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BOR - Papers in Press, published online ahead of print March 3, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.023226
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BIOLOGY OF REPRODUCTION 71, 89–96 (2004)
DOI: 10.1095/biolreprod.103.023226
© 2004 by the Society for the Study of Reproduction, Inc.

Embryo

Localization of Janus Kinase 2 to the Nuclei of Mature Oocytes and Early Cleavage Stage Mouse Embryos

Masahiko Ito2, Makoto Nakasato2, Tomonori Suzuki3, Senkiti Sakai3, Masao Nagata2, and Fugaku Aoki1,2

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, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Jak2, which is a member of the Janus tyrosine kinase family, plays essential roles in cytokine signal transduction and in the regulation of cell growth and gene expression. To investigate the involvement of Jak2 in the regulation of early preimplantation development, we examined the expression of Jak2 in mouse embryos. Reverse transcription-polymerase chain reaction assays revealed that the relative amount of Jak2 mRNA was highest in unfertilized oocytes, gradually decreased until the four-cell stage, and remained at low levels until the blastocyst stage. Immunocytochemistry showed that Jak2 was localized predominantly to the female pronucleus in one-cell embryos. The immunofluorescence signal was very weak or undetectable in the male pronucleus. In unfertilized oocytes and one-cell embryos at M phase, Jak2 was localized to the chromosomes. After cleavage to the two-cell stage, the intensity of the immunofluorescence signal decreased in the nucleus while the embryos were in late G2. This decrease was independent of DNA synthesis because it was not affected by inhibition of DNA replication. However, inhibition of protein synthesis repressed the disappearance of Jak2 from the nucleus. These results suggest a novel function for Jak2 in the regulation of early preimplantation development.

early development, embryo, gamete biology, kinases, signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein tyrosine kinases (PTKs) play important roles in numerous cellular functions, e.g., differentiation, proliferation, and morphogenesis, in various types of cells. Certain oncogene products and receptors for growth factors possess intrinsic tyrosine kinase activities and transduce signals to their respective substrates [1, 2]. Most PTKs and serine/ threonine kinases are activated by phosphorylation and participate in cascades of protein phosphorylation, in which PTKs often take part in the initial stage of the cascade. In the transduction of hormone and cytokine signals, the cascades start with the association of ligands with their receptors, which either possess intrinsic PTK activity or bind PTK.

In preimplantation mouse embryos, it has been shown that several peptide growth factors, the receptors of which possess intrinsic PTK activity, are involved in the regulation of development. The receptors for insulin, insulin-like growth factor (IGF-I), epidermal growth factor (EGF), and transforming growth factor {alpha} (TGF{alpha}) are expressed in mouse preimplantation embryos and regulate cell proliferation and differentiation [3–6]. Various oncogenes, e.g., c-raf-1, rasII, rasK, rasN, c-fos, and c-myc, which are downstream of PTKs in the signal transduction cascade, are expressed in preimplantation embryos as well as in unfertilized oocytes [7, 8].

It appears that these peptide growth factors are involved in the regulation of development after the four-cell stage. For instance, although the addition of exogenous insulin and IGF-I to the culture medium increases the cell number in blastocysts [9, 10], the receptors for insulin and IGF-I are not detectable until the compacted eight-cell stage [11]. The EGF ligand is involved in the formation of hatched blastocysts [12], but its receptor remains at low levels until the four-cell stage [13]. Although TGF{alpha} reduces the incidence of apoptosis in mouse blastocysts [14], it is not detected before the four-cell stage [15]. Thus, receptor PTKs for peptide growth factors do not seem to be involved in the regulation of development before the four-cell stage. However, it is possible that nonreceptor PTK but not receptor PTK is involved. There are many nonreceptor PTKs that bind to cytokine receptors to transduce the signal for cell proliferation. Although there is no information about the expression of those nonreceptor PTKs in early preimplantation embryos, it has been reported that various nonreceptor PTKs are expressed and activated in sea urchin eggs and embryos [16–19].

