Hum. Reprod. Advance Access originally published online on September 30, 2005
Human Reproduction 2006 21(1):202-209; doi:10.1093/humrep/dei286
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Adverse effects of retinoic acid on embryo development and the selective expression of retinoic acid receptors in mouse blastocysts
1 Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, Kaohsiung, Taiwan, 2 Chang Gung University School of Medicine and 3 Animal Science, National Ping-Tung University of Science Technology
4 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: huangfj{at}seed.net.tw
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Abstract |
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BACKGROUND: All-trans retinoic acid (RA), the oxidative metabolite of vitamin A, is essential for normal development. In addition, high levels of RA are teratogenic in many species. We have previously shown that excess RA results in immediate effects on the preimplantation embryo and on blastocyst development. This study was conducted to clarify the long-term survival of mouse blastocyst and the effect of RA on gene expression. METHODS AND RESULTS: Using an in vitro model, we identified the immediate adverse impact of RA on mouse blastocyst development. This involved an inhibition of cell proliferation and growth retardation. Using an in vivo model, we also identified the resorption of postimplanted blastocysts that had been treated with excess RA. Analysis of RA-mediated gene induction was also included. The retinoic acid receptors RAR
and RAR
were constitutively expressed in the blastocyst and the inner cell mass, whereas RAR
was induced upon RA treatment. CONCLUSIONS: This is the first evidence to show the impacts of RA on mouse blastocysts in vitro and any carry-over effects in the uterus. There is a retardation of early postimplantation blastocyst development and then subsequent blastocyst death. Our findings also show that there is some degree of selective induction of retinoic acid receptors when excess RA is administered to the blastocysts.
Key words: blastocyst/embryo development/retinoic acid/retinoic acid receptor
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Introduction |
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Vitamin A and its physiological metabolites, retinoic acid (RA) and other retinoids, exhibit a striking effect on pattern formation and may be one of the morphogens that control embryonic development (Dencker et al., 1987
; Eichele et al., 1989; Maden et al., 1982; Gudas et al., 1992; Hofmann and Eichele, 1994). While normal embryo development requires retinoids, too much or too little, at the wrong stage or at the wrong time, can adversely affect the developing embryo (Sporn and Roberts, 1991). Embryos exposed to low concentrations of RA (0.0001–0.1 µmol/l) develop normally, whereas those exposed to higher concentrations (0.5–100 µmol/l) develop characteristic dose-dependent defects (Vandersea, 1998). Embryonic exposure to RA causes a wide spectrum of severe malformation in the offspring of humans (Lammer et al., 1985), rodents (Shenefelt, 1972; Kessel and Gruss, 1991), chickens (Thaller and Eichele, 1987) and Xenopus (Durston et al., 1989; Sive et al., 1990), and these include neural tube and central nervous system defects, skeletal defects, cleft palate, ear, and other craniofacial malformations, defects of the heart, thymus, and urogenital system, and limb and digit reduction or duplication.
