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Reproduction (2007) 133 1213-1221
DOI: 10.1530/REP-07-0085
Copyright © 2007 Society for Reproduction and Fertility
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RESEARCH

The conceptus increases secreted phosphoprotein 1 gene expression in the mouse uterus during the progression of decidualization mainly due to its effects on uterine natural killer cells

Jennifer L Herington and Brent M Bany

Department of Physiology, Southern Illinois University School of Medicine, Carbondale, Illinois 62901, USA

Correspondence should be addressed to B M Bany; Email: bbany{at}siumed.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Within the mouse endometrium, secreted phosphoprotein 1 (SPP1) gene expression is mainly expressed in the luminal epithelium and some macrophages around the onset of implantation. However, during the progression of decidualization, it is expressed mainly in the mesometrial decidua. To date, the precise cell types responsible for the expression in the mesometrial decidua has not been absolutely identified. The goal of the present study was to assess the expression of SPP1 in uteri of pregnant mice (decidua) during the progression of decidualization and compared it with those undergoing artificially induced decidualization (deciduoma). Significantly (P<0.05) greater steady-state levels of SPP1 mRNA were seen in the decidua when compared with deciduoma. Further, in the decidua, the majority of the SPP1 protein was localized within a subpopulation of granulated uterine natural killer (uNK) cells but not co-localized to their granules. However, in addition to being localized to uNK cells, SPP1 protein was also detected in another cell type(s) that were not epidermal growth factor-like containing mucin-like hormone receptor-like sequence 1 protein-positive immune cells that are known to be present in the uterus at this time. Finally, decidual SPP1 expression dramatically decreased in uteri of interleukin-15-deficient mice that lack uNK cells. In conclusion, SPP1 expression is greater in the mouse decidua when compared with the deciduoma after the onset of implantation during the progression of decidualization. Finally, uNK cells were found to be the major source of SPP1 in the pregnant uterus during decidualization. SPP1 might play a key role in uNK killer cell functions in the uterus during decidualization.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
In most mammals, implantation of the conceptus begins with the attachment of the embryo to the uterine wall and ends in the formation of the definitive placenta. One of the first major processes that begins to occur in the rodent uterus after the onset of implantation is the proliferation then differentiation of the endometrial fibroblast-like cells into large polyploid decidual cells (Das & Martin 1978, Lejeune et al. 1982). This process, called decidualization, results in the formation of tissue that is referred to as the decidua and occurs in response to an implantation stimulus provided by the implanting blastocyst in rodents. However, due to an observation first reported almost a century ago in the guinea pig (Loeb 1908), then in several other species (Krehbiel 1937), molecular signals from the conceptus appear not to be required for decidualization to occur. This is because the uterus can undergo decidualization in response to an artificial stimulus such as an intra-luminal injection of sesame oil (Finn & Martin 1972) or transfer of blastocyst-sized agarose beads (Sakoff & Murdoch 1994) into ovariectomized hormonally sensitized or pseudo-pregnant animals (Finn & Martin 1974). In order to discern the tissue that forms in response to artificial stimuli from the one that forms in response to an implanting blastocyst, we refer to it as a deciduoma (Krehbiel 1937).

