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Regular Article |
4 Integrin in Mouse Blastocysts and Uterus During Implantation1
a Department of Biochemistry, All India Institute of Medical Sciences, Ansari Nagar, New Delhi-110029, India
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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4ß1 integrin by steroid hormones in the blastocyst and the receptive uterus using an in vivo, delayed-implantation mouse model system. The dormant and activated blastocysts as well as the uteri were recovered from ovariectomized mice after progesterone-alone and progesterone-plus-estrogen therapy, respectively. Immunolocalization of protein expression of 4 and ß1 integrin subunits indicate that steroids modulate the expression of 4ß1 integrin receptor in the mouse blastocyst as well as the uterus and that a differential expression is observed with exposure to progesterone and estrogen. Intrauterine blocking of 4 integrin by specific antibody resulted in implantation failure in normal as well as in delayed-implantation mice. Based on our data, we propose here, to our knowledge for the first time, that 4ß1 integrin, which is responsible for binding to fibronectin and vascular cell adhesion molecule-1, is induced by estradiol and is down-regulated by progesterone in mice during implantation. Furthermore, the results also indicate the direct role of 4 integrin in the process of implantation.
blastocyst, implantation/early development, integrin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1, 2, 4, 5, 6, 7, and ß3 have been shown to be expressed in trophoblast cells of preimplantation and implanting mouse embryos [10]. Abnormal expression of 4, v, and ß1 are reported to be associated with implantation failure and abnormal placental development [2].
Mouse embryos seem to produce a broad repertoire of ECM proteins, such as fibronectin, laminin, vitronectin, collagen, and so on, as well as ECM protein receptors, such as 5ß1, 6Bß1, and vß3, from the onset of development [10]. A variable pattern of expression of certain integrins in the murine uterus during the reproductive cycle and pregnancy suggests that these proteins may be directly involved in the process of implantation and in normal placental formation and function [9]. Although abnormal expression of 4ß1 integrin has been associated with certain unexplained cases of infertility [11, 12], the factors involved in the regulation of integrin expression during blastocyst activation and implantation are not known.
The present study aims to elucidate the regulation of 4ß1 integrin expression by steroid hormones during mouse embryo implantation. Estrogen and progesterone are necessary for implantation of the blastocyst, and the preimplantation estrogen surge is essential for implantation in mice. Ablating the surge of estrogen that occurs just before implantation prevents attachment of the blastocyst, which remains in a state of diapause [13]. Administration of estrogen induces implantation of the dormant blastocyst (i.e., delayed implantation). Therefore, we have utilized this system to study hormonal modulation of 4ß1 integrin receptor expression in mouse blastocysts and uterus during implantation and, thereby, to try to delineate the complex cascade of molecular events modulating the key signals for successful implantation. We hypothesize that the modulation of 4 integrin by estrogen and progesterone has a direct role in the process of implantation, and that it could be one of the many critical factors facilitating embryo implantation at the fetomaternal interface. We further correlate the hormonal modulation of 4ß1 integrin receptor expression with blastocyst implantation using functional in vivo neutralization studies.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Virgin, 6- to 8-wk-old, female Swiss Albino mice were housed in the animal care facility having constant light and dark cycles (photoperiod, 14L:10D). The rooms were provided with a controlled temperature range (22–24°C), and the mice were fed ad libitum. The female mice were mated with males of the same strain to induce pregnancy and were checked the following morning for vaginal plug formation, which was considered to be Day 1 of pregnancy.
Blastocyst Collection
For blastocyst collection, the mice were divided into 3 groups: normal, dormant, and activated. Normal blastocysts were recovered from the pregnant mice on the afternoon of Day 4 of pregnancy (Day 1 = vaginal plug) in 0.1 M PBS (pH 7.2) by flushing their reproductive tracts. Delayed implantation was induced by conducting ovariectomy on the afternoon of Day 3 of pregnancy according to the protocol of Paria et al. [1]. To maintain delayed implantation, ovariectomized mice were injected s.c. with progesterone (2 mg/mouse in 0.1 ml of olive oil) once daily from Day 3 to Day 6. Dormant blastocysts were recovered from ovariectomized mice on the morning of Day 6 of pregnancy. Delayed implantation was terminated by administration of a single s.c. injection of 17ß-estradiol (25 ng/mouse in 0.1 ml olive oil) in conjunction with progesterone on the third day of delay (afternoon of Day 6 of pregnancy). Activated blastocysts were recovered within 18 h of estradiol-plus-progesterone injection on Day 7 of pregnancy.
