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BOR - Papers in Press, published online ahead of print December 27, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.012948
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BIOLOGY OF REPRODUCTION 68, 1951–1958 (2003)
DOI: 10.1095/biolreprod.102.012948
© 2003 by the Society for the Study of Reproduction, Inc.

Pregnancy

Osteopontin Expression in Uterine Stroma Indicates a Decidualization-Like Differentiation During Ovine Pregnancy

Greg A. Johnson1,2,3, Robert C. Burghardt2,3, Margaret M. Joyce2,3, Thomas E. Spencer2,4, Fuller W. Bazer2,4, Christiane Pfarrer5, and C. Allison Gray2,4

Center for Animal Biotechnology and Genomics,2 Department of Veterinary Anatomy and Public Health, College of Veterinary Medicine,3 Department of Animal Science,4 College of Agriculture and Life Sciences, Texas A&M University, College Station, Texas 77843 Department of Veterinary Anatomy, Histology, and Embryology,5 Justus-Liebig-University, 35392 Giessen, Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Osteopontin (OPN) is a component of the extracellular matrix that interacts with cell surface receptors, including integrins, to mediate cell adhesion, migration, differentiation, survival, and immune function. In pregnant mice and primates, OPN has been detected in decidualized stroma and is considered to be a gene marker for decidualization. Decidualization involves transformation of spindle-like fibroblasts into polygonal epithelial-like cells that are hypothesized to limit conceptus trophoblast invasion through the uterine wall during invasive implantation. Decidualization is not considered characteristic of species with noninvasive implantation, such as domestic animals. However, the extent of trophoblast invasion between sheep and pigs differs, with sheep exhibiting erosion of the uterine luminal epithelium (LE) and fusion of trophectoderm with LE to form syncytia, and pigs maintaining an intact LE throughout pregnancy. Therefore, the present study measured changes in the decidualization marker genes OPN, desmin, and alpha smooth muscle actin ({alpha}SMA) in ovine and porcine uterine stroma throughout pregnancy. The morphology of endometrial stromal cells in pregnant ewes changes following conceptus attachment, with cells increasing in size and becoming polyhedral in shape by Day 35 of pregnancy. Expression of OPN mRNA and protein, as well as desmin and {alpha}SMA proteins, was observed in this same uterine stromal compartment. In contrast, no morphological changes in uterine stroma nor induction of OPN mRNA and protein, or desmin protein, were detected during porcine pregnancy. Interestingly, {alpha}SMA protein was absent on Day 20, but prominent in uterine stroma of pregnant pigs on Day 45. Collectively, these results indicate that the uterine stroma of sheep undergoes a program of differentiation similar to decidualization in invasive implanting species, whereas porcine stroma exhibits differentiation that is more limited than that in sheep, rodents, or primates. Results suggest that uterine stromal decidualization is common to species with different types of placentation, but the extent is variable and correlates with the depth of trophoblast invasion during implantation.

Decidua, implantation, pregnancy, trophoblast, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Osteopontin (OPN) is an acidic single chain phosphorylated glycoprotein component of the extracellular matrix (ECM) that ranges in length from 264 to 301 amino acids (for a review, see [1]). The Arg-Gly-Asp (RGD) sequence of OPN interacts with cell surface receptors, including integrins, to mediate cell adhesion, migration, differentiation, survival, and immune function [2–5]. Expression of OPN by various cells in the uterus has been demonstrated in sheep, pigs, cows, mice, baboons, and humans [6–13]. Recent studies have focused on the role of epithelial-derived OPN as a progesterone-induced secretory product of uterine glandular epithelium (GE) that binds receptors on the apical surface of uterine luminal epithelium (LE) and conceptus trophectoderm to stabilize adhesion between uterus and conceptus for implantation [8, 14–17]. However, OPN mRNA and protein have also been detected in decidualized stroma of species that undergo invasive implantation, including mice, baboons, and humans [10–12]. Thus OPN is one of a number of proteins that may serve as markers of decidualization [11].

