Biol Reprod Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print October 30, 2002.
Biol Reprod 2002, 10.1095/biolreprod.101.001347
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
68/5/1484    most recent
biolreprod.101.001347v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Apparao, K.B.C.
Right arrow Articles by Lessey, B. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Apparao, K.B.C.
Right arrow Articles by Lessey, B. A.
Agricola
Right arrow Articles by Apparao, K.B.C.
Right arrow Articles by Lessey, B. A.
BIOLOGY OF REPRODUCTION 68, 1484–1490 (2003)
DOI: 10.1095/biolreprod.101.001347
© 2003 by the Society for the Study of Reproduction, Inc.

Pregnancy

Regulated Expression of Osteopontin in the Peri-Implantation Rabbit Uterus1

K.B.C. Apparao3, Maria J. Illera4, Stan A. Beyler3, Gary E. Olson5, Kevin G. Osteen6, Martha H. Corjay7, Kim Boggess8, and Bruce A. Lessey2,3

Department of Obstetrics and Gynecology,3 Division of Reproductive Endocrinology and Fertility, the University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7570 Department of Physiology,4 School of Veterinary Medicine, Universidad Complutense, 28040 Madrid, Spain Department of Cell Biology,5 Vanderbilt University School of Medicine, Nashville, Tennessee 37232 Department of Obstetrics and Gynecology,6 Women's Reproductive Health Research Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 DuPont Pharmaceuticals Research Laboratories,7 Experimental Station, Wilmington, Delaware 19880-0400 Department of Obstetrics and Gynecology,8 Division of Maternal and Fetal Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7570


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Blastocyst attachment to the lining of the mammalian uterus during early implantation involves the initial apposition of the trophoblast to the uterine epithelial surface. Osteopontin (OPN) is a glycoprotein component of the extracellular matrix that is secreted by the glandular epithelium of mammalian uteri at the time of implantation. This protein is recognized by several members of the integrin family and promotes cell-cell attachment and adhesion. In the present study, rabbit uteri were examined using Northern and in situ hybridization to evaluate the temporal and spatial distribution of OPN mRNA during early pregnancy. Northern blot analysis demonstrated a dramatic increase in OPN expression on Days 4–7 of pregnancy, corresponding to the rise in circulating progesterone and the time of initial embryo attachment in this species. In situ hybridization analysis revealed OPN mRNA expression on Day 6.75 of pregnancy, which was most prominent on endometrial epithelium. Using immunofluorescence, OPN protein was present on the glandular epithelium on Day 6.75 of pregnancy, but was absent on blastocysts. Further, no expression of OPN mRNA or protein was found in the nonpregnant endometrium. Induction of endometrial OPN expression was observed in unmated rabbits treated with progesterone alone and was prevented by cotreatment with the antiprogestin ZK137.316. Estradiol-17ß had no effect on OPN expression by itself, and estrogen priming was not necessary to demonstrate the stimulatory effect of progesterone. In The rabbit uterus, as in other mammalian species studied, OPN is expressed in a stage-specific manner by the endometrial glands during the peri-implantation period and is regulated by progesterone.

embryo, implantation, pregnancy, progesterone, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The initial adherence of the nascent embryo to uterine surface epithelium is an integral part of successful implantation [1]. The surface epithelium appears to play a critical role in embryonic adhesion and invasion during the process of implantation. However, the mechanism by which uterine epithelia acquire the structural and functional properties necessary for successful implantation remains elusive. During early pregnancy, endometrial receptivity to blastocyst implantation is temporally restricted [2] and the transition from a nonreceptive to receptive state is regulated by ovarian steroids [3]. Based on a receptor-mediated concept of implantation [4], endometrial receptivity involves the acquisition of membrane components required for receptor-ligand interaction with the embryo as well as the loss of antiadhesive apical properties [5, 6]. Thus implantation likely involves complementary expression of receptors and ligands on the maternal and embryonic surfaces.

The steroid hormone-dependent establishment of endometrial receptivity is associated with a complex set of morphological transitions of the uterine lining. Interaction between trophectoderm and luminal epithelium likely initiates the implantation process. Both soluble signals and association between molecules on opposing epithelial surfaces appear to be involved [7]. A number of cell surface molecules have been implicated in the initial attachment reaction between trophectoderm and maternal surface epithelium. These include heparin-binding epidermal growth factor (HB-EGF) [8, 9], trophinin [10], CD44 [11], integrins [12, 13], and extracellular matrix molecules such as osteopontin (OPN) [14], each of which exhibit elevated levels of expression at the time of implantation.

OPN is a phosphorylated glycoprotein containing an arginine-glycine-aspartic acid (RGD) cell adhesion sequence [15]. Although originally identified in bone, OPN expression is found in a number of different tissues and has been shown to exert multifunctional biological effects [16]. In the reproductive tract, OPN expression by secretory phase endometrial epithelial cells, invading cytotrophoblasts, and placentae is temporally correlated with blastocyst invasion and placentation [17]. Recent studies in humans [14] and ewes [18, 19] have shown that OPN is abundantly expressed in the glandular epithelium of luteal-phase endometrium and its expression is regulated by progesterone [20].

Osteopontin has been shown to bind primarily to {alpha}vß3 integrin on tissues/cells via its RGD sequence and may promote cell attachment and/or cell spreading [21, 22]. The presence of {alpha}vß3 integrin both on trophoblast cells of mouse and human embryos [23, 24] and endometrium [12, 25, 26] at the time of implantation led to the hypothesis that this integrin mediates some of the molecular aspects of adhesion between the nascent embryo and the endometrium.

