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BOR - Papers in Press, published online ahead of print June 14, 2006.
Biol Reprod 2006, 10.1095/biolreprod.105.047860
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BIOLOGY OF REPRODUCTION 75, 508–517 (2006)
DOI: 10.1095/biolreprod.105.047860
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Involvement of the STAT5 Signaling Pathway in the Regulation of Mouse Preimplantation Development

Makoto Nakasato 2, Yoshiyuki Shirakura 2, Masatoshi Ooga 2, Masato Iwatsuki 2, Masahiko Ito 2, Shun-ichiro Kageyama 2, Senkiti Sakai 3, Masao Nagata 2, and Fugaku Aoki 1 2

Department of Integrated Biosciences,2 Graduate School of Frontier Sciences, University of Tokyo, Chiba 277-8562, Japan Department of Animal Breeding,3 Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan

ABSTRACT

The signal transducer and activator of transcription 5 (STAT5) is an essential factor in the signal transduction pathways for a number of cytokines that regulate the growth and differentiation of mammalian cells. In this study, we investigated the STAT5 signaling pathway in mouse embryos, to elucidate the mechanism of cytokine signal transduction that regulates preimplantation development. The results of the RT-PCR analysis showed that both STAT5A and B were expressed throughout preimplantation development. Immunocytochemistry revealed that the STAT5A/B proteins were located in the nucleus from the early 1-cell stage to the blastocyst stage. STAT5 activation appeared to be regulated by Janus kinases (JAKs) and SRC family kinases (SFKs), since inhibitors of these kinases inhibited the localization of STAT5 proteins to the nucleus. The JAK inhibitor Ag490 reduced both the developmental rate of the embryos and the expression levels of the downstream genes of the JAK-STAT5 signaling pathway. These findings suggest that STAT5 proteins function in preimplantation development by mediating the signals from cytokines.

developmental biology, early development, embryo, growth factors, signal transduction

INTRODUCTION

In somatic cells, signaling pathways stimulated by cytokines are essential for the control of gene expression and cell growth. Cytokines have been implicated in the regulation of development of preimplantation embryos. Growth hormone (GH) has been shown to stimulate the development of mouse preimplantation embryos cultured in vitro; it increased the percentage of embryos that developed to the blastocyst stage by 25%, as well as the number of cells in those blastocysts [1]. Csf2–/- (previously known as Gm-csf) mice show reduced rates of preimplantation development [2]. Prolactin receptor (Prlr)-null mice also exhibit reduced rates of fertilization and preimplantation development [3]. Although there have been numerous reports regarding the involvement of cytokines in the regulation of preimplantation development, the mechanisms that underlie downstream signaling remain unclear.

The signal transducer and activator of transcription 5 (STAT5) is an essential factor in signal transduction pathways for a number of cytokines and growth factors [4–6]. Mammalian STAT5 consists of two highly related homologs, STAT5A and B [7], which form a homodimer or heterodimer [8] to coregulate cell growth and differentiation in various cell types [9–13]. Stat5a–/–/Stat5b–/– double-knockout mice exhibit female infertility [14]. Knockout mice with the Prlr gene, which is involved in the activation of STAT5s, also show reproductive abnormalities [3]. Thus, STAT5s may participate in oogenesis and/or embryo development.

The activation of STAT5 proteins is regulated by the phosphorylation of tyrosine residues at the C-terminus. Activated STAT5s are dimerized through interaction of the phosphorylated tyrosine residues in the SRC-homology 2 (SH2) domains. The STAT5 dimer translocates into the nucleus and activates the transcription of downstream genes [15]. Many protein tyrosine kinases phosphorylate the tyrosine residues of STAT5s, which leads to activation, and most of these kinases are classified into two groups. The first group is the JAK kinase family, which includes JAK1, JAK2, JAK3, and TYK2. These factors activate STAT5 in the signaling pathways of a variety of cytokines, e.g., prolactin (PRL), GH, and CSF2 [16–18]. The second group contains the SRC family kinases (SFKs), such as CSK, YES1, FYN, and LYN. SFKs activate STAT5 in the cytokine signaling pathways for erythropoietin and epidermal growth factor (EGF) [19–21].

In this study, we analyzed the STAT5 signaling pathway, to understand the mechanism of cytokine signal transduction that regulates preimplantation development. The results show that both STAT5A/B are expressed and activated during preimplantation development. STAT5s activation appears to be controlled by JAKs and SFKs, since inhibitors of these kinases inhibited the localization of STAT5s to the nucleus. The JAK inhibitor Ag490 reduced the developmental rate of embryos and the expression of the downstream genes of the JAK-STAT5 signaling pathway. These findings suggest that STAT5s participate in preimplantation development via the regulation of gene expression.

MATERIALS AND METHODS

Animals

Three-week-old female ddY mice and mature male ICR mice were purchased from SLC Japan (Shizuoka, Japan). They were maintained on a constant light/dark cycle (14L:10D), with standard mouse food and water available. The animals were housed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.

In Vitro Fertilization

Mature male ICR mice were killed by cervical dislocation, and the cauda epididymidis was removed. It was punctured with a 22G needle, and a mass of sperm was squeezed into a 5% CO2-saturated 200-µl drop of Whitten medium (WM) [22] covered with paraffin oil. The sperm were incubated in 5% CO2-95% air with 100% humidity at 38°C for 2 h before use.

