Human Reproduction Vol.16, No.10 pp. 2171–2176, 2001
Effect of retinoic acid on implantation and postimplantation development of mouse embryos in vitro Fu-Jen Huang1,2,4, Tsung-Chieh J.Wu3 and Meng-Yin Tsai1,2 1Department
of Obstetrics and Gynecology, Chang Gung Memorial Hospital, Kaohsiung, Taiwan, 2Chang Gung University School of Medicine and 3Department of Obstetrics and Gynecology, UCLA School of Medicine, California, USA 4To
whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, 123, Ta-Pei Road., Niao-Sung Hsiang, Kaohsiung County, Taiwan. E-mail:
[email protected]
BACKGROUND: This study was designed to examine the embryotoxic potential of retinoic acid (RA) at the blastocyst stage and during early post-implantation development of mouse embryos in vitro. METHODS AND RESULTS: All-trans retinoic acid (t-RA) was administered to ICR mice embryos at a dose level of 0, 0.001 µmol/l, 0.1 µmol/l and 10 µmol/l throughout in-vitro culture. A total of 404 embryos was randomly assigned to all different dose groups. The percentage of embryos in later stages of development changed depending upon the dose of RA used. Exposure to 10 µmol/l of t-RA at the blastocyst stage, implanted blastocyst stage or early oocyte stage was also found to cause different degrees of retardation of embryo development and embryo death. These findings demonstrate, for the first time, that RA exerts an adverse effect on embryo growth during the early post-implantation stages of development, in comparison with day 3 to day 8 of gestation in vivo. CONCLUSIONS: Although isotretinoin (13-cisretinoic acid) is effective for the therapy of cystic acne and other dermatological disorders, retinoid treatment should be avoided at the early post-implantation stage of gestation. Key words: implantation/in-vitro exposure/mouse embryo development/post-implantation/retinoic acid
Introduction Retinoic acid (RA), an active metabolite of vitamin A, plays an important role in normal embryogenesis and differentiation of normal and malignant cells (Dencker et al., 1987; Ruberte et al., 1991; Wu et al., 1992b; Wan et al., 1995). RA also participates in pattern formation and organ development during embryo growth (Maden et al., 1982; Paulsen et al., 1988; Eichele, 1989; Gudas, 1992). RA is a valuable compound in the therapy of cystic acne and numerous other dermatological disorders (Bollag, 1983; Becherel et al., 1996; Duell et al., 1996). In a series of 59 pregnant women exposed to retinoids in the form of isotretinoin during the first 28 days of gestation, 12 out of 59 (20%) had a spontaneous abortion and 20 out of 47 (43%) of newborns had congenital malformation (Lammer et al., 1985). Excess amounts of RA are also teratogenic in animals. Several animal studies have demonstrated a potential adverse effect of RA when administered during mid and late pregnancy in mice. Administration of RA on day 7 or day 8 of gestation caused retardation of general development, abnormal differentiation of the cranial neural plate and abnormal development of the hindbrain (Morriss-Kay et al., 1991). Administration of RA on day 9 of gestation induced dysmorphogenensis of the inner ear in mice (Frenz et al., 1996). Abnormalities of limb and neural plate development were induced when RA was administered between day 10 and day 16 of gestation (Kochhar, 1973; Kochhar et al., 1984; © European Society of Human Reproduction and Embryology
Stafford et al., 1995). However, the effects of RA on preimplantation and early post-implantation embryo development remain unclear. We previously found mRNA expression of all three RA receptors (RAR) α, β and γ in mouse blastocysts and in early post-implantation mouse embryos (Wu et al., 1992a). These findings suggest that RA may be involved in the developmental process of early mammalian embryos. The aim of this study was to examine this possibility using a culture of mouse embryos in vitro during and beyond implantation. This model allows us to observe the first steps of embryonic differentiation, and is the key to the assessment of certain critical developmental phases. It has the advantage of being independent of maternal influences and is more sensitive than assessment of early post-implantation development in vivo. Using this model, we investigated the effect of RA on the implantation and early post-implantation development of mice embryos.
