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Endocrinology Vol. 144, No. 4 1464-1473
Copyright © 2003 by The Endocrine Society

ARTICLE

Enhanced Expression of Uterine Stathmin during the Process of Implantation and Decidualization in Rats

Kazuhiro Tamura, Takahiko Hara, Mikihiro Yoshie, Shinya Irie, Andre Sobel and Hiroshi Kogo

Department of Pharmacology (K.T., M.Y., S.I., H.K.), Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji, Tokyo 192-0392, Japan; Tokyo Metropolitan Institute of Medical Science (T.H.), Honkomagome, Bunkyo-ku, Tokyo 113-0032, Japan; and Institut National de la Santé et de la Recherche Médicale (A.S.), Unite-440, Institut du Fer a Moulin, Paris 75005, France

Address all correspondence and requests for reprints to: Dr. Kazuhiro Tamura, Department of Pharmacology, Tokyo University of Pharmacy and Life Science, Horinouchi 1432-1, Hachioji, Tokyo, Japan. E-mail: hiro{at}ps.toyaku.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We used the library subtraction technique to identify genes specifically expressed in the rat uterus during early pregnancy. One such gene was that for stathmin, a factor that is associated with tubulin binding and the destabilization of microtubules. Stathmin was expressed at higher levels in implantation sites than in interimplantation sites on d 6 and 7 of pregnancy; the levels on d 6 and 7 were higher in implantation sites than in the entire uterus on d 3–5 of pregnancy or in nonpregnant uteri. Intense expression of stathmin mRNA was primarily limited to the subluminal stromal cells at the implantation site. Expression was also detected in the decidual zones and was accentuated during the period of decidualization (d 7–12). In the delayed implantation pregnant rat model, uterine stathmin expression was low, but increased after implantation induced by administration of 17ß-estradiol to the progesterone-primed animal. Further, decidualization in the pseudopregnant rat, induced by intrauterine infusion of oil, enhanced stathmin expression. Stathmin expression clearly increases in the uterus when stimulated by embryo implantation and decidualization and may play a role in the early stages of pregnancy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
COORDINATION of a developing conceptus and a receptive uterine epithelium is necessary for successful implantation (1, 2, 3, 4, 5). The uterus undergoes dramatic morphological and molecular changes during the preimplantation period, which are controlled by the ovarian steroid hormones estradiol (E2) and progesterone (P4) (1, 2, 3, 4, 5, 6, 7). In rats, a nidatory surge of estrogen on the afternoon of d 5 of pregnancy is essential for initiating implantation later that evening or in the early morning of d 6 (1, 5). Once this estrogen surge occurs, blastocysts are activated, and the receptive uterus allows them to attach to its epithelial layers (3, 7). Upon stimulation first by E2 and then by P4, uterine epithelial cells differentiate and undergo apoptosis, whereas stromal cells underneath the implantation sites proliferate and transform into decidual cells (6, 7). Increased vascular permeability in the uterine epithelium is necessary for and is the first obvious indication of the implantation process (1, 6). Thus, extravasation of an intravascularly administered macromolecular dye on the morning of d 6 indicates the location of the implantation sites (1, 6).

Because of the complex tissue reorganizations required for implantation, it is assumed that a unique set of genes involved in preparing the uterus for accepting invading blastocysts is induced during the early stages of pregnancy (Ref. 5 and references therein). Gene targeting has proven useful for understanding the genes essential for the implantation process in the mouse (reviewed in Ref. 5). For example, female mice deficient in leukemia inhibitory factor (LIF) are infertile, but administration of exogenous LIF restores fertility and can even replace the need for the nidatory estrogen surge (8). The genes encoding IGF-I and Hoxa-10 are expressed extensively at the implantation site, and mice deficient in these genes have implantation and decidualization failures (reviewed in Ref. 5). Recently, Yoshioka et al. (9) and Reese et al. (10), using gene chip arrays, reported on global gene expression profiles during implantation in mice. Although normal blastocyst implantation and decidualization are still poorly understood, the analysis of gene products that are expressed selectively and abundantly in the implantation site is one way to find the factors that participate in the process.

