polyamine-related genes in mouse implantation_2008_endocrinology

Polyamines Are Essential in Embryo Implantation:Expression and Function of Polyamine-Related Genes inMouse Uterus during Peri-Implantation Period

Yue-Chao Zhao, Yu-Jing Chi, Yong-Sheng Yu, Ji-Long Liu, Ren-Wei Su, Xing-Hong Ma, Chun-Hua Shan,and Zeng-Ming Yang

Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering (Y.-C.Z., Z.-M.Y.), College of LifeScience, 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

Polyamines are key regulators in cell growth and differenti-ation. It has been shown that ornithine decarboxylase (Odc)was essential for post-implantation embryo development, andoverexpression of spermidine/spermine N1-acetyltransferasewill lead to ovarian hypofunction and hypoplastic uteri. How-ever, the expression and function of polyamine-related genesin mouse uterus during early pregnancy are still unknown. Inthis study we investigated the expression, regulation, andfunction of polyamine-related genes in mouse uterus duringthe peri-implantation period. Odc expression was stronglydetected at implantation sites and stimulated by estrogen

treatment. The expression of Odc antizyme 1 and spermidine/spermine N1-acetyltransferase was also highly shown at im-plantation sites and regulated by Odc or polyamine level inuterine cells. Embryo implantation was significantly inhib-ited by �-difluoromethylornithine, an Odc inhibitor. More-over, the reduction of Odc activity caused by �-difluorometh-ylornithine treatment was compensated by the up-regulationof S-adenosylmethionine decarboxylase gene expression. Col-lectively, our results indicated that the coordinated expres-sion of uterine polyamine-related genes may be important forembryo implantation. (Endocrinology 149: 2325–2332, 2008)

THE POLYAMINES, PUTRESCINE, spermidine, andspermine, exist in almost all living species. Through

binding different molecules, polyamines are involved in cellgrowth and differentiation. A complicated network isformed to sustain polyamine homeostasis in cells (1, 2).

Ornithine decarboxylase (Odc) is the key regulator of thepolyamine biosynthetic pathway and decarboxylates L-or-nithine to form putrescine. Spermidine and spermine can besynthesized from putrescine and decarboxylated S-adeno-sylmethionine by S-adenosylmethionine decarboxylase(Amd) in two aminopropyltransferase reactions via spermi-dine synthase (Srm) and spermine synthase (Sms), respec-tively. However, putrescine and spermidine can also be pro-duced from spermidine and spermine under the catalysis ofspermidine/spermine N1-acetyltransferase (Sat) and perox-isomal N1-acetyl-spermine/spermidine oxidase (Paox), re-spectively (3). Recently, spermine oxidase (Smox), an induc-ible oxidase, is shown to be able to convert spermine backinto spermidine directly without acetylation (4). Odc is con-sidered as the rate-limiting enzyme of polyamine synthesis.

The rapid turnover of Odc protein is mediated by Odc an-tizyme (Oaz) (2). The pathway and relationship in polyaminebiosynthesis and metabolism are shown in supplemental Fig.1, which is published as supplemental data on The EndocrineSociety’s Journals Online web site at http://endo.endojour-nals.org. Although the pathway of polyamine biosynthesishas been well characterized, and polyamines are crucial tothe growth and proliferation of mammalian cells, the cellularfunctions of natural polyamines are still largely unidentified(5).

Embryo implantation is a mutual interaction between blas-tocyst and uterus. Successful implantation is dependent onthe cellular and molecular dialogue between competent em-bryos and receptive uterus (6, 7). Although many specificfactors have been identified and characterized during em-bryo implantation, the molecular mechanism underlyingembryo implantation still remains unknown. Odc enzymeactivity was shown to be essential for post-implantation em-bryo development in the mouse and hamster using �-di-fluoromethylornithine (DFMO), an irreversible inhibitor ofOdc (8, 9). Odc-deficient mouse embryos failed to developthrough the stage of gastrulation (10). Transgenic femalemice overexpressing Sat were infertile due to ovarian hypo-function and hypoplastic uteri (11). In our microarray anal-ysis, Odc expression was significantly higher in mouse uterusat implantation sites than that at interimplantation sites (ourunpublished data). However, the expression, regulation, andfunction of polyamine-related genes in mouse uterus duringembryo implantation are still unknown. We assumed thatpolyamines should be important for mouse implantation.This study was to investigate the expression, regulation, and

