The Estrous Cycle Modulates Small Leucine-Rich Proteoglycans Expression in Mouse Uterine Tissues

Authors

  • Renato M. Salgado,

    1. Laboratory of Reproductive and Extracellular Matrix Biology, Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil
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  • Rodolfo R. Favaro,

    1. Laboratory of Reproductive and Extracellular Matrix Biology, Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil
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  • Sebastian San Martin,

    1. Centro de Investigación en Biología de la Reproducción, Facultad de Medicina, Universidad de Valparaíso, Valparaíso, Chile
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  • Telma M.T. Zorn

    Corresponding author
    1. Laboratory of Reproductive and Extracellular Matrix Biology, Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil
    • Department of Cell and Developmental Biology, ICB-USP, Av. Lineu Prestes, 1524. CEP-05508-900, São Paulo, Brazil
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    • Fax: 55 (11) 3091-7309.


Abstract

In the pregnant mouse uterus, small leucine-rich proteoglycans (SLRPs) are drastically remodeled within a few hours after fertilization, suggesting that ovarian hormone levels modulate their synthesis and degradation. In this study, we followed by immunoperoxidase approach, the presence of four members of the SLRP family (decorin, lumican, biglycan, and fibromodulin) in the uterine tissues along the estrous cycle of the mouse. All molecules except fibromodulin, which predominates in the myometrium, showed a striking modulation in their distribution in the endometrial stroma, following the rise in the level of estrogen. Moreover, notable differences in the distribution of SLRPs were observed between superficial and deep stroma, as well as between the internal and external layers of the myometrium. Only biglycan and fibromodulin were expressed in the luminal and glandular epithelia. All four SLRPs were found in cytoplasmic granules of mononucleated cells. The pattern of distribution of the immunoreaction for these molecules in the uterine tissues was found to be estrous cycle-stage dependent, suggesting that these molecules undergo ovarian hormonal control and probably participate in the preparation of the uterus for decidualization and embryo implantation. In addition, this and previous results from our laboratory suggest the existence of two subpopulations of endometrial fibroblasts that may be related to the centrifugal development of the decidua. Anat Rec, 2008. © 2008 Wiley-Liss, Inc.

The four stages of the estrous cycle (proestrus, estrus, metaestrus, and diestrus) can be easily identified by a colpocytologic examination of the female tract (Allen, 1927; Shorr, 1941). Diestrus, a quiescent period influenced by progesterone, presents low uterine growth. At proestrus, anabolic growth begins through proliferation of uterine cells until the end of estrus, characterized by high levels of estrogen. Metaestrus is a catabolic phase accompanied by shrinkage of the uterus (Kim et al., 2005), probably caused by extracellular matrix (ECM) degradation by matrix metalloproteinases (MMPs) and apoptosis (see review in Curry and Osteen, 2001). In addition, estrogen (E2) increases the uterine metabolic activity, influencing the synthesis of mRNA of different molecules, such as specific enzymes, growth factors, and ECM components. Small amounts of progesterone are produced by the granulosa cells of the Graafian Follicle during the estrous cycle, before ovulation and prior to the corpus luteum formation (Guttenberg, 1961; Drummond et al., 2007). Both estrogen and progesterone promote differentiation of uterine tissues, preparing them to blastocyst implantation. In the adult animal, E2 produced during estrus stimulates epithelial cell proliferation and synthesis of progesterone receptors (PR). Progesterone, however, inhibits epithelial proliferation while stimulates the multiplication of stromal cells that characterizes the beginning of decidualization (Martin and Finn, 1969).

The ECM is a complex structure of macromolecules capable of self-assembly and is composed predominantly of collagens, noncollagenous multiadhesive glycoproteins, elastin, hyaluronan, and proteoglycans (PGs) (Kresse and Schönherr, 2001). The endometrial ECM plays important roles in endometrial decidualization, embryo implantation, trophoblast cell invasion, and the maintenance of gestation (Iwahashi et al., 1996). Previous reports have documented the remodeling of ECM molecules in the mouse endometrium during early pregnancy as for example, the accumulation of heparan sulfate proteoglycans in the uterine epithelium, immediately before blastocyst implantation (Morris et al., 1988).

Decorin, biglycan, lumican, and fibromodulin are members of a family of small leucine-rich-proteoglycans (SLRPs) of the extracellular matrix (Hocking et al., 1998; Iozzo, 1999). The SLRP family comprises about 17 genes that share structural homologies, such as cysteine residues, leucine rich repeats, and at least one glycosaminoglycan side chain. These proteoglycans are divided into five distinct classes. Decorin and biglycan belong to class I, presenting similarities in their amino acid sequence, in the chondroitin or dermatan sulfate side chains and a typical cluster of cysteine residues at the N-termini that form two disulfide bonds. Fibromodulin and lumican belong to class II, both presenting keratan sulfate and polylactosamine side chains, as well as clusters of tyrosine-sulfate residues at their N-termini (Schaefer and Iozzo, 2008). Previous data showed that some of these molecules, besides participating in the process of fibrillogenesis, act as a growth factors reservoir in the ECM, modulating cell proliferation (Vogel et al., 1984; Hocking et al., 1998).

