Disruption Of Embryonic Development Research Articles

Protective Effects of Liquiritigenin against Citrinin-Triggered, Oxidative-Stress-Mediated Apoptosis and Disruption of Embryonic Development in Mouse Blastocysts.

The mycotoxin citrinin (CTN), a natural contaminant in foodstuffs and animal feeds, exerts cytotoxic and genotoxic effects on various mammalian cells and embryos. A previous investigation by our group revealed potentially hazardous effects of CTN on mouse oocyte maturation and pre- and post-implantation embryo development via the induction of apoptosis. The present study showed that CTN induces apoptosis and inhibits cell proliferation in the inner cell mass of mouse blastocysts. Notably, we observed for the first time that both these effects are suppressed by liquiritigenin (LQ). LQ is a type of flavonoid isolated from Glycyrrhiza radix with several biochemical and pharmacological activities, including antioxidant and anti-inflammatory properties. The preincubation of blastocysts with LQ clearly prevented CTN-induced disruption of pre- and post-implantation embryonic development and fetal weight loss, both in vitro and in vivo. CTN-induced damage processes directly promoted reactive oxygen species (ROS) generation, loss of mitochondrial membrane potential (MMP) and activation of caspase-9 and caspase-3, which were effectively blocked by LQ. Moreover, in an animal model, intravenous injection of dams with CTN (3 mg/kg/day) triggered apoptosis of blastocysts, disruption of embryonic development from the zygote to the blastocyst stage and a decrease in fetal weight. Pre-injection with LQ (5 mg/kg/day) effectively reduced apoptosis and impaired the cytotoxic effects of CTN on development. Our in vivo findings further confirm that CTN exposure via injection has the potential to impair pre- and post-implantation development, leading to apoptosis and the suppression of sequent embryonic development, which can be effectively prevented by LQ.

Wolbachia infections of the butterfly Eurema mandarina interfere with embryonic development of the sawfly Athalia rosae

Although maternally transmitted microorganisms such as Wolbachia are well known to have a variety of effects on the reproduction of diverse insect species, little is known about the underlying mechanisms of actions. Artificial transfer of Wolbachia between taxonomically distant host species may provide insights into Wolbachia-induced manipulations of hosts. Here we performed a cross-order transfer of feminizing Wolbachia derived from a butterfly, Eurema mandarina. The Wolbachia were propagated in the Eurema hecabe cell line, called NTU-YB, and then used to inject prepupal/pupal females of a Wolbachia-free hymenopteran sawfly, Athalia rosae. The 14 females that emerged as adults looked morphologically and behaviorally healthy, and ovarian development appeared normal on dissection. However, in contrast to the control, none of the 333 eggs harbored by the seven Wolbachia-injected females developed successfully. Similarly, none of the 140 eggs laid on host plant by the four Wolbachia-injected females, which were mated with males, showed any signs of development. Wolbachia infection was detected from whole-body samples of the inoculated individuals, but not from the eggs they produced. Disruption of embryonic development despite the absence of Wolbachia in the egg cytoplasm may represent a new phenotype involving maternal effects that result in female sterility.

Resveratrol Protects against 2-Bromopropane-Induced Apoptosis and Disruption of Embryonic Development in Blastocysts

2-Bromopropane (2-BP) is used as an alternative to ozone-depleting cleaning solvents. Previously, we reported that 2-BP has cytotoxic effects on mouse blastocysts and is associated with defects in subsequent development. In the present work, we show that 2-BP induces apoptosis in the inner cell mass of mouse blastocysts, and inhibits cell proliferation. Both effects are suppressed by resveratrol, a grape-derived phytoalexin with known antioxidant and anti-inflammatory properties. 2-BP-treated blastocysts displayed lower levels of implantation (compared to controls) when plated on culture dishes in vitro, and a reduced ability to proceed to later stages of embryonic development. Pretreatment with resveratrol prevented 2-BP-induced disruption of embryonic development, both in vitro and in vivo. Further investigation of these processes revealed that 2-BP directly promotes ROS generation, loss of mitochondrial membrane potential (MMP), and activation of caspase-3, whereas resveratrol effectively blocks 2-BP-induced ROS production and the accompanying apoptotic biochemical changes. Our results collectively imply that 2-BP triggers the mitochondrion-dependent apoptotic pathway via ROS generation, and the antioxidant activity of resveratrol prevents 2-BP-induced toxicity.