Stathmin is a member of a highly conserved cytosolic protein family that has been studied in relation to cell growth and differentiation (11). This 19-kDa protein has recently been proposed to function solely by sequestering tubulin and not by changing the structures of microtubules (12). Phosphorylation of stathmin is triggered by a large variety of regulatory agents, including hormones and growth factors whose signaling involves kinases (13). Stathmin has been suggested to participate in both oocyte maturation and preimplantation embryonic development (14, 15) because it is expressed in inner cell mass cells of expanded and hatched blastocysts. In the present study we systematically isolated genes that are expressed in the rat uterus on the day of implantation (d 6) and further characterized their expression patterns to determine whether they are associated with implantation and decidualization. We show in the present study increased expression of stathmin in the uterus at the time of implantation and decidualization and therefore conclude that stathmin should be considered an implantation-associated gene in the rat.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and protocols
The animal care and surgery protocols used in these studies were reviewed and approved by the institutional animal care committees of Tokyo University of Pharmacy and Life Science in compliance with institutional guidelines for experimental animal care. Eight-week-old female rats of the Wistar-Imamichi strain (Imamichi Institute for Animal Reproduction, Ibaraki, Japan) were mated at proestrus with 10-wk-old males of the same strain. Day 1 of pregnancy was determined by the presence of a vaginal plug or sperm. To construct two uterine cDNA libraries from d 5 and 6 of pregnancy, uteri were excised on the morning (0900–0930 h) of each day and flushed with sterilized PBS to remove blastocysts. Injection via the tail vein of 0.2 ml of a 1% solution of Chicago Sky Blue dye in saline 5 min before killing was used to identify implantation sites, which appear as blue bands around the uterus. The tissue between the blue bands was defined as interimplantation sites. The implantation sites from three rats on d 6 were separated from the interimplantation sites by careful dissection, and the tissues were pooled for RNA extraction. Complete uteri collected on d 5 from two rats were pooled for RNA extraction.

Pseudopregnancy was induced by mating females with vasectomized males of the same strain. On d 5 of pseudopregnancy, sesame oil (100 µl) was infused into one uterine horn to induce decidualization. The noninfused horn was used as a control. Delayed implantation was induced by hypophysectomy on d 3 as previously described (16, 17), and the pregnancy was maintained by daily sc injection of P4 (3 mg/rat) dissolved in sesame oil. Six days after the surgery, each rat was given 0.5 µg E2 sc to initiate implantation, and the uterus was isolated 6 or 24 h later. To determine the effects of E2 and P4 on stathmin levels in the rat uterus, animals were ovariectomized 14 d before a single administration of E2 (0.5 µg, sc) and/or P4 (3 mg, sc). The uteri were collected 24 h after the injection of the steroids.

Library subtraction and sequencing of cDNAs
cDNA library construction and library subtraction were performed as previously described by Hara et al. (18). mRNA from d 5 or 6 uteri was used to generate the driver cDNA and the tracer cDNA, respectively. Each cDNA library consisted of size-selected (>1 kb) cDNA fragments in pME18S vectors. One hundred micrograms of purified plasmid DNA from the d 5 library were digested with EcoRI, NotI, and ScaI before biotinylation using Photoprobe biotin (Vector Laboratories. Inc., Burlingame, CA). Five micrograms of plasmid DNA from the d 6 library (implantation sites) was digested with EcoRI and NotI, followed by hybridization with the biotinylated driver DNA in hybridization buffer [0.75 M NaCl, 5 mM EDTA, 25 mM HEPES buffer (pH 7.5), and 0.1% sodium dodecyl sulfate] containing 0.25 mg/ml Escherichia coli tRNA at 68 C for 20 h. Biotinylated DNA was removed by incubation with streptavidin, followed by four extractions with phenol/chloroform/isoamyl-alcohol (25:24:1). The subtracted DNA was further hybridized for 2 h with the same biotinylated driver DNA. After removing biotinylated double-stranded DNAs, residual DNA was ligated into a pCRII vector (Invitrogen, Groningen, The Netherlands), digested with EcoRI and NotI, and used to transform electrocompetent E. coli (ElectroMAX DH10B, Life Technologies, Inc., Grand Island, NY) for generation of the subtracted library. Randomly isolated clones were further screened by direct sequencing and Southern hybridization using both cDNA libraries and/or Northern hybridization using rat uterine RNA preparations from d 5 and 6 of pregnancy. Each clone was sequenced and analyzed using the BLAST program to search public DNA databases.

