| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering (Y.-C.Z., Z.-M.Y.), College of Life Science, Xiamen University, Xiamen 361005, China; and College of Life Science (Y.-C.Z., Y.-J.C., Y.-S.Y., J.-L.L., R.-W.S., X.-H.M., C.-H.S., Z.-M.Y.), Northeast Agricultural University, Harbin 150030, China
Address all correspondence and requests for reprints to: Zeng-Ming Yang, College of Life Science, Xiamen University, Xiamen 361005, China. E-mail: zmyang{at}xmu.edu.cn.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-difluoromethylornithine, an Odc inhibitor. Moreover, the reduction of Odc activity caused by -difluoromethylornithine treatment was compensated by the up-regulation of S-adenosylmethionine decarboxylase gene expression. Collectively, our results indicated that the coordinated expression of uterine polyamine-related genes may be important for embryo implantation. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Ornithine decarboxylase (Odc) is the key regulator of the polyamine biosynthetic pathway and decarboxylates L-ornithine to form putrescine. Spermidine and spermine can be synthesized from putrescine and decarboxylated S-adenosylmethionine by S-adenosylmethionine decarboxylase (Amd) in two aminopropyltransferase reactions via spermidine synthase (Srm) and spermine synthase (Sms), respectively. However, putrescine and spermidine can also be produced from spermidine and spermine under the catalysis of spermidine/spermine N1-acetyltransferase (Sat) and peroxisomal N1-acetyl-spermine/spermidine oxidase (Paox), respectively (3). Recently, spermine oxidase (Smox), an inducible oxidase, is shown to be able to convert spermine back into spermidine directly without acetylation (4). Odc is considered as the rate-limiting enzyme of polyamine synthesis. The rapid turnover of Odc protein is mediated by Odc antizyme (Oaz) (2). The pathway and relationship in polyamine biosynthesis and metabolism are shown in supplemental Fig. 1, which is published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org. Although the pathway of polyamine biosynthesis has been well characterized, and polyamines are crucial to the growth and proliferation of mammalian cells, the cellular functions of natural polyamines are still largely unidentified (5).
Embryo implantation is a mutual interaction between blastocyst and uterus. Successful implantation is dependent on the cellular and molecular dialogue between competent embryos and receptive uterus (6, 7). Although many specific factors have been identified and characterized during embryo implantation, the molecular mechanism underlying embryo implantation still remains unknown. Odc enzyme activity was shown to be essential for post-implantation embryo development in the mouse and hamster using -difluoromethylornithine (DFMO), an irreversible inhibitor of Odc (8, 9). Odc-deficient mouse embryos failed to develop through the stage of gastrulation (10). Transgenic female mice overexpressing Sat were infertile due to ovarian hypofunction and hypoplastic uteri (11). In our microarray analysis, Odc expression was significantly higher in mouse uterus at implantation sites than that at interimplantation sites (our unpublished data). However, the expression, regulation, and function of polyamine-related genes in mouse uterus during embryo implantation are still unknown. We assumed that polyamines should be important for mouse implantation. This study was to investigate the expression, regulation, and function of polyamine-related genes in mouse uterus during the peri-implantation period.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To induce delayed implantation, pregnant mice were ovariectomized under ether anesthesia at 0830–0900 h on d-4 pregnancy. Delayed implantation was maintained by daily sc injection of progesterone (1 mg/mouse; Sigma-Aldrich) on d 5–7. To terminate delayed implantation, progesterone-primed delayed-implantation mice were treated with estradiol-17β (25 ng/mouse, sc; Sigma-Aldrich) on d 7. The mice were killed to collect uteri 24 h after estrogen treatment. Delayed implantation was confirmed by flushing blastocysts from one horn of the uterus.
