathogenic agents of bovine embryos is unable to eliminate these bacteria in caprine embryos. Nevertheless, the finding of C. burnetii DNA by conventional PCR does not imply that the bacteria found remain infective. Further studies are required to investigate whether enzymatic or antibiotic treatment of caprine embryos infected by C. burnetii would eliminate or inactivate the bacteria from the ZP of in vitro-produced goat embryos.

Attachment of Coxiella burnetii to the zona pellucida of in vitro produced goat embryos

Previous work demonstrated that after infection of in vivo derived caprine embryos, Coxiella burnetti (C. burnetii) showed a strong tendency to adhere to the zona pellicida (ZP). To investigate the risk of C. burnetii transmission via embryo transfer of in vitro-produced goat embryos the aim of this study was, (i) to evaluate the ability of C. burnetii to adhere to the intact zona pellicida of in vitro-produced goat embryos and to determine by confocal microscopy the location of the bacteria, (ii) to test the efficacy of IETS recommended rules for the washing of bovine embryos to eliminate C. burnetii. One hundred ZP-intact caprine embryos, produced in vitro, at the 8 to 16 cell stage, were randomly divided into 11 batches of eight to nine embryos. Nine batches were incubated for 18 h with 109Coxiella/ml of CbB1 strain (IASP, INRA Tours). The embryos then were recovered and washed in batches in 10 successive baths following the IETS guidelines. In parallel, two batches of embryos were subjected to similar procedures but without exposure to C. burnetii, to serve as the control group. One of the nine batches of infected embryos and one of the two non-infected control batches were separated to perform immunolabeling to locate the bacteria.C. burnetii DNA was detected by C-PCR in all eight batches of infected embryos after 10 successive washings. However, bacterial DNA was not detected in the embryo control batch. The first five washing media of the infected group were consistently found to be positive and Coxiella DNA was detected in the wash bath up to the 10th wash for two batches.After immunolabeling, the observation of embryos under confocal microscopy allowed C. burnetti to be found on the external part of the zona pellucida without deep penetration.This study clearly demonstrates that C. burnetii, after in vitro infection at 109Coxiella/ml, stick strongly to the external part of the zona pellucida of in vitro produced caprine embryos without deap penetration and that the 10 washings protocol recommended by IETS to eliminate the pathogenic agents of bovine embryos is unable to eliminate these bacteria from in vitro-produced goat embryo.

108 IS THE BINDING OF COXIELLA BURNETII TO THE ZONA PELLUCIDAE FOLLOWING IN VITRO INFECTION OF IN VITRO-PRODUCED GOAT EMBRYOS CONCENTRATION DEPENDENT?

Previous experiments using in vitro infection have shown that at concentrations of 109 bacteria/mL, Coxiella burnetii strongly adheres to the zona pellucidae (ZP) of caprine embryos produced in vitro or in vivo (Alsaleh et al., 2013). However, spontaneous infection results in bacterial concentrations of between 106 and 107 bacteria/mL (Rodolakis, 2006; Alsaleh et al., 2011). The aim of this study was to determine whether the concentration of Coxiella burnetii affected its ability to bind to the ZP in vitro. A total of 120 ZP-intact 8- to 16-cell embryos, produced in vitro from ovaries collected at slaughter, were infected with Coxiella burnetti (strain CbC1) produced via ovoculture at 109 mL−1 (3 batches of 10 embryos), 107/mL (5 batches of 10 embryos), 105 mL−1 (3 batches of 8 embryos). After overnight incubation at 37°C in 5% CO2, the embryos were recovered and washed in batches, in 10 successive baths of PBS with 5% FCS, in accordance with International Embryo Technology Society guidelines. The 10 wash baths were collected separately and centrifuged for 1 h at 13,000 × g. The presence of C. burnetii was determined by conventional PCR in each batch of embryos and in the pellets of the 10 wash baths (Table 1). As demonstrated previously, Coxiella DNA was detected in embryo batches after 10 washes when a concentration of 109 bacteria/mL was used for in vitro infection, but this binding property did not occur at lower bacterial concentrations. We can conclude that the attachment of Coxiella burnetii to the zona pellucida of in vitro-produced embryos is concentration dependent. This finding illustrates the limitations of in vitro experiments to study the risk of pathogen transmission via embryo transfer. Table 1. Detection of Coxiella burnetii (CB) in successive embryo washing baths and batches of 8 to 10 infected ZP-intact 8- to 16-cell embryos after 10 wash cycles, using conventional PCR (C-PCR), as a function of the concentration of CB used for in vitro infection and determined by quantitative PCR (Q-PCR)

Can Chlamydia abortus be transmitted by embryo transfer in goats?