Verification of implantation-selective expression by Southern blotting and RT-PCR
Each cDNA library (1 µg) from d 5 uterus or d 6 implantation sites was digested with EcoRI and NotI, loaded on a 1.2% agarose gel, and transferred to a nylon membrane. The cDNA fragments obtained by library subtraction were labeled with digoxigenin (DIG) using a DIG-DNA labeling kit (Roche, Mannheim, Germany). For Southern hybridization with cloned individual cDNA fragments, hybridization was performed for 17 h at 47 C with high sodium dodecyl sulfate buffer containing the DIG-labeled cDNA probe (10 ng/ml) as previously described (19).

Northern blot analysis
Uterine tissues were homogenized in 10 vol 4 M guanidine isothiocyanate using a Polytron (Brinkmann Instruments, Inc., West Orange, NY). Extraction of polyadenylated [poly(A)+] RNA from total RNA was performed using the Oligotex-dT30<super>mRNA purification kit (TaKaRa, Siga, Japan) according to the manufacturer’s instructions. Ten to 20 µg total RNA or 100 ng poly(A)+ RNA were separated on 1.5% agarose gels containing 2.4% formaldehyde and transferred to positively charged nylon membranes (Roche). For preparation of cDNA probes, a pCRII vector carrying rat stathmin cDNA was digested with EcoRI, NotI, and PstI, and the digested cDNA (~300 bp) was subcloned into the plasmid vector pBluescript. After fixation of the RNA on the membranes, prehybridization was carried out for 4 h in high sodium dodecyl sulfate buffer containing the DIG-labeled cDNA. The blot was reacted with alkaline phosphatase-labeled anti-DIG antibody (Roche) and a chemiluminescent substrate (CDP-star) according to the manufacturer’s instructions. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) or ß-actin was used as an internal control. The bands on the Kodak scientific imaging film (X-OMAT XB-1, Eastman Kodak Co., Rochester, NY) were analyzed using NIH Image, and each value was normalized against that of the G3PDH band in the corresponding lane.

In situ hybridization
Uteri from rats on d 3–7 were directly fixed for 8 h in 0.1 M phosphate buffer (pH 7.45, room temperature) containing 4% paraformaldehyde. Paraffin-embedded sections (6 µm) were prepared for in situ hybridization and immunocytochemistry. DIG-labeled antisense and sense riboprobes were prepared using the stathmin cDNA fragment (nucleotide 252–653) in the pBluescript vector and an RNA transcription kit (Toyobo, Tokyo, Japan) using the manufacturer’s instructions. As described previously (20), hybridization was performed in a humidified chamber at 42 C for 18 h, and bound probes were visualized using an alkaline phosphatase-conjugated anti-DIG antibody with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Promega Corp., Madison, WI) as substrates.

