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BOR - Papers in Press, published online ahead of print December 26, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.023283
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BIOLOGY OF REPRODUCTION 70, 1270–1276 (2004)
DOI: 10.1095/biolreprod.103.023283
© 2004 by the Society for the Study of Reproduction, Inc.

Embryo

Leukemia Inhibitory Factor Antisense Oligonucleotide Inhibits the Development of Murine Embryos at Preimplantation Stages1

Tzu-Chun Cheng4,5,6, Chun-Chia Huang4,6, Chung-I Chen6, Chung-Hsien Liu7, Yih-Shou Hsieh4, Chih-Yang Huang4, Maw-Sheng Lee3,5,6,7, and Jer-Yuh Liu2,4

Institute of Biochemistry,4 College of Medicine, Chung-Shan Medical University, Taichung 402, Taiwan, Republic of China Department of Medicine,5 China Medical University, Taichung 402, Taiwan, Republic of China Division of Infertility Clinic,6 Lee Women's Hospital, Taichung 402, Taiwan, Republic of China Department of Obstetrics and Gynecology,7 Chung-Shan Medical University Hospital, Taichung 402, Taiwan, Republic of China


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leukemia inhibitory factor (LIF) is an essential factor for implantation and establishment of pregnancy. However, its role in the development of preimplantation embryos remains controversial. In this study, changes in preimplantation embryos were determined after microinjection of LIF antisense oligonucleotide at the two-pronucleus stage. Although no significant differences were found in the percentages between the untreated group and the 0.25-fmol-treated group, the 0.5- or 1.0-fmol-treated groups had significantly lower percentages of embryos developed to the morula or blastocyst stage and the 2.0-fmol-treated group had significantly lower percentages of embryos developed to the four-cell, morula, or blastocyst stage. No embryos developed to the four-cell stage in the 4.0-fmol-treated group. Moreover, there was a decreasing trend in the levels of LIF immunoactivity with the increasing amount of LIF antisense oligonucleotide injected. The diameter of blastocysts in the 2.0-fmol-treated group was significantly smaller than that in the untreated group. The blastocysts in this group had significantly lower numbers of blastomeres and cells in the inner cell mass (ICM) or trophectoderm (TE) and ICM:TE ratio. The 1.0- or 2.0-fmol-treated groups had significantly lower implantation rates than their corresponding control groups. In the 2.0-fmol groups with supplementing exogenous LIF, significantly lower percentages were also observed in the four-cell, morula, and blastocyst stages. However, blastocysts treated with 50 ng/ml LIF had a significantly higher percentage than those in the LIF gene-impaired group without LIF supplement. These results indicate that LIF is a critical factor for the normal development of embryos at the preimplantation stages.

cytokines, developmental biology, early development, embryo, growth factors


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leukemia inhibitory factor (LIF) is a multifunctional cytokine. It has been considered to be an essential factor for implantation and establishment of pregnancy [1, 2]. Although LIF is mainly provided by maternal uterus for preimplantation embryos [3], the expression of this cytokine is only dependent on the embryo in in vitro fertilization. Because blastocyst implantation depends on maternal expression of LIF [4], LIF gene mutation may give rise to decreased availability or biological activity of LIF in the uterus and cause implantation failure [5]. Moreover, recombinant LIF in standard medium may not enhance in vitro human blastocyst formation but may play a role at later stages of human embryogenesis and during implantation [6]. LIF added to embryo culture medium has no major beneficial effect on the proportion of bovine embryos reaching the blastocyst stage [7]. However, LIF not only augments blastocyst formation and hatching in the embryos of mice [8, 9] and cattle [10] but also enhances the blastocyst formation rates of human embryos in a serum-free medium [11] or in human tubal fluid [12]. These findings lead to the controversy regarding the necessity of supplementing LIF in the culture medium to emulate the conditions of maternal uterus for in vitro fertilization. To distinguish the precise role of LIF at the preimplantation stages of embryogenesis in vitro, we employed antisense oligonucleotides [13] to attenuate the function of the LIF gene in the in vitro embryos and determined the effects of this treatment on preimplantation development and implantation. Moreover, effects of supplementing exogenous LIF to the LIF gene-impaired embryos were also investigated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Oligonucleotides

