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Regular Article |
a Interdisciplinary Concentration in Animal Molecular and Cell Biology, Department of Animal Sciences, University of Florida, Gainesville, Florida 32611-0910
b Department of Molecular & Integrative Physiology, University of Illinois, Urbana-Champaign, Illinois 61801
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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64-kDa protein corresponding in size to human ovarian granulosa cell ER-ß, respectively. In Day 12 filamentous embryos, ER-ß expression was immunolocalized to trophoblastic cell nuclei, coincident with that of proliferative cell nuclear antigen (PCNA). The developmental ontogeny of ER-ß mRNA was evaluated in embryos of different morphologies (spherical, tubular, and filamentous) by semiquantitative RT-PCR, along with those for other steroid hormone receptors (ER-
and progesterone receptor) and known embryonic genes associated with cell differentiation (cytochrome P450 aromatase type III) and growth (cyclin D1). ER-ß mRNA levels varied with embryo morphology (filamentous maximum at Day 12), coincident with that of cyclin D1. Progesterone receptor mRNA levels were maximal in tubular embryos, similar to that of P450 aromatase, whereas the expression of the ER- gene was barely detectable and appeared constitutive for all developmental stages examined. Estradiol-17ß treatment of Day 12 filamentous embryos in culture up-regulated ER-ß and P450 aromatase (type III) mRNA levels, respectively, but decreased those of PCNA, and had no effect on cyclin D1 mRNA levels. These studies taken together suggest that embryonic ER-ß likely mediates the autocrine functions of estrogens in the dynamic regulation of embryonic growth and development at periimplantation.
embryo, estradiol receptor, implantation, pregnancy, steroid hormone receptors
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-hydroxylase, the last 2 enzymes in the estrogen biosynthetic pathway [3, 4]. Peak levels of embryonic estrogen occur around Days 11 and 12, a period correlated with maternal recognition of pregnancy [5]. The main target of this estrogen is likely the endometrium, which exhibits functional estrogen receptors (ERs) [6] and which, upon estrogen and progesterone stimulation, secretes a myriad of growth factors, cytokines, proteases, protease inhibitors, and other yet-unknown molecules, which are requisite for implantation and pregnancy success [7, 8].
The biological effects of estrogen are mediated by two ER subtypes, ER- and ER-ß [9]. Although the distribution of both ER isoforms has been widely documented in female reproductive tissues of numerous mammalian species, and their respective functions have been inferred from the use of specific ER- and ER-ß knockout mouse models [10], studies that address the embryo as a direct target for estrogen action via ER-mediated mechanisms are limited [11–13] and, in some cases, conflicting [14, 15]. Moreover, the functionality of the reported embryonic ER has not been extensively evaluated, specifically as it relates to potential regulatory roles in embryonic growth, development, or both.
The pig, like the human, suffers from high rates (30%–40%) of periimplantation embryo mortality [16]. The cause for this condition in humans is not well understood; in the pig, however, asynchronous development, in which the larger, more developed embryos (tubular and filamentous) impair the development of their smaller (spherical) counterparts, has been postulated to underlie embryonic death [17]. The possibility that estrogen is involved in this asynchrony was borne from the findings that estrogen is toxic to porcine embryos when administered before the time of maternal recognition of pregnancy [18]. The more developed porcine embryos also exhibit greater estrogen production than less developed counterparts [2, 4], and a line of pigs (Meishan) exhibiting increased prolificacy and greater within-litter synchrony in embryo development displayed lower levels of estrogen production than the Large White line, which suffers from high embryo mortality [19].
ER-mediated estrogen action has long been implicated in the control of cell growth [20]. Moreover, recent data have provided increasing support to the notion that positive or negative regulation of cell growth by estrogen is a function, in part, of the ER subtype expressed in target cells [21–24]. Thus, the identification of the predominant ER isoform in porcine developing embryos, if present, is necessary for determining whether and how estrogen plays an essential embryotrophic role in early pregnancy events. Indeed, a finding of distinct expression of ER- and ER-ß by porcine embryos at discrete developmental stages will likely provide important insights into possible molecular mechanisms through which estrogen may be correlated with embryonic asynchrony.