Immunoblot and immunohistochemical analyses
Uterine tissues were homogenized with a Polytron in ice-cold lysis buffer [50 mM Tris-HCl buffer (pH 7.5) containing 0.15 M NaCl, 10 mM EDTA, 0.1% Tween-20, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 0.1 mM phenylmethylsulfonylfluoride, and 0.1% (vol/vol) ß-mercaptoethanol]. The homogenate was centrifuged twice at 13,000 x g at 4 C to remove the insoluble protein pellets. The crude supernatants were used for Western blotting analyses. The protein concentration was determined using the Bio-Rad Laboratories, Inc. protein dye reagent (Richmond, CA), and 15 µg of the protein samples were subjected to electrophoresis on 7.5–15% gradient SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (Immobilon, Millipore Corp., Bedford, MA) using a semidry transfer system (Horiseblot, ATTO Co., Tokyo, Japan). After blocking overnight with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20 (at 4 C), membranes were incubated for 2 h with an antistathmin antiserum (1:1000) (21). Membranes were incubated for 1 h with goat antirabbit IgG conjugated to horseradish peroxidase (2 µg/ml) and after extensive washing were visualized using enhanced chemiluminescence (Renaissance, NEN Life Science Products, Boston, MA). Each lysate was subjected to analysis at least twice. The bands on the blots were quantified using NIH Image. All incubations except blocking were performed at room temperature. For the immunohistochemical analysis, paraffin sections of 4% paraformaldehyde-fixed uterus were deparaffinized and rehydrated. After treatment with PBS containing 0.5% Tween 20 and 0.2% gelatin, the sections were incubated with 3% H2O2 for 30 min to inactivate endogenous peroxidases. The sections were exposed for 2 h to 10% normal goat serum. The rabbit primary antistathmin antiserum was diluted (1:500) in PBS containing 2% normal goat serum and incubated with the sections for 2 h at room temperature. After washing and incubation with the biotinylated secondary antibody, a peroxidase-conjugated streptavidin complex was applied. Signals were visualized with 3,3'-diaminobenzidine (Kirkegaard \|[amp ]\| Perry Laboratories, Gaithersburg, MD), and samples were counterstained with methyl green. Antibody diluent alone was used as a negative control.

Statistical analysis
The results of densitometric analyses were obtained from two or three independent experiments. Data are expressed as the mean ± SEM, and statistical significance (P < 0.05) was determined using t test and ANOVA. The results of Northern and Western blots that are shown represent single experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of stathmin as a gene highly expressed during implantation and verification of its implantation-selective expression
In the present study subtraction cloning was carried out using implantation sites from d 6 pregnant rats and the entire uterus from d 5 pregnant rats to identify genes that are highly expressed at the time of implantation. In the subtracted uterine cDNA library, 226 independent clones were sequenced. Among them, 9 genes (7.0%) were unknown whereas 119 clones were known genes, including several cDNAs that have previously been reported to be expressed in the uterus during the periimplantation period (22). For example, cathepsin, whose expression is known to be associated with embryo development and uterine decidualization (23), was one of the clones identified in the subtracted library (Table 1Go). Among the clones sequenced, mRNAs for collagen I type I, {gamma}-enteric smooth muscle actin isoform, collagen {alpha}1 type III, and lipoprotein lipase were found in the subtracted library with a high frequency. cDNAs encoding some constituents of the extracellular matrix, such as collagen {alpha}1 type 3, pro-{alpha}-2(I) collagen, and dermatan sulfate proteoglycan as well as collagen {alpha}1 type 1, were isolated as implantation-associated genes. To analyze the expression of known genes whose physiological functions in the uterus have not been reported, the individual cDNA inserts in the subtracted library were used as probes for Southern blot analyses of the cDNA libraries (d 5 and 6). We observed increased expression of mRNAs for cathepsin S, cyclophilin, ferritin-H subunit, nonhistone chromosomal high mobility group 1 protein, and stathmin. Of these, stathmin exhibited the biggest change, showing a distinct signal in the cDNA from the d 6 uterine library, in contrast to its faint band on d 5 (Fig. 1AGo).


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  		Table 1. Summary of known and homologous genes isolated from implantation site-derived cDNA library

 


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  		Figure 1. Uterine stathmin expression in the periimplantation period in rats. A, Southern blot analysis of stathmin cDNA in two cDNA libraries. Two cDNA libraries were constructed from poly(A)+ RNAs of the entire uterus on d 5 of pregnancy and of the implantation sites on d 6. Each library (1 µg) was digested with EcoRI and NotI and analyzed by Southern blotting as described in Materials and Methods. B and C, Representative Northern blot analysis of uterine stathmin mRNA expression during early pregnancy. The blue bands indicating the implantation sites on d 6 and 7 of pregnancy were removed and used for RNA extraction. Numbers in the figure indicate the day of pregnancy. 6-IS, Implantation site on d 6; 6*, interimplantation site on d 6. D, Densitometric analysis of stathmin mRNA levels detected by three independent experiments (n = 3 on each day) including data in C. Quantitation of stathmin mRNA levels was conducted using NIH Image, and the values obtained were normalized against G3PDH mRNA levels. Data include the mRNA levels in nonpregnant animals (n = 2) over the estrous cycle (D, diestrus; E, estrus). The levels are expressed relative to the density of the band on d 5. *, P < 0.05; **, P < 0.01.

