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Embryo |
Departments of Obstetrics and Gynecology,3
Biology,4
Medicine,5 McGill University, Montreal, QC Canada H3A 1A1
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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embryo, estradiol, gene regulation, implantation, signal transduction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The blastocyst also appears to play a key role in regulating at least some of the events required for implantation. Transcription of the genes encoding integrins 
2, 6A, and 7, as well as activity of the receptors for HB-EGF (encoded by the ErbB gene family), are upregulated in blastocysts, and these could, in a paracrine manner, modulate gene expression in the surrounding uterine tissue [11, 17]. Indeed, several genes, including HB-EGF, epiregulin, and beta-cellulin become transcriptionally activated at implantation specifically in the region of the uterine luminal epithelium and underlying stroma that are adjacent to the implanting blastocyst [18, 19]. Moreover, these genes are not expressed in ovariectomized mice, but are induced at the site of implantation following injection of estradiol. This strongly suggests that their expression requires estradiol-dependent signals emanating from the blastocyst. Further support for a role for the blastocyst derives from the following observations. When blastocysts have been exposed to the estradiol surge in vivo, they are able to implant in ovariectomized mice up to 16 h after the hosts have received an injection of estradiol. In contrast, blastocysts not exposed to the estradiol surge are able to implant into such recipients only within the first hour after estradiol injection [10]. Thus, exposure to estradiol in the uterine environment appears to induce changes in the blastocyst that are required for implantation. Taken together, these results suggest that implantation requires factors secreted by the blastocyst that modulate gene expression in uterine cells and that expression of at least some of these factors is dependent on estradiol (or its metabolite, 4-hydroxy-estradiol). However, the identity of the blastocyst-derived factors is unknown.
Wnt genes encode a large family of cysteine-rich secreted glycoproteins that function as signaling molecules and play key roles in a wide variety of cellular and developmental processes, (reviewed in [20–22]). To date, 19 Wnt genes have been identified in humans and mice. Wnt proteins act by binding to members of the Frizzled protein family [23–25]. These transmembrane receptors contain an N-terminal, cysteine-rich extracellular domain that is believed to bind Wnts, a putative seven-transmembrane domain, and a variable-length cytoplasmic C-terminus [22, 26]. Nine Frizzled proteins have been identified in humans and mice. Within the cell, multiple pathways have been identified through which Wnt signals may be transduced. In the best known, so-called canonical pathway, Wnt signaling leads to stabilization and nuclear translocation of ß-catenin, which then acts as a transcriptional coactivator of target genes (reviewed in [20, 22]). More recent work has shown that Wnts also signal through pathways that do not involve ß-catenin, including the planar cell polarity pathway and a pathway involving Ca2+ signaling and activation of protein kinase C [27]. Current evidence suggests that the Wnts may be grouped into families that preferentially signal through different pathways. Thus, the Wnt1 family (including Wnt1, Wnt2, Wnt3a, Wnt8a, and Wnt8b) is believed to signal through the canonical pathway, whereas the Wnt5a family (including Wnt5a, Wnt4, and perhaps Wnt11) appears to signal through the Ca2+ pathway (reviewed in [22]).
Despite their widespread role in many developmental processes, the potential role of Wnt genes in the implantation process has not been extensively explored. A recent report of Wnt3a and Wnt4 expression at the 8-cell stage in the mouse [28] is, to our knowledge, the sole report concerning Wnt gene expression in preimplantation embryos. Here, we have investigated the expression of Wnt family members in morulae and blastocysts, and have studied the role of uterine factors, including estradiol, in regulating expression of certain of these Wnt genes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Embryos were obtained as previously described [29]. Briefly, CD-1 females (Charles River, Montréal, QC Canada) were superovulated by an injection of 7.5 IU of eCG (Sigma Chemical Company, St. Louis, MO) followed 44–48 h later by 5 IU of hCG (Sigma) and caged individually with CD-1 males overnight. Mating was indicated by the presence of a vaginal plug the following morning. Morula-stage embryos were flushed using Hepes-buffered KSOM medium from the oviducts of Day 3-pregnant females (Day 1 is Plug Day), 72 h after hCG injection. They were either lysed immediately in a 0.5-ml microfuge tube containing 100 µl of lysing buffer (Trizol; Invitrogen, Burlington, ON Canada) and stored at –80°C until the time of RNA extraction, or placed in 10-µl droplets of bicarbonate-buffered KSOM medium under mineral oil (Sigma) at 37°C in a humidified atmosphere of 5% CO2 in air. After 24 h of incubation, blastocyst-stage embryos (as judged by the formation of blastocoelic cavity) were selected, lysed, and stored as described above. Blastocysts that developed in vivo were flushed from uteri of Day 4-pregnant females at the times indicated in Results, and lysed and stored as described above. All experiments were conducted according to established ethical guidelines for the care and use of laboratory animals.