Morpholino oligonucleotides were provided by Gene Tools, LLC (Philomath, OR). The LIF antisense oligonucleotide and non-sense oligonucleotide were 5'-GACCTTCATTATGGGCTGGACTCTA-3' and 5'- CCTCTTACCTCAGTTACAATTTATA-3', respectively. There were no sequences with significant similarity to the non-sense control. The LIF antisense oligo sequence was determined to be in the region (156–180) of murine LIF mRNA (GenBank Accession Number: NM_008501). It was formed within the translational starting target. The sequence had no more than four contiguous intrastrand base pairs or four contiguous G:C pairs. Moreover, it did not contain over 36% guanines or more than three contiguous guanines for increasing water solubility. The antisense oligo was confirmed by the NCBI FASTA BLAST databases that the sequence does not correspond to any other transcripts. The stability of these oligos was determined by injecting fluorescein isothiocyanate-labeled preparations into mouse embryo and observing the results under a fluorescence microscope.

Animals

All mice were obtained from the National Laboratory Animal Center (Taipei, Taiwan) and were housed in humidity- (40–60%)- and temperature- (22 ± 2°C) controlled rooms and were maintained on a 12L:12D photoperiod. Mice were given food and water ad libitum. All procedures were approved by the Chung-Shan Medical University Institutional Animal Care and Use Committee and were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.

Preparation of Embryos

Superovulation was induced in virgin mice (6–8 wk old) of the B6CBF1 strain (C57BL/6 x CBA) by intraperitoneal injection of eCG (5 IU) (Sigma, St. Louis, MO) and hCG (5 IU) (Serono, Rome, Italy) 48 h later. Each superovulated mouse was then placed overnight in a cage with a sexually mature male of the same strain. Successful mating was determined by the presence of a copulation plug in the vagina. The mated female mice were killed 20 h after hCG injection and zygotes were collected from the oviducts. The cumulus cells of the zygotes were removed by exposure to hyaluronidase (80 IU/ml) (Sigma). The zygotes were then placed into wells with fresh human tubal fluid medium [14]. Embryos in the two-pronucleus (2PN) stage were obtained by incubating the zygotes in an atmosphere with 5% CO2 at 37°C for 4 h.

Microinjection of Oligonucleotides

In the experimental groups, embryos at the 2PN stage were injected with 1 pl of 0.25, 0.5, 1.0, 2.0, or 4.0 fmol LIF antisense oligonucleotide. In the positive control groups, the embryos were injected with the non- sense oligonucleotide (2 or 4 fmol) or normal saline. Embryos in the negative control group remained untreated.

Injection pipettes (inner diameter 2 µm, outer diameter 5 µm) and holding pipettes (inner diameter 15 µm, outer diameter 80 µm) were produced using a micropipette puller (Sutter Instrument Co., Novato, CA) and a microforge (Narishige Co., Ltd., Tokyo, Japan). Microinjections were performed under a phase-contrast microscope (Nikon, Ltd., Tokyo, Japan) with micromanipulators (Narishige). The oligonucleotide or normal saline was injected into the male pronucleus. The embryo was than incubated in an atmosphere of 5% CO2 at 37°C and monitored daily using an optical microscope.

Immunocytochemistry

Embryos were recovered from the culture medium and freed of zona pellucida by brief exposure to acidic Tyrode solution [15]. After washing three times with phosphate-buffered saline (PBS), the embryos were placed three times onto microscope slides and fixed in 2% formalin for 15 min. The embryos were washed in PBS and incubated in a blocking solution (10% fetal calf serum, 0.5% Tween 20, 0.02% sodium azide in PBS) for 1 h. After incubating with an affinity-purified rabbit antipeptide antibody preparation specific to LIF (1 µg/ml) (Chemicon, VIC, Australia) at 4°C overnight, the embryos were washed with the blocking solution for 10 min.

Immunostaining was performed using the VECTASTAIN ABC kit (Vector Laboratories, Burlingame, CA). The embryos were incubated with biotinylated anti-rabbit IgG (1 µg/ml) for 1 h. The embryos were then incubated with avidin-biotinylated horseradish peroxidase for 1 h. After washing with TBST buffer (50 mM Tris-HCl, 0.025% Tween 20, pH 7.8) five times, the embryos were treated with 3,3'-diaminobenzidine substrate (Sigma) for 20 min. The embryos were dehydrated through graded alcohol and mounted with glycerol. Results of immunostaining were observed using phase-contrast microscopy. The visible staining indicated the immunoreactive LIF protein sites. Corresponding nonspecific binding of embryos was shown by parallel incubation with the antibody preneutralized with excess antigenic peptide.