The objectives of this study were to determine (i) whether Chlamydia abortus would adhere to or penetrate the intact zona pellucida (ZP-intact) of early in vivo-derived caprine embryos, after in vitro infection; and (ii) the efficacy of the International Embryo Transfer Society (IETS) washing protocol for bovine embryos. Fifty-two ZP-intact embryos (8–16 cells), obtained from 14 donors were used in this experiment. The embryos were randomly divided into 12 batches. Nine batches (ZP-intact) of five embryos were incubated in a medium containing 4 × 107Chlamydia/mL of AB7 strain. After incubation for 18 hours at 37 °C in an atmosphere of 5% CO2, the embryos were washed in batches in 10 successive baths of a phosphate buffer saline and 5% fetal calf serum solution in accordance with IETS guidelines. In parallel, three batches of ZP-intact embryos were used as controls by being subjected to similar procedures but without exposure to C. abortus. The 10 wash baths were collected separately and centrifuged for 1 hour at 13,000 × g. The washed embryos and the pellets of the 10 centrifuged wash baths were frozen at −20 °C before examination for evidence of C. abortus using polymerase chain reaction. C. abortus DNA was found in all of the infected batches of ZP-intact embryos (9/9) after 10 successive washes. It was also detected in the 10th wash fluid for seven batches of embryos, whereas for the two other batches, the last positive wash bath was the eighth and the ninth, respectively. In contrast, none of the embryos or their washing fluids in the control batches were DNA positive. These results report that C. abortus adheres to and/or penetrates the ZP of in vivo caprine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS for bovine embryos, failed to remove it. The persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from infected donor goats to healthy recipients and/or their offspring. Nevertheless, the detection of C. abortus DNA by polymerase chain reaction does not prove that the bacteria found was infectious. Further studies are required to investigate whether enzymatic and/or antibiotic treatment of caprine embryos infected by C. abortus would eliminate the bacteria from the ZP.

In vivo DNA mismatch repair measurement in zebrafish embryos and its use in screening of environmental carcinogens

Impairment of DNA mismatch repair (MMR) function leads to the development and progression of certain cancers. Many environmental contaminants can target DNA MMR system. Currently, measurement of MMR activity is limited to in vitro or in vivo methods at the cell line level, and reports on measurement of MMR activity at the live organism level are lacking. Here, we report an efficient method to measure DNA MMR activity in zebrafish embryos. A G-T mismatch was introduced into enhanced green fluorescent protein (EGFP) gene. Repair of the G-T mismatch to G-C in the heteroduplex plasmid generates a functional EGFP expression. The heteroduplex plasmid and a similarly constructed homoduplex plasmid were injected in parallel into the same batch of embryos at 1-cell stage and EGFP expression in EGFP positive embryos was quantified at 24h after injection. MMR efficiency was calculated as the total fluorescence intensity of embryos injected with the heteroduplex construct divided by that of embryos injected with the homoduplex construct. Our results showed 73% reduction of MMR activity in embryos derived from MMR-deficient mlh1 mutant fish (positive control) when compared with embryos from MMR-competent wild type AB line fish, indicating feasibility of in vivo MMR activity measurement in zebrafish embryos. We further applied this novel assay for measurement of MMR efficiency in embryos exposed to environmental chemicals such as cadmium chloride (CdCl2), benzo[a]pyrene (BaP), and perfluorooctanesulphonic acid (PFOS) from 6hpf to 24hpf. We observed significant reductions of MMR efficiency in embryos exposed to 0.1μM CdCl2 (52%) and 0.5μM BaP (34%), but no effect in embryos exposed to PFOS. Our study for the first time provides a model system for in vivo measurement of DNA MMR activity at the organism level, which has important implications in risk assessment of various environmental carcinogens.