To investigate the involvement of PTK in the regulation of development before the four-cell stage in mice, we tried to identify PTKs that are expressed in unfertilized oocytes because the maternal mRNA that is accumulated in unfertilized oocytes is important for early embryonic development before the four-cell stage [20]. Reverse transcription-polymerase chain reaction (RT-PCR) using primers for the highly conserved regions of the PTK family [21] have revealed that Jak2, which is a member of the Janus protein tyrosine kinase family, is expressed in unfertilized oocytes. It is known that Jak2 binds to the receptors for members of the growth hormone superfamily that includes interleukin-6, erythropoietin, and prolactin, which leads to the transduction of their signals [22–24]. In the present study, we show that the Jak2 protein localizes to the chromosome in unfertilized oocytes. Interestingly, in one-cell embryos, the concentration of Jak2 is much higher in the female pronucleus than in the male pronucleus, which suggests that Jak2 plays a novel function in preimplantation development.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Collection and Culture of Oocytes and Embryos

All procedures described within 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. Female ddY mice, 21–23 days of age, and mature male ICR mice were purchased from SLC Japan (Shizuoka, Japan). The female mice were superovulated by injection with 5 IU of eCG. To obtain ovulated metaphase II-stage oocytes, the eCG-primed mice were injected 48 h later with 5 IU of hCG. Ovulated oocytes were collected from the oviducts into modified Whitten medium (109.51 mM NaCl, 4.78 mM KCl, 1.19 mM KH2PO4, 1.19 mM MgSO4, 22.62 mM NaHCO3, 5.55 mM glucose, 0.31 mM sodium pyruvate, 1.49 mM calcium lactate, 0.075 mg/ml sodium penicillin G, 0.05 mg/ml streptomycin sulfate, and 3 mg/ml BSA) [25] 15–16 h after hCG injection. Sperm were collected from the cauda epididymis of the male mice. The oocytes were fertilized with capacitated sperm, which had been incubated for 2 h at 38°C in an atmosphere of 5% CO2 and 95% air. Two hours after insemination, the embryos were washed with glucose-free CZB medium (81.62 mM NaCl, 4.83 mM KCl, 1.18 mM KH2PO4, 1.18 mM MgSO4, 25.12 mM NaHCO3, 0.27 mM sodium pyruvate, 1.7 mM CaCl2, 31.30 mM sodium lactate, 6.9 mM taurine, 0.11 mM EDTA [disodium salt], 0.075 mg/ml sodium penicillin G, 0.05 mg/ml streptomycin sulfate, and 3 mg/ ml BSA) [26] and cultured at 38°C in an atmosphere of 5% CO2 and 95% air. Synchronized embryos were obtained by removing those embryos that had not yet formed a pronucleus 5.5 h after insemination.

Treatment with Inhibitors

The embryos were incubated in the presence of 10 µg/ml cycloheximide or 3 µg/ml aphidicolin from 3 h after insemination. Nocodazole was used at a final concentration of 0.5 µg/ml to arrest the cell cycle at M phase.

Semiquantitative RT-PCR

RT-PCR was used to quantify changes in the relative amounts of Jak2 mRNA [27, 28]. Total RNA was isolated from either 30 oocytes or 30 embryos by the acid guanidinium phenol-chloroform method, and reverse-transcribed using AMV reverse transcriptase (Invitrogen Corp., Carlsbad, CA) with the oligo(dT) 12-18 primer (Invitrogen), according to the manufacturer's instructions. PCR was performed on a volume of template cDNA that was equivalent to three oocytes or embryos. The following primers were used: Jak2 sense, 5'-CTCAGATATGCAAGGGCATG-3'; Jak2 antisense, 5'-ATAAATTCCACGGGTGGACT-3'; rabbit globin sense, 5'-GCAGCCACGGTGGCGAGTAT-3'; rabbit globin antisense 5'-GTGGGACAGGAGCTTGAAAT-3'.

The PCR reaction comprised 35 cycles and used the AmpliTaq Gold enzyme (Applied Biosystems, Foster City, CA). Each cycle consisted of 20 sec of denaturation at 94°C, 20 sec of annealing at 55°C, and 30 sec of extension at 72°C, followed by a final step of 4 min at 72°C. The PCR products were separated by electrophoresis in a 2% agarose gel and stained with ethidium bromide. For semiquantitative analysis, rabbit globin mRNA was added before the isolation of total RNA. This served as an external standard to evaluate the efficiency of RNA extraction and reverse transcription. The ratios of the density values for the PCR products of Jak2 relative to that of rabbit globin were calculated.

