MOLECULAR REPRODUCTION AND DEVELOPMENT 55:83–88 (2000)
Estrogen Receptor Is Expressed in Pig Embryos During Preimplantation Development CHINGWEN YING,1* WEI-LU HSU,1 WEI-FONG HONG,1 WINSTON T.K. CHENG,2 AND Y.-C. YANG1 1Department of Microbiology, Soochow University, Taipei, Taiwan, Republic of China 2Department of Animal Sciences, National Taiwan University, Taipei, Taiwan, Republic of China
ABSTRACT Although estrogen is recognized as essential for embryonic development and maintenance of pregnancy, it remains unclear whether it has a direct role in the embryos themselves. The aim of this study was to investigate whether estrogen can have any effect in pig embryos during preimplantation development. Since the function of estrogen is mediated through its specific receptor, estrogen receptor (ER), the presence of ER mRNA and protein in pig embryos collected in vivo at different stages of preimplantation development was determined and compared. Using reverse transcription polymerase chain reaction, ER RNA was detected at the one-cell, two-cell, and four-cell stages. The level became undetectable at the five- to eight-cell stages and the morula stages and then reappeared again at the blastocyst stage. To determine whether the ER message observed in the embryos was translated into ER protein, immunocytochemical analysis was performed and the presence of ER protein was detected in oocytes at one-cell and four-cell stages. However, the amount of ER protein in porcine embryos at the blastocyst stage was still below the detection limit. The presence of ER mRNA at the blastocyst stages suggests that estrogen may start to act directly on pig embryos afterwards, and our results provide a basis for determining the direct role of estrogen in preimplantation pig embryos. Mol. Reprod. Dev. 55:83– 88, 2000. r 2000 Wiley-Liss, Inc. Key Words: embryonic gene expression; estrogen; implantation
INTRODUCTION Estrogen is required for normal preimplantation, embryonic development in the reproductive tract, and the uterine changes induced by estrogen have been well documented (Weitlauf, 1994). Elimination of estrogen from pregnant animals causes deleterious effects on the development and implantation of the embryos. Hypophysectomy of pregnant rats results in the delayed entry of eggs into the uterus, expulsion of eggs from the uterus and retarded development of eggs. In pigs, estrogen is essential for the transformation of the compacted morula to the cavitated blastocyst stage (Niemann and Elsaesser, 1986). Administration of antiestrogen in culture medium impaired the transforma-
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tion of pig morulae to blastocyst (Niemann and Elsaesser, 1987). The cleavage rate of porcine embryos was significantly enhanced by coculturing fertilized eggs with estrogen-treated oviductal epithelial cells (Xia et al., 1996). The maternal recognition of pregnancy in pigs, which is established about 11–12 days after the start of estrus, was influenced by estrogen (Geisert et al., 1990). Implantation in pig has also been found to be preceded by synthesis of estrogen by the conceptus to maintain functional corpora lutea throughout pregnancy (Geisert and Yelich, 1997; Gadsby et al., 1980) while preimplantation mouse blastocyst showed no ability to synthesize estrogen (Stromstedt et al., 1996). The exact means by which estrogen regulates embryo development is still not well understood and remains controversial. Wu et al. (1992) have suggested that the effects of estrogen on embryos were indirect and preimplantation embryogenesis may be controlled by certain paracrine factors originating from the reproductive tract under the influence of estrogen. In addition, the growth rate of preimplantation embryos grown in vitro is retarded compared to embryos in vivo (Wu et al., 1971). On the other hand, subsequent studies on mouse preimplantation embryos have suggested that estrogen may act directly on the embryos (Hou and Gorski, 1993; Hou et al., 1996). Estrogen is thought to regulate female reproductive functions primarily through the nuclear estrogen receptor–alphas (ER-alpha), and only after hormone binding does the ER-alpha protein become activated and serve as a transcription factor that modulates the expression of target genes (Beato, 1989; Das et al., 1997; Rissman et al., 1997). Mice lacking ER-alpha showed severe reproductive and behavior phenotypes, including complete infertility of both male and female mice and inhibition of the induction of female sexual behaviors by estrodiol and progesterone (Moffatt et al., 1998). The ER-alpha knockout mouse model also revealed that ER-alpha is required for successful ovulation and estrogen-induced epithelial mitogenesis in female reproductive organs (Cooke et al., 1998; Schomberg et al., 1999).