The treatments of steroid hormones were initiated 2 wk after mature female mice were ovariectomized. Ovariectomized mice were treated with estradiol-17β (100 ng/mouse) or progesterone (1 mg/mouse) for 24 h. To examine whether nuclear receptors for estrogen or progesterone are involved in steroid hormonal regulation, ovariectomized mice were also treated with estraodiol-17β plus ICI 182,780 (30 mg/kg; Tocris Cookson, Inc., Ballwin, MO) or progesterone plus RU-486 (25 mg/kg; Cayman Chemical, Ann Arbor, MI). Estraodiol-17β, progesterone, ICI 182,780, and RU-486 were dissolved in sesame oil and injected sc, respectively. Controls received the vehicle only (0.1 ml/mouse).
DFMO was kindly provided by Dr. Patrick M. Woster (Wayne State University, Detroit, MI). Trans-4-methylcyclohexylamine (4MCHA), a specific inhibitor for Srm (12), was provided by Dr. Keijiro Samejima (Josai University, Sakado, Saitama, Japan). DFMO and 4MCHA were dissolved in saline and injected sc, respectively. Controls received saline (0.1 ml/mouse). Implantation sites were identified by iv injection of Chicago blue solution. Ovulation was confirmed by counting the number of corpus luteum on the morning of d 5. The pregnant rate was calculated as the ratio of the number of females with implantation sites to the number of total females in each group.
In situ hybridization
Total RNAs from mouse placenta were reverse transcribed and amplified with the corresponding primers (the results can be found in supplemental Table 1). The amplified fragment of each gene was cloned into pGEM-T plasmid (pGEM-T Vector System 1; Promega, Madison, WI) and verified by sequencing. Each recombinant plasmid was amplified with the primers for T7 and SP6 to prepare templates for labeling sense or antisense probes. Digoxigenin-labeled antisense or sense cRNA probe was transcribed in vitro using the digoxigenin RNA labeling kit (Roche Diagnostics GmbH, Mannheim, Germany).
Uteri were cut into 4- to 7-mm pieces, flash frozen in liquid nitrogen, and stored at –80 C. Frozen sections (10 µm) were mounted on 3-aminopropyltriethoxy-silane (Sigma-Aldrich) treated slides and fixed in 4% paraformaldehyde solution in PBS. Hybridization was performed as previously described (13). Endogenous alkaline phosphatase activity was inhibited with 2 mM levamisole (Sigma-Aldrich). Sections were counterstained with 1% methyl green (Sigma-Aldrich). The positive signal was visualized as a dark-brown color. The sense probe for each gene was also hybridized and served as a negative control. There was no detectable signal from sense probes.
Western blot
Proteins were extracted from uterine tissues by homogenization in lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.25% sodium deoxycholate, and complete protease inhibitor cocktail (Roche Diagnostics)]. The concentration of proteins was measured by Bradford reagent (Sigma-Aldrich). Uterine proteins were run on an 8% PAGE and transferred onto nitrocellulose membranes. After blocked with 5% low-fat milk in PBST (PBS containing 0.1% Tween 20) for 1 h, the membranes were incubated with mouse anti-Odc monoclonal antibody (1:1000; Thermo Fisher Scientific Inc., Fremont, CA) or rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (1:2000, sc-25778; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4 C. After washing in PBST, the membranes were incubated in goat antimouse antibody or goat antirabbit antibody conjugated with horseradish peroxidase (1:5000) for 1 h, followed by three washes in PBST. The signals were visualized by an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Arlington Heights, IL).
Isolation and culture of uterine epithelial and stromal cells
Uteri from estrous mice were split longitudinally, and incubated with 0.1% trypsin (AMRESCO Inc., Solon, OH), 1.2 mg/ml dispase (Roche Diagnostics), and penicillin/streptomycin (Hyclone, Logan, UT) in Hanks’ balanced salt solution (HBSS) (Sigma-Aldrich) for 1 h at 4 C, 1 h at 22 C, and 10 min at 37 C, respectively. The digested uteri were vortexed gently and rinsed three times with HBSS. After passing the cell suspension through a 100-µm nylon mesh, epithelial sheets were collected from cell suspension by natural settling. Epithelial sheets were resuspended in complete medium consisting of DMEM (Sigma-Aldrich) with 10% fetal bovine serum and penicillin/streptomycin. Each 35-mm culture dish was seeded with 2 ml cell suspension. After a short attachment of 30 min to remove contaminated stromal cells, the medium containing suspended epithelial cells was transferred to new dishes for further culture.