First Published Online January 17, 2008Abbreviations: Amd, S-adenosylmethionine decarboxylase; DFMO,

�-difluoromethylornithine; GAPDH, glyceraldehyde-3-phosphate de-hydrogenase; HBSS, Hanks’ balanced salt solution; Oaz, ornithine de-carboxylase antizyme; Odc, ornithine decarboxylase; Paox, polyamineoxidase; 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.Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving theendocrine community.

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function of polyamine-related genes in mouse uterus duringthe peri-implantation period.

Materials and MethodsAnimals and treatments

Mature mice (Kunming White outbred strain) were caged in a con-trolled environment with a 14-h light, 10-h dark cycle. All animal pro-cedures were approved by the Institutional Animal Care and Use Com-mittee of Xiamen University. To induce pregnancy or pseudopregnancy,adult female mice were mated with fertile or vasectomized males of thesame strain by co-caging, respectively (d 1 � day of vaginal plug). Ond 1–4, pregnancy was confirmed by recovering embryos from the ovi-ducts or uterus. The implantation sites on d 5 were identified by ivinjection of 0.1 ml 1% Chicago blue dye (Sigma-Aldrich, St. Louis, MO)in saline.

To induce delayed implantation, pregnant mice were ovariectomizedunder ether anesthesia at 0830–0900 h on d-4 pregnancy. Delayed im-plantation 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 es-tradiol-17� (25 ng/mouse, sc; Sigma-Aldrich) on d 7. The mice werekilled to collect uteri 24 h after estrogen treatment. Delayed implantationwas confirmed by flushing blastocysts from one horn of the uterus.

The treatments of steroid hormones were initiated 2 wk after maturefemale mice were ovariectomized. Ovariectomized mice were treatedwith estradiol-17� (100 ng/mouse) or progesterone (1 mg/mouse) for24 h. To examine whether nuclear receptors for estrogen or progesteroneare involved in steroid hormonal regulation, ovariectomized mice werealso treated with estraodiol-17� plus ICI 182,780 (30 mg/kg; TocrisCookson, Inc., Ballwin, MO) or progesterone plus RU-486 (25 mg/kg;Cayman Chemical, Ann Arbor, MI). Estraodiol-17�, progesterone, ICI182,780, and RU-486 were dissolved in sesame oil and injected sc, re-spectively. Controls received the vehicle only (0.1 ml/mouse).

DFMO was kindly provided by Dr. Patrick M. Woster (Wayne StateUniversity, Detroit, MI). Trans-4-methylcyclohexylamine (4MCHA), aspecific inhibitor for Srm (12), was provided by Dr. Keijiro Samejima(Josai University, Sakado, Saitama, Japan). DFMO and 4MCHA weredissolved in saline and injected sc, respectively. Controls received saline(0.1 ml/mouse). Implantation sites were identified by iv injection ofChicago blue solution. Ovulation was confirmed by counting the num-ber of corpus luteum on the morning of d 5. The pregnant rate wascalculated as the ratio of the number of females with implantation sitesto the number of total females in each group.

In situ hybridization

Total RNAs from mouse placenta were reverse transcribed and am-plified with the corresponding primers (the results can be found insupplemental Table 1). The amplified fragment of each gene was clonedinto pGEM-T plasmid (pGEM-T Vector System 1; Promega, Madison,WI) and verified by sequencing. Each recombinant plasmid was am-plified with the primers for T7 and SP6 to prepare templates for labelingsense or antisense probes. Digoxigenin-labeled antisense or sense cRNAprobe 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 �m) were mounted on 3-amin-opropyltriethoxy-silane (Sigma-Aldrich) treated slides and fixed in 4%paraformaldehyde solution in PBS. Hybridization was performed aspreviously described (13). Endogenous alkaline phosphatase activitywas inhibited with 2 mm levamisole (Sigma-Aldrich). Sections werecounterstained with 1% methyl green (Sigma-Aldrich). The positivesignal was visualized as a dark-brown color. The sense probe for eachgene was also hybridized and served as a negative control. There wasno detectable signal from sense probes.