Grant sponsor: Agricultural Council Republic of China; Grant numbers: 85-AST-1.1-FAD-49(30) and 86-AST-1.1-AID-06. *Correspondence to: Chingwen Ying, Dept. of Microbiology, Soochow University, Taipei, Taiwan, R.O.C. E-mail:
[email protected] Received 13 May 1999; Accepted 27 July 1999
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ER-beta, a novel steroid receptor that is expressed in rat prostate and ovary, shares many of the functional characteristics of ER-alpha, but the molecular mechanisms regulating the transcriptional activity of ERbeta may be distinct from those of ER-alpha (Tremblay et al., 1997). Observation of mice lacking ER-beta indicated that ER-beta is essential for normal ovulation efficiency but is not essential for female or male sexual differentiation, fertility, or lactation (Krege et al., 1998). In this study, we detected the presence of ER-alpha mRNA and ER protein assuming that for estrogen to have direct effects on the pig embryos at the early developmental stage, ER must be present in these embryos. Our results showed that ER protein is indeed present in preimplantation pig embryos and provides a basis for the direct effect of estrogen on the embryos. These results also suggest that both maternal and embryonic sources of mRNA are involved. MATERIALS AND METHODS Animals Gilts at the age of 12–15 months old were treated with 0.4% altrenogest Regumate porcine (Hoechst U.K. Ltd., Bucks, UK) for 18 days and then superovulated by intraperitoneal injection of 1,500 units of pregnant mare’s serum gonadotrophin, followed by 1,000 units of human chorionic gonadotophin (hCG) 72–80 hr later. The pigs were artificially inseminated twice (at 24 hr and 30 hr after the last intraperitoneal injection). The preimplantation embryos were collected 52 hr (one-cell stage), 64 hr (two-cell stage), 80 hr (four-cell stage), 100 hr (five- to eight-cell stage), 120 hr (morular stage) or 144 hr (blastocyst stage) after the last hCG injection by flushing the oviducts and uterine horns of the pregnant female pigs with Dulbecco’s phosphate buffer saline (PBS). After flushing, the uterine horns were surgically removed from the female pigs, washed with Dulbecco’s phosphate buffer saline, and then snap-frozen and stored at ⫺70°C until use. RNA Extraction Total embryonic RNA was isolated from a pool of embryos at the same stage of development (each pool consisted of about 100 cells equivalent embryos) by microadaptation of the guanidine/cesium chloride method. Cells were lysed with 150 µl of RNAzol containing 15 µg of Escherichia coli rRNA as a carrier, and the resulting lysate was precipitated, washed, and resuspended in water. Total uterine RNA was prepared according to the method of Chomocyznski and Sacchi (1987). RT-PCR For reverse transcription, extracted total RNA equivalent to 38 cells was reverse transcribed with Superscript II reverse transcriptase (BRL, Gibco, Grand Island, NY) in a MicroAmp tube (Perkin Elmer, Foster City, CA) as described by Hsu et al. (1999a). One tenth of the RT product was then subjected to PCR in a DNA thermal cycler. Each tube contained 2 µl 10⫻ PCR