Preimplantation genetic diagnosis (PGD) for maternal dominant disorders: is polar body (PB) better than blastomere biopsy?

OBJECTIVE: Preimplantation diagnosis (PGD) for maternally inherited dominant or X-linked disorders may be performed by direct genetic analysis of the blastomere(s) or by sequential analysis of the first and second polar bodies. If blastomere biopsy is performed, it is recommended to analyze 2 blastomeres for preventing misdiagnosis due to allele drop-out (ADO). With PB analysis, the genotype of the embryo is predicted by deduction. The objective of this study was to compare polar body biopsy with blastomere biopsy, in PGD for maternally inherited disorders, regarding accuracy of the analysis as well as the effect of the biopsy method on embryo development.DESIGN: A prospective study, comparing 31 PGD cycles for maternal dominant disorders analyzed by PB biopsy, to 51 PGD cycles for dominant disorders, analyzed by 2 blastomeres biopsy.MATERIALS AND METHODS: A total of 241 embryos, analyzed by PB biopsy, were evaluated according to the following parameters: biopsy failure rate, amplification failure rate, rate of non-informative first PB, ADO and contamination rates, and the proportion of embryos in which a blastomere biopsy was performed also. Cleavage rate, degree of fragmentation and development parameters of the biopsed embryos on day 3 were assessed too.RESULTS: Failure of biopsy of 1st and 2nd PBs was encountered in 7.5% and 15.7%, respectively. Blastomere Biopsy after attempted PB biopsy was performed in 117 (48.5%) of the embryos due to homozygosity of the 1st PB and a lack of informative markers that could excluded ADO (n=110) and contamination (n=7). Cleavage rate, degree of fragmentation and development parameters of embryos on day 3 were similar in both groups. Ongoing pregnancy rates per embryo transfer were 22.5% for PB biopsy and 17.6% for 2-blastomere biopsy.CONCLUSIONS: We conclude that PB biopsy is more difficult to perform and genetic analysis of PB is hampered by inability to detect maternal contamination in the 1st PB, and inability to distinguish between homozygousity of 1st PB and ADO. On the other hand, PB biopsy may be associated with less disruption of embryonic development. Therefore, we are recommending on PB biopsy only in analyses of maternal disorders that are based on more than 3 informative maternal polymorphic markers. This reduces the need for an additional blastomere biopsy when 1st PB is homozygote. OBJECTIVE: Preimplantation diagnosis (PGD) for maternally inherited dominant or X-linked disorders may be performed by direct genetic analysis of the blastomere(s) or by sequential analysis of the first and second polar bodies. If blastomere biopsy is performed, it is recommended to analyze 2 blastomeres for preventing misdiagnosis due to allele drop-out (ADO). With PB analysis, the genotype of the embryo is predicted by deduction. The objective of this study was to compare polar body biopsy with blastomere biopsy, in PGD for maternally inherited disorders, regarding accuracy of the analysis as well as the effect of the biopsy method on embryo development. DESIGN: A prospective study, comparing 31 PGD cycles for maternal dominant disorders analyzed by PB biopsy, to 51 PGD cycles for dominant disorders, analyzed by 2 blastomeres biopsy. MATERIALS AND METHODS: A total of 241 embryos, analyzed by PB biopsy, were evaluated according to the following parameters: biopsy failure rate, amplification failure rate, rate of non-informative first PB, ADO and contamination rates, and the proportion of embryos in which a blastomere biopsy was performed also. Cleavage rate, degree of fragmentation and development parameters of the biopsed embryos on day 3 were assessed too. RESULTS: Failure of biopsy of 1st and 2nd PBs was encountered in 7.5% and 15.7%, respectively. Blastomere Biopsy after attempted PB biopsy was performed in 117 (48.5%) of the embryos due to homozygosity of the 1st PB and a lack of informative markers that could excluded ADO (n=110) and contamination (n=7). Cleavage rate, degree of fragmentation and development parameters of embryos on day 3 were similar in both groups. Ongoing pregnancy rates per embryo transfer were 22.5% for PB biopsy and 17.6% for 2-blastomere biopsy. CONCLUSIONS: We conclude that PB biopsy is more difficult to perform and genetic analysis of PB is hampered by inability to detect maternal contamination in the 1st PB, and inability to distinguish between homozygousity of 1st PB and ADO. On the other hand, PB biopsy may be associated with less disruption of embryonic development. Therefore, we are recommending on PB biopsy only in analyses of maternal disorders that are based on more than 3 informative maternal polymorphic markers. This reduces the need for an additional blastomere biopsy when 1st PB is homozygote.

