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Regular Article |
a Departments of Physiological Sciences
b Veterinary Clinical Sciences,
c College of Veterinary Medicine, and Department of Animal Science, Division of Agricultural Science and Natural Resources, Oklahoma State University, Stillwater, Oklahoma 74078-2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, RXRß, RXR
, alcohol dehydrogenase I (ADH-I), retinaldehyde dehydrogenase 2 (RALDH2), peroxisome proliferator activated receptor gamma (PPAR), and glyceraldehyde-3-phosphate dehydrogenase. Transcripts for RXR, RXRß, RALDH2, and PPAR were detected in all stages beginning from the oocyte through to the hatched blastocyst. Whole mount in situ hybridization performed using digoxigenin-labeled antisense probes detected all 4 transcripts in both the inner cell mass and the trophectoderm of hatched blastocysts. PCR products obtained for ADH-I exhibited very low homology to known human and mouse sequences. Immunohistochemistry was performed using polyclonal anti-rabbit antibodies against RXRß and PPAR to investigate whether these embryonic mRNAs were translated to the mature protein. Strong immunostaining was observed for both RXRß and PPAR in the trophectoderm and inner cell mass cells of intact and hatched blastocysts. Messenger RNA was not detected at any stage for RXR. Expression of mRNA for RXR, RXRß, RALDH2, and PPAR suggests that the early embryo may be competent to synthesize retinoic acid and regulate gene expression during preattachment development in vitro.
developmental biology, embryo
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Retinol (vitamin A) metabolism in the cell can give rise to many physiologically active compounds [3]. Retinol is secreted into the circulation bound to a specific protein called retinol-binding protein (RBP), which is mainly responsible for its intercellular transport. Because retinol lacks appreciable water solubility, it is bound intracellularly to a second binding protein called cellular RBP (CRBP), which helps solubilize it in the aqueous cellular environment. Before exerting its biological effects, retinol is oxidized to retinaldehyde by a group of enzymes called retinol dehydrogenases; retinaldehyde is then oxidized to retinoic acid (RA) by a second group of enzymes called aldehyde dehydrogenases. RA and its isoforms are believed to interact with 2 separate subgroups of nuclear receptors, retinoic acid receptors (RAR, RARß, RAR) and retinoid X receptors (RXR, RXRß, RXR). The formation of ligand-receptor complexes will either activate or repress specific target genes by binding to specific response elements present in the vicinity of the promoter region.
Approximately a decade ago, the RXRs, the second class of nuclear retinoid receptors responsible for transduction of the differentiating properties of 9-cis RA, was discovered [4]. Similar to the RARs, 3 separate RXR genes (, ß, and ) are known to exist. Retinoid receptors can give rise to isoforms through differential splicing or promoter usage; however, unlike the RARs, receptor isoforms have been isolated only for RXR. RXRs play a crucial role in several nuclear receptor signaling pathways by homodimerizing or heterodimerizing with RARs, thyroid hormone receptors, vitamin D3 receptors, peroxisomal proliferator activated receptors (PPARs), and a number of orphan receptors [5]. Thus, RXRs may be key players in several hormonal pathways.
The extreme sensitivity of embryonic development to vitamin A is clear; both hypovitaminosis and hypervitaminosis A can lead to abortion and embryonic malformation. Important vitamin A-mediated events occur during very early stages in the quail and around the first 2–3 wk of gestation in the human [6]. Retinol supplementation enhanced embryo survival in polytoccus species such as mice [7], rabbits [8], and swine [9]. Retinol administered at the time of superovulation increased the number of transferable embryos in cattle [10] and increased blastocyst formation and hatching rates in sheep [11]. Recently, in the pig, administration of vitamin A to sows before ovulation enhanced embryonal survival by advancing meiotic resumption and altering follicular hormonal environment during follicle maturation [12]. Promotion of embryonic development in these studies suggested a possible direct or indirect interaction with the developing embryo. Hypothesizing a direct interaction, as a first approach, we detected the mRNAs for RBP, RAR, and RAR and the RAR and RAR proteins in bovine preattachment embryos fertilized in vitro [13]. To better understand the retinoid signaling pathway, we used reverse transcription polymerase chain reaction (RT-PCR), whole mount in situ hybridization, and immunohistochemistry to characterize the expression and spatial distribution of RXRs, alcohol dehydrogenase I (ADH-I), retinaldehyde dehydrogenase 2 (RALDH2), and PPAR in preattachment bovine embryos produced in vitro.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovaries were collected from cows at a local abattoir and transported to the laboratory in 0.9% normal saline supplemented with penicillin-G (100 IU/ml) and streptomycin sulfate (0.2 µg/ml) (Sigma Chemical Co., St. Louis, MO) at 26–30°C within 5 h. Oocytes were aspirated from follicles ranging in diameter from 2 to 5 mm using an 18-gauge needle into modified PBS solution (Life Technologies Inc., Rockville, MD). In vitro maturation, fertilization, and culture were performed according to protocols described by Mohan et al. [13].