 
Stathmin mRNA is abundantly expressed at implantation sites
To evaluate the pattern of expression during the periimplantation period, the levels of stathmin mRNA were analyzed by Northern hybridization on gestational d 3–7. Expression of the 1.1-kb stathmin transcript was detected on all 5 d. However, the expression level was low in uteri until d 5 and markedly increased at implantation sites on d 6 and 7 (6-IS and 7-IS in Fig. 1BGo). Furthermore, on d 6 there was a distinct difference in the intensity of the stathmin mRNA signals in preparations from implantation sites compared with interimplantation sites (6IS vs. 6* in Fig. 1CGo). Densitometric analyses are shown in Fig. 1DGo. Uterine RNA from nonpregnant rats at various stages of the estrous cycle was compared with that from pregnant animals. Only faint signals were found in uteri from nonpregnant rats and were similar to those found on d 3 of pregnancy.

In situ hybridization of stathmin mRNA in the periimplantation rat uterus
Figure 2Go shows the temporal and cell-specific localization of stathmin expression using in situ hybridization. On d 3 and 4, signals were detected in glandular epithelial cells and endometrial cells close to the luminal epithelium (Fig. 2Go, A and B). On d 5 just before implantation, an intense signal was detected in uterine stromal cells underlying epithelial cells (Fig. 2Go, C and D). mRNA was also detected in blastocysts that were unattached in the uterine lumen. At the site of implantation on d 6 (Fig. 2Go, E and F), stathmin mRNA was observed primarily in subluminal stromal and smooth muscle cells. When longitudinal sections of uteri were examined, expression of stathmin mRNA was never limited to a specific region of endometrial cells beneath the luminal epithelium, but higher levels were always found in the area surrounding the implanting blastocyst. Signals were found in both mesometrial and antimesometrial areas as well as in the decidual zone on d 7 (Fig. 2GGo). The specificity of hybridization was confirmed by the lack of signals in tissue sections hybridized with a stathmin sense probe (Fig. 2HGo).



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  		Figure 2. Localization of uterine stathmin mRNA during the early stage of pregnancy in rats (A, d 3; B, d 4; C and D, d 5; E and F, H, d 6; G, d 7). Original magnification, x63 (except C, x315; and E, x3.2). The section in H was hybridized with a sense cRNA probe. ge, Glandular epithelium; le, luminal epithelium; s, stroma; bl, blastocyst; am, antimesometrial; m, mesometrial; em, embryo; pdz, primary decidual zone; sdz, secondary decidual zone; IS, implantation sites.

 
Expression of stathmin protein in the rat uterus
We next analyzed the localization of stathmin protein during the periimplantation period. On d 3 and 4, staining of the protein was detected in the glandular epithelial and stromal cells underlying the luminal epithelium (Fig. 3Go, A and B). On d 5 the area of staining had expanded in the endometrium compared with that on d 4 (Fig. 3DGo). At implantation sites on d 6, intense staining was detected in uterine stromal cells, especially those underlying the luminal epithelium (Fig. 3FGo). The intensity of staining of the stromal cells lying just below the luminal epithelium on d 6 was stronger than that found on d 4 or 5 (Fig. 3FGo vs. Fig. 3Go, B or D). Stathmin protein was also found in decidual cells (Fig. 3GGo). The lack of staining in control slides confirmed the specificity of the staining pattern (Fig. 3HGo).