Materials and methods Collection of mouse blastocysts ICR mice, 6–8 weeks old, were induced to superovulate by injecting 5 IU pregnant mares’ serum gonadotrophin (PMSG; Sigma Chemical Co., St Louis, MO, USA) followed 48 h later by injecting 5 IU human chorionic gonadotrophin (HCG, Serono, NV Organon Oss, The Netherlands). They were then mated overnight with a single
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fertile male of the same strain. Mating was confirmed by the presence of a copulatory plug the following day. ICR albino virgin mice and male mice were kept under a 12 h day, 12 h night regimen, with food and water available ad libitum. All animals received humane animal care as outlined in the Guidelines for Care and Use of Experimental Animals (Canadian Council on Animal Care, Ottawa, 1984). The morning after an overnight mating period, female mice with vaginal plugs were isolated and used for the experiment. The day a vaginal plug was found was defined as day 0 of pregnancy. The pregnant mice were maintained on mouse breeder chow and water ad libitum. Embryos were obtained by flushing the uterine horn and Fallopian tubes on the afternoon of day 3 of gestation with CMRL-1066 culture medium (Gibco Life Technologies, Grand Island, NY, USA) containing 1 mmol/l glutamine and 1 mmol/l sodium pyruvate (Sigma). The embryos were collected in uncoated plastic 35 mm Falcon culture dishes and washed a minimum of three times. Expanded blastocysts from different females were pooled and selected randomly for the various experiments. 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 4well 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.
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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 CMRL1066 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.
Results In the first series of experiments blastocysts were cultured in regular medium using routine methods modified from established methods (Hsu, 1979). The purpose of these experiments was to determine the embryonic changes in the developing oocyte. In a subsequent series of experiments, the effects of treatment with different doses of RA in different stages of embryonic development were determined. 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). The blastocysts initiated hatching behaviour, i.e. shedding of the zona pellucida (Figure 1B), on the first day of culture. By the end of the second day, 100 of the 104 embryos (96.2%) had hatched (Table I). All of the hatched embryos attached to the substratum of the culture dish (Figure 1C). During days 3–4 of culture the cells of the ICM and polar trophectoderm proliferated to form the advanced ICM stage of development (Figure 1D, E). The EEC stage was attained by 60 of the 104 embryos (57.7%) by the end of day 4 of culture (Table I). By the end of day 6 of culture, 44 of the 104 embryos (42.3%)
Effect of retionic acid on mouse embryos in vitro
Figure 1. The sequence of mouse embryo development in vitro from blastocyst to the somite stage. (A) Blastocysts with zona pellucida (arrow) (Witschi stage 6, original magnification ⫻100). (B) Free blastocysts shed the zona pellucida and attached lightly (arrow) to the surface of the culture dishes (Witschi stage 7, original magnification ⫻100). (C) Implanted blastocysts left an inner cell mass (ICM) (arrowhead) on the outgrowth trophoblasts (arrow) (Witschi stage 8, original magnification ⫻100). (D) The ICM protruded from the outgrowth trophoblasts and formed a small cavity (arrow) in the centre (gastrula) (Witschi stage 9, original magnification ⫻200). (E) Embryos with gastrula increased in mass and elongated (arrow) (Witschi stage 10, original magnification ⫻200). (F) LEC with two distinguishable parts; the proximal part of the extra-embryonic region (arrow) and the distal part of the embryonic region (arrowhead) (snowman shape) (Witschi stage 11, original magnification ⫻100). (G) LEC (arrow) with primitive streak or neurula (Witschi stage 12–13, original magnification ⫻200). (H) Somite-stage embryos with yolk sac (arrow) enclosing the embryonic shield (Witschi stage 14–15, original magnification ⫻40). The scale bar represents 40 µm in A, B, C and F; 20 µm in D, E and G; 100 µm in H.
Table I. Development of mouse blastocysts exposed to retinoic acid (RA) throughout in-vitro culture Developmental stagec
RA group 10 µmol/l
Blastocysts Hatched/implanted blastocysts (7–8) Early egg cylinder stage (9–10) Late egg cylinder stage (11–13) Early somite stage (14–15) Logistic regression
Control group 0.1 µmol/l
0.001 µmol/l
100 98 102 88 (88.0) 92 (93.9) 94 (92.2) 0 (0) 14 (14.3) 40 (39.2) 0 (0) 4 (4.1) 36 (35.3) 0 (0) 1 (1.0) 15 (14.7) Effect of concentration P ⫽ 0.0001 Development stage P ⫽ 0.0001 Interaction P ⫽ 0.0001
104 100 60 44 22
(96.2) (57.7) (42.3) (21.2)
aFive experiments/concentration. bAll values are numbers with percentages cIn
in parentheses. parentheses: developmental stage (Witschi, 1972).
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Table II. Development of mouse embryos exposed to retinoic acid (RA) at the blastocyst stagea,b Developmental stagec
RA group (10 µmol/l)
Control group
Blastocyst Hatched/implanted blastocysts (7–8) Early egg cylinder stage (9–10) Late egg cylinder stage (11–13) Early somite stage (14–15) Logistic regression
104 96 (92.3) 2 (1.9) 0 (0) 0 (0) Effect of RA P ⫽ 0.0001 Development stage P ⫽ 0.0001 Interaction P ⫽ 0.0001
106 100 60 42 220
aFive
(94.3) (56.7) (39.6) (18.9)
experiments/concentration.
bAll values are numbers with percentages in parentheses. cIn parentheses: developmental stage (Witschi, 1972).