RNA Extraction
Tubes containing embryos in Trizol were removed from the –80°C freezer and were allowed to stand for 5 min at room temperature to thaw. Ten micrograms of glycogen was added to the tubes, which were mixed and allowed to stand for another 5 min. Thirty microliters of chloroform was then added and the tubes were vigorously shaken, then allowed to stand for 3 min. Following centrifugation at 4°C for 15 min at 13 000 x g, the aqueous phase was transferred to a clean tube, to which was added 100 µl of isopropanol. After a 10-min incubation at room temperature, the tubes were centrifuged at 4°C for 15 min at 13 000 x g, and the supernatant was withdrawn. The RNA pellet was washed with 70% ethanol, allowed to dry for 10–15 min, and dissolved in 10 µl of diethyl pyrocarbonate-treated water.
Complementary DNA Synthesis
Complementary DNA synthesis was carried out using standard procedures. Briefly, each reaction mixture contained 10 µl of RNA solution, 27 units of RNase inhibitor (Pharmacia, Montréal, QC Canada), 3 µl of a dNTP mix of 10 mM concentration, 6 µl of 5x reverse buffer (Invitrogen), 3 µl of 10 mM dithiothreitol, 1 µl of dimethyl sulfoxide (Sigma), 500 ng of oligo-d(T)12–18 (Invitrogen), and 200 units of Moloney murine leukemia virus reverse transcriptase (RT) (Invitrogen). Complementary DNA synthesis was allowed to proceed for 3 h at 37°C, after which the mixture was heated at 85°C for 10 min.
Polymerase Chain Reaction (PCR) Amplification
The primers used are shown in Table 1. Amplification was carried out using cDNA from 5 embryo-equivalents in a buffer consisting of 10 mM Tris (pH 8.3), 50 mM KCl, 200 mM dNTPs, 60–100 pmol of each primer, and 2.5 units of Taq polymerase (Invitrogen) in a total volume of 50 µl. The concentration of MgCl2 was as indicated for each set of primers. Each cycle consisted of 1 min at 94°C, 1 min at a gene-specific temperature (as indicated for each set of primers), and 1 min at 72°C. Forty cycles were run for each reaction. To visualize the amplified products, 15 µl of each PCR reaction was electrophoresed through a 2% (w/v) agarose gel containing 0.025% ethidium bromide.
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Semiquantitative Analysis of RT-PCR Products
The signal intensity of each PCR product in ethidium bromide-stained gels was measured using a FluorChem 8800 imaging system (Alpha Innotech Corporation, San Leandro, CA). The size of the area selected for measurement was the same for each band in a gel, and values obtained were subtracted from a background value obtained from an area of the same size in the same gel. The value obtained for each product was then normalized to a designated control product for each PCR reaction, as indicated in the figure legends. Means and standard deviations were calculated. Statistical tests were carried out as indicated in the figure legends.
