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BIOLOGY OF REPRODUCTION 68, 766–771 (2003)

© 2003 by the Society for the Study of Reproduction, Inc.

Embryo

A Role for {alpha}vß3 Integrin During Implantation in the Rabbit Model1

Maria J. Illeraa, P.L. Lorenzoa, Yao-ting Guib, Stanley A. Beylerc, K.B.C. Apparaoc, and Bruce A. Lessey2,c

a Departamento de Fisiologia, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Spain b Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada R2E 3J7 c Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The study of implantation has been facilitated by the identification of specific biomarkers that are associated with uterine receptivity. The {alpha}vß3 integrin is a cell surface adhesion receptor, whose expression has been shown to be elevated in the endometrium at the time of implantation in both humans and other mammalian species; however, the distribution of {alpha}vß3 in the rabbit model is unknown. The rabbit has been shown to be an excellent model for the study of implantation. As an obligate ovulator, the timing of pregnancy can be precisely established, and embryonic attachment occurs through specialized trophoblast-endometrial structures known as trophoblastic knobs. In the present study, the expression of {alpha}vß3 integrin subunit in the rabbit uterus was examined by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and in situ hybridization. Expression of the {alpha}vß3 integrin was examined in Day 6.5 embryos, flushed from pregnant does. Immunofluorescence demonstrated strong immunostaining on the rabbit blastocyst (Day 6.5). RT-PCR analyses showed higher levels of mRNA for ß3 subunit at the implantation site, with reduced expression in nonimplantation sites and in nonpregnant adult and immature endometrium. Immunohistochemistry demonstrated little, if any, ß3 immunoreactivity on the endometrial epithelium. In contrast, in situ hybridization showed expression of the ß3 integrin subunit mRNA in the uterine myometrium and on the trophoblast. To further determine the functional significance of {alpha}vß3 integrin expression during implantation, pregnant female rabbits that underwent ventral laparotomy on the morning of Day 6 received intrauterine injection of the following into the right uterine horn: 1) the monoclonal {alpha}vß3 neutralizing antibody (LM609), 2) arg-gly-asp (RGD) hexapeptides (GRGDSP), 3) non-RGD hexapeptides (GRGESP), and 4) IgG isotype matched control antibody. The left horn served as a control and received only saline injections. A significant reduction in the number of implantation sites was observed in the horns receiving anti-{alpha}vß3 antibody (P < 0.001) and the RGD peptides (P = 0.03). In the rabbit, the {alpha}vß3 integrin is present on the embryo and trophoblast and appears to be involved in early embryo-maternal interaction.

embryo, implantation, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Despite many years of investigation, factors responsible for successful implantation have remained undefined. Attachment of the embryo to the maternal endometrium is thought to be an active process, facilitated by the attainment of a period of uterine receptivity. This interval, now known as the window of implantation, was first suggested by McLaren and Michie [1], Psychoyos [2], and Finn and Martin [3]. Subsequent studies in animal models greatly refined this concept [4–6]. Indeed, considerable progress has been made based on studies on comparative placentation [7], and in some instances, the differences between species has been very informative [8]. Recent studies have identified several key factors that may contribute to this period of maximal uterine receptivity, including uterine ultrastructural components called pinopods [9], cytokines and growth factors such as LIF (leukemia inhibitory factor) [10], CSF-1 [11], prostaglandins [12], and other factors [13–15]. In addition, molecules have been identified that appear to interfere with implantation, such as the mucin MUC-1 [16, 17].

Based on a receptor-mediated model of implantation [18] there has been a search for receptor-ligand molecules that could account for embryo-maternal cell interactions. We and others have examined integrins as markers of uterine receptivity [19–21] and have found alterations in the endometrial integrin profile of women with infertility [22–24]. The {alpha}vß3 integrin is a promiscuous receptor that recognizes and binds several different extracellular matrix ligands, including fibronectin, vitronectin, osteopontin, and thrombospondin, through the three amino-acid sequence arg-gly-asp (RGD). RGD peptides have been shown to play a role in trophoblast attachment and outgrowth [25] and have been postulated to play a role in implantation [26, 27]. In addition, this {alpha}vß3 integrin appears to be critical for angiogenesis [28] and has been implicated in placental invasion into the maternal vasculature [29].