153 ALLEVIATIVE EFFECTS OF ANTIOXIDANT ADMINISTRATION ON MATERNAL HYPERTHERMIA-INDUCED EARLY EMBRYONIC DEATH IN MICE

Hyperthermia-induced early embryonic death is generally ascribed to the high susceptibility of early embryos to elevated maternal body temperature. However, recent studies have indicated that the disruption of embryonic development by maternal hyperthermia has relevance to not only high temperature exposure to the embryo, but also hyperthermia-associated changes in the maternal body. Hyperthermia enhances physiological production of reactive oxygen species (ROS) systemically. Early embryos are susceptible to oxidative stress and it becomes easy to arrest their development when the oxidative stress is exposed. These findings led us to speculate that maternal hyperthermia-induced early embryonic death is caused by an increase in oxidative stress to the embryo. Vitamin E and melatonin are both well known to function as antioxidants in vivo when they are administered exogenously. In this study, we administered vitamin E or melatonin to heat-stressed pregnant mice, aiming to alleviate the hyperthermia-induced embryonic death. Female mice were heat-stressed (35�C with 60% relative humidity) for 12 h on the day of mating. In Experiment 1, vitamin E (1000 mg/kg body weight) was injected intraperitoneally just before stress treatment, or melatonin (3 mg/kg body weight) was injected subcutaneously for every 2 h during heat exposure. Then, zygotes were collected and in vitro developmental ability was assessed. In Experiment 2, intracellular glutathione (GSH) content in the zygote, ROS level and free radical scavenging activity (FRSA) in the oviduct, and lipid peroxidation level in the liver were measured to evaluate the effects of melatonin administration on physiological redox status. All data were analyzed using one-way ANOVA followed by Fisher's protected least-significant test. The percentage of embryos that developed to the morula or blastocyst stage was significantly (P < 0.01) increased by administration of either vitamin E (58.6%) or melatonin (47.9%) compared with that in heat-stressed mice that were administered placebos (14.8%). Intracellular GSH content in zygotes derived from melatonin-administered mice was not significantly different from that in unstressed mice (1.67 and 1.82 pmol/zygote, respectively), whereas in heat-stressed mice that were administered placebo, intracellular GSH content was significantly decreased (1.48 pmol/zygote, P < 0.05) compared to that in unstressed mice. There were no significant differences in lipid peroxidation levels in the liver and in ROS levels in the oviduct between melatonin-administered stressed mice and unstressed mice, although these parameters in heat-stressed mice with placebo were significantly higher than in unstressed mice (P < 0.05). Furthermore, FRSA in the oviduct was significantly (P < 0.05) higher in the melatonin-administered mice than in the heat-stressed mice. These findings suggest that antioxidant administration to heat-stressed mice alleviates the hyperthermia-induced early embryonic death, and this may be accomplished in part by maintaining a neutral redox status within the mother.