Several mouse 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 causes 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 induces dysmorphogenesis of the inner ear in mice (Frenz et al., 1996). Abnormalities of limb and neural plate development have been induced when RA is administered between day 10 and day 16 of gestation (Kochhar, 1973; Kochhar et al., 1984; Stafford et al., 1995). In our previous studies (Huang et al., 2001, 2003), the dose-dependent effects of RA on blastocysts have been evaluated in the presence of 0, 0.001, 0.1 and 10 µmol/l RA. An adverse effect of RA was detected in the presence of 10 µmol/l RA. It has previously been demonstrated that in utero exposure to excess RA during the preimplantation period (early cleavage stage) does not result in any adverse effect on preimplantation embryo development (blastocyst formation) (Huang and Lin, 2001a). However, in vitro exposure to excess RA during the peri-implantation (blastocyst stage) and early postimplantation period does result in early growth retardation and later embryo death in postimplantation (comparable to day 3 to day 8 of gestation) embryos (Huang et al., 2001). Cellular responses to RA in vitro range from cell death to cell differentiation. Some teratogenic effects of retinoids may be due to their ability to induce apoptosis. RA-induced cell death with the characteristics of apoptosis has been observed during our blastocyst studies (Huang et al., 2003, 2005). The expression of mRNA encoding all three RA receptors (RAR) -, - and - has been found in mouse blastocysts and in early postimplantation mouse embryos (Wu et al., 1992a). These nuclear receptors regulate gene transcription in a ligand-dependent fashion through binding to specific DNA sequences, generally located upstream of the promoter of the target gene (Umesono et al., 1988; de The et al., 1989, 1990). Furthermore, these nuclear RA receptors act as RA-inducible enhancer factors (Evans, 1988; Green and Chambon, 1988; Vasios et al., 1989) in mouse and human. These findings suggest that RA may be involved in the developmental process of early mammalian embryos through interaction with the RAR and the marked effects of retinoic acid on morphogenesis result from the activation of development control genes. Thus, the differential expression of RAR seems to be an important step in RA signalling of the embryonic cells.
Although the short-term effects of RA on blastocysts (around day 8–9 of gestation) have been demonstrated in in vitro studies (Huang et al., 2001), maternal compensation for RA-treated blastocyst has not been demonstrated. In order to investigate the long-term effects of RA on blastocysts, we examined whether incubating mouse embryos with RA during the blastocyst stage resulted in an immediate effect of RA on their developmental ability in vitro, and whether there is a long-term effect of RA in vivo. We also investigated the expression of RAR, RAR or RAR in the system to detect the RA signalling in the blastocysts.
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Materials and methods |
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Blastocyst collection and culture with RA
ICR mice (6 ± 8 weeks old) were induced to superovulate by injecting 5 IU pregnant mare’s serum gonadotrophin (PMSG; Sigma Chemical Co., St Louis, MO, USA) and this was followed 48 h later by the injection of 5 IU HCG (Serono, NV Organon Oss, The Netherlands). They were then mated overnight with a single fertile male of the same strain. Mating was confirmed by the presence of a copulatory plug the following day. The ICR virgin albino mice, male mice and pregnant mice were maintained on breeder chow and 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, 1984). The morning after an overnight mating period, female mice with vaginal plugs were separated and used experimentally. The day a vaginal plug was found was defined as day 1 of pregnancy. Embryos were obtained by flushing the uterine horn and Fallopian tubes on the afternoon of day 4 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. Expended blastocysts from different females were pooled and randomly selected for the various experiments. Expended blastocysts were exposed to 0, 0.1 or 10 µmol/l all-trans RA without serum supplementation for 24 h. This covers the concentrations expected in embryos after oral administration (Kraft et al., 1989
) and forms 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% dimethylsulphoxide (DMSO) and a similar solution of DMSO alone was used as the control.
Development of blastocyst in vitro
Outgrowing embryo culture
Blastocysts from ICR mice were collected on day 4 of pregnancy and incubated in the presence or absence of RA (10 µmol/l) as described in a previous study (Huang et al., 2001). After 24 h of incubation, blastocysts were transferred to fibronectin-coated culture wells and cultured individually in the absence of RA for 72 h in CMRL-1066 medium supplemented with 20% FBS (CMRL–FBS) (Armant et al., 1986). Under these culture conditions, the hatched blastocysts will attach onto the fibronectin and outgrow to form a cluster of inner cell mass (ICM) cells over the trophoblastic layer [trophectoderm (TE) outgrowth].