We have previously shown (San Martin et al., 2003a) that decorin, biglycan, lumican, and fibromodulin vary in the endometrium according to the period of pregnancy. These changes certainly require exposure to a defined hormonal regimen, followed by a triggering stimulus provided by the implanting embryo (Finn, 1977; Parr and Parr, 1989; Abrahamsohn and Zorn, 1993).

Most available data about the relationship between proteoglycans and the sexual hormones were obtained from studies in human, rat, bovine, and ovine uteri. Germeyer et al. (2007) studied four syndecan isoforms in the human uterus during the menstrual cycle and showed that syndecan-1 and syndecan-4 increased in the secretory phase in comparison with the proliferative phase. Unexpectedly, however, there are few studies on proteoglycans in the mouse uterus during the estrous cycle. Potter and Morris (1992) showed that syndecan was differentially expressed on uterine epithelial cells in the different phases of the murine estrous cycle, and Julian et al. (2001) demonstrated the expression of heparan sulfate interacting protein/ribosomal protein L29 (HIP/RPL29) on the surface of different uterine cell types.

The aim of this work is to characterize the distribution of the SLRPs decorin, lumican, biglycan, and fibromodulin in the endometrium and myometrium in the different stages of the murine estrous cycle. Our hypothesis is that the structural and functional changes observed in the uterine tissues during the estrous cycle are probably orchestrated by the ovarian hormonal profile, which may influence the expression and organization of these SLRPs in the mouse uterus during the estrous cycle. In addition, the present results will be correlated with those already obtained by our group during early pregnancy in the mouse.

MATERIALS AND METHODS

Tissue Collection

Twenty Swiss mice, aged 2–4 months, were used in this experiment. Animals were housed in a 12-hr light: 12-hr dark, temperature-controlled (22°C) environment, with free access to food and water. The stages of the estrous cycle were determined by vaginal smears. The secretion was smeared on a glass slide, fixed in ether/absolute ethanol (1:1), air-dried, and stained by Shorr method (Shorr, 1941). Animals in proestrus, estrus, metaestrus, and diestrus were anesthetized with an intraperitoneal injection of tribromoethanol (Avertin®) (Aldrich Chemical Company, Milwaukee, WI; 0.025 mL/g body weight). The uteri were subsequently removed, cut with razor blades, and immediately immersed in a fixative solution. National guidelines for laboratory animal care were followed, and all experiments were approved by the Institute of Biomedical Sciences Animal Ethics Committee (authorization number, 144/2002).

Light Microscopy Processing

Samples were fixed at 4°C for 3 hr in Methacarn (absolute methanol, chloroform, and glacial acetic acid; 6:3:1), rinsed with absolute ethanol, and embedded in Paraplast (Oxford, St. Louis, MO) at 60°C. Samples were cut into 5 μm sections, adhered to glass slides using 0.1% poly-L-lysine (Sigma, St. Louis, MO), and then dried at 37°C. To confirm the stages of the estrous cycle, sections were stained with haematoxylin-eosin.

Antibodies

Table 1 lists the antibodies and enzymes used in this study. Decorin, biglycan, and fibromodulin antibodies (LF-113, LF-159, and LF-150, respectively) were raised in rabbit and recognize the core protein of each macromolecule. These antibodies were previously tested by Western blot by the producers. Detail of the procedures made by Larry Fisher (National Institute of Dental and Craniofacial Research, NIH, Bethesda, USA) may be found in Fisher et al. (1995).

Table 1. The antibodies and enzymes used in this study
Primary ABSpecificityEnzymatic pretreatmentWorking dilution primary ABWorking dilution secondary AB
  • 1, 2

    Dr. Larry Fisher (National Institute of Dental and Craniofacial Research, NIH, Bethesda, MD).

  • 2

    Seikagaku Corp. (Tokyo, Japan).

  • 3

    R&D Systems (Minneapolis, MN).

Polyclonal LF-1131Anti-decorin (IIPYDPDNPLISMC-[LPH])0.2 U Chase ABC21:3,000Anti-rabbit IgG-1:2,000
Polyclonal LF-1591Anti-biglycan (VPDLDSVTPTFSAMS-[LPH])0.2 U Chase ABC21:1,000Anti-rabbit IgG-1:2,000
Polyclonal LF-1501Anti-fibromodulin (CDKVGRKVFSKLRHLER-[KLH]) and (CDPYDPYPYEPSEPYPYGVEE-[KLH])0.2 U Chase ABC21:400Anti-rabbit IgG-1:2,000
Polyclonal3Anti-lumican (whole molecule)0.2 U Chase ABC21:1,500Anti-goat IgG-1:2,000

The anti-mouse lumican antibody (R&D Systems, #AF2745) is an IgG produced in goats, immunized with purified, NS0-derived, recombinant mouse Lumican (rmLumican). The mouse lumican specific IgG was purified by affinity chromatography. The specificity was tested by direct ELISA and Western blot.