Immunostaining and Semiquantification of Fluorescence Intensity

Oocytes and embryos for immunocytochemistry were fixed in 3.7% paraformaldehyde in PBS for 1 h after removal of the zona pellucida with acid minimum essential medium compatible buffer [29]. After washing three times in PBS that contained 1 mg/ml of BSA (PBS-BSA), the fixed embryos were permeabilized for 10 min with PBS that contained 0.1% (v/v) Triton X-100, and then incubated for 60 min in RNase reaction buffer (40 mM Tris-Cl [pH 8.0], 10 mM NaCl, 6 mM MgCl2) that contained 0.5 mg/ml RNase. Following a 1-h incubation in the RNase stop solution (15 mM EDTA in PBS), the oocytes and embryos were washed in PBS-BSA and then incubated for 60 min in PBS-BSA that contained a 1:200 dilution of either of the primary antibodies against the peptide that contains the C-terminal or N-terminal sequence of Jak2 (C-terminal peptide; Santa Cruz Biotechnology, Inc., Santa Cruz, CA and N-terminal peptide; Upstate Biotechnology Inc., Charlottesville, VA). After washing three times in PBS-BSA, the oocytes and embryos were incubated for 60 min in the dark in PBS-BSA that contained a 1:50 dilution of the fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA). After washing three times in PBS-BSA, the oocytes and embryos were incubated for 10 min in PBS that contained 0.5 mg/ml propidium iodide [14] and then washed three times in PBS-BSA. The cells were mounted on a glass slide in VectaShield (Vector Laboratories, Burlingame, CA) and observed with a confocal laser-scanning microscope (LSM510; Carl Zeiss Inc., Oberkochen, Germany). To confirm specificity, the primary antibody was neutralized with 1 µM of antigen peptide (sc294-p; Santa Cruz Biotechnology) or with a control peptide that was unrelated to the antigen (12-148; Upstate Biotechnology) for 1 h at 4°C before use. As a negative control, unfertilized oocytes were incubated with normal rabbit IgG instead of the primary antibody (Jackson ImmunoResearch). The intensity of fluorescence was quantified as previously described [30]. In brief, the pixel value/unit area was measured for the nucleus, and the average value for two different regions of the cytoplasm was subtracted as background. This value was multiplied by the pronuclear volume to yield the total amount of fluorescence. In the experiment, the average value calculated for the female pronucleus was set at 100% and the values obtained for the male pronucleus were expressed relative to this value.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression Levels of Jak2 mRNA in Preimplantation Embryos

The temporal expression pattern of Jak2 during preimplantation development was examined by RT-PCR using Jak2-specific primers (Fig. 1, A and B). The results show that the relative amount of Jak2 mRNA was highest in unfertilized oocytes, decreased by the two-cell stage, and remained at low levels until the blastocyst stage. This is typical of the expression patterns observed for many maternal mRNAs that are degraded following the onset of meiotic maturation [11].



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  		FIG. 1. Expression pattern of Jak2 transcripts during preimplantation development. The relative amounts of Jak2 and {alpha}-globin transcripts were determined by RT-PCR in mature metaphase II oocytes (MII), one-cell embryos (1-c), two-cell embryos (2-c), four-cell embryos (4-c), morulae (mor), and blastocysts (blast). Similar results were obtained in three independent experiments; a representative result is shown in (A). The integrated optical densities of the bands are shown in (B). The value obtained in the oocyte, which had the highest relative amount of transcript, is set at 100%, and the values obtained for the other developmental stages are expressed relative to this value. The experiment was performed three times and the results are presented as the mean ± SEM

Localization of Jak2 Protein in Oocytes and Preimplantation Embryos

The expression of Jak2 protein was examined by immunocytochemistry, using an antibody against a peptide with the C-terminal amino-acid sequence of Jak2. As shown in Figure 2, Jak2 was localized to the chromosomes of the unfertilized oocytes. This staining was specific because preincubation of the antibody with the immunizing peptide but not a peptide with an unrelated sequence completely abolished the staining. In addition, no chromosome-localized staining was observed when normal rabbit IgG was used. These results were confirmed by immunocytochemistry with another anti-Jak2 antibody, which was raised against a peptide that comprised part of the N-terminal sequence of the Jak2 protein (data not shown). The antigen N-terminal peptide also abolished the staining. Previous studies have shown that these two antibodies specifically recognize the Jak2 protein in immunoblotting experiments [31–34].