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  		Figure 3. Immunostaining of uterine stathmin during early pregnancy in rats (A, d 3; B, d 4; C and D, d 5; E, F, and H, d 6; G, d 7). Original magnification, x63. H, Negative control (d 6), only secondary antibody was added in the procedure. ge, Glandular epithelium; le, luminal epithelium; s, stroma; am, antimesometrial; m, mesometrial; em, embryo; pdz, primary decidual zone; sdz, secondary decidual zone; IS, implantation sites.

 
To further examine the changing expression of stathmin protein during the periimplantation period, protein extracts were analyzed by Western blotting. The stathmin protein was identified as a 19-kDa single band in uterine lysates (Fig. 4AGo). The levels at the implantation sites on d 6 and 7 were prominently higher than those on d 3–5. Densitometric analysis (Fig. 4CGo) indicated a significant increase in stathmin levels on d 6 (1.32 ± 0.09) and d 7 (1.60 ± 0.11) compared with that on d 5 (1.00 ± 0.07). Further, levels were higher in implantation sites recovered on d 6 or 7 than in interimplantation sites on the same days (data not shown). To determine the fate of this protein as pregnancy progressed, levels of stathmin were analyzed in uteri between d 8 and 20 (Fig. 4Go, B and C). The expression increased markedly on d 6 and remained elevated through d 12 (P < 0.05 vs. d 5), but decreased on d 14–16. However, the levels gradually increased again between d 16 and 20.



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  		Figure 4. Western blot analysis of uterine stathmin expression during pregnancy in rats. A and B, Numbers at the top of the figures indicate the day of pregnancy. 6-IS and 7-IS, Implantation sites on d 6 and 7; 6*, interimplantation site on d 6. A, Each lane shows the data from an individual animal (n = 2). C, Densitometric analysis of stathmin levels detected by two independent experiments (n = 4), including the data in B. Quantitation of stathmin protein contents was conducted using NIH Image. The levels are expressed relative to the density of the band on d 5. *, P < 0.05; **, P < 0.01.

 
Effects of ovarian hormones on stathmin expression in the delayed implantation and ovariectomized nonpregnant rats
To examine whether stathmin expression is associated with the induction of implantation and is regulated by ovarian hormones, especially the nidatory surge in estrogen, Northern blot analyses were carried out using the P4-treated delayed implantation model (Fig. 5Go). Low levels of the stathmin transcript were found in the uteri of rats treated only with P4, similar to those found in hypophysectomized rats (data not shown). After the termination of delayed implantation by an injection of E2, only faint expression of stathmin mRNA was seen within 6 h, but the expression was pronounced after 24 h. Similar patterns in the levels of stathmin protein were observed in the delayed implantation model using Western blot (data not shown). However, a significant influence of ovarian steroids on stathmin expression was not observed in ovariectomized rats under the same doses as those used in delayed implantation model (Fig. 6Go).



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  		Figure 5. Effects of P4 and E2 on the expression of stathmin mRNA in the delayed implantation model. A, P4 (3 mg) was administered sc for 6 d to hypophysectomized rats with or without injection of E2 (0.5 µg) on the final day (d 8 of pregnancy). The uterus was removed on the morning of d 6 of normal pregnancy, 24 h after the final injection of P4 without E2 (Hypox + P4), or 6 and 24 h after the final injection of P4 and E2 (Hypox + P4/E2). Each lane shows representative data obtained from three individual animals. B, Quantitation of stathmin mRNA levels (n = 6) was conducted using NIH Image, and the values obtained were normalized against G3PDH mRNA levels. The levels are expressed as a ratio to the density of the band on Hypox + P4. *, P < 0.05; ***, P < 0.001.

 


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  		Figure 6. Effects of P4 and E2 on the expression of stathmin protein in ovariectomized rats. Fourteen days after ovariectomy, rats were administered E2 (0.5 µg) alone or in combination with P4 (3 mg). The uterus was removed 24 h after the injection of steroids. After Western blotting, the levels of stathmin were determined with NIH Image. Each column shows the data obtained from four individual animals.