Uterine Tissue Collection
Normal uteri were recovered on Day 4 of pregnancy between 1530 and 1600 h (standardized time just before implantation) from 6- to 8-wk-old pregnant mice. Dormant (i.e., progesterone-treated) uteri were recovered on the morning of Day 6 of pregnancy, whereas activated (i.e., estrogen-treated) uteri were recovered within 18 h of estrogen-plus-progesterone injection on the morning of Day 7 of pregnancy. All tissues were snap-frozen in liquid nitrogen for cryosectioning.
Antibodies
Purified rat anti-mouse CD49d (integrin 4 subunit) monoclonal antibody and purified rat anti-mouse CD29 (integrin ß1 subunit) monoclonal antibody were obtained from Pharmingen (San Diego, CA). Vectastain Avidin Biotinylated Complex (ABC) Alkaline Phosphatase kit, Vectastain ABC peroxidase kit, containing normal serum, biotinylated secondary antibody and ABC reagent, Vector red substrate kit, and Vector Diaminobenzidine (DAB) Brown substrate kit were procured from Vector Laboratories, Inc. (Burlingame, CA).
Immunocytochemistry
Blastocysts attached to poly-L-lysine-coated microscopic slides were initially fixed in cold acetone for 10 min, followed by chilled paraformaldehyde (4%, v/v) in phosphate buffer for 15 min. The blastocysts were then washed in PBS for 15 min. Endogenous alkaline phosphatase activity was quenched by incubating the blastocysts in levamisole solution (an alkaline phosphatase inhibitor; DAKO, Glostrup, Denmark) for 10 min at room temperature, followed by washing in PBS for 10 min. Subsequently, the blastocysts were incubated in normal serum of the species in which the secondary antibody had been raised for 20 min at room temperature. Thereafter, they were incubated with primary antibodies for 4 or for ß1 at a dilution of 1:500 (v/v) for 2 h at room temperature. The control set of blastocysts were incubated with isotype-matched, normal rat immunoglobulin (Ig)G for 2 h at room temperature. Subsequently, the blastocysts were washed with PBS and incubated with biotinylated anti-rat IgG secondary antibody for 30 min at room temperature. After washing with PBS, the embryos were incubated in ABC reagent for 30 min, followed by several washings with PBS and incubation with the red substrate. The reaction was allowed to proceed until the color was apparent and then terminated by rinsing in water. Blastocysts were mounted on Glycergel mounting medium (DAKO) and photographed with a Nikon FX camera (Nikon, Tokyo, Japan) connected to a Leica Image Analyzing System (Leica, Wetzlar, Germany). All photographs were recorded on Kodak Color film (Kodak, Rochester, NY).
Cryosections (thickness, 6 µm) were obtained from the uteri of normal (Day 4), dormant (ovariectomized, progesterone-treated), and activated (estrogen-treated) mice and then processed for immunocytochemical localization of 4 integrin. The sections were fixed with acetone for 10 min, followed by washing in 0.1 M PBS. They were then treated with 0.1% (w/v) saponin, followed by PBS washing. The sections were blocked with normal serum for 30 min, followed by incubation with the primary antibody for 4 for 2 h at room temperature. The sections were further washed in PBS and incubated with secondary antibody for 45 min at room temperature. After several washings with PBS, the sections were finally incubated with ABC solution for 30 min at room temperature, washed, and the substrate (DAB) added. The reaction was allowed to proceed until the color developed. The slides were washed, counterstained with hematoxylin, and mounted, and the photographs were recorded on Kodak