The major objective of the present studies was to evaluate whether distinct ER isoforms are expressed by periimplantation embryos. Toward this end, the sequence of the complete open reading frame of porcine embryo ER-ß mRNA was determined. Further, the developmental expression of ER-ß, relative to ER-, and in concert with those of progesterone receptor (PR) and other genes (P450arom type III, cyclin D1) known to be involved in growth and differentiation, was evaluated in embryos of different morphologies by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Finally, in vitro cultures of Day 12 filamentous embryos were used to examine the effects of estrogen on ER-ß, P450arom, cyclin D1, and proliferative cell nuclear antigen (PCNA) gene expression.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cross-bred gilts with regular estrous cycles were used in accordance with procedures approved by the University of Florida Institutional Animal Care and Use Committee (A463). Animals were killed at the Meats Processing Facility on Pregnancy (Px) Day 10, 12, or 14. Porcine embryos were collected and separated according to size (diameter for spherical embryos) and morphology (spherical, tubular, or filamentous) as previously described [4]. Total cellular RNA was isolated from tissues using TRIzol reagent (Gibco BRL, Grand Island, NY) following the manufacturer's instructions.
Porcine ER-ß cDNA Cloning and Nucleotide Sequence Analysis
Complementary DNA was transcribed from total RNA (5 µg) prepared from porcine Day 12 filamentous embryos following the manufacturer's protocol (cDNA Cycle kit; Invitrogen, Carlsbad, CA). The strategy used for PCR cloning is shown in Figure 1. Several primer sets based collectively on conserved sequences from rat, human, bovine, and ovine ER-ß mRNAs were synthesized (Gemini Biotech, The Woodlands, TX), and employed for PCR amplification (Table 1) using an Eppendorf Mastercycler Gradient (Eppendorf Scientific Inc., Westborg, NY) under optimized MgCl2 concentrations and pH in 1x PCR buffer (5x = 300 mM Tris-HCl, 75 mM ammonium sulfate). The PCR products were electrophoresed on 1.5% agarose gels, and the expected DNA fragments were subcloned into TOPO vector (Invitrogen). The nucleotide sequences of the generated clones were determined at the nucleotide sequencing facility of the Interdisciplinary Center for Biotechnology Research of the University of Florida, and compared with those of other mammalian ER-ß sequences to confirm their identities and locations relative to other ER-ß mRNA sequences previously reported in GenBank. The porcine embryonic ER-ß reported here has GenBank accession number AF164957.
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Northern and Western Blot Analyses
Total cellular RNA isolated from Day 12 filamentous embryos, and poly(A)+ RNA purified on oligo(dT) columns (Qiagen Inc., Valencia, CA) from total cellular RNA prepared from Day 12 pregnant pig uterine endometrium were fractionated on a 1% agarose-formaldehyde-MOPS (3-N-morpholino-propanesulfonic acid) gel, and transferred onto a Biotrans nylon membrane (ICN Biotech, Irvine, CA) by capillary transfer. The membrane was hybridized to nick-translated porcine ER-ß cDNA (gel-purified, 491 base pair [bp] insert from the 5' end of the porcine embryo ER-ß mRNA; Fig. 1) at 42°C overnight in UltraHyb buffer (Ambion Inc., Austin, TX). After hybridization, the membrane was washed twice for 15 min each at 42°C in 2x saline sodium citrate (SSC), 0.1% SDS, and then in 0.1x SSC, 0.1% SDS for 30 min at the same temperature. The presence of ER-ß transcripts was detected by autoradiography at -80°C. The sizes of the transcripts were calculated based on their migration positions relative to those of 18S and 28S ribosomal RNAs.