In species with invasive implantation, decidua constitutes the maternal side of the maternal-fetal interface. As such, the decidua provides an intricate balance between maternal support and fetal invasion/development that is maintained to ensure successful completion of gestation. Decidualization involves hyperplasia and hypertrophy of small spindle-like fibroblast stroma cells into enlarged polygonal epithelial-like cells [18, 19]. Functionally, decidualized stroma synthesizes and secretes many endocrine and paracrine factors that are not present in nondecidualized tissue [20, 21] and exhibits marked accumulation of desmin [22, 23], alpha smooth muscle actin ({alpha}SMA) [24], and ECM components. The end result is the formation of a morphologically and functionally distinct tissue that is a source of hormones, promotes embryo nutrition, and prevents fetal allograft rejection. In addition, decidua regulates placentation by limiting conceptus trophoblast invasion through generation of a local cytokine environment that promotes conceptus attachment over invasion [25, 26].

Conceptuses of all mammals are inherently invasive and can attach to and invade a diverse array of artificial ectopic sites and biological matrices without discrimination or need for hormonal priming [27, 28]. However, the uterine LE is unique in that it serves as a barrier to conceptus invasion until it is cyclically transformed to a receptive state that responds to embryonic signals and permits adhesive contact with conceptus epithelium [29]. Invasive implantation in humans is characterized by trophoblast adhesion to the apical glycocalyx of LE, local ECM degradation by matrix metalloproteinases, conceptus cell migration into stroma, and eventual inhibition of conceptus invasion and migration by decidua [30–32].

Decidualization is characteristic of primates and rodents with invasive implantation, but is not thought to be a property of species with central and noninvasive implantation, including livestock. One of the primary roles of decidua in invasive implanting species is to restrain conceptus trophoblast invasion to a circumscribed region of the uterine wall within a defined period of pregnancy. Both sheep and pigs have noninvasive implantation, but the extent of trophoblast invasion into the uterine wall differs between these species. Pigs have true epitheliochorial placentation in which the uterine LE remains intact throughout pregnancy [33] and maternal tissue is not penetrated by the conceptus [34]. Sheep have synepitheliochorial placentation where LE cell disintegration results in areas of epithelial erosion onto which trophectoderm binucleate cells migrate and eventually fuse with remaining epithelial cells to form syncytia; however, stromal invasion does not occur [35]. Mossman [36] and Kellas [37] described decidual cell characteristics in placentomal crypts of sheep and antelope in 1937 and 1966, respectively, but these reports have been largely ignored.

In support of these studies, we recently reported that OPN mRNA and protein expression increases in endometrial stromal cells during early implantation in the ewe [38]. Therefore, this study was to determine if, in addition to OPN, other classical markers of decidualization, including {alpha}SMA and desmin, are induced as part of a stromal response to conceptus implantation and placentation in the ewe. The expression of OPN, {alpha}SMA, and desmin were also examined and compared with true epitheliochorial placentation in the pig.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Tissue Collection

Experimental and surgical procedures complied with the Guide for Care and Use of Agricultural Animals and were approved by the Texas A&M University Institutional Agricultural Animal Care and Use Committee.