 
Effect of artificial decidual stimulation on stathmin expression in pseudopregnant uterus
The levels of stathmin expression in uteri of pseudopregnant rats, which lack both implantation sites and decidualization, were analyzed on d 5 and 6 (Fig. 7AGo). The same levels of stathmin protein were detected in uteri from d 5 pregnant or pseudopregnant animals. On d 6, however, stathmin did not increase in pseudopregnant uteri and were significantly below the level found in pregnant animals on d 6. In fact, on d 6 the stathmin levels in pseudopregnant animals had significantly decreased from d 5 (d 6, 0.53 ± 0.002; d 5, 0.93 ± 0.070; P < 0.05; Fig. 7CGo, left panel). Decidualization is initiated immediately in the endometrial cells adjacent to the implantation sites during blastocyst attachment and invasion (7). To examine the relationship between decidualization and stathmin expression, an artificial stimulus was given to the pseudopregnant rats (Fig. 7BGo). After injection of sesame oil into the uterine lumen, stathmin levels started to increase within 24 h (P < 0.01 vs. 0 h, Fig. 7CGo, right panel), peaked at 48 h, and continued to be expressed for at least 120 h. Thus, there were increases in stathmin expression during the development of deciduoma under conditions that lacked blastocysts, in contrast to the time-dependent decreases in the levels in nontreated intact uteri.



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  		Figure 7. Expression of uterine stathmin on d 5 and 6 of normal pregnancy and pseudopregnancy, and effect of artificial decidualization on stathmin expression in the pseudopregnant rats. A, The protein levels of stathmin in the uteri on d 5 and 6 of pseudopregnancy were determined by Western blot analysis. Each lane shows an individual animal (n = 5). B, Uterine stathmin levels were measured 0–120 h after sesame oil infusion into one uterine horn between 1300 and 1330 h on d 5 of pseudopregnancy [oil (+)]. As a control, the intact horn was subjected to the same analysis [oil (-)]. C, Quantitation of stathmin protein was conducted using NIH Image. The relative levels from two independent experiments (n = 4–6) are expressed relative to the density of the band on d 5 of normal pregnancy (left panel) or in oil (-) at 0 h (right panel). *, P < 0.05; **, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study was undertaken in an attempt to identify genes in the rat that are associated with implantation, and therefore may be related to the establishment of pregnancy. Based on the results obtained by Southern blot analyses, attention was focused on the profile of stathmin expression in the uterus during the periimplantation period. The uterus undergoes dramatic developmental changes during early pregnancy (1, 2, 3, 4, 5, 6, 7). With the increase in progestin that follows the formation of the ovarian corpora lutea, the endometrial luminal epithelium ceases to proliferate and undergoes differentiation in preparation for embryo implantation. After the cell to cell reactions associated with the attachment of blastocysts to epithelial cells, the underlying stromal cells undergo proliferation and differentiation into decidual cells (decidualization; Refs. 1, 2, 3, 4, 5, 6, 7). Recent studies using mice lacking estrogen receptor {alpha} (ER{alpha}) (24) have emphasized that implantation, but not decidualization, requires the action of estrogen. In contrast, the action of the P4 receptor is essential for both implantation (1, 2, 3, 4) and decidualization (25). The latter study established that increased expression of the P4 receptor in the uterus could be accomplished by the action of either ER{alpha} or ERß as well as by a mechanical stimulus in the presence of P4.