Morphology of outgrowing embryos
The morphological scores for outgrowth were assessed after a total of 96 h of incubation. The outgrowing embryos were classified as attached or as outgrowth, in which they develop a cluster of ICM over the trophoblastic layer. The scores of ICM clusters were given according to the shape of the ICM clusters from compact-rounded ICM (+++) to a few scattered cells (+) over the trophoblastic layer as described previously (Pampfer et al., 1994). The surface area of the trophoblastic layer (trophectoderm outgrowth) was photographed and measured by weighing the cut-out images, and then indirectly evaluating the surface of the images from a standard curve of weight versus area. The morphological distribution was expressed as a percentage and compared by 
2-analysis.
Cell proliferation of outgrowths
The proliferation of outgrowths was estimated by counting the nuclei directly on the dish using Giemsa stain. This method was based on nuclear labelling by Giemsa stain (Wuu et al., 1999) and the cell spreading technique used for rat implanting embryos (Pampfer et al., 1994).
The culture medium was carefully removed and replaced with 0.05% hypotonic sodium citrate (30 µl/well) at room temperature for 5 min. This solution was evaporated under partial vacuum (200 torr) at 50–55°C for 60–90 min. Outgrowths were fixed with 50 µl per well of FixDenat fixative at room temperature for 30 min. The total number of nuclei in the outgrowths was examined by staining with a 4% Giemsa solution at room temperature for 30 min. The proliferation of an individual outgrowth was expressed as the number of Giemsa stained nuclei. The dead cells of outgrowths appeared either as fragmented nuclei (karyorrhexis) detected by bisbenzimide stain or as DNA strand breaks (karyolysis) detected by the TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) method, as described in the previous studies (Huang et al., 2003). Data were represented as a mean ± SEM, and the difference between the control and RA-treated groups was analysed by one-way analysis of variance (ANOVA).
Development of blastocysts in vivo
Embryo transfer
The ability of expanded blastocysts to implant and develop in vivo was evaluated by transferring the embryos to a recipient mouse. The ICR females were mated with vasectomized males (C57BL/6J) to produce pseudopregnant mothers as recipients for the embryo transfer experiments. The impact of RA on postimplantation growth in vivo was addressed by exposing blastocysts to 0 or 10 µmol/l of RA for 24 h and transferring them (five to eight embryos) in parallel into pairs of uterine horns in day 4 pseudopregnant mice. The surrogate mouse was killed on day 18 of the pregnancy. The frequency of implantation was calculated as the number of implantation sites per number of embryos transferred. The frequency of resorption or surviving fetuses was calculated as the number of resorptions or of surviving fetuses per number of implantations. The weight of the surviving fetuses and placentas was measured immediately after dissection. The results were represented as mean ± SEM and the difference between control and RA-treated groups was analysed by one-way analysis of variance (ANOVA).
Quantitative expression RA receptor , , in the blastocyst and ICM
Collection of ICM and in vitro culture from the blastocysts
The ICM clump was isolated by immunosurgery as described for rat blastocysts (Pampfer et al., 1990) and mouse blastocysts (Huang et al., 2005). Using the hanging drop culture, the ICM were isolated from the blastocysts in aliquots of 20 µl Earle’s balanced salt solution containing one ICM, in the presence of 0, 0.1, 10 µmol/l of all-trans RA and placed on the lids of Petri dishes. The lids were inverted, placed onto sterile water-filled dishes and incubated at 37°C for 24 h. In hanging drop culture, the ICM moves to the bottom of the drop and does not attach to the culture dish. After the 24 h culture, the ICM is washed and collected into the individual test groups for treatment over another 24 h culture.
Primers and PCR consumables
Four sets of primers were designed from the available mouse RAR, RAR and RAR sequences and the -actin sequence found in GenBank. The primers were chosen with the assistance of two computer programs, Oligo 4.0 (National Biosciences, Plymouth, MN, USA) and Primer Express (Perkin–Elmer Applied Biosystems, Foster City, CA, USA). The nucleotide sequences of the primers are shown in Table I.