Western blot

Proteins were extracted from uterine tissues by homogenization inlysis 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 byBradford reagent (Sigma-Aldrich). Uterine proteins were run on an 8%PAGE and transferred onto nitrocellulose membranes. After blockedwith 5% low-fat milk in PBST (PBS containing 0.1% Tween 20) for 1 h,the membranes were incubated with mouse anti-Odc monoclonal an-tibody (1:1000; Thermo Fisher Scientific Inc., Fremont, CA) or rabbitanti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonalantibody (1:2000, sc-25778; Santa Cruz Biotechnology, Inc., Santa Cruz,CA) overnight at 4 C. After washing in PBST, the membranes wereincubated in goat antimouse antibody or goat antirabbit antibody con-jugated with horseradish peroxidase (1:5000) for 1 h, followed by threewashes in PBST. The signals were visualized by an enhanced chemilu-minescence 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 with0.1% trypsin (AMRESCO Inc., Solon, OH), 1.2 mg/ml dispase (RocheDiagnostics), and penicillin/streptomycin (Hyclone, Logan, UT) inHanks’ balanced salt solution (HBSS) (Sigma-Aldrich) for 1 h at 4 C, 1 hat 22 C, and 10 min at 37 C, respectively. The digested uteri werevortexed gently and rinsed three times with HBSS. After passing the cellsuspension through a 100-�m nylon mesh, epithelial sheets were col-lected from cell suspension by natural settling. Epithelial sheets wereresuspended in complete medium consisting of DMEM (Sigma-Aldrich)with 10% fetal bovine serum and penicillin/streptomycin. Each 35-mmculture dish was seeded with 2 ml cell suspension. After a short attach-ment of 30 min to remove contaminated stromal cells, the mediumcontaining suspended epithelial cells was transferred to new dishes forfurther culture.

To isolate stromal cells, the digested uteri after the removal of epi-thelial cells were incubated in 2 ml HBSS containing 0.5% collagenaseI (Invitrogen Corp., Carlsbad, CA) and penicillin/streptomycin for 30min at 37 C. The digested uteri were vigorously shaken and filteredthrough a 40-�m nylon mesh. The resultant cell suspension was washedwith HBSS and centrifuged at 500 rpm for 5 min. Cells were resuspendedin culture medium and seeded onto 35-mm culture dishes at the con-centration of 1 � 106 cells per ml. After 30-min adherence to the dish,the medium was changed for the removal of epithelial sheets. Bothprimary epithelial and stromal cells were incubated at 37 C with 5% CO2for 24 h and rinsed with the fresh complete medium before treatmentsor transfections.

Odc overexpression

A 1385-bp Odc cDNA fragment was amplified by RT-PCR frommouse placenta using the following primers: 5�-CTCGGATCCATGA-GCAGCTTTACTAAGGAC-3� and 5�-TCTGATATCCTACACATTGA-TCCTAGCAG-3�, in which the digestion sites for BamH I and EcoRVwere underlined, respectively. The PCR product was digested with EcoRV and BamH I and subcloned into pcDNA3.1 expression vector(Promega) (pc-Odc). An empty pcDNA3.1 expression vector served asa control.

Transfection was performed according to the manufacturer’s instruc-tions (LipofectAMINE 2000; Invitrogen). Briefly, 4 �g pc-Odc or emptyvector was mixed with 250 �l Opti-MEM (Invitrogen) for each 35-mmcell culture dish. This mixture was gently added to the solution con-taining 10 �l LipofectAMINE 2000 diluted with 250 �l Opti-MEM. Thesolution was incubated for 20 min at room temperature and gentlyadded onto 70–80% confluent primary endometrial stromal or epithelialcells in 2 ml Opti-MEM. After 24 h for transfection, cells were lysed forfurther analysis. The overexpression of Odc in cultured endometrial cellswas confirmed by real-time PCR.

Real-time PCR

Total RNAs from uteri or cultured cells were isolated using TRIzolreagent according to the manufacturer’s instructions (Invitrogen). cDNAwas reverse transcribed from 1 �g total RNA using the ExScript RTReagents Kit (Perfect Real Time; Takara, Dalian, China).