The rabbit appears to be an excellent model to study the molecular events of implantation. As an obligate ovulator, pregnancy can be precisely timed, and several biochemical markers have been described that define the period of receptivity in this species [30–32]. In addition, the points of blastocyst attachment to the uterine epithelium are unique structures, known as trophoblastic knobs, and are readily identifiable during early pregnancy [33]. Recently, this model has been studied to examine the expression of several endometrial biomarkers during implantation, including MUC-1 [17] and VEGF [34]. The present study was undertaken to investigate whether the {alpha}vß3 integrin is present in the embryo-maternal environment during the implantation window in the rabbit and to further determine whether perturbation of this cell adhesion molecule would disrupt implantation in this species.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

With the approval of our respective institutional guidelines, adult New Zealand female rabbits (n = 18) were housed in the animal care facility at the School of Veterinary Medicine in Madrid, Spain, or in the animal care facility at the University of North Carolina in Chapel Hill. Animals were maintained on a 12L:12D schedule and fed ad libitum. Females were naturally inseminated with a buck of proven fertility. The day of coitus was counted as Day 0 of pregnancy. Unmated female rabbits (both immature and mature) were used as nonpregnant controls. Uteri were harvested from some animals by laparotomy at the time they were killed on Day 7 of pregnancy. Animals underwent midventral laparotomy on the morning of Day 6 and were given intrauterine injections of 0.5 ml of saline containing one of the following: 1) 100 µg/ml of LM609 (monoclonal IgG1 anti-{alpha}vß3 integrin neutralizing antibody, generously provided by Dr. David Cheresh, Scripps Research Institute, La Jolla, CA; n = 6), 2) 500 µg/ml of RGD peptide (arg-gly-asp) containing the hexapeptides (GRGDSP; Gibco-BRL, Grand Island, NY; n = 4), 3) 500 µg/ml of a non-RGD peptide (GRGESP; n = 4), 4) 100 µg/ml IgG isotype-matched control antibody against human ß1 integrin (provided by Dr. David Cheresh; n = 4). Injections were performed close to the uterotubal junction into the right uterine horn; the left horn in each animal served as a control, receiving 0.5 ml of saline (0.9% NaCl). All animals were killed on Day 10 of pregnancy by i.v. injection of pentobarbital. The uterus was removed and the number of implants counted in each uterine horn. LM609 is an IgG1 monoclonal antibody that has been shown to block the intact {alpha}vß3 integrin and prevent binding to ligand. It was initially developed against human integrin but cross-reacts with rabbit [35].

Endometrial samples were obtained from pregnant and nonpregnant, mature and immature controls by opening the uterine horn and scraping the endometrium. Samples collected for RNA were weighed and immediately snap frozen in liquid N2 and stored at -70°C until use. Intact tissues were used for immunohistochemistry and in situ hybridization and were immediately immersed in liquid N2 and stored at -70°C until cryosectioning. Formalin-fixed paraffin-embedded samples were also processed for histologic examination. Collection of embryos was performed from mature pregnant female rabbits (n = 2) killed by lethal i.v. injection of pentobarbital 6.5 days 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).

Immunohistochemistry

To investigate the localization of {alpha}vß3 integrin expression in the rabbit endometrium, we performed immunohistochemistry as previously described [19] using {alpha}vß3-specific monoclonal antibody (LM609). Briefly, 6-µm cryosections were placed on poly-L-lysine-coated glass microscopic slides and fixed in formaldehyde (4% v/v) in PBS. Nonspecific binding sites were blocked with 2% normal goat serum for 30 min at room temperature. After washing three times with PBS, the slides were incubated with primary antibody (LM609; 10 µg/ml) overnight at 4°C. Immunoreactive protein was then detected using a fluorescein isothiocyanate (FITC)-conjugated anti-mouse secondary antibody (Vector Laboratories, Burlingame, CA) for 1 h at room temperature at a dilution of 1:100. Following multiple washes, coverslips were applied and the resulting staining was evaluated on a Nikon microscope (Tokyo, Japan) with or without fluorescence at 200x magnification.

Embryos were collected from Day 6.5 pregnant does as described above and transferred to organ culture dishes (Falcon; Becton Dickinson, Lincoln Park, NJ) that contained culture medium with 1 mg/ml protease (Sigma Chemicals, St. Louis, MO). Following a 20-min incubation at room temperature to digest the outer mucin coat, the hydrolyzed mucin was 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. After washing three times with PBS, the embryos were put in 2% normal goat serum for blocking nonspecific binding before being incubated with anti-{alpha}vß3 antibody (LM609) or an equal concentration of nonimmune mouse serum (control) for 18 h at 4°C. Embryos were then washed three times with PBS prior to further incubation with FITC-labeled secondary anti-mouse antibody for 1 h at room temperature 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 (Olympus Corp., Tokyo, Japan) at standard FITC settings. Images were captured digitally with the aid of NIH videomicrograpy software (NIH Image Software, version 1.61; National Institutes of Health, Bethesda, MD)).

