1 Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, Kaohsiung, Taiwan, 2 Chang Gung University School of Medicine and 3 Department of Obstetrics and Gynecology, UCLA School of Medicine, California, USA
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Abstract |
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Key words: implantation/in-vitro exposure/mouse embryo development/post-implantation/retinoic acid
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Introduction |
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Materials and methods |
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Embryo culture
Blastocysts were cultured for assessment of implantation in vitro and further embryonic differentiation according to an established modified method (Hsu, 1979). Incubation conditions included maintenance of a constant temperature (37°C) and atmosphere (5% CO2 and 95% air). The embryos were cultured in 1 mol/l of culture medium in 4-well Multidishes (Nunc, Roskilde, Denmark) at 37°C under 5% CO2 in air. For group culture, five embryos were cultured per well. CMRL-1066 was used as the basic culture medium. It was supplemented with 1 mmol/l glutamine and 1 mmol/l sodium pyruvate plus 50 IU/ml penicillin and 50 mg/ml streptomycin (Gibco). During the first 4 days the culture medium was supplemented with 20% fetal calf serum (FCS) (Gibco) and thereafter with 20% heated-inactivated human placental cord serum (HCS). All blastocysts were initially cultured for the first 2 days without media change. After the first 2 days, when the blastocysts had attached, fresh medium was renewed daily until day 8 of cultivation. Embryos were inspected daily under a dissecting microscope and classified according to an established method (Witschi, 1972
). In the following 8 days, developmental parameters, such as hatching through the zona pellucida, attachment to the culture dishes, trophoblastic outgrowth, and differentiation of the embryo proper into early or late egg cylinders (germ layer stage) or primitive streak to early somite stage were recorded daily. Embryonic development was observed through a phase-contrast microscope (Olympus IMT-2, Tokyo, Japan). To decrease observer bias, all the data were analysed using the following criteria. Attachment was defined as the condition that the blastocyst attached to the culture dish. An early egg cylinder (EEC) embryo was defined as an embryo that had reached stages 9 or 10 by day 4. A late egg cylinder (LEC) embryo was defined as an embryo that reached stages 11, 12 or 13 by day 6 of culture. An early somite (ES) embryo was defined as an embryo that had reached stages 14 or 15 by day 8.
Experimental design
Experiment 1 [exposure to 0, 0.001 µmol/l, 0.1 µmol/l and 10 µmol/l all trans retinoic acid (t-RA) throughout in-vitro culture]
In order to investigate the possible dose effects of t-RA on implantation and post-implantation development, embryos were randomly assigned into four dose groups, and cultured with 20% FBS in CMRL-1066 media for the first 4 days and 20% HCS for the next 4 days in the presence of 0, 0.001 µmol/l, 0.1 µmol/l and 10 µmol/l t-RA, the concentrations expected in embryos after oral administration, (Kraft et al., 1989) and 0.01% of dimethyl sulphoxide (DMSO) as control. t-RA(Sigma) was prepared in an aqueous solution of DMSO.
Experiment 2 (short-term exposure to 10 µmol/l t-RA at the blastocyst stage)
Expanded blastocysts were exposed to 10 µmol/l of RA in the treated group, washed in RA-free medium and further cultured in CMRL-1066 medium in order to assess their further development as outlined above. Briefly 10 µmol/l of t-RA was added in the first 2 days of culture in the treated group. In the following 6 days of culture the culture medium was changed to CMRL-1066 in both the treatment and control groups.
Experiment 3 (short-term exposure to 10 µmol/l t-RA at the implanted blastocyst stage)
Implanted blastocysts, cultured from the blastocyst for 2 days, were allocated into two groups and 10 µmol/l of t-RA was added in the following 2 days of culture in the treatment group. In the resting 4 days of culture, the culture medium was changed to CMRL-1066 in both the treatment and control groups.
Experiment 4 (short-term exposure to 10 µmol/l t-RA at the EEC stage)
Embryos at the EEC stage, which were cultured from blastocysts for a further 4 days, were allocated into two groups, and 10 µmol/l of t-RA was added in the following 2 days of culture in the treatment group. In the resting 2 days of culture, the culture medium was changed to CMRL-1066 media in both the treatment and control groups.
Statistical analysis
Repeated measures analysis of variance was used to compare the developmental rates to EEC, LEC, and ES stages according to culture day and to check the dose-effect and time-effect of RA on the success rates of development to EEC, LEC, and ES stages. All data were analysed by logistic regression using the SAS statistical package (SAS Inc. 1988). Differences of P < 0.05 were considered to be significant.