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  		FIG. 2. Confocal immunofluorescence microscopy images of metaphase II oocytes stained with the anti-Jak2 antibody (A1/B1), anti-Jak2 antibody that was preincubated with the antigen peptide (A2/B2), anti-Jak2 antibody that was preincubated with the control peptide of a sequence unrelated to the antigen (A3/B3), and normal rabbit IgG (A4/B4). A1–A4) Immunofluorescence using the FITC-conjugated anti-rabbit IgG antibody; (B1–B4) DNA stained with propidium iodide. The experiment was performed three times with similar results; representative examples are shown. Original magnification x280

Temporal changes in the localization of the Jak2 protein were examined during preimplantation development (Fig. 3). After fertilization, Jak2 immunofluorescence was detected in the pronucleus and a polar body 6 h after insemination (Fig. 3, A2). Interestingly, the intensity of fluorescence was always much higher in the female pronucleus, i.e., the smaller pronucleus, which was located closer to the second polar body. In the male pronucleus, fluorescence was very weak or below the level of detection. Semiquantification of fluorescence showed that the intensity of fluorescence was much higher in the female pronucleus than in the male pronucleus. Even after correcting for the pronuclear volume, a 10-fold difference was detected (Fig. 4). Jak2 appeared to be localized on the chromatin, but not in the nucleoplasm, except possibly at a very low level because chromatin is not evenly distributed in the pronucleus. Confocal images of Jak2 immunofluorescence could be superimposed on those of DNA staining in embryos that were double-stained with the anti-Jak2 antibody and propidium iodide. Supporting this idea, Jak2 was localized on the chromosomes at the first M phase (Fig. 3, A3). Note that the fluorescence was not distributed evenly on the chromosomes: one half was stained intensely and the other half was stained weakly. The half with intense fluorescence probably originated from the female pronucleus. After cleavage to the two-cell stage, Jak2 was localized evenly throughout the nucleus (Fig. 3, A4). Because chromosomes of different parental origin remain topologically separated in the nucleus during the two-cell stage [35], these results suggest that Jak2 accumulates on the chromosomes of paternal origin during the M phase. To confirm this, changes in Jak2 localization were examined during the M phase of one-cell embryos. As shown in Figure 5A, the detected immunofluorescent signal for Jak2 was highly asymmetric, i.e., differentially localized in two chromosome masses at prophase, and this asymmetry was maintained until anaphase. During the telophase of mitosis, Jak2 was distributed uniformly on the chromosomes, which indicates that Jak2 accumulates during this period. To confirm the accumulation of Jak2 during M phase, the embryos were arrested at M phase by treatment with nocodazole. In the arrested embryos 32 h after insemination, Jak2 immunofluorescence was detected on both chromosome masses (Fig. 5B). Therefore, paternal chromosomes apparently acquired as much immunofluorescence as that of the maternal chromosomes during M phase.



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  		FIG. 3. Immunolocalization of Jak2 in mouse oocytes and embryos. Metaphase II oocytes (A1/B1) and embryos that were collected 12 h (A2/B2), 15 h (A3/B3), 21 h (A4/B4), 32 h (A5/B5), 48 h (A6/B6), 72 h (A7/B7), and 96 h (A8/B8) after insemination were immunostained with the antibody against the Jak2 C-terminal peptide (A1–A8) and stained for DNA with propidium iodide (B1–B8). The arrows indicate male (m) and female (f) pronuclei. The experiment was performed three times with similar results; representative examples are shown. Note that only the upper half of the chromosomes of the one-cell embryos (A3) were stained with the anti-Jak2 antibody compared with the DNA staining (B3). Original magnification x360



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  		FIG. 4. Semiquantification of Jak2 protein in the pronuclei of one-cell-stage mouse embryos. Embryos that were collected 12 h after insemination were immunostained with the antibody against the Jak2 C-terminal peptide. The intensity of fluorescence was quantified as described in the Materials and Methods section. Male (M PN) and female pronuclei (F PN) in 20 fertilized eggs were analyzed. The amount of fluorescence in the female pronucleus was set as 100%, and the values for the male pronucleus were expressed relative to this value. The experiments were performed four times and the data were combined