For real-time PCR, cDNA was amplified using SYBR Premix Ex Taqkit (Takara) according to the manufacturer’s instructions. PCR was per-

2326 Endocrinology, May 2008, 149(5):2325–2332 Zhao et al. • Polyamine-Related Genes in Mouse Implantation


formed with the Real Time PCR System (ABI PRISM 7500 Real-time PCRSystem; Applied Biosystems, Foster City, CA). After analysis using the�Ct method, data were normalized to Gapdh expression (14). Primersequences of Oaz1 used for real-time PCR were 5�-GACGAGCGGCT-GAATGTGA-3� and 5�-CCGTGAGCGTGGACTGGAT-3�. Other primersequences 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 con-ducted with MATLAB 7.0 software (The MathWorks, Inc., Natick, MA).

ResultsExpression pattern of polyamine-related genes duringperi-implantation

The expression of polyamine-related genes in mouseuterus during d 1–5 was examined by in situ hybridizationand is shown in Fig. 1. From d 1–4, there was a low level ofOdc expression in the uterine epithelium. A weak Odc ex-pression was also detected in the subluminal stroma on d 3and 4. On d 5, Odc expression was strongly shown in thesubluminal stroma surrounding the implanting blastocystcompared with interimplantation sites.

From d 1–4, there was a low level of Oaz1 expression in theluminal and glandular epithelia on d 3. Compared withinterimplantation sites, Oaz1 expression in subluminalstroma was stronger at the implantation site on d 5.

From d 1–4, there was a basal expression for Oaz2, Srm,Sms, Amd1, Sat, Smox, and Paox. On d 5, no evident expres-

sion was seen in both implantation and interimplantationsites for Oaz2, Sms, and Paox. However, significantly strongersignals for Amd1, Srm, Sat, and Smox were detected in thesubluminal stroma at implantation sites compared with in-terimplantation sites.

To confirm further the expression level of polyamine-re-lated genes in the uterus on d 5 between implantation andinterimplantation sites, we chose four highly expressedgenes at implantation sites for real-time PCR analysis. Com-pared with interimplantation sites, the expression level ofOdc, Srm, and Oaz1 was significantly higher at implantationsites (Fig. 2).

Because Odc was the rate-limiting and key enzyme forpolyamine biosynthesis, Odc protein expression was exam-ined by Western blot (Fig. 3). Although Odc protein wasdetected in the uterus on d 5 at both implantation and in-terimplantation sites, Odc signal was obviously stronger atimplantation sites than that at interimplantation sites.

Expression of polyamine-related genes duringpseudopregnancy

To address whether the expression of polyamine-relatedgenes was dependent upon embryos, in situ hybridizationwas performed to examine their expression during d 3–5 ofpseudopregnancy (Fig. 4). A low level of Odc expression wasdetected in the subluminal stroma on d 3 and 4, but noexpression was seen on d 5. There was a low level of Oaz1expression in the luminal and glandular epithelium from d3–5. During d 3–5 there were no detectable signals for Oaz2,Srm, Sms, Amd1, Sat, Smox, and Paox.

Expression of polyamine-related genes under delayedimplantation

To see whether the expression of polyamine-relatedgenes was dependent on the presence of an active blas-tocyst, in situ hybridization was performed to examinetheir expression under delayed implantation (Fig. 4). Un-der delayed implantation no Odc expression was detected.After delayed implantation was terminated by estrogen

FIG. 2. Quantification of Odc, Srm, Amd1, and Oaz1 mRNA expres-sion in mouse uterine endometrium on d 5 of pregnancy by real-timePCR. After the endometrium was squeezed out of the whole uterus,the interimplantation and implantation sites were separately col-lected for RNA isolation and cDNA synthesis. Real-time PCR wasperformed as described in Materials and Methods. The expressionlevel of Odc, Srm, Amd1, and Oaz1 at implantation sites was nor-malized with Gapdh expression and determined relative to that ofinterimplantation sites. Compared with interimplantation sites, theexpression level of Odc, Oaz1, and Srm was significantly higher atimplantation sites. Data are given as mean � SD (*, P � 0.05).