Reverse Transcription-Polymerase Chain Reaction

Total RNA isolated from rabbit endometrium using the Tri Reagent (Molecular Research Center, Cincinnati, OH) was reverse transcribed and cDNA was subjected to PCR using primers specific for ß3 integrin subunit (sense: 5'-GGAAAGATTGGCTGGAGGAA-3'; antisense 5'-GGCATACCCCACACTCAAAG-3'). The housekeeping gene glyceraldehyde phosphate dehydrogenase (GAPDH)-specific primers (sense: 5'-TCACCATCTTCCAGGAGCGA-3'; antisense: 5'-CACAATGCCGAAGTGGTCGT-3') were used as an internal control. Reverse transcription (RT) was performed in a final volume of 20 µl with 5 µg of total RNA using a RT system (Promega, Madison, WI). RT mainly included an incubation period of 15 min at 42°C with oligo(dT) primer followed by incubation for 5 min at 99°C to denature the enzyme. The cDNA templates were then diluted (1:10) with nuclease-free sterile water, and 10 µl of diluted cDNA was PCR amplified in a 50 µl volume using ß3-specific primers in one tube, and GAPDH-specific primer was added in an another tube to serve as an internal control. In preliminary experiments, we optimized the amount of cDNA subjected to PCR as well as the number of cycles and showed that the cDNAs of interest were amplified linearly between 15 and 35 cycles of PCR. PCR amplification using ß3-specific primers resulted in an amplicon size of 312 base pairs (bp). The PCR reaction mixture consisted of 1x PCR buffer, 2.0 mM MgCl2, 200 µM each dNTPs, 1.25 U Taq DNA polymerase (Promega), and 50 pM of each primer. PCR amplification was carried out as follows: After an initial denaturation at 94°C for 5 min, 35 cycles consisting of 94°C (1 min), 55°C (1 min), and 72°C (2 min) were followed by 10 min of final extension at 72°C. The PCR products were electrophoretically resolved on 1% agarose gel and photographed.

In Situ Hybridization

In situ hybridization was performed essentially as described previously [36]. Briefly, frozen sections (10 µm) were mounted onto poly-L-lysine-coated slides and stored at -70°C until use. Upon removal from -70°C, slices were placed on a slide warmer (37°C) for 2 min and then fixed in 4% paraformaldehyde in PBS for 15 min at 4°C. A 683-bp cDNA fragment of human ß3 integrin subunit cloned into pCR2.1 vector (Invitrogen) was used as a template for the SP6-directed (sense) or T7-directed (antisense) cRNA probes. Following prehybridization, hybridization was carried out in a humidified chamber using 35S-labeled antisense complementary RNA probes specific for ß3 integrin subunit for 4–5 h at 42°C. After hybridization, the coverslips were removed by washing in 4x SSC (standard saline citrate) 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, Rochester, NY). Exposure was carried out for 7–14 days at 4°C. The slides were counterstained with hematoxylin and eosin. Representative dark and bright fields were photographed at 200–400x magnification on a microscope (Olympus).

Statistics

A null hypothesis was developed to test the major research question of the study. The number of implantation sites was compared between the experimental uterine horn and the control horn using a paired t-test. An alpha level of 0.05 was used in testing the hypothesis that the perturbation of the {alpha}vß3 integrin would not alter implantation efficiency in this model system. Based on power analysis and the assumption that perturbation of {alpha}vß3 would reduce the implantation rate by 50%, we calculated that four animals per group would be the minimum number necessary to reject the null hypothesis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
RT-PCR primers specific for the ß3 subunit of the {alpha}vß3 integrin were used to investigate the expression of ß3 integrin subunit in the rabbit endometrium obtained from different stages of the reproductive cycle. Little to no expression was observed in immature and nonpregnant endometrium (Fig. 1). In contrast, increased ß3 subunit expression was seen in pregnant endometrium, with greatest expression at the implantation site. The identity of this PCR product was confirmed to be ß3 by DNA sequence analysis (data not shown), with 89% homology to the human gene. This sequence was submitted to GeneBank (accession no. AF184591). The amount of RNA used in RT-PCR was normalized by GAPDH (lower panel).