To isolate stromal cells, the digested uteri after the removal of epithelial cells were incubated in 2 ml HBSS containing 0.5% collagenase I (Invitrogen Corp., Carlsbad, CA) and penicillin/streptomycin for 30 min at 37 C. The digested uteri were vigorously shaken and filtered through a 40-µm nylon mesh. The resultant cell suspension was washed with HBSS and centrifuged at 500 rpm for 5 min. Cells were resuspended in culture medium and seeded onto 35-mm culture dishes at the concentration of 1 x 106 cells per ml. After 30-min adherence to the dish, the medium was changed for the removal of epithelial sheets. Both primary epithelial and stromal cells were incubated at 37 C with 5% CO2 for 24 h and rinsed with the fresh complete medium before treatments or transfections.
Odc overexpression
A 1385-bp Odc cDNA fragment was amplified by RT-PCR from mouse placenta using the following primers: 5'-CTCGGATCCATGAGCAGCTTTACTAAGGAC-3' and 5'-TCTGATATCCTACACATTGATCCTAGCAG-3', in which the digestion sites for BamH I and EcoRV were underlined, respectively. The PCR product was digested with EcoR V and BamH I and subcloned into pcDNA3.1 expression vector (Promega) (pc-Odc). An empty pcDNA3.1 expression vector served as a control.
Transfection was performed according to the manufacturer’s instructions (LipofectAMINE 2000; Invitrogen). Briefly, 4 µg pc-Odc or empty vector was mixed with 250 µl Opti-MEM (Invitrogen) for each 35-mm cell culture dish. This mixture was gently added to the solution containing 10 µl LipofectAMINE 2000 diluted with 250 µl Opti-MEM. The solution was incubated for 20 min at room temperature and gently added onto 70–80% confluent primary endometrial stromal or epithelial cells in 2 ml Opti-MEM. After 24 h for transfection, cells were lysed for further analysis. The overexpression of Odc in cultured endometrial cells was confirmed by real-time PCR.
Real-time PCR
Total RNAs from uteri or cultured cells were isolated using TRIZOL reagent according to the manufacturer’s instructions (Invitrogen). cDNA was reverse transcribed from 1 µg total RNA using the ExScript RT Reagents Kit (Perfect Real Time; Takara, Dalian, China).
For real-time PCR, cDNA was amplified using SYBR Premix Ex Taq kit (Takara) according to the manufacturer’s instructions. PCR was performed with the Real Time PCR System (ABI PRISM 7500 Real-time PCR System; Applied Biosystems, Foster City, CA). After analysis using the 
Ct method, data were normalized to Gapdh expression (14). Primer sequences of Oaz1 used for real-time PCR were 5'-GACGAGCGGCTGAATGTGA-3' and 5'-CCGTGAGCGTGGACTGGAT-3'. Other primer sequences for real-time PCR were described previously (15).
Statistics
All the experiments were independently repeated at least three times. The significance of difference was assessed by the 
2 test or t test. P < 0.05 was considered statistically significant. Statistical analysis was conducted with MATLAB 7.0 software (The MathWorks, Inc., Natick, MA).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. From d 1–4, there was a low level of Odc expression in the uterine epithelium. A weak Odc expression was also detected in the subluminal stroma on d 3 and 4. On d 5, Odc expression was strongly shown in the subluminal stroma surrounding the implanting blastocyst compared with interimplantation sites.
|
From d 1–4, there was a basal expression for Oaz2, Srm, Sms, Amd1, Sat, Smox, and Paox. On d 5, no evident expression was seen in both implantation and interimplantation sites for Oaz2, Sms, and Paox. However, significantly stronger signals for Amd1, Srm, Sat, and Smox were detected in the subluminal stroma at implantation sites compared with interimplantation sites.