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RNA extraction. Blastocyts were exposed to 0, 0.1, 10 µmol/l of RA in serum supplement for 24 h. ICM were also exposed to the same dose of RA in a separate experiment. Following the treatments, 40 blastocysts and 40 ICM in each group for one experiment were lysed in Trizol Reagent (Invitrogen, Life Technologies, Inc., Gaithersburg, MD, USA) by repetitive pipetting. At the end of a series of procedures, the RNA pellet was briefly dried and then finally the RNA was dissolved in 15 µl of diethyl pyrocarbonat (DEPC) water by passing the solution a few times through a pipette tip followed by incubation for 10 min at 60°C. The quality of the RNA samples was determined by agarose gel electrophoresis with ethidium bromide staining. The 18S and 28S RNA bands were easily visualized under UV light.
cDNA synthesis. RNA was reverse-transcribed in a final volume of 25 µl containing 5xreverse transcriptase buffer, 10 mmol/l each dNTP, 15 mmol/l MgCl2, 375 mmol/l KCl, and 250 mmol/l Tris–HCl (pH 8.30), 25 IU of Rnasin Rnase inhibitor (Promega, Madison, WI, USA), 50 mmol/l dithiothreitol, 200 IU of M-MLV (Moloney Murine Leukemia Virus) Reverse Transcriptase) (Promega, Madison, WI, USA), 0.5 µg oligo-dT primer (Promega), and 2 µg total RNA.
PCR amplification. All of the PCR reactions were performed using an ABI Prism 7700 Sequence Detection System (Perkin–Elmer Applied Biosystems). PCR was performed using the SYBR Green PCR Core Reagents kit (Perkin–Elmer Applied Biosystems). Specific PCR amplification products were detected using the fluorescent double-stranded DNA-binding dye, SYBR Green (Schmittgen et al., 2000). Experiments were performed in triplicate for each data point. All of the samples with a coefficient of variation for the Ct value >1% were retested.
Theoretical basis of real-time RT–PCR. Quantitative values are obtained from the threshold cycle (Ct) number at which the increase in signal associated with an exponential growth of PCR products starts to be detected (using Perkin–Elmer Biosystems analysis software), according to the manufacturer’s manual. The precise amount of total RNA added to each reaction (based on absorbance) and its quality (i.e. lack of extensive degradation) are both difficult to assess. We therefore also quantified transcripts of the gene -actin as an endogenous RNA control, and each sample was normalized on the basis of the -actin content. Final results, expressed as logtarget with target gene expression relative to the -actin gene, are termed ‘logtarget’, and were determined as follows: log target = log10 (105x2
Ct sample), where Ct values of the sample are determined by subtracting the average Ct value of the target gene from the average Ct value of the -actin gene in each sample.
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Results |
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Development in vitro
In order to determine whether a 24 h exposure to RA would impair their subsequent growth in vitro, blastocysts were incubated with 0, 0.1 or 10 µmol/l RA and then transferred (in the absence of RA) onto fibronectin-coated dishes for 72 h. At the end of the incubation period, 96.5% of the control blastocysts had attached and outgrown compared with 92.1% of the RA-pretreated group. Compared with control outgrowths, fewer implants derived from RA-pretreated blastocysts (10 µmol/l of RA) featured a compact and structured ICM at the centre of the trophectoderm (TE) layer, and more of them showed an apparently normal TE expansion surrounding a limited cluster of ICM cells (P < 0.05, Table II). Further analysis of the outgrowths showed that the number of nuclei per outgrowth decreased significantly after RA pretreatment (143 ± 20 versus 118 ± 24 versus 108 ± 19; control versus 0.1 versus 10 µmol/l, Figure 1). To know whether this decrease of nuclei in outgrowth was associated with cell death, we applied the TUNEL method and bisbenzimide stain to analyse the index of nuclear labelling. Results showed that neither the number of fragmented nuclei cells nor the number of TUNEL-positive cells in outgrowths was increased by RA pretreatment (not significant, Table III).