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Results |
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Embryology of developing blastocysts
Blastocysts obtained from mice on day 3 of gestation were composed of a single layer of trophectoderm enclosing the cells of the inner cell mass (ICM) and the blastocoele cavity. The trophectoderm could be divided into two regions; the mural trophectoderm, which covered the blastocoele cavity, and the polar trophectoderm, which bordered the ICM. The blastocyst was surrounded by the zona pellucida (Figure 1A).
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Effect of t-RA on blastocyst stage
Overall, 210 blastocysts were further cultured for implantation in vitro as shown in Table II. Implantation was similar in the treatment and control groups, but in the treatment group, formation of a 2-layer ICM and the formation of an ectoplacental cone were reduced significantly. In the treatment group, fewer embryos developed to the advanced egg cylinder stages (LEC and ES stages) in comparison with the control group (logistic regression: effect of concentration, P = 0.001; effect of development, P = 0.001; interaction, P = 0.001).
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Discussion |
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Since RA is an important morphogen inducing pattern formation, it is reasonable to interpret these results as suggesting that excess of RA disrupts the normal developmental programme and leads to serious retardation, as demonstrated in the present study. In previous studies the adverse effects of RA were found to be dose-dependent at the different stages of embryo development. Mouse embryos exposed to excess RA (12 mg/kg) on day 7 of gestation showed retardation of general development, abnormal differentiation of the cranial neural plate and abnormal development of the hindbrain (Morriss-Kay et al., 1991
). The morphological features of embryos from treated mice were characterized by reduced somite numbers, reductions in pharyngeal arch size and number, a rostrally displaced otocyst, delayed closure of the anterior neuropore, as well as retardation of heart development. These effects of RA were related to the expression of the Hox-2.9 and Krox-20 genes, which in turn reduced the expression of transforming growth factor (TGF) ß1 and TGF ß2 proteins. Maternal exposure to RA (20 mg/kg) on day 9 of gestation has been found to induce dysmorphogenensis of the inner ear in mouse embryos (Frenz et al., 1996
). Similarly, a congenital limb anomaly can be induced following exposure of pregnant Swiss-Webster mice to a non-physiological concentration of RA (120 mg/kg) on day 10 and day 11 of gestation (Kochhar, 1973
; Kochhar et al., 1984
). In our previous study, RA (30 mg/kg) adversely affected early post-implantation embryogenesis on day 3 or day 4 of gestation, yet 50 mg/kg of RA did not affect early or late pre-implantation embryos adversely, and even higher doses of RA (100 mg/kg) did not adversely affect the development of late pre-implantation embryos (Huang and Lin, 2001
). These findings suggest that embryos at different stages have different tolerances for increases in t-RA concentration. In the present study, we showed that t-RA affected germ layer and subsequent neurula development from day 3 to day 8 of gestation. It has been shown that RA suppressed mesodermal gene expression in mouse embryonic stem cells (Bain et al., 1996
) and induced endodermal specific gene expression in F9 embryonal carcinoma cells (Wu et al., 1992b
). These findings suggest that the teratogenic effects of RA on early post-implantation embryos may be mediated by disrupting germ layer specific gene activities.
Retinoids have been used in the treatment of a variety of hyperproliferative diseases in humans, including acute promyelocytic leukaemia, squamous cell carcinoma of the skin, cervical intra-epithelial neoplasia, bladder papilloma and leukoplakia of the oral cavity and larynx (Breitman et al., 1980; Markowska et al., 1994
; Toma et al., 1996
; Vahlquist et al., 1996
). Isotretinoin (13-cis-RA) is an effective therapy for cystic acne and other dermatological disorders (Bollag, 1983
; Becherel et al., 1996
; Duell et al., 1996
). Unfortunately, retinoids are highly teratogenic in humans, even in the therapeutic dose range of 0.5 to 1.5 mg/kg/day. The major malformations found among isotretinoin-exposed infants involved the cranium and face, the heart, the thymus, and the brain (Lammer et al., 1985
). In this study, we have shown that in-vitro exposure of mice embryos to excess RA at the blastocyst stage and during the early post-implantation period results in adverse effects on development. Retinoid treatment should be avoided at the early post-implantation stage of gestation.
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Acknowledgements |
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Notes |
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References |
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Submitted on February 2, 2001; accepted on June 15, 2001.