To confirm further the expression level of polyamine-related genes in the uterus on d 5 between implantation and interimplantation sites, we chose four highly expressed genes at implantation sites for real-time PCR analysis. Compared with interimplantation sites, the expression level of Odc, Srm, and Oaz1 was significantly higher at implantation sites (Fig. 2).
|
). Although Odc protein was detected in the uterus on d 5 at both implantation and interimplantation sites, Odc signal was obviously stronger at implantation sites than that at interimplantation sites.
|
). A low level of Odc expression was detected in the subluminal stroma on d 3 and 4, but no expression was seen on d 5. There was a low level of Oaz1 expression in the luminal and glandular epithelium from d 3–5. During d 3–5 there were no detectable signals for Oaz2, Srm, Sms, Amd1, Sat, Smox, and Paox.
|
). Under delayed implantation no Odc expression was detected. After delayed implantation was terminated by estrogen treatment, a strong signal for Odc expression was seen in the subluminal stroma at implantation sites. Compared with delayed implantation, a stronger expression for Oaz1, Srm, Sat, Smox, and Amd1 was also observed after the activation of delayed implantation. There were no detectable signals for Oaz2, Sms, and Paox in delayed uterus or activated implantation sites.
Steroid hormonal regulation of polyamine-related genes in ovariectomized mice
Because estrogen and progesterone were essential for the establishment of mouse pregnancy, in situ hybridization was performed to determine whether steroid hormones could regulate the expression of polyamine-related genes in the uterus of ovariectomized mice (Fig. 5). In ovariectomized mice there was no Odc expression in the uterus. A weak signal of Odc expression was detected in the subluminal stroma after progesterone treatment. When ovariectomized mice were treated with both progesterone and RU-486, there was only a basal level of Odc expression. Estrogen stimulated a strong Odc expression in the luminal epithelium. However, there was no detectable Odc expression after a cotreatment of both estrogen and ICI 182,780. Only a weak Odc expression was seen in the luminal epithelium after the treatment by both estrogen and progesterone. When the Odc antisense probe was replaced with the sense probe, there was no detectable signal in all the treatments (Fig. 5).
|
). After ovariectomized mice were treated with both estrogen and ICI 182,780, only a basal level of Oaz1 expression was detected. A strong Oaz1 expression was also strongly induced by a cotreatment of estrogen and progesterone. Similarly, there was no detectable signal when the Oaz1 antisense probe was replaced by the Oaz1 sense probe (Fig. 5). In ovariectomized mice, either estrogen or progesterone had any obvious effects on the expression of Oaz2, Srm, Sms, Amd1, Sat, Smox, and Paox in the uteri (data not shown).
Regulation of Oaz1 expression by Odc and polyamine level
Because the rapid turnover of Odc protein is mediated by Oaz (2), we would like to see whether Oaz1 expression was regulated by Odc expression and polyamine level.
Odc overexpression in cultured endometrial cells was confirmed by real-time PCR (Fig. 6A). Odc expression in the stromal and epithelial cells was significantly increased after Odc cDNA transfection. Odc overexpression led to an obvious increase of Oaz1 expression in the stromal and epithelial cells, respectively. However, exogenous putrescine had no effects on the Oaz1 expression (Fig. 6B).
|
). In the cultured stromal cells, Sat expression was significantly stimulated by spermidine (Fig. 7A). Because stromal cells were sensitive for spermine treatment, the effects of spermine on Sat expression were not examined. In the cultured epithelial cells, Sat expression was sharply stimulated by either spermidine or spermine. Paox and Smox expression was slightly down-regulated by spermidine, but not by spermine (Fig. 7B).
|
). When the mice were treated with 500 mg/kg DFMO twice on d 4 of pregnancy, the pregnant rate was significantly reduced compared with the control (54.6 vs. 85%; P < 0.05). After pregnant mice were treated with 500 mg/kg DFMO three times on both d 3 and 4, the pregnant rate was further reduced to 36.4% compared with the control (87.5%). However, a higher dosage of DFMO (1000 mg/kg) had no further inhibitory effect compared with 500 mg/kg DFMO (42.9 vs. 36.4%; P > 0.05).