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Development in vivo
Assessment of the uterine content was performed 14 days after transfer. Control blastocysts (88 of 129 embryos in 15 recipients) implanted at a rate of 
70.8% (Table IV), and RA pretreatment induced a 10% decrease in that proportion (76 of 129 embryos) (P = 0.314). Early resorption resembled small dark moles without distinct structure. Late resorption resembled placenta without fetal structure. Late resorption was found in the RA-pretreated group. The proportion of implanted embryos that failed to develop normally (early and late resorptions) was 68.7% higher in the RA-pretreated group (50 of 76 implants) in comparison to the control group (39 of 88 implants) (P = 0.017). The external examination of surviving fetuses did not reveal any gross morphological alterations in both groups. The external examination of organs (including nervous system, heart, lung, thymus, gastrointestinal system and urological system) also showed no gross morphological alterations. Although a comparison of fetal weight did not show a significant difference between the two groups (528 ± 92 versus 511 ± 71, control versus RA-treated), there was a 28% decrease in the fetuses that were >600 mg in weight among the RA-pretreated blastocysts (P = 0.043, Figure 2). No difference was found in placental weights between the two groups (138 ± 27 versus 126 ± 25, control versus RA-treated).
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Effect of RA on the expression of RAR, RAR, RAR in the blastocyst and ICM
The data in each group were averaged from three experiments. Forty blastocysts or ICM in each group were pooled to analyse gene expression. The expression levels of RAR, RAR, and RAR were quantified in the three groups of blastocysts (Figure 3). The expression levels were determined as described in Materials and methods. Comparison of the various RAR and the reference -actin gene was used to correct for variations in the amounts of RNA. The average expression levels of RAR did not vary significantly across the three groups (1.72 ± 0.09, 10 mmol/l of RA versus 1.67 ± 0.03, 0.1 mmol/l of RA versus 1.63 ± 0.06, control; not significant). The average expression level of RAR also did not vary significantly (1.67 ± 0.11, 10 mmol/l of RA versus 1.86 ± 0.05, 0.1 mmol/l of RA versus 1.61 ± 0.11, control). However, the expression levels of RAR in the group treated with RA were significantly higher than for the control (1.76 ± 0.02, 10 mmol/l of RA versus 0.92 ± 0.03, control, P < 0.001; 1.68 ± 0.08, 0.1 mmol/l of RA versus 0.92 ± 0.03, control, P < 0.001). The expression levels of RAR, RAR and RAR were also quantified in the three groups of ICM (Figure 4). The average expression levels of RAR did not differ significantly between the three groups (2.84 ± 0.08, 10 mmol/l of RA versus 2.69 ± 0.01, 0.1 mmol/l of RA versus 2.75 ± 0.23, control). The average expression levels of RAR were also not significantly different (2.04 ± 0.06, 10 mmol/l of RA versus 2.03 ± 0.05, 0.1 mmol/l of RA versus 2.04 ± 0.23, control). However, the expression levels of RAR in the group with RA-treatment were significantly higher than for the control (2.78 ± 0.10, 10 mmol/l of RA versus 1.78 ± 0.12, control, P < 0.001; 2.60 ± 0.02, 0.1 mmol/l of RA versus 1.78 ± 0.12, control, P < 0.001).