The rabbit appears to be an outstanding model in which to study implantation [27]. Both pseudopregnancy and pregnancy can be precisely triggered, and rabbit endometrium resembles the physiology of human endometrium of the secretory phase [28, 29]. This animal model has been used to study the expression of several endometrial proteins thought to be associated with implantation including cytokines [30, 31], haptoglobin [32], vascular endothelial growth factor (VEGF) [33], and the mucin MUC-1 [34]. We recently demonstrated that the {alpha}vß3 integrin is expressed by the rabbit embryo but not by the peri-implantation endometrium [35]. To date, OPN expression has been shown to be regulated during the menstrual cycle in humans [14], the estrous cycle in sheep [18], and early pregnancy in the mouse [36], but its expression patterns in rabbit endometrium and embryo are unknown. The present study was therefore undertaken to compare the temporal and spatial pattern of OPN expression during early pregnancy in the rabbit and to study its hormonal regulation in this species. Based on these results, we hypothesize that OPN plays a critical role in the endometrial receptivity with possible roles as a ligand for the blastocyst integrin receptor {alpha}vß3 during attachment to the surface epithelium.


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

Sexually mature New Zealand white female rabbits were used for this study. Animal protocols conformed to NIH guidelines for humane animal care, and their use in research was approved by institutional animal care and use committees at the Universidad Complutense in Madrid, Spain, and the University of North Carolina and Vanderbilt University. Rabbits used in these studies were housed in individual cages in a controlled environment with a 12L:12D cycle. Food and water were provided ad libitum. Nonpregnant rabbits were used to study the basal and regulated expression of OPN. To study the expression of OPN during early pregnancy, females were naturally mated twice to a fertile buck; the day of mating was designated as Day 0 of pregnancy. Samples of endometrium were obtained from nonmated and mated rabbits on Days 1–8 of pregnancy. Uterine tissues from these animals were used for Northern blot analyses, in situ hybridization, and immunohistochemistry. Prior to collection of the tissues, rabbits were killed by a lethal i.v. injection of pentobarbital. Endometrial samples were obtained by immediately opening the excised uterus and scraping the uterine lumen with a razor blade. Tissue for RNA and cross-sections of uteri for immunohistochemistry or in situ hybridization was immediately snap frozen in liquid N2 and stored at -70°C until use.

Embryos were collected from mature pregnant female rabbits (n = 2) killed by lethal i.v. injection of pentobarbital 6.5 d after breeding. The entire uterus was immediately excised and the uterine horns were cut below the oviduct. A 16-gauge catheter was inserted in each horn, and the embryos were flushed into a 100 mm Petri dish with 10 ml HEPES buffered Ham F-10 culture medium (Irvine Scientific, Irvine, CA) supplemented with human serum albumin 10 mg/ml (Sage Biopharma, Badmister, NJ).

RNA Isolation and Northern Blot Analysis

Total RNA was isolated from tissue using TRIzol Reagent (Life Technologies, GIBCO-BRL, Gaithersburg, MD) and quantified by absorbance spectroscopy at 260 nm, and integrity of the RNA was confirmed by electrophoretic fractionation through 0.8% agarose gel. Twenty micrograms of total RNA from each stage was aliquotted and stored at -70°C until use. Total RNA (20 µg per lane) was glyoxylated followed by fractionation through 1.0% agarose gels and blotted by capillary action onto a nylon membrane. RNA was cross-linked to the membrane by UV radiation (Stratalinker 1800, Stratagene, La Jolla, CA) using 12 x 104 µJ radiation. The membrane was prehybridized with NorthernMax-Gly hybridization solution (Ambion, Inc., Austin, TX) at 42°C for 2 h. Approximately 1.4 kb cDNA fragment encoding a full-length human OPN gene served as the template for synthesis of a labeled DNA probe using a random priming method (Random Primed DNA labeling kit, Boehringer Mannheim Biochemicals, Indianapolis, IN). The membrane was subsequently hybridized with ({alpha}-32P)dCTP (Amersham Pharmacia Biotech, Piscataway, NJ) labeled OPN probe for 16 h at 42°C. After hybridization, the membrane was washed twice at room temperature for 15 min in 2 x SSC and 0.1% SDS, and twice further, 15 min each at 42°C with 0.1 x SSC and 0.1% SDS. Autoradiography was performed using Hyperfilm (Amersham) for 24–72 h at -70°C until the desired exposure was obtained. Further, the integrity and relative amount of RNA loaded into each lane were confirmed by using a GAPDH 32P-labeled cDNA probe as a constitutively expressed marker.

Immunohistochemistry

The polyclonal antibody used to detect rabbit OPN was raised in guinea pig against the human-OPN fusion protein. This antibody has been previously characterized and shown by Western analysis and immunohistochemistry (IHC) to efficiently and specifically detect rabbit OPN [37]. Immuno-localization of OPN protein expression in endometrial cell types was performed on endometrium obtained from estrous and Day 6.75 of pregnancy. Uterine segments were placed in histologic embedding medium (Histo Prep, Fisher Scientific, Chicago, IL), frozen in liquid N2, and stored at -70°C. Five-micrometer-thick cryosections were fixed with 4% formaldehyde in 0.1 M phosphate buffer, pH 7.2, at 4°C for 30 min. Sections were rinsed in Tris-buffered saline (TN: 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) and then blocked in TN containing 2.5% BSA and 5% normal donkey serum. Sections were then incubated for 1 h in guinea pig antiserum to osteopontin diluted 1:1000 in blocking solution. Control specimens were incubated in an equivalent dilution of nonimmune guinea pig serum. After three washes in TN containing 1% serum, specimens were incubated in Cy3-donkey anti-guinea pig IgG (Jackson ImmunoResearch, West Grove, PA) diluted 1:500 in blocking solution. Specimens were then washed three times in TN and examined by phase contrast and fluorescence microscopy.