For Western blot analysis, nuclear extracts prepared from different pig tissues, following previously described protocols [25] and whole cell extracts from Day 12 filamentous porcine embryos and human ovarian granulosa cells were fractionated on 10% polyacrylamide-SDS gels under reducing conditions. Proteins were electrophoretically transferred onto a nylon membrane (Dot Scientific, Inc., Lippincott Borton, MI), and immunoblotting was performed with a mouse monoclonal antibody against human ER-ß (CWK-F12) for 24 h at 4°C [26]. Following incubation with anti-mouse secondary antibody (immunoglobulin G [IgG] fraction), the filter was rinsed in 5 changes of TBS buffer (10 mM Tris, 150 mM NaCl pH 7.4) containing 0.2% Tween-20, and binding was detected by enhanced chemiluminescence (Amersham-Pharmacia Biotech Inc., Piscataway, NJ). The blots were exposed to x-ray films (Fuji; Fisher Scientific, Pittsburgh, PA) to detect the anti-ER-ß immunoreactive band.
Immunolocalization of ER-ß and PCNA
Immunohistochemistry of Day 12 filamentous embryos using anti-ER-ß polyclonal antibody was performed as previously described [27]. In brief, filamentous embryos were fixed in 4% paraformaldehyde for 24 h and then processed and embedded in paraffin. Sections of 5 µm were cut, deparaffinized, and subsequently rehydrated through a series of alcohol dilutions. Sections were placed in 0.01 M citric acid (pH 6) and microwaved for 15 min to expose the cellular antigens [28]. Nonspecific binding and endogenous peroxidase activity were blocked by incubation of sections in 1% hydrogen peroxide and then in normal horse serum (2% final concentration). The slides were then incubated overnight at 4°C with polyclonal anti-ER-ß antibody (10 µg/ml; Upstate Biotechnology, Lake Placid, NY) generated in rabbits against the N-terminal region (amino acids 54 to 71; YAEPQKSPWCEARSLEHT) of the rat ER-ß protein sequence [29]. The PCNA antibody used (3 µg/ml) was an anti-rat PCNA mouse monoclonal antibody (Roche Diagnostics Corp., Indianapolis, IN). As negative controls, parallel tissue sections were incubated with normal rabbit or mouse IgG at the same concentrations as the corresponding test antibodies. The respective biotinylated anti-rabbit (Biomeda Corp., Foster City, CA) or anti-mouse (Vector Laboratories Inc., Burlingame, CA) IgGs were used as secondary antibodies. Sections were placed into a substrate solution of diaminobenzidine (Sigma Chemical Company, St. Louis, MO) for staining. Photomicroscopy was performed using a Zeiss Axioplan 2 microscope with an RGB Spot Digital camera system with corresponding software (Carl Zeiss Inc., New York, NY).
RT-PCR Analysis of Embryo Gene Expression
Complementary DNA was synthesized from equal amounts of total RNA (5 µg) prepared from porcine embryos of different sizes and morphologies as previously described [4]. PCR amplification was performed in a total reaction volume of 50 µl containing 1 µl of cDNA template, 50 pmol of the forward and reverse primers (Table 2), 10 nM of each dinucleotide triphosphate, 1x PCR buffer, and 1 unit of Taq polymerase (Boehringer-Mannheim, Mannheim, Germany). To eliminate the possibility of PCR amplification of genomic DNA that may be present in RNA samples used for cDNA synthesis, the set of primers for each target gene (except ER-ß, because the intron/exon organization of its porcine chromosomal gene is currently lacking) were designed to span introns. In addition, another set of primers that detected a larger size PCR product when traces of genomic DNA were present in the sample, as compared with the predicted smaller sized PCR fragment in samples free of genomic DNA, was used to screen embryo samples; those deemed to contain genomic DNA by this methodology were eliminated from further analysis. For each PCR reaction, the number of cycles used was optimized so that the amplification process was carried out within the exponential (linear) range. All PCR products (20 µl of 50 µl total reaction volume) were electrophoresed in 1.5% agarose gels containing ethidium bromide, and were visualized over UV light. DNA fragments were quantified using an Alpha Imager 2000 Documentation and Analysis System (Alpha Innotech Co., San Leandro, CA).