Female ddY mice were superovulated by injection with 5 IU pregnant mare serum gonadotropin (Teikoku Zouki, Tokyo, Japan), followed 48 h later by 5 IU human chorionic gonadotropin (hCG; Sankyo, Tokyo, Japan). Sixteen hours after hCG injection, the female mice were killed by cervical dislocation and their oviducts were removed. The oocyte-cumulus cell complexes were isolated in a 5% CO2-saturated 200-µl drop of WM covered with paraffin oil. For in vitro fertilization, 5 µl of the sperm suspension was added to the medium that contained the oocytes. Inseminated oocytes were incubated in 5% CO2-95% air at 38°C. Six hours after insemination, the embryos were detached from the surrounding cumulus cells. Embryos that had two pronuclei were collected, washed three times in a 100-µl drop of CZB medium [23], and incubated in a 200-µl drop of CZB medium. Forty hours after insemination, the embryos were transferred and incubated in a 200-µl drop of CZB medium that contained 1 mM glucose. All procedures described herein were reviewed and approved by the University of Tokyo Institutional Animal Care and Use Committee and were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.

Reverse Transcription (RT)-PCR

Total RNA was isolated from MII stage oocytes and embryos at various developmental stages. The cells were collected into Isogen (Wako, Osaka, Japan) and kept at –80°C until use. The samples were thawed at room temperature, and 100 pg of rabbit {alpha}-globin mRNA was added as an external control before RNA isolation.

The RNA pellets obtained were resolved in 27 µl of DEPC-treated water and added to 2 µl Oligo(dT)12–18 primer (Invitrogen, Carlsbad, CA) and 2 µl of 10 mM dNTP mix (Takara Bio Inc., Kyoto, Japan). After incubation at 70°C for 5 min, the sample mixture was supplemented with 4 µl of 10x RT reaction buffer, 4 µl dithiothreitol (DTT), 0.5 µl ReverScript II (Wako), and 0.5 µl RNasin ribonuclease inhibitor (Promega, Madison, WI), and incubated at 42°C for 90 min, followed by incubation at 75°C for 15 min. The sample solution was cooled to 4°C and used as the cDNA template for PCR.

Each PCR mixture (25 µl) consisted of 18.45 µl double-distilled water, 2.5 µl 10x PCR buffer, 0.5 µl of 10 mM dNTP mix, 0.3 µl of 250 mM MgCl2, 0.25 µl ExTaq HS (Takara Bio), 0.5 µl each of 10 µM gene-specific sense and antisense primers, and 2 µl of cDNA template. PCR was carried out in cycles that consisted of denaturation at 94°C for 30 sec, annealing at the indicated temperature for 30 sec, extension at 72°C for 1 min, and a final extension step at 72°C for 10 min. The annealing temperatures and sequences of the primers for each gene are shown in Table 1.


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  		TABLE 1. PCR primers used in this study

For the analysis of Stat5 transcripts, the relative amounts of PCR products were determined by measuring the densities of the bands in electrophoresis gels. The PCR products of the Stat5 and {alpha}-globin genes that were obtained after 40 and 27 cycles of PCR, respectively, were separated by electrophoresis in a 2% agarose gel and stained with ethidium bromide. The gel image was obtained using the UV illuminator DT-20MP (Atto, Tokyo, Japan) and the densities of the bands were measured using NIH Image software (National Institutes of Health, Bethesda, MD). The values were normalized for the rabbit {alpha}-globin gene.

For the analysis of Lta (previously known as Tnf-ß), Cdkn1a (p21Cip/Waf1), Eif1ax, and Csn2 (ß-Casein) mRNAs, their relative amounts were determined by real-time PCR using the Smart Cycler System (Takara Bio). With the exception of Csn2, the reaction mixture was formulated as described above. For the detection of double-strand DNA, 1/30 000 (v/v) of SYBR Green I (BioWhittaker Molecular Applications, Rockland, ME) was added to the PCR mixtures. For Csn2 detection, the reaction mixture (25 µl) contained 9.25 µl double-distilled water, 2.5 µl of 10x PCR buffer, 2 µl of 2.5 mM dNTP mix, 1.5 µl of 25 mM MgCl2, 0.25 µl of ExTaq HS (Takara Bio), 2.5 µl each of 3 pM gene-specific sense and antisense primers, 2.5 µl of 2 pM gene-specific TaqMan probe, and 2 µl of cDNA template. The sequences of the PCR primers and TaqMan probe used are shown in Table 1. PCR was performed for 35 cycles of denaturation at 95°C for 15 sec, annealing at the indicated temperature for 15 sec, and extension at 72°C for 20 sec, with measurement of the fluorescence for 6 sec. The annealing and measurement temperatures are shown in Table 1. To test the specific amplification, PCR products were subjected to agarose gel electrophoresis. In the PCR products, only a single band was observed in their expected base-pair sizes. The relative amounts of cDNA products were determined using cDNA standard curves, which were obtained by amplification of a 3-fold dilution series of a cDNA that had been prepared by PCR. In all samples, PCR products were detected in the linear range of this standard curve, except for Eif1ax. For detection of Eif1ax transcripts, the product of one sample could not be detected on the standard curve. The amount of this sample was assumed to be less than 1/100th the amount of the DNA sample of control 2-cell embryos, since at least 1/100th of the control embryos could be detected on the standard curve.