Ovariectomy and Drug Treatments
Mice were ovariectomized on the morning of Day 4 of pregnancy between 0700 and 0900 h. Immediately after surgery, mice received an s.c. injection of progesterone (2 mg/mouse) only or progesterone with estradiol-17ß (50 ng/mouse), or progesterone with 4-hydroxy-estradiol (4-OH-estradiol) (50 ng/mouse). Mock-treated animals were anesthetized and surgically opened, and the ovaries were exposed outside, and then returned in place. Animals treated with ICI 182,780 received an s.c. injection (0.5 mg/mouse or 1 mg/mouse) of the drug at 0700 h on Day 4 of pregnancy. Animals used for the study of trapping blastocysts in the oviduct were exposed to surgery in the afternoon of Day 3 of pregnancy. The junction between the oviduct and the uterus was tightly tied using 6-0 silk thread (Ethicon, Somerville, NJ). Animals were killed on Day 4 at 2000 h, and the blastocysts were recovered from the oviducts. All experiments were conducted according to established ethical guidelines for the care and use of laboratory animals.
Drugs
Progesterone (4-pregnen-3,20-dione) (Q2600-000; Steraloids, Newport, RI) was dissolved in sesame oil at a concentration of 20 mg/ml. Estrogen (1,3,5[10]-estratriene-3,17ß-diol) was purchased from Sigma (E-8875) and was dissolved in sesame oil at a concentration of 500 ng/ml. 4-OH-estradiol (E2500,000; Steraloids) was dissolved in sesame oil at a concentration of 500 ng/ml. ICI 182,780 (AstraZeneca, Wilmington, DE) was dissolved in sesame oil at a concentration of 10 mg/ml. For in vitro studies, embryos were cultured in 10-µl microdrops of KSOM containing either estradiol-17ß or 4-OH-estradiol at a concentration of 2 ng/ml.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To investigate the potential involvement of Wnts in the implantation process, we examined the expression of Wnt genes in developing embryos. Morulae were flushed from the oviducts of pregnant females on the afternoon of Day 3 and processed for RT-PCR using specific primers for hypoxanthine guanine phosphoribosyl transferase (HPRT) and 15 Wnt genes (Table 1). As shown in Figure 1A, products corresponding to 6 of the 15 Wnt genes tested were observed. Next, we flushed blastocysts, which were now in the uteri, between 1900 h and 2000 h on Day 4 of pregnancy, and processed them for RT-PCR. All of the Wnt genes expressed in morulae were also expressed in blastocysts (Fig. 1B), certain of them at a higher level (e.g., Wnts 4, 5b, 7b, and 10b). In addition, several Wnt genes (1, 5a, 7a, 11, and 13) were newly detectable in the blastocysts. These results establish that expression of a large number of Wnt genes is upregulated in blastocysts near the time of implantation.
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To investigate the mechanism by which the expression of these Wnt genes was upregulated, we collected morulae from the oviducts of pregnant females on Day 3. These were cultured in vitro until the evening of Day 4, and those that reached the blastocyst stage were processed for RT-PCR as previously described. Several of the Wnt genes (7a, 7b, 10b, and 13) that were upregulated in the blastocysts that developed in vivo were similarly upregulated in blastocysts that developed in vitro (Fig. 1C). In contrast, Wnt5a and Wnt11 were expressed at relatively low levels in the in vitro blastocysts. Figure 1D represents the expression of each Wnt gene in blastocysts that developed in vivo compared with those that developed in vitro. These results imply that upregulated expression of certain Wnt genes in blastocysts required exposure to the uterine environment during the morula-to-blastocyst transition.
Upregulation of Wnt11 Expression by the Blastocyst Requires an Ovarian Factor That Can be Replaced by Administration of Estradiol-17ß
Based on these results, we examined the timing of Wnt11 gene upregulation in vivo. Because implantation is preceded by an obligatory surge in estradiol [10, 30, 31], we focused on this event. Blastocysts were flushed from uteri of pregnant females on Day 4 in the morning (before the estradiol surge), at midday (just after the estradiol surge), and in the early evening, and processed for RT-PCR using primers for Wnt6 and Wnt11, and HPRT as a control. As shown in Figure 2, Wnt11 expression was modestly higher at midday as compared to the morning, and by the evening was considerably higher. In contrast, Wnt6 expression did not change substantially during this period. These results establish a correlation between the onset of the estradiol surge and the upregulation of Wnt11 expression. A similar time course was observed for Wnt5a expression (not shown).
