Molecular Human Reproduction, Vol. 6, No. 6, 498-503,
June 2000
© 2000 European Society of Human Reproduction and Embryology
Ovary and oogenesis |
TGFß receptor types I and II and the substrate proteins Smad 2 and 3 are present in human oocytes
Reproductive Medical Center, Division of Obstetrics and Gynecology, Department of Women and Child Health, Karolinska Hospital, S-171 76 Stockholm, Sweden
Abstract
We have recently found that values of the transforming growth factor (TGF)ß1 in human ovarian follicular fluid obtained during ovarian stimulation for IVF were higher in women who subsequently became pregnant following embryo transfer. We therefore postulated that TGFß1 may have a beneficial effect on the preimplantation embryo and improve the chances of a successful implantation. We have used reverse transcription–polymerase chain reaction (RT–PCR) and immunohistochemistry to investigate the presence in human oocytes and preimplantation embryos of the essential components of the TGFß signalling pathway, TGFß receptors type I and II and the substrate proteins Smad 2 and 3. We found that both receptors, as well as Smad 2 and 3, were present in the unfertilized oocyte, whereas only the type I receptor and Smad 2 and 3 were present at the blastocyst stage. At the 4-cell and 8-cell stages neither of the receptors was present, but Smad 2 and 3 were present at both stages. These findings support our hypothesis that the TGFß1 in follicular fluid may interact with the oocyte and preimplantation embryo via TGFß receptors, and that TGFß signalling may be important for the development of the oocyte and the preimplantation embryo.
IVF/oocyte/signal transduction/Smad/TGFß
Introduction
We have recently shown that the multifunctional growth factor transforming growth factor ß1 (TGFß1) is present in human follicular fluid during ovarian stimulation for IVF, and that the concentrations of TGFß1 were higher in women who subsequently became pregnant following embryo transfer (Fried and Wramsby, 1998
). We therefore postulate that TGFß1 may have a beneficial effect on the preimplantation embryo and that it might contribute to improve the chances of a successful implantation. One reported effect of TGFß on mouse preimplantation embryos is to increase total cell number (Lim et al., 1993). One possibility whereby follicular fluid TGFß1 may increase total cell number, and perhaps confer upon the embryo improved chances of a successful implantation, is by a receptor-mediated interaction with the oocyte and preimplantation embryo. The possible presence of TGFß receptors on human oocytes is previously unknown.
Currently, there are three types of TGFß receptors known, TGFß receptor types I, II and III (TGFßR-I, TGFßR-II and TGFßR-III) (Massagué et al., 1992, 1994; Lin and Lodish, 1993; Henis et al., 1994; Lin and Moustakas, 1994). It is believed that for signalling to occur, heterodimerization between the type I and type II receptor is necessary (Wrana et al., 1992; Moustakas et al., 1993; Okadome et al., 1994). Type II receptor binding of TGFß causes conformational changes leading to binding of type I receptor to the type II receptor–TGFß complex (Ventura et al., 1994; Wrana et al., 1994). Both receptors are serine-threonine kinases (ten Dijke et al., 1994), and signalling is initiated by phosphorylation of the type I receptor by the type II receptor. The type III receptor, a proteoglycan, is not directly involved in signalling but presents the ligand to the signalling receptor.
The substrates for the type I receptor kinase activity have recently been identified as belonging to the so-called Smad family of proteins (Liu et al., 1996; Heldin et al., 1997). There are currently nine Smad proteins reported, all essential intracellular signalling components for members of the TGFß superfamily (Heldin et al., 1997; Nakao et al., 1997a). Smad 2 and 3 are structurally similar, and mediate TGFß and activin signals (Nakao et al., 1997b). Smad 1 and 5, and probably also 9, mediate bone morphogenetic protein (BMP) signals (Kretzschmar et al., 1997; Suzuki et al., 1997). Smad 4 (distantly related to Smad 2 and 3) acts as a `common partner' molecule forming a heteromeric complex with Smad 2–3 after TGFß or activin stimulation and with Smad 1–5 after BMP stimulation (Heldin et al., 1997).
To examine the possibility of TGFß signalling in the human oocyte and preimplantation embryo, we have therefore, in the present study, examined oocytes and preimplantation embryos for the presence of TGFß receptor types I and II (TGFßR-I, TGFßR-II) and Smad 2 and 3 using reverse transcription–polymerase chain reaction (RT–PCR) and confocal immunohistochemistry.