Mature western range ewes and crossbred gilts were observed daily for estrous behavior. After experiencing at least two estrous cycles of normal duration (16–18 day for ewes; 18–21 day for gilts), animals were assigned randomly on Day 0 (estrus/mating) to cyclic or pregnant status. Eighty-four ewes were ovariohysterectomized (n = 4 ewes/day) on Days 11, 13, 14, or 15 of the estrous cycle, and Days 11, 13, 14, 15, 17, 20, 25, 30, 35, 40, 45, 50, 55, 60, 80, 100, or 120 of pregnancy. Twenty-seven gilts were ovariohysterectomized (n = 3 gilts/day) on either Day 10, 15, 20, 25, 30, 35, 40, 60, or 85 of pregnancy. Days of hysterectomy were chosen to represent previously defined morphological and developmental changes in intercaruncular and caruncular regions of the uterus during pregnancy in sheep, and changes in GE development (i.e., hyperplasia followed by hypertrophy) in pigs. At hysterectomy, uteri from Days 11, 13, 15, and 17 of ovine pregnancy and uteri from Days 10 and 15 of porcine pregnancy were flushed with 0.9% NaCl to verify pregnancy by recovery of apparently normal conceptuses. Pregnancy was verified on all later days by visual observation of conceptus tissues undergoing attachment and placentation. Several sections (~1–1.5 cm) from the middle of each uterine horn were fixed in fresh 4% paraformaldehyde in PBS (pH 7.2) for 24 h and then embedded in Paraplast Plus (Oxford Labware, St. Louis, MO), whereas other sections were embedded in Tissue-Tek Optimal Cutting Temperature (OCT) compound (Miles, Oneonta, NY), snap-frozen in liquid nitrogen, and stored at -80°C.

Histological Analysis

Ovine and porcine uteri were sectioned (5 µm), deparaffinized in CitraSolv (Fisher Scientific, Fairlain, NJ), and rehydrated through a graded ethanol series to distilled water. Tissues were then exposed to a Masson trichrome staining procedure as previously described [39]. This staining procedure results in black nuclei, red cytoplasm and muscle fibers, and blue ECM components.

In situ Hybridization Analyses

Because OPN is a secretory protein that can bind to cells that do not make the protein, OPN mRNA expression in ovine and porcine uterine tissues were localized by in situ hybridization using methods previously described [6]. Deparaffinized, rehydrated, and deproteinated uterine cross-sections (5 µm) were hybridized with [35S]-radiolabeled antisense or sense cRNA probes for ovine OPN [6]. Following washes and RNAse A digestion, slides were dipped in Kodak NTB-2 liquid photographic emulsion (Eastman Kodak, Rochester, NY); stored at 4°C for 5 day; developed in Kodak D-19 developer; counterstained with Harris modified hematoxylin (Fisher Scientific); dehydrated; and protected with coverslips.

Dark-field images of endometrial sections from three different animals on Days 13, 25, 30, 35, and 40 were used to quantify silver grains as an indication of mRNA expression levels. Images of compact stroma immediately underlying LE cells were recorded with a Zeiss Axioplan2 microscope (Carl Zeiss, Thornwood, NY) fitted with a Hamamatsu chilled 3CCD color camera (Hamamatsu Corporation, Bridgewater, NJ) and Adobe Photoshop 4.0 software (Adobe Systems, Seattle, WA) and converted to grayscale. All images were recorded with the same instrument and image capture settings. The mean number of silver grains in microscopic fields of compact stroma measuring 5 x 105 µm2 from a total compact stroma area of approximately 5 x 106 µm2 from each slide were quantified using Metamorph (Universal Imaging Corp., Dowington, PA). Fields for quantitation were chosen irrespective of the presence or absence of OPN-producing immune cells. Nonspecific binding was corrected by subtracting the average number of silver grains observed in sense controls (n = 3). Differences in grain density were analyzed using ANOVA followed by Bonferroni multiple comparison test.

Immunofluorescence Analyses

Antibodies used for immunofluorescence staining included rabbit anti-OPN amino terminus (LF-123) [40]; a series of mouse monoclonal antibodies (Sigma Chemical Company, St. Louis, MO) against desmin (clone DE-U-10), {alpha}SMA (clone 1A4), vimentin (clone V9), and cytokeratin 8.13 (clone K8.13); a fluorescein-conjugated goat antibody against rabbit IgG (Chemicon International, Temecula, CA); and a fluorescein-conjugated goat antibody against mouse IgG from Zymed (San Francisco, CA).