Immunoperoxidase Procedure

The immunoperoxidase staining was performed as previously described (San Martin et al., 2003a). Sections were treated with 3% (vol/vol) H2O2 in PBS (30 min) to block endogenous peroxidase activity. Each of the succeeding steps was followed by a thorough rinse with PBS. All steps were performed in a humidity chamber. As shown in Table 1, samples were pretreated with Chondroitinase ABC from Proteus vulgaris (Seikagaku, Tokyo, Japan), diluted in 20 mM pH 6.0 Tris-HCl buffer (1 hr at 37°C). Nonspecific staining was blocked by incubating the sections for 1 hr with normal rabbit serum (for lumican) or goat serum (for the other molecules), diluted 1:1 (vol/vol) in PBS–10% BSA (wt/vol) (room temperature). Sections were then incubated with primary antibodies diluted in PBS containing 0.3% (vol/vol) Tween 20, overnight (4°C). After extensive rinsing in PBS, all sections were incubated for 1 hr at room temperature with biotin-conjugated secondary antibodies (see Table 1) diluted in PBS, for 1 hr at room temperature. After rinsing in PBS, sections were incubated with Vectastain ABC kit (Vector Labs, Burlingame, CA) for 1 hr at room temperature. Peroxidase reaction was visualized using 0.03% (wt/vol) 3,3′-diaminobenzidine in PBS with 0.03% (vol/vol) H2O2. To achieve standardization of the immunoreactions, for each antibody, the slides from all four estrous cycle stages were simultaneously incubated with DAB and the reaction was immediately interrupted with PBS after a specific period (1–5 min, depending on the antibody). After immunostaining, sections were lightly counterstained with Mayer's haematoxylin (Merck, Darmstadt, Germany). For each immunocytochemical reaction, controls were performed by replacing the primary antibodies with the respective nonimmune serum at similar concentrations or by omitting the primary antibody step from the protocol. In addition, sections of mouse embryos were used as positive and negative control. Sections were examined in a Nikon Eclipse E600 microscope and the images were captured using a digital camera (Cool SNAP-Procf color; Roper Scientific, Trenton, NJ) and Image Pro Plus software (Media Cybernetics, Silver Spring, MD).

RESULTS

Immunolocalization

In general, two morphologically distinct compartments were identified in the endometrial stroma in all estrous cycle stages, herein denominated superficial and deep stroma. The superficial stroma underlying the luminal epithelium was formed by four or five layers of round shaped and compactly arranged endometrial fibroblasts. The deep stroma, situated between the superficial stroma and the myometrium, was composed by elongated and loosely arranged endometrial fibroblasts.

Decorin

In proestrus, decorin was detected as a brownish reaction underlining the luminal epithelium and no immunoreaction was detected in the intercellular spaces surrounding the endometrial fibroblasts of the superficial stroma. Contrarily, in the deep stroma decorin immunoreaction was observed as a network of thin filaments surrounding the stromal cells. In the myometrium, the immunoreaction was strong in the internal layer, surrounding smooth muscle cells and inside the cytoplasm. In the external layer, however, the immunoreaction was exclusively present surrounding muscle cells. Decorin was also detected in the connective tissue between myometrium layers, particularly around blood vessels. One unexpected result was the presence of immunoreactive granules in the cytoplasm of round-shaped mononucleated cells, probably mast cells, present in the myometrium (Fig. 1a–d).

Figure 1.

Immunoperoxidase for Decorin. (ad) Proestrus: (a) immunolabeling is seen underlining the luminal epithelium (arrows); (b) contrarily, strong immunoreactivity is present in the deep stroma (DS), forming a network of immunoreactive fibrils surrounding blood vessels and glands; (c) interface between DS and the IML. Immunoreactivity is present in the cytoplasm (arrow-head) and surrounding some smooth muscle cells (arrows); (d) in EML, the immunoreaction surrounds bundles of muscle cells (arrows) and is present in the connective tissue (arrow-heads); (eh) Estrus: (e) except by the luminal and glandular epithelia, the entire SS is intensely immunoreactive. Note the network of brownish fibrils filling the extracellular spaces; (f) in the DS, the immunoreaction is maintained strong. Note, however, that the immunostained fibrils become parallel and loosely arranged toward the myometrium (arrows); (g) as in proestrus, the immunoreaction is maintained inside (arrow-head) and surrounding muscle cells (arrow) of the myometrium; (h) in EML, the immunoreactivity is observed exclusively surrounding muscle cells (arrows). LE, luminal epithelium; UG, uterine gland; BV, blood vessel; SS, superficial stroma; DS, deep stroma; IML, internal layer of the myometrium; EML, external layer of the myometrium. Scale bars: 20 μm.

In estrus, the immunoreaction was strong and broadly distributed in the whole stroma, as a network of thin fibrils, particularly in the deep stroma, where it is observed as loosely arranged fibrilar structures. In the myometrium, reaction for decorin was maintained exclusively in the cytoplasm of smooth muscle cells of the internal layer. Nevertheless, immunoreaction was found surrounding muscle cells of both layers, as well as in the connective tissue between them. Immunoreaction was present in the cytoplasm of round-shaped mononucleated cells (Fig. 1e–h).