RNA Extraction
Immature bovine oocytes and embryos at the 2-, 4-, 8-, and 16- to 20-cell, morula, and blastocyst stages were frozen in 250 µl of denaturing solution (4 M guanidium isothiocyanate [Promega, Madison, WI], 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl, 0.1 M 2-ß mercaptoethanol; Sigma). Total RNA was extracted from a pool of 25 embryos at each stage according to the method described by Mohan et al. [13].
Reverse Transcription Polymerase Chain Reaction
To maximize the sensitivity of detection, RT-PCR was the method of choice for investigating gene expression from the small quantities of RNA obtained from bovine embryos. Ten microliters of the eluted total RNA was denatured by heating to 70°C and reverse transcribed in the presence of random hexamers (pdN6, 100 pmole; Pharmacia, Piscataway, NJ), dATP, dTTP, dCTP, and dGTP (dNTPs; Pharmacia), MgCl2, RNase inhibitor (20 U per reaction; Promega), and reverse transcriptase (Superscript, 200 U/reaction; Gibco-BRL, Gaithersburg, MD) at 42°C for 1 h. The RT was terminated by heating to 70°C.
Reverse transcribed cDNA (1–2 µl) was denatured by heating to 95°C and subjected to PCR in the presence of picomole quantities of specific primers, MgCl2, dNTPs, and Amplitaq DNA polymerase (0.5 U/reaction; Perkin-Elmer, Foster City, CA). Specific primers and the PCR conditions used to generate target cDNA fragments using a 2-step PCR procedure are shown in Tables 1 and 2. Because of the variation in published sequences for ADH-I, we used degenerate primers to amplify this target (Table 1). Products of RT-PCR were resolved on 1.5% agarose-TAE (40 mM Tris-acetate, 1 mM EDTA) gels and visualized on an ultraviolet transilluminator following ethidium bromide staining. Representative RT-PCR products from each primer were excised from agarose gels, subcloned, and subjected to dideoxy chain termination sequencing (Model 373A Automated Sequencer; Applied Biosystems, Foster City, CA). The identity of each product was confirmed in a sequence homology analysis using the basic local alignment search tool [14]. This analysis was repeated on 4 separate groups of embryos.
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Whole Mount In Situ Hybridization
All RT-PCR products were cloned into pCR II vector (Invitrogen, Carlsbad, CA). The cDNA-containing plasmids were linearized with BamHI or EcoRV, depending on the orientation, to generate either the antisense or sense probe. The resulting fragments were phenol-chloroform extracted, ethanol precipitated, and used as plasmid templates for riboprobe synthesis (Roche Diagnostics Corporation, Indianapolis, IN). In vitro transcription was performed with 1 µg of plasmid template in a final volume of 21 µl containing the digoxigenin RNA labeling mix, transcription buffer, 10 mM dithiothreitol, RNase inhibitor (1 unit), and SP6 RNA polymerase or T7 RNA polymerase incubated for 2 h at 37°C. The template cDNAs were digested with RNase-free DNase (5 units) for 15 min at 37°C, and the reaction was stopped by adding 0.2 M EDTA, pH 8.0 (2 µl). The riboprobes were ethanol precipitated in the presence of 4 M LiCl and quantified with a series of digoxigenin-labeled control RNAs according to the manufacturer's instructions (Roche Diagnostics).