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Embryo In Vitro Culture
Porcine Day 12 filamentous embryos were preincubated in Medium 199 (M199; Gibco BRL) containing 1% antibiotic/antimycotic solution at 37°C in an atmosphere of 50% N2, 47.5% O2, 2.5% CO2 for 2 h, immediately after removal from the uterus. Medium was then replaced with fresh M199 containing vehicle alone (ethanol) or estradiol-17ß (E2; 50 nM final concentration; Sigma) in vehicle, and embryos were further incubated for 24 h under the same conditions. The dose of E2 used was chosen based on the reported range of estrogen levels [2] to which periimplantation embryos are exposed in utero at Days 11–12 of pregnancy. The embryos were then collected for RNA isolation and subsequent RT-PCR analysis.
Statistical Analysis
Data from in vitro embryo culture gene expression analysis (n = 5 experiments, in which each experiment represents pooled embryos from a different Day 12 pregnant pig) were subjected to least squares ANOVA using the General Linear Models procedures of the Statistical Analysis Systems [30]. Values were considered significant at P < 0.05, and are presented as least squares means ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Analysis of the published nucleotide sequences of ER-ß mRNAs from human [31], rat [32], mouse [33], ovine (GenBank accession number AF177936), and bovine [27] allowed the design of multiple primer sets within conserved regions, which were then used to generate overlapping DNA fragments using porcine Day 12 filamentous embryo RNAs as a template for RT-PCR. The 4 overlapping fragments of variable sizes (491, 540, 409, and 492 bp; Fig. 1) were sequenced. The composite sequence of 1578 nucleotides encodes a 526 amino acid porcine ER-ß protein. Alignment of this deduced amino acid sequence with those reported for other mammalian species revealed an overall identity, on average, of greater than 90% (Fig. 2). The highest identity of the porcine ER-ß was observed to the bovine and ovine homologs (92% and 91%, respectively), with lesser identity demonstrated to human (86%), rat (87%), and mouse (86%) proteins. The N-terminal (amino acid residues 1–40) and ligand binding (amino acids residues 228–526) domains of pig ER-ß were less conserved among mammalian ER-ß proteins. By contrast, the region encompassing amino acid residues 141 to 227, which spans the DNA binding domain of this nuclear receptor, exhibited 100% identity among these species.
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Characterization of Porcine ER-ß mRNA and Protein
Total RNA prepared from filamentous embryos collected at Day 12 of pregnancy and poly(A)+ RNA isolated from Day 12 pregnant pig endometrium were used in Northern blot analysis using a radiolabeled probe corresponding to the N-terminal region (nt 1 to 491) of porcine embryo ER-ß mRNA. As shown in Figure 3A, multiple transcripts ranging from 9.5 to 3.5 kilobases (kb) were observed in Day 12 filamentous embryos, in agreement with the sizes described for ER-ß mRNA in other species [33]. Pregnant porcine endometrium also displayed these same multiple transcripts (Fig. 3A). Whereas the 4.9-kb transcript was, interestingly, predominant in Day 12 filamentous embryos, the expression of the 3 major transcripts were comparable in the corresponding endometrium.
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Western blot analysis was used to determine the size and expression of the ER-ß protein in pig tissues using a mouse anti-human ER-ß monoclonal antibody (CWK-F12; [26]). This antibody detects a region (amino acids 273–285) within the hormone binding domain that is highly conserved (except for one conservative amino acid substitution) between the human and porcine sequences. A protein of 64 kDa was detected in whole tissue extracts from Day 12 filamentous embryos and in nuclear extracts prepared from pregnant pig endometrium (Day 12 Px) and myometrium (Day 14 Px). The size of this protein corresponds to that of the ER-ß determined for human granulosa cells (Fig. 3B), as described previously [26].
Inmunolocalization of ER-ß and PCNA
Immunocytochemistry of Day 12 filamentous porcine embryos with anti-ER-ß antibody revealed immunostaining of trophoblastic cells (Fig. 4B). This staining was found to be predominantly nuclear in location (Fig. 4, B and D). Positive immunostaining for ER-ß was also demonstrated in endometrial tissue sections prepared from a Day 60 pregnant pig (Fig. 4F). In the latter, immunostaining was restricted to endometrial luminal and glandular epithelial cells, and largely undetected in stromal cells. Parallel serial sections of Day 12 filamentous embryo and Day 60 pregnant uterine endometrial tissues did not show any staining when incubated with normal rabbit IgG at concentrations (10 µg/ml) equal to that of anti-rat ER-ß antibody (Fig 4, A, C, and E).