For immunofluorescent detection of OPN and {alpha}vß3 on rabbit embryos, we used blastocysts collected on Day 6.5 as described above. Embryos were transferred to organ culture dishes (Falcon, Becton Dickinson and Co., Lincoln Park, NJ) that contained culture medium with 1 mg/ml protease (Sigma Chemicals Co., St. Louis, MO). Following a 20-min incubation at room temperature, the outer mucin coat was digested and completely removed by repeated pipetting through a wide-tip transfer pipette. The embryos were then washed three times in culture medium and fixed for 20 min in 2% paraformaldehyde in PBS. Embryos were rinsed three times with PBS and incubated in 2% of normal goat serum to block nonspecific binding before being incubated with anti-{alpha}vß3 antibody (1:500; LM609; Chemicon International Inc., Temecula, CA), guinea pig antiosteopontin antibody (1:1000), or an equal concentration of nonimmune serum (control) for 18 h at 4°C. Embryos were then washed three times with PBS prior to further incubation with fluorescein isothiocyanate-labeled secondary anti-mouse or anti-guinea pig antibody (Vector Laboratories Inc., Burlingame, CA) for 1 h at room temperature (RT) at a working dilution of 1:100. Following multiple washes, embryos in each group were then examined for fluorescence immunostaining with an inverted Olympus fluorescence microscope at standard fluorescein isothiocyanate settings. Images were captured digitally with the aid of NIH videomicrography software (NIH Image Software, version 1.61, Bethesda, MD).

In Situ Hybridization

The human osteopontin cDNA clone encompassing the entire coding sequence was linearized with XbaI and XhoI for a generation of antisense and sense (control) cRNA probes using T7 and T3 polymerases, respectively. Uterine sections obtained from secretory phase human endometrium and rabbit endometrium on Day 6.75 of pregnancy were used in this study. The in situ hybridization method using 35S-labeled probes was derived from the research of Cox et al. [38] and adapted for application with formalin-fixed, paraffin-embedded tissues essentially as described previously [39]. Briefly, formalin-fixed, paraffin-embedded tissues were sectioned (7 µm) and placed onto poly-L-lysine coated slides. Tissues were deparaffinize with xylene and rehydrated through a graded series of ethanols. Hybridization was carried out in a humidified chamber using 35S-labeled sense and antisense complementary RNA probes specific for OPN overnight at 56°C. After hybridization, the coverslips were removed by washing in 4 x SSC followed by incubation with 20 µg/ml of RNase A for 30 min at 37°C. After a series of washes, the RNase A resistant hybrids were detected by autoradiography using Kodak NTB-2 liquid emulsion (Eastman Kodak Co., Rochester, NY). Exposure was carried out for 3 wk at 4°C. The slides were counterstained with hematoxylin, dehydrated through a graded series of alcohol, cleared in xylene, and coverslipped. Representative dark and bright fields were photographed at 200x magnification on a microscope (Olympus Corp., Tokyo, Japan).

Steroid Hormone Effects on Uterine Osteopontin Expression

In vivo steroid hormonal treatments were carried out on nonpregnant does, as previously described [31]. Nonmated animals were used in this study. Steroids comprising estradiol-17ß, progesterone (Sigma), and antiprogesterone ZK137.316 (Schering AG, Berlin, Germany) were first dissolved in a minimal amount of absolute ethanol and diluted to final concentration in vegetable oil. Doses of steroids were chosen on the basis that they would maintain pregnancy in ovariectomized animals. The unmated rabbits were distributed into six groups comprising three rabbits per group. The rabbits received subcutaneous injections of 0.5 ml of vegetable oil alone or with steroid hormones as follows: 1) controls; vehicle only/rabbit/d for 3 d; 2) 5µg of estradiol-17ß/rabbit/d for 3 d; 3) 5 mg progesterone/rabbit/d for 3 d; 4) 5µg of estradiol-17ß plus 5 mg progesterone/rabbit/d for 3 d; 5) 5 mg progesterone plus 15 mg of antiprogesterone ZK137.316/rabbit/d for 3 d; 6) 5µg of estradiol-17ß plus 5 mg progesterone plus 15 mg of anti-progesterone ZK137.316/rabbit/d for 3 d. Rabbits were killed by i.v. injection of pentobarbital, and the endometrium was collected for RNA isolation and Northern blot analysis as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of Osteopontin Messenger RNA in Nonpregnant and During Early Pregnancy in Rabbit Endometrium

To monitor uterine OPN expression through early pregnancy, Northern blot analysis was performed in the endometrium obtained prior to mating and from successive days of early pregnancy (Days 1–8). A representative Northern blot demonstrates that a single OPN mRNA transcript was detectable in the rabbit endometrium (Fig. 1, upper panel). OPN mRNA was present at very low levels in nonpregnant rabbits and during Days 1–3 of pregnancy. OPN expression increased on Days 4 and 5 and decreased thereafter. Very little, if any, OPN mRNA transcripts were detected by Day 8 of pregnancy. RNA integrity was confirmed by rehybridizing the same blot to a GAPDH probe (Fig. 1). These results indicate that OPN is expressed in the uterus during early pregnancy in a highly stage-specific manner between Days 4 and 7 of gestation, corresponding to the mid to late luteal phase in the human.



View larger version (54K):
[in this window]
[in a new window]
  		FIG. 1. Northern blot analysis of osteopontin (OPN) mRNA in the rabbit endometrium in nonpregnant uteri and during early pregnancy. Twenty micrograms of total RNA extracted from endometrium was probed with 32P-labeled OPN cDNA as described in Materials and Methods. The day of pregnancy (Day 0 = day of mating) is shown at the top. Note the maximum expression of OPN mRNA was detected on Days 4–5 of pregnancy (upper panel). As a control for the RNA integrity and loading, the same filter was reprobed with the constitutively expressed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA after stripping (lower panel)

In Situ Localization of Osteopontin mRNA in the Uterus

Cell-specific distribution of OPN mRNA within human secretory endometrium and from rabbits on Day 6.75 of pregnancy was examined by in situ hybridization analyses (Fig. 2). As a comparison, human endometrium expresses an OPN message during the midsecretory phase confined to both the glandular and luminal epithelium (Fig. 2, top panel). Antisense OPN probing of rabbit endometrium also defines expression in the glandular elements (Fig. 2, lower panel). Hybridization with the sense strand demonstrated no specific labeling in either human or rabbit endometrium. Tissues were counterstained with hematoxylin to enhance visualization by the light field (Fig. 2, far right).