The results of the present study reveal that stathmin expression, as evidenced by increases in its mRNA and protein, is a characteristic of embryo implantation in the rat. Uterine stathmin expression does not increase in the delayed implantation model, which has intraluminal blastocysts and is exposed to P4, but not E2. However, the increases in expression found in pregnant animals were mimicked in the delayed model after providing an estrogen stimulus to induce implantation. During normal pregnancy, increased expression of stathmin was seen between d 5 and 6, particularly in implantation sites. In the delayed implantation model, increased expression was found 24 h after exposure to E2, which would be equivalent to d 6 of pregnancy. Therefore, the hormonal environment associated with implantation, i.e. estrogen acting on a P4-primed uterus (1, 2, 3, 4, 5, 6), is necessary to cause increased expression of stathmin. However, the hormones are not sufficient, because the interimplantation sites, which are exposed to the same hormonal milieu as the implantation sites, express significantly less stathmin. Furthermore, the uteri of pseudopregnant rats, exposed only to the action of P4, actually showed decreased, rather than increased, stathmin expression between d 5 and 6. However, intraluminal instillation of sesame oil, an artificial stimulus to induce decidualization, increased expression of the stathmin gene in the pseudopregnant rat. Therefore, the important physiological changes in the endometrium that regulate the expression of stathmin appear to be those that signal decidualization. Several gene products could be involved in this signaling, including IGF-I, cyclooxygenase-2, LIF, HOX-10, HOX-11, or the IL-11 receptor (reviewed in Ref. 5), all of which are essential for implantation in the mouse. Whether increased stathmin is a cause or an effect of implantation/decidualization, and whether it has any specific effects on these processes remain to be determined. The function of stathmin in cell division and differentiation is unresolved, but is generally considered to involve microtubule stabilization (12, 13). Recent studies have been interpreted to show that most, if not all, of the effects of stathmin can be accounted for by its sequestering of tubulin, which controls the microtubule dynamics of the cell cycle (12). Phosphorylation of 4 serines (serines 16, 25, 38, and 63) in stathmin inactivates its microtubule-destabilizing activity and is necessary for progression through the cell cycle (12). This phosphorylation appears to be controlled by both cell cycle- and cell membrane-regulated kinases (Ref. 26 and references therein). However, during hepatectomic regeneration, the levels of stathmin expression increase dramatically without detectable changes in its phosphorylation state (27). The morphological and physiological changes that occur during pregnancy would seem to offer several possibilities for altering the phosphorylation pattern of stathmin during pregnancy.

The cell cycle is controlled by the interaction of cyclins, cyclin-dependent kinases (cdk), and cyclin-dependent kinase inhibitors (see Refs. 28 and 29 for recent discussion). The G1 cyclins (D type) are associated with cdk4 or cdk6 and are involved with the entry of cells into the S (DNA synthesis) phase of the cycle. The retinoblastoma proteins negatively regulate the G1 cyclins and are inactivated by phosphorylation through the action of the cyclin/cdk complex. Increased expression of cyclin D3 has been associated with decidualization at implantation sites in mice and interpreted as indicating increased cell proliferation of the decidualizing stromal cells (30, 31). This increase in proliferation and particularly the inhibition of cyclin B/cdk1 for the production of polyploid decidual cells might involve the action of stathmin. However, there is currently no evidence to support this view. The localization of cyclin D3 in the uterus resembles that of stathmin in rats, but there are some differences. Cyclin D3 is primarily expressed in decidualizing stroma, but cyclin D3 signals have also been found in epithelial cells, whereas we did not detect distinct signals for stathmin in epithelial cells during the periimplantation period.

Stathmin appears to be unnecessary for the establishment or maintenance of pregnancy, because female mice deficient in the stathmin gene are not infertile nor do they have any major disorders in the nervous system, which normally expresses stathmin (32, 33). However, no detailed examination of reproductive function in stathmin-deficient animals has been made. Stathmin is a member of a phosphoprotein family that includes the developmentally regulated neuronal protein SCG10, rat homolog of XB3 (RB3), and SCG10-like protein (SCLIP); Ref. (34); thus, other members of the gene family might substitute for it in stathmin-deficient animals.

In conclusion, our data suggest that stathmin is an implantation- and decidualization-associated gene that may be involved in the proliferation and differentiation of endometrial stromal cells during early pregnancy. Further studies will be required to determine whether the stathmin expression induced by implantation of embryos is controlled by the same signaling system as that occurring after artificially induced decidualization.


   Acknowledgments
 
The authors are grateful to D. C. Johnson (University of Kansas Medical Center) for his critical reading of the manuscript.


   Footnotes
 
Abbreviations: cdk, Cyclin-dependent kinase; DIG, digoxigenin; E2, estradiol; ER, estrogen receptor; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; LIF, leukemia inhibitory factor; P4, progesterone; poly(A)+, polyadenylated.

Received August 12, 2002.

Accepted for publication December 17, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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