Immunoblotting

After washing three times in PBS that contained 3 mg/ml polyvinylpyrrolidone, 150 MII stage oocytes and embryos at various developmental stages were collected into 12 µl of modified RIPA buffer [100 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholic acid, 50 mM Tris-HCl (pH 8.0 at 12°C), 15 mM ethyleneglycotetraacetic acid (EGTA), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml aprotinin, 10 µg/ml leupeptin, 1 µg/ml pepstatin A]. The samples were stored at –80°C until use. The samples were thawed at room temperature, added to 12 µl of 2x SDS sample buffer [12% ß-mercaptoethanol, 4% SDS, 20% glycerol, 0.1 M Tris-HCl (pH 6.8 at 12°C)], and incubated at 95°C for 3 min. The samples were separated by electrophoresis in a 10% polyacrylamide gel, followed by transfer to a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated overnight at 4°C in the primary antibody solution. The primary antibody solutions were prepared by diluting the antibodies to a concentration of 0.4 µg/ml in Tris-buffered saline (TBS; 137 mM NaCl, 2.68 mM KCl, 25 mM Tris) that contained 0.1% (v/v) Tween-20 and 0.2% ECL Advance blocking agent (Amersham Biosciences, Piscataway, NJ). The anti-STAT5A rabbit polyclonal antibody (L-20; Santa Cruz Biotechnology, Santa Cruz, CA) or the anti-STAT5B mouse monoclonal antibody (G-2; Santa Cruz Biotechnology) was used as the primary antibody. After three washes with TBS that contained 0.1% (v/v) Tween-20 (TBS-T), the membranes were incubated in the secondary antibody solution at room temperature for 1 h. The secondary antibody solutions were prepared by diluting the antibodies to 1:1000 (v/v) in TBS-T that contained 0.2% ECL Advance blocking agent. The horseradish peroxidase-conjugated anti-rabbit IgG antibody derived from a donkey (Amersham Biosciences) or the peroxidase-conjugated anti-mouse IgG antibody derived from a sheep (Amersham Biosciences) was used as the secondary antibody. The adsorbed antibodies were reacted with the ECL Advance Western Blotting Detection Kit (Amersham Biosciences) and detected using the Fujifilm LAS-1000 Plus luminoimager (Fuji Photo Film, Kanagawa, Japan).

To examine the specificities of the antibodies, 50 embryos at the 2-cell stage were subjected to immunoblotting with 0.4 µg/ml primary antibodies that had been preincubated with 0.4 or 1 µg/ml antigen peptides of STAT5A or STAT5B, respectively, for 1 h before use.

Treatment with Tyrosine Kinase Inhibitors

Sixty embryos at each developmental stage were collected, transferred, and incubated in 200 µl CZB medium with 0.2% (v/v) of tyrosine kinase inhibitor that had been prepared by adding the stock solution of Ag490 (Merck Biosciences, Darmstadt, Germany) or SU6656 (Merck Biosciences) to CZB medium to a final concentration of 20 µM or 5 µM, respectively. The stock solutions of the inhibitors were prepared by dissolving in dimethylsulfoxide (DMSO). As a control, 0.2% DMSO was added to the medium. The embryos were observed every day, and the percentages of embryos that had developed to the 2-cell, 4-cell, morula, and blastocyst stages at 31 h (Day 2), 48 h (Day 3), 72 h (Day 4), and 96 h (Day 5) postinsemination, respectively, were determined.

Immunocytochemistry

The oocytes and embryos were fixed in 3.7% paraformaldehyde in PBS overnight at 4°C after removal of the zona pellucida with acid MEMCO [24]. After washing three times in PBS that contained 1 mg/ml BSA (PBS/BSA), the fixed cells were permeabilized for 15 min with PBS that contained 0.5% (v/v) Triton X-100. After washing four times in PBS/BSA, the cells were incubated for 60 min in PBS/BSA that contained 2 µg/ml primary antibody (anti-STAT5A rabbit polyclonal antibody L-20; Santa Cruz Biotechnology) or the anti-STAT5B mouse monoclonal antibody (Zymed Laboratories Inc., San Francisco, CA). After washing four times in PBS/BSA, the cells were incubated with a 1:100 dilution of the secondary antibody. For the detection of STAT5A/B, FITC-conjugated donkey anti-rabbit IgG antibody (Jackson ImmunoResearch, West Grove, PA) and FITC-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch), respectively, were used as the secondary antibodies. After washing four times in PBS/BSA, the cells were mounted on a glass slide with Vectashield antibleaching solution (Vector Laboratories, Burlingame, CA). Fluorescence was detected using the Leica TCS SP2 laser-scanning confocal microscope (Leica AG, Solms, Germany).

Semiquantitative analysis of the fluorescence intensities from the images obtained by laser-scanning confocal microscopy was conducted using the NIH Image program. The average pixel value/unit area of the nucleus was subtracted by the average cytoplasmic pixel value/unit area and multiplied by the nuclear dimensions to yield the relative values of the fluorescence intensity. In each quantification procedure, the averaged value of the control embryo was set at 100% and the fluorescence intensity observed for each sample was expressed relative to this value.