Proteins were localized in frozen ovine and porcine uterine cross-sections using methods previously described [7]. Briefly, tissues were fixed in -20°C methanol, permeabilized with 0.3% Tween 20 in PBS, blocked in 10% normal goat serum, incubated overnight at 4°C with 2 µg/ml primary antibody, and detected with fluorescein-conjugated secondary antibodies. Slides were overlaid with a coverglass and Prolong antifade mounting reagent (Molecular Probes, Eugene, OR). Photomicrographs of representative fields of in situ hybridization and immunofluorescence staining were evaluated using a Zeiss Axioplan2 microscope as described previously. Photographic prints were prepared electronically using a Kodak DS8650 color printer (Eastman Kodak). In the interest of brevity in presenting in situ hybridization and immunofluorescence results, only a single representative control image from one of the days studied is present in the figures. The control panel is representative of the background signal detected by sense cRNA probes or irrelevant IgGs on all of the days examined.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Histoarchitecture of Ovine Uterine Stroma

A decrease in ECM was observed in the endometrial stromal compartment of pregnant ewes between Days 13 and 35 of pregnancy (Fig. 1A). Fibroblastic cells within the stratum compactum stroma of Day 35 uteri exhibited classic morphological characteristics of decidualization, including increased size and rounded or polyhedral shape. Similar changes were not observed on Day 40 of porcine pregnancy (Fig. 1B).



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  		FIG. 1. Representative photomicrographs of endometrium from (A) Day 13 and Day 35 pregnant ewes, and (B) Day 20 and Day 40 pregnant pigs subjected to Masson trichrome stain. This procedure stains nuclei black, cytoplasm and muscle fibers red, and ECM blue. LE, luminal epithelium; ST, stroma. Width of each field is 250 µm

Expression of Decidualization Marker Genes in Ovine Stroma

Expression of OPN mRNA was low to undetectable in fibroblastic cells of the stratum compactum stroma from all cyclic (data not shown) and Day 11–20 pregnant ewes (Fig. 2A). In addition, OPN mRNA was detected in scattered immune cells. OPN mRNA was visibly increased in stratum compactum stroma by Day 25, and detectable in stroma through Day 120 of pregnancy (Fig. 2A). OPN mRNA was present in stroma of both intercaruncular and caruncular regions of the uterus after Day 40 of pregnancy (Fig. 2A). An index of the increase in OPN mRNA levels in endometrium from in situ hybridization data is indicated by the increase in silver grains in the overall compact stromal compartment of the uterus (Fig. 2B). OPN mRNA was also present in placental immune cells and GE, but not present in vasculature.



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  		FIG. 2. In situ hybridization analysis of OPN mRNA in endometrial stroma of pregnant (P) ewes. A) Corresponding bright-field and dark-field images of endometrium from different days of pregnancy are shown. A representative section from Day 40 hybridized with radiolabeled sense cRNA probe (Sense) serves as a negative control. scST, Stratum compactum stroma; ssST, stratum spongiosum stroma; cST, caruncular stroma; icST, intercaruncular stroma; Car, caruncle; Cot, cotyledon; PL, placenta; I, placental immune cells; V, vasculature. Width of each field is 940 µm. B) Quantification of silver grains from in situ hybridization analysis of OPN mRNA in endometrial stroma. Data represents mean ± SEM silver grains per unit area (5 x 105 µm2) of compact stroma from three different animals. The number of silver grains on Days 25–40 was greater than that for Day 13 (P < 0.0001)

Immunofluorescence staining detected the presence of OPN protein in uterine stroma on Day 20 of pregnancy (Fig. 3). Immunoreactive OPN levels increased in stroma on Day 30 and remained high in both intercaruncular and caruncular regions of the uterus through Day 120 of pregnancy (Fig. 3).