FIG. 1. In situ hybridization of Odc, Oaz1, Oaz2, Srm, Sms, Amd1,Sat, Smox, and Paox mRNA expression in mouse uterus on d 1–5 ofpregnancy. Compared with interimplantation sites on (N-IM) on d 5of pregnancy (D5), Odc, Oaz1, Srm, Amd1, Smox, and Sat werestrongly expressed at implantation (IM) sites. Bar, 300 �m.

Zhao et al. • Polyamine-Related Genes in Mouse Implantation Endocrinology, May 2008, 149(5):2325–2332 2327


treatment, a strong signal for Odc expression was seen inthe subluminal stroma at implantation sites. Comparedwith delayed implantation, a stronger expression for Oaz1,Srm, Sat, Smox, and Amd1 was also observed after theactivation of delayed implantation. There were no detect-able signals for Oaz2, Sms, and Paox in delayed uterus oractivated implantation sites.

Steroid hormonal regulation of polyamine-related genes inovariectomized mice

Because estrogen and progesterone were essential forthe establishment of mouse pregnancy, in situ hybridiza-tion was performed to determine whether steroid hor-mones could regulate the expression of polyamine-relatedgenes in the uterus of ovariectomized mice (Fig. 5). Inovariectomized mice there was no Odc expression in theuterus. A weak signal of Odc expression was detected inthe subluminal stroma after progesterone treatment.When ovariectomized mice were treated with both pro-gesterone and RU-486, there was only a basal level of Odcexpression. Estrogen stimulated a strong Odc expression inthe luminal epithelium. However, there was no detectableOdc expression after a cotreatment of both estrogen andICI 182,780. Only a weak Odc expression was seen in theluminal epithelium after the treatment by both estrogenand progesterone. When the Odc antisense probe was re-placed with the sense probe, there was no detectable signalin all the treatments (Fig. 5).

Oaz1 expression was at a basal level in the luminal epi-thelium of ovariectomized mice. Progesterone had little ef-fects on Oaz1 expression. However, a high level of Oaz1expression in the luminal and glandular epithelium wasdetected after estrogen treatment (Fig. 5). After ovariecto-

FIG. 5. In situ hybridization of Odc and Oaz1 mRNA expression inovariectomized mouse uterus after hormonal treatments. There wasno detectable signal for Odc and Oaz1 expression in the uteri ofovariectomized mice. Estrogen (E) strongly stimulated the expressionof both Odc and Oaz1 in the luminal epithelium. However, there wasa basal level of Odc and Oaz1 expression in the luminal epitheliumafter the cotreatment of both estrogen and ICI 182,780 (ICI), sug-gesting the involvement of estrogen nuclear receptors. Odc expressionin the subluminal stroma was stimulated by progesterone (P), whichwas also abolished by the cotreament with progesterone and RU-486(RU). Bar, 200 �m.

FIG. 3. Western blot analysis of Odc protein in mouse uterus on d 5of pregnancy. After the endometrium was squeezed out of the wholeuterus, the nonimplantation (N-IM) and implantation (IM) sites werecollected separately for protein extraction. A, A single band (�53 kDa)was detected with monoclonal Odc antibody. B, The protein level ofOdc was quantitated by measuring OD of each band and normalizedwith Gapdh protein level. The Odc protein level at implantation siteswas significantly higher than that at interimplantation sites. Dataare given as mean � SD (*, P � 0.05).

FIG. 4. In situ hybridization of polyamine-related gene expression ond 3–5 of pseudopregnancy, and under delayed implantation and ac-tivation. On d 5 of pseudopregnancy (PD5), except for a low level ofOaz1 expression in the uterine epithelium, there was no detectablesignal for other genes. Compared with delayed implantation, Odc,Oaz1, Srm, Amd1, Sat, Smox, and Paox were strongly expressed afterdelayed implantation was activated by estrogen treatment. Bar,300 �m.



mized mice were treated with both estrogen and ICI 182,780,only a basal level of Oaz1 expression was detected. A strongOaz1 expression was also strongly induced by a cotreatmentof estrogen and progesterone. Similarly, there was no de-tectable signal when the Oaz1 antisense probe was replacedby the Oaz1 sense probe (Fig. 5).