In Vitro Transcription Assay

The transcriptional activity was measured in vitro as described previously [25]. Briefly, the embryos, the plasma membranes of which had been permeabilized with Triton X-100, were incubated with BrUTP. The amount of incorporated BrU was measured by immunostaining with a primary antibody recognizing BrU (Roche Diagnostics Co., Indianapolis, IN) and a Cy5-conjugated secondary antibody (Jackson ImmunoResearch). Fluorescence was detected using the Leica TCS SP2 laser-scanning confocal microscope and the intensity of the fluorescence signal was quantified using the NIH Image program.

RESULTS

Expression Levels of Stat5 mRNAs

The temporal changes in the expression levels of Stat5a/b mRNAs were examined during preimplantation development (Fig. 1A). The relative expression levels of Stat5s are shown in Fig. 1B. The level of Stat5a mRNA decreased by 80% between the MII and 4-cell stages (P < 0.05) and then increased 8-fold (P < 0.01) during the 4-cell to blastocyst stage. The expression pattern of Stat5b mRNA was similar to that of Stat5a mRNA, although the increase in level after the morula stage was more prominent. The relative expression level of Stat5b mRNA decreased by 70% from the MII to 4-cell stage and then increased 20-fold (P < 0.01) during the 4-cell to blastocyst stage.


Figure 1
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  		FIG. 1. Expression of Stat5 transcripts during preimplantation development in mice. Total RNA was isolated from the oocytes at the MII stage (MII) and from embryos at the 2-cell (2), 4-cell (4), morula (M), and blastocyst (B) stages collected 31, 48, 72, and 96 h postinsemination, respectively, and subjected to RT-PCR analysis for the expression of Stat5a/b. Rabbit {alpha}-globin mRNA was added as an external control. The images of the PCR bands on agarose gels (A) and the results of quantification of the PCR products (B) are shown. For quantification, the relative fluorescence intensities of the PCR bands were determined. The value obtained for the MII oocyte was set at 100%, and the values for the other stages were calculated relative to this value. The experiment was performed three times and the results are presented as the mean ± SEM

Expression of STAT5 Proteins

The amounts of STAT5A/B proteins were examined by immunoblotting using specific antibodies. The changes in the levels of these proteins were similar during preimplantation development (Fig. 2), while the levels of both proteins decreased slightly between the MII and 1-cell stage. In the 1-cell stage, the expression levels of the STAT5 proteins did not change between 6 h and 12 h postinsemination. After cleavage into the 2-cell stage, the protein levels increased abruptly and persisted at high levels until the blastocyst stage.


Figure 2
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  		FIG. 2. Expression of STAT5 proteins during preimplantation development in mice. The extracts of oocytes at MII stage (MII) and embryos at the 1-cell (1 C; 6 h and 12 h), 2-cell (2), 4-cell (4), morula (M), and blastocyst (B) stages collected 6, 12, 31, 48, 72, and 96 h postinsemination, respectively, were subjected to immunoblotting with antibodies directed against STAT5A/B. Arrowheads indicate the molecular weights of STAT5A/B

In the immunoblotting experiments, preincubation of the primary antibodies with peptide antigens resulted in the disappearance of the bands that corresponded to STAT5A/B from the immunoblot (Supplemental Figure S1, available online at http://www.biolreprod.org/, which indicates that the antibodies are specific.

Localization of STAT5 Proteins in the Preimplantation Embryos

The locations of the STAT5A/B proteins during preimplantation development were examined by immunocytochemistry (Fig. 3). Neither STAT5A nor STAT5B was observed in the MII stage oocytes. However, these proteins were localized in nuclei of the 1-cell stage embryos 6 h after insemination. Between the 1-cell and 2-cell stages, the concentrations of STAT5s in the nuclei increased. Thereafter, the STAT5s were localized in the nuclei until the blastocyst stage.


Figure 3
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  		FIG. 3. Localization of STAT5 proteins in preimplantation mouse embryos. Oocytes at the MII stage (MII) and embryos at the 1-cell (6 h and 12 h), 2-cell (2), 4-cell (4), morula (M), and blastocyst (B) stages collected 6, 12, 31, 48, 72, and 96 h postinsemination, respectively, were stained with antibodies directed against STAT5A (A) and STAT5B (B). Bars = 20 µm

Ag490 and SU6656 Inhibit the Localization of STAT5s to the Nucleus

Since it is known that the activation and nuclear localization of Stats are regulated by JAK and SRC family kinases [15, 21], the effects of inhibiting JAKs and SFKs on the nuclear localization of STAT5s were examined by treating the embryos with Ag490 and SU6656, which are selective inhibitors of JAK and SFKs, respectively. The embryos were incubated with Ag490 or SU6656 starting 6, 31, 48, and 72 h postinsemination, and then examined for nuclear localization of STAT5s at 31, 48, 72, and 96 h, respectively. The embryos that had been incubated with inhibitors from 6 h after insemination were also examined at 12 h.

In the embryos that were incubated with Ag490, different localization patterns were observed for STAT5A/B (Fig. 4). Following treatment with Ag490, STAT5A localization in the nucleus decreased significantly in the 2-cell (P < 0.001), morula (P < 0.05), and blastocyst (P < 0.001; trophectoderm [TE] and inner cell mass [ICM]) stages, but not in the 1-cell or 4-cell stage, when compared to the untreated control embryos. On the other hand, STAT5B localization decreased significantly in the 1-cell (P < 0.001; male and female pronucleus; Fig. 4 and Fig. 5), 2-cell (P < 0.001), and blastocyst (P < 0.001; ICM only) stages, but not in the 4-cell or morula stages. Thus, Ag490 inhibited the localization of STAT5B but not of STAT5A in the 1-cell embryos. In contrast, inhibition was observed for STAT5A but not for STAT5B in the morula stage embryos. Interestingly, although the localization of STAT5A was inhibited in both the ICM and TE of blastocysts, the inhibition of STAT5B was observed in the ICM but not in the TE (Fig. 4 and Fig. 5).