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  		FIG. 3. OPN protein expression in ovine endometrial stroma of cyclic (C) and pregnant (P) ewes. OPN was detected using immunofluorescence staining of frozen sections. Compare the absence of antibody staining in Day 120 endometrium when rabbit IgG (IgG) was used to detect immunoreactive proteins. scST, Stratum compactum stroma; ssST, stratum spongiosum stroma; cST, caruncular stroma; icST, intercaruncular stroma; LE, luminal epithelium; Cot, cotyledon; PL, placenta. Width of each field is 940 µm

Desmin protein was detected at low levels in stroma beginning on Day 15 of the estrous cycle and pregnancy and was maintained through Day 25 of pregnancy (Fig. 4). Intensity of desmin immunostaining increased markedly in stroma by Days 35 and 45, and remained prominent in intercaruncular and caruncular uterine stroma through Day 120 of pregnancy (Fig. 4). {alpha}SMA protein was absent in stratum compactum stroma of the estrous cycle and early pregnancy (Fig. 5). However, {alpha}SMA expression paralleled that of desmin in uterine stroma after Day 25; i.e., staining was intense from Days 35 to 45 and prominent in both intercaruncular and caruncular stroma through Day 120 of pregnancy (Fig. 5). The induction of OPN, desmin, and {alpha}SMA occurred in all stratum compactum stroma of both intercaruncular and caruncular regions of the uterus, with no correlation to areas of direct contact between conceptus trophectoderm and uterine stromal tissues. Both desmin and {alpha}SMA were uniformly present in smooth muscle cells from endometrium and placenta.



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  		FIG. 4. Detection of desmin protein in ovine endometrial stroma from cyclic (C) and pregnant (P) ewes using immunofluorescence staining of frozen sections. ST, Stratum compactum stroma; LE, luminal epithelium; PL, placenta; V, vasculature. Width of each field is 940 µm



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  		FIG. 5. Detection of {alpha}SMA protein in ovine endometrial stroma from cyclic (C) and pregnant (P) ewes using immunofluorescence staining of frozen sections. ST, Stratum compactum stroma; LE, luminal epithelium; PL, placenta; V, vasculature. Width of each field is 940 µm

Expression of Decidualization Marker Genes in Porcine Stroma

In contrast to sheep, no changes in the stratum compactum stroma of porcine uterus were detected on Day 40 of pregnancy (Fig. 1B). OPN mRNA was not present in the stratum compactum stroma during either the estrous cycle or pregnancy in pigs (Fig. 6). OPN mRNA was only expressed in porcine uterine LE and GE during pregnancy as previously reported [8].



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  		FIG. 6. In situ hybridization analysis of OPN mRNA in endometrial stroma of pregnant gilts. Corresponding bright-field and dark-field images of endometrium from Day 40 of pregnancy are shown. A representative section from Day 40 hybridized with radiolabeled sense cRNA probe (Sense) serves as a negative control. scST, Stratum compactum stroma; LE, luminal epithelium; GE, glandular epithelium; PL, placenta. Width of each field is 940 µm

Immunofluorescence staining for OPN, desmin, and {alpha}SMA in pregnant gilts revealed protein expression patterns that diverged from one another and collectively differed from those observed in ewes (Fig. 7). As previously reported [8], OPN protein was present on the apical surface of uterine LE on Day 20 and at the uterine-placental interface on Day 45 of pregnancy. OPN protein was not detected in Day 45 pregnant stratum compactum stroma (Fig. 7). Both desmin and {alpha}SMA proteins were detected in uterine vasculature, but not in fibroblastic cells of the stratum compactum stroma on Day 20 of pregnancy (Fig. 7). By Day 45 of pregnancy prominent immunostaining for stromal {alpha}SMA but not OPN or desmin was evident in stromal cells (Fig. 7).