Materials and methods
Clinical IVF protocol
All women involved in this study underwent ovarian stimulation after pituitary suppression with a gonadotrophin-releasing hormone (GnRH) agonist (Suprefact; Svenska Hoechst AB, Stockholm, Sweden) as previously described (Csemiczky et al., 1995; Fried et al., 1996). After verifying down-regulation, ovarian stimulation was performed with FSH (Gonal-F or Fertinorm-HP; Serono Nordic AB, Sollentuna, Sweden). Follicular development was monitored by vaginal ultrasound measurements of follicles combined with blood samples for oestradiol analysis. The s.c. administration of 10 000 IU human chorionic gonadotrophin (HCG, Profasi; Serono Nordic AB) was followed ~35 h later with oocyte retrieval, performed by transvaginal ultrasound-guided follicle aspiration.
IVF and preimplantation embryo culture
In this study, surplus preimplantation embryos were donated by couples undergoing IVF treatment. For insemination and sperm swim-up, Earle's balanced salt solution (Sigma, Stockholm, Sweden) was used with the addition of pyruvate, 0.1 mmol/l (Sigma), penicillin, 10 000 IU/ml (Sigma) and 10% heat inactivated patient's serum. For cleavage culture the same medium with 15% serum was used. At 3–5 h after oocyte retrieval, 100 000 motile spermatozoa were added to each oocyte in 1 ml of culture medium. Insemination and cleavage were carried out at 37°C in an incubator, with a humidified mixture of 5% CO2 and 95% air. Fertilization was assessed by the presence of pronuclei 16–18 h after insemination followed by cleavage observed 2 days after oocyte retrieval. The ova were thoroughly denuded from granulosa cells. Those preimplantation embryos with highest scores (Puissant et al., 1987), were selected for replacement. During the present study period not more than two preimplantation embryos were replaced. To achieve different developmental stages, a number of donated preimplantation embryos were cultured in cleavage medium for up to 6 additional days. As a result, 8-cell and blastocyst stages became available. This work was approved by the Local Ethics Committee of the Karolinska Hospital
Preparation of mRNA
mRNA (poly-A mRNA) was prepared from individual embryos (Rappolee et al., 1988, 1989). As a positive control, mRNA was also prepared from human placenta (Mitchell et al., 1992). For the mRNA preparation, the QuickPrep Micro mRNA Kit (Pharmacia, Uppsala, Sweden) was used according to the manufacturer's instructions. Briefly, the preimplantation embryos were lysed, homogenized and mixed with oligo(dT)-cellulose followed by repetitive washing steps and elution of total poly-A mRNA.
Primers
The sequence of the oligonucleotide primers used in the RT and PCR reactions are given in Table I. The primers for Smad2 and 3 were designed to pick up both Smad2 and Smad3.
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Reverse transcription
RNA was transcribed into cDNA using the Pharmacia First-Strand cDNA Synthesis kit. The mRNA was incubated at 37°C for 60min with a mixture of 45mmol/l Tris (pH8.3), 68mmol/l KCl, 15 mnol/l dithiothreitol, 9 mmol/l MgCl2, 0.08 mg/ml bovine serum albumin (BSA) and 1.8 mmol/l of each dNTP in a total volume of 15 µl. The reaction was primed with 0.2 µg Not-I-d(T)18 primer (Pharmacia). The reaction was terminated by placing the reaction tube at 90°C for 5 min and then chilled on ice for immediate use in PCR. As a control, parallel RT reactions in the absence of reverse transcriptase were performed to rule out genomic DNA contamination in the mRNA preparation, as a cause of false positive results.
Polymerase chain reaction
PCR was performed on the resulting cDNA (5 µl) using 2 IU of Dynazyme DNA polymerase (In Vitro AB, Solna, Sweden) in a 50µl reaction mixture containing 0.2 mmol/l each of dNTP, 0.3 µmol/l of specific primers, 1–1.5 mmol/l MgCl2, 0.1% Tween 20, 45 mmol/l KCl and 10 mmol/l Tris–HCl.