Day 9–10 hatched blastocysts produced in vitro were fixed overnight in 4% paraformaldehyde, washed in PBS containing 0.1% Tween-20 (PBST), and dehydrated by an ascending methanol concentration series immediately followed by rehydration in the reverse order on ice. Rehydrated embryos were washed 3 times with PBST at room temperature. Embryos were permeabilized by 3 incubations in a cocktail of ionic and nonionic detergents (RIPA: 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 50 mM Tris, pH 8.0), with each incubation lasting 10 min. Embryos were then postfixed with 4% paraformaldehyde/0.2% electron microscopy grade glutaraldehyde in PBS for 20 min. Prehybridization, hybridization, and posthybridization were performed at 60°C for PPAR, 65°C for RXRß, and 70°C for RXR and RALDH2. Embryos were washed 5 times in PBST and incubated for 15 min with a 1:1 mix of hybridization mixture (HB: 50% deionized formamide, 5x saline-sodium citrate [SSC], pH 7.0, 50 µg/ml heparin, 0.1% Tween 20) and PBST followed by a brief wash with HB at room temperature. Embryos were incubated for 1–3 h in prehybridization mixture (HB containing 100 µg/ml tRNA and 100 µg/ml sheared denatured herring sperm DNA). The probes were denatured at 95°C for 10 min and added to the HB mix at the following concentrations: 1 µg/100 µl for PPAR, RXRß, and RALDH2 and 0.5 µg/100 µl for RXR. Hybridization was carried out overnight in a box saturated with 50% formamide/5x SSC to prevent evaporation at the temperatures used for each probe. Posthybridization washes included 50% formamide in 2x SSCT (SSCT: SSC + 0.1% Tween 20) (30 min), 2x SSCT containing 0.5% SDS (2 x 15 min), 0.2x SSCT containing 1% SDS (2 x 15 min), and 0.1x SSCT containing 2% SDS (2 x 20 min).
Specific in situ hybridization signals were detected by incubation in a peroxidase-conjugated anti-DIG antibody solution at a dilution of 1:100 (Roche Diagnostics) and 3,3' diaminobenzidine (DAB; Sigma) for color development. The color reaction was stopped by moving the embryos into PBS. Hatched blastocysts subjected to hybridization with the sense probe served as controls. This procedure was repeated for 4 separate groups of embryos.
Whole Mount Immunohistochemistry
In vitro-produced bovine blastocysts and hatched blastocysts were washed in PBS and fixed in 4% paraformaldehyde overnight at 4°C. Fixed embryos were dehydrated in an ascending methanol series (5 min) and permeabilized in PBS containing 0.1% Triton X-100 (PBST) at room temperature for 40 min. Embryos were then incubated in blocking solution (PBST containing 1% casein) for 1 h. Embryos were incubated with the polyclonal primary antibody (either RXRß or PPAR; Affinity Bioreagents, Golden, CO) at a 1:500 dilution in blocking solution overnight at 4°C. Embryos were washed 5 times in PBST, with the final wash lasting 4 h. Detection of RXRß and PPAR primary antibody was performed using a goat anti-rabbit IgG conjugated to horseradish peroxidase (Kirkegaard & Perry Laboratories, Gaithersburg, MD) diluted at 1:500 in blocking solution and incubated for 2.5 h at room temperature. Embryos were again washed 5 times in PBST, with the final wash lasting 4–5 h. Embryos were then transferred to a dish containing DAB tablets dissolved in 5 ml of water. Embryos were frequently observed for the appearance of a reddish brown color. The color reaction was stopped by moving the embryos into PBS. Control embryos were prepared by omitting primary antibody, secondary antibody, or both primary and secondary antibodies. This procedure was repeated for 2 separate groups of embryos.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in the Early Bovine Embryo
Primer sequences and PCR conditions used to amplify RXR, RXRß, RXR, ADH-I, RALDH2, PPAR, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are given in Tables 1 and 2. Products of the predicted size (Table 1) were detected for each target cDNA (Figs. 1–3). Because mRNA was extracted from a small sample of 25 embryos, a second round of amplification was needed to visualize PCR amplicons. The identity of the PCR products was verified by sequence analysis. The isolated bovine cDNA sequences exhibited a very high (>90%) homology to published mouse and human RXR, RXRß, RALDH2, and PPAR cDNA sequences.