In ovariectomized mice, either estrogen or progesteronehad 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 byOaz (2), we would like to see whether Oaz1 expression wasregulated by Odc expression and polyamine level.

Odc overexpression in cultured endometrial cells was con-firmed by real-time PCR (Fig. 6A). Odc expression in thestromal and epithelial cells was significantly increased afterOdc cDNA transfection. Odc overexpression led to an obvi-ous increase of Oaz1 expression in the stromal and epithelialcells, respectively. However, exogenous putrescine had noeffects on the Oaz1 expression (Fig. 6B).

Regulation of Sat, Paox, and Smox expression by polyaminelevel

Under the catalysis of Sat and Paox, spermidine andspermine could be back-converted into putrescine and sper-midine, respectively (3). Spermine could also be oxidizedinto spermidine directly under the catalysis of Smox (4). Wewould like to examine whether exogenous spermidine or

spermine had any effects on the expression of Sat, Paox, andSmox (Fig. 7). In the cultured stromal cells, Sat expression wassignificantly stimulated by spermidine (Fig. 7A). Becausestromal cells were sensitive for spermine treatment, the ef-fects of spermine on Sat expression were not examined. In thecultured epithelial cells, Sat expression was sharply stimu-lated by either spermidine or spermine. Paox and Smox ex-pression was slightly down-regulated by spermidine, but notby spermine (Fig. 7B).

Effects of DFMO treatment on embryo implantation

Because Odc was the limiting step for polyamine syn-thesis and highly expressed at implantation sites in ourstudy, DFMO was used to see whether Odc was requiredfor embryo implantation (Table 1). When the mice weretreated with 500 mg/kg DFMO twice on d 4 of pregnancy,the pregnant rate was significantly reduced comparedwith the control (54.6 vs. 85%; P � 0.05). After pregnantmice were treated with 500 mg/kg DFMO three times onboth d 3 and 4, the pregnant rate was further reduced to36.4% compared with the control (87.5%). However, ahigher dosage of DFMO (1000 mg/kg) had no furtherinhibitory effect compared with 500 mg/kg DFMO (42.9vs. 36.4%; P 0.05).

Because implantation could not be inhibited completely byDFMO, and Srm was also highly expressed at implantationsites, we assumed that Srm might also be essential for em-bryo implantation. A specific inhibitor for Srm, 4MCHA, wasused to determine whether Srm was required for embryoimplantation (Table 1). When pregnant mice were treatedwith 4MCHA (100 mg/kg), no obvious inhibition was ob-served compared with the control (77.7 vs. 85%; P 0.05).

FIG. 6. Regulation of Oaz1 expression by Odc or putrescine level. A,Confirmation of Odc overexpression in endometrial cells by real-timePCR. B, Effects of Odc overexpression or exogenous putrescine onOaz1 expression. Endometrial cells were transfected with pc-Odc ortreated with 1 mM putrescine at the presence of 1 mM aminoguanidinefor 24 h. The expression level of Oaz1 was normalized by Gapdh. Dataare given as mean � SD (*, P � 0.05).

FIG. 7. Effects of exogenous spermidine or spermine on Sat, Paox,and Smox expression. Endometrial cells were treated with 10 �Mspermidine or spermine at the presence of 1 mM aminoguanidine for24 h. The expression level of each gene was normalized by Gapdh. A,Stromal cells. B, Epithelial cells. Data are given as mean � SD (*, P �0.05).



Even if pregnant mice were treated by both 4MCHA andDFMO, the pregnant rate was 41.2%, similar to that treatedby DFMO alone.

In the mouse with estrous cycles, DFMO administrationcauses the reduction of progesterone level in serum andthe inhibition of angiogenesis in the corpus luteum (16).Therefore, the model of delayed implantation was used todetermine whether ovarian factors were involved in theinhibition of implantation by DFMO. As described previ-ously, pregnant mice were ovariectomized on d 4 andinjected daily with progesterone from d 5–7 to maintaindelayed implantation. The mice were treated with DFMO(500 mg/kg) three times from the afternoon on d 6 to theafternoon on d 7, and delayed implantation was activatedby estrogen on d 7. Embryo implantation was examined24 h after estrogen treatment. Compared with the control(83.3%), the pregnant rate of the DFMO-treated group wassignificantly reduced to 38.9% (P � 0.05), similar to thattreated by DFMO on d 3 and 4 of pregnancy. This indicatedthat DFMO mainly acted on uterine factors to block im-plantation during early pregnancy.