Table III. Development of mouse embryos exposed to retinoic acid (RA) at the implanted blastocyst stagea,b Developmental stagec
RA group (10 µmol/l)
Control group
Implanted blastocyst (8) Early egg cylinder stage (9–10) Late egg cylinder stage (11–13) Early somite stage (14–15) Logistic regression
59 26 (44.1) 1 (1.7) 0 (0) Effect of RA P ⫽ 0.0001 Development stage P ⫽ 0.024 Interaction P ⫽ 0.0001
57 35 (61.4) 25 (43.8) 10 (17.5)
aThree
experiments/concentration.
bAll values are numbers with percentages in parentheses. cIn parentheses: developmental stage (Witschi, 1972).
had formed a LEC (Figure 1F, G). By the end of day 8 of culture, 22 of the 104 embryos (21.2%) had formed an early somite (Figure 1H). Dose effect of t-RA throughout in-vitro culture Embryos were cultured in regular medium with a dose of 0, 0.001, 0.1 and 10 µmol/l RA. A total of 404 blastocysts was evaluated for their ability to develop in the different doses of RA. As indicated in Table I, the percentage of embryos that reached the later stages of development decreased with increased dose of RA. Furthermore, 10 µmol/l RA treatment resulted in a failure in the proliferation of cells of the ICM and polar trophectoderm so that by the end of day 4 of culture no embryos could develop to the EEC and advanced stage. 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). Effect of t-RA on implanted blastocyst stage A total of 116 implanted blastocysts collected after 2 days of in-vitro culture was included (Table III). Differentiation from 2174
the EEC stage occurred in 61.4% of embryos in the control group, while in the treatment group only 44.1% of embryos reached this stage. However, differences became obvious upon subsequent culturing. Only one out of the 59 embryos (1.7%) in the treatment group developed to the LEC stage in comparison to 25 out of the 57 embryos (43.8%) in the control group. Effect of t-RA on early egg stage Of the embryos at the EEC stage in this experiment (Table IV), 19 out of 30 (63.3%) differentiated from the LEC stage in the control group, while only eight out of 25 (32.0%) of embryos in the treatment group reached this stage. Nine embryos in the control group (30.0%) and no (0%) embryos in the treatment group reached the early somite stage (Witschi stage 13–15). Discussion Although cleavage and development from the blastocyst to the early post-implantation stage are critical endpoints in toxicological studies on peri-implantation, they are not as sensitive towards previous exposure as subsequent implantation (Morriss-Kay et al., 1991; Tournaye et al., 1993; Frenz et al., 1996). In the present study the dose and stage effects of RA were assessed throughout the in-vitro culture and throughout the early developmental stage in order to determine the actual effects of RA on implantation and during the post-implantation period. The most striking findings of this study are that t-RA
Effect of retionic acid on mouse embryos in vitro
Table IV. Development of mouse embryos exposed to retinoic acid (RA) at the early egg cylinder stagea,b Developmental stageb
RA group (10 µmol/l)
Control group
Early egg stage (9–10) Late egg cylinder stage (11–13) Early somite stage (14–15) Logistic regression
25 8 (32) 0 (0) Effect of RA P ⫽ 0.001 Development stage P ⫽ 0.053 Interaction P ⫽ 0.11
30 19 (63.3) 9 (30)
aThree experiments/concentration. bAll values are numbers with percentages cIn
in parentheses. parentheses: developmental stage (Witschi, 1972).
exposure at the peri-implantation stage and during the early post-implantation stage adversely affected development in a dose-dependent manner. The adverse effects of RA on periimplantation embryos and early post-implantation embryos observed in the present study show that RA has a direct impact on embryos. In addition, excess RA administered at the blastocyst stage or at different stages of early post-implantation also resulted in different effects on development. These findings show that the degree of development induced by t-RA differs depending upon the time of administration and dose. Thus, the effects of RA on embryogenesis appear to be stage-specific and dose-specific. 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 734 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) β1 and TGF β2 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 tRA 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.
Acknowledgements We thank Mr Zong-Xian Lin, who has assisted in this study in the animal laboratory. We are also grateful to Hsueh-Wen Chang, who is at the Department of Biological Science, National Sun Yat-sen University, for his assistance in statistical analysis. This work was supported by grant NSC 89–2314-B-182A-051 from the National Science Council of the Republic of China.
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