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  		FIG. 5. Accumulation of Jak2 on male-derived chromosomes. A) Immunolocalization of Jak2 during the M phase of the one-cell stage. Embryos were collected 15 h after insemination. The images are arranged in the order of progression of mitosis: A1–3, prophase; B1–3, metaphase; C1–3, anaphase; D1–3, telophase. B) Immunolocalization of Jak2 in embryos arrested at the M phase of the one-cell stage. The embryos were transferred to medium that contained nocodazole 12 h after insemination and were fixed in paraformaldehyde 32 h after insemination. Confocal microscopy images of the embryos immunostained with the antibody against the Jak2 C-terminal peptide (Jak2) and stained with propidium iodide (DNA), and merged images of these two images (Merged) are shown. The experiments were performed three times with similar results; representative examples are shown. A) original magnification x600; (B) original magnification x430

During the two-cell stage, the intensity of immunofluorescence decreased and was weak in the late G2 phase (Fig. 3, A5). Thereafter, it was not detected in the four-cell embryos, morulae, or blastocysts (Fig. 3, A6–8). To investigate the mechanism that regulates the disappearance of Jak2, the embryos were treated with aphidicolin (Fig. 6). When DNA synthesis was inhibited with aphidicolin in one-cell embryos, the embryos arrested at the one-cell stage 32 h after insemination, at which time untreated embryos had reached the G2 phase of the two-cell stage. In the arrested one-cell embryos, Jak2 had disappeared from the nucleus. Thus, the disappearance of Jak2 was independent of DNA synthesis. However, Jak2 did not disappear in embryos that were treated with cycloheximide. When protein synthesis was inhibited with cycloheximide, an equivalent intensity of immunofluorescence was detected in the female pronucleus 32 h after insemination as in embryos 6 h after insemination. This result suggests that newly synthesized protein is involved in the disappearance of Jak2 from the nucleus. Thus, the disappearance of Jak2 is dependent on de novo protein synthesis, but not DNA synthesis, during the first cell cycle.



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  		FIG. 6. Disappearance of Jak2 localization in the late G2 phase of two-cell embryos. Control embryos were fixed 6 h (A), 20 h (B), and 32 h (C) after insemination. The embryos were cultured in media that contained aphidicolin (D) or cycloheximide (E) and fixed in paraformaldehyde 32 h after insemination. The collected embryos were immunostained with the antibody against the Jak2 C-terminal peptide. The experiments were performed three times with similar results; representative examples are shown. Original magnification x460


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown that Jak2 is expressed in early preimplantation embryos. Jak2 is a member of the Janus family of nonreceptor tyrosine kinases, such as Jak1, Jak3, and Tyk2. These proteins are approximately 130 kDa in mass, and they contain seven conserved Jak homology domains (JH1–JH7) [36, 37]. In general, these kinases are associated with the cytoplasmic domain of receptor chains and are catalytically inactive in resting cells [23, 38]. They are rapidly activated by ligand-stimulated phosphorylation of tyrosine residues within the kinase domain [8, 39], which results in the phosphorylation, dimerization, and nuclear translocation of latent cytoplasmic Stat transcription factors [40, 41]. Therefore, in this pathway, Jak2 would be expected to localize to the cellular membrane, where it functions as a signal transducer.

The results of this study, however, show that Jak2 is localized to the nucleus at the one- and two-cell stages (Fig. 3), although it disappears from the nucleus during the two-cell stage via a mechanism that is dependent on protein synthesis (Figs. 3 and 6). The nuclear localization of Jak2 suggests that Jak2 may have a special function during these developmental stages because Jak2 cannot bind to the membrane receptors as long as it is restricted to the nucleus. A few reports have demonstrated the nuclear localization of Jak2. Nucleoplasmic distribution of Jak2 has been shown by immunogold electron microscopy of rat liver hepatocytes and Chinese hamster ovary cells [42]. In islet of Langerhans and Nb2 cells, Jak2 was also present in the nucleus [43]. However, these studies did not reveal the function(s) of Jak2 when localized to the nucleus.