In metaestrus, the immunoreaction for decorin was strong in the entire stroma, forming a dense network around cells and endometrial glands, mainly in the deep stroma. In the internal layer of the myometrium, the pattern of distribution for decorin was similar to the previous stage. Apparently, the immunostaining increased surrounding bundles of smooth muscle cells of the external layer, as well as in the connective tissue between layers. Surprisingly, round-shaped immunoreactive mononucleated cells were not found in this stage (Fig. 2a–d).

Figure 2.

Immunoperoxidase for Decorin. (ad) Metaestrus: (a) in the SS, the immunoreaction is strong. However, the immunostained network of fibrils observed in estrus is no longer present; (b) in the DS, the immunoreaction is maintained strong and compact, occupying the entire extracellular spaces; (c) the IML was maintained similar to estrus; (d) In the EML, the immunoreaction is significantly increased compared with estrus. However, the immunoreaction is restricted to the connective tissue surrounding sheaths of muscle cells (arrows); (eh) Diestrus: (e) no immunoreaction is observed in the most superficial stroma; (f) DS is intensely immunoreactive, showing a network of brownish fibrils, particularly surrounding blood vessels; (g) in the IML, some muscle cells are immunoreactive to decorin (arrows); (h) in the EML, the immunoreaction pattern is similar to metaestrus, although moderate. LE, luminal epithelium; UG, uterine gland; BV, blood vessels; SS, superficial stroma; DS, deep stroma; IML, internal muscle layer; EML, external muscle layer. Scale bars: 20 μm.

In diestrus, decorin was concentrated in the deep stroma and only traces of immunoreaction were found in the superficial stroma. Strong immunoreaction was observed around uterine glands and blood vessels, as a network of thin filaments. In the myometrium, similarly to proestrus and estrus, the immunoreactivity was found in the cytoplasm of smooth muscle cells, exclusively in the internal layer. Moderate immunoreactivity was present surrounding cells of the external layer. In the connective tissue between layers, the immunostaining was seen as fragmented brownish fibrils. In addition, the immunoreactive round-shaped cells were again found scattered in the connective tissue of the myometrium (Fig. 2e–h).

Epithelial cells were non immunoreactive to decorin in all stages of the estrous cycle (Figs. 1a,e and 2a,e).

Lumican

In proestrus, lumican was strongly stained in the superficial stroma, observed as loosely arranged delicate filaments. The deep stroma was also strongly immunoreactive, showing a dense network of brownish fibrils around cells, glands, and blood vessels, and displaying a parallel arrangement to the myometrium. In the myometrium, the immunoreaction was strong and formed an extensive network of thin filaments surrounding muscle cells in both internal and external layers, as well as in the cytoplasm of these cells. Strong immunoreaction was found in the connective tissue. Lumican was immunodetected in granules of round-shaped mononucleated cells, present in the connective tissue of the myometrium (Fig. 3a–d).

Figure 3.

Immunoperoxidase for Lumican. (ad) Proestrus: (a) In the SS, the immunoreaction is strong and observed as thin brownish fibrils; (b) The reaction is maintained strong in the DS and is observed as bundles of parallel reactive fibrils that fulfill the extracellular spaces; (c) in the IML, immunoreaction is seen in the cytoplasm (arrows) and surrounding some muscle cells (arrow-head); (d) In the EML, the immunoreactivity is strong and is also present in the cytoplasm of muscle cells (arrow-head); (eh) Estrus: strong immunoreaction is present in both SS (e) and DS (f). In the DS, the immunoreaction reveals a loose network of thin brownish fibrils; (g) in the IML, the immunoreaction is present in the cytoplasm of some cells (arrow-head), as well as filaments surrounding muscle cells (arrows); (h) in the EML, the immunoreaction is high inside (arrow-head) and outside (arrow) muscle cells. LE, luminal epithelium; UG, uterine gland; BV, blood vessels; SS, superficial stroma; DS, deep stroma; IML, internal muscle layer; EML, external muscle layer. Scale bars: 20 μm.

In estrus, the immunoreaction was strong in the entire endometrial stroma, as a conspicuous network, particularly around uterine glands. The immunoreactive fibrils were loosely arranged in the deep stroma. In the myometrium, the immunoreaction appeared stronger than in proestrus and was present in the cytoplasm of muscle cells in both layers, particularly strong in the external layer. The immunoreaction was also strong surrounding smooth muscle cells and in the connective tissue. Immunoreactivity remained in the granules of mononucleated cells (Fig. 3e–h).

In metaestrus, the immunoreaction was abundant in the entire stroma, particularly around glands and the luminal epithelium. In the myometrium, a moderate immunoreaction was detected surrounding muscle cells, as well as in the connective tissue. In contrast, no immunoreaction was found inside granules of mononucleated cells (Fig. 4a–d).

Figure 4.

Immunoperoxidase for Lumican. (ad) Metaestrus: Except by the luminal and glandular epithelia, lumican was intensely immunoreactive in both SS (a) and DS, fulfilling the extracellular spaces (b). Note in (b) immunoreactive fibrils surrounding uterine glands, which show mitosis figures (arrows); (c) immunoreaction in the cytoplasm of muscle cells of the IML; (d) Contrarily, no immunoreactivity is observed in the cytoplasm of cells of EML (arrow), surrounded by an immunoreactive connective tissue (arrows); (eh) Diestrus: (e) the immunoreaction is present in both SS and DS. However, it is stronger in the DS (f), where it forms a network surrounding the endometrial cells; (g) in the IML, the immunoreaction is weak and some reaction is surrounding muscle cells (arrow); (h) In EML, however, the immunoreaction is strong and is present in both cytoplasm (arrow-head) and surrounding muscle cells (arrows). A network of stained fibrils is connecting both muscle layers (asterisk). LE, luminal epithelium; UG, uterine gland; BV, blood vessels; SS, superficial stroma; DS, deep stroma; IML, internal muscle layer; EML, external muscle layer. Scale bars: 20 μm.

In diestrus, although present in the entire stroma, the immunoreaction was stronger in the deep region, forming a network around cells. In the myometrium, a weak and discontinuous reaction was observed surrounding muscle cells of the internal layer. Nevertheless, a strong immunoreaction was found in the cytoplasm and surrounding the muscle cells of the external layer. Similar to estrus, a network of immunostained fibrils was found in the connective tissue. Curiously, again immunoreaction was detected in granules of mononucleated cells (Fig. 4e–h).

Similarly to decorin, lumican was never detected in epithelial cells in any stage of the estrous cycle (Figs. 3a,e and 4a,e).

Biglycan

In proestrus, the immunoreaction for biglycan was restricted to the deep stroma, where it was observed as brownish thin fibrils, concentrated around blood vessels. In the myometrium, immunoreaction was strong in the cytoplasm of muscle cells of the internal layer and absent in cells of the external layer. The connective tissue was weakly immunoreactive. As observed for the other molecules, biglycan was immunodetected in granules of mononucleated cells present in the myometrium (Fig. 5a–d).

Figure 5.

Immunoperoxidase for Biglycan. (ad) Proestrus: (a) traces of immunoreaction are seen in SS; (b) in the DS, the immunoreaction is prominent surrounding blood vessels (BV); (c) Strong immunoreaction is seen in the cytoplasm of cells of the IML (arrows); (d) In the EML, the immunoreaction is restricted to the connective tissue between layers (arrows). Note two round-shaped immunolabeled cells between layers (arrow-heads); (eh) Estrus: (e) biglycan is observed on the surface of luminal epithelial cells (LE) (arrow-head) and underlining the epithelium (arrow). The reaction is present in both SS (e) and particularly in the DS (f), where the immunoreaction is seen as thin fibrils. (g) The immunoreaction is strong in the IML, particularly outside the cells (arrows); (h) Compared with proestrus, the immunoreaction is stronger in the EML, where it is surrounding the bundles of muscle cells (arrows). LE, luminal epithelium; UG, uterine gland; BV, blood vessels; SS, superficial stroma; DS, deep stroma; IML, internal muscle layer; EML, external muscle layer. Scale bars: 20 μm.

In estrus, reaction for biglycan was observed in the apical cytoplasm of luminal epithelial cells. Contrarily to proestrus, biglycan was immunodetected in the entire endometrial stroma. The reaction was, however, weak in the superficial stroma and stronger in the deep stroma. In the myometrium, the immunoreaction was strong in the internal layer. However, in this stage, most of the reaction appeared to be outside the cells. A weaker immunoreaction was found in the external layer, particularly surrounding bundles of muscle cells, and in the connective tissue (Fig. 5e–h).

In metaestrus, the reaction was maintained in the apical cytoplasm of luminal epithelial cells and was observed as a brownish line underlining the luminal epithelium. The immunoreaction was still strong in the deep stroma, particularly around uterine glands. In the myometrium, the immunoreaction was present in both layers surrounding smooth muscle cells. In the connective tissue between layers, the immunoreaction seemed to be stronger than that observed in estrus. Staining for biglycan was observed in mononucleated cells (Fig. 6a–d).

Figure 6.

Immunoperoxidase for Biglycan. (ad) Metaestrus: (a) immunoreactivity is seen in the apical cytoplasm of luminal epithelial cells (LE) (arrow-head), and as a brownish line underlining the luminal epithelium (arrow). In the SS, the immunoreaction is rare, increasing toward the DS; (b) Note immunoreactive fibrils surrounding an uterine gland (UG) (arrow-head); (c) Immunoreaction is seen in the IML, surrounding smooth muscle cells (arrows); (d) in the EML, immunoreactivity is observed surrounding sheaths of muscle cells (arrows), as well as in the connective tissue between layers (asterisk); (eh) Diestrus: (e) Immunoreaction is observed in the apical cytoplasm of the luminal epithelium (arrow-head) and is very faint in the SS; (f) Note that the immunoreactivity increases toward the deep stroma. Stained fibrils are seen surrounding blood vessels (BV); (g) the immunoreaction is intense, especially surrounding the cells of the IML (arrows); (h) the connective tissue between layers (arrows) is strongly labeled. LE, luminal epithelium; UG, uterine gland; BV, blood vessels; SS, superficial stroma; DS, deep stroma; IML, internal muscle layer; EML, external muscle layer. Scale bars: 20 μm.

In diestrus, immunoreactivity for biglycan was also found in the apical cytoplasm of luminal epithelial cells. In the deep stroma, the immunostained area was significantly enlarged toward the lumen, and only traces of immunoreaction were found in the most superficial stroma. The immunoreaction was notable around blood vessels. In the myometrium, the immunoreaction was found in the cytoplasm and surrounding muscle cells, and it was stronger in the internal layer. Similar strong reaction was present in the connective tissue between layers, especially around blood vessels. Mononucleated cells were immunoreactive to biglycan (Fig. 6e–h).

Fibromodulin

Fibromodulin was not detected in the stroma during the four stages of the estrous cycle.

In proestrus, immunostaining for fibromodulin was weakly detected in the apical cytoplasm of both luminal and glandular epithelia. Leukocytes scattered between the fibroblasts were immunoreactive to fibromodulin. In this stage, fibromodulin was moderately expressed inside muscle cells of the external layer. Only a weak reaction was seen in the internal layer, and in the connective tissue, mainly around blood vessels. Immunolabeled mononucleated cells were observed (Fig. 7a–d).

Figure 7.

Immunoperoxidase for Fibromodulin. (ad) Proestrus: (a) The apical cytoplasm of luminal epithelial cells (arrow-head) is weakly labeled; (b) in the DS, only some leukocytes (arrows) are immunoreactive and traces of reaction are seen in the glandular epithelium (arrow-head); (c) weak immunoreaction is seen inside muscle cells of the IML (arrow); (d) moderate immunoreaction is present inside muscle cells of the EML (arrow-heads), as well in the connective tissue between layers (asterisk), where immunolabeled round-shaped mononucleated cells are observed (arrows); (eh) Estrus: (e–f) immunoreaction is strong in the apical cytoplasm of luminal and glandular epithelial cells (arrow-heads); (g-h) Note the strong immunoreactivity inside muscle cells of both internal and external layers of the myometrium (arrow-heads). Observe that in the connective tissue the immunoreactive fibrils seem to be fragmented (asterisk). LE, luminal epithelium; UG, uterine gland; BV, blood vessels; SS, superficial stroma; DS, deep stroma; IML, internal muscle layer; EML, external muscle layer. Scale bars: 20 μm.

In estrus, the immunoreaction was markedly strong in the apical cytoplasm of luminal and glandular epithelial cells. Fibromodulin was strongly reactive in the cytoplasm of smooth muscle cells of the internal and external layers of the myometrium. In the connective tissue, the immunoreaction was seen as a network of thin fibrils, which seemed fragmented. Interestingly, in the myometrium but not in the endometrium, immunoreaction was detected in round-shaped mononucleated cells (Fig. 7e–h).

In metaestrus, the reaction was weak in luminal and glandular epithelia. In the myometrium, the immunoreaction appeared less intense compared with estrus. In this region, a moderate and diffuse reaction was found mostly outside muscle cells, in both internal and external layers. Immunoreactive cells were observed neither in the myometrium nor in the endometrial stroma (Fig. 8a–d).

Figure 8.

Immunoperoxidase for Fibromodulin. (ad) Metaestrus: (a) apical cytoplasm of luminal epithelial cells (arrow-head) shows immunoreactivity for fibromodulin; (b) glandular epithelium shows a weak reaction for fibromodulin (arrow-head); (c-d) moderate immunoreactivity is observed, mostly surrounding muscle cells of both internal and external layers of the myometrium (arrows); (eh) Diestrus: (e–f) the immunoreaction is present in the cytoplasm of luminal and glandular epithelial cells (arrow-heads); (g-h) immunoreaction is observed inside muscle cells, particularly intense in the external layer (arrow-heads). LE, luminal epithelium; UG, uterine gland; BV, blood vessels; SS, superficial stroma; DS, deep stroma; IML, internal muscle layer; EML, external muscle layer. Scale bars: 20 μm.

In diestrus, the immunoreaction increased in luminal and glandular epithelia and was maintained absent in the stroma. In the myometrium, strong immunoreaction reappeared inside smooth muscle cells, particularly in the external layer. Interestingly, the immunoreaction was neither detected in the connective tissue between layers nor surrounding muscle cells. Some labeled granulated cells were observed in the connective tissue (Fig. 8e–h).

DISCUSSION

SLRPs in the Endometrium

Our data show for the first time the distribution of the SLRPs decorin, lumican, biglycan, and fibromodulin in the uterus of mice in each stage of the estrous cycle. Moreover, the organization and distribution of these molecules in the uterine tissues were found to be estrous cycle-stage dependent, suggesting that these molecules undergo ovarian hormonal control and probably participate in the preparation of the uterus for decidualization and embryo implantation.

We found that decorin and lumican were expressed in the whole stroma in estrus and metaestrus, stages of estradiol predominance. Interestingly, in proestrus and diestrus—high progesterone levels—decorin was restricted to the deep stroma, and lumican was diminished in the superficial stroma. Similar distribution was observed by San Martin et al. (2003a) in the preimplantation period in the mouse. This result supports our hypothesis that the deposition of these proteoglycans appears to be related with both hormonal profile and the region of the endometrial stroma.

We have previously documented that, in mice, remodeling of the extracellular matrix begins early in pregnancy, characterized by synthesis, degradation, and alteration of collagen fibrillogenesis, accompanied by pregnancy-stage dependent expression of collagen, glycosaminoglycans and proteoglycans. Interestingly, decorin is abolished from the superficial stroma after decidualization and lumican is maintained, although in very low levels (Zorn et al., 1995; San Martin et al., 2003a, b; Spiess and Zorn, 2007). In fact, studies from Vogel and Trotter (1987) showed that decorin and lumican link to fibrilar collagens in vitro, stabilizing thin fibrils. During pregnancy, the loss of decorin and reduction of lumican are related to the appearance of thick collagen fibrils in the mature decidua. We do not know yet whether these molecules participate in collagen fibrillogenesis during the estrous cycle.

Decorin is also known to control cell division and to stop cell growth of neoplastic cells of different origins by its ability to bind TGF-β (Hildebrand et al, 1994; Santra et al., 1997). We speculate that the absence of decorin in the superficial stroma in diestrus may be favorable to the cellular proliferation in this region. Furthermore, overexpression of decorin regulates the distribution of several matrix metalloproteinases (MMPs) and cytokines by gingival fibroblasts, evidencing its role in tissue metabolism (Al Haj Zen et al., 2003).

Biglycan was found exclusively in the deep stroma in all stages of the estrous cycle, except in estrus, where it is present in both superficial and deep stroma. Similarly, biglycan is distributed preferentially in the deep stroma of non decidualized interimplantation sites. However, in the implantation sites, biglycan appears in the decidualized stroma on day 4 of pregnancy, remaining around decidual cells until day 8 (San Martin et al., 2003a). These data may suggest that the extracellular matrix architecture is spatial, temporal, and functionally modulated in the uterus of mice.

Among the studied SLRPs, fibromodulin was the only one to be absent in the endometrial stroma. Fibromodulin was absent in the mouse antimesometrial pregnant stroma until the beginning of decidualization when it is weakly expressed in the deep stroma (San Martin et al., 2003a). Fibromodulin has been found mainly in dense connective tissues. Interestingly, in the connective tissue of papillary gingiva, fibromodulin was detected, along with biglycan and lumican, mainly in the deep region (Alimohamad et al., 2005). These results highlight differences between distinct types of connective tissues, showing that the deposition of these molecules is tissue especific as previously shown by our group in developing embryonic tissues (Miqueloto and Zorn, 2007). Moreover, our results on the distribution of proteoglycans reinforce the regional differences between superficial and deep stroma.

Similar distinct regional distribution was found for other ECM molecules in the endometrial stroma of mice during the preimplantation period. Grecca et al. (1998) showed differences in safrannin O positive network, when superficial stroma is compared with deep endometrial stroma; Stumm and Zorn (2007) showed that fibrilin-1 declines in the superficial stroma only in diestrus; Spiess and Zorn (2007) studied the distribution of collagens I, III, and V in the mouse pregnant endometrium, showing that collagen III was the only one present at the maternal-fetal interface, suggesting that this interface needs a specific molecular composition, favorable to embryo implantation and development. Those and the present results suggest a predefined regionalization of the mouse endometrium into superficial and deep stroma that may be related to the centrifugal development of the decidua (reviewed by Oliveira et al., 1998).

Studies by Lee and Jeung (2007) showed the distribution of TRPV6, a calcium-regulating protein, in the mouse uterus during the estrous cycle and pregnancy. TRPV6 was highly expressed in estrus, an E2-dominant stage. On the contrary, CAPB-9K, another calcium-regulating protein, was found to increase in diestrus and to be induced after progesterone replacement therapy (Kim et al., 2006). All these data together reinforce the idea of a dynamic organization of uterine tissues, including their extracellular matrices. They also demonstrate that ECM molecules are under control of the ovarian steroid hormones, which alternate periodically during the estrous cycle, regulating uterine structure and function. High resolution autoradioautography demonstrated a highly differential binding of 3H-estradiol to luminal and glandular epithelia with region- and time-specific changes of related effects on cell proliferation, differentiation, and secretion, probably involving involution and remodeling (Zorn et al., 2003). Similar region-and time specific changes are observed in the endometrial compartments (manuscript in preparation). These regional differences on estradiol binding may be related with the differential synthesis of proteoglycans exhibited by cells of the superficial and deep stroma.

SLRPs in the Myometrium

We found that all four SLRPs were present in the myometrium. However, we observed notable changes in their expression and degradation, according to the estrous cycle stage. Fascinatingly, we also observed that each muscle layer of the myometrium has a particular behavior concerning the expression of proteoglycans and the estrous cycle stage.

Decorin had an intriguing distribution in the myometrium. In all stages of the estrous cycle, it was present inside muscle cells of the internal layer, whereas immunoreaction was never present inside cells of the external layer, suggesting that only the cells of the internal layer are committed with synthesis and secretion of decorin. Interestingly, the immunoreaction for decorin around bundles of cells of the external layer seems to be stronger in metaestrus than in proestrus and estrus. However, in diestrus, the immunoreaction was weakly detected. These results indicate an intense remodeling of decorin in the myometrium that is probably modulated by the hormonal levels. Differently from decorin, biglycan was immunolocalized inside of cells from the internal or the external layer only in proestrus and diestrus, respectively. This is a very intriguing behavior that suggests the existence of a fine control specific for each one of the myometrium layers. Both decorin and biglycan are known as TGF-β binding molecules modulating the proliferative capability of this growth factor (Hildebrand et al., 1994). It is possible that synthesis and degradation of these molecules may be related with proliferation of muscle cells. In fact, the morphological observation shows intense modifications in the thickness of the myometrium layer along the estrous cycle (data not shown).

An interesting and dynamic relationship between fibromodulin and lumican was observed. These two molecules were alternately expressed inside and outside smooth muscle cells according to the stage of the estrous cycle, showing a dynamic synchrony between synthesis, secretion, and degradation of both molecules. In fact, Svensson et al. (1999) studied the ratio between lumican and fibromodulin and estimated it to be 1:3, in normal mice tendons. In absence of fibromodulin, the amount of lumican was multiplied by four. This suggests that lumican and fibromodulin compete for the same binding site on collagen fibrils and that these proteoglycans may exert important roles in smooth muscle biological processes, such as cell proliferation and differentiation, possibly under control of ovarian hormones.

A previous study conducted by Levens et al. (2005) showed that gonadotropin-releasing hormone analogue (GnRHa) influences the differential expression of fibromodulin in the myometrium at the proliferative and secretory phases of the menstrual cycle, suggesting again that this proteoglycan is under the control of ovarian hormones. Notwithstanding, the functional relevance of fibromodulin and the other proteoglycans in the myometrium needs to be better understood.

SLRPs in Glandular and Luminal Epithelia

Curiously, only biglycan and fibromodulin were immunodetected in the epithelial tissues. Proteoglycans have been previously observed in epithelial cells in other models. Schaefer et al. (2000) evidenced the presence of biglycan in glomerular endothelial cells and in distal tubular cells of the kidney, whereas Qian et al. (2003) showed that fibromodulin was strongly expressed in gingival epithelia. These data suggest that these molecules might be synthesized or internalized by some epithelial cells via specific receptors.

In addition to playing structural roles, SLRPs have been reported to interact with molecular regulators, such as EGF, TGF-β, and TNFα. Their glycosaminoglycan chains enable these proteoglycans to provide a sink for growth factor accumulation, thus modulating cell metabolism. By binding to growth factors, SLRPs influence and regulate cell functions, such as adhesion, migration, proliferation, differentiation, and apoptosis. Therefore, they may induce intracellular signaling cascades through cell-ECM interactions (Yamaguchi and Ruoslahti, 1988; Hildebrand et al., 1994; Roughley, 2006). Fibromodulin is known to be responsible for TGF-β retention in the ECM. In fact, Levens et al. (2005) demonstrated that TGF-β increased five-fold the expression of fibromodulin in the myometrium of human uteri. Burton-Wurster et al. (2003) reported that TGF-β modulates the synthesis and accumulation of decorin, biglycan, and fibromodulin in cartilage. Moreover, there are indications that the interaction between decorin and TGF-β is competitively inhibited by biglycan (Hocking et al., 1998). Decorin binding is thought to neutralize TGF-β biological activity. Nevertheless, due to the reversibility of this interaction, decorin also acts as a local reservoir for TGF-β in tissues (Ruoslahti and Yamaguchi, 1991).

CONCLUDING REMARKS

In summary, we showed that remodeling of proteoglycans occurs in the mouse uterus and may be modulated by the dynamic cycle of ovarian hormones production. Furthermore, we may expect that cycles of synthesis and degradation of SLRPs may be correlated with inhibition and activation of growth factors, consequently modulating uterine growth along the estrous cycle. These results support previous ones from our group, which showed remodeling of proteoglycans in the mouse uterus a few hours after fertilization. Finally, this and previous results from our laboratory suggest the existence of two subpopulations of endometrial fibroblasts that may be related to the centrifugal development of endometrial decidualization. Experiments using ovariectomized animals submitted to hormonal replacement therapy are ongoing to understand whether ovarian hormones really modulate the expression of these molecules in the uterine tissues.

Acknowledgements

This study was carried out as partial fulfillment of a PhD degree by Renato M. Salgado (advisor: T.M.T. Zorn). The authors are grateful to L. Fisher (National Institute of Dental and Craniofacial Research, NIH, Bethesda, USA) for granting them with anti-decorin, anti-biglycan, and anti-fibromodulin antibodies. The authors are also thankful to Mrs. Fernanda C. Barrence and Mrs. Cleusa R. Pellegrini for the excellent technical assistance.

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