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  		FIG. 1. RT-PCR analysis of the ß3 integrin subunit expression (upper panel) in rabbit endometrium from pregnant and nonpregnant animals. The lower panel represents the amplification of GAPDH, which was used as an internal loading control in each lane. Endometrial tissues were obtained from different stages, as indicated above the gel. The Control lane represents PCR in the absence of cDNA, with no specific product detected

Immunohistochemical staining for the {alpha}vß3 integrin in rabbit endometrium demonstrated no staining on the glandular or luminal epithelium (not shown). In contrast, rabbit embryos stained strongly for the {alpha}vß3 integrin (Fig. 2). Further, in situ localization revealed that ß3 mRNA expression is restricted to the myometrium and to the embryo. As shown in Figure 3, A and B, Day 8 trophoblast cells expressed the ß3 integrin subunit with expression along the embryo and at the sites of uterine epithelial and trophoectoderm contact (trophoblastic knobs; arrowheads). Low and high power of these structures are shown in Figure 3, C and D.



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  		FIG. 2. Photomicrographs of immunofluorescence for the {alpha}vß3 integrin subunit in a rabbit embryo flushed from uterine horns on Day 6.5 of pregnancy. Note that the immunofluorescence for the {alpha}vß3 integrin subunit was strong on the rabbit embryos incubated with anti-{alpha}vß3 monoclonal antibody, LM609 (D; x200). A control embryo, incubated in the absence of primary antibody, showed no specific fluorescent staining for this integrin (C). Phase microscopy for each embryo is included (A and B)



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  		FIG. 3. In situ localization of the ß3 integrin subunit mRNA at the implantation site on Day 8 of pregnancy. A, B) Bright-field and dark-field images of the same section, depicting the hematoxylin-eosin (H-E)-stained section and in situ localization, respectively. Silver grains are localized along the embryo, including the points of attachment known as trophoblastic knobs (arrowheads). C, D) Low (x200)- and high (x400)-power H-E sections in paraffin-embedded, formalin-fixed sections in a similar view to better demonstrate the histology at the implantation site

To investigate the functional role of this integrin during early implantation, we injected bioactive compounds that have been shown to disrupt the function of {alpha}vß3 integrin [37] into the right uterine horn on Day 6.5 of pregnancy. This is thought to be the time when embryos attach in this species. The results of these experiments are shown in Figure 4. As noted, we found a significant reduction in the number of implantation sites in LM609-injected horns, with a mean of 1.2 implants, compared with 4.5 implants in the saline-injected horn (P < 0.001). This reduction was more dramatic than that seen in the RGD-injected animals. In this group, a mean of 2.3 implants was found in the right horn (RGD-injected horn) compared with 4.7 implants in the left horn, reaching statistical significance (P = 0.03). There was no apparent effect of non-RGD peptides or IgG control antibody on implantation rates in similarly treated animals. When implants did occur in the LM609- or RGD-treated horns, they always appeared distal to the site of injection (closer to the cervix), suggesting that the volume of the injected substances (0.5 ml) may not have been sufficient to uniformly neutralize endometrial-embryo interaction along the entire uterine horn (Fig. 5).



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  		FIG. 4. Comparison of implantation rates in rabbit model receiving intrauterine injections of {alpha}vß3 integrin function-blocking factors on Day 6.5 of pregnancy. We injected 0.5 ml of two different bioactive substances that have been shown to disrupt the function of the {alpha}vß3 integrin [37] into the right uterine horn on the day of implantation. 1, LM609 antibody (100 µg/ml); 2, RGD peptide (500 µg/ml GRGDSP; Gibco). As a control, we injected a non-RGD peptide at a concentration of 500 µg/ml (GRGESP; Gibco) and IgG isotype-matched control antibody at a concentration of 100 µg/ml



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  		FIG. 5. A representative photomicrograph of a resected uterus from a pregnant rabbit on Day 10 following injection of blocking {alpha}vß3 integrin antibody (LM609). Note the reduced number of implantation sites in right horn, which were located distal to the site of injection as compared with the left horn, where normal implantation occurred. Even though the left horn received the same volume of saline solution, it had the expected number and distribution of embryos implanted


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our null hypothesis that there would be no difference between the implantation rates in LM609-injected versus saline-injected horns was rejected. Perturbation of the {alpha}vß3 integrin using a neutralizing antibody or RGD peptides resulted in fewer embryos successfully implanting in the experimental versus the control horn. As was reported previously in the mouse [37], disruption of this integrin at the time of implantation also appears to reduce implantation efficiency in that species. In those studies, we demonstrated a dose responsiveness to both neutralizing antibodies and to the RGD peptides. Unlike the mouse and human [19], the endometrium of the rabbit does not appear to express this integrin during the window of implantation. This was a fortuitous finding and may provide further insight into the potential role of the {alpha}vß3 integrin during implantation.

