Molecular Human Reproduction, Vol. 7, No. 11, 1047-1056,
November 2001
© 2001 European Society of Human Reproduction and Embryology
Embryology |
Quantification of transforming growth factor ß1 (TGFß1) mRNA expression in mouse preimplantation embryos and determination of TGFß receptor (type I and type II) expression in mouse embryos and reproductive tract
1 Department of Obstetrics and Gynaecology and 2 Department of Zoology, The University of Hong Kong, Hong Kong, China
Abstract
We hypothesized that transforming growth factor ß1 (TGFß1) and its receptors play a role in the interaction between the preimplantation embryo and the reproductive tract. To investigate this hypothesis, TGFß 1 mRNA in mouse embryos was quantified by competitive reverse transcription–polymerase chain reaction using an RNA mimic. TGFß 1 was first detected in the unfertilized oocyte, disappeared after fertilization and was expressed again at the 2-cell stage (4410 ± 1330 transcripts/embryo). Its expression increased gradually, peaked at the 8-cell stage (58 600 ± 17 300 transcripts/embryo) and declined rapidly after the morula stage reaching a concentration of 1520 ± 546 transcripts/embryo at the blastocyst stage. The mRNA levels of TGFß 1 at the 8-cell and morula stages were significantly higher than that at other cell stages (P < 0.05). The expression of TGF receptors in embryos and in the reproductive tract was also investigated. Both TGFß1 type I (ALK-5) and type II TGFß receptors were detected in embryos from 1-cell to blastocyst stage by immunohistochemistry. Northern hybridization and immunohistochemistry showed a constant expression of both TGFß receptors in the oviduct from day 1 to day 4 of pregnancy, whilst in the uterus there was a marked increase in the expression of TGFß type I receptor on day 3. Expression of TGFß type II receptor in the uterus remained unaltered throughout the study period. This study has shown that preimplantation mouse embryos produce TGFß1 and that both the embryos and the reproductive tract are responsive to TGFß1 in the preimplantation period.
embryo/oviduct/receptor/TGFß1/uterus
Introduction
The interaction between preimplantation embryos and the reproductive tract (i.e. the oviduct and uterus) is widely believed to play an integral role in preimplantation embryo development. The involvement of a growth factor signalling system between the embryos and the maternal physiology has been demonstrated (Kaye and Harvey, 1995
). For example, tubal epithelial cells secrete high molecular weight glycoproteins that enhance mouse embryo development (Liu et al., 1995, 1998). Transforming growth factors and their receptors at the uterine luminal epithelium are likely to play important paracrine/autocrine roles during the peri-implantation period (Gupta et al., 1998). The embryo in turn produces transforming growth factor-ß1 (TGFß1) which induces apoptosis in the endometrium at the site of implantation (Kamijo et al., 1998). There are also indications that the intra-oviductal embryo can exert a biological effect on the uterus, enhancing endometrial receptivity (Wakuda et al., 1999). In any event, the reproductive tract provides a complex yet poorly understood environment required for gamete transport, maturation, fertilization, early pre-embryo development and implantation.
TGFß1, one of the growth factors detected in the mouse embryo and reproductive tract, is a multifunctional polypeptide hormone that influences numerous physiological processes (Akhurst et al., 1991; Lyons et al., 1991). The expression of TGFßs in the ovary, testis, pre- and post-implantation uterus as well as in the embryo (Kane et al., 1997) suggest the involvement of this growth factor in various reproductive functions. Furthermore, a knockout mouse study showed that intercrosses of heterozygous animals carrying one wild type and one disrupted TGFß1 allele resulted in a significant decrease in the production of homozygous mutant animals (Shull et al., 1992). Another knockout study showed that on a predominantly CF-1 (Albino c origin) genetic background, lack of TGFß1 caused a pre-morula lethality in 
50% of the null embryos (Kallapur et al., 1999). Thus, TGFß1 is believed to play an important role in preimplantation development of embryo.