Differential Staining of Trophectoderm and Inner Cell Mass

Cells in the trophectoderm (TE) and inner cell mass (ICM) of the blastocysts were counted after differential staining of the nuclei using a modified method of Piekos et al. [16]. The zona-free blastocysts were incubated for 10 min at 5°C in M16 medium (Sigma) containing 10 mM trinitrobenzenesulphonic acid, 4.0 mg/ml polyvinylpyrrolidone, and 0.015% Triton X-100. After washing in M2 medium (Sigma), the blastocysts were incubated in 0.1 mg/ml anti-dinitrophenol-BSA at 37°C for 15 min and washed again (three times) with the M2 medium. The blastocysts were then incubated in M2 medium containing a 1:10 dilution of guinea pig complement serum (Sigma) and 10.0 µg/ml propidium iodide (Sigma) at 37°C for 15 min and washed three times in Dulbecco PBS (Gibco). After fixing in absolute ethanol containing 22.0 µg/ml bisbenzimide (Sigma) at 5°C overnight, individual blastocysts were mounted in glycerol on microscope slides and compressed manually before visualization by epi- fluorescence using the Nikon filter blocks UV-2A and G-2A.

Embryo Transfer

Recipient female mice (8–12 wk old, ICR strain) were prepared by mating with vasectomized males of the same strain 4 days before embryo transfer. The procedures of embryo transfer were performed according to Nagy et al. [17]. In the same recipient, 5–7 blastocysts injected with LIF antisense oligonucleotide (0.5, 1.0, or 2.0 fmol) were transferred to the right uterine horn and the same number of blastocysts treated with the non-sense oligonucleotide to the left uterine horn. The mice were killed 2 days after embryo transfer. Successful implantation was verified and determined by injection of Chicago Sky Blue 6B (Sigma).

Supplement of LIF to LIF Antisense Oligonucleotide-Treated Embryos

After microinjection with 2.0 fmol LIF antisense oligonucleotide, 5, 10, or 50 ng/ml LIF (Sigma) was added to the culture medium of the treated embryos. These embryos were incubated in an atmosphere of 5% CO2 at 37°C and monitored daily using an optical microscope.

Statistical Analysis

Rates were expressed as percentages. Differences between blastocyst formation rates were determined using the chi-square test. The numbers and dimensions of cells were expressed as mean ± standard deviations and were compared using Student t-tests. Changes in the implantation rates were determined using the Kruska-Wallis test followed by a Mann-Whitney U-test. P < 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of LIF Antisense Oligonucleotide on Preimplantation Development

Table 1 shows the percentages of murine embryos developing into different preimplantation stages after various treatments. There were no significant differences in the percentages of embryos developing to the two-cell, four-cell, morula, and blastocyst stages among the four control groups (P > 0.05). No significant differences were found in the percentages between the untreated group and the group treated with 0.25 fmol of LIF antisense oligonucleotide (P > 0.05). In the groups treated with 0.5 or 1.0 fmol of LIF antisense oligonucleotide, significantly lower percentages of embryos were found to develop to the morula or blastocyst stages (P < 0.05). Significantly lower percentages of embryos treated with 2.0 fmol of LIF antisense oligonucleotide developed to the four-cell, morula, or blastocyst stage. No embryos developed to the four-cell stage in the group treated with 4.0 fmol of LIF antisense oligonucleotide.


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  		TABLE 1. Percentage of murine embryos developing into different preimplantation stages after microinjection of LIF antisense oligonucleotide at the two-pronucleus stage

The untreated embryos reached the four- to eight-cell stage on Day 3, morula stage on Day 4, and blastocyst stage on Day 5. The groups treated with non-sense oligonucleotide had similar developmental stages as the untreated group. However, in the embryos treated with 1.0 or 2 fmol of LIF antisense oligonucleotide, the number developing into the morula or blastocyst stage was greatly reduced on Days 4–5. Among those treated with 4.0 fmol of LIF antisense oligonucleotide, only two-cell-stage embryos were found on Days 2–5 (Fig. 1).