Regulation of Amd1 expression by DFMO

In our study the incomplete inhibition of embryo implan-tation by DFMO injection might suggest the presence of a

compensational mechanism for the loss of Odc activity in theuterus. Implantation sites of DFMO-treated mouse uteriwere checked for the expression of Amd1, Srm, and Sms.According to real-time RT-PCR analysis, Amd1 expressionwas significantly higher compared with the control. Theexpressions of Srm and Sms were not changed after DFMOinjection (Fig. 8).

In vitro culture was also performed to examine further theregulation of Amd1 expression by Odc or polyamine level. Inthe cultured uterine stromal and epithelial cells, Amd1 ex-pression was significantly stimulated by DFMO, whereas theexpressions of Srm and Sms were not changed by DFMO. Odcoverexpression caused the down-regulation of Amd1 andSms expressions, and an increase of Srm expression (Fig. 9).

FIG. 8. Effects of DFMO treatment on the expression of Amd1, Srm,and Sms at implantation sites in the uterus on d 5. After 500 mg/kgDFMO was injected on d 3 and 4, the uteri at implantation sites werecollected from treated or control mice, respectively. The relative ex-pression level of Amd1, Srm, and Sms was quantified by real-timePCR and normalized by Gapdh. Amd1 expression was significantlyhigher after DFMO treatment. Results are given as mean � SD (*, P �0.05).

FIG. 9. Effects of Odc or putrescine on the expression of Amd1, Srm,and Sms. A, Stromal cells. B, Epithelial cells. Endometrial cells weretransfected with pc-Odc, or treated with 5 mM DFMO or 1 mM pu-trescine for 24 h. The expression level of each gene was normalizedby Gapdh. In both stromal and epithelial cells, Amd1 expression wassignificantly up-regulated by DFMO and down-regulated by Odc over-expression. Data are given as mean � SD (*, P � 0.05).

TABLE 1. Influences of DFMO and/or 4MCHA injections on embryo implantation

Groups Time of injection Treatment and dosage (mg/kg) ofeach inhibitor

No. of pregnant mice/No. of total mice (%)

1 d 4 (0800 h) d 4 (1600 h) Control 17/20 (85)DFMO (500) 12/22 (54.6)a

2 d 3 (1600 h) d 4 (0800 h) d 4 (1600 h) Control 21/24 (87.5)DFMO (500) 8/22 (36.4)a

3 d 3 (1600 h) d 4 (0800 h) d 4 (1600 h) Control 21/24 (87.5)DFMO (1000) 6/14 (42.9)a

4 d 3 (1600 h) d 4 (0800 h) d 4 (1600 d) Control 17/20 (85)4MCHA (100) 7/9 (77.7)

5 d 3 (1600 h) d 4 (0800 h) d 4 (1600 h) Control 17/20 (85)DFMO (500) 4MCHA (100) 7/17 (41.2)a

The implantation sites were identified by injection of Chicago blue dye on the morning of d 5.a P � 0.05, � 2 test.



In the epithelial cells, Amd1 and Sms expressions were down-regulated by exogenous putrescine (Fig. 9B).

DiscussionThe specific expression of Odc at implantation sites

In our study Odc expression was significantly higher atimplantation sites than that at interimplantation sites. More-over, Odc expression was dependent upon the presence of anactive blastocyst, suggesting that Odc may play a key roleduring embryo implantation.

DFMO can irreversibly and specifically inactivate Odc (1).We showed that embryo implantation was significantly in-hibited by DFMO either in normal pregnancy or under de-layed implantation, suggesting that DFMO mainly acted onuterine factors in implantation. However, embryo implan-tation was not completely inhibited by DFMO treatment. Thehalf-life of DFMO under ip administration is around 126 min(17). The half-life of DFMO after sc injection should be similarwith the intraperitoneal route. Thus, the relative short half-life might be one of the reasons for the incomplete implan-tation inhibition effect by DFMO after sc injection becausethere is not enough DFMO present in the uterus duringembryo implantation. Fozard et al. (8) reported that the post-implantation development of mouse embryo was blocked byDFMO treatment on d 5–8 during early pregnancy, whereasthere was no obvious development or implantation inhibi-tion effect after DFMO was provided in drinking water on d1–4. However, the results from pharmacological researchproved that after treated in drinking water, DFMO accumu-lation was much more obvious in the organs responsible forreabsorption or metabolism, including intestine, liver, andkidney, than in other tissues (17). Therefore, the efficiency ofDFMO absorption in the uterus might not be effectualenough after treated in drinking water.

