Retinoic acid influences the embryoid body formation in mouse embryonic stem cells by induction of caspase and p38 MAPK/JNK-mediated apoptosis.

Although all-trans retinoic acid (RA), the oxidative metabolite of vitamin A, is essential for normal development, high levels are teratogenic in many species. RA results in immediate effects on the preimplantation embryo and on blastocyst development in vitro and in vivo. To further elucidate the cellular mechanisms of early postimplantation embryo development induced by RA, we present an embryonic cell line, B5, as a candidate system for the investigation of these processes. We used undifferentiated ES cells as the model, which is from the undifferentiated status to differentiated status [embryoid body (EB) formation] mimicking postimplantation embryo development (egg-cylinder stage of embryo formation) to clarify the cellular mechanism of action of RA in the implanted blastocysts and cell apoptosis following the series of exposures to differing RA concentrations. Using an in vitro model, we identified the impact of RA on undifferentiated embryonic stem (ES) cells, including inhibition of cell proliferation and induction of cell apoptosis. JNK, P-38 and caspase activation were shown in the nature of RA-triggered apoptotic signaling in ES cells. The carry-on influences of RA on the ES cell were shown in the formation of EB from the pretreated ES cells. RA resulted in apparent impact on undifferentiated ES cells in vitro, with increased numbers of apoptotic cells initially and inhibited cell proliferation, which led to decreased size of EB. The process of EB formation (mimicking the early postimplantation embryo development) is regulated by RA-induced apoptosis through the activation of caspase and P38 MAPK/JNK pathway.

Effect of curcumin on in vitro early post-implantation stages of mouse embryo development.

OBJECTIVES: This study was designed to examine the embryotoxic potential of the curcumin at the blastocyst stage and during early post-implantation development of mouse embryos in vitro. STUDY DESIGN: Curcumin was administered to ICR mice embryos at a dose of 0, 6, 12, 24 µM throughout in vitro culture. A total of 1015 embryos were randomly assigned to the different dosage groups. The embryotoxic effects were studied by the exposure of curcumin at the blastocyst, implanted blastocyst and early egg cylinder stages, respectively. For assessment of implantation in vitro and further embryonic differentiation, blastocysts were cultured for 8 days. The cell proliferation of outgrowth blastocysts was analysed by Giemsa staining. RESULTS: Exposure to 24 µM of curcumin at the implanted blastocyst stage or early egg stage cause adverse effects on development. The percentage of embryos in the later stages of development was changed depending upon the dose of curcumin used. Furthermore, exposure to 24 µM of curcumin at the blastocyst stage was lethal to all embryos. The number of nuclei per outgrowth of the blastocyst decreased significantly after curcumin pre-treatment. The percentage of trophoblastic giant cells per outgrowth increased significantly after curcumin pre-treatment. CONCLUSIONS: These findings demonstrate that curcumin exerts an adverse effect on mouse embryos during the early post-implantation stages of development, equivalent to day 3-day 8 of gestation in vivo. Curcumin treatment or administration should be used carefully at the early post-implantation stage of gestation.

Pregnancy outcome and neonatal data of children born after intracytoplasmic sperm injection with a different duration of cryopreservation of spermatozoa obtained through testicular sperm extraction.

OBJECTIVE: To evaluate the impact of the duration of cryopreservation of testicular sperm on clinical and neonatal outcomes after intracytoplasmic sperm injection (ICSI) for patients experiencing azoospermia. MATERIALS AND METHODS: This study included 132 azoospermic men who participated in 212 ICSI cycles. The participating men underwent testicular biopsy for the cryopreservation of tissue to be used for subsequent ICSI cycles. The duration of the storage of testicular sperm was determined by the time of oocyte retrieval. Fertilization, embryo development in vitro, pregnancy rates, and neonatal outcomes were assessed. RESULTS: Although the mean percentage of viability decreased from 73.2% prior to freezing to 50.2% after thawing, viable spermatozoa were visualized subsequent to thawing of the tissue samples for all patients. The potential for fertilization and subsequent embryonic development was not influenced by the duration of sperm cryopreservation up to 2 years longer. The pregnancy outcomes also varied independently of the duration of sperm cryopreservation. The duration of storage did not appear to affect the neonatal outcomes adversely, including the Apgar score and intensive care unit admission rates, although neonatal outcomes were influenced by advanced maternal age. It also has no obvious impact on the major and minor congenital malformation rate of the newborns. CONCLUSION: ICSI outcomes, pregnancy outcome, neonatal outcome, and congenital malformation rate appear not to be affected by the duration of the period of cryostorage. An earlier start of the ICSI cycle following the testicular sperm cryopreservation is preferable because longer preservation is associated with more advanced maternal age.

Ginkgolide B induces apoptosis via activation of JNK and p21-activated protein kinase 2 in mouse embryonic stem cells.

Ginkgolide B (GKB), the major active component of Ginkgo biloba extracts, can both stimulate and inhibit apoptotic signaling. We previously showed that ginkgolide treatment of mouse blastocysts induces apoptosis, decreases cell numbers, retards the proliferation and development of mouse embryonic stem cells and blastocysts in vitro, and causes developmental injury in vivo. However, the precise molecular mechanisms underlying its actions are currently unknown. Here, our study further revealed that GKB induced apoptotic biochemical changes, including activation of JNK, caspase-3, and p21-activated protein kinase 2 (PAK2), in ESC-B5 mouse embryonic stem cells. Treatment of ESC-B5 cells with a JNK-specific inhibitor (SP600125) reduced GKB-induced activation of both JNK and caspase-3, indicating that JNK activity is required for GKB-induced caspase activation. Experiments using caspase-3 inhibitors and antisense oligonucleotides against PAK2 showed that caspase-3 activation is required for PAK2 activation and both of these activations are required for GKB-induced apoptosis in ESC-B5 cells.