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To test whether upregulation of Wnt11 expression depended on the estradiol surge, we performed the following experiments. First, we ovariectomized pregnant females between 0700 h and 0900 h of Day 4 (i.e., before the estrogen surge). Control animals were mock-ovariectomized or left untreated. Blastocysts were collected between 2000 h and 2200 h on Day 4 from all groups and processed for RT-PCR. Figure 3 shows that ovariectomy did not detectably affect the expression of Wnt1, Wnt5a, Wnt5b, and Wnt6. This suggests that upregulation of Wnt5a expression, although it occurs only in embryos that developed from the morula-to-blastocyst stage in vivo, does not require ovarian factors produced during this time. In contrast, Wnt11 expression was much lower in blastocysts collected from ovariectomized animals compared with those obtained from control mock-ovariectomized and intact animals. Thus, upregulation of Wnt11 expression requires one or more ovarian factors that are secreted during the period just preceding implantation.
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To study the potential role of estradiol more directly, we tested the effect on Wnt11 expression of administering ICI 182,780, an estradiol antagonist that competes for binding to nuclear estrogen receptors [32, 33]. Pregnant females received an injection of ICI 182,780 on the morning of Day 4, before the estrogen surge, and blastocysts were collected in the evening of that day and processed for RT-PCR. Upregulation of Wnt11 expression was blocked in blastocysts collected from animals injected with ICI 182,780, whereas expression of Wnt5a was unaffected. Next, we ovariectomized pregnant females before the estrogen surge, and then administered either estradiol-17ß or a metabolite, 4-OH-estradiol, which is a component of uterine fluid [11], to these females. Blastocysts were collected on the evening of Day 4, and Wnt expression was assayed. Both estradiol-17ß and 4-OH-estradiol restored Wnt11 expression to near control levels (Fig. 4C). These results indicate that upregulation of Wnt11 expression is estradiol-dependent and that the effect of estradiol is mediated through an ICI-sensitive receptor.
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Upregulation of Wnt11 Expression by the Blastocyst Requires the Uterine Environment
To determine whether estradiol was the sole component of the in vivo environment required for upregulation of Wnt11 expression by blastocysts, we recovered morulae from the oviducts of Day 3 pregnant females and cultured them in the presence of estradiol-17ß or 4-OH-estradiol. At 2000 h of Day 4 the embryos (which were now at the blastocyst stage) were collected and processed for RT-PCR. Figure 5 shows that Wnt11 was not upregulated under these conditions.
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The failure of estradiol to upregulate Wnt11 expression in blastocysts cultured in vitro suggested that this activity of estradiol might require the uterine environment. To test this idea, we ligated the utero-tubal junction of pregnant females on Day 3, thus trapping the embryos in the oviduct. We reasoned that the embryos in the oviduct would witness the systemic estradiol surge, but would be isolated from the uterine environment. On the evening of Day 4, blastocysts were collected from the oviducts and processed for RT-PCR. Wnt11 expression was not upregulated in the blastocysts trapped in the oviduct. Rather, it was similar to that observed in blastocysts following ovariectomy (Fig. 4A, lower; Fig. 4B). These results imply that upregulation of Wnt11 expression by the blastocyst requires both estradiol and the uterine environment. Unexpectedly, expression of Wnt5a also was significantly reduced in blastocysts that were trapped in the oviduct (Fig. 4A, middle; Fig. 4B), although expression of Wnt5b (Fig 4A, upper) and Wnt6 (not shown) were unaffected. This suggests that upregulation of Wnt5a expression, although not estrogen-dependent, also requires one or more uterine factors.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2, 6A, and 7 become more abundant in late blastocysts; however, this increase also occurs in embryos that develop to blastocysts in vitro and thus in the absence of estradiol [17]. Perlecan is a heparan sulfate proteoglycan present in extracellular matrix. Although the encoding mRNA is upregulated in blastocysts, it is also present in "delayed" blastocysts recovered from ovariectomized animals [14]. The protein, however, is induced in delayed blastocysts following estradiol injection into the mother, suggesting that estradiol may act post-transcriptionally in this case [14]. Blastocysts also express members of the ErbB family of EGF receptors. Moreover, addition of EGF to embryos cultured in vitro increases both the blastocyst cell number and the fraction of embryos that hatch from the zona pellucida [1, 34, 35]. This implies that functional EGF receptors are expressed in the absence of estradiol. However, estradiol may maintain ErbB expression, as ErbB mRNA levels fall in delayed blastocysts and are restored by estradiol injection [36]. The results reported here provide direct evidence that the uterine environment, and estradiol in particular, regulate expression of specific genes in the blastocyst.