buffer, 1.6 µl 50 mM MgCl2, 12.7 µl DEPC-treated water, 1.5 µl RT-reaction mixture, 1 µl 10 mM dNTPs, 0.2 µl Taq DNA polymerase (5 units/µl), and 1 µl of estrogen receptor primers (500 µg/ml). The primers used here, pER734 (58-TACGAAGTGGGCATGATG-38) and pER846 (58-CAGATCTCATGTCTCCAG-38), amplify a region that encodes the DNA binding domain and the hinge region of the ER-alpha and yield a PCR product of 112 base pairs (bp) (Koike et al., 1987; Bokenkamp et al., 1994). The -actin primers (58GAGCTATGAGCTGCCTGACG-38 and 58-AGCACTTGCGGTCCACGATG-38) were used as described previously (Wu et al., 1992). The expected size of the amplified -actin cDNA fragment is 410 bp. All solutions were kept on ice to prevent nonspecific primer annealing and extension. Samples were denatured at 94°C for 4 min, followed by 35 cycles of denaturation at 94°C for 1 min, 60°C for 1 min, 72°C for 1 min. The reaction was postextended for 10 min at 72°C. PCR products were fractionated on 2% agarose gel stained with ethidium bromide. Identification of the amplified DNA fragment was confirmed by Southern blot analysis using a biotin-labeled rat ER cDNA probe. The sensitivity and detection limit of the above procedure were determined by reverse transcribed and PCR amplified pig uterine RNA prediluted to various concentrations by five serial dilution (i.e., 1:50 to 1:57 ). The extent of any interference due to the E. coli carrier rRNA was also determined by adding E. coli rRNA (0.4 and 4 µg) to pig uterine RNA prior to RT reaction. Immunocytochemistry Embryos were washed in buffer M (25% glycerol, 50 mM KCl, 0.5 mM MgCl2, 0.1 mM EDTA, 50 mM immidazole hydrochloride, 0.5 mM EGTA, 1 mM 2-mercaptoethanol, pH 6.8) twice and fixed in chilled methanol for 10 min. They were then permeabilized with PBS containing 0.1% Triton X-100 for 3–5 min. This was followed by incubating first in blocking solution (PBS containing 0.1% Triton X-100 and 3 µg/ml BSA) for 40 min at 37°C, and then in PBS containing 3 µg/ml BSA and diluted ER715 (or its controls) for 40 min at 37°C. ER715 is an ER-specific antibody that was developed in rabbit against a synthetic peptide corresponding to the hinge region of the ER (Furlow et al., 1990). The controls were: blocking solution without primary antibody; nonimmune purified rabbit IgG; and ER715 antibody preabsorbed with ER peptide. After incubation, embryos were washed with PBS containing 0.1% Triton X-100 and 3 µg/ml BSA, and developed with Histostain-Plus according to the manufacturer’s recommendations (Zymed Laboratories Inc., San Francisco, CA). Immunoprecipitation and Western Analysis To confirm the specificity of ER715 antibody against pig ER, cell extracts prepared from the pig uterine horn tissues were immunoprecipitated with ER715 overnight at 4°C, and this was followed by incubation for 12 hr at 4°C with 50 µl of protein A agarose that has
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Fig. 1. Amplification of ER mRNA from pig uterus. (A) Various amounts of total uterine RNA as indicated were reversed transcribed and the ER cDNA was amplified by PCR for 35 cycles. (B) Pig uterine RNA was reverse transcribed in the presence of E. coli rRNA. One tenth of the RT product was used as the template for ER cDNA amplification. The size of the amplified ER cDNA fragment is 112 bp (arrowhead). M, 1 kb DNA ladder used as molecular weight marker; H2O, water instead of RNA was added in the RT reaction as a negative control.