The up-regulation of Amd1 expression may be compensatoryafter DFMO treatment

In our study embryo implantation was not completelyinhibited by a high dose of DFMO. Moreover, Odc nullembryos were competent for implantation even if theyfailed to survive from the stage of gastrulation (10). Thesedata may suggest a compensational mechanism for poly-amine biosynthesis. Because Srm expression was specifi-cally enhanced at implantations sites, we assumed that theSrm activity might also be important during implantation.4MCHA, a specific inhibitor for Srm, was proved to de-crease the in vivo activity of Srm effectively and specifically(12). However, 4MCHA alone had no effects on implan-tation. Moreover, 4MCHA had no further inhibitory ef-fects on embryo implantation when pregnant mice weretreated by a combination of DFMO and 4MCHA, suggest-ing that Srm should not be the key factor.

In our study Amd1 expression was significantly up-regu-lated by DFMO treatment, but down-regulated by Odc over-expression in the cultured uterine cells. In the DFMO-treatedmice, Amd1 expression was also up-regulated at implanta-tion sites. Our data indicated that Amd1 expression should bea factor for compensating the reduction of Odc activity. Theup-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 Amdinhibitor (20). The combined use of DFMO and CGP 48664may be required for a complete inhibition of embryoimplantation.

Tight control on uterine polyamines during embryoimplantation

Polyamines cannot only promote cell growth but alsotrigger the death process in many studies (3, 21). Undernormal circumstances polyamine concentrations regulatetheir own biosynthesis and prevent overproduction bydeveloping complex systems consisting of a series of en-zymes and polyamine transporters. However, in abnormalcases a high concentration of exogenous polyamines, es-pecially spermine, leads to cell death (22–24). To ensurecell growth and avoid potential toxic effects, intracellularpolyamine needed to be maintained within a narrowlylimited concentration (25). Although Odc was stronglyexpressed at implantation sites, Oaz1 expression was alsohighly detected in these areas. Oaz can associate with anddirect Odc protein to the proteasome without ubiquitina-tion. The rapid turnover of Odc protein is mediated by Oaz(2). The colocalization of Oaz1 and Odc expression sug-gested that Odc activity may be tightly controlled by Oaz1.

In addition, spermine is highly toxic for cells and canlead to cell death (24). It is important to avoid spermineoverproduction in the uterus. Sat is the rate-limiting en-zyme in polyamine metabolism and can be induced bypolyamines or their analogs in several cell lines (3, 26, 27).In our study Sat was also highly expressed at implantationsites, and was promoted by exogenous spermidine andspermine. Furthermore, spermine could be directly con-verted into spermidine by Smox (4), which was alsostrongly expressed at implantation sites. Our data sug-gested that the strong expression of Sat and Smox at im-plantation sites might relate to the tight control of sperm-ine level at implantation sites.

In conclusion, Odc expression was strongly detected at im-plantation site and dependent on the presence of an activeblastocyst. The coordinate expression of polyamine-relatedgenes during the peri-implantation period was important tomaintain the homeostasis of uterine polyamines for facilitatingendometrial cell proliferation and establishing a suitable envi-ronment for embryo implantation.

Acknowledgments

Received October 17, 2007. Accepted January 9, 2008.Address all correspondence and requests for reprints to: Zeng-Ming

Yang, College of Life Science, Xiamen University, Xiamen 361005, China.E-mail: [email protected].

This work was supported by the National Basic Research Program ofChina (2006CB504005 and 2006CB944006), and Chinese National Nat-ural Science Foundation Grants 30330060, 30570198, and 30770244.

Disclosure Statement: The authors have nothing to disclose.

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