TGFß1 exerts its effects through binding to specific cell surface receptors. Three types of TGFß receptors, namely type I [Tß RI, (ALK-5) and (Tsk 7L)], type II (Tß RII) and type III (Tß RIII), have been identified (Massagué, 1992; Lin and Lodish, 1993). Signal propagation for TGFß1 is dependent upon heteromeric [Tß RI (ALK-5) and Tß RII] complex formation and transphosphorylation of Tß RI by Tß RII (Anders et al., 1998). The function of Tß RIII remains elusive; it has been suggested that it increases the affinity of Tß RI–RII complex towards TGFß2 (Wang et al., 1991; López-Casillas et al., 1993), or may also store and regulate the availability of TGFß towards an unidentified Tß RII subtype (Lawler et al., 1994). Studies on the expression of TGFß receptors could manifest the capability of the cells to respond to TGFß1 stimulation.
Quantification of transcripts in preimplantation embryos is important because it casts light on the importance of certain genes at specific stages of development. However, quantification of transcripts in the preimplantation embryo is difficult because the amount of mRNA present is scarce. Competitive reverse transcription–polymerase chain reaction (RT–PCR) using an RNA mimic (Becker-André and Hahlbrock, 1989) is one of the most sensitive methods used to quantify rare mRNA species in tissues and samples. Although TGFß1 mRNA can be detected in mouse embryos at different stages of development (Paria et al., 1992), quantification of the message has not been demonstrated. In this study, we reported, for the first time, a sensitive quantitative competitive RT–PCR method using an RNA mimic to quantify TGFß1 expression in preimplantation mouse embryos. We also examined the mRNA and protein expression of TGFß receptors in preimplantation embryos. To study the role of TGFß1 in the interaction between the embryo and the reproductive tract, the temporal expression of TGFß receptors, at both mRNA and protein levels, in the uterus and oviduct was also investigated. Our findings, when considered together, are consistent with the hypothesis that there is a close interaction between the preimplantation embryo and the reproductive tract via TGFß1.
Materials and methods
Collection of embryos, oviducts and uteri
Female F1 (
C57BL/6Jx
A2G) mice underwent ovarian stimulation with intraperitoneal injections of 5 IU pregnant mare's serum gonadotrophin (Sigma, St Louis, MO, USA) followed 48 h later by 5 IU human chorionic gonadotrophin (HCG; Sigma). The animals were mated with BALB/C males. One-cell, 2-cell, 4-cell and 8-cell embryos, morulas and blastocysts were obtained by flushing the oviduct or uterus at 18–20, 42–44, 50–52, 66–72 and 90–92 h post-HCG respectively. Embryos of identical cell stage at each specified period were pooled for RNA extraction. Unfertilized oocytes were obtained from the oviduct of unplugged mice at 18–20 h post-HCG. The morning with the presence a vaginal plug was defined as day 1 of pregnancy and the mice were killed between 14:00 and 15:00 on the indicated day of pregnancy (days 1–4). The oviducts and the uteri of the stimulated animals were flash-frozen in liquid nitrogen immediately after being flushed free of embryos, and stored at –70°C until use.
Isolation of mRNA
Messenger RNA from mouse embryos was extracted by Dynabeads mRNA Direct Kit (Dynal AS, Oslo, Norway) according to the manufacturer's protocol. In brief, the zona pellucidas of mouse embryos were dissolved by acid Tyrode treatment as we failed to extract mRNA with the mRNA extraction kit from embryos with intact zona pellucidas (unpublished data). A known number (usually 15–40) of embryos was lysed and mixed with Dynabeads oligo (dT)25 followed by repetitive washing and elution of poly(A)+ RNA. mRNA was finally eluted with 10–20 µl of diethyl pyrocarbonate (DEPC)-treated water.
Frozen oviducts (n = 3–6, for each day) and uteri were first homogenized in stainless steel grinders pre-chilled at –70°C and total RNA was prepared by Trizol Reagent (Gibco/BRL, Gaithersburg, MD, USA) according to the manufacturer's protocol. The quality of total RNA was determined by both the ratio of A260/A280 and RNA gel electrophoresis.