101 RISK OF COXIELLA BURNETII TRANSMISSION BY EMBRYO TRANSFER USING IN VITRO EARLY BOVINE EMBRYOS

Coxiella burnetii, an obligate intracellular bacterium of worldwide distribution, is responsible for Q fever. Domestic ruminants are the main sources of infection for humans. In cattle, infection is frequently asymptomatic, but it may cause abortion, reproductive failure (metritis, placentitis, and infertility), and economic losses. A previous study in goats showed that Coxiella burnetii had a strong tendency to cling to the zona pellucida (ZP) after in vitro infection and the washing procedure recommended by IETS for bovine embryos failed to remove it (Alsaleh et al. 2013 Theriogenology). The aims of this study were to determine (1) whether Coxiella burnetii would adhere to the intact ZP (ZP-intact) of early in vitro-produced bovine embryos, (2) whether the bacteria would adhere to or infect the embryo cells (ZP-free) after in vitro infection, and (3) the efficiency of the washing protocol recommended by the IETS. One hundred and sixty 8- to 16-cell bovine embryos produced in vitro were randomly divided into 16 batches of 10 embryos each. Twelve batches (8 ZP-intact and 4 ZP-free) were incubated in medium containing C. burnetii CbB1 (IASP, INRA Tours, France). After 18 h of incubation at 37°C and 5% CO2 in air, the embryos were washed in 10 successive baths of a phosphate buffer saline (PBS) and 5% FCS solution in accordance with the IETS guidelines. In parallel, 4 batches (2 ZP-intact and 2 ZP-free) were subjected to similar procedures but without exposure to C. burnetii to act as controls. The 10 washing fluids for all batches were collected and centrifuged for 1 h at 13 000 × g. Embryo and pellet washing were tested by C-PCR. Coxiella burnetii DNA was found in all ZP-intact and ZP-free embryo batches after 10 successive washes. It was also detected in the first 4 washing fluids for ZP-intact embryos and in the 10th washing fluid for 2 of the 4 batches of ZP-free embryos. In contrast, none of the embryos or their washing fluids in the control batches were DNA positive. These results demonstrate that C. burnetii adhere and (or) penetrate the early embryonic cells as well as the ZP of in vitro bovine embryos after in vitro infection and the standard washing protocol recommended by the IETS for bovine embryos failed to remove it. The persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from infected donor cows to healthy recipients or their offspring, or both. Further studies are needed to investigate whether enzymatic or antibiotic treatment of bovine embryos infected by C. burnetii would eliminate the bacteria from the ZP.

Genetic Origins of Birth Defects Revealed by New Animal Model

​CorneliaCornelia de Lange syndrome is a rare congenital disorder marked by malformations of many different body parts, including the face, head, heart, and intestines. Most cases are caused by mutations that reduce levels of the Nipped-B-like (NIPBL) protein, whose best known role is to help ensure that chromosomes separate properly during cell division through its interactions with a protein complex called cohesin. Even a slight decrease in the amount of NIPBL can cause deformities in humans, and an ongoing puzzle in this and other diseases is how such a subtle change in the levels of one protein can give rise to devastating effects. Gut bifurcation, with mirror-image duplication of liver and pancreas, are among the phenotypes of nipbl-deficient fish embryos reproduced by combined knockdown of two Nipbl-downstream genes, sox17 and foxa2. Given that Nipbl regulates the expression of hundreds of genes, a minor deficiency in this protein could result in many small changes that interact to produce severe and widespread defects. Zebrafish (Danio rerio) is a convenient model organism for testing this possibility because its transparent body during early development allows scientists to examine its internal anatomy. As reported in this issue of PLoS Biology, a team led by Arthur Lander and Thomas Schilling, developmental biologists at the University of California at Irvine, has established zebrafish as an animal model of Cornelia de Lange syndrome. The researchers found that a shortage of Nipbl in zebrafish embryos leads to modest declines in the expression of genes involved in organ development, resulting in malformations that characterize the disorder. This finding provides an alternative to single-gene models commonly used to explain a variety of birth defects. The researchers first searched the zebrafish genome database and identified two genes that code for proteins similar to human NIPBL. They then blocked the production of these proteins, Nipbla and Nipblb, in zebrafish embryos shortly after fertilization. The loss of both proteins led to various heart and gut abnormalities about one to two days later. The heart and the gut did not form normal loops in most embryos, and the co-occurrence of these defects suggests that they originated from shared deficiencies in the arrangement of organ parts. In support of this idea, several genes involved in creating asymmetric patterns in organs were found to be expressed at low levels and on the wrong side of the body. In addition, many other embryos lacked both an intact heart tube and a complete gut tube. Before these anomalies appeared, Nipbl depletion reduced the expression of genes required for the formation of the endoderm, a cell layer that gives rise to the gastrointestinal tract. These genes included sox17, foxa2, sox32, and gata5. When the researchers partially restored the expression of either gata5 or sox32, the heart tube fused correctly and the entire gut tube formed, indicating that these abnormalities arose from impairments in endoderm development. Some cases of Cornelia de Lange syndrome are caused by mutations in genes that encode cohesin subunits. Because a loss of Nipbl could also interfere with cohesin activity, the researchers tested whether impaired cohesin function could account for the anomalies seen in Nipbl-depleted embryos. When they lowered the expression of cohesin subunit genes in a new batch of embryos, the expression of sox32, sox17, and foxa2 did not change. This result suggests that Nipbl and cohesin regulate distinct sets of genes, and that the disorder is not caused simply by a decrease in cohesin activity. Taken together, the findings reveal that Nipbl controls a network of genes that contribute to endoderm development and organ asymmetry, and to deformities resembling those of Cornelia de Lange syndrome. Because the gene interactions seen in this condition share features with those presumed to underlie many different genetic disorders, this research may provide new insights into a range of human birth defects. Muto A, Calof AL, Lander AD, Schilling TF (2011) Multifactorial Origins of Heart and Gut Defects in nipbl-Deficient Zebrafish, a Model of Cornelia de Lange Syndrome. doi:10.1371/journal.pbio.1001181