Implantation is a complex process involving a myriad of biomolecular markers, including growth factors, cytokines, enzymes, receptors, extracellular matrix molecules, and cell-adhesion molecules [38]. Based on a receptor-mediated model of implantation [18], researchers have long postulated a cell-adhesion receptor(s) may mediate embryo attachment and subsequent invasion. Many candidate molecules have been suggested to be involved in human implantation, including HB-EGF/EGF receptor [39], trophinin [13], CD44 [40], and integrins [19, 20], to name a few. Integrins are well-characterized markers of uterine receptivity that have been reported in several species, including human [19, 21, 41], mouse [37], and sheep [42]. Integrins are heterodimeric glycoproteins that are present on virtually all cells [43] and have been localized to both maternal and embryonic surfaces during implantation [7, 27, 41, 44–47]. In the present study, we expand on our previous work that has focused on the temporal and spatial distribution of endometrial integrins. In the rabbit, we encountered differences in the expression of one key integrin, {alpha}vß3, compared with that reported in other species.

In the rabbit, embryos normally implant on Day 6 to 7 of pregnancy. Unlike humans, the up-regulation of {alpha}vß3 integrin could not be detected on the endometrial surface during the time of uterine receptivity. In sheep, this integrin is constitutively expressed on the conceptus and on the luminal and glandular endometrial epithelium in cyclic ewes and in early pregnancy [42]. In the mouse, this integrin is also expressed in a cycle-dependent fashion but is present in the subepithelial stroma and on the mouse blastocyst [37]. In this study, we report for the first time the expression pattern of {alpha}vß3 in rabbit endometrium and embryo. In contrast with these other models studied, we find that this integrin is only expressed on the embryo and trophoblast during early implantation.

This study may provide insight into the potential mechanisms by which embryo attachment may occur. In humans and primates, {alpha}vß3 and its ligand osteopontin are coexpressed at the time of implantation [19, 48, 49]. This RGD-containing extracellular matrix molecule is expressed in the sheep uterus as well [42] and was recently shown to be stimulated in response to progesterone in the rabbit during the time of implantation [50]. It is interesting to note that, in each species studied, osteopontin (OPN) is expressed in the glandular epithelium in response to progesterone, while its receptor, {alpha}vß3, is variably present on endometrial surfaces. In every species studied, however, the {alpha}vß3 integrin is always expressed by the embryo and trophoblast. OPN could mediate adhesion during implantation, but unequivocal demonstration of this will require a suitable model. The rabbit endometrium has been shown to express another cell-adhesion molecule on its surface at the time of implantation, namely CD44 [51]. Because CD44 has been shown to bind to OPN [52, 53], it is possible that OPN could serve as a bridging molecule between coordinately expressed receptors on the embryo and maternal epithelium. The rabbit is a unique model, with {alpha}vß3 and CD44 on opposing epithelial surfaces, and therefore becomes a valuable animal system in which to further explore this hypothesis regarding the sandwich theory of implantation.

In summary, these studies in the rabbit underscore the variability of molecular interactions that may occur during implantation between species. Using this model, we demonstrate for the first time that the {alpha}vß3 integrin is important for implantation in this species. The precise role for embryonic {alpha}vß3 and its ligands or counterreceptors is not yet known but may include cell signaling, attachment, or regulation of innate immunity during implantation. We believe that the rabbit is a useful model to study these potential roles of this integrin and its complementary adhesion molecules (i.e., CD44) and extracellular matrix ligands (i.e., OPN) during this process. Such studies will likely facilitate our understanding of implantation in the human and provide new avenues for research into both contraception and infertility diagnosis and treatment.


   ACKNOWLEDGMENTS
 
The authors would like to thank Jining Zhang and Palestrina Truong for their technical assistance.


   FOOTNOTES
 
1 This research was supported by NICHD/NIH through cooperative agreement U54 HD-35041 (B.L.) 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.L.), and the Fogerty International Fellowship award (Y.G. and K.B.C.A.). Back

2 Correspondence: Bruce A. Lessey, Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, CB #7570, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599. FAX: 919 966 5214; e-mail: lessey{at}med.unc.edu Back

Received: 11 September 2001.

First decision: 3 October 2001.

Accepted: 2 September 2002.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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