Synthesis of RNA mimic for TGF ß1 (cRNA)
TGFß1 mimic (mTGFß1), created by inserting a 152 bp fragment from human oviduct-specific glycoprotein (accession no. U09550, nt 1067–1220) into the TGFß1 gene, was cloned downstream to the T7 promoter of pBluescript SK+ vector (Stratagene, La Jolla, CA, USA). RNA of mTGFß1 (cRNA) was synthesized by in-vitro transcription using MEGAscript T7 in-vitro transcription kit (Ambion, Austin, TX, USA). cRNA was further confirmed to be free of DNA template by PCR without reverse transcription and was finally diluted to ~100 fmol/µl with glycogen (50 µg/ml) in DEPC-treated water and stored in aliquots at –70°C.
RT–PCR
Reverse transcription was done using First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech., Uppsala, Sweden) according to manufacturer's protocol. A negative control was included by replacing Bulk First-Strand cDNA Reaction Mixes with water. All cDNA was kept on ice until used for PCR in the same day. Prolonged storage of cDNA for RT–PCR was avoided, as this was prone to give inconsistent results that may have been due to the adherence of minute amounts of mRNA to the wall of the tube.
All oligonucleotide primers, except those for Tß RI (ALK-5), were designed by software Primer Premier (Premier Biosoft International, Palo Alto, CA, USA) using published cDNA sequences retrieved from GenBank. All primers were synthesized from Gibco/BRL. The sequences of primers are listed in Table I. For competitive RT–PCR between TGFß1 and mTGFß1, duplicated PCR reactions were performed for each concentration of RNA mimic. For each PCR reaction (final volume 50 µl), one-third of the cDNA was mixed with PCR components to a final concentration of 1x PCR buffer (10 mmol/l Tris–HCl, 1.5 mmol/l MgCl2, 50 mmol/l KCl, pH 8.3), 0.2 mmol/l of dNTP (Boehringer Mannheim) and 0.4 µmol/l of each forward and reverse primer. After a first denaturation step at 95°C for 5 min, 2 units of Taq polymerase was added. The PCR mixture was then subjected to 40 cycles of amplification with a programme as follows: 94°C for 20 s, 60°C for 30 s and 72°C for 1 min. An extension step at 72°C for 5 min was added at the end of amplification. TGFß receptor mRNA transcripts in embryos were amplified as above, but the reaction mixtures were cycled for 60 cycles with steps that consisted of 94°C for 45 s, 60°C for 40 s and 72°C for 45 s. PCR products for TGFß1, Tß RI (ALK-5), and Tß RII were confirmed by AatII, XhoI and PstI digestion respectively. All PCR products were separated on 2.5% Nusieve 3:1 agarose gel (FMC Bioproducts, Rockland, ME, USA) with 0.5 µg/ml ethidium bromide in 1xTBE buffer (90 mmol/l Tris–borate, 2 mmol/l EDTA, pH 8.0).
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Quantification of PCR products
Gel images were captured on Polaroid 667 film (Polaroid Co., Cambridge, MA, USA) after UV illumination, converted into digitized signal by a densitometer and quantified by ImageQuant Program (Molecular Dynamics, Sunnyvale, CA, USA) as described (Lee et al., 1999
).
Hybridization probes
PCR products of Tß RI (ALK-5), Tß RII and ß-actin were obtained by PCR with specific primers as listed in Table I
. They were gel-purified and radiolabelled by [
-32P]dCTP with Rediprime II DNA labelling system (Amersham Pharmacia Biotech.). All radiolabelled probes were purified by spin columns (Princeton Separations, Adelphia, NJ, USA). The specific activities of the probes were 2x109 d.p.m./µg.
Northern blot analysis
Total RNA (2.0 µg) was denatured, size-fractionated on 1% agarose–2.2 mol/l formaldehyde gel electrophoresis, and transferred to Hybond N+ membrane (Amersham Pharmacia Biotech.). RNA was cross-linked to the membranes by UV irradiation (Spectrolinker XL-1000; Spectronics Corp., Westbury, NY, USA) at 120 mJ/cm2. Hybridization was carried out at 65°C overnight in the presence of Rapid hybridization buffer (Amersham Pharmacia Biotech.). Northern blots were washed twice in 2xSSC, 0.1% SDS (0.3 mol/l NaCl, 0.03 mol/l sodium citrate, pH 7.2, 0.1% sodium dodecyl sulphate) at room temperature and then once with 1xSSC, 0.1% SDS (0.15 mol/l NaCl, 0.015 mol/l sodium citrate, pH 7.2, 0.1% SDS). Stripping was achieved by soaking the blots in boiled 0.1% SDS for 30 min twice. Hybridization signal was detected by autoradiography at –70°C with intensifying screens. Autoradiographs were converted into digitized signal by a densitometer and quantified by ImageQuant Program (Molecular Dynamics, Sunnyvale, CA, USA).