Secreted phosphoprotein 1 gene (Spp1, also referred to as osteopontin) encodes a 44 kDa protein and is expressed in several tissue types. Spp1 expression is found in many types of cells and might play physiological and pathological roles (Okamoto 2007). Studies examining the role of Spp1 in early mouse development revealed that it is expressed in the uterus during pregnancy (Nomura et al. 1988, Waterhouse et al. 1992). In these studies it was speculated that Spp1 expression was localized to immune cells at the onset of implantation. This speculation was confirmed in a recent study which shows that Spp1 expression occurs in macrophages in the mouse uterus at the onset of implantation (White et al. 2006). After the onset of implantation, during the progression of decidualization, Spp1 expression is found in the mesometrial decidua (Nomura et al. 1988, Waterhouse et al. 1992). However, to our knowledge, work providing the precise identity of the cells expressing Spp1 has not been reported. Therefore, the goal of the present study was to examine Spp1 expression during the progression of decidualization in the mouse. Further, we determined whether the conceptus possibly influences uterine Spp1 expression during the progression of decidualization by comparing its expression in pregnant uteri (conceptus present) with those undergoing artificially induced decidualization (conceptus absent).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Animals
All procedures involving mice were approved by the Southern Illinois University Institutional Animal Care and Use Committee. Unless otherwise noted, experiments were carried out using 12- to 16-week-old CD1 mice (Charles River Breeding Laboratories, Wilmington, MA, USA). In some cases, mice with a targeted deletion of the Il15 gene (Il15–/–; C57BL/6-Il15tm1Imx) plus their wild-type (Il15+/+; C57BL/6-Tac) controls (National Institute of Allergy and Infectious Diseases Emerging Models Program, Taconic Farms, Germantown, NY, USA) were used. Females were placed with fertile males and the morning a vaginal plug was detected was considered to be day 0.5 of pregnancy. Mice were killed at 0900 h on days 6.5, 7.5, or 8.5 of pregnancy which approximately corresponds to days 2, 3, and 4 after the onset of decidualization respectively (Fig. 1Go). Segments of uteri containing implanting conceptuses, implantation sites (IS), were dissected and then used for either histological or RT-real-time-PCR experiments. For samples used in the latter, the conceptuses were carefully dissected out of the IS tissues as described elsewhere (Nagy et al. 2003). To generate artificially induced deciduomas, ovariectomized CD1 mice were treated exactly as described previously (Bany & Cross 2006). Briefly, after mice were allowed a 1-week recovery after ovariectomy, they were injected subcutaneously with a regimen of estradiol-17ß (E2) and/or progesterone (Sigma) at 0900 h as outlined in Fig. 1Go. This regimen serves to adequately sensitize the uterus for an artificial deciduogenic stimulus. Once sensitized, an intra-luminal injection of 10–15 µl of sesame oil (Sigma) was used as an artificial deciduogenic stimulus between 1100–1300 h and mice were maintained on daily s.c. injections (0900 h) of progesterone thereafter until killed (Fig. 1Go). Mice were killed at approximately 48, 72, or 96 h after artificially inducing decidualization, which correspond to days 2, 3, and 4 after the onset of decidualization respectively. The resulting uterine horns undergoing artificially induced decidualization, called stimulated (ST) uterine horns, were dissected and processed for histological or RT-real-time-PCR work.


Figure 1
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  		Figure 1 Time line showing the preparation and collection of mouse uteri during decidualization. Decidual tissue samples were collected on days 6.5, 7.5, and 8.5 of pregnancy corresponding to days 2, 3, and 4 after the onset of decidualization respectively (A). Deciduomal tissue samples from ovariectomized mice treated with estradiol-17ß (E2) and/or progesterone (P4) were collected on days 2, 3, and 4 after the onset of decidualization in response to an intra-luminal injection of sesame oil (B).

 
RT-real-time-PCR
Total RNA was extracted from the uterine tissue using TRIzol reagent (Invitrogen Corp.), according to the manufacturer’s instructions. The method of RT-real-time-PCR was then used to evaluate the relative steady-state levels of Spp1 mRNA in the total RNA samples. Briefly, total RNA (1 µg) was reverse transcribed into cDNA using ImpromII reverse transcriptase (Promega) according to manufacturer’s instructions. Each RT reaction was carried out in a 20 µl volume and contained downstream primers for both 18 S rRNA and Spp1 mRNA (Integrated DNA Technologies Inc., Coralville, IA, USA). Real-time-PCR for 18 S rRNA and Spp1 mRNA was then carried out using IQ SYBR Green Supermix (Bio-Rad) as suggested by the manufacturer. Briefly, 2 µl (for Spp1 mRNA) or 2 µl of a 50-fold dilution (for 18S rRNA) of the RT reactions were combined with 13 µl supermix, 1 µl of each upstream and downstream primers, and 9 µl RNase-free water. These reaction mixes, in 96-well plates (Bio-Rad), were then placed in an iCycler Thermal Cycler coupled to a MyIQ real-time detection system (Bio-Rad). Primer sequences for Spp1 (upstream 5'-AGCAAGAAACTCTTCCAAG-CAA-3'; downstream 5'-GTGAGATTCGTCAGATTCATCCG-3') and 18S rRNA (upstream 5'-TCAAGAACGAAAGTCG-GAGGTT-3'; downstream 5'-GGACATCTAAGGGCAT-CACAG-3') were obtained from Primer Bank Database (Wang & Seed 2003) and designed using software respectively. The conditions of the RT-real-time-PCR were 40 repetitive cycles of melting (94 °C), annealing (61.8 °C) and extension (72 °C) for 15, 15, and 30 s respectively. The cycle threshold (Ct) values provided by the MyIQ software were used to calculate the relative steady-state levels of Spp1 mRNA in the samples normalized to 18S rRNA. Briefly, for each of the four independent samples from each time point and tissue type, the {Delta}Ct values (CtSPP1–CtrRNA) were calculated where CtSpp1 and CtrRNA are the Ct values for Spp1 mRNA and 18S rRNA respectively. Next, the average {Delta}Ct for the values found for the deciduomas on day 2 after the onset of decidualization was subtracted from all individual {Delta}Ct values to normalize them to that tissue type and time point. Finally, the normalized {Delta}Ct values for each of the samples were then transformed using the following: 2–({Delta}Ct). These data were then analyzed using a two-way ANOVA to determine overall effects of time and tissue source. This was followed by the use of Duncan multiple range test to determine differences between means for each given day after the onset of decidualization. At the end of the RT-real-time-PCR, a melt curve confirmed the existence of a single amplicon as did agarose gel electrophoresis (data not shown). For further verification, the amplicons were also sequenced (University of Illinois Urbana-Champaign Core Sequencing Facility, Urbana-Champaign, IL, USA).

SPP1 and uterine natural killer (uNK) cell double-fluorescent staining
Dolichos biflorus agglutinin (DBA) lectin histochemistry can be used to identify uNK cells in mouse uterine sections (Paffaro et al. 2003). Combining this histochemical technique with immunofluorescence, we co-localized uNK cells and SPP1 protein within uterine cross-sections from 4–7 independent samples per time sampling. After both perfusion and immersion fixation as previously described (Herington & Bany 2006), the tissue was embedded in paraffin blocks using routine histo-logical methods. Uterine cross-sections (5 µm) were mounted onto silanized glass slides and stored until use. For double-fluorescent staining, the sections were deparaffinized in xylene (Fisher Scientific, Pittsburg, PA, USA), hydrated in decreasing concentrations of ethanol (MIDSCI, St Louis, MO, USA), and then washed in PBS. The slides were then placed in blocking solution containing 2% (w/v) normal donkey serum (Biomeda Corporation, Foster City, CA, USA) in PBS containing 0.05% Tween-20 (DS–PBST) for 1 h. This was followed by incubation of the sections overnight in 0.5 µg/ml anti-SPP1 immunoglobulin G (IgG) (Assay Design Inc., Ann Arbor, MI, USA) in DS–PBSTat 4 °C. After washing in PBS containing 0.05% Tween-20 (PBST), the sections were incubated for 3 h in DS–PBST containing both 7.5 µg/ml donkey anti-rabbit IgG-cyanine 3 (Cy3) conjugate (Jackson Immuno Research Laboratories, Inc., West Grove, PA, USA) and 62.5 µg/ml DBA lectin-fluorescein conjugate (Biomeda Corporation) at room temperature. After washing with PBST, the sections were incubated for 20 min in PBS containing 5 µg/ml 4',6-diamidino-2-phenylindole and dihydrochloride (DAPI; Pierce Biotechnology, Rockford, IL, USA) to stain nuclei. To reduce lipofuscin-like autofluorescence, the sections were then incubated in a cupric sulfate solution as previously described (Schnell et al. 1999). Finally, after washing in PBS, coverslips were mounted over the sections using Fluoromount-G mounting medium (Southern Biotechnology Associates Inc., Birmingham, CA, USA). Cy3 and fluorescein fluorescent signals were not detected in control sections incubated in DS–PBST containing 0.5 µg/ml rabbit IgG (in place of anti-SSP1 IgG), DBA lectin (as above), and 0.1 M N-acetyl-D-galactosamine (Sigma) competitor (data not shown).

All microscopy work was conducted using a Leica MZFLIII stereomicroscope (North Central Instruments, Maryland Heights, MO, USA) or Nikon light/fluorescence microscope (Hitschfel Instruments Inc., St Louis, MO, USA), each equipped with Retiga digital cameras (QImaging, Burnaby, Canada). Images were captured using QCapture Pro software (QImaging). Only cells in the cross-sections that had DAPI-stained nuclei within them were counted and used in the analysis of DBA lectin and/or SPP1 double fluorescence in the entire mesome-trial region of deciduomas and deciduas. DBA lectin-positive uNK cells were classified according to their stage of maturation (Paffaro et al. 2003) as types I–IV, based on the localization of DBA lectin binding, presence or absence of granules, cell size plus shape, and nuclear morphology exactly as previously described (Herington & Bany 2006). The types I–IV represent immature, intermediate, fully mature, and senescent uNK cells respectively. An ANOVA on arcsine-transformed data was performed to determine whether the percentage of total cells in the entire mesometrial area that were SPP1 negative plus DBA lectin positive, SPP1 positive plus DBA lectin positive, and SPP1 positive plus DBA lectin negative was different between the deciduomas and deciduas on each day examined. Similarly, a one-way ANOVA on arcsine-transformed data was also used to determine whether the proportion of SPP1-positive cells that stained negative for DBA lectin was different between the two tissue types on each day examined.

SPP1 and EMR1 double-immunofluorescent staining
Epidermal growth factor (EGF)-like containing mucin-like hormone receptor-like sequence 1 protein (EMR1, originally referred to as F4/80) is a membrane protein that is commonly used to localize macrophages within the mouse tissues (Austyn & Gordon 1981). Although, this protein is also localized to some dendritic cells and eosinophils in other tissues (McGarry & Stewart 1991, Peters et al. 1996), it has also been the most commonly used marker for localizing macrophages specifically in uterine tissue (De et al. 1991, Pollard et al. 1991, 1998, Hunt 1994, Robertson et al. 1999, Tibbetts et al. 1999, White et al. 2006). After using the same antigen retrieval methods described above, sections were blocked with DS–PBST for 1 h. Sections were then incubated overnight in DS–PBST containing 0.5 µg/ml anti-SPP1 IgG (Assay Design Inc.) and 50 µg/ml rat anti-mouse EMR1 (eBioscience, San Diego, CA, USA) at 4 °C. After washing in PBST, the sections were incubated for 3 h in DS–PBST containing 7.5 µg/ml donkey anti-rabbit IgG-Cy3 and anti-rat IgG-fluorescein conjugates (Jackson Immuno Research Laboratories Inc.) at room temperature. After washing with PBST, the sections were covered in PBS containing DAPI for 20 min to stain the nuclei. Sections were then treated with cupric sulfate solution, and then coverslips were mounted as described above. Cy3 or fluorescein fluorescent signals were not detected in control sections incubated in DS–PBST containing 0.5 µg/ml normal rabbit and 50 µg/ml normal rat IgG (Sigma) in place of the primary antibodies (data not shown).

Statistical analyses
All statistical analyses described above were carried out using either SAS (SAS Institute Inc., Cary, NC, USA) or SigmaStat (Systat Software Inc., Point Richmond, CA, USA) software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
Steady-state Spp1 mRNA levels
Utilizing the method of RT-real-time-PCR, we measured the relative steady-state level of Spp1 mRNA in the deciduoma and decidua on days 2–4 after the onset of decidualization (Fig. 2Go). Although the levels in the deciduomas were not different between days 2 and 3, there was a significant (P < 0.05) increase in the steady-state level of Spp1 mRNA on day 4 after the onset of decidualization. On the other hand, a significant (P < 0.05) increase in the steady-state Spp1 mRNA levels occurred in the decidua between days 2 and 3 and also days 3 and 4 after the onset of decidualization. Finally, the steady-state levels of Spp1 mRNA were significantly (P < 0.05) greater in the decidua when compared with deciduoma by approximately 8-, 17-, and 3-fold on days 2, 3, and 4 after the onset of decidualization respectively.


Figure 2
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  		Figure 2 RT-real-time-PCR analysis showing relative steady-state Spp1 mRNA levels in the mouse decidua and deciduoma on days 2, 3, and 4 after the onset of decidualization. Bars represent the mean ( ± S.E.M.; N=4) and those with different letters on a given day are significantly different (P < 0.05).

 
Double-fluorescent localization
Since the steady-state mRNA levels differed between the deciduas when compared with deciduomas, we co-localized SPP1 protein and uNK cells in uterine cross-sections on days 2, 3, and 4 after the onset of decidualization. On these days, SPP1-positive cells along with DBA lectin-positive uNK cells were localized almost exclusively in the mesometrial region of both deciduomas (Fig. 3A, B, E, F, I, and JGo) and deciduas (Fig. 3C, D, G, H, K, and LGo). The cells staining positive for DBA lectin and/or SPP1 appeared to increase in number from days 2 to 4 after the onset of decidualization in both tissues. Although not all uNK cells stained positive for SPP1 (Fig. 4AGo), this protein was localized to a subpopulation of only the granulated (types II–IV) forms (Fig. 4BGo). No SPP1-positive immature type I uNK cell could be found. Finally, there was a subset of unknown cells that stained positive for SPP1 but were non-uNK cells (DBA lectin-negative; Fig. 4CGo). Since EMR1-postive dendritic/macrophage cells have previously been shown to stain positive for SPP1 earlier in pregnancy (White et al. 2006), we next determined whether these unknown cell(s) were EMR1 positive. As shown in Fig. 4DGo, the unknown SPP1-positive DBA lectin-negative cells were not EMR1 positive. Finally, the localization of the SPP1 protein in all cells, regardless of type, appeared to be intracellular and cytoplasmic in nature. The staining seemed to be localized to either small or large granules and close inspection of the granulated DBA lectin- and SPP1-positive cells using regular fluorescence (Fig. 4BGo) and confocal (data not shown) microscopy revealed that the SPP1 protein was not localized to the DBA lectin-positive granules.


Figure 3
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  		Figure 3 Double-fluorescent co-localization of SPP1 and DBA lectin binding in cross-sections of the mouse uterus during the progression of decidualization. Representative photomicrographs of the deciduoma (A, B, E, F, I, and J) and decidua (C, D, G, H, K, and L) on days 2 (upper row), 3 (middle row), and 4 (lower row) after the onset of decidualization. Green, red, and blue fluorescence localizes DBA lectin-positive uNK cells, SPP1 localization, and nuclei respectively. Scale bars=0.5 mm. For all photomicrographs, sections are oriented with the antimesometrial (AM) side up and mesometrial (M) side down.

 

Figure 4
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  		Figure 4 Cell types in the mesometrial region of the mouse decidua and deciduoma that contain SPP1 during the progression of decidualization. DBA lectin-positive uNK cell types (I–IV) that do not (A) and do contain (B) SPP1 were seen. Some SPP1 was localized to DBA lectin-negative cells (C) but these cells were not EMR1 positive (D). Green, red, and blue fluorescence shows DBA lectin-positive uNK cells or EMR1, SPP1, and nuclei respectively. Graph representing the mean percentage of uNK cell types (I–IV) that do not (SPP1–) or do (SPP1+) stain positive for SPP1 in the total mesometrial region of the deciduomas and deciduas on days 2 (upper graph), 3 (middle graph), and 4 (lower graph) after the onset of decidualization (E). Bars represent the mean ( ± S.E.M.; N=4–7) and ND denotes none detected.

 
For days 2–4 after the onset of decidualization, all DBA lectin-positive uNK cells in the mesometrial region of the deciduomas and deciduas were counted, typed (types I–IV), and grouped by whether they were SPP1-negative or positive (Fig. 4EGo). Further, of the uNK cell types that were SPP1 positive in the deciduoma and decidua, the majority were type 2 on day 2 and type 3 on both days 3 and 4. Overall, on each day examined, there were no differences in the percentage of each uNK cell type between deciduomas and deciduas regardless if they were SPP1 negative or positive.

To statistically evaluate the different cell types, we counted all SPP1-negative plus -positive uNK cells regardless of type as well as SPP1-postitive non-uNK (DBA lectin-negative) cells throughout the mesometrial region in cross-sections from the deciduomas and deciduas (Fig. 5Go). On day 2, there was no difference in the percentage of uNK cells in the deciduoma that were SPP1 positive when compared with SPP1 negative. However, higher percentages of uNK cells were SPP1 positive on days 3 and 4 in the deciduomas. In the decidua, unlike the deciduoma, there were higher percentages of SPP1 positive uNK cells when compared with SPP1-negative uNK cells at all days examined. Next, for the deciduoma on days 2 and 3, there were no differences in the percentages of SPP1-positive uNK cells relative to SPP1-positive non-uNK cells. However, by day 4, there was a significantly (P < 0.05) greater percentage of the SPP1-positive cells that were uNK cells. Unlike the deciduoma, significantly (P < 0.05) greater percentages of SPP1-positive uNK cells when compared with SPP1-positive non-uNK cells were found in the decidua on all days examined. Finally, we compared differences in the percentages of SPP1-positive non-uNK cells between the deciduoma and decidua on each day after the onset of decidualization. There were significantly (P < 0.05) higher percentages of these cells in the deciduoma when compared with decidua on days 2 (approximately sixfold) and 3 (approximately threefold). However, the complete opposite was seen on day 4 where there was a significantly (P < 0.05) lower (~0.5-fold) percentage in the deciduoma when compared with decidua.


Figure 5
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  		Figure 5 Graphs showing the percentage of cells in the deciduoma and decidua staining positive for DBA lectin binding (DBA+) and/or SPP1 (SPP1+/SPP1–) in the mesometrial region of the mouse uterus during the progression of decidualization on days 2 (upper graph), 3 (middle graph), and 4 (lower graph) after the onset of decidualization. Bars represent the mean ( ± S.E.M.; N=4–7) and those with different letters are significantly different (P < 0.05).

 
SPP1 expression in Il15–/– mice
Il15–/– mice lack uNK cells in the uterus during implantation (Ye et al. 1996, Kennedy et al. 2000, Barber & Pollard 2003). We performed RT-real-time-PCR on decidual total RNA from Il15–/– and Il15+/+ mice to measure the steady-state level of Spp1 mRNA in these tissues. There was a significant (P < 0.01) decrease in the steady-state levels of Spp1 mRNA in the IS of Il15–/– mice when compared with those from Il15+/+ mice by approximately sevenfold on day 3 after the onset of decidualization (Fig. 6AGo). To provide further confirmation that the major site of SPP1 localization was to uNK cells, we also carried out double-fluorescent staining of uNK cells and SPP1 protein in uterine cross-sections from Il15–/– and Il15+/+ mice. Surprisingly, a complete loss of SPP1 localization in the sections from Il15–/– mice (Fig. 6BGo) was seen, while localization in Il15+/+ mice was normal (Fig. 6CGo).


Figure 6
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  		Figure 6 SPP1 expression in the decidua on day 3 after the onset of decidualization in Il15+/+ when compared with Il15–/– mice. Bar graph summarizing the RT-real time-PCR analysis showing relative steady-state levels of Spp1 mRNA in the decidua of Il15+/+when compared with Il15–/–mice on day 3 after the onset of decidualization (A). Representative photomicrographs of DBA lectin- and SPP1-stained cross-sections of the mouse decidua from Il15+/+ (B) and Il15–/– mice (C). Green and red fluorescent colors indicate DBA lectin binding and SPP1 localization respectively. Bars represent the mean ( ± S.E.M.; N=4) and those with different letters are significantly different (P < 0.01). For both photomicrographs, sections are oriented with the mesometrial (M) side down and also shown is the conceptus (C). Scale bars = 0.5 mm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
Spp1 expression increases in the pregnant mouse uterus during the progression of decidualization mainly due to an influx of uNK cells. In tissues outside the uterus, Spp1 gene expression is localized to activated lymphocytes including T-cells and a subset of NK cells (Pollack et al. 1994). Further, in vitro experiments show that Spp1 expression increases in NK cells after activation with IL2 (Patarca et al. 1989). Past studies have shown the presence of Spp1 mRNA and an increase in its levels in the mesometrial region of the mouse uterus during the progression of decidualization after the onset of implantation (Nomura et al. 1988, Waterhouse et al. 1992). Although they also provided some evidence that suggested Spp1 mRNA might be present in uNK cells, to the best of our knowledge, there is no definitive proof of this. In this current study, we confirmed that there is an increase in steady-state levels of Spp1 mRNA during the progression of decidualization in the decidua. This increase occurs at a similar time that uNK cells have been previously shown to dramatically increase in the mesometrial region of the mouse decidua (Stewart & Peel 1978, Peel 1989, Herington & Bany 2006). The present study also shows that a major localization of SPP1 protein is within a subpopulation of the granulated uNK cell types in the uterus during the progression of decidualization. Taken together, these observations provide evidence that uNK cells are a major source of Spp1 expression in the uterus during the progression of decidualization in the decidua. This is strongly supported by the additional observation of this study that there is a dramatic decrease in Spp1 gene expression in the decidua of uNK cell-deficient Il15–/– (Ye et al. 1996, Ashkar et al. 2003) when compared with that of normal Il15+/+ mice.

The localization of Spp1 expression in the uterus dramatically changes during early pregnancy in mice. Recently, it has been shown that all Spp1 is expressed in the luminal epithelia and EMR1-positive immune cells in the mouse endometrium around the onset of implantation (White et al. 2006). As discussed above, Spp1 expression is localized mainly to DBA lectin-positive granulated uNK cell types in the endometrium after the onset of implantation as it undergoes the process of decidualization. The dominant uNK cell types where this protein is localized changes from type 2 to type 3 in both the deciduoma and decidua during the progression of decidualization. Notably, this correlates well with previous work (Herington & Bany 2006) showing these are the dominant types of uNK cells present in the uterus at these times. Therefore, the changes seen in SPP1 localization appear to depend on changes in the proportion of uNK cell types during decidualization. Further, since the majority SPP1-expressing cells in the stroma of the endometrium around the onset of implantation have been shown to be EMR1-positive cells, we hypothesized that the non-uNK (DBA lectin-negative) SPP1-positive cells found in this study were also EMR1 positive during the progression of decidualization. However, we found that SPP1 protein was not co-localized to the EMR1-positive immune cells in the decidua and deciduoma at this time. To complicate matters, all SPP1-positive cells appeared to be absent in the decidua of Il15–/– mice during the progression of decidualization, including the SPP1-positive non-uNK cells. This suggests uNK cells may regulate the presence of the SPP1-positive non-uNK cells. It has been shown that SPP1 is a chemotactic factor for many immune cell types in other tissue (Denhardt & Guo 1993, Patarca et al. 1993). Thus, we still speculate the SPP1-positive non-uNK cells observed in the present study are immune cells. However, more work is required to identify the exact identity of these cells in the future.

The level of Spp1 expression in mouse uterus during the progression of decidualization is enhanced in the presence of a conceptus, at least in part, due to its influence on uNK cells. This study shows that SPP1 protein is localized to a majority of the granulated uNK cell types in the mouse deciduoma and decidua during the progression of decidualization. In a similar fashion to the decidua, previous work (Nomura et al. 1988) in combination with the work in this study indicates that Spp1 expression increases in the deciduoma. However, to our knowledge, there have been no reports where the level and localization of Spp1 gene expression has ever been compared between the deciduoma (conceptus absent) and decidua (conceptus present) during the progression of decidualization. Indeed, one interesting aspect of the expression of Spp1 in the deciduomas found in this study is that its levels were significantly lower when compared with the deciduas at similar times after the onset of decidualization. This decreased level of Spp1 expression in the deciduoma when compared with decidua correlates well to a previous finding that there are less uNK cells in the deciduoma when compared with decidua during the progression of decidualization (Herington & Bany 2006). That study shows uNK cells appear in the endometrium during the progression of decidualization regardless of whether it is a deciduoma or decidua. However, if a conceptus is present, there are significantly more uNK cells. Therefore, the lower level of Spp1 expression during the progression of decidualization in the deciduoma appears to be a consequence of the reduction in uNK cell numbers when compared with that of the decidua.

Although many different functions have been attributed to SPP1 protein in other tissues, we know very little about its function in the mouse uterus during decidualization. A survey of the current literature reveals several potential functions of SPP1 protein in other tissues and include such things as cell–cell adhesion (Leali et al. 2003), angiogenesis (Denhardt & Guo 1993, Prols et al. 1998, Shijubo et al. 1999, Takano et al. 2000), immune cell chemoattraction (Liaw et al. 1995), and immune cell function (O’Regan et al. 2000, Denhardt et al. 2001). Further, this is complicated by the fact that SPP1 function may (Nemir et al. 1989, Ek-Rylander et al. 1994) or may not (Weber et al. 1996) change depending on its phosphorylation status. Notably, SPP1 protein that is secreted by uterine epithelium has been suggested to provide a substrate for integrin-mediated interactions at the conceptus–maternal interface at the onset of implantation in several species (Johnson et al. 1999, Apparao et al. 2001, von Wolff et al. 2001), including mice (White et al. 2006). Although these are potential roles of SPP1 in the mouse uterus during the progression of decidualization, a great deal of work will be required to confirm this. One logical approach may be to conduct an in-depth investigation of Spp1-deficient mice (Liaw et al. 1998) to determine whether there are abnormalities in the utero-placental vascular changes during implantation since it was found that these mice experience decreased pregnancy rates and an intrauterine growth restriction during pregnancy when compared with their wild-type counterparts (Weintraub et al. 2004). Since uNK cells do play a role in changes in the maternal vasculature during implantation in the mouse (Croy et al. 2003) and appear to be a major source of SPP1, such a role is plausible.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
This study was supported by NIH Grant HD049010. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.


   Footnotes
 
Received 16 February 2007
First decision 27 February 2007
Accepted 27 February 2007


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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