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To correlate expression of embryonic ER-ß with growth status, immunolocalization of the growth-associated marker protein PCNA was evaluated in Day 12 filamentous porcine embryos. Intense staining in nuclei of trophectoderm cells was observed in Day 12 filamentous embryo tissue sections incubated with anti-PCNA antibody (Fig. 5B). Parallel sections incubated with normal mouse serum IgG (Fig. 5A) showed no detectable immunostaining.
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ER-ß and ER- Gene Expression by Periimplantation Embryos
Primer sets for the detection of porcine ER- and ER-ß transcripts in periimplantation embryos were synthesized (Table 2), and the products from each PCR reaction were confirmed for authenticity by nucleotide sequence analysis. Primers sets were also designed to detect transcripts for PR, cytochrome P450arom type III, cyclin D1, and the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in the same samples. Porcine embryos collected at different stages of pregnancy (Days 11, 12, 13, and 14), were separated according to size and morphology and analyzed for expression of the specific mRNAs by semiquantitative RT-PCR. To ensure that the amounts of the starting material for the PCR reactions were the same for all samples, the yields from the RT reactions were equalized using GAPDH (Fig. 6). In this strategy, the amount of cDNA that would yield an equivalent amount of GAPDH PCR product was determined for each cDNA preparation, and this aliquot was then used in all PCR reactions for each cDNA sample.
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As shown in Figure 6A, the levels of ER-ß mRNA varied with embryo morphology. ER-ß gene expression was low in spherical embryos (5–9 mm), increased progressively in embryos at the tubular stages, and reached maximal levels in filamentous embryos at Day 12 of pregnancy. A significant decline in ER-ß gene expression was subsequently observed in filamentous embryos at Pregnancy Day 14 (Fig. 6, A and C). By contrast, ER- gene expression was barely detectable (and only after Southern blot hybridization analysis), and remained relatively constant for all embryos of different morphologies (Fig. 6A). PR mRNA expression was low in spherical embryos, increased in tubular embryos, and was significantly diminished in filamentous embryos (Fig. 6, A and C). The expression of cyclin D1, a gene positively correlated with growth, was low at spherical stages, increased significantly by the tubular stage, and reached maximal levels in Day 12 filamentous embryos, with a significant decline observed in Day 14 filamentous embryos (Fig. 6, A and C). The pattern of P450arom type III gene expression was consistent with results of a previous study from this laboratory [4], with detectable expression in spherical embryos, and peak levels of expression for tubular embryos, which moderately decreased in Day 12 filamentous embryos to nearly undetectable levels by Day 14 (Fig. 6B).
Embryo Gene Expression in Response to E2 Treatment In Vitro
To evaluate whether the ER-ß gene expressed in porcine Day 12 filamentous embryos is functional, experiments were conducted to analyze the effects of added E2 on the pattern of gene expression of these embryos by RT-PCR (Fig. 7). Embryos exposed to E2 (50 nM for 24 h) had increased levels of ER-ß transcripts over those of control embryos receiving only vehicle. This increase in ER-ß transcript levels was accompanied by an increase in P450arom type III gene expression, which was normally barely detectable in Day 12 filamentous embryos in culture. In contrast, a decrease in PCNA gene expression was observed in embryos treated with E2 compared with control embryos. Cyclin D1 gene expression in embryos exposed to E2, however, did not differ from those of control embryos. In these studies, gene expression levels for all samples were normalized to that of GAPDH.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and to a very limited extent ER-ß, in embryos of mouse and pig [12, 13, 15, 34], a direct comparison of the expression levels of these steroid receptors as a function of periimplantation development in these species, or for that matter, any mammalian species, has not been reported. Moreover, the functional implication of the expression of either receptor subtype to embryonic developmental events has not been specifically evaluated. In the present study, we addressed the developmental expression patterns of both ER subtypes in porcine embryos of differing sizes and morphologies by semiquantitative RT-PCR, and correlated these with other embryonic genes whose expression serves as important parameters for tissue growth, differentiation, or both. Our results indicate that expression of the ER-ß gene is significantly higher and more tightly regulated than that of ER- in periattachment porcine embryos, and that this pattern of expression for ER-ß mRNA parallels that of cyclin D1. Morever, we found that the ER ligand E2 can potentially autoregulate ER-ß gene expression, with the demonstrated increase in ER-ß transcript levels with added E2 occurring coincident with diminished PCNA gene expression. Taken together, these data suggest a functional linkage between ER-ß and embryonic growth, with estrogen produced by developing embryos through the embryonic (type III)-specific P450arom [35] exercising a major role in the initiation or propagation of these events.