View larger version (72K):
[in this window]
[in a new window]
  		FIG. 2. Localization of OPN mRNA expression in human (upper panel) and rabbit endometrium (lower panel) by in situ hybridization. Transverse sections of midsecretory endometrium and Day 6.75 of pregnancy from rabbits were hybridized with antisense and sense riboprobes to osteopontin. Additional serial sections were stained with hematoxylin and eosin (far right panels). Magnification x100

Immunolocalization of Osteopontin in the Rabbit Uterus

To examine the spatial patterns of expression of OPN protein in the rabbit uterus, we performed immunofluorescence staining from samples obtained from nonpregnant uteri and from Day 6.75 of pregnancy. Photomicrographs showing red immunofluorescence indicated the sites of immunostaining for OPN protein (Fig. 3, A–D). There is no specific fluorescence noted in nonpregnant rabbits (Fig. 3B). Intensive staining was noted in the uterine section of Day 6.75 of pregnancy and was limited to the glandular epithelium (Fig. 3D). No staining was detected in any cell types within the stroma or myometrium during either phase.



View larger version (135K):
[in this window]
[in a new window]
  		FIG. 3. Immunofluorescent localization of OPN protein in rabbit uterus using sections from nonpregnant endometrium and on Day 6.75 of pregnancy. Frozen sections were stained with anti-OPN as described in Materials and Methods. Red deposits indicate positive immunostaining of OPN. Day 6.75 of pregnancy sections (D) show an intense staining for OPN specifically localized on glandular epithelium. No staining is evident in underlying stroma and myometrium. In contrast very little if any staining for OPN is observed on any cell types in endometrium from nonpregnant rabbits (B). A) and C) The corresponding phase contrast photomicrographs from a nonpregnant uterus and Day 6.75 of pregnancy, respectively. Magnification x200

Steroid Hormone Regulation of Osteopontin Expression in the Rabbit Uterus

As uterine OPN expression peaked during the progesterone-dominated secretory phase in early pregnancy, it was considered that ovarian steroids were responsible for its regulation. Unmated rabbits were treated with physiologic concentration of either estradiol-17ß or progesterone alone or in combination for 3 d in the presence or absence of the antiprogestin ZK137.316 as described above. RNA extracted from the endometrium of these hormonally treated animals was subjected to Northern blot analysis for OPN. As shown in Figure 4, progesterone stimulated OPN mRNA expression. In contrast, estrogen alone had little or no effect on the expression of OPN. In animals treated with a combination of estrogen and progesterone, there was no additive effect, but OPN expression was slightly lower than that found in rabbits receiving progesterone alone. No OPN expression was detected in control animals that were injected with vegetable oil alone. The antiprogesterone ZK137.316 completely inhibited the stimulatory effects of progesterone in these studies. Taken together, the results suggest that the OPN expression in the rabbit uterus is strictly regulated by progesterone, and these effects are mediated through its nuclear receptor.



View larger version (46K):
[in this window]
[in a new window]
  		FIG. 4. Northern blot analysis of uterine OPN expression in untreated rabbits (nonmated controls) or rabbits treated for 3 d with estradiol-17ß (E2) and progesterone (P4) alone or in combinations, as well as further combination with antiprogestin ZK137.316. After 3 d, uteri were collected for RNA extraction and subjected to Northern blot analysis as described in Materials and Methods. Note that clear induction of OPN expression by P4 and the stimulatory effect is completely ablated by administration of antiproteins ZK137.316 (upper panel). Duplicate samples were obtained in some cases from different animals receiving similar treatments. As a control for the RNA integrity and loading, the same filter was reprobed with the constitutively expressed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA after stripping (lower panel). Relative absorbance normalized for loading (OPN/GAPDH) is presented in a bar graph format (upper panel)

Osteopontin and Its Receptor {alpha}vß3 Integrin in Rabbit Preimplantation Embryos

Expression of OPN and its receptor, {alpha}vß3, was studied in Day 6.5 rabbit blastocysts by immunofluorescence (Fig. 5). Relatively intense cell-surface staining for {alpha}vß3 integrin was detected on the embryo (Fig. 5F). In contrast, no immunofluorescence was noted for OPN on preimplantation embryos (Fig. 5E) and no staining was seen when primary antibody was omitted (Fig. 5D).



View larger version (101K):
[in this window]
[in a new window]
  		FIG. 5. Immunofluorescent localization of OPN and the {alpha}vß3 integrin receptor in rabbit embryos. Rabbit embryos collected on Day 6.5 of pregnancy were stained with either polyclonal guinea pig antibodies against OPN or monoclonal antibodies against the {alpha}vß3 integrin receptor (LM609) as detailed in Materials and Methods. Strong immunofluorescence staining for {alpha}vß3 integrin receptor was noted on the embryo (F) compared with very little if any staining for OPN (E) in rabbit embryos. No immunofluorescence was observed in controls when preimmune serum was used in place of the primary antibody (D). The corresponding phase contrast photomicrographs of embryos used for the {alpha}vß3 integrin receptor, OPN, and control are (C), (B), and (A), respectively. Magnification x200


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The rabbit endometrium undergoes a series of dramatic changes after ovulation in preparation for implantation. These changes include rapid proliferation of both epithelial and stromal cells under the influence of estrogen in the first 0–4 d of pregnancy. During this interval, there is a transition of steroid hormone milieu from estrogen to an increasingly progesterone dominance [40]. Progesterone levels rise during the secretory phase 4–7 d after ovulation, concomitant with epithelial cell differentiation, including the appearance of stage-specific uterine secretions and epithelial cell surface modifications [41]. These two stages, Days 0–4 and Days 4–7 of pregnancy, are analogous to the proliferative and secretory phase of the primate menstrual cycle [42]. Further, the structural and biochemical parallels between rabbit and human uteri have supported the use of rabbit, over other smaller mammals, as a unique model for the luteal phase of human endometrium [43].

