Changes in repair pathways of radiation-induced DNA double-strand breaks at the midblastula transition in Xenopus embryo.

Ionizing radiation (IR) causes DNA damage, particularly DNA double-strand breaks (DSBs), which have significant implications for genome stability. The major pathways of repairing DSBs are homologous recombination (HR) and nonhomologous end joining (NHEJ). However, the repair mechanism of IR-induced DSBs in embryos is not well understood, despite extensive research in somatic cells. The externally developing aquatic organism, Xenopus tropicalis, serves as a valuable model for studying embryo development. A significant increase in zygotic transcription occurs at the midblastula transition (MBT), resulting in a longer cell cycle and asynchronous cell divisions. This study examines the impact of X-ray irradiation on Xenopus embryos before and after the MBT. The findings reveal a heightened X-ray sensitivity in embryos prior to the MBT, indicating a distinct shift in the DNA repair pathway during embryo development. Importantly, we show a transition in the dominant DSB repair pathway from NHEJ to HR before and after the MBT. These results suggest that the MBT plays a crucial role in altering DSB repair mechanisms, thereby influencing the IR sensitivity of developing embryos.

Regulation of zygotic genome activation and DNA damage checkpoint acquisition at the mid-blastula transition.

Following fertilization, oviparous embryos undergo rapid, mostly transcriptionally silent cleavage divisions until the mid-blastula transition (MBT), when large-scale developmental changes occur, including zygotic genome activation (ZGA) and cell cycle remodeling, via lengthening and checkpoint acquisition. Despite their concomitant appearance, whether these changes are co-regulated is unclear. Three models have been proposed to account for the timing of (ZGA). One model implicates a threshold nuclear to cytoplasmic (N:C) ratio, another stresses the importance cell cycle elongation, while the third model invokes a timer mechanism. We show that precocious Chk1 activity in pre-MBT zebrafish embryos elongates cleavage cycles, thereby slowing the increase in the N:C ratio. We find that cell cycle elongation does not lead to transcriptional activation. Rather, ZGA slows in parallel with the N:C ratio. We show further that the DNA damage checkpoint program is maternally supplied and independent of ZGA. Although pre-MBT embryos detect damage and activate Chk2 after induction of DNA double-strand breaks, the Chk1 arm of the DNA damage response is not activated, and the checkpoint is nonfunctional. Our results are consistent with the N:C ratio model for ZGA. Moreover, the ability of precocious Chk1 activity to delay pre-MBT cell cycles indicate that lack of Chk1 activity limits checkpoint function during cleavage cycles. We propose that Chk1 gain-of-function at the MBT underlies cell cycle remodeling, whereas ZGA is regulated independently by the N:C ratio.

Intracellular signal transduction in mouse oocytes and irradiated early embryos.

In order to determine the effect of X-irradiation on intracellular signal transduction in mouse oocytes and embryos, JNK, ERK and p38 kinase activities were measured by the state of phosphorylation of their respective substrates (c-Jun, Elk-1 and ATF-2, respectively) in two mouse strains differing in radiation sensitivity, namely C57BL and BALB/c. In a first step, control oocytes and embryos were compared for their respective kinase activities at various stages of oocyte maturation (germinal vesicle and metaphases of 1st and 2nd meiosis stages) and early embryonic development (1-, 2-, 4-, 8- and 16-cell, morula and blastula stages). Levels of p38, ERK or JNK kinase activities were shown to vary with the stage of oocyte maturation and embryo development. In a second step, 1- and 2-cell embryos were X-irradiated with 2.5 Gy during the S-phase of the 1st or the 2nd cell-cycle, respectively. There were no significant differences in p38, ERK and JNK kinase activities between control and irradiated embryos, whatever the stage or mouse strain was considered. In conclusion, p38, ERK and JNK kinase activities were shown to vary during oocyte maturation and early embryonic development. Apparently, X-irradiation did not affect these kinase activities at the 1- and 2-cell stages in either mouse strains regardless of their difference in radiation sensitivity.

Chk2/Cds1 protein kinase blocks apoptosis during early development of Xenopus laevis.

Early Xenopus laevis embryos possess cell cycles that do not arrest at checkpoints in response to damaged DNA. At the midblastula transition (MBT), embryos with damaged DNA undergo apoptosis. After the MBT, DNA damage triggers cell cycle arrest rather than apoptosis. The transition from checkpoint-unregulated to checkpoint-regulated cycles makes Xenopus embryos compelling for studying mechanisms regulating response to genomic damage. The DNA damage checkpoint is mediated by the Chk2/Cds1 kinase. Conflicting evidence implicates Chk2 as an inhibitor or promoter of apoptosis. To better understand the developmental function of Chk2, we expressed wild-type (wt) and dominant-negative (DN) Chk2 in Xenopus embryos. Wt-Chk2 created a pre-MBT checkpoint due to degradation of Cdc25A and phosphorylation of cyclin-dependent kinases. Embryos expressing DN-Chk2 developed normally until gastrulation and then underwent apoptosis. Conversely, low doses of wt-Chk2 blocked radiation-induced apoptosis. Therefore, Chk2 operates at a switch between cell cycle arrest or apoptosis in response to genomic assaults.

Routine laser assisted hatching results in significantly increased clincal pregnancies.

PURPOSE: To elucidate the appropriateness of current indications for assisted hatching (AH) in cleavage stage human embryos and to confirm our preliminary findings that only young patients (about 67%) benefit from AH. METHODS: Prior to transfer, 2 of 3 embryos selected for ET were subjected to laser assisted hatching (LAH). Control group consisted of patients matched by similar characteristics and protocol except LAH was not performed. RESULTS: The clinical pregnancy rate in women < or = 36 years was 64.9% (24/37) for embryos subjected to LAH but was significantly lower (p = 0.029) in the control (33.3%; 10/30). The implantation rate in women < or = 36 years in the test group was 38.1% (40/105) that was significantly higher than that of the control group (17.5%, 14/80; p = 0.0039). CONCLUSIONS: LAH is beneficial for women < or = 36 years but not for women > or = 37 years, for embryos with thin zonae (< or = 16 micron) but not with thick zonae (> or = 17 micron), and for those with repeated failures (37-50%).