The present study provides the first report of the complete coding sequence for porcine embryo ER-ß. Sequence analysis of the isolated overlapping porcine ER-ß cDNA fragments revealed an open reading frame of 1578 bp, which encoded a protein of 526 amino acids, and which displayed significant (92%) homology with ovine (GenBank accession number AF110402) and bovine (GenBank accession number AF177936) ER-ß molecules. A difference noted between the porcine cDNA sequence and those of the other two is the 3-nucleotide gap at position 1241 in the pig, which corresponds to a missing proline in the deduced protein sequence. The high sequence homology (90% overall) of porcine embryonic ER-ß protein with those previously reported for other mammals, coupled with the exact correspondence of this sequence with that recently described for porcine ovarian ER-ß (GenBank accession number AF267736), confirm the authenticity of the embryonic ER-ß reported here. Moreover, these data validate the results of the biological assays described herein that employed heterologous antibodies (anti-human and anti-rat) to determine the location (trophoblast compartment, nuclear) and size (64 kDa) of the porcine embryo protein.
The use of primer sets designed within a region of the ER-ß mRNA that had no homology with that of ER- to detect and quantify embryonic ER-ß gene expression during development provided the requisite stringency and specificity to the assessment of one or more bona fide ER-ß transcripts. However, our finding by Northern blot analysis that multiple transcripts exist for embryonic ER-ß, similar to that found for the maternal uterus, is not entirely consistent with our identification of only one cDNA sequence corresponding to porcine embryonic ER-ß. The presence of several ER-ß subtypes with variations in the carboxy-terminal regions has been reported for other species [36, 37]. One likely explanation for our observation may be related to the strategy by which the overlapping cDNA fragments reported here were isolated. Because primer sets were designed to optimize the isolation of the anticipated full-length transcript, based on the reported sequences of other mammalian ER-ß cDNAs, the detection of lesser abundance or shorter cDNA fragments could have been inadvertently omitted. Thus, it is not possible to conclude from the present studies whether other ER-ß isoforms previously reported for the human exist for the pig as well, although the recent report of a unique porcine ovarian ER-ß cDNA sequence (GenBank accession number AF267736) of exact sequence and size is in agreement with the findings reported here.
The dynamic regulation of ER-ß gene expression at periimplantation, occurring coincident with the rapid changes in embryo morphology from spherical to tubular to filamentous, and with the production levels of estrogen via P450arom (type III), suggests a correlation between ER-ß mediated action of embryonic-derived estrogen and control of embryo growth. Several lines of evidence obtained in the present study support this postulated linkage. First, peak P450arom type III gene expression of these embryos occurred in Day 11 tubular embryos, immediately preceding maximal expression of ER-ß gene in Day 12 filamentous embryos. Second, E2 increased embryonic expression of P450arom gene in Day 12 filamentous embryos in vitro. Third, peak levels of ER-ß mRNA immediately preceded the significant reduction of cyclin D1 transcript levels in Day 13/Day 14 filamentous embryos in vivo. Fourth, addition of E2 to Day 12 filamentous embryos in vitro resulted in a significant diminution of PCNA gene expression and did not alter basal cyclin D1 mRNA levels. Finally, ER- gene expression during this developmental period appears to be low and constitutive, arguing against its active participation in the observed dynamic changes in specific gene expression. Data from other laboratories using various other model systems support a role for ER-ß in mediating inhibition of growth [22–24]. Indeed, using a recently described porcine trophoblastic cell line derived from Day 12 filamentous embryos [38], we demonstrated the inhibition of labeled thymidine incorporation into cellular DNA, indicative of diminished cellular proliferation, upon exposure of these trophoblastic cells to a dose of E2 (50 nM) used in the in vitro embryo culture experiments presented here [39]. Although the mechanism for this effect of ER-ß, distinct from the proliferative action of ER-, in growth processes remains unclear, the recent demonstration of a physical interaction between ER-ß protein and a nuclear protein, termed MAD2, which serves as a mitotic checkpoint, allowing cells to divide when chromosomes are perfectly aligned, may in part underlie this observation [40]. The interaction of MAD2 with ER-ß could, conceivably, interfere with the function of MAD2, resulting in the inhibition of mitogenesis.