Developmental competence and glutathione content of maternally heat‐stressed mouse oocytes and zygotes

ABSTRACTThe loss of developmental competence and the glutathione (GSH) content of maternally heat‐stressed mouse oocytes and zygotes were determined. In experiment 1, zygotes were collected from female mice that were heat‐stressed at 35°C for 10 h after hCG injection (oocyte maturation stage), or for 12 h on Day 1 of pregnancy (zygote stage), followed by in vitro culture. To minimize the effects of heat stress on the fertilization process, heat‐stressed oocytes that were fertilized in vitro were also included in this experiment. In experiment 2, heat‐stressed oocytes and zygotes were assayed for GSH content. The application of heat stress to the oocytes resulted in a significant decrease in the percentage of zygotes that developed to morulae or blastocysts, both for naturally fertilized oocytes (56.9% for heat‐stressed vs 85.4% for control) or in vitro‐fertilized oocytes (54.5%vs 73.6%). In the heat‐stressed zygotes, the disruption of embryonic development was more drastic (24.3%vs 90.3%), with the majority of zygotes being arrested at the two‐cell stage. In contrast, the GSH content decreased significantly in heat‐stressed zygotes, but not in heat‐stressed oocytes. These results demonstrate that the loss of developmental competence of early embryos is associated with a decrease in the GSH content of maternally heat‐stressed zygotes, but not of maternally heat‐stressed oocytes.

Inhibition of Retroviral Pathogenesis by RNA Interference

Background: RNA interference (RNAi) is a newly discovered cellular defense system that is known to suppress replication of genomic parasites in model organisms. It has been widely conjectured that RNAi may also serve as an antiviral system in vertebrates.Results: Retroviral infection could be initiated by electroporation of cloned Rous sarcoma virus (RSV) proviral DNA into the developing chick neural tube. Coelectroporation of proviral DNA and short double-stranded RNAs matching sequences of avain retroviruses, which were designed to induce RNAi against RSV, inhibited viral replication. Replication of RSV after electroporation resulted in disruption of embryonic development and early death, but this, too, could be suppressed by RNAi against the RSV genome. RNAi could also inhibit the growth of RSV and HIV in cell culture. Analysis of the step of the retroviral life cycle that is inhibited by RNAi revealed that it primarily prevented accumulation of the viral RNAs synthesized late during infection. RNA genomes introduced in viral particles early during infection were less sensitive.Conclusions: RNAi can block retroviral infection in vertebrates. The tissue electroporation method described here should allow RNAi to be used widely to study gene function and control of infection in vertebrate animals.

Disruption of embryonic development by juvenile hormone and its mimics in Dysdercus fasciatus Sign. (Hemiptera, Pyrrhocoridae)

AbstractThe embryonic development of Dysdercus fasciatus Sign. is described in terms of 15 arbitrary stages based on external morphological characters. The effects of treating eggs early in development with juvenile hormone and a mimic (JH) are then described in terms of the stages at which embryonic development is arrested and the resulting morphological aberrations are summarised. The effects of the treatment of two strains of D. fasciatus with two JH (Law's mimic and Ayerst synthetic juvenile hormone) are described and compared in terms of both the hatchability of the eggs and the stage distributions of the arrested embryos. Law's mimic was more effective than Ayerst SJH in preventing egg hatch, and the Malawi strain was less susceptible than the Reading strain. Embryonic development was never arrested earlier than the commencement of blastokinesis (Stage VIII). After treatment with Ayerst SJH most embryos of both strains were arrested during dorsal closure (Stages XI-XII) or at the time of eclosion (Stages XHI-XIV). Treatment of the Reading strain with Law's mimic resulted in the majority of embryos being arrested during blastokinesis (Stages VIII-X) at all doses. Examination of the timetable of development in both control and treated eggs reveals a dramatic reduction in developmental rate in treated eggs at the commencement of blastokinesis. It is suggested that JH may act on eggs of D. fasciatus in three ways: interference with eclosion, differentiation and the embryonic movements during blastokinesis.