Figure 4
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  		FIG. 4. Inhibition of STAT5 localization to the nucleus by tyrosine kinase inhibitors Ag490 and SU6656 in preimplantation mouse embryos. The embryos were incubated with Ag490 or SU6656 starting from 6, 31, 48, and 72 h postinsemination, and then examined for the localization of Stat5s at 31 h (2-cell), 48 h (4-cell), 72 h (morula), and 96 h (blastocyst), respectively. The embryos that had been incubated with inhibitors from 6 h postinsemination were also examined at 12 h (1-cell). The embryos were subjected to immunocytochemistry with anti-STAT5A or anti-STAT5B antibody, and the relative fluorescence intensities of the nuclei were determined. Sixty nuclei were examined at the various stages, except for the 1-cell and 2-cell stages, for which only 15 and 20 nuclei, respectively, were examined. The mean value for the control (untreated) embryos was set at 100%, and the values obtained for the inhibitor-treated embryos were calculated relative to this value. The results are presented as the mean ± SEM. *P < 0.05; ***P < 0.001


Figure 5
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  		FIG. 5. Localization of STAT5s in mouse preimplantation embryos treated with Ag490 and SU6656. The embryos were treated with Ag490 or SU6656 starting from 6 or 72 h postinsemination, and subjected to immunocytochemistry with anti-STAT5A or anti-STAT5B antibody at 12 h (1-cell) or 96 h (blastocyst), respectively. The embryos that had been cultured without inhibitors served as the control. The laser-scanning confocal microscopy images are shown. Bars = 20 µm

SU6656 did not affect the nuclear localization of STAT5A or STAT5B between the 1-cell and morula stages (Fig. 4). However, in the blastocyst stage embryos, the patterns of inhibition were similar to those seen with Ag490 (Fig. 5). Although SU6656 significantly inhibited the nuclear localization of STAT5A in both the ICM and TE (P < 0.001; Fig. 4), decreased localization of STAT5B was observed for the ICM (P < 0.001) but not for the TE (Fig. 4).

Ag490 and SU6656 Inhibit the Development of Embryos at Specific Stages During Preimplantation Development

The embryos were incubated with Ag490 or SU6656 starting 6 h postinsemination and examined for development up to 96 h postinsemination (Fig. 6A). Although Ag490 did not affect the first cleavage, it significantly inhibited the second cleavage (P < 0.001); only 10% of the embryos that were treated with the inhibitor developed to the 4-cell stage on Day 3.


Figure 6
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  		FIG. 6. Effects of treatment with Ag490 or SU6656 on preimplantation development in mice. The embryos were cultured with Ag490 or SU6656 from 6 to 96 h (A), 31 to 72 h (B), 48 to 96 h (C), and 72 to 96 h (D). The embryos that had been cultured without the inhibitors served as the control. The percentages of embryos that developed to the 2-cell, 4-cell, morula, and blastocyst stages at 31 h (Day 2), 48 h (Day 3), 72 h (Day 4), and 96 h (Day 5) postinsemination, respectively, were determined. The experiment, in which 60 embryos were used for each treatment, was performed four times. The results are presented as the mean ± SEM. The asterisk designations indicate significant differences, as compared to the corresponding controls at *P < 0.05, **P < 0.01, and ***P < 0.001

Thereafter, none of the treated embryos developed to the blastocyst stage on Day 5, by which time >60% of the control embryos had developed to the blastocyst stage. The treatment with SU6656 did not affect development before the blastocyst stage. However, the percentage of embryos that had developed to the blastocyst stage on Day 5 was significantly lower (P < 0.05) in the inhibitor-treated group than in the control group.

Since the effects of the inhibitors on the nuclear localization of the STAT5s were dependent on the developmental stage (Fig. 4), the embryos were incubated with Ag490 or SU6656 starting from 6, 31, 48, and 72 h postinsemination and examined for development at 31, 48, 72, and 96 h. Initially, the embryos were incubated with Ag490 or SU6656 starting from 31 h (Day 2) postinsemination and examined for development until the morula stage. Treatment with either Ag490 or SU6656 had no effect on the cleavage of embryos between the 2-cell and morula stage (Fig. 6B). The embryos were also incubated with the inhibitors starting from 48 h (Day 3) and examined for development until the blastocyst stage. Although treatment with either Ag490 or SU6656 had no effect on the development until morula stage on Day 4, treatment with either of these inhibitors significantly inhibited development to the blastocyst stage on Day 5 (P < 0.05). The percentage of embryos that developed to the blastocyst stage was lower by 25% in the Ag490-treated group than in the control group and lower by 20% in the SU6656-treated group than in the control group (Fig. 6C). Finally, the embryos were incubated with the inhibitors starting from 72 h (Day 4) postinsemination, and examined for development until the blastocyst stage. Both Ag490 and SU6656 significantly inhibited cleavage between days 4 and 5 (P < 0.05; Fig. 6D).

