1 Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, Kaohsiung, Taiwan, 2 Chang Gung University School of Medicine, 3 Department of Nutrition and Health Science, Fooyin Institute of Technology and 4 Department of Obstetrics and Gynecology, UCLA School of Medicine, California, USA 5 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, 123, Ta-Pei Rd., Niao-Sung Hsiang, Kaohsiung County, Taiwan. e-mail: s10d1{at}mail.st1917.com.tw
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Abstract |
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Key words: apoptosis/blastocyst/implantation/postimplantation/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 a modification of an established method (Huang, 2001). The embryos were cultured in 1 ml 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). Following the 24 h culture using different dose of retinoic acid without serum supplementation, all groups of embryos were cultured in the same condition (RA-free) for 7 days. During the first 3 days the culture medium was supplemented with 20% fetal calf serum (Gibco) and thereafter with 20% heated-inactivated human placental cord serum. Embryos were inspected daily under a dissecting microscope and classified according to an established method (Witschi, 1972
). 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 throughout the 8 day culture period. 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. Implanted blastocyst was defined as the attachment and outgrowth of the blastocyst 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 (24 h exposure to all-trans retinoic acid at the blastocyst stage in all experiments)
Experiment 1 (morphological development in vitro)
Expanded blastocysts were exposed to 0 (n = 150), 0.1 (n = 145) or 10 (n = 165) µmol/l all-trans RA without serum supplementation for 24 h. This covers concentrations expected in embryos after oral administration (Kraft et al., 1989) in the treated group. They were then washed in RA-free medium and further cultured in CMRL-1066 medium in order to assess their further development as outlined above. All-trans RA (Sigma) was prepared in an aqueous solution of 0.01% dimethyl sulphoxide (DMSO) and a similar solution of DMSO alone was used as the control. Briefly, 0, 0.1 or 10 µmol/l of RA was added on the first day of culture to the treated group. Over the following 7 days of culture, the culture medium was changed to CMRL-1066 without RA in both the treatment and control groups.
Experiment 2 (effect of RA on cell proliferation in blastocysts)
A total of 208 blastocysts was incubated in 0, 0.1 or 10 µmol/l RA for 24 h and washed in RA-free medium following the incubation. The proliferation of blastocysts was evaluated by counting separately the number of ICM and TE cells identified by dual differential staining (Pampfer et al., 1990). This method is based on immunosurgery, considering the impermeability of the TE layer, which protects the ICM cells from exposure to the antibody and the complement reaction. The two cell lineages can be distinguished following fluorochrome staining (Figure 1). According to the protocol, the zona pellucida was removed by incubating the blastocysts in 0.4% pronase in M2 medium supplemented with 0.1% bovine serum albumin (M2-BSA). The denuded blastocysts were then exposed to 1 mmol/l of trinitrobenzenesulphonic acid (TNBS) in BSA-free M2 medium (M2) containing 0.1% PVP at 4°C for 30 min (Hardy et al., 1989
) and washed with M2. Further, the blastocysts were treated with 30 µg/ml anti-dinitrophenol-BSA complex antibody in M2-BSA at 37°C for 30 min. After rinsing, the blastocysts were incubated in M2 supplemented with 10% whole guinea-pig serum as a source of complement, 20 µg/ml bisbenzimide and 10 µg/ml propidium iodide at 37°C for 30 min. The immunolysed blastocysts were gently transferred onto a slide and protected from light before observation. Under appropriate UV light excitation, the ICM cells, which take up bisbenzimide but exclude propidium iodide (PI) were stained blue, whereas the TE cells, which are labelled with both fluorochromes, were stained orangered. Because multinucleated cells have been shown to be infrequent in preimplantation embryos (Gardner and Davies, 1993
), the number of nuclei was considered as an accurate measure of the number of cells.
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Annexin staining: A total of 183 blastocysts was incubated in 0, 0.1 or 10 µmol/l of RA for 24 h and washed in RA-free medium following the incubation. The blastocysts following the 24 h treatment were used in this study. An Annexin VFLUOS staining kit (Roche) was used to stain these embryos according to the manufacturers instructions. According to the protocol, the zona pellucida was removed by incubating the blastocysts in M2-BSA. Embryos were washed well with PBS plus 0.3% BSA. They were incubated with a mixture of 100 µl binding buffer, 1 µl fluorescein isothiocyanate (FITC) conjugated Annexin V and 1 µl PI for 60 min. After incubation, the embryos were washed well before photographic images were taken under a fluorescence microscope (Zeiss) with bright light and under UV light with DAPI/FITC/Ron Daman triple filters. This kit contains PI to determine the integrity of the plasma membrane and Annexin V to detect the translocation of phosphatidylserine (PS) from the inner face to the outer surface of the plasma membrane. In apoptotic cells, the cells translocate PS from the inner face of the plasma membrane to the cell surface while maintaining membrane integrity. Once on the cell surface, PS can easily be detected by staining with FITC-conjugated Annexin V, a protein that will specifically bind to PS. However, in necrotic cells, the broken plasma membrane allows PI, a DNA binding dye, to enter the cell through broken membranes to bind to the cellular DNA. Thus, apoptotic, necrotic and viable cells can be distinguished. Apoptotic cells stain positive for Annexin V (green) and necrotic cells stain positive for PI (red), while viable cells with an intact plasma membrane and no translocated PS are negative for both Annexin V and PI (colourless). The illustration is shown in Figure 1DH.