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  		FIG. 7. Immunofluorescence staining for OPN, desmin (Des), {alpha}SMA (SMA), cytokeratin (Cyt), and vimentin (Vim) proteins in serial frozen endometrial cross-sections from Day 20 and Day 45 pregnant gilts. Cytokeratin staining identifies the location of epithelia at the uterine-placental interface of Day 45 sections, and vimentin shows the location of uterine and placental stroma. LE, Luminal epithelium; ST, uterine stroma; PL, placenta; V, vasculature. Width of each field is 940 µm


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Sheep and pigs have noninvasive implantation; however, they differ in type of placentation. Pig conceptuses undergo true epitheliochorial placentation in which LE remains morphologically intact throughout pregnancy and the conceptus trophectoderm simply attaches to the apical LE surface without contacting uterine stromal cells [34, 35]. In contrast, synepitheliochorial placentation in sheep involves extensive erosion of the LE due to syncytium formation with trophectoderm binucleate cells. After Day 19 of pregnancy, conceptus tissue is apposed to, but does not penetrate, ovine uterine stroma. Results from this study provide evidence for a stromal decidualization-like response in the pregnant ovine and porcine uterus. Differences in endometrial OPN, desmin, and {alpha}SMA expression in these species suggest that the extent of decidualization correlates positively with the degree of conceptus invasiveness.

Because domestic animals have a central noninvasive implantation, few studies have focused on the role of and changes in the uterine stroma that underlie LE. As a result, a considerable gap has developed in knowledge of events in uterine function that may be critical for conceptus survival. Epithelial-stromal interactions have been implicated in development, growth, differentiation, and adult function of the uterus. The stroma is crucial for maintaining hormonal responsiveness, morphogenesis, and secretory function of the uterine epithelium [41, 42]. Uterine epithelial-stromal interactions are also crucial for placental growth and development [43], and establishment and maintenance of pregnancy in sheep and pigs appear to involve interdependent paracrine conceptus-endometrial and epithelial-stromal interactions [44–47]. Recently, several genes have been shown to be induced in uterine stroma of ruminants in response to conceptus-derived interferon tau (IFN{tau}). The temporal and spatial expression of these genes may be mechanistically involved with the sequential exposure of the sheep uterus to estrogen, progesterone, IFN{tau}, placental lactogen, and growth hormone hypothesized to be critical for establishment and maintenance of pregnancy [47, 48–51]. Likewise, changes in ECM and integrin expression within the uterine stroma have been correlated with time of implantation in the cow. MacIntyre et al. [52] reported increased collagen IV and laminin in subepithelial stroma through Day 24 of gestation. Interestingly, expression of the {alpha}1 integrin subunit becomes more broadly distributed in stroma, exhibiting an inverse relationship between staining intensity and distance from uterine LE [52]. Results of the present study extend our limited understanding of the uterine stromal response to conceptus implantation and placentation to implicate a decidualization-like transformation in sheep and, to a lesser extent, in pigs.

On a morphological basis, decidua is defined as tissue made up of endometrial fibroblast cells that have enlarged and become rounded or polyhedral as the result of the accumulation of glycogen or lipids within the cytoplasm and occur either in pregnancy, pseudo-pregnancy, or in artificially stimulated deciduomas [36]. A comparison of histoarchitecture between ovine endometrial sections from Days 13 and 35 of pregnancy clearly indicates a stromal transformation that conforms to this definition. In addition, the decidualization markers OPN, desmin, and {alpha}SMA are induced in pregnant uterine stroma of sheep, whereas only {alpha}SMA is induced in pigs. This provides the first molecular evidence for a decidualization-like response in these species. Although decidualization is generally not recognized in domestic animals, a few reports have suggested that this physiological phenomenon may not be limited to species with invasive implantation. Mossman [36] described rounded or polyhedral endometrial connective tissue cells within ovine placentomes. Similar morphological alterations were later reported for antelope endometrial stromal fibroblasts [37]. In contrast to sheep, there are no reports of hypertrophy and glycogen accumulation indicative of decidualization within stratum compactum stroma of pregnant gilts, even when blastocysts were transferred through the uterine wall into pockets within the endometrial stroma [53]. However, syncytial formation was observed, and stroma in the vicinity of the conceptus exhibited increased collagen deposition in this experimental model for ectopic pregnancy in pigs [53].