Western blotting
Samples of oviduct and uterine tissue (n = 3 samples per day) were homogenized in stainless steel grinders pre-chilled at –70°C and lysed in 1xSDS protein sample buffer (50 mol/l Tris–HCl, pH 6.8, 2% SDS, 0.1% Bromophenol Blue, 10% glycerol, 1% ß-mercaptoethanol). The lysates were denatured for 5 min at 95°C, fractionated by 12% SDS–polyacrylamide gel electrophoresis and then transferred to a PVDF membrane (Sambrook et al., 1989). The membranes were blocked with 5% skim milk in TBST (10 mmol/l Tris–HCl, pH 7.5, 150 mmol/l NaCl, 0.1% Tween 20) and probed with rabbit anti-Tß RI, anti-Tß RII (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or mouse anti-ß-actin antibodies (Sigma) at 1:1000 dilutions. The membranes were washed three times with TBST for 15 min each and incubated with either anti-rabbit IgG conjugated with horseradish peroxidase (HRP) or anti-mouse IgG conjugated with HRP at 1:1000 dilutions for 1 h at room temperature. The membranes were washed and the signals were visualized by enhanced chemiluminescence (ECL), according to manufacturer's recommendations (Amersham Pharmacia Biotech.).
Immunohistochemistry
Frozen oviducts (n = 3 samples per day) and uteri (n = 6–8 samples per day) were sectioned at 6 µm and mounted onto Tespa (3-aminopropyltriethoxysilane; Fluka)-coated microscope slides. The sections were fixed in acetone at –20°C for 20 min and then hydrated in TN (0.05 mol/l Trizma base, 0.5 mol/l NaCl, pH 8.6). After blocking for 30 min in 3% bovine serum albumin (BSA)/TNT (0.05 mol/l Trizma base, 0.5 mol/l NaCl, 0.5% Triton X-100, pH 8.6), the sections were treated with the primary anti-Tß RI antibody (rabbit IgG, 4 µg/ml; Santa Cruz Biotechnology) or anti-Tß RII antibody (rabbit IgG, 4 µg/ml; Santa Cruz Biotechnology), diluted in TNT overnight at 4°C. After being carefully rinsed in TNT, sections were incubated at 37°C for 1 h with the secondary antibody, sheep anti-rabbit IgG conjugated with Cy3 (Sigma), diluted 1/200 with TNT. Control sections were incubated with normal rabbit IgG (4 µg/ml; Santa Crutz Biotechnology) or primary antibody pre-neutralized with an excess of blocking peptides according to manufacturer's protocol (February 99, Santa Crutz Biotechnology). Immunohistochemical staining with each primary antibody was repeated at least twice.
All embryos were fixed in 3.7% formaldehyde/phosphate-buffered saline (PBS) for 30 min at room temperature after thorough washing in 0.3% BSA/PBS. Embryos were then permeabilized in PBS solution containing 0.1% Triton X-100 (Sigma) for 2 min on ice. After blocking for 30 min in 3% BSA/PBS, separate embryos were allowed to react with anti-Tß RI antibody (Santa Crutz Biotechnology) or anti-Tß RII antibody (Santa Crutz Biotechnology) diluted in 0.3% BSA/PBS/0.1% Tween 20 (Tß RI, 0.4 µg/ml; Tß RII, 0.2 µg/ml) overnight at 4°C. A sheep anti-rabbit IgG conjugated with Cy3 diluted 1/1000 in 0.3% BSA/PBS/0.1% Tween 20 was used as the secondary antibody. To control for the non-specific immunofluorescence, embryos were either reacted with normal rabbit IgG antibody (0.4 µg/ml) or with primary antibody pre-neutralized with excess blocking peptide, followed by incubation with Cy3-conjugated secondary antibody. All immunofluorescent images were observed in a Nikon epifluorescent microscope and captured by the software Metamorph (Version 3.51; Universal Imaging Corp., West Chester, PA, USA). The relative intensity of images was adjusted according to their corresponding negative control.