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  		FIG. 1. Morphology of murine embryos on Days 2–5 after microinjection of 1.0, 2.0, or 4 fmol LIF antisense oligonucleotide. The untreated group and the 2-fmol and 4-fmol non-sense-treated groups were the controls. Bar = 50 µm

Effects of LIF Antisense Oligonucleotide on the Expression of LIF Protein at Different Preimplantation Stages

Except the blank control, the densities of immunoreactive LIF protein sites were similar in all groups on Day 1. Similar densities were also observed among the untreated embryos and those injected with 2 or 4 fmol non-sense oligonucleotide from Day 1 to Day 5. The groups treated with 1, 2, or 4 fmol of LIF antisense oligonucleotide had apparently lower densities than the control groups from Day 2 to Day 5. Moreover, there was a decreasing trend in the densities with increasing amount of LIF antisense oligonucleotide injected (Fig. 2).



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  		FIG. 2. Immunocytochemical analysis of LIF protein expression on Days 1–5 in murine embryos. The three control groups consist of untreated, non-sense (2 fmol), and non-sense (4 fmol). The three LIF antisense oligonucleotide-treated groups consist of LIF antisense (1 fmol), LIF antisense (2 fmol), and LIF antisense (4 fmol). The blank group consists of embryos incubated with normal rabbit serum, and the neutralization group consists of embryos incubated with the antibody preneutralized with excess antigenic peptide. Bar = 50 µm

Effects of LIF Antisense Oligonucleotide on Morphology of Blastocysts

The diameter of blastocysts derived from embryos treated with 2.0 fmol of LIF antisense oligonucleotide was significantly smaller than that in the untreated group (P < 0.01). Moreover, these blastocysts also had significantly lower numbers of blastomeres and cells in ICM or TE. A significantly lower ICM:TE ratio was also found in these embryos (P < 0.01). Although embryos treated with 1.0 fmol of LIF antisense oligonucleotide had significantly lower numbers of blastomeres and cells in ICM or TE, no significant difference was found in the ICM:TE ratio between this group and the untreated embryos (Table 2).


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  		TABLE 2. Changes in the number of cells in the ICM and TE of the blastocysts derived from murine embryos after microinjection of LIF antisense oligonucleotide at the two-pronucleus stage

Effects of LIF Antisense Oligonucleotide on Implantation Rate

There was no significant difference between the implantation rate of the blastocysts derived from the untreated and the non-sense oligonucleotide-treated embryos and between the embryos treated with 0.5 fmol of LIF antisense oligonucleotide at the two-pronucleus stage and those treated with the non-sense oligonucleotide (P > 0.05). However, the embryos treated with 1.0 and 2.0 fmol of LIF antisense oligonucleotide had significantly lower implantation rates than their corresponding control embryos treated with the non-sense oligonucleotide (P > 0.01) (Table 3).


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  		TABLE 3. Changes in the implantation rate of murine blastocysts derived from murine embryos after microinjection of LIF antisense oligonucleotide at the two-pronucleus stage

Effects of Supplementing Exogenous LIF to LIF Gene-Impaired Embryos

Although there was no significant difference between the percentages of untreated 2PN embryos and those treated with 2.0 fmol of LIF antisense oligonucleotide developing into the two-cell stage, significantly lower percentages were found in the treated group for the development into the four-cell, morula, and blastocyst stages (P < 0.01). In the groups with supplementing exogenous LIF, significantly lower percentages were also observed in these preimplantation stages (P < 0.01). However, blastocysts treated with 50 ng/ml LIF had a significantly higher percentage than those in the LIF gene-impaired group without LIF supplement (P < 0.05) (Table 4).


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  		TABLE 4. Percentages (%) of murine embryos developing into different preimplantation stages after microinjection of 2.0 fmol LIF antisense oli gonucleotide at the two-pronucleus stage and supplemented with LIF