Several specific endometrial gene products have been studied in the rabbit during estrous and early pregnancy, including uteroglobin [44], leukemia-inhibitory factor (LIF) [30], MUC-1 [34], and VEGF [33]. In the present study, we examined the pattern of OPN expression in this species. The results of Northern blot analysis demonstrate that OPN mRNA is expressed in a stage-dependent fashion in rabbit endometrium, increasing during the peri-implantation period. Consistent with observations in mouse [36], sheep [18, 45], and human [14], OPN mRNA levels are highest during the secretory phase, corresponding to the time of epithelial differentiation under the influence of progesterone. As demonstrated in the human [46], progesterone is essential for successful pregnancy in the rabbit [47]. Further, the rabbit uterus has been shown to be receptive to embryo attachment 5–7 d postovulation [48]. Maximal uterine OPN expression correlates well with the rise in maternal progesterone concentration at this time in pregnancy.

Cellular localization of OPN mRNA and protein demonstrated that OPN expression is restricted to the glandular epithelium of the secretory phase endometrium. No expression of OPN was observed prior to mating, and little if any OPN expression was present on the blastocyst. Similar expression patterns have been reported in the sheep [18, 44] and human [14], with OPN expression restricted to the glandular epithelium during the secretory phase in both species. Like LIF [30], haptoglobin [32], MUC-1 [34], and uteroglobin [49] in the rabbit uterus, OPN is modulated by progesterone and appears during the secretory phase of endometrial development. In the nonpregnant rabbit model, we demonstrate that OPN is dependent on progesterone, but not estrogen, and its expression in progesterone-treated animals was blocked by the addition of the antiprogestin ZK137.316.

In the human, OPN is also dependent on progesterone [14], acting perhaps through the progesterone receptor (PR)-B, rather than the PR-A, progesterone receptor isoform [50]. Interestingly, the rabbit endometrium expresses only the PR-B isoform [51], which declines on glandular and luminal epithelium around Day 5 of pregnancy [43, 44], consistent with reports in other mammalian species that down-regulation of epithelial estrogen receptor (ER) and/or PR correlates well with the time of implantation, including mouse [52], dog [53], pig [54], and human [55, 56]. Regulation of OPN by PR-B is consistent with the recent study by Mote et al. [57], which showed that PR-A and PR-B were differentially expressed in the endometrium during the menstrual cycle. Regulation of OPN by PR-B in human and rabbit endometrium is further supported by observations made using PR-A null mutant mice [58].

The timing and localization of OPN expression during the peri-implantation period suggests a potential role of OPN during early embryo attachment in both rabbits and humans. The existence of an RGD tripeptide sequence in OPN provides the binding sequence for interaction with cell surface receptors such as {alpha}vß3. Indeed, OPN has been shown to bind to a variety of integrins, including {alpha}vß1, {alpha}vß5, {alpha}4ß1, and {alpha}vß3, which is a primary receptor to promote cell adhesion and migration [22, 59]. Previous reports have demonstrated cycle-dependent expression of the {alpha}vß3 integrin receptor in the human endometrium [12, 60] and outer surface of the embryo [24]. Recent in vitro studies have further demonstrated that OPN binds to the {alpha}vß3 integrin receptor in the endometrial epithelium [14]. In the present study, we now demonstrate that this integrin is expressed only on the rabbit blastocyst at the time of pregnancy. These data suggest that the rabbit model is slightly different compared with the human, although OPN is secreted by uterine epithelial cells in both species. Unlike the human endometrium that expresses {alpha}vß3 on both the endometrial and embryonic surface epithelium [12, 24], the rabbit uterus lacks expression of {alpha}vß3 integrin on the surface or glandular epithelium [35] but maintains expression on the embryo.

Other adhesion molecules on the surface epithelium of the rabbit uterus at the time of implantation that might serve as a receptor that binds to OPN have been reported. The most likely candidate includes the hyaluronate receptor CD44 [61]. This receptor has been shown to bind OPN through a non-RGD site, providing a compelling model for cell-cell interaction involving OPN as the bridging molecule between {alpha}vß3 and CD44. Introduction of antibodies that block the {alpha}vß3 receptor or RGD peptides that could compete for OPN was previously shown to reduce implantation efficiency in the rabbit model [35]. Future studies will be focused on CD44 to test whether neutralization of this adhesion molecule also perturbs implantation in the rabbit.

In conclusion, these results indicate that OPN is expressed in a stage-specific manner in the rabbit uterus, correlating temporally with increasing concentration of plasma progesterone during the luteal phase. Further, OPN expression is induced by progesterone and antagonized by the antiprogestin ZK137.316 in vivo, indicating that OPN expression is regulated through PR. The presence of {alpha}vß3 integrin expression on the embryo and the reported expression of CD44 on the maternal surface suggests that OPN may serve as a bridging molecule between maternal and embryonic surfaces at the time of implantation. Roles for OPN in embryo signaling may not be excluded. The simplicity of the rabbit model with unilateral expression of key adhesion molecules makes this species particularly attractive for the study of embryo attachment during early pregnancy.


   ACKNOWLEDGMENTS
 
We thank Ms. Wendy Eisel for her expertise in the in situ hybridization technique.


   FOOTNOTES
 
1 Part of this work was presented at the 47th Annual Meeting of the Society for Gynecologic Investigation in Chicago, Illinois, March 22–25, 2000. This research was supported by NICHD/NIH through cooperative agreement U54 HD-35041 (B.A.L.) and HD-37321 (K.G.O., G.E.O.) as part of the Specialized Cooperative Centers Program in Reproduction Research, the National Cooperative Program on Markers of Uterine Receptivity for Blastocyst Implantation (HD-34824; B.A.L.), and the Fogerty International Fellowship award (K.B.C.A.). Back

2 Correspondence: Bruce A. Lessey, Department of OB/GYN, Division of Reproductive Endocrinology and Fertility, CB #7570, Old Clinic Building, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7570. FAX 919 966 5214; lessey{at}med.unc.edu Back

Received: 12 November 2001.