Effects of Ag490 on Gene Expression in the 2-cell Embryos

We showed that Ag490 significantly inhibited the development of the 2-cell stage embryos (Fig. 6A). Since STAT5s function as transcription factors in somatic cells, it seems likely that Ag490 inhibits the transcription of genes that are targeted by the JAK-STATs signaling pathway in the 2-cell stage embryos. During the 2-cell stage, a burst of zygotic gene activation that is critical for the development of mouse embryos occurs [26]. Therefore, we examined the total transcriptional activity and the expression levels of specific genes regulated by the JAK-STATs signaling pathway in 2-cell stage embryos that had been incubated with or without Ag490.

Total transcriptional activity was measured by the in vitro BrUTP incorporation assay. Since the JAK-STATs signaling pathway stimulates the transcription of particular target genes, it was expected that the inhibition of JAKs by Ag490 would affect the expression of some specific genes, but not that of global genes. The results of the in vitro transcriptional activity assay were consistent with this hypothesis in that they showed that embryos treated with Ag490 from 6 h to 29 h postinsemination did not exhibit any differences in total transcriptional activity as compared to the control embryos (Fig. 7). Thus, Ag490 did not affect the total transcriptional activity of the 2-cell stage embryos.


Figure 7
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  		FIG. 7. Total transcriptional activity of 2-cell stage mouse embryos treated with Ag490. Embryos that were incubated with (+Ag490) or without (–Ag490) Ag490 from 6 h postinsemination were collected at 29 h, and then subjected to the in vitro transcription assay. (A) Confocal microscopic images of embryos in which the incorporation of BrUTP was detected by immunocytochemistry. Two blastomeres in the 2-cell stage embryos fused after treatment with Triton X-100 in the assay procedure. Bar = 20 µm. (B) Quantification of incorporated BrU. For quantification, the relative fluorescence intensities of the nuclei were determined. The averaged value for the control (untreated) embryos was set at 100%. The experiment was performed three times and the data were accumulated. The results are presented as the mean ± SEM. The numbers of embryos examined were 36 and 44 in the control (–Ag490) and experimental (+Ag490) groups, respectively

To examine the effect of Ag490 on the transcription of genes whose expression levels are regulated by the JAK-STATs signaling pathway, quantification of the transcripts was performed using real-time PCR. In this experiment, we selected four monitoring genes: Csn2, Lta, Cdkn1a, and Eif1ax. The expression levels of Csn2, Lta, and Cdkn1a mRNAs are regulated by the JAK-STAT5 signaling pathway in somatic cells [27–30]. The Eif1ax gene was used as a control gene that is not regulated by the JAK-STAT5 signaling pathway.

The results of the real-time PCR analysis showed that the expression levels of all four genes were significantly increased between the MII and 2-cell stages (P < 0.05; Fig. 8). Comparing the expression levels of the untreated and treated 2-cell embryos, the expression levels of the Csn2 and Lta genes were significantly inhibited by treatment with Ag490 (Fig. 8, A and B). The expression levels of Cdkn1ax transcripts were slightly lower in the embryos that were treated with Ag490 (Fig. 8C), although this difference was not significant. The levels of mRNAs for Csn2, Lta, and Cdkn1a were decreased by 70%, 60%, and 15%, respectively, by treatment with Ag490. In contrast, the expression level for Eif1ax mRNA was not affected by this treatment (Fig. 8D).


Figure 8
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  		FIG. 8. Effect of Ag490 on the expression of genes targeted by the JAK-STATs signaling pathway in the 2-cell mouse embryos. The transcripts for the Csn2, Lta, Cdkn1a, and Eif1ax genes were quantified by real-time, fluorescence-monitored RT-PCR of the MII stage oocytes and 2-cell stage embryos. The 2-cell stage embryos were treated with (+Ag490) or without (–Ag490) Ag490 from 6 h postinsemination and collected at 31 h. For quantification, the value obtained for the untreated 2-cell embryos was set at 100%, and the values for the treated embryos were calculated relative to this value. The experiments were performed four and three times for ß-casein (A) and the other genes (B–D), respectively. The results are presented as the mean ± SEM. The asterisk designations indicate significant differences at *P < 0.05, **P < 0.01, and ***P < 0.001, respectively

DISCUSSION

In this study, we examined STAT5 expression and deduced a potential mechanism for the activation of STAT5s in preimplantation mouse embryos. Previous reports have shown that STAT5s play important roles in transcriptional regulation in various somatic cells [31, 32].

RT-PCR analysis revealed that the expression levels of Stat5a/b transcripts per embryo were dynamically changed during preimplantation development (Fig. 1). However, previous microarray analyses did not show evident changes of Stat5 transcripts during preimplantation development [33–35]. Since these analyses were performed after normalizing the total amount of amplified cDNA, the results did not necessarily reflect the changes in the mRNA quantity per whole embryo, which may be why the increase in Stat5s transcripts in the morula and blastocyst stages was not detected in the microarray analyses; the quantity of total mRNA also increased up to the blastocyst stage during preimplantation development [36], and the normalization of this increase seemed to counterbalance the increase in Stat5s transcripts. In our results, the relative expression levels of Stat5 transcripts decreased between the MII and 4-cell stages, and then increased from the morula stage, which suggests that maternally derived mRNAs are gradually degraded until the 4-cell stage, and that the expression of Stat5s from embryonic genomes starts at the morula stage. It has been reported for the goat mammary gland that Stat5 expression is induced by treatment with GH [37]. In the case of mouse preimplantation embryos, it is known that GH mRNA is expressed from the morula stage [38]. Therefore, the expression of Stat5s may also be induced by autocrine GH after the morula stage.