An interesting finding from these studies is the demonstrated up-regulation by E2 of ER-ß gene expression by Day 12 filamentous embryos in vitro, which was consistent with the developmental increase in ER-ß transcript levels observed in Day 12 filamentous embryos occurring immediately after peak P450arom gene expression by Day 11 tubular embryos. Under these conditions, a parallel increase in ER- gene expression was not observed (Fig. 6 and data not shown). This positive regulation by E2 of ER-ß gene expression contrasted with the direction observed for PCNA, highlighting the specificity of the E2 effect. Moreover, this finding is also consistent with published data documenting the induction by E2 of ER-ß mRNA levels in rat decidual cells [41], and the presence in the human ER-ß gene promoter region of estrogen-responsive sequences that may mediate this regulatory role of E2 at the level of gene transcription [42]. The additional observation that E2 induces P450arom type III gene expression in vitro, suggests that embryonic sensitivity to estrogen may be regulated at two distinct levels; namely, P450arom and ER-ß gene expression, respectively, to ensure enhanced embryonic estrogen effects.
In a recent study, Kao et al. [43] demonstrated that the major steroid product of the stably expressed porcine P450arom type III (blastocyst) isoform is not estradiol, but rather, 19-nortestosterone (19-nor). The relevance of such an observation in vivo is not clear at the present time, because measurements of 19-nor concentrations in uterine luminal fluids from periimplantation uteri have not been extensively evaluated. However, 19-nor has been shown to bind ER-ß, albeit with low affinity [44]; thus, if indeed high concentrations of 19-nor are present within the uterine microenvironment, these could compensate for its low affinity for ER-ß and could conceivably influence ER-ß signaling pathways within the embryo. The distinct functional consequences on embryonic growth and development of ER-ß bound to 19-nor from that bound to estradiol is an interesting question that requires further investigation.
The results presented here highlight a natural selection process possibly occurring in utero, whereby more developed embryos, through their estrogen production, inhibit the growth of less developed embryos expressing ER-ß, leading to their ultimate demise. In this regard, a breed of pigs known for its low embryonic mortality (i.e., Meishan) is characterized by lower embryonic estrogen production [45] and lesser asynchrony in development [19]. Indeed, the potential ability of embryonic-derived estrogen to coordinate the inhibition of embryonic growth through ER-ß on one hand, and the induction of uterine endometrial activity through production of growth factors on the other [46], the latter possibly occurring through ER-, suggest the versatility of embryonic estrogen action, which could conceivably be manipulated for increasing pregnancy success. Thus, further elucidation of the potential involvement of ER-ß on the mechanism underlying embryo mortality using the pig system may provide clues to the etiology of implantation defects during early pregnancy in humans, whose embryos produce estrogens, albeit at lower levels than those of pigs [47].
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by grants from the U.S. Department of Agriculture (98-35205-6739) and National Institutes of Health (HD-21961) to F.A.S. and R.C.M.S.; and by a grant from the National Institutes of Health (CA-18119) to B.S.K. This is journal series R-08390 from the Florida Agricultural Experiment Station. 
2 Correspondence. FAX: 352 392 7652; simmen{at}animal.ufl.edu
Accepted: October 18, 2001.
Received: July 19, 2001.
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