First decision: 28 November 2001.

Accepted: 18 October 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Carson DD, Bagchi I, Dey SK, Enders AC, Fazleabas AT, Lessey BA, Yoshinaga K. Embryo implantation. Dev Biol 2000 223:217-237[CrossRef][Medline]
  2. Tabibzadeh S, Babaknia A. The signals and molecular pathways involved in implantation, a symbiotic interaction between blastocyst and endometrium involving adhesion and tissue invasion. Hum Reprod 1995 10:1579-1602[Abstract/Free Full Text]
  3. Psychoyos A. Uterine receptivity for nidation. Ann N Y Acad Sci 1986 476:36-42[Medline]
  4. Yoshinaga K. Receptor concept in implantation research. In: Yoshinaga K, Mori T (eds.), Development of Preimplantation Embryos and their Environment. New York: Alan Liss; 1989: 379–387
  5. Lessey BA. Integrins and uterine receptivity. In: Carson DD (ed). Embryo Implantation: Molecular, Cellular and Clinical Aspects. New York: Springer; 1999: 210–222
  6. Surveyor GA, Gendler SJ, Pemberton L, Spicer AP, Carson DD. Differential expression of Muc-1 at the apical cell surface of mouse uterine epithelial cells. FASEB J 1993 7:1151a
  7. Kimber SJ. Molecular interactions at the maternal-embryonic interface during the early phase of implantation. Semin Reprod Med 2000 18:237-253[CrossRef][Medline]
  8. Raab G, Klagsbrun M. Heparin-binding EGF-like growth factor. Biochim Biophys Acta 1997 1333:F179-F199[Medline]
  9. Yoo HJ, Barlow DH, Mardon HJ. Temporal and spatial regulation of expression of heparin-binding epidermal growth factor-like growth factor in the human endometrium: a possible role in blastocyst implantation. Dev Genet 1997 21:102-108[CrossRef][Medline]
  10. Fukuda MN, Sato T, Nakayama J, Klier G, Mikami M, Aoki D, Nozawa S. Trophinin and tastin, a novel cell adhesion molecule complex with potential involvement in embryo implantation. Genes Dev 1995 9:1199-1210[Abstract/Free Full Text]
  11. Behzad F, Seif MW, Campbell S, Aplin JD. Expression of two isoforms of CD44 in human endometrium. Biol Reprod 1994 51:739-747[Abstract]
  12. Lessey BA, Damjanovich L, Coutifaris C, Castelbaum A, Albelda SM, Buck CA. Integrin adhesion molecules in the human endometrium. Correlation with the normal and abnormal menstrual cycle. J Clin Invest 1992 90:188-195
  13. Tabibzadeh S. Patterns of expression of integrin molecules in human endometrium throughout the menstrual cycle. Hum Reprod 1992 7:876-882[Abstract/Free Full Text]
  14. Apparao KBC, Murray MJ, Fritz MA, Meyer WR, Chambers AF, Truong PR, Lessey BA. Osteopontin and its receptor {alpha}{nu}ß3 integrin are coexpressed in the human endometrium during the menstrual cycle but regulated differentially. J Clin Endocrinol Metab 2001 86:4991-5000[Abstract/Free Full Text]
  15. Prince CW, Oosawa T, Butler WT, Tomana M, Bhown AS, Bhown M, Schrohenloher RE. Isolation, characterization, and biosynthesis of a phosphorylated glycoprotein from rat bone. J Biol Chem 1987 262:2900-2907[Abstract/Free Full Text]
  16. Denhardt DT, Guo X. Osteopontin: a protein with diverse functions. FASEB J 1993 7:1475-1482[Abstract]
  17. Brown LF, Berse B, Van de Water L, Papadopoulos Sergiou A, Perruzzi CA, Manseau EJ, Dvorak HF, Senger DR. Expression and distribution of osteopontin in human tissues: widespread association with luminal epithelial surfaces. Mol Biol Cell 1992 3:1169-1180[Abstract]
  18. Johnson GA, Burghardt RC, Spencer TE, Newton GCR, Ott TL, Bazer FW. Ovine osteopontin. II. Osteopontin and {alpha}vß3 integrin expression in the uterus and conceptus during the peri-implantation period. Biol Reprod 1999 61:892-899[Abstract/Free Full Text]
  19. Johnson GA, Bazer FW, Jaeger LA, Hakhyun K, Garlow JE, Pfarrer C, Spencer TE, Burghardt RC. Muc-1, integrin, and osteopontin expression during the implantation cascade in sheep. Biol Reprod 2001 65:820-828[Abstract/Free Full Text]
  20. Johnson GA, Spencer TE, Burghardt RC, Taylor KM, Gray CA, Bazer FW. Progesterone modulation of osteopontin gene expression in the ovine uterus. Biol Reprod 2000 62:1315-1321[Abstract/Free Full Text]
  21. Ross FP, Chapper J, Alvarez JI, Sander D, Butler WT, Farach-Carson MC, Mintz KA, Robey PG, Teitelbaum SL, Cheresh DA. Interactions between the bone matrix proteins osteopontin and bone sialoprotein and osteoclast integrin {alpha}vß3 potentiate bone resorption. J Biol Chem 1993 263:19433-19436
  22. Liaw L, Skinner MP, Raines EW, Ross R, Cheresh DA, Schwartz SM, Giachelli CM. The adhesive and migratory effects of osteopontin are mediated via distinct cell surface integrins. Role of {alpha}vß3 in smooth muscle cell migration to osteopontin in vitro. J Clin Invest 1995 95:713-724
  23. Sutherland AE, Calarco PG, Damsky CH. Developmental regulation of integrin expression at the time of implantation in the mouse embryo. Development 1993 119:1175-1186[Abstract]
  24. Campbell S, Swann HR, Seif MW, Kimber SJ, Aplin JD. Cell adhesion molecules on the oocyte and preimplantation human embryo. Hum Reprod 1995 10:1571-1578[Abstract/Free Full Text]
  25. Lessey BA, Ilesanmi AO, Sun J, Lessey MA, Harris J, Chwalisz K. Luminal and glandular endometrial epithelium express integrins differentially throughout the menstrual cycle: implications for implantation, contraception, and infertility. Am J Reprod Immunol 1996 35:195-204
  26. Illera MJ, Cullinan E, Gui YT, Yuan LW, Beyler SA, Lessey BA. Blockade of the {alpha}vß3 integrin adversely affects implantation in the mouse. Biol Reprod 2000 62:1285-1290[Abstract/Free Full Text]
  27. Hoffman LE, Breinan DR, Blaeuer GL. The rabbit as a model for implantation. In: Carson DD (ed.), Embryo Implantation: Molecular, Cellular and Clinical Aspects. New York: Springer; 1999: 151–160
  28. Beier HM. Uteroglobin and other endometrial proteins: biochemistry and biological significance in beginning pregnancy. In: Beier HM, Karlson P (eds.), Proteins and Steroids in Early Pregnancy. Berlin: Springer; 1982: 311–334
  29. Miller JB, Keyes PL. Transition of the rabbit corpus luteum to estrogen dependence during early luteal development. Endocrinology 1978 102:31-38[Abstract/Free Full Text]
  30. Yang Z-M, Le S-P, Chen D-B, Harper MJK. Temporal and spatial expression of leukemia inhibitory factor in rabbit uterus during early pregnancy. Mol Reprod Dev 1994 38:148-152[CrossRef][Medline]
  31. Yang Z-M, Chen D-B, Le S-P, Harper MJK. Differential hormonal regulation of leukemia inhibitory factor (LIF) in rabbit and mouse uterus. Mol Reprod Dev 1996 43:470-476[CrossRef][Medline]