|
). When pregnant mice were treated with 4MCHA (100 mg/kg), no obvious inhibition was observed compared with the control (77.7 vs. 85%; P > 0.05). Even if pregnant mice were treated by both 4MCHA and DFMO, the pregnant rate was 41.2%, similar to that treated by DFMO alone. In the mouse with estrous cycles, DFMO administration causes the reduction of progesterone level in serum and the inhibition of angiogenesis in the corpus luteum (16). Therefore, the model of delayed implantation was used to determine whether ovarian factors were involved in the inhibition of implantation by DFMO. As described previously, pregnant mice were ovariectomized on d 4 and injected daily with progesterone from d 5–7 to maintain delayed implantation. The mice were treated with DFMO (500 mg/kg) three times from the afternoon on d 6 to the afternoon on d 7, and delayed implantation was activated by estrogen on d 7. Embryo implantation was examined 24 h after estrogen treatment. Compared with the control (83.3%), the pregnant rate of the DFMO-treated group was significantly reduced to 38.9% (P < 0.05), similar to that treated by DFMO on d 3 and 4 of pregnancy. This indicated that DFMO mainly acted on uterine factors to block implantation during early pregnancy.
Regulation of Amd1 expression by DFMO
In our study the incomplete inhibition of embryo implantation by DFMO injection might suggest the presence of a compensational mechanism for the loss of Odc activity in the uterus. Implantation sites of DFMO-treated mouse uteri were checked for the expression of Amd1, Srm, and Sms. According to real-time RT-PCR analysis, Amd1 expression was significantly higher compared with the control. The expressions of Srm and Sms were not changed after DFMO injection (Fig. 8).
|
). In the epithelial cells, Amd1 and Sms expressions were down-regulated by exogenous putrescine (Fig. 9B).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
DFMO can irreversibly and specifically inactivate Odc (1). We showed that embryo implantation was significantly inhibited by DFMO either in normal pregnancy or under delayed implantation, suggesting that DFMO mainly acted on uterine factors in implantation. However, embryo implantation was not completely inhibited by DFMO treatment. The half-life of DFMO under ip administration is around 126 min (17). The half-life of DFMO after sc injection should be similar with the intraperitoneal route. Thus, the relative short half-life might be one of the reasons for the incomplete implantation inhibition effect by DFMO after sc injection because there is not enough DFMO present in the uterus during embryo implantation. Fozard et al. (8) reported that the post-implantation development of mouse embryo was blocked by DFMO treatment on d 5–8 during early pregnancy, whereas there was no obvious development or implantation inhibition effect after DFMO was provided in drinking water on d 1–4. However, the results from pharmacological research proved that after treated in drinking water, DFMO accumulation was much more obvious in the organs responsible for reabsorption or metabolism, including intestine, liver, and kidney, than in other tissues (17). Therefore, the efficiency of DFMO absorption in the uterus might not be effectual enough after treated in drinking water.
The up-regulation of Amd1 expression may be compensatory after DFMO treatment
In our study embryo implantation was not completely inhibited by a high dose of DFMO. Moreover, Odc null embryos were competent for implantation even if they failed to survive from the stage of gastrulation (10). These data may suggest a compensational mechanism for polyamine biosynthesis. Because Srm expression was specifically enhanced at implantations sites, we assumed that the Srm activity might also be important during implantation. 4MCHA, a specific inhibitor for Srm, was proved to decrease the in vivo activity of Srm effectively and specifically (12). However, 4MCHA alone had no effects on implantation. Moreover, 4MCHA had no further inhibitory effects on embryo implantation when pregnant mice were treated by a combination of DFMO and 4MCHA, suggesting that Srm should not be the key factor.
In our study Amd1 expression was significantly up-regulated by DFMO treatment, but down-regulated by Odc overexpression in the cultured uterine cells. In the DFMO-treated mice, Amd1 expression was also up-regulated at implantation sites. Our data indicated that Amd1 expression should be a factor for compensating the reduction of Odc activity. The up-regulation of Amd (encoded by Amd1) activity caused by DFMO administration was also shown by other groups (8, 18, 19). CGP 48664 (SAM 468A) is a newly developed Amd inhibitor (20). The combined use of DFMO and CGP 48664 may be required for a complete inhibition of embryo implantation.
Tight control on uterine polyamines during embryo implantation
Polyamines cannot only promote cell growth but also trigger the death process in many studies (3, 21). Under normal circumstances polyamine concentrations regulate their own biosynthesis and prevent overproduction by developing complex systems consisting of a series of enzymes and polyamine transporters. However, in abnormal cases a high concentration of exogenous polyamines, especially spermine, leads to cell death (22, 23, 24). To ensure cell growth and avoid potential toxic effects, intracellular polyamine needed to be maintained within a narrowly limited concentration (25). Although Odc was strongly expressed at implantation sites, Oaz1 expression was also highly detected in these areas. Oaz can associate with and direct Odc protein to the proteasome without ubiquitination. The rapid turnover of Odc protein is mediated by Oaz (2). The colocalization of Oaz1 and Odc expression suggested that Odc activity may be tightly controlled by Oaz1.
In addition, spermine is highly toxic for cells and can lead to cell death (24). It is important to avoid spermine overproduction in the uterus. Sat is the rate-limiting enzyme in polyamine metabolism and can be induced by polyamines or their analogs in several cell lines (3, 26, 27). In our study Sat was also highly expressed at implantation sites, and was promoted by exogenous spermidine and spermine. Furthermore, spermine could be directly converted into spermidine by Smox (4), which was also strongly expressed at implantation sites. Our data suggested that the strong expression of Sat and Smox at implantation sites might relate to the tight control of spermine level at implantation sites.
In conclusion, Odc expression was strongly detected at implantation site and dependent on the presence of an active blastocyst. The coordinate expression of polyamine-related genes during the peri-implantation period was important to maintain the homeostasis of uterine polyamines for facilitating endometrial cell proliferation and establishing a suitable environment for embryo implantation.
|
Footnotes |
|---|
Disclosure Statement: The authors have nothing to disclose.
First Published Online January 17, 2008
Abbreviations: Amd, S-adenosylmethionine decarboxylase; DFMO, -difluoromethylornithine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HBSS, Hanks’ balanced salt solution; Oaz, ornithine decarboxylase antizyme; Odc, ornithine decarboxylase; Paox, polyamine oxidase; PBST, 0.1% Tween 20 in PBS; Sat, spermidine/spermine N1-acetyltransferase; Smox, spermine oxidase; Sms, spermine synthase; Srm, spermidine synthase; 4MCHA, trans-4-methylcyclohexylamine.
Received October 17, 2007.
Accepted for publication January 9, 2008.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase. Eur J Pharmacol 65:379–391[CrossRef][Medline]-difluoromethylornithine in mice. Cancer Chemother Pharmacol 19:30–34[CrossRef][Medline]This article has been cited by other articles:
 |
|
 |
|
  H. Gao, G. Wu, T. E. Spencer, G. A. Johnson, and F. W. Bazer Select Nutrients in the Ovine Uterine Lumen. V. Nitric Oxide Synthase, GTP Cyclohydrolase, and Ornithine Decarboxylase in Ovine Uteri and Peri-Implantation Conceptuses Biol Reprod, July 1, 2009; 81(1): 67 - 76. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Lassala, F. W. Bazer, T. A. Cudd, P. Li, X. Li, M. C. Satterfield, T. E. Spencer, and G. Wu Intravenous Administration of L-Citrulline to Pregnant Ewes Is More Effective Than L-Arginine for Increasing Arginine Availability in the Fetus J. Nutr., April 1, 2009; 139(4): 660 - 665. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
53 kDa) was detected with monoclonal Odc antibody. B, The protein level of Odc was quantitated by measuring OD of each band and normalized with Gapdh protein level. The Odc protein level at implantation sites was significantly higher than that at interimplantation sites. Data are given as mean ± SD (*, P < 0.05).