previously been equilibrated with lysis buffer (Ying et al., 1999). The reaction mixture was then centrifuged and washed with ice-cold dilution buffer (0.1% Triton X-100, 1% bovine hemoglobin, 20 mM Tris-HCl, 0.14 M NaCl) three times, and with TSA buffer (20 mM TrisHCl, 0.14 M NaCl) and 0.005 M Tris-HCl (pH 6.8) once each at 4°C. The resulting precipitated immune complexes were solubilized at 100°C for 3–5 min in 20 µl of Laemmli sample buffer. The solubilized proteins were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane by electroblotting. After blocking overnight at 4°C, the membrane was incubated with ER715 antibody and then detected with an enhanced chemiluminescence (ECL) western blotting system (Amersham) as described previously (Hsu et al., 1999b). Blots were quantified using an LAS-1000 Luminescent Image Analyzer (Fujifilm, Tokyo, Japan). RESULTS Sensitivity, Detection Limits and E. coli rRNA Interference Fig. 1A shows the results from PCR using pig uterine RNA prediluted to various concentrations. A bright band of 112 bp was detected in all lanes except the control, which had no detectable signal. The lowest detection limit for the presence of ER expression reproducibly ranged from 32 to 1.3 pg of total RNA. We were able to detect the presence of ER message in as little as 0.3 pg of total RNA in some experiments. Potential interference due to the presence of E. coli rRNA (i.e., the carrier) was also determined (Fig. 1B). The inten-
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Fig. 2. Detection of ER mRNA in pig embryos during preimplantation stages. Total embryonic RNA was reverse transcribed, and PCR amplified with (A) pER846 and pER734 or (B) -actin primer set. The position of amplified ER and -actin cDNA on the agarose gel is indicated with an arrowhead, respectively. M, 1 kb DNA ladder (A) or 50 bp DNA ladder (B) used as molecular weight marker; H2O, water instead of RNA was added in the RT reaction as a negative control.
sity of amplified ER product was reduced in the samples containing 4 µg of E. coli rRNA. Surprisingly, the 112 bp PCR band that appeared more intense than that of the control (compare Fig. 1B, lanes 1 and 2) was also sometimes seen when 0.4 µg of E. coli rRNA was added to the pig uterine RNA. This effect was not always seen in our usual results, and the actual cause for this phenomenon is not clear. ER mRNA Detection in Pig Embryonic Cells Total RNA extracted from embryos at different developmental stages was reverse transcribed and amplified with the same set of ER primers. The presence of 112-bp fragments in the one-cell, two-cell and four-cell lanes (Fig. 2) indicates that ER was expressed in pig embryos at these stages. In the five- to eight-cell embryo and morular lanes, however, no 112-bp fragments were detected in any of the assays we performed. The reoccurrence of the 112-bp fragment in the blastocyst lane was most interesting since it indicates that the ER gene is expressed once again at this stage. The identify of this 112-bp band was confirmed by Southern blot hybridization using rat ER cDNA fragment as the probe (data not shown). Corresponding quality control for each stage of embryos with the -actin primer set is shown in Fig. 2B. Detection of ER Protein in Pig Embryos To determine whether the ER mRNA present in the pig embryos is indeed translated into ER protein during preimplantation stages, we used immunocytochemistry to visualize the presence of ER protein in these embryos. The specificity of ER715 antibody against pig ER was first confirmed by the immunoprecipitation followed by western blot analysis. The ER715 antibody was generated against a synthetic peptide of rat ER protein and has been shown to cross-react with the
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Fig. 3. Determination of ER protein in pig uterine cell extracts. The pig uterine cell extracts were immunoprecipitated by ER715 antibody and the supernatant (lane 1) and immunoprecipitates (lane 2) were then western blot analyzed in 10% SDS-PAGE with ER715 antibody. The molecular weight markers are indicated on the right.
mouse and human ER protein. However, it remains to be proven that whether ER715 react to the ER-alpha and ER-beta protein equally well. Fig. 3 shows that ER715 was able to readily precipitate pig ER protein from the uterine cell extracts and only one protein band corresponding to the size of ER protein (approximately 65–67 kDa) was observed. The immunocytochemistry protocol was also validated by the ER-containing GH3 cells: immunostaining with the ER715 antibody produced staining in the GH3 cells, but no staining was observed when nonimmune rabbit IgG was used instead of ER715 or when the secondary antibody was omitted from the incubation (data not shown). Pig oocytes and embryos at the preimplantation developmental stage that were immunostained with ER715 antibody produced strong staining in oocytes, one-cell and four-cell stages (Fig. 4; data not shown). Staining with reduced intensity was also detected in the five- to eight-cell embryos. Surprisingly, no staining was observed in the embryos at the blastocyst stage (data not shown) in spite of the fact that the expression of ER mRNA was detected at this developmental stage. In the positive embryos, all the cells exhibited positive staining, and staining intensity was uniform throughout the embryos; staining did not appear to concentrate in the nucleus. All immunostaining experiments were replicated four times and similar results were obtained in each replication. Embryos incubated in nonimmune rabbit IgG or with ER715 antibody preabsorbed with ER peptide gave consistently negative results.