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The coculture technique has been used to determine the effects of LIF on implantation [18, 19] or preimplantation development of murine embryos in vitro [8, 9]. Although the enhancing effects of LIF may be observed, it is difficult to reveal the changes in embryos with LIF-gene impairment in vitro using this technique. Knockout experiments have demonstrated that endometrial LIF is essential for in vivo murine implantation [4]. However, preparation of LIF knockout mice is a laborious task. Antisense RNA/DNA inhibition of gene expression has been documented as a feasible approach to the elucidation of mechanisms regulating the development of preimplantation mammalian embryos [20]. Murine embryos at the pronuclear stage treated with c-myc antisense oligo may inhibit development to the blastocyst stage, especially at the first cleavage of zygotes to the two-cell stage [13]. Treatment at the two-cell stage may lead to developmental arrest at the eight-cell or morula stage [21]. Disruption of Na/K-ATPase gene expression by antisense oligodeoxynucleotide may abolish blastocyst formation [22]. Morpholino antisense oligonucleotides have been demonstrated to be effective tools for down-regulating gene expression during mammalian preimplantation development [23]. Moreover, the 2'-methoxyethoxy-modified antisense oligonucleotides are candidates for effective examination of roles of large numbers of genes during early embryonic development [24]. In this study, we applied morpholino antisense oligonucleotide to inhibit the expression of LIF. Although LIF protein was detectable in the untreated embryo from the 2PN to the blastocyst stage by immunocytochemistry, signals of the protein expression were reduced in those treated with 1.0, 2.0, or 4.0 fmol of LIF antisense oligonucleotide from the two-cell stage to blastocyst stage. These findings indicate that our design is useful in investigating the effects of LIF on the preimplantation development of murine embryos in vitro.

In the four experimental groups, 2PN embryos treated with various dosages of LIF antisense oligo were able to develop to the two-cell stage. Although the inhibitory effects of the antisense oligo may commence immediately after microinjection, the embryo may have sufficient amounts of LIF for developing into the next stage. Because embryonic genome activation in mice occurs at the two- cell stage [25], it is possible for the treated 2PN embryos to develop into the next stage. Moreover, in embryos treated with 4.0 fmol LIF antisense oligonucleotide, development was arrested at the two-cell stage. It has been reported that antisense oligonucleotides may lead to unpredictable activities within cells and morpholino modification of the oligos may avoid these adverse effects [26]. Because we employed morpholino oligos in this study, the arrest of the developing embryos at the two-cell stage may be mainly due to the effects of LIF antisense oligo at the high dose.

In this study, we observed the inhibitory effects of LIF antisense oligonucleotide on the treated embryos, and microinjection of 1.0 or 2.0 fmol led to a significant reduction in the percentage of embryos that developed from the morula into blastocysts. It has been reported that PN-stage morphology is related to blastocyst development [27]. There are a number of stage-specific genes expressed at the different stages of the preimplantation embryos. Lex (Galbeta1-4(Fucalpha1-3)GlcNAc) and Ley (Fucalpha1-2Galbeta1-4(Fucalpha1-3)GlcNAc) are stage-specific embryonic antigens. The former is first detected on the blastomeres of the eight-cell-stage embryo and correlates with the onset of blastomere compaction. The latter is highly expressed on the surface of the blastocyst and has been shown to be involved in blastocyst attachment in the mouse. By in situ hybridization, mRNAs of these two enzymes were detected only in the morula and blastocyst embryos [28]. Other genes, such as the glucose transporter GLUT3, growth factor, EGF (epidermal growth factor), and EGF receptor, are also detected during and after the morula stage [29, 30]. It is possible that LIF may collaborate with these genes to regulate the blastocyst formation. However, LIF has been found to prevent leptin-induced apoptosis in embryo development [31]. Therefore, LIF may also associate with some other factors to modulate the embryo growth.

Numbers of cells in the TE and ICM and the ICM:TE ratio in Day 5 blastocysts are important predictive variables of in vitro fertilization and preimplantation embryonic development in the mouse [32]. These variables may change under different culturing conditions. The ICM at the blastocyst stage has been demonstrated to be more sensitive to high temperature than the TE [33]. The numbers of TE and total nuclei are higher in embryos cultured in an atmosphere with 5% CO2 in air than in those developed under 5% CO2:5% O2:90% N2 [34]. Changes in the concentrations of insulin or glucose in the culture medium have been reported to affect the numbers of cells in the ICM and TE [35]. The number of cells in the ICM may be altered by addition of cytokines in the culture medium. The incidence of blastulation in human embryos may increase by 2-fold in the presence of granulocyte-macrophage colony-stimulating factor [36]. The specific impact of tumor necrosis factor alpha on the ICM of blastocysts has also been reported [37]. In this study, we found that the numbers of cells in the ICM and TE were significantly decreased in the groups treated with 1.0 or 2.0 fmol of LIF antisense oligonucleotide. Moreover, in the group treated with 2.0 fmol of the oligo, the blastomeres had a significantly smaller size and a significantly lower ICM:TE ratio. These findings indicate that microinjection of the oligo to the embryos at the 2PN stage may have significant influence on the morphological characteristics of the blastocysts, which in turn decreases the rate of implantation [38]. Because changes in the number of cells in the ICM or TE as well as the ICM: TE ratio have been observed in the blastocysts with impairments at the gene level [39, 40], the LIF antisense oligonucleotide may block the translation of the selected mRNAs (the sense strand) and lead to the morphological changes in the blastocysts.