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Transcript for GAPDH was detected at all stages of embryo development examined except the 16- to 20-cell stage (Fig. 1), suggesting that RNA populations suitable for RT-PCR amplification of specific cDNAs were produced. Although methods employed in this study are not quantitative, under similar conditions the level of the message was apparently decreased from 2 cells to below the limit of detection in the 16- to 20-cell stage, increased to the morula stage, and remained elevated through the blastocyst stage. Disappearance of the message between the 8- to 16-cell and the 16- to 20-cell stages suggests that utilization and/or degradation of all maternally derived transcripts had occurred followed by reappearance at the initiation of transcription from the embryonic genome. This finding is in agreement with the model for transition from maternal to embryonic genome control in the cow occurring around the 8- to 16-cell stage [19].
Transcripts for RXR (Fig. 2A) and RXRß (Fig. 2B) were detected in stages from the 2-cell embryo through the hatched blastocyst. However, expression of RXR was not detected at any stage examined (data not shown). As in the case of GAPDH expression, the level of the message for both RXR and RXRß was apparently at or below the limit of detection at the 16- to 20-cell stage and was elevated again at the morula stage. RXR message was very low from the 2-cell through the morula stages until the blastocyst stage, when the transcript levels apparently increased. On the contrary, RXRß expression was clearly evident from the 2-cell to the 8- to 16-cell stage.
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Transcripts for PPAR (Fig. 3A) and RALDH2 (Fig. 3B) decreased from the 2-cell to the morula stage. The level of the message was lower at the 8- to 16-cell and 16- to 20-cell stages and was undetectable at the morula stage. The message levels were elevated again at the blastocyst stage. PCR products obtained for ADH-I agreed in terms of the predicted size. Their sequence exhibited very low homology to known mouse and human sequences. Transcripts for RXR, RXRß, RALDH2, and PPAR were also identified in immature oocytes (data not shown).
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In Situ Localization of Transcripts for RXRs, RALDH2, and PPAR in the Early Bovine Embryo
Identifying the cell type that expressed the RXR and PPAR genes was essential in determining the possible combinations of RXR homo/heterodimers that formed in the embryo during preattachment embryogenesis. RXR transcripts were present in the trophectoderm cells and inner cell mass cells. The hybridization signal obtained with the antisense probe was much stronger than that seen with the sense probe (Fig. 4A). The sense probe, however, produced a mild background signal in the inner cell mass (Fig. 4B). RXRß, RALDH2, and PPAR had a distribution similar to that of RXR. All 3 transcripts were detected in both trophectoderm cells and inner cell mass cells (Fig. 4, C, E, and G). Detection was more specific compared with that of RXR; no signal was obtained with the control sense probes (Fig. 4, D, F, and H). The hybridization signal for all 4 transcripts appeared stronger in the inner cell mass cells than in the trophectoderm cells.
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Presence of RXR Proteins in the Early Bovine Embryo
Whole mount immunohistochemistry revealed immunoreactive RXRß and PPAR proteins in the trophectoderm and the inner cell mass of blastocysts and hatched blastocysts (Figs. 5 and 6). Both RXRß and PPAR proteins were expressed in the same regions as their corresponding transcripts.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and RAR and the mature protein for RAR and RAR [13]. In the present study, we investigated the importance of vitamin A during preattachment development more specifically by characterizing the expression patterns of RXRs, ADH-I, RALDH2, and PPAR in preattachment bovine embryos.
Although RXRß and RXR are ubiquitously expressed in every embryonic tissue during mouse development, RXR shows a restricted pattern during embryogenesis [21, 22]. In earlier studies on Xenopus laevis, RXR and RXR were detected in unfertilized eggs and embryos until gastrulation [24]. Later, in the same species, a third RXR more closely resembling mammalian RXRß was identified and shown to be expressed throughout early development [25]. Extending these studies to preattachment embryos of farm animals, in the present study we detected mRNA for RXR and RXRß in the unfertilized bovine oocyte (data not shown), indicating that these transcripts are synthesized and accumulated during oogenesis. These transcripts showed a steadily declining pattern through early cleavage, indicating usage or degradation until the 16- to 20-cell stage, at which time the embryonic genome becomes activated. Messenger RNA for both RXR subtypes reappeared at the morula and persisted at higher levels until the hatched blastocyst stage. In the blastocyst, both the trophectoderm and inner cell mass cells expressed both RXR subtypes (Fig. 4, A and C). The results of immunohistochemistry suggest that, at least in the blastocyst stages, expression of transcripts for RXRß translates into protein expression (Fig. 5A). Expression of the protein may also enable all trans RA/9-cis RA to exert effects at the level of the nuclear RXRs in both trophectoderm and inner cell mass cells. Messenger RNA for RXR was not detected, suggesting that RXR may not be important during very early stages of mammalian embryo development and that its lack can be compensated for by the presence of the other 2 RXRs. Expression of these RXR subtypes suggests some essential function for these receptors during maturation, fertilization, early cleavage, blastocyst development, and hatching, most likely regulation of gene expression through nuclear receptor-mediated pathways.