Statistical analysis
2-test and one-way ANOVA were used as appropriate. Differences of P < 0.05 were considered to be significant.
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Results |
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Discussion |
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Embryonic development is a complex process that is extremely sensitive to vitamin A as both deficiency and excess can lead to abortion and embryonic malformation. It is reasonable to interpret these results as suggesting that an excess of RA disrupts the normal developmental programme and leads to a 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 development 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 a reduced somite number, reductions in the 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 the TGF-ß1 and TGF-ß2 proteins. Maternal exposure to RA (20 mg/kg) on day 9 of gestation has been found to induce dysmorphogenesis of the inner ear in mouse embryos (Frenz et al., 1996
). Similarly, a congenital limb anomaly can be induced following exposure of pregnant SwissWebster mice to non-physiological levels of RA (120 mg/kg) on days 10 and 11 of gestation (Kochhar, 1973
; Kochhar et al., 1984
). Additionally, elevated RA doses (30 mg/kg) adversely affected early postimplantation embryogenesis on days 3 or 4 of gestation (F.J.Huang et al., unpublished results). However, 50 mg/kg of RA does not affect early or late preimplantation embryos adversely and even higher doses of RA (100 mg/kg) did not adversely affect the development of late preimplantation embryos (Huang and Lin, 2001
). In contrast, RA in vitro (10 µmol/l) does affect germ layer and subsequent neurula development from day 3 to day 8 of gestation (Huang et al., 2001
). These findings suggest that embryos at different stages have different tolerances for increases in RA concentration. It has been shown that RA suppresses mesodermal gene expression in mouse embryonic stem cells (Bain et al., 1996
) and induces endodermal gene expression in F9 embryonal carcinoma cells (Cho et al., 1999
). These findings suggest that the teratogenic effects of RA on early postimplantation embryos may be mediated by disruption of germ layer-specific gene activities.
Embryonic cells in embryo development are often poised between proliferation and apoptosis. It has been shown that RA-induced apoptosis is involved in embryonic development. For example, RA inhibited the proliferation and induced apoptosis of ectomesenchymal stem cells. These influences may contribute to the formation of cleft palate and other orofacial congenital malformation (Lu et al., 1999; Suwa et al., 2001
). RA has an important role in digit separation with the increase of apoptotic cell death, as shown in limb development (Stewart et al., 2000
; Kochhar et al., 1993
; Lee et al., 1994
; Tamagawa et al., 1995
). RA also induced down-regulation of Wnt-3a, involving induction of widespread apoptosis in the tail bud cells of mouse embryo (Shum et al., 1999
). In the present study, excess RA inhibited the proliferation (reduced cell number) and induced apoptosis in the ICM of mouse blastocysts. This may result in the postimplantation embryo retardation in vitro.
The TE is a sphere of epithelial cells surrounding the ICM and the blastocoel. Trophoblast cells arising at the blastocyst stage as the TE are required for development in the mammalian conceptus and contribute to the placenta (Cross et al., 1994), which is critical for the survival of mammalian embryos. Trophoblast stem cells treated with RA were compromised in their ability to proliferate and exhibited properties of differentiation into giant cells (Yan et al., 2001
). This may implicate a function for RA in giant-cell formation during placentation. Furthermore, in-vitro studies performed with human trophoblastic cells demonstrated that the secretion of hCG and trophoblast differentiation are stimulated by retinoids (Tarrade et al., 2001
). Although RA inhibited the proliferation (reduced cell number) and induced apoptosis in the TE of mouse blastocyst in the present study, this did not inhibit the embryo implantation in vitro. However, giant-cell formation and differentiation were difficult to demonstrate in the present in-vitro model.
The molecular mechanisms underlying the induction of developmental apoptosis by RA are beginning to be understood. A stress protein of the HSP 100/Clp family (HSP 110) could represent a biochemical event of apoptotic cell death induced by RA, administered to embryos at day 9 post coitum (Evrard et al., 2000). This effect resulted in craniofacial malformation similar to those of mandibulofacial dysostosis in man. Retinoic acid and bone morphogenetic protein 4 (BMP4) together induce p27 protein leading to retinoblastoma (Rb) protein activation and ultimately apoptosis in P19 embryonal carcinoma cells (Glozak and Rogers, 2001
). Apoptosis induced by RA may operate by a mechanism activated by reactive oxygen species in embryonic stem cell death (Castro-Obregón and Covarrubias 1996
).
There are several reports regarding the effects of RA on different stages of postimplantation embryos, embryonal stem cell, embryonal carcinoma cells, or trophoblast stem cells. As a follow-up to the previous studies (Huang and Lin, 2001; Huang et al., 2001
), this study is the first to show that RA induced cell death (apoptosis) in blastocysts, which can result in subsequent retardation of postimplantation developmental and/or death. However, the molecular mechanism of apoptosis by RA in blastocysts and the interaction between ICM and TE need to be determined in further studies.
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Acknowledgements |
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References |
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Submitted on April 2, 2002; resubmitted on July 29, 2002. accepted on September 6, 2002