All mammalian conceptuses are inherently invasive [54] and share many phenotypic properties with carcinomas, including altered expression of adhesion molecules [55] and elevated expression of matrix-degrading proteinases [56, 57]. Our working hypothesis is that differential expression of OPN, desmin, and {alpha}SMA in the uterine stroma of sheep and pigs defines a decidualization-like response that correlates with the degree of conceptus invasiveness and functions to protect and limit trophoblast invasion beyond the compromised LE barrier in sheep. Transient but direct exposure of stratum compactum stroma to invading conceptus results in a decidualization-like transformation that restrains further conceptus invasion and maintains superficial implantation in sheep. There were no obvious morphological changes in porcine uterine stroma through Day 40 of pregnancy. However, limited stromal differentiation, as evidenced by stromal induction of {alpha}SMA but not OPN or desmin, occurs in pigs where conceptuses do not invade the uterine wall. Indeed, in primates and rodents trophoblast cells penetrate the uterine LE barrier to invade and confront a stromal compartment that initially has no barrier functions. However, conceptus transition from an epithelial to stromal environment triggers a series of responses within the stroma that are collectively termed decidualization [18, 19]. Stromal cells differentiate into epithelial-like decidual cells that form a chamber surrounding the conceptus [58] and restrain trophoblast invasion to a circumscribed region of the uterine wall within a defined period of pregnancy [25].

Interestingly, OPN is up-regulated in ovine (but not porcine) stratum compactum stroma that has undergone considerable loss of ECM by Day 35 of pregnancy. Although speculative, a possible explanation for this concomitant increase in OPN and decrease in overall ECM might be that stromal OPN is intracellular. OPN has generally been characterized as a protein secreted into biological fluids and luminal spaces, such as the uterine lumen [6, 7], or into the ECM where it binds receptors to mediate various cell-cell and cell-ECM interactions [1]. However, an intracellular form of OPN associated with a hyaluronin-CD44-ERM (ezrin/radixin/moesin) attachment complex and implicated in migration of embryonic fibroblasts, activated macrophages, and invasive cancer cells has been described. As such, intracellular OPN has been hypothesized to modulate CD44-mediated changes in cytoskeletal architecture involved in cell differentiation and migration [59, 60]. Analysis of the OPN species (i.e., intracellular or extracellular) associated with decidualized ovine stromal cells will require the high spatial resolution provided by electron microscopy. The nature of the potential intracellular and extracellular OPN interactions with stromal cells will be critical to understanding the function of OPN during decidualization.

It is generally accepted that the trophoblast aggressively drives the invasive process of implantation in primates and rodents. Ovine and porcine conceptuses are also inherently invasive; therefore, it is logical to surmise that maternal stromal receptivity to conceptus invasion plays a role in limiting, modulating, and accommodating this invasion in domestic animals. Collectively, recent reports from several laboratories highlight the idea that uterine stroma differentiates in response to conceptus implantation in superficially implanting species. Results from this study provide evidence for stromal decidualization-like response in the pregnant ovine uterus and suggest a heretofore unknown stromal role in limiting trophoblast invasion during a vulnerable period of placental development when the integrity of uterine LE is compromised. However, results from pigs, which maintain an intact LE, do not support the phenomenon of decidualization but suggest pregnancy-related stromal changes in the form of {alpha}SMA expression. A better understanding of stromal decidualization, as observed between humans, rodents, sheep, and pigs, may be exploited to elucidate the mechanistic nature of the various genes that increase in expression in uterine stroma as its functions change during pregnancy.


   FOOTNOTES
 
1 Correspondence: Greg A. Johnson, Department of Veterinary Anatomy and Public Health, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4458. FAX: 979 847 8981; gjohnson{at}cvm.tamu.edu Back

Received: 4 November 2002.

First decision: 22 November 2002.

Accepted: 19 December 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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