Several observations in this study suggest that estradiol does not act directly on the blastocyst, but rather indirectly via uterine cells. First, neither estradiol-17ß nor its metabolite, 4-OH-estradiol, could upregulate Wnt11 expression when applied to blastocysts in vitro under our conditions. Second, Wnt11 expression was not upregulated in blastocysts that were prevented from migrating from the oviduct to the uterus. Although we cannot be certain that these blastocysts were exposed to estradiol, this observation suggests that Wnt11 upregulation requires an additional factor specific to the uterine environment. Third, Wnt11 expression in blastocysts was not upregulated when the mothers received ICI 182,780, implying that this effect is mediated through a nuclear estradiol receptor. Therefore, the effect of injected estradiol-17ß and 4-OH-estradiol on Wnt11 expression is likely mediated through these receptors. In contrast, the previously documented activity of 4-OH-estradiol toward blastocysts is not inhibited by ICI 182,780 [11]. Thus, the estradiol effect described here and the previously reported effect of 4-OH-estradiol on the blastocyst [11] appear to be mediated through different signaling pathways. Consistent with the notion that estradiol does not act directly on the blastocyst to upregulate Wnt expression, mouse embryos lacking the estrogen receptor- gene are viable, although persistence of maternal gene product to the blastocyst stage cannot be formally excluded [37, 38]. Estradiol is known to trigger expression of leukemia inhibitory factor (LIF) from uterine glandular cells, and LIF can largely or completely replace the nidatory function of estradiol surge [16]. LIF did not upregulate Wnt11 expression in vitro in our hands (unpublished data), which could reflect the absence of necessary cofactors.
The function of the Wnts expressed by the blastocyst remains to be identified. Expression of numerous genes, including HB-EGF, epiregulin, and beta-cellulin becomes upregulated only in the region of the uterine epithelium adjacent to the blastocyst [1, 19]. This strongly suggests that upregulation depends on signals emanating from the blastocyst. The molecular nature of these signals, however, remains unknown. Although formal demonstration that the Wnt mRNAs expressed in blastocysts are translated to produce Wnt proteins awaits the production of effective antibodies, this is a reasonable inference. Thus, the Wnts are attractive candidates to be signaling molecules that mediate the blastocyst effect on uterine gene expression. We propose that estradiol acts on uterine cells to trigger the expression of diffusible molecules that in turn act on the blastocyst to upregulate Wnt11 expression (Fig. 6). Secreted Wnts may modulate uterine differentiation, or, in an autocrine manner, blastocyst differentiation. This model reinforces the concept [12] that implantation depends on reciprocal and mutually dependent interactions between the blastocyst and uterine environment.
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Finally, it may be noted that mice lacking individual Wnt genes, including Wnt11 and Wnt5a, do not show implantation defects [39–47]. As defined by the absence of an implantation defect in knockout animals, other genes that are expressed specifically in the uterus at the site of the implanting blastocyst, such as HB-EGF and beta-cellulin, do not seem to be essential for implantation [48]. Given the tightly regulated pattern of expression of these genes, however, it would be surprising if they played no role in this process. In this case of the Wnts, we observed that both Wnt5a and Wnt11 are upregulated in blastocysts that develop in vivo. It may be speculated that the expression of several genes whose products serve the same biochemical function means that absence of a single gene product has no detectable effect under laboratory conditions.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Hugh J. Clarke and Daniel Dufort, Room F3.36, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, QC Canada H3A 1A1. FAX: 514 843 1662; hugh.clarke{at}muhc.mcgill.ca
Received: 20 November 2003.
First decision: 7 December 2003.
Accepted: 16 March 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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