  32. Hoffman LH, Winfrey VP, Blaeuer GL, Olson GE. A haptoglobin-like glycoprotein is produced by implantation-stage rabbit endometrium. Biol Reprod 1996 55:176-184[Abstract]
  33. Das SK, Chakraborty I, Wang J, Dey SK, Hoffman LH. Expression of vascular endothelial growth factor (VEGF) and VEGF-receptor messenger ribonucleic acids in the peri-implantation rabbit uterus. Biol Reprod 1997 56:1390-1399[Abstract]
  34. Hoffman LH, Olson GE, Carson DD, Chilton BS. Progesterone and implanting blastocysts regulate Muc-1 expression in rabbit uterine epithelium. Endocrinology 1998 139:266-271[Abstract/Free Full Text]
  35. Illera MJ, Lorenzo PL, Gui L-T, Beyler SA, Apparao KBC, Lessey BA. A role for {alpha}{nu}ß3 integrin during implantation in the rabbit model. Biol Reprod 2003; 68:766–771.
  36. Waterhouse P, Parhar RS, Guo X, Lala PK. Regulated temporal and spatial expression of the calcium-binding proteins calcyclin and OPN (osteopontin) in mouse tissues during pregnancy. Mol Reprod Dev 1992 32:315-323[CrossRef][Medline]
  37. Corjay MH, Diamond SM, Schlingmann KL, Gibbs SK, Stoltenborg JK, Racanelli AL. {alpha}vß3, {alpha}vß5, and osteopontin are coordinately upregulated at early time points in a rabbit model of neointima formation. J Cell Biochem 1999 75:492-504[CrossRef][Medline]
  38. Cox KH, DeLeon DV, Angerer LM, Angerer RC. Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev Biol 1984 101:485-502[CrossRef][Medline]
  39. Rodgers WH, Osteen KG, Matrisian LM, Navre M, Giudice LC, Gorstein F. Expression and localization of matrilysin, a matrix metalloproteinase, in human endometrium during the reproductive cycle. Am J Obstet Gynecol 1993 168:253-260[Medline]
  40. Browning JY, Keyes PL, Wolf RC. Comparison of serum progesterone, 20{alpha}-dihydroprogesterone and estradiol-17ß in pregnant and pseudopregnant rabbits: evidence for post-implantation recognition of pregnancy. Biol Reprod 1980 23:1014-1019[Abstract]
  41. Anderson TL, Olson GE, Hoffman LH. Stage specific alterations in the apical membrane glycoproteins of endometrial epithelial cells related to implantation in rabbits. Biol Reprod 1986 34:701-720[Abstract]
  42. Davies J, Hoffman LH. Studies on the progestational endometrium of the rabbit. I. Light microscopy, day 0 to day 13 of gonadotrophin-induced pseudopregnancy. Am J Anat 1973 137:423-426[CrossRef][Medline]
  43. Hegele-Hartung C, Chwalisz K, Beier HM. Distribution of estrogen and progesterone receptors in the uterus: an immunohistochemical study in the immature and adult pseudopregnant rabbit. Histochemistry 1992 97:39-50[CrossRef][Medline]
  44. Gutierrez Sagal R, Perez Palacios G, Langley E, Pasapera AM, Castro I, Cerbon MA. Endometrial expression of progesterone receptor and uteroglobin genes during early pregnancy in the rabbit. Mol Reprod Dev 1993 34:244-249[CrossRef][Medline]
  45. Johnson GA, Spencer TE, Burghardt RC, Bazer FW. Ovine osteopontin. I. Cloning and expression of messenger ribonucleic acid in the uterus during the peri-implantation period. Biol Reprod 1999 61:884-891[Abstract/Free Full Text]
  46. Baulieu EE. Contragestion and other clinical applications of RU-486, an antiprogesterone at the receptor. Science 1989 245:1351[Abstract/Free Full Text]
  47. Pickworth S, Lamming GE. Observations on pregnancy in intact rabbits and in ovariectomized rabbits treated with 17{alpha}-hydroxyprogesterone caproate. J Reprod Fertil 1967 13:457-468[Abstract/Free Full Text]
  48. Chang MC. Developmental and fate of transferred rabbit ova or blastocysts in relation to the ovulation time of recipients. J Exp Zool 1950 114:197-225[CrossRef]
  49. Beier HM. Uteroglobin and related biochemical changes in the reproductive tract during early pregnancy in the rabbit. J Reprod Fertil 1976 25:suppl53-69
  50. Apparao KBC, Chambers AF, Lessey BA. Progesterone receptor isoform-B modulates the expression of osteopontin in human endometrial epithelium during the window of implantation. Ann Meet Soc Gynecol Invest 2001 43:60A
  51. Loosfelt H, Logeat F, Vu Hai MT, Milgrom E. The rabbit progesterone receptor. J Biol Chem 1984 259:14196-14202[Abstract/Free Full Text]
  52. Tan J, Paria BC, Dey SK, Das SK. Differential uterine expression of estrogen and progesterone receptors correlates with uterine preparation for implantation and decidualization in the mouse. Endocrinology 1999 140:5310-5321[Abstract/Free Full Text]
  53. Vermeirsch H, Simoens P, Hellemans A, Coryn M, Lauwers H. Immunohistochemical detection of progesterone receptors in the canine uterus and their relation to sex steroid hormone levels. Theriogenology 2000 53:773-788[CrossRef][Medline]
  54. Geisert RD, Pratt TN, Bazer FW, Mayes JS, Watson GH. Immunocytochemical localization and changes in endometrial progestin receptor protein during the porcine oestrus cycle and early pregnancy. Reprod Fertil Dev 1994 6:749-760[CrossRef][Medline]
  55. Lessey BA, Killam AP, Metzger DA, Haney AF, Greene GL, McCarty KS Jr. Immunohistochemical analysis of human uterine estrogen and progesterone receptors throughout the menstrual cycle. J Clin Endocrinol Metab 1988 67:334-340[Abstract/Free Full Text]
  56. Garcia E, Bouchard P, De Brux J, Berdah J, Frydman R, Schaison G, Milgrom E, Berrot-Applanat M. Use of immunocytochemistry of progesterone and estrogen receptors for endometrial dating. J Clin Endocrinol Metab 1988 67:80-87[Abstract/Free Full Text]
  57. Mote PA, Balleine RL, McGowan EM, Clarke CL. Colocalization of progesterone receptors A and B by dual immunofluorescent histochemistry in human endometrium during the menstrual cycle. J Clin Endocrinol Metab 1999 84:2963-2971[Abstract/Free Full Text]
  58. Mulac-Jericevic B, Mullinax RA, DeMayo FJ, Lydon JP, Conneely OM. Subgroup of reproductive functions of progesterone mediated by progesterone receptor-B isoform. Science 2000 289:1751-1753[Abstract/Free Full Text]
  59. Hu DD, Lin ECK, Kovach NL, Hoyer JR, Smith JW. A biochemical characterization of the binding of osteopontin to integrins {alpha}vß1 and {alpha}vß5. J Biol Chem 1995 270:26232-26238[Abstract/Free Full Text]
  60. Lessey BA, Castelbaum AJ, Buck CA, Lei Y, Yowell CW, Sun J. Further characterization of endometrial integrins during the menstrual cycle and in pregnancy. Fertil Steril 1994 62:497-506[Medline]
  61. Hohn H-P, Huch G, Tlolka U, Denker H-W. Differential expression of CD44 in rabbit uterine epithelium during early pregnancy. Acta Anat (Basel) 1995 152:185-194[Medline]