Although the function of Jak2 in the nucleus is currently unclear, Jak2 may be activated and transduce signals in the nucleus by a mechanism that is similar to the one that operates on the plasma membrane, because the pertinent receptors and polypeptide ligands that are upstream of the Jak2-Stat pathway are present in the nucleus. Growth hormone, prolactin, insulin, and interferon gamma and their corresponding receptors have been reported to undergo internalization or endocytosis followed by translocation into the nucleus [44–51]. However, the downstream substrates of Jak2 are unknown. It seems unlikely that Stats represent the substrate in the nucleus because, in the known mechanism, they are phosphorylated by Jak2 on the plasma membrane, which enables them to translocate into the nucleus. Thus, the Stats that are translocated into the nucleus are already phosphorylated. Therefore, Jak2 may transduce the signal to unknown substrates. Several proteins may exist in the nucleus as downstream targets. Further studies are required to examine these candidates. On the contrary, it is possible that Jak2 has dual functions depending on its localization, i.e., it functions as an authentic protein tyrosine kinase in the plasma membrane and as an inactive structural protein in the nucleus. For instance, PHGPx exists as an active peroxidase in spermatids but persists in the mature spermatozoa as an enzymatically inactive structural protein [52]. The nuclear localization of Jak2 suggests a possible function for this protein during early preimplantation development.

Another striking result in this study is that Jak2 is localized on the chromosomes during meiosis and mitosis. There is no other report in the literature that shows Jak2 localization to the chromosomes at M phase. In general, transcription is silenced abruptly when eukaryotic cells enter mitosis. Earlier studies have indicated that most transcription factors and RNA polymerase II are displaced when chromatin is condensed into mitotic chromosomes [53]. Therefore, Jak2 does not seem to function as a transcription factor bound to chromatin. Further investigation is required to uncover its function during M phase.

It is also interesting to note that the intensity of Jak2 staining was much higher in the female pronuclei than in the male pronuclei of one-cell embryos. Although these pronuclei reside in the same cytoplasm, they are heterogeneous in many aspects. The temporal and spatial distributions of the sites of DNA replication are asynchronous between the two pronuclei in that the female pronucleus requires a longer time to complete replication in the intranuclear region than in the peripheral regions [54]. Transcriptional regulation also differs; the male pronucleus supports a higher level of transcription than the female pronucleus [55–57]. This discrepancy has been attributed to differences in the chromatin structures of the two pronuclei and the fact that the chromatin of the female pronucleus (but not that of the male pronucleus) is in a transcriptionally repressed state [30, 57, 58]. The two pronuclei also show asymmetric DNA methylation, which may be responsible for the functional differences between the parental genomes during development [35, 59]. Because the male and female pronuclei show different features in the same cytoplasmic environment, it appears that some component in the nucleus differs between male and female nuclei. Jak2 is a candidate for such a component.

The observations made in this study present challenges for understanding the known function of Jak2. What is the role of Jak2 in the nucleus? What regulates its nuclear localization? What controls the different localization patterns in male and female pronuclei? Jak2 is composed of several domains, the functions of which are not yet fully understood. A recent study showed that the pseudokinase domain of Jak2 negatively regulates the activity of Jak2, probably through an interaction with the kinase domain, and that this regulation is required to keep Jak2 inactive in the absence of ligand stimulation [60]. Because the mechanism for regulating the activation of Jak2 is complicated, analyses of the various Jak2 domains may elucidate novel functions. Further investigation of Jak2 may allow us to define a specific mechanism by which Jak2 functions in development.


   FOOTNOTES
 
1 Correspondence: Fugaku Aoki, Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba 277-8562, Japan. FAX: 81 4 7136 3698; aokif{at}k.u-tokyo.ac.jp Back

Received: 12 September 2003.

First decision: 9 October 2003.

Accepted: 9 February 2004.


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TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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M. Nakasato, Y. Shirakura, M. Ooga, M. Iwatsuki, M. Ito, S.-i. Kageyama, S. Sakai, M. Nagata, and F. Aoki
Involvement of the STAT5 Signaling Pathway in the Regulation of Mouse Preimplantation Development
Biol Reprod, October 1, 2006; 75(4): 508 - 517.
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