The results obtained in this in vitro study were consistent with those studies on in vitro models that indicate that this protein is a critical factor for embryos at various preimplantation stages [8–12]. In contrast with our findings, embryos of knockout mouse have been shown to develop to the blastocyst stage in the absence of LIF in vivo [4]. The success in this preimplementation development may be due to the effects of the other growth factors produced by the cells of the reproductive tract. A number of growth factors and cytokines from the reproductive tract have been shown to promote blastocyst formation [41]. The insulin-like growth factor-I (IGF-I) produced by the fallopian tube is present in the oviduct and uterine fluid but is not expressed in the embryo [42]. Addition of IGF-I to the culture medium may increase the percentage of embryos developing into the blastocyst stage [43]. In addition, heparin-binding epidermal growth factor and granulocyte-macrophage colony-stimulating factor produced in the reproductive tract [44, 45] may also improve the preimplantation development [36, 46].

The effects of microinjection of LIF antisense oligonucleotide to embryos at the 2PN stage were mainly observed in the development of morula to blastocysts. Although there were no significant differences in the formation of morula among the groups treated with 0.5–2.0 fmol of the oligo, formation of blastocysts was observed to be significantly affected in the 1.0- and 2.0-fmol groups. Moreover, these effects were dose-dependent in this range. Coculture of the treated group (2.0 fmol) with 50 ng/ml of LIF was able to recover the formation of blastocysts to a significantly higher level than the group without supplement. These findings are consistent with those reported previously that LIF has beneficial effects on preimplantation embryos, especially from morula to blastocyst stage [9, 12]. However, this level remained significantly lower than that of the untreated control. The effective concentrations of LIF supplement by coculture may not be sufficient for the normal development of these embryos. It is also possible that microinjection of LIF antisense oligonucleotide to embryos at the 2PN stage may cause irreversible changes in the embryos from the two-cell to morula stage. It has been reported that microinjection of DNA fragments, including the yeast centromeric element sequence, into one-cell murine embryos results in an early arrest of development with abnormal nuclei containing variable amounts of DNA [47]. The sequence of LIF antisense oligonucleotide used in this study includes a region similar to the DNA binding site CATT(A/T) of the transcription factor YY1. This binding site has been located in the sequence of the promotor of a number of cytokines [48, 49]. Although this region may affect development of the preimplantation embryo through its unspecified reactions, the expression of LIF in two-cell treated embryos has been altered. We have also found that supplementing LIF to the treated embryos at the two-cell stage did not improve the percentage of embryos developing into the blastocyst stage (unpublished data). These findings confirmed that LIF is a critical factor for normal development of embryos at the preimplantation stages.


   ACKNOWLEDGMENTS
 
We thank Dr. Chuan-Mu Chen, Institute of Biochemistry, National Chung Hsing University, for his valuable technical help in this study.


   FOOTNOTES
 
1 This study was supported in part by grants from the National Science Council, Republic of China (NSC 91-2745-B-040-001 and NSC 92-2314- B-039-021). Back

2 Correspondence: Jer-Yuh Liu, Institute of Biochemistry, Medical College, Chung-Shan Medical University, No. 110, Sec. 1, Chien-Kuo N. Road, Taichung 402, Taiwan, Republic of China. FAX: 886 4 2324 8185; jyl{at}csmu.edu.tw Back

3 Correspondence: Maw-Sheng Lee, Division of Infertility Clinic, Lee Women's Hospital, No. 263, Pei-Tun Road, Taichung 406, Taiwan, Republic of China. FAX: 886 4 22384602; msleephd{at}giga.nettw Back

Received: 15 September 2003.

First decision: 9 October 2003.

Accepted: 15 December 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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