Statistical analysis
All statistical analysis was carried out by one-way analysis of variance followed by Student–Newman–Keuls Test (Armitage and Berry, 1994). P < 0.05 was regarded as statistically significant.
Results
Quantitative competitive RT–PCR of TGFß1 in mouse embryos
The insertion mutant of TGFß1 (mTGFß1) shares the same primer sequences with TGFß1, with a size difference of only 152 bp. Our kinetic study showed that TGFß1 and mTGFß1 have similar amplification efficiencies under competitive conditions (data not shown). In this study, mRNA, extracted from a pool of embryos at the same stage of development, was divided into five equal portions and mixed with four different concentrations of mTGFß1 (1.08, 0.36, 0.12, 0.04 amol, for 4-cell, 8-cell and morula; 0.36, 0.12, 0.04, 0.013 amol for 2-cell and blastocyst). A negative control for cDNA synthesis was included. Each competitive RT–PCR was carried out in duplicate and each experiment was repeated at least three times using different pools of embryo. The sensitivity of this assay was 7800 transcripts per reaction (unpublished data). In order to determine the minute expression level of TGFß1 in embryos, 15–40 embryos, depending on the developmental stages, were pooled for analysis. With the use of an appropriate and reasonable number of embryos, we detected down to 1520 TGFß1 mRNA transcript per blastocyst.
The calculated amount of TGFß1 mRNA transcripts in embryos at different stages is shown in Figure 1A. TGFß1 mRNA was first detected in unfertilized oocytes; however, the expression level was too low to be quantified by this method. No TGFß1 mRNA was detected after fertilization at the 1-cell stage. Then TGFß1 mRNA was detected again after first cleavage, and the expression level increased gradually and reached a maximum at the 8-cell stage, then declined rapidly to the blastocyst stage (Figure 1B). The levels of TGFß1 mRNA expression at the 8-cell and morula stages were significantly higher than at all other cell stages (P < 0.05).
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Expression of TGFß receptors in preimplantation mouse embryos
Figure 2
shows the RT–PCR products with the predicted sizes of 315 bp for Tß RI (ALK-5) and 279 bp for Tß RII using respective specific primers. The mRNA for Tß RI (ALK-5) was detected at all cell stages studied, while mRNA expression of Tß RII was only detected at the blastocyst stage. Each experiment was repeated at least three times using different pools of embryos.
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Tß RI and Tß RII antibodies bound to the blastomeres, but not the zona pellucida, of 1-cell (Figure 3A
and E), 2-cell (B and F), morula (C and G), and blastocyst stage (D and H) mouse embryos. In contrast with the restricted Tß RII mRNA expression at only the blastocyst stage, Tß RII immunoreactivity was detected at all the embryonic stages studied. Incubation of embryos with rabbit normal IgG (I) or primary antibody pre-neutralized with excess blocking peptide (data not shown), followed by Cy3-conjugated secondary antibody, gave no staining. Computer image analysis of the embryos that had been stained simultaneously showed that the fluorescence intensity due to Tß RII immunoreactivity relative to background signal decreased as the embryo developed from the 1-cell to the blastocyst stage.
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Northern blot analysis of TGFß receptors in the mouse oviduct and uterus
The relative levels of Tß RI (ALK-5) and Tß RII mRNA in the oviduct and uterus were examined by Northern blot hybridization using 32P-labelled DNA probes (Figure 4A
). In this experiment, the housekeeping gene, ß-actin, served as an internal standard for size and relative abundance of mRNA. After correction of the expression levels of mRNA against that of ß-actin, the relative abundance of mRNA at difference stages is expressed graphically in Figure 4B. A single Tß RI (ALK-5) transcript (5.4 kb) was detected in both the oviduct and uterus on days 1–4 of pregnancy (Figure 4A, the uppermost panel). The expression levels of this transcript in the oviduct remained virtually unaltered within this period (Figure 4B, uppermost panel). However, its expression in the uterus was low on day 1 of pregnancy (Figure 4B, second panel) and increased gradually on day 2. There was a nearly 3.5-fold increase in expression on day 3, but the level dropped on day 4. A single mRNA transcript of Tß RII (4.2 kb) was detected in the oviduct and uterus on days 1–4 of pregnancy (Figure 4A, second panel). As shown in the last two panels of Figure 4B, the expression levels of this transcript did not change much in either the oviduct or uterus throughout the studied period.