This article has been cited by other articles:


Home page
 ReproductionHome page
D. W Erikson, A. L Way, D. A Chapman, and G. J Killian
Detection of osteopontin on Holstein bull spermatozoa, in cauda epididymal fluid and testis homogenates, and its potential role in bovine fertilization
Reproduction, May 1, 2007; 133(5): 909 - 917.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
F. J White, R. C Burghardt, J. Hu, M. M Joyce, T. E Spencer, and G. A Johnson
Secreted phosphoprotein 1 (osteopontin) is expressed by stromal macrophages in cyclic and pregnant endometrium of mice, but is induced by estrogen in luminal epithelium during conceptus attachment for implantation.
Reproduction, December 1, 2006; 132(6): 919 - 929.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
F. J. White, J. W. Ross, M. M. Joyce, R. D. Geisert, R. C. Burghardt, and G. A. Johnson
Steroid Regulation of Cell Specific Secreted Phosphoprotein 1 (Osteopontin) Expression in the Pregnant Porcine Uterus
Biol Reprod, December 1, 2005; 73(6): 1294 - 1301.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
M. H. Melner, N. A. Ducharme, A. R. Brash, V. P. Winfrey, and G. E. Olson
Differential Expression of Genes in the Endometrium at Implantation: Upregulation of a Novel Member of the E2 Class of Ubiquitin-Conjugating Enzymes
Biol Reprod, February 1, 2004; 70(2): 406 - 414.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
G. A. Johnson, R. C. Burghardt, F. W. Bazer, and T. E. Spencer
Osteopontin: Roles in Implantation and Placentation
Biol Reprod, November 1, 2003; 69(5): 1458 - 1471.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
68/5/1484    most recent
biolreprod.101.001347v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Apparao, K.B.C.
Right arrow Articles by Lessey, B. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Apparao, K.B.C.
Right arrow Articles by Lessey, B. A.
Agricola
Right arrow Articles by Apparao, K.B.C.
Right arrow Articles by Lessey, B. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS