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BOR - Papers in Press, published online ahead of print December 6, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.056630
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BIOLOGY OF REPRODUCTION 76, 579–588 (2007)
DOI: 10.1095/biolreprod.106.056630
© 2007 by the Society for the Study of Reproduction, Inc.

research-article

Effect of the Conceptus on Uterine Natural Killer Cell Numbers and Function in the Mouse Uterus During Decidualization1

Jennifer L. Herington , and Brent M. Bany 2 

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

ABSTRACT

Uterine natural killer (uNK) cells are the most abundant lymphocytes in the uterus during early pregnancy and play a role in spiral arteriole modifications. In the present study, we investigated whether uNK cell populations differed between mouse decidua and deciduoma. Histochemical staining using the Dolichos biflorus agglutinin (DBA) lectin was used to identify uNK cells and classify their stages of maturation. We found differences in the pattern of localization and density of uNK cells between the decidua and deciduoma at Days 2–4 after the onset of decidualization. The cells were more distributed and the densities were significantly greater in the mesometrial region of the decidua than in the deciduoma. Using double-labeling for DBA lectin binding and bromodeoxyuridine incorporation, we found that the higher number of uNK cells in the decidua was not due to an increase in uNK cell proliferation. Western blot analyses revealed that the increase in uNK cell number was accompanied by significant increases in the levels of interferon gamma (IFNG) and prointerleukin 18 when a conceptus was present. Vascular morphometry revealed that modifications of the spiral arterioles occurred in the mesometrial decidua but not in the deciduoma, which could be attributed to the differences observed in uNK cell number and IFNG production. The present study demonstrates that differences exist in uNK cell populations between the decidua and deciduoma, providing evidence that the conceptus generates signals that regulate uNK cell number and function in the uterus during implantation.

decidua, immunology, implantation, pregnancy

INTRODUCTION

Two key processes for the advancement of mammalian pregnancy are implantation and formation of the placenta. Implantation 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 events to occur in the uterus during this time is the proliferation and subsequent differentiation of the endometrial fibroblast-like cells into large polyploid decidual cells [1, 2]. This process, called decidualization, results in the formation of tissue that is referred to as the decidua, and it occurs in response to the implanting conceptus in rodents. However, based on an observation first reported almost a century ago [3], molecular signals from the conceptus do not appear to be required for decidualization to occur. This is because the uterus can undergo decidualization in response to an artificial stimulus, such as an intraluminal injection of sesame oil or transfer of beads into ovariectomized hormonally sensitized or pseudopregnant animals, respectively [4]. In order to distinguish it from the decidua that forms in pregnant animals, the tissue that develops in response to an artificial stimulus is termed a deciduoma [5]. In the present study, both pregnant uteri (conceptus present) and those undergoing artificially induced decidualization (conceptus absent) were used to determine whether the conceptus plays a role in regulating natural killer (NK) cell populations in the mesometrial region of the uterus during decidualization.

NK cells are a part of the innate immune system and defend against allogenic cells and cells under stress, such as virally infected or tumor cells [6]. Although the conceptus is a semiallogenic graft that expresses both maternal and paternal antigens, it is not rejected by the maternal immune system under normal conditions [7]. It is well known that a special set of NK cells are present in the uterus during implantation and later in pregnancy [8–11]. However, instead of being harmful to the conceptus, recent evidence shows these cells play a key role in maintaining decidual integrity and evoking changes in the spiral arterioles in the mesometrial region of the mouse uterus during pregnancy [12]. Therefore, these NK cells seem to play a role in the process of normal implantation.

NK cells are derived from pluripotent hematopoeitic stem cells that differentiate into common lymphoid progenitor cells in the bone marrow [13]. These cells then become progenitor NK (pre-NK) cells in the secondary lymphoid tissues and migrate to the various tissues of the body to differentiate further [14]. Once these small agranular pre-NK cells home to and are located within the uterus, they become uterine NK (uNK) cells and begin the maturation process into large granulated cells [15]. The processes of pre-uNK cell recruitment into the uterus and uNK cell maturation within the uterus have been shown to involve interleukin 15 (IL15) [16, 17], as demonstrated by the establishment of IL15-deficient mice, which completely lack NK cells, including uNK cells [18, 19].

Uterine NK cells are the most abundant lymphocyte and a major source of cytokines in the uterus during early pregnancy [20]. They secrete both interferon gamma (IFNG) and interleukin 18 (IL18) [21], which appear to play roles in the uterus during implantation. Paracrine IL18 signaling is responsible, at least in part, for stimulating IFNG synthesis by uNK cells. In turn, the uNK cell-derived IFNG plays a key role in the spiral arteriole modifications [12]. Although maternal hormones have been suggested to play a key role in regulating uNK cell populations in the uterus during pregnancy [22], a role for the conceptus has not been confirmed. In fact, since uNK cells are present in the deciduoma [23], one could argue that the conceptus is not required for the appearance of all the uNK cells in the uterus during implantation. In the present study, we set out to determine whether the conceptus has an effect on uNK cell numbers, localization, and maturation in the mouse uterus in the first 4 days after the onset of decidualization. The results provide evidence that the conceptus does have an influence on uNK cell populations and their functions.

MATERIALS AND METHODS

Animals

For each of the experiments in the present study, treatment and time-point data were from obtained for 3 to 6 independent animals (n = 3–6). All procedures that involved mice were approved by the Southern Illinois University Institutional Animal Care and Use Committee. CD1 mice (6–8 wk old) were purchased from Charles River Breeding Laboratories (Wilmington, MA), maintained under controlled light conditions (lights on from 0700 h to 1900 h), and allowed free access to food and water. Females were placed with fertile males and the morning on which a vaginal plug was detected was considered to be Day 0.5 of pregnancy. Mice were killed at 0900 h on Days 3.5 to 9.5 of pregnancy, which correspond approximately to –1 to 5 days after the onset of decidualization, respectively (Fig. 1A).


Figure 01
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  		FIG. 1. Time-lines for uteri collected on days after the onset of decidualization. A) Pregnant uteri were collected on Days –1 to 5, corresponding to days of pregnancy (DOP) 3.5 to 9.5. B) Uteri undergoing artificially-induced decidualization in ovariectomized mice injected with estradiol-17ß (E2) and progesterone (P4), to sensitize the uterus for a deciduogenic stimulus. Pseudopregnant females received a transfer of concavalin A-coated agarose beads (C) or injection of sesame oil into the uterine lumen to induce artificially decidualization (D). Uteri were collected on Days 1–4 after the onset of artificially-induced decidualization (B–D).

Three models of artificially induced decidualization were utilized in the present study. In the first model (Fig. 1B), animals were ovariectomized, allowed 1 wk of recovery, then injected subcutaneously with estradiol and/or progesterone in order to sensitize adequately the uterus for an artificial deciduogenic stimulus [24]. The ovariectomized mice were injected with estradiol and/or progesterone at 0900 h, and an intraluminal injection of 10–15 µl of sesame oil was used as an artificial deciduogenic stimulus between 1100–1300 h. The mice were killed exactly 24, 48, 72 or 96 h after artificial induction of decidualization, corresponding to Days 1 to 4 after the onset of decidualization. The tissue collected is referred to as ovariectomized oil-induced (Ovx-OID) deciduoma. Samples were not collected from Day 5 onwards, since the deciduoma undergoes massive regression at this time [25]. The second and third models of artificially induced decidualization utilized pseudopregnant females generated by mating to vasectomized males (the day on which a vaginal plug was detected was considered as Day 0.5 of pseudopregnancy). Uterine horns were obtained either after transfer of 12 concavalin A-coated blastocyst-sized agarose beads (Pseudo-BID; Fig. 1C) at 1300–1500 h [26] or intraluminal injection of 15 µl of sesame oil (Pseudo-OID; Fig. 1D) at 1100–1300 h on days 2.5 and 3.5, respectively. In both cases, the mice were killed on Days 6.5–9.5 of pseudopregnancy at 0900 h, corresponding to Days 1–4 after the onset of decidualization.

Tissue Processing for Paraffin Section Analyses

For samples collected for histochemical and immunohistochemical analyses, the mice were anesthetized with an i.p. injection of a ketamine hydrochloride (200 mg/kg) plus xylazine (20 mg/kg) (Henry Schein, Melville, NY) mixture. The mice were then perfused with 10 ml of PBS that contained 1% sodium nitrate, followed by 50–100 ml of 4% paraformaldehyde (Fisher, Pittsburg, PA) (w/v) in PBS (PFA-PBS) at a pressure of 120 mmHg. After dissection, uterine tissues were placed in 4% PFA-PBS for 24 h, and then in 70% ethanol for 24 h at 4°C. Tissues were then dehydrated, cleared in xylene, and embedded in paraffin using routine histological procedures. Cross-sections (5-µm thickness) of the uteri were prepared and mounted onto silanized glass slides.