The functional significance of RXR expression during embryogenesis has been investigated by creating entire subtype, double, and compound mutant mice. Although RXRß knockout mice appeared morphologically normal [26], homozygous RXR mutant mice died around 13.5–16.5 days postcoitum [27]. Double (RXRß-/-/RXR-/-) and triple (RXR+/-/RXRß-/-/RXR-/-) RXR mutant mice were postnatally normal [28], indicating that early developmental processes could proceed normally provided a single copy of RXR was available to heterodimerize with RARs and other nuclear receptors such as PPARs. Further, RXR-/-/RXRß-/- mutant mice died between 9.5 and 10.5 days of gestation [29], again implicating RXR as the main RXR during early development. Previously, we showed that the preattachment bovine embryo fertilized and cultured in vitro expressed both mRNA and the mature protein for RAR subtypes and [13]. This expression suggests possibilities for a functional interaction between RARs and RXRs in transduction of the retinoid signal around this critical period of development. Detailed analysis of RAR/RXR double-null mutant phenotypes revealed that RXR/RAR heterodimers are the most common functional units responsible in transduction of the retinoid signal during embryogenesis [30]. Messenger RNA for RXR is strongly expressed in the placenta of the mouse [31] and human [32]. Abnormalities of the chorioallantoic placenta have been the hallmark of RXR-/- homozygous [33] and RXR-/-/RXRß-/- compound mutant mice [29], suggesting a critical role for RXR during placentation. During the formation of the blastocyst, retinoid signaling utilizing RXR may contribute to the process of differentiation as loss of RXR alters morphological endodermal differentiation of F9 cells [34].
Presence of the mRNA and protein for the nuclear receptors of the retinoid superfamily prompted us to investigate how retinoid metabolism was normally regulated during preattachment development to provide the biologically active ligand. ADH-I and ADH-IV along with several other enzymes, i.e., 3 forms of microsomal retinol dehydrogenase and short-chain dehydrogenase/reductases, can oxidize all trans-retinol to all trans-retinal [35–38]. We investigated gene expression for ADH-I using degenerate primers in a nested PCR. Despite obtaining a PCR product for ADH-I very close to the required size in all preattachment stages examined, homology searches revealed extremely low similarity to known alcohol dehydrogenases. Therefore, the conversion of retinol to retinaldehyde in bovine embryos needs further scrutiny. Retinaldehyde generated from retinol is oxidized to RA by another group of enzymes called aldehyde dehydrogenases (ALDHs) [39], among which ALDH-I is known to efficiently perform this function [40]. Recently, RALDH2, an NAD-dependant dehydrogenase known to exhibit the greatest specificity for retinaldehyde, was cloned and its expression patterns characterized throughout embryogenesis [41]. RALDH2 is essential for embryo survival and morphogenesis in the mouse, and its complete absence or knockout results in embryonic mortality around midgestation [42]. Transcripts for RALDH2 also occur in immature bovine oocytes. Although not quantitative, the message level started to diminish as early cleavage progressed, indicating that these transcripts were either translated or degraded, and then disappeared but reemerged at the morula stage and remained constant all the way through to the hatched blastocyst stages. Using in situ hybridization, we localized mRNA for RALDH2 to both the inner cell mass and the trophectoderm in the present study (Fig. 4E). The bovine unfertilized oocyte does encounter retinol because of its presence in higher concentrations in healthy follicles [43, 44]. The bovine oocyte also contains transcripts for RBP, which may aid in the uptake of retinol in the follicle [13]. These observations and the detection of RALDH2 in the present study suggest possible RA synthesis and, together with the presence of nuclear receptors, the presence of a retinoid signaling pathway from the oocyte through the hatched blastocyst stage.