DISCUSSION In this study, we examined the expression of the ER gene in preimplantation pig embryos by RT-PCR and immunochemistry techniques. Estrogen receptor mRNA found in the one-cell, two-cell, and four-cell embryos was absent in the five- to eight-cell and morula stages, and then reappeared in the blastocyst. The ER protein was also detected in embryos at the oocytes and one-cell through to the five- to eight-cell developmental stages, but despite the presence of ER mRNA, ER protein was not detected in the blastocyst. To our knowledge, this study is the first to demonstrate the expression of ER in pig embryos at preimplantation stages. Recurrence of ER gene expression at the blastocyst stage was also observed in a previous study on mouse embryos (Hou and Gorski, 1993); the expression of ER gene reappeared at the blastocyst stage. Wu et al. (1992), on the other hand, detected ER messages in two-cell mouse embryos, which is consistent with our findings, but they failed to detect any ER message at the blastocyst stage. The difference between our results and theirs may be due to the sensitivity of the respective RT-PCR techniques used. Although Hou et al. (1996) found ER protein in mouse blastocyst, no ER protein was detected by our immunocytochemistry analysis of pig blastocyst. We did, however, detect ER mRNA, which suggests that there is a lag period between the transcription of the ER gene into mRNA and the translation of ER mRNA into proteins in pig embryos. It is also possible that ER protein is in fact present in the blastocyst but only at levels that are still below the detection limit of our immunocytochemistry assays. If this is so, it would also be interesting to determine with direct measurement of ER protein by western blot analysis. However, at present the sensitivity of available methods is a limiting factor. Even assuming that the background protein level does not interfere, to obtain satisfactory results with western blot analysis, at least 20 pg of ER protein would be required. This in turn means that an impractically large number of embryos (we estimate 10,000 or more) would have to be extracted. Early development of mammalian embryos is directed by the maternal genome until the zygotic genome assumes control over synthesis of RNA and protein. In pig embryos, uptake and incorporation of 35S-methionine into protein decreased from the time of fertilization to the four-cell stage and then increased at the blastocyst stage of development (Freitag et al., 1988; Tomanek et al., 1989; Jarrell et al., 1991). The decreased rates of methionine uptake and incorporation at the four-cell stage may reflect the period when control of development switches from the maternal to the zygotic genome. Tomanek et al. (1989) suggested that zygotic genome is activated at the four-cell stage in pigs because renewed incorporation of uridine was first detected at this stage of embryogenesis. Since ER mRNA was detected here in the one-cell, two-cell, and four-cell embryos, we likewise contend that this ER mRNA was carried over from the oocytes where mater-
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Fig. 4. Immunocytochemical detection of ER protein in pig oocytes and four-cell preimplantation embryos. Pig embryos were immunostained with ER715 antibody (B, E), rabbit IgG (C), or ER715 antibody preabsorbed with ER peptide (F). Bar ⫽ 10 µm (A–B, D–F; shown in F) or 50 µm (C). For reference, embryos are also shown before immunostaining (A, D). Photos B and E were developed to similar background brightness corresponding to their control C and F, respectively, to insure easy comparison within each embryo group.
nally synthesized mRNA is stored. After the five- to eight-cell and morula stages when ER mRNA was absent or undetectable, its reappearance in the blastocyst must therefore be a result of embryonic RNA synthesis. ACKNOWLEDGMENTS The ER 715 antiserum was obtained through the National Hormone & Pituitary Program, National Insti-
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