Uterine NK Cell Staining Using DBA Lectin Histochemistry

Dolichos biflorus agglutinin (DBA) lectin histochemistry was used to identify uNK cells in uterine cross-sections, essentially as described previously [15]. Briefly, the cross-sections were deparaffinized in xylene, rehydrated, and then treated with 1% hydrogen peroxide for 30 min to block endogenous peroxidase activity. Sections were then washed with PBS, blocked with 1% (w/v) BSA (Fisher) in PBS (BSA-PBS) for 1 h, followed by an overnight incubation with 2 µg/ml biotinylated-DBA lectin (ICN Biomedicals Inc., Aurora, OH) in 1% BSA-PBS at 4°C. After washing in PBS, the sections were incubated with 10 µg/ml ExtrAvidin Peroxidase conjugate (Sigma, St. Louis, MO) in 1% BSA-PBS for 30 min at room temperature. After washing with PBS, the slides were incubated with the peroxidase substrate 3,3'-diaminobenzidine tetrahydrochloride (ICN Biomedicals), yielding a brown stain. In order to visualize the nuclei, sections were counterstained with Harris hematoxylin (Statlab Medical Products, Lewisville, TX). Control sections were stained as described above, with the addition of 0.1 M N-acetyl-D-galactosamine (Sigma) to the DBA lectin incubation. In every case, this displaced all of the DBA lectin binding in the sections, thereby verifying the specificity (data not shown). Microscopy was conducted using a Leica MZFLIII stereomicroscope (North Central Instruments, Maryland Heights, MO) and a Nikon microscope (Hitschtel Instruments Inc., St. Louis, MO), each of which was equipped with a Retiga digital camera (QImaging, Burnaby, BC, Canada).

DBA-lectin-positive immunohistochemical staining of uNK cells was used for the classification of maturation stages, essentially as described previously [15]. The stages of uNK cell maturation include subtypes I (immature), II (intermediate), III (fully mature), and IV (senescent) (Fig. 2A). Uterine NK cells were classified into subtypes I-IV based on several the characteristics of cell shape, nuclear morphology, number of granules present, cell size, and location of the DBA-lectin reactivity (Table 1). The three experiments described below were conducted to determine whether the conceptus has an effect on uNK cells in the uterus on Days 2–4 after the onset of decidualization.


Figure 02
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  		FIG. 2. Uterine NK cell maturation in the mouse. A) Uterine NK cells found in the mesometrial region of the decidua and deciduoma by DBA lectin histochemistry were classified as subtypes I-IV. B) Day 4 after the onset of decidualization: a uterine cross-section illustrating subregions 1–3 (central axis toward conceptus from the myometrium) and subregion 4 (lateral region). AM, antimesometrial region; C, conceptus; M, mesometrial region; MLAp, mesometrial lymphoid aggregate of pregnancy. Bar = 10 µm (A) and 1 mm (B).


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  		TABLE 1. Uterine natural killer subtypes within the uterus during decidualization.

Total mesometrial uNK cell density. The total numbers of uNK cells in the mesometrial regions of the uteri were counted and normalized to the mesometrial tissue area using the Image J software [27]. Two-way ANOVA was conducted to detect effects of the type of decidual tissue and the time after onset of decidualization on uNK cell density. This was followed by the Duncan multiple range test, to determine differences between the means for each day after the onset of decidualization.

Regional uNK cell density. The uNK cells were classified as subtypes I-IV (Fig. 2A) in subregions 1–4 (Fig. 2B) of the mesometrial area of the decidua and Ovx-OID deciduoma, essentially as described previously [15]. Briefly, subregion 1 is the area that eventually becomes the mesometrial lymphoid aggregate of pregnancy. Subregion 2 is located in the central area, at the midpoint between the myometrium and conceptus. Subregions 3 and 4 are found in areas adjacent to the conceptus centrally and laterally, respectively. Uterine NK cell subtypes I-IV were counted in subregions 1–4 of the decidua and the Ovx-OID deciduoma in order to determine the density of each uNK cell subtype per test area (25 600 µm2). A minimum of nine random test areas and at least 800-4000 cells were counted in each of the mesometrial subregions of the decidua and Ovx-OID deciduoma of each independent sample. Two-way ANOVA was performed to detect overall differences in the uNK cell subtype densities for each individual day and subregion. This was followed by the use of Duncan multiple range tests to determine differences between the means.

Regional uNK cell maturation. The percentages of each uNK cell subtype in mesometrial subregions 1–4 on Days 2, 3, and 4 after the onset of decidualization were determined. Chi-square tests were conducted to determine if there was a significant difference in the uNK cell subtype profile between the decidua and Ovx-OID deciduoma.

Measuring the uNK Cell Proliferation Index

In order to visualize those uNK cells undergoing proliferation, mice were injected i.p. with 1 mg bromodeoxyuridine (BrdU) dissolved in 0.1 ml Dulbecco PBS (Invitrogen, Grand Island, NY) exactly 4 h prior to perfusion and uterine collection [28]. BrdU was incorporated into those cells in the S phase of the cell cycle for 4 h prior to tissue collection. Paraffin sections of these samples were then subjected to histochemical and immunohistochemical staining for DBA lectin and BrdU, respectively. All incubations were carried out at room temperature, except where noted. Briefly, after deparaffinization and hydration, sections were digested with 0.2% trypsin in PBS for 10 min at 37°C for antigen retrieval. After washing in PBS, the sections were incubated in 1.5 M HCl for 15 min at 37°C, washed with PBS, and then incubated in borate buffer (0.1 M boric acid [pH 8.5]) for 10 min. Next, the sections were blocked with 1% BSA-PBS for 1 h prior to incubation with the primary antibody (sheep anti-BrdU; Biodesign, Sako, ME) at a concentration of 8 µg/ml IgG in BSA-PBS for 1 h. Control sections were incubated with 8 µg/ml normal IgG (Sigma) in 1% BSA-PBS. After washing with PBS, the sections were incubated with the secondary antibody (biotinylated donkey anti-sheep IgG; Jackson Immunoresearch, West Grove, PA) at a concentration of 4 µg/ml in BSA-PBS for 1 h. Next, the sections were washed in PBS and covered with 2.5 µg/ml alkaline phosphatase-conjugated streptavidin (Vector Laboratories, Burlingame, CA) in BSA-PBS for 15 min. After washing the sections with Tris-buffered saline that contained 0.6 mg/ml levamisole (Acros Organics, Morris Plains, NJ), the sections were incubated with the Vector Blue substrate (Vector Laboratories) that contained 1 mg/ml levamisole. The sections were washed with water, followed by PBS, and covered with 3% hydrogen peroxide diluted in PBS for 10 min, to block endogenous peroxidase activity. The sections were then washed with PBS and blocked in 1% BSA-PBS for 10 min prior to incubation with 2 µg/ml biotinylated DBA lectin in BSA-PBS for 1 h. After washing with PBS, the sections were covered with horseradish peroxidase-conjugated streptavidin (2.5 µg/ml) (Vector Laboratories) in BSA-PBS for 15 min. After washing with PBS, the sections were incubated with aminoethyl carbozole substrate (ZyMed Laboratories Inc., South San Francisco, CA) and washed with water. Finally, the sections were placed in 4',6-diamidino-2-phenylindole dihydrochloride (Pierce Biotechnology, Rockford, IL), washed in PBS, and mounted with Fluoromount-G (Southern Biotechnology Associates Inc., Birmingham, AL). The proliferation index was measured as the proportion of uNK cells (brown color) that stained positive for BrdU (blue color); a minimum of 500 cells was assessed in 2–3 sections from three or more independent samples. Two-way ANOVA was performed to detect overall differences in proliferation index, followed by the use of Duncan multiple range tests to determine the significance of differences between the means.

Western Blot Analyses

Protein extracts were collected from decidua and Ovx-OID plus Pseudo-BID deciduomas from four independent samples. Briefly, uteri were collected and homogenized in Tissue Protein Extraction Reagent (TPER; Pierce Biotechnology). After the homogenate was centrifuged at 12 000 x g for 20 min at 4°C, the supernatants were collected and the concentration of protein was determined using the BCA Protein Assay Kit (Pierce Biotechnology). Protein samples (18 µg per lane) were then subjected to reducing SDS-PAGE using the method of Laemmli [29] with 15% Tris-glycine PAGEr Gold Precast gels (Pierce Biotechnology). The proteins were then transferred to an Immunobilon-FL membrane (Millipore, Billerica, MA) using the method of Towbin et al. [30]. The remaining steps were carried out at room temperature with gentle agitation. The membranes were washed in PBS and blocked for 1 h in Odyssey Blocking Buffer (LI-COR Biosciences, Lincoln, NE) diluted 1:1 with PBS. Next, the membranes were incubated in blocking buffer with primary antibodies against IL15, IL18, IFNG or beta-actin (ACTB). The primary antibodies were added for 60 min at the following concentrations: 0.4 µg/ml for goat anti-IL15 IgG (Santa Cruz Biotechnology Inc., Santa Cruz, CA), 0.1 µg/ml for goat anti-IFNG IgG (Abcam, Inc., Cambridge, MA), 2 µg/ml for rabbit anti-IL18 IgG (Rockland Inc., Gilbertsville, PA), and rabbit anti-ACTB antiserum was used at a dilution recommended by the manufacturer (Biolegend, San Diego, CA). For controls, purified IgG and normal serum at the same concentrations and dilution were used in place of the primary antibody. The membranes were washed in 1% PBS that contained 0.05% Tween-20 (PBST), and then incubated with the appropriate secondary antibody at a concentration of 0.06 µg/ml (IRDye700DX-conjugated donkey anti-goat IgG or IRDye800DX-conjugated donkey anti-rabbit IgG; Rockland) for 60 min, followed by washing with PBST and then with PBS. Finally, the infrared fluorescent signals were measured using the Odyssey Infrared Imaging System and software (LI-COR Biosciences). All the fluorescent intensities were normalized to ACTB. Where possible, two-way ANOVA was performed to determine the overall differences in relative fluorescent intensities, followed by the use of t-tests to determine differences between the means on a given day after the onset of decidualization.

Vascular Morphometry

Previous work has shown that uNK cells play a key role in the spiral arteriole modifications that normally occur during implantation, which includes thinning of the wall and dilation [12]. Therefore, an experiment was conducted to determine whether these modifications occur by Day 4 after the onset of decidualization in the decidua and Ovx-OID plus Pseudo-BID deciduomas. Cross-sections adjacent to those used for DBA-lectin staining were stained with Harris hematoxylin and eosin, to visualize the spiral arterioles in the mesometrial region. Photomicrographs were taken and calibrated using QCapture Pro (QImaging) and a stage micrometer, respectively. At the narrowest point, the vessel (V) and lumen (L) diameters of the spiral arterioles were measured, and the vessel wall thickness was determined using the formula (V-L)/2. Vessels were considered to be spiral arterioles based on size (10–100 µm in diameter) and the presence of a smooth muscle layer. Spiral arteriole wall thickness and lumen diameter were obtained not only for the implantation sites or areas undergoing artificially induced decidualization from each animal, but also from adjacent nonimplantation sites or nonstimulated areas (controls), respectively. Data were collected for a total of 50–200 vessels per tissue type (from six independent samples) and two-way repeated measures ANOVA was used to analyze the data.

Statistical Analyses

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

RESULTS

Total Mesometrial uNK Cell Density

In our initial studies of DBA lectin-stained cross-sections, it appeared that the uNK cell numbers in the mesometrial region of the decidua differed dramatically from those found in all types of deciduomas on the same days after onset of decidualization. On Day 3 after the onset of decidualization, it was most apparent that there was a dramatic difference in the pattern of localization of uNK cells in the cross-sections of all types of deciduoma examined as compared to the decidua (Fig. 3A). Uterine NK cells were distributed more diffusely throughout the mesometrial subregions of the decidua, while those in the deciduoma were more centrally located in the mesometrial region of the deciduoma.


Figure 03
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  		FIG. 3. Uterine NK cell distribution and density. A) Photomicrographs of DBA lectin-stained cross-sections of the mouse decidua and deciduomas on Day 3 after the onset of decidualization. The brown color indicates DBA lectin binding to uNK cells. B) Graph showing density of uNK cells normalized to the total area of mesometrial subregion in the mouse decidua and deciduomas at various days after the onset of decidualization. Bars represent the mean (± SEM; n = 6) and those with different letters on a given day are significantly different (P < 0.01). An asterisk (*) denotes values only determined for the decidua. Bar = 500 µm.

Very few DBA lectin-positive uNK cells were found in the uterine sections of the decidua and deciduoma on Day 1 after the onset of decidualization (Fig. 3B). No significant differences in total uNK cell density were detected between the decidua and deciduomas on this day. In contrast, uNK cells were plentiful and readily seen in the mesometrial region thereafter during decidualization (Fig. 3A). As shown in Figure 3B, the total density of uNK cells within the mesometrial decidua was significantly higher (P < 0.01) compared to that of all types of deciduomas on Days 2, 3, and 4 after the onset of decidualization. Notably, the total uNK cell densities of all three deciduoma models on each day after the onset of decidualization were not significantly (P > 0.05) different (Fig. 3B). Since there were no differences between all of the types of deciduomas examined, in subsequent studies, we only compared the decidua to Ovx-OID and Pseudo-BID deciduomas.

Regional Cell Density

Since the total uNK cell densities were dramatically different between the decidua and Ovx-OID deciduoma, we looked at the density of each of the uNK cell subtypes (Fig. 2A) in subregions 1–4 (Fig. 2B) of the mesometrial area of the uterus (Fig. 4). On Day 2 after the onset of decidualization, there were significantly (P < 0.05) higher densities of subtypes II and III in subregion 2, and of subtype III in subregion 3 of the decidua compared to the Ovx-OID. Similar differences in density were seen on Day 3 after the onset of decidualization for subtype II cells in subregion 1 and for subtype III plus IV cells in subregion 4. On Day 4, the densities of subtypes II and III in subregion 1, subtypes III and IV in subregion 2, and subtype IV in subregion 4 were significantly (P < 0.05) higher in the decidua than in the Ovx-OID deciduoma. Finally, small but significantly higher uNK cell densities were seen on Day 2 in subregion 1 subtype I and on Day 3 in subregion 2 subtype II in the decidua compared to the Ovx-OID deciduoma. The lower uNK cell densities found in subregions 1 and 4 of the deciduoma in Figure 4 are correlated to the more central distribution of cells compared to that of the decidua, which were more diffusely spread throughout the mesometrial region. Similar to the Ovx-OID deciduomas, decreased uNK cell subtype densities were also seen in several subregions of the Pseudo-BID deciduomas on Days 2, 3, and 4 after the onset of decidualization relative to that of the decidua (data not shown).


Figure 04
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  		FIG. 4. Graphs summarizing regional uNK cell subtype (I-IV) densities per test area in subregions 1–4 of the mouse Ovx-OID deciduoma and decidua on Days 2, 3, and 4 after the onset of decidualization. An asterisk (*) indicates a significant difference (P < 0.05) between the Ovx-OID deciduoma and decidua for a given subtype. Bars represent the mean and error bars denote the standard error of the mean (n = 6).

Regional uNK Cell Maturation

To investigate further the differences in uNK cell populations between the decidua and Ovx-OID deciduoma, we measured regional uNK cell maturation. We determined the percentage of each uNK cell subtype (Fig. 2A) in the four subregions (Fig. 2B) of the mesometrial region of the uterus. On Day 2 after the onset of decidualization, there was a significant (P < 0.05) difference in the percentage of uNK cell subtypes in subregions 2 and 3 of the decidua compared to the Ovx-OID deciduoma (Fig. 5). In subregion 2, this was due to a higher percentage of subtype III, with fewer subtype I uNK cells in the decidua compared to the Ovx-OID deciduoma. In subregion 3, this was due to a higher percentage of subtypes III and IV, with fewer subtype I and II uNK cells in the decidua compared to the Ovx-OID deciduoma. A significant difference (P < 0.05) was also seen on Day 3 in subregion 1, with the majority being subtype II in the decidua and subtype I in the Ovx-OID deciduoma, respectively. However, by Day 4, there were no significant (P < 0.05) differences in the percentages of uNK cell subtypes found in the decidua compared to the Ovx-OID deciduoma. Similar to the Ovx-OID deciduomas, some differences in the percentages of uNK cell subtypes were also seen in the subregions of the Pseudo-BID deciduomas relative to those of the decidua (data not shown).


Figure 05
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  		FIG. 5. Graphs summarizing the percentages of uNK cells subtype (I-IV) in mesometrial subregions 1–4 of the mouse Ovx-OID deciduomas and decidua on Days 2, 3, and 4 after the onset of decidualization. Bars represent the mean and error bars denote the standard error of the mean (n = 6).

Uterine NK Cell Proliferation

In order to determine whether the increase in uNK cell numbers in the decidua was a result of uNK cell proliferation, we compared the proliferation indices (the proportions of uNK cells staining positive for BrdU) of the decidua and Ovx-OID plus Pseudo-BID deciduomas on Days 2, 3, and 4 after the onset of decidualization (Fig. 6). No significant increase in the uNK cell proliferation index was seen for the decidua as compared to the deciduomas.


Figure 06
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  		FIG. 6. Graph showing the mean (± SEM, n = 5–6) uNK cell proliferation index (proportion of uNK cells staining positive for BrdU) in the mouse Ovx-OID plus Pseudo-BID deciduomas and decidua on Days 2, 3, and 4 after the onset of decidualization. Bars with different letters are significantly different (P < 0.01).

Western Blot Analyses of IL15, IFNG, and IL18

Previous work has indicated that certain cytokines are important in the modulation of uNK cell function [12]. Therefore, Western blot analyses were performed to investigate whether the levels of some of these cytokines differed between the decidua and deciduoma (Fig. 7A). IL15 has been shown to be important for the maturation of uNK cells [12]. Our results revealed that the IL15 levels did not differ significantly (P > 0.05) between the decidua and Ovx-OID plus Pseudo-BID deciduomas examined on each day after the onset of decidualization (Fig. 7B). Both IFNG and IL18 have been shown to be produced by uNK cells [21]. IFNG exists in both glycosylated and nonglycosylated forms [31]. The level of glycosylated IFNG was significantly (P < 0.05) increased in the decidua compared to the Ovx-OID and Pseudo-BID deciduomas on Days 2–4 after the onset of decidualization (Fig. 7, A and C). There was an 80-fold increase on Day 3 and a 30-fold increase on Day 4 after the onset of decidualization. IL18 exists in an unprocessed proform and a processed active form [32]. Pro-IL18 was detected in the decidua but not in the Ovx-OID plus Pseudo-BID deciduomas on Days 2–4 after the onset of decidualization (Fig. 7, A and D). Finally, no fluorescent bands were seen for the negative controls in Western blots in which the primary antibodies were replaced with rabbit or goat IgG (Fig. 7, E and F).


Figure 07
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  		FIG. 7. Western blot analyses of IL15, IFNG, and IL18 in the mouse Ovx-OID plus Pseudo-BID deciduomas and decidua on Days 1–4 after the onset of decidualization. A) Representative Western blot analysis. ACTB was used as a control to ensure equal protein loading. Graph bars represent the mean (± SEM, n = 4) relative fluorescence normalized to ACTB for IL15 (B), IFNG (C) and IL18 (D). An asterisk (*) indicates a significant difference (P < 0.05) between the deciduomas and decidua. N indicates no fluorescence signal detected above the background. E) Rabbit IgG (2 µg/ml) negative control. F) Goat IgG (0.4 µg/ml) negative control.

Vascular Morphometry

Previous studies have indicated that uNK cells and the IFNG that they secrete are involved in the thinning and dilation of the spiral arterioles by midpregnancy [12]. Since we found significantly more uNK cells and IFNG in the decidua compared to the Ovx-OID plus Pseudo-BID deciduomas, we examined the spiral arterioles in the mesometrial region of these tissues. As shown in Figure 8A, on Day 4 after the onset of decidualization, spiral arterioles in the mesometrial region of the decidua appear to be both thinner and more dilated compared to those present in the mesometrial regions of the deciduomas. Using a more quantitative approach, we confirmed that the spiral arteriole wall thickness was significantly (P < 0.05) decreased in the areas of implantation stimulus (implantation site) relative to the control areas (nonimplantation site) of the decidua, without any corresponding change in the deciduomas (Fig. 8B). Finally, the lumen diameters of the spiral arterioles were significantly (P < 0.05) increased in the areas of implantation stimulus of the decidua relative to the control areas, again without any corresponding change in the deciduomas (Fig. 8C).


Figure 08
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  		FIG. 8. Mouse spiral arteriole morphometry on Day 4 after the onset of decidualization. A) Photomicrographs of representative spiral arterioles in the Ovx-OID plus Pseudo-BID deciduomas and decidua. B) Mean (± SEM, n = 4–6) spiral arteriole thickness. C) Lumen diameters of nonstimulated (control) plus stimulated (implantation stimulus) areas and nonimplantation (control) plus implantation (implantation stimulus) sites of the deciduomas and decidua, respectively. V, vessel thickness; L, lumen diameter. Star denotes a significant (P < 0.05) difference between the control and areas undergoing decidualization in response to an implantation stimulus. Bar = 25 µm.

DISCUSSION

Uterine NK cells play roles in vascular changes and in maintaining decidual integrity during pregnancy, although very little is known about what factors control their presence and maturation [20, 33, 34]. Previous studies have shown that uNK cells appear in the rodent decidua and increase in number during decidualization [11, 35]. It has been known for some time that uNK cells are present in artificially induced deciduomas [23]. Furthermore, in the human uterus, there is evidence that the presence of uNK cells is associated with endometrial decidualization [36]. Therefore, current data suggest that regardless of whether or not an embryo is present, uNK cells appear in the uterus during decidualization [37]. One could conclude from this that signals from the conceptus are not required for the presence of uNK cells and the changes that occur in the uNK cell population during decidualization. However, this conclusion appears to be premature given the limited data. Therefore, in the present study, we examined closely the uNK cell populations in the decidua and compared them to those in the deciduomas during the process of decidualization. Although many of the uNK cell changes that occur in response to decidualization are independent of the conceptus, we detected for the first time a conceptus-dependent increase in uNK cell numbers and cytokine production in uteri that were undergoing decidualization.

A focal decidualization response is observed in response to the implanting blastocyst. However, a decidualization response is seen along the entire length of the uterine horn in an oil-induced deciduoma. Therefore, it is possible that the both types of uteri contain the same total number of uNK cells, although the uNK cells are spread out along the entire length of the uterine horn in the deciduoma as compared to their distribution focally in the decidua. This suggests that the results of these experiments are due to effects of the model used for induction of the deciduomas and not the conceptus. In order to rule out this possibility, a model of artificially induced decidualization, in which the decidual response is a response to a focal deciduogenic stimulus, was incorporated into the present study. Indeed, when uNK cells were enumerated in this deciduoma, the uNK cell number was more similar to that of oil-induced deciduoma than to that of the decidua. This indicates that the decreased number of uNK cells in the deciduoma is not related to the nature of the artificial deciduogenic stimulus.

Uterine NK cells proliferate in the uterus during implantation, but little is known about how this is controlled in the rodent. Previous studies indicate that the proliferation of uNK cells in the uterus require the presence of progesterone [38–40]. We hypothesized that the conceptus-dependent increase in uNK cell numbers seen in the present study might be due to increased proliferation. However, we did not see any increase in the proliferation of uNK cells in the decidua compared to the deciduoma. Therefore, the effect of the conceptus on uNK cell number is not mediated by proliferation. An alternative explanation is that it is due to a conceptus-dependent effect on the recruitment of uNK cells into uteri that are undergoing decidualization. This is consistent with previous data that the increasing number of uNK cells in the mouse uterus is mainly due to the recruitment of uNK cell precursors from secondary lymphoid tissues and not to self-renewal of an existing uNK cell population [14]. Recently, there has been great interest in examining factors associated with leukocyte recruitment into areas of uteri that are undergoing decidualization [41, 42], and clearly more work is needed to determine the effect of the conceptus on these factors.

IL15 has been suggested to play a role in the regulation of uNK cell proliferation and is present in decidual macrophages and uNK cells [43]. In the present study, there were no differences in the levels of IL15 between the decidua and the deciduomas. This suggests that the conceptus-dependent increase in uNK cell numbers does not involve IL15 signaling. A more important and highly researched function of IL15 is its involvement in the maturation of uNK cells from immature subtype I to senescent subtype IV uNK cells [16, 17]. This was demonstrated by the use of IL15 knockout mice, which completely lack uNK cells [18, 19]. In addition, when bone marrow from IL15-deficient mice was injected into alymphoid Rag 2–/-yc–/- recipients, which do express IL15, the uNK cell populations were completely rescued [44]. When mice that lack IL15 are given exogenous IL15, the uNK cell populations are restored, which confirms the role of this cytokine in the maturation of uNK cells [18]. In the present study, we found that there were no differences in the IL15 levels between the decidua and deciduomas. In addition, very little difference was observed in uNK cell maturation between the decidua and deciduomas. These two pieces of evidence support the hypothesis that signals from the conceptus do not play a role in the maturation of uNK cells. This is consistent with previous work that suggests that uNK cell maturation is directed by maternal- but not fetal-derived factors associated with pregnancy [35, 45].

Uterine NK cells modify spiral arterioles by controlling both the dilation and thinning of the wall in spiral arterioles present in the mesometrial region of the uterus during decidualization [12]. IFNG is a major product of uNK cells and it has been established that it is responsible for these changes in the uterine vasculature associated with pregnancy [20, 46]. By Day 6 of pregnancy, IL18 is produced exclusively by uNK cells [21]. Recently, it has been demonstrated that IL18 is at least partially responsible for the induction of uNK cell IFNG synthesis leading to the pregnancy-induced spiral arteriole modifications [21]. In the present study, we found that when a conceptus was present there were significantly higher levels of both glycosylated IFNG and pro-IL18, and these higher levels correlated with the observed increase in uNK cell number. Therefore, although uNK cells are present in the deciduomas, there are fewer uNK cells that secrete glycosylated IFNG and pro-IL18 than are found in the decidua. The increased levels of glycosylated IFNG may explain, at least in part, why spiral arteriole modifications were seen only in the decidua. However, since only the biologically inactive pro-IL18 and not the active IL18 levels differed between the decidua and deciduomas, it seems unlikely that IL18 is regulating the increased glycosylated IFNG levels. Further work is necessary to determine if the conceptus has an effect on uNK cell Ifng and Il18 gene expression.

The present study strongly supports the hypothesis that the conceptus plays a critical role in regulating uNK cell populations in the uterus undergoing decidualization. These effects on uNK cell number and cytokine production may be modulated either directly or indirectly. Currently, it is unknown what signal comes from the conceptus. Notably, although prolactin-like protein A is believed to be a uNK cell regulatory signal from the conceptus [47, 48], mice that are deficient for this protein do not show differences in the distribution of uNK cells [49]. Therefore, it is unlikely that this is the signal and more research is needed to determine what signals are involved.

In conclusion, the conceptus probably has an effect on uNK cell recruitment. It has been shown that the conceptus is not involved during the initial recruitment process that precedes implantation in rodents [37]. However, past studies have shown that the increase in uNK cell number during pregnancy in the mouse is mainly due to the recruitment of uNK cell precursors from secondary lymphoid tissues [14]. Therefore, we are currently investigating the role of the conceptus in the process of recruitment of uNK cells during mouse pregnancy.

ACKNOWLEDGMENTS

The Division of Statistics and Research Consulting, Southern Illinois University School of Medicine provided recommendations regarding the use of the chi-square analyses.

FOOTNOTES

1Supported by NIH grant HD049010. Back

Correspondence: 2FAX: 618 453 1517; e-mail: bbany{at}siumed.edu

Received: 19 August 2006.

First decision: 7 September 2006.

Accepted: 5 December 2006.

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