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Protein expression of TGFß receptors in mouse oviduct and uterus
Figure 5
shows the Western blot analysis of Tß RI and Tß RII in mouse oviduct and uterus. A major single immunoreactive band was observed for Tß RI and Tß RII respectively throughout the studied period. Although ß-actin was used as a loading control, the Western blot data were interpreted only qualitatively as the amount of receptor proteins relative to ß-actin fluctuated between replicated experiments.
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In order to characterize the temporal and spatial protein expressions of the receptors, immunohistochemical staining was performed in the oviduct (Figure 6
) and uterus (Figure 7). Control sections were incubated with normal IgG (Figures 6F and 7L) or with primary antibodies pre-neutralized with an excess of specific blocking peptides (data not shown). These sections showed greatly reduced or no immunostaining.
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In the oviduct, anti-Tß RI (ALK-5) exclusively stained the apical region of tubal epithelial cells (Figure 6B
). Anti-Tß RII also intensively stained the tubal epithelial cells (Figure 6D). The intensity of staining for both receptors in the oviduct remains virtually the same from days 1–4 of pregnancy. Therefore, only a day 1 section is depicted in Figure 6.
In the uterus, weak Tß RI immunostaining was observed in the glandular epithelia on day 1 and day 2 (Figure 7E and F). More intense staining was detected on day 3 (Figure 7G), mainly restricted to the luminal epithelia. In contrast to the Western blot result, no immunopositive signal was detected on day 4 (Figure 7H). Immunoreactive Tß RII was located to the luminal and glandular epithelia of the uterus on all the days examined. As the intensity of staining for Tß RII remained similar from days 1–4, only a day 2 section is depicted in Figure 7J.
Discussion
A number of studies have reported mRNA expression of growth factors in embryos (Kane et al., 1997), but most of them have only demonstrated the expression qualitatively in an all-or-none fashion. Quantification of gene expression in embryos is critical in order to identify the importance of certain growth factors during development. However, there are only a few reports showing the quantification of transcripts in embryos. One study quantified the mRNA of interleukin-1 by RT–PCR using ß-actin for normalization (Huang et al., 1997); however, it has been reported that expression of ß-actin rapidly undergoes changes during embryonic development (Bachvarova et al., 1989). Another study used heterologous cDNA as a mimic in competitive RT–PCR (Johnson et al., 1997). This method may underestimate the transcript level for two reasons. First, using a DNA mimic neglects the suboptimal reverse transcription efficiency; usually 40–50% of mRNA is reverse transcribed (Berger et al., 1983; Bouaboula et al., 1992). Second, a heterologous sequence mimic may have different denaturation characteristics with the target and may result in a different amplification efficiency (Siebert and Larrick, 1993). In order to alleviate the above limitations, we performed competitive quantitative RT–PCR using a TGFß1 mimic with a sequence homologous to the wild type TGFß1. This is the first report on the use of such a technique for quantifying gene expression in mouse preimplantation embryos. The PCR products for TGFß1 and mTGFß1 have a similar molecular weight (only 152 bp difference) and share identical primer sequences. This should minimize the discrepancy in their amplification efficiency. In fact, our kinetics study showed that TGFß1 and mTGFß1 were amplified with similar efficiency (data not shown). The sensitivity of this quantification method is ~1520 transcripts per embryo.
In this study, the TGFß1 transcript was first detected by competitive RT–PCR in unfertilized oocytes but disappeared in the 1-cell embryo. This suggests that the TGFß1 mRNA is maternal in origin and is degraded after fertilization. However, Paria and co-workers previously reported a low but detectable TGFß1 immunoreactivity in 1-cell embryos. This could be due to a faster degradation of mRNA than its corresponding protein (Paria et al., 1992). It is also possible that the discrepancy could be due to the use of different strains of mice in the two studies. When the zygotic genome is activated at the 2-cell stage (Telford et al., 1990; Schultz, 1993), a low level of TGFß1 expression was found, suggesting that the observed transcript was derived from embryonic genome. The expression of TGFß1 continued to increase through the 4-cell stage to a maximum at the 8-cell stage. The expression declined rapidly thereafter.
Tß RI mRNA was detected in the oocyte as well as the embryo at all stages of development. This is consistent with the detection of Tß RI immunoreactivity from 1-cell to blastocyst stage. On the other hand, Tß RII mRNA was present only in the blastocyst whereas its immunoreactive signal dropped from the 1-cell to blastocyst stage. The presence of both Tß RI and Tß RII proteins is in line with the observation that iodinated TGFß1 and TGFß2 ligands bind to embryos from the 8-cell stage onward (Paria et al., 1992).
The exact reason for the discrepancy between Tß RII mRNA and protein expression profiles in this study is unknown. It is possible that mRNA degrades much faster than its corresponding protein. Although we have failed to demonstrate the presence of Tß RII mRNA in oocytes, and this may be due to fast degradation of mRNA at the time of oocyte collection, we are of the opinion that it is present in the oocyte for two reasons. First, Tß RI and Tß RII immunoreactivities are present in unfertilized human oocytes (Osterlund and Fried, 2000). Second, TGFß has been implicated to play a regulatory role in follicular development, oocyte maturation and ovulation (Juneja et al., 1996). We believe that the maternal Tß RII mRNA degrades rapidly after fertilization. Roelen and co-workers also reported the presence of Tß RII mRNA only in the fertilized oocyte and blastocyst but not in other intermediate developmental stages in another strain of mouse (Roelen et al., 1998). In the present study, Tß RII protein persisted but dropped gradually from the 1-cell to the blastocyst stage. In connection with this, it has been reported that a number of major and minor proteins persist in preimplantation embryos at all stages of development (Sasaki et al., 1999). Some of these proteins have an expression profile similar to the protein expression pattern of Tß RII reported here. Tß RII protein has been detected on the cell surface of 1-cell, 2-cell and blastocyst stage embryos of another mouse strain (Roelen et al., 1998). Tß RII synthesis starts again at the blastocyst stage. Thus, the Tß RII protein detected in the blastocyst could be of both maternal and embryonic origins.
In a knockout study, severe embryonic lethality has been shown to occur in TGFß1 knockout embryos on a predominantly CF-1 genetic background, with ~50% of the 129xCF1 Tgfb1 –/– embryos dying prior to the morula stage (Kallapur et al., 1999). Furthermore, transient expression of truncated Tß RII in fertilized oocytes stops the embryo from dividing at the 2-cell stage, an effect that can be rescued by co-injecting with constitutively active Tß RI (Roelen et al., 1998). These findings indicate that TGFß signalling is necessary for embryos to pass the 2-cell stage when the embryonic genome is activated. The increased production of TGFß1 mRNA at the 8-cell stage, the presence of immunoreactive TGFß1 (Paria et al., 1992) and the appropriate TGFß receptors in the embryo suggest that autocrine TGFß signalling is important in early embryo development. Furthermore, oviductal TGFß1 may also modulate embryo development in a paracrine manner.
TGFß1 expression in the mouse oviduct is virtually unaltered throughout the oestrous cycle in mouse (Dalton et al., 1994). We demonstrate for the first time the presence of Tß RI and Tß RII mRNA and protein expression in the oviduct. Similar to their ligand, their expression remains fairly constant in the first 4 days of pregnancy. These findings suggest that the oviduct is responsive to TGFß1 and that the growth factor may have an autocrine function in the oviduct.
Early studies have shown that there is a continuously changing requirement in the embryo during early development (Leese, 1995). Alteration of oviduct biochemistry by the embryo has been demonstrated (Stein and O'Neill, 1994; Murray, 1995; Tadokoro et al., 1995). A recent report identified a number of genes, including TGF- and TGFß-binding protein II, that are differentially expressed in porcine oviduct containing early embryos compared with control oviduct (Chang et al., 2000). We postulate that the increased production of TGFß1 by the 8-cell embryo and morula acts additionally or synergistically with the oviductal TGFß1 to modulate the function of the oviduct. It is known that the oviductal cells enhance mouse embryo development (Liu et al., 1995) via the production of high molecular weight glycoproteins (Liu et al., 1998). In this connection, it is possible that the paracrine action of TGFß1 on the oviduct may in turn induce the expression of embryotrophic factors.
In this study, we found TGFß receptors in the epithelium of mouse oviduct and uterus. Similar observations have been reported previously in other tissues, e.g. human Fallopian tube (Zhao et al., 1994). In all these studies, receptor immunoreactivities were localized to the cytoplasm of epithelial cells. However, one cannot exclude the presence of immunoreactivity in the membrane of these cells, as light microscopy cannot distinguish cytoplasmic immunoreactivity from membranous immunoreactivity when the former has a positive signal. Future studies using isolated membrane fractions or immunohistochemical staining at electron microscopic levels will be helpful to confirm the presence of membranous TGFß receptors in these tissues.
To the best of our knowledge, this is the first report on the up-regulation of Tß RI mRNA and protein in mouse uterus on day 3 of pregnancy. The mRNA and protein levels of Tß RII are constant for the first 4 days of pregnancy according to our Northern blot analysis and immunostaining respectively. Immunostaining localized Tß RI and Tß RII mainly to the luminal or glandular epithelia where TGFß1 and TGFß2 are also located (Tamada et al., 1990). Although we failed to detect Tß RI immunohistochemically in day 4 uterus, we believe that Tß RI protein is still expressed in this period according to the Western blot result. The discrepancy between the two experiments is probably due to the higher sensitivity of the Western blotting method. In the Western blot, all the Tß RI molecules are concentrated in a single band, whereas it is diffused along the whole epithelium in the immunohistochemical method. Moreover, more tissue, and hence more Tß RI, are used in the former method. Therefore, these results suggest that there is a decrease in the amount of the receptor in the day 4 uterus. The presence of embryos that were not flushed out of the uterus before protein extraction for the Western blot was unlikely to affect the present result because the amount of Tß RI in embryos is low and would not contribute significantly to the results of the blotting analysis.
The above results, along with the previous reported increases in TGFß1 and TGFß2 protein expression in the day 3 and day 4 uterus (Tamada et al., 1990), suggest that TGFß modulates uterine biology during the peri-implantation period. Decreases in TGFß receptor expression (type I, II and III) in mouse uterus interrupts TGFß signalling and results in delayed implantation (Das et al., 1997). Apart from the uterine epithelium, the blastocyst in the uterine cavity is another source of TGFß1. Despite a decrease in mRNA expression of TGFß1 at the blastocyst stage compared with the 8-cell and morula stage, immunoreactive TGFß1 is still detected in the blastocyst (Rappolee et al., 1988). Mouse blastocysts have been demonstrated to produce TGFß1 that induces apoptosis in uterine epithelial cells (Kamijo et al., 1998). Whether the TGFß1 produced by the 8-cell embryos and morulas is responsible for the enhancement of endometrial receptivity by intra-oviductal embryos (Wakuda et al., 1999) remains to be investigated.
The co-expression of Tß RI and Tß RII at mRNA and protein levels suggests that the blastocyst is responsive to TGFß. This is in line with the observation that TGFß1 promotes blastocyst outgrowth by increasing the endogenous production of parathyroid hormone-related protein (Nowak et al., 1999) and modulates gene expression of the early cavitating blastocyst (Babalola and Schultz, 1995). However whether the source of TGFß1 is from the embryo, from the uterus or a combination of both is unclear.
In conclusion, there is a complex interaction between preimplantation mouse embryos and the reproductive tract. TGFß1 derived from both the embryos and the reproductive tract may act as an autocrine/paracrine factor for embryo development and for modulating the micro-environment within which the embryo develops and implants. The detailed mechanisms of interaction remain to be elucidated.
Acknowledgements
We are grateful to Mr S.K.Chan and Mr J.S.Xu for their invaluable collection of mouse embryos and tissues. We are also grateful to Miss P.C.Wong for her technical assistance.
Notes
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, China. E-mail: wsbyeung{at}hkucc.hku.hk 
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Submitted on May 15, 2001; accepted on August 10, 2001.
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