Exposure to ultraviolet radiation causes proteomic changes in embryos of the purple sea urchin, Strongylocentrotus purpuratus

We performed experiments to determine how environmentally relevant ultraviolet radiation (UVR) affects protein expression during early development in the sea urchin, Strongylocentrotus purpuratus. To model the protein-mediated cell cycle response to UV-irradiation, six batches of embryos were exposed to UVR, monitored for both delays in the first mitotic division and changes in the proteome at two specific developmental time points. Embryos were exposed to or protected from artificial UVR (11.5 W/m 2) for 25 or 60 min. These levels of UVR are within the range we have measured in coastal waters between 0.5 and 2 m. Embryos treated with UVR for 60 min cleaved an average of 23.2 min (± 1.92 s.e.m.) after UV-protected embryos. Differential protein spot migration between UV-protected and UV-treated embryos was examined at 30 and 90 min post-fertilization using two-dimensional SDS-PAGE (2D GE). A total of 1306 protein spots were detected in all gels, including differences in 171 protein spots (13% of the detected proteome) in UV-treated embryos at 30 min post-fertilization and 187 spots (14%) at 90 min post-fertilization (2-way ANOVA, P = 0.03, n = 6). The majority of the proteins affected by UVR were subsequently identified using matrix assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI-TOF–TOF MS). Our results indicate UVR affects proteins from multiple cellular pathways and indicate that the mechanisms involved in UV-stress and UV-induced developmental delay in sea urchin embryos are integrated among multiple pathways for cellular stress, protein turnover and translation, signal transduction, cytoskeletal dynamics, and general metabolism.

Female reproductive biology of the bluemouth Helicolenus dactylopterus dactylopterus: spawning and fecundity

The bluemouth Helicolenus dactylopterus dactylopterus is a zygoparous species that spawns multiple batches of embryos enclosed within a gelatinous matrix. Oocyte development is asynchronous, and the recruitment of secondary growth oocytes occurs continuously during the developing phase, but stops before the start of the first spawning (i.e. fecundity is determinate). The number of developing oocytes can be estimated as a function of the total length of the fish, its ovary mass and its gonado-somatic index. Only at the onset of spawning, when potential fecundity is determined, does condition also have a significant effect. The low levels of atresia detected during most of the spawning season show that this mechanism does not substantially affect the process. There is variability both in the spawning interval (with a mean of 2 days) and in the number of embryos comprising every single batch (up to 37,000). Expected effect of fisheries on the reproductive traits of this deep-sea species is also discussed.

Fertilization capacity with rainbow trout DNA-damaged sperm and embryo developmental success

Mammalian spermatozoa undergo a strong selection process along the female tract to guarantee fertilization by good quality cells, but risks of fertilization with DNA-damaged spermatozoa have been reported. In contrast, most external fertilizers such as fish seem to have weaker selection procedures. This fact, together with their high prolificacy and external embryo development, indicates that fish could be useful for the study of the effects of sperm DNA damage on embryo development. We cryopreserved sperm from rainbow trout using egg yolk and low-density lipoprotein as additives to promote different rates of DNA damage. DNA fragmentation and oxidization were analyzed using comet assay with and without digestion with restriction enzymes, and fertilization trials were performed. Some embryo batches were treated with 3-aminobenzamide (3AB) to inhibit DNA repair by the poly (ADP-ribose) polymerase, which is an enzyme of the base excision repair pathway. Results showed that all the spermatozoa cryopreserved with egg yolk carried more than 10% fragmented DNA, maintaining fertilization rates of 61.1+/-2.3 but a high rate of abortions, especially during gastrulation, and only 14.5+/-4.4 hatching success. Furthermore, after 3AB treatment, hatching dropped to 3.2+/-2.2, showing that at least 10% DNA fragmentation was repaired. We conclude that trout sperm maintains its ability to fertilize in spite of having DNA damage, but that embryo survival is affected. Damage is partially repaired by the oocyte during the first cleavage. Important advantages of using rainbow trout for the study of processes related to DNA damage and repair during development have been reported.