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Discussion |
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Embryonic levels of RA and the response of cells to RA are critical for normal development in vertebrates. However, the administration of pharmacological doses of RA and its analogues (retinoids) during pregnancy results in a wide range of cranio-facial and limb malformations (Sulik et al., 1988
; Alles and Sulik, 1989; Satre and Kochhar, 1989), as well as defects in brain development (Langman and Welch, 1967; Lammer et al., 1985). This provides evidence, albeit indirect, of the role of RA in mammalian embryogenesis. 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 Swiss–Webster mice to non-physiological levels of RA (120 mg/kg) on day 10 and day 11 of gestation (Kochhar, 1973; Kochhar et al., 1984). Additionally, RA (30 mg/kg) adversely affects early postimplantation embryogenesis on day 3 or day 4 of gestation (Huang et al., unpublished results). However, 50 mg/kg of RA, and even higher doses of RA (100 mg/kg), do not affect the development of early or late preimplantation embryos adversely in vivo (Huang and Lin, 2001). According to general pharmacokinetics, it seems to be higher in oviduct or uterine fluids if mice are exposed to high level of RA in vivo. But there are still some factors to counteract the possibility. For example, the fluid in oviduct or uterine cavity is hydrophilic but the RA is hydrophobic. It will determine the final concentration of RA in the oviduct or uterine cavity. Another possible factor is the cells and zona pellucida surrounding the embryo. They may protect the preimplantation embryos in the oviduct or uterine cavity. However that may be, it i needs to be confirmed by measuring the concentration of RA in oviduct and uterine fluids of pregnant mice. The maternal compensation mechanism for the RA-damaged preimplantation embryo could be explained in a further study. However, RA in vitro (10 µmol/l) has an adverse effect on 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 sensitivities to an increase in RA concentration and the adverse effects of RA are different at different stages of embryo development.
In previous experiments, blastocysts with an ICM population reduced to an average of 70% of the normal cell population gave a high incidence of fetal loss, in spite of a normal TE population and a normal implantation rate (Tam et al., 1988). It has also been suggested that a sufficient number of ICM cells is essential to the process of implantation and an excessive reduction in this cell lineage may impair embryo viability (Pampfer et al., 1990; Kelly et al., 1992). Morphogenesis of the postimplantation mouse embryo involves the transformation of the solid ICM of the late blastocyst into a hollow structure known as the ‘egg cylinder’ (Huang et al., 2001). It is possible that the selective effect of RA on the ICM cell population may have an impact on subsequent embryo development, implantation and embryogenesis. The effects of RA on the blastocyst, especially the ICM, have been demonstrated in our previous studies (Huang et al., 2001, 2003, 2005) and its effects are dependent on the concentration of RA. However, maternal compensation for RA-pretreated blastocysts in utero was not demonstrated in these studies. To clarify this question, we aimed to collect adequate data in the present study. Here, RA-pretreated blastocysts were shown to have an apparent reduction in cell growth at 72 h of culture. If the blastocysts were transferred into the maternal uterus, damaged blastocysts could not be rescued in the maternal environment. Although implantation could be reached, the postimplantation embryo development was highly impacted and this led to a higher incidence of embryo resorption in the RA-treated blastocysts. It has been shown that the impact of RA on the ICM is deeper than only on the trophectoderm of blastocysts (Huang et al., 2003, 2005). In the present study, we observe that the pretreatment of blastocyst with RA reduced its ability to develop into outgrowth with a well-structured ICM cluster. Those outgrowths derived from RA-pretreated blastocysts contained fewer nuclei, suggesting that the pretreatment of blastocysts with RA had a long-lasting effect on embryo proliferation in vitro. In contrast, a previous study based on morphological observation showed that a long period of exposure of outgrowing embryos to RA alter the embryo development in vitro, regardless of the aspect of the ICM cluster over the outgrowth (Huang et al., 2001). However, TE as well as trophoblast cells have been shown to be resistant to RA in the study, which might explain that the effect of RA on TE outgrowth is small or absent. Because these outgrowths did not present signs of persisting occurrence of cell death in the study, it was assumed that the cell number difference was secondary to the irreversible loss of cells detected immediately after RA treatment (Huang et al., 2003). Considering our previous data that RA has a major inhibition on the ICM cell population (Huang et al., 2003, 2005), we may believe that the high rate of resorption is due to a RA-mediated deficiency of ICM cells in embryos. Although the rate of implantation was not affected by RA pretreatment, our data suggest that the deleterious effect of RA on ICM cells can result in a potential risk of fetal death and embryogenesis arrest in spite of a normal TE invasion.