PCR conditions for the nested primer assay were two three-temperature cycles of 95°C for 1 min, 55°C for 1 min and 72°C for 1 min (30–50 cycles each). In the second amplification, 2 µl of the first amplification product was used. The PCR was performed on a Perkin Elmer DNA Thermal Cycler (Perkin Elmer, Norwalk, CT, USA).
Gel electrophoresis
PCR products (6 µl mixed with loading buffer dye) were separated on a 1.5% agarose gel (Agarose NA; Pharmacia) at 140 V and visualized by ethidium bromide staining.
Verification of RT–PCR products
The identities of the RT–PCR products were verified by sequencing. The RT–PCR products were gel purified using the Qiaex II Agarose gel extraction kit (Qiagen Inc, USA) and sequenced using the ABI Prism Dye terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer) and analysed at the automated sequencing facility at Molecular Genetics, Department of Cell and Molecular Biology, The Karolinska Institute, Sweden.
Immunohistochemistry
Embryos were fixed in 1% paraformaldehyde for 2–8 h at 4°C followed by washing (three times) in cold phosphate-buffered saline (PBS). Non-specific binding was blocked by incubating the embryos overnight at 4°C in PBS with 3% BSA, 0.2% Triton. The embryos were then incubated with a 1:100 dilution of primary antibody (rabbit-anti-human TGFßR-I or II, goat-anti-human Smad2/3; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature, followed by incubation with a 1:100 dilution of fluorescein isothiocyanate (FITC)-conjugated secondary antibody [swine-anti-rabbit immunoglobulin (Ig); Dako; Copenhagen, Denmark and rabbit-anti-goat; Southern Biotechnology; Birmingham, AL, USA]. Embryos were whole-mounted, and then viewed through a Zeiss confocal laser scanning microscope, LSM 410. The thickness of the optical sections was 8µm. The images were further processed on a Silicon Graphics Indy computer. Negative staining controls were included in which: (i) only the FITC-conjugated secondary antibody was used; and (ii) where the oocytes were not incubated with the primary antibody but instead with undiluted rabbit IgG control serum, followed by washing and incubation with secondary antibody as described. These controls were included to demonstrate that the observed fluorescence was not due to: (i) non-specific binding of the secondary antibody; or (ii) non-specific binding of antibodies from normal rabbit IgG serum.
Results
Type I receptor
The calculated size of the RT–PCR product was 207 bp, using the outer primer pair in the first amplification and the inner pair in the second amplification (Table I
). We obtained a RT–PCR product of the expected size 207 bp in the human unfertilized oocytes and preimplantation embryos at the blastocyst stage but not at the 4-cell or 8-cell stages (Figure 1a). As a positive control of the RT–PCR reaction we also included human placenta which exhibited a RT–PCR product of the same size (Figure 1a). The RT–PCR product of 207 bp was by sequencing confirmed to be TGFßR-I (data not shown). These results are summarized in Table II.
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Immunohistochemistry performed on unfertilized oocytes showed strong TGFßR-I immunostaining in single sharply defined areas close to or in the plasma membrane (Figure 2a
). A lower intensity of immunostaining was also present further from the plasma membrane, surrounding circular structures. There was no clear indication of a selective distribution of the receptor in certain parts of the oocyte. Incubation of oocytes with undiluted rabbit IgG serum followed by FITC-conjugated secondary antibody gave no staining (Figure 2d). The negative control, in which only the FITC-conjugated secondary antibody was used, showed no staining (data not shown).
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Type II receptor
The calculated size of the RT–PCR product was expected to be 439 bp, using the outer primer pair in the first amplification and the inner pair in the second amplification (Table I
). We obtained a RT–PCR product of the expected size 439 bp in the human unfertilized oocytes but not at the 4-cell, 8-cell or blastocyst stages (Figure 1b). The human placenta exhibited a RT–PCR product of the same size (Figure 1b). The RT–PCR product of 439 bp was confirmed by sequencing to be TGFßR-II (data not shown). These results are summarized in Table II.
By immunohistochemistry performed on unfertilized oocytes TGFßR-II staining was observed in discrete peripheral patches, suggestive of a regional plasma membrane localization (Figure 2b). Incubation of oocytes with undiluted rabbit IgG serum followed by FITC-conjugated secondary antibody gave no staining (Figure 2d). The negative control, in which only the FITC-conjugated secondary antibody was used, showed no staining (data not shown).
Smad2/3
The calculated size of the RT–PCR product was expected to be 429 bp, using the outer primer pair in the first amplification and the inner pair in the second amplification (Table I). We obtained a RT–PCR product of the expected size 429 bp in the human unfertilized oocytes, as well as in preimplantation embryos at the 4-cell, 8-cell and blastocyst stages (Figure 1c). The human placenta exhibited a RT–PCR product of the same size (Figure 1c). The RT–PCR product of 429 bp was confirmed by sequencing to be Smad2 (data not shown). These results are summarized in Table II. Smad2/3 immunostaining in unfertilized oocytes (n = 5) showed varying intensity throughout the cytoplasm. Numerous rounded immunonegative areas with a diameter of 5–10 µm were seen in all oocytes examined, suggesting the presence of small organelles negative for Smad2/3, but surrounded by immunopositive material (Figure 2c). There were no structures correlating to this pattern visible using light microscopy. Incubation of oocytes with undiluted rabbit IgG serum followed by FITC-conjugated secondary antibody gave no staining (Figure 2d). The negative control, in which only the FITC-conjugated secondary antibody was used, showed no staining (data not shown).
Discussion
We have shown in this study, to our knowledge for the first time, the presence of mRNA and proteins for TGFß receptors type I and II in the human oocyte and receptor type I in blastocysts, as well as Smad 2/3 in both oocytes and preimplantation embryos. The findings indicate a selective expression of transcripts for TGFß receptors in oocytes and blastocysts, with no expression in early cleavage stages. Furthermore, the results show that all components for an active cellular signalling via TGFß are present in the oocyte, and thus support our hypothesis that TGFß present in human follicular fluid (Ruegsegger Veit and Assoian, 1988; Mulheron et al., 1992) may interact via TGFß receptors in the oocyte and that TGFß signalling may be important for the development of the oocyte (Fried and Wramsby, 1998; Fried et al., 1998).
Our RT–PCR results suggested that, in unfertilized oocytes, the TGFß receptor types I and II are maternal transcripts which are subsequently lost during the early cleavage stages. We performed immunohistochemistry in order to determine whether the detected transcripts were translated into proteins. Using polyclonal antibodies against TGFß receptor types I and II and Smad 2 and 3, we could confirm (using confocal microscopy) the presence of both receptors as well as Smad 2 and 3 at the oocyte stage. We did not examine the later stages of pre-embryo development by immunocytochemistry, primarily because of the limited number of available human pre-embryos, especially blastocysts, but also because of the fact that TGFßR-II was not detected by RT–PCR in later cleavage stages.
The Smad 2/3 transcripts were present in oocytes, early cleavage stages as well as in blastocysts. The expression pattern as visualized by confocal microscopy was very conspicuous, and much different from that of TGFßR-I and TGFßR-II. In contrast to the regional, plasmalemmal/sub- plasmalemmal localization of TGFßR-I and -II, Smad 2 and 3 protein was found throughout the cytoplasm, and was only excluded from multiple small circular areas. The pattern suggests a soluble, as opposed to membrane-bound subcellular localization. The presence of Smad 2/3 throughout all cleavage stages examined, in parallel with absence of TGFß receptor expression, indicate that there is a possibility for other molecules than TGFß to regulate early cleavage stage processes through Smad 2/3. For instance, activins also signal through Smad 2/3 by way of the activin receptors ActR-I and ActR-II (Lebrun et al., 1999). Interestingly, activin receptors (but not activin), have recently been detected by RT–PCR in early cleavage stages and blastocysts of human preimplantation embryos (He et al., 1999), indicating a possibility for exogenous activin to activate Smad 2/3. Furthermore, the potential for a cross-talk or partial overlap between activin and TGFß signalling in cell stages where receptors for both are present is illustrated by the report that activin receptors may form heteromeric kinase complexes with TGFß receptors (Attisano et al., 1993).
The localization of Smad 2/3 immunostaining was distinctly non-regional, in contrast to TGFßR-I and TGFßR-II, which showed staining in large plasmalemmal/subplasmalemmal regions, unevenly distributed. However, as far as we could discern, there was no clear polarization of TGFßR-I and TGFßR-II staining in the surplus oocytes we studied, as has recently been described for several regulatory proteins including leptin, signal transducer and activator of transcription 3 (STAT3), BaX, BcL-X, TGFß2, vascular endothelial growth factor (VEGF), c-kit and epidermal growth factor receptor (EGF-R) (Antczak and Van Blerkom, 1997, 1999). However, this does not exclude that a polarized expression for TGFß-receptors may occur in normal oocytes and preimplantation embryos with full developmental potential, since polarity and axis formation is fundamental for early cell determination and differentiation (Edwards and Beard, 1997)
The presence of TGFß-receptors and their transducer proteins Smad 2/3 in the oocyte indicate a possibility for TGFß to regulate oocyte processes. In the human ovary, TGFß1 has been detected by immunohistochemistry in oocytes, granulosa and theca cells from small ovarian follicles, and TGFß2 has been detected in theca cells (Chegini and Flanders, 1992). This means that ovarian TGFß may have effects on the oocyte both via autocrine and paracrine mechanisms.
However, locally produced TGFß may also act on ovarian cells other than the oocyte. There are TGFß receptors in mouse thecal cells (Schmid et al., 1994) and recently both TGFßR-I and TGFßR-II were reported in human granulosa-, theca- and interstitial cells (Roy and Kole, 1998).
The possibility that TGFß1 may influence processes related to successful fertilization and embryo development should be taken into consideration in view of our observation that TGFß1 concentrations in follicular fluid were higher in IVF-treated patients who subsequently became pregnant (Fried and Wramsby, 1998a). Also in support of this hypothesis is the recent observation that high concentrations of TGFß1 in individual follicles were correlated with `high quality' (grade 1) embryos derived from the same follicle (Kurtz et al., 1997; Roy et al., 1998).
The possibility that receptor-mediated actions of TGFß can improve the fertilizability and developmental capacity of oocytes is in agreement with previous results (Feng et al., 1988), which showed that TGFß stimulates maturation of follicle-enclosed oocytes and cumulus–oocyte complexes in rats. However, this contrasts with findings in vitro, where it has been reported that addition of TGFß did not increase cumulus expansion or germinal vesicle breakdown in swine (Singh et al., 1993) and that TGFß suppressed porcine oocyte maturation in vitro (Coskun and Lin, 1994). TGFß has no reported effect on bovine oocyte fertilizability or cumulus expansion (Kobayashi et al., 1994).
At the blastocyst stage, we only detected TGFß receptor type I mRNA. Since both receptors are believed to be necessary for signalling (Wrana et al., 1992; Moustakas et al., 1993; Okadome et al., 1994) this indicates that TGFß signalling may be quiescent during the early cleavage stages of the human preimplantation embryo. This contrasts to recent findings on bovine oocytes and preimplantation embryos, where both TGFßR-I and -II were found in 2-, 4-, 8-cell, morula and blastocyst stages (Roelen et al., 1998).
As discussed above for activins, it is possible that, during these early cleavage stages, TGFß may act in synergy with other growth factors present in the human pre-embryo. The platelet-derived growth factor (PDGF) receptor, for instance, is present in the human pre-embryo from the 4-cell stage and onward (Osterlund et al., 1996). This receptor is down regulated upon binding its ligand (Heldin et al., 1982) and TGFß has been reported to significantly enhance the recovery of the PDGF receptor in human embryonic fibroblasts (Psarras et al., 1994). TGFß is also known to act synergistically with EGF (Paria and Dey, 1990).
In conclusion, we have identified the main required components for signalling via TGFß in the human oocyte, indicating that TGFß signalling may be of importance for oocyte development, thereby possibly influencing successful fertilization and implantation.
Acknowledgments
This study was supported by grants from the Swedish Medical Research Council (14X-07164) and Karolinska Institutets Fonder. We thank Dr H.Wramsby and Dr Å.Pousette for support and advice, the staff at the Reproductive Medical Center, Karolinska Hospital, and the patients who donated excess oocytes and fragmented embryos.
Notes
1 To whom correspondence should be addressed at: Division of Obstetrics and Gynecology, Reproductive Medical Center, Department of Woman and Child Health, Karolinska Hospital, S-171 76 Stockholm, Sweden. E-mail: gabriel.fried{at}fyfa.ki.se 
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Submitted on December 1, 1999; accepted on March 15, 2000.
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