Bayesian classification for the selection of in vitro human embryos using morphological and clinical data

In vitro fertilization (IVF) is a medically assisted reproduction technique that enables infertile couples to achieve successful pregnancy. Given the uncertainty of the treatment, we propose an intelligent decision support system based on supervised classification by Bayesian classifiers to aid to the selection of the most promising embryos that will form the batch to be transferred to the woman’s uterus. The aim of the supervised classification system is to improve overall success rate of each IVF treatment in which a batch of embryos is transferred each time, where the success is achieved when implantation (i.e. pregnancy) is obtained. Due to ethical reasons, different legislative restrictions apply in every country on this technique. In Spain, legislation allows a maximum of three embryos to form each transfer batch. As a result, clinicians prefer to select the embryos by non-invasive embryo examination based on simple methods and observation focused on morphology and dynamics of embryo development after fertilization. This paper proposes the application of Bayesian classifiers to this embryo selection problem in order to provide a decision support system that allows a more accurate selection than with the actual procedures which fully rely on the expertise and experience of embryologists. For this, we propose to take into consideration a reduced subset of feature variables related to embryo morphology and clinical data of patients, and from this data to induce Bayesian classification models. Results obtained applying a filter technique to choose the subset of variables, and the performance of Bayesian classifiers using them, are presented.

Soluble tubulin complexes, γ-tubulin, and their changing distribution in the zebrafish ( Danio rerio) ovary, oocyte and embryo

Tubulin dynamics, i.e., the interchange of polymeric and soluble forms, is important for microtubule (MTs) cellular functions, and thus plays essential roles in zebrafish oogenesis and embryogenesis. A novel finding in this study revealed that there were soluble pools of tubulins in zebrafish oocytes that were sequestered and maintained in a temporary “oligomeric” state, which retained assembling and disassembling potential (suggested by undetected acetylated tubulin, marker of stable tubulin), but lacked abilities to assemble into MTs spontaneously in vivo. Using differential centrifugation, gel chromatography and DM1A-probed western blot, soluble α-tubulin was found to be associated with large molecular weight complexes (MW range to over 2 MDa) which were reduced in amount by the blastula stage, especially in some batches of embryos, with a concomitant decrease in soluble tubulin. Complexes (MW range less than 2 MDa) then increased in the gastrula with an increase in soluble α-tubulin. Two different anti-γ-tubulin monoclonal antibodies, GTU 88 and TU 30, revealed the existence of soluble γ-tubulin in both zebrafish oocytes and embryos, which also decreased by the blastula stage and increased in the gastrula stage. Soluble α-tubulin and γ-tubulin extracted from zebrafish ovaries, oocytes and embryos co-localized in fractions on three different columns: S-200 Sephacryl, DEAE and Superose-6b. The soluble tubulin complexes were competent to assemble into MTs in vitro induced by taxol, and γ-tubulin was co-localized with assembled MTs. These soluble tubulin complexes were stable during freeze–thaw cycles and resisted high ionic interaction (up to 1.5 M NaCl). Furthermore, some ovarian soluble α-tubulin could be co-immunoprecipitated with γ-tubulin, and vice versa. Two antibodies specific for Xenopus γ-tubulin ring complex proteins (Xgrip 109 and Xgrip 195) detected single bands from ovarian extracts in western blots, suggesting the existence of Xgrip 109 and Xgrip 195 homologues in zebrafish. These findings, together with recent work on γ-tubulin ring complexes in oocytes, eggs and embryos of other species, suggest that soluble γ-tubulin-associated protein complexes may be involved in regulating tubulin dynamics during zebrafish oogenesis and embryogenesis.

Proteomics of early zebrafish embryos

BackgroundZebrafish (D. rerio) has become a powerful and widely used model system for the analysis of vertebrate embryogenesis and organ development. While genetic methods are readily available in zebrafish, protocols for two dimensional (2D) gel electrophoresis and proteomics have yet to be developed.ResultsAs a prerequisite to carry out proteomic experiments with early zebrafish embryos, we developed a method to efficiently remove the yolk from large batches of embryos. This method enabled high resolution 2D gel electrophoresis and improved Western blotting considerably. Here, we provide detailed protocols for proteomics in zebrafish from sample preparation to mass spectrometry (MS), including a comparison of databases for MS identification of zebrafish proteins.ConclusionThe provided protocols for proteomic analysis of early embryos enable research to be taken in novel directions in embryogenesis.