RA signalling is mediated by two distinct classes of nuclear receptors, RAR and retinoid X receptors (RXR). These receptors are members of the nuclear receptor family and function by directly activating transcription via binding to promoter elements of the target genes (Petkovich et al., 1987). This process influences several developmental processes by interacting with different types of nuclear receptors. Each family is composed of three genes, namely, RAR, RAR and RAR, and RXR, RXR and RXR. There is both convergence and divergence in the signal transduction pathways between the RAR and RXR families and between the different members of each family. For example, RAR, RAR and RAR bind to elements that activate transcription in response to all-trans-RA and 9-cis-RA. In contrast, the retinoid X receptors (RXR, RXR and RXR) bind and activate transcription in response to 9-cis-RA (Heyman et al., 1992; Levin et al., 1992; Zhang et al., 1992; Allenby et al., 1993). These nuclear receptors regulate gene transcription in a ligand-dependent fashion through binding to specific DNA sequences, generally located upstream of the promoter of the target gene (Umesono et al., 1988; de The et al., 1989, 1990). The discovery of specific nuclear receptors for retinoic acid provides strong evidence that the marked effects of retinoic acid on morphogenesis result from the activation of development control genes. These nuclear RA receptors act as RA-inducible enhancer factors (Evans et al., 1988; Green et al., 1988; Vasios et al., 1989) in mouse and human and provide a basis for understanding how RA signals are transduced at the level of gene expression. However, they have been shown to result in differential expression, depending upon the RA concentration in different systems. For example, RA differentially regulates the expression of different transcripts for RAR, RAR and RAR depending upon the duration and dose of the treatment in F9 teratocarcinoma cells (Hu et al.,1990; Martins et al., 1990; Wu et al., 1992b). In Harnish’s studies, RAR mRNA levels in 9 day gestation mouse conceptuses are increased as early as 3 h after administration of a completely teratogenic dose of retinoic acid (100 mg/kg body weight).
Given the limited information available concerning RA-mediated RAR expression, particularly in the blastocyst or the ICM other than embryonic stem cells, this study was performed to gain an insight into the possible role of RAR, RAR, RAR expression in the blastocyst and the ICM using a quantitative real-time RT–PCR assay. Our results demonstrate that an in vitro teratogenic dose (10 µmol/l) of retinoic acid increases the level of RAR mRNA, but not RAR or RAR mRNA in the blastocysts and the ICM. This is consistent with a report on RAR expression in hepatoma cells in vitro (de The et al., 1990) and for 9 day gestation mouse conceptuses (Harnish et al., 1990). Thus RAR, - or - mRNA levels in the blastocyst, especially in the ICM, are influenced differentially by RA treatment in vitro and these are closely related to over-expression of the RAR gene. However, the present data could not explain by which mechanism RA exerts a deleterious effect. It seems to be an intranuclear signalling in the embryonic cell, which RAR- is increased in the presence of high concentrations of RA.
In conclusion, excess RA resulted in adverse impact on the blastocysts, especially on the ICM in vitro and this led to the resorption of postimplanted blastocysts or retardation of the surviving fetus in vivo. As part of the RA signalling in the embryonic cells, the differential expression of RAR, RAR and RAR was noted in RA-treated blastocysts and the ICM of blastocysts. These results further confirm the concept that the ICM is the major target of RA impact and plays a very important role in postimplantation development. This agrees with our previous findings that excess RA affects the postimplantation development of blastocysts adversely. The data reported here, combined with previously reported results, re-emphasize the role of RA in the ICM of blastocysts and its effects on early embryogenesis. RA can have profound effects on the mouse ICM making it important to define how RA receptor expression changes in a RA-induced ICM.
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Acknowledgement |
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This work was supported by grant NSC-91-2314-B-182A-150 and NSC-92-2314-B-182A-176 from the National Science Council of Taiwan.
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References |
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