The expression patterns of Stat5a/b mRNAs were similar (Fig. 1), which suggests that the expression levels of Stat5s are regulated by a common mechanism during preimplantation development. In humans, these genes map to the adjacent locus on chromosome 17. The functions of the human Stat5a/b promoters are identical [39]. The mouse Stat5 genes also map to the adjacent locus on chromosome 11 [40]. Therefore, it is likely that the mechanisms that regulate the transcription of mouse Stat5a/b are comparable, which ensures similar patterns of Stat5a/b expression during preimplantation development. However, the rates of increase in Stat5a/b expression after the 4-cell stage were different. During the 4-cell to blastocyst stage, the expression level of Stat5a mRNA increased 8-fold, while that of Stat5b increased 20-fold. This difference may have been due to differences in mRNA stability. Stat5a mRNA may be more labile than Stat5b mRNA. Indeed, the rate of degradation of maternally stored Stat5a mRNA seemed to be faster than that of Stat5b mRNA postfertilization (Fig. 1B).

The localization of STAT5 proteins in the nucleus was observed throughout preimplantation development and even in the embryos at the early 1-cell stage (Fig. 3). It is known that activated STAT5s localize to the nuclei of somatic cells, where they can induce transcription [15]. Thus, STAT5 localization in the early 1-cell stage nuclei suggests that STAT5s are activated and involved in the regulation of transcription during the early 1-cell stage. However, in mouse embryos, embryonic transcription starts at the late 1-cell stage [41, 42]. Therefore, STAT5s appear to be localized to the nucleus so as to prepare the initiation of transcription during the early 1-cell stage, and STAT5s are functional in transcription after the late 1-cell stage. After the 1-cell stage, the STAT5s were always located in the nucleus until the blastocyst stage, which suggests that STAT5s are continuously activated during all the stages of preimplantation development.

The changes in the amounts of STAT5 proteins in the nucleus were not consistent with the changes in the total amounts of proteins detected by immunoblotting. Although the total levels increased greatly between the 1-cell and 2-cell stages (Fig. 2), only slight increases in the nuclear concentrations of the proteins were observed (Fig. 3). This inconsistency may reflect the fact that the STAT5 proteins were expressed excessively after the 2-cell stage, while only some of the proteins were localized to the nucleus.

The tyrosine kinase inhibitors Ag490 and SU6656 inhibited the translocation of STAT5s into the nucleus during preimplantation development (Fig. 3). However, the patterns of inhibition differed according to the stage of preimplantation development between STAT5A/B and between TE and ICM in the blastocysts. These patterns of inhibition are summarized in Table 2.


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  		TABLE 2. Inhibition of nuclear localization of STAT5s in preimplantation mouse embryos by Ag490 and SU6656.*

Ag490 was originally shown to inhibit JAK2 specifically in pre-B cells. It was effective at the range from 1 to 50 µM, and its effect reached a plateau at 20 µM in hematopoietic progenitor cells [43]. Similar results were obtained in the dose-response experiment in mouse embryos (data not shown). At the range of 2 to 20 µM, the percentages of cleaved embryos into the 4-cell stage decreased with the dose of the inhibitor. SU6656 is a selective inhibitor of SRC family kinases [44]. It has been shown that 5 µM SU6656 was effective in inhibiting fertilization in rat eggs [45]. Therefore, we used this concentration of SU6656 in the mouse preimplantation embryos used in this study.

In terms of developmental stage, Ag490 inhibited the nuclear localization of STAT5A in the 2-cell, morula, and blastocyst stages but not in the 1-cell or 4-cell stage, and that of STAT5B in the 1-cell, 2-cell, and blastocyst stages but not in the 4-cell or morula stage (Fig. 4 and Table 2). These results suggest that JAK kinase regulates the activation of STAT5A and/or STAT5B at specific stages during preimplantation development. Moreover, treatment with SU6656 inhibited the activation of STAT5s in the blastocyst stage only. These results suggest that the mechanisms for activation of STAT5s change during preimplantation development. The activities of the upstream kinases involved in STAT5 signaling are regulated by cytokines and cytokine receptors in the signaling pathways. Therefore, it seems likely that the expression of cytokines and their receptors that are involved in STAT5s signaling pathways are modulated during preimplantation development. Indeed, we have previously reported dynamic changes in the expression of cytokine receptors that activate JAK2-mediated STAT5 signaling pathways in cells during preimplantation development [46].

On the other hand, STAT5A/B differed in terms of their activation mechanisms during preimplantation development. In the 1-cell, morula, and blastocyst stages, the patterns of inhibition of nuclear localization were different for STAT5A/B (Fig. 4 and Table 2). In the 1-cell stage, Ag490 inhibited the nuclear localization of STAT5B but not that of STAT5A. In contrast, in the morula and TE of the blastocysts, Ag490 decreased the localization of STAT5A but not that of STAT5B. SU6656 also inhibited the nuclear localization of STAT5A, but not that of STAT5B, in the TE of blastocysts. Although in somatic cells, STAT5A/B are activated by common mechanisms that involve many cytokine signaling pathways, some cytokine receptors activate only one of these two species. Several receptors for peptide growth factors such as TGFBRE (previously known as TGFR) and EGFR selectively activate STAT5B in epithelial cells [47]. The insulin receptor (INSR) also activates STAT5B, but not STAT5A, in rhabdomyosarcoma cells [48]. In contrast, the CSF2 receptor preferentially activates STAT5A in human blood monocytes [49]. It seems that the signaling pathways that act via cytokine receptors to activate only one of the STAT5s, in addition to the pathways that activate both STAT5s, are functional during preimplantation development.

In terms of the cell lineage, the difference observed for the inhibition of STAT5 nuclear localization between the TE and ICM suggests that the mechanisms for the activation of STAT5s are different for these two lineages in blastocysts. In the Ag490-treated blastocysts, the localization of STAT5B in the nucleus was decreased in the ICM, but not in the TE (Fig. 5 and Table 2). This difference may have been caused by mechanisms that regulate the differentiation of cell lineages at the blastocyst stage.

The stages at which the inhibitors exerted their effects on development and the nuclear localization of STAT5s coincided well with each other. Ag490 affected development between the 2-cell and 4-cell stages and between the morula and blastocyst stages (Fig. 6), and it affected the nuclear localization of STAT5s at similar stages (Table 2). Although SU6656 inhibited neither the development nor the nuclear localization of STAT5s before the blastocyst stage, it affected both the nuclear localization of STAT5s and development at the blastocyst stage (Fig. 6 and Table 2). Thus, JAK- and SFK-mediated signaling pathways that involve STAT5s seem to be activated and regulate the growth of embryos during preimplantation development. However, Ag490 and SU6656 did not affect the translocation of STAT5A into the nucleus in the 1-cell and 4-cell stage embryos or that of STAT5B in the 4-cell, morula, and TE of the blastocyst stage embryos. During these stages, STAT5s are probably redundantly activated by both JAKs and SFKs and/or other upstream kinases that are not inhibited by Ag490 and SU6656, e.g., JAK1 and TYK2 [15]. A number of reports have demonstrated signaling pathways that involve JAK1 and TYK2 in the regulation of somatic cell growth [50–52]; they have shown that several cytokines, e.g., GH and interleukins, activate STAT5s via these tyrosine kinases [53].

Our results suggest that the JAK signaling pathway plays a critical role in development between the 2-cell and 4-cell stages, since treatment with Ag490 almost completely arrested the embryos at the 2-cell stage (Fig. 7). During the 2-cell stage, maternal RNA is drastically degraded [54], while the amounts of mRNAs transcribed from zygotic genome increase. In mice, the transition from maternal to embryonic control of development occurs at the 2-cell stage [27, 55–57]. Therefore, those transcripts that are regulated by the JAK-STAT5 signaling pathway probably play a role in the regulation of development at the 2-cell stage. A recent report has shown that JAK2 is expressed in mouse embryos at the 1-cell and 2-cell stages [58]. Therefore, JAK2 is a candidate for the mediator of STAT5 activation during these developmental stages.

In the activated STAT5 signaling pathway, phosphorylated STAT5 proteins are dimerized and translocated into the nucleus. They bind to the target sequences, IFN-gamma activation site (GAS)-like elements, which contain the TTCNNNGAA sequence and lie in the promoters of specific genes, and this leads to the activation of transcription [54]. Thus it is likely that inhibition of the STAT5 signaling pathway decreases the expression of specific genes that have GAS-like elements in their promoters. The in vitro transcription assay showed that treatment with Ag490 did not affect total transcriptional activity (Fig. 8), which suggests that the fraction of transcripts from the downstream genes of the JAK-mediated signaling pathway is relatively small in 2-cell embryos. On the other hand, the real-time PCR analysis showed that the expression levels of several downstream genes of the JAK-STAT5 signaling pathway, i.e., Csn2 and Lta, were selectively suppressed by treatment with Ag490 (Fig. 8). These results confirm that the JAK-STAT5 signaling pathway functions in the regulation of expression of downstream genes at the 2-cell stage.

Of the genes whose promoter sequences contain a GAS-like region, the expression levels of Csn2 and Lta, but not Cdkn1a, were inhibited by treatment with Ag490 (Fig. 8, A–C). This difference may be explained by the finding that the expression of the Cdkn1a gene is stimulated not only by the JAK-STAT5 signaling pathway, but also by another signaling pathway. In somatic cells, the expression of Cdkn1a mRNA is also stimulated by EGFR-CSK (c-SRC tyrosine kinase) [58] and PDTC-p38MAPK signaling pathway [59]. These pathways may function in the 2-cell embryos.

In conclusion, both of STAT5A/B are activated throughout preimplantation development and seem to function in the regulation of gene expression. The JAK- and SFK-mediated signaling pathways are involved in embryo growth, and STAT5s are candidate components of these signaling pathways. Thus STAT5s may play several important roles in the regulation of preimplantation development.

FOOTNOTES

1 Correspondence. FAX: 81 4 7136–3698; aokif{at}k.u-tokyo.ac.jp Back

Received: 26 September 2005.

First decision: 24 October 2005.

Accepted: 23 May 2006.

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