Because PPAR is a well-established heterodimeric partner for RXRs, we also examined the embryonic expression and localization of this isoform of PPAR using PCR, in situ hybridization, and immunohistochemistry. The PPAR family of nuclear receptors, which consists of , ß, and isoforms, has received much attention mainly because of its role in the regulation of lipid and glucose metabolism [45]. The 3 isoforms, which are encoded by different genes, differ in their metabolic effects, tissue specific expression, and response to pharmacological agents. PPAR is specifically expressed in adipose tissue and acts to suppress adipocyte differentiation. It has 2 isoforms, 1 and 2, which are derived from the same gene through differential splicing and promoter usage. PPAR was given attention in this study because it is essential for placental development and differentiation in the mouse [46] and is expressed in synctiotrophoblasts and cytotrophoblasts in human placental villi [47], suggesting a similar function. We detected mRNA encoding for PPAR in all stages of preattachment development in vitro, from the 2-cell stage to the hatched blastocyst. Transcripts were also detected in the oocytes, suggesting a likely role during in vitro maturation and fertilization. Expression of PPAR would indicate the presence of a heterodimeric partner for RXRs during preattachment development. With the exception of PPAR, mRNA encoding PPAR and PPARß have been detected in Xenopus oocytes and embryos [48].
All 3 isoforms of PPAR can be activated by many of the same ligands and bind to the same peroxisome proliferator response element in the promoter regions of their target genes. They exert similar influence on transcriptional regulation of several enzymes involved in fatty acid oxidation in vitro [49]. Under in vitro conditions, all 3 PPAR subtypes can interact with RXR, RXRß, or RXR [50, 51]. Thus, under specific conditions regulation of PPAR target gene transcription is contingent on its heterodimerization with RXRs. The occurrence of the PPAR-RXR/RXRß heterodimer can be expected because in the present study PPAR mRNA was coexpressed with RXR and RXRß in both trophectoderm and inner cell mass cells (Fig. 4, A, C, and G). The immunolocalization of PPAR and RXRß protein in both trophectoderm and inner cell mass cells (Figs. 5 and 6) indicates that at least in the blastocyst these proteins are available for interaction.
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The simultaneous expression of RARs [13], RXRs, and RALDH2 provides a compelling argument for the existence of a functional retinoid signaling pathway and support for the hypothesis that the transduced signal functions in the regulation of a subset of genes important during preattachment development in the cow. Providing more support to this argument is the presence of significant concentrations of RA in Day 10 spherical blastocysts and trophectoderm cell lines in the pig, as revealed by HPLC and reporter assays [52]. A specific enzyme, 9-cis retinol dehydrogenase [53], can oxidize 9-cis retinol to 9-cis retinaldehyde, which may then be oxidized to 9-cis RA, a specific ligand that can activate RXRs. However, nothing is known about the activity of this enzyme in bovine preattachment embryos. In Xenopus embryos, RXRs respond to various natural vitamin A metabolites, including RA, albeit at higher concentrations than those of RARs [24]. In the Xenopus study, the presence of both RARs and RXRs in a single cell was advantageous because it gave the embryo an option to respond to a shallow gradient of RA through differential activation of either RARs or RXRs. Whether the expression of RXRs and RARs along with PPAR would encourage homodimerization or heterodimerization, thereby activating some putative retinoid responsive target genes during preattachment development in the cow, is largely unknown and needs future attention.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was supported by the Oklahoma Agricultural Experiment Station. 
2 Correspondence: J.R. Malayer, Department of Physiological Sciences, Oklahoma State University, 264 McElroy Hall, Stillwater, OK 74078–2006. FAX: 405 744 8263; malayer{at}okstate.edu
Accepted: October 18, 2001.
Received: August 2, 2001.
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in disease of pancreatic ß cells. Proc Natl Acad Sci U S A 1998; 95:8898-8903+/-/RXRß-/-/RXR-/- mutant mice are viable. Proc Natl Acad Sci U S A 1996; 93:9010-9014 null fetuses. Dev Biol 1997;; 191:29-41[CrossRef][Medline]-null F9 embryonal carcinoma cells are resistant to the differentiation, anti-proliferative and apoptotic effects of retinoids. EMBO J 1996; 15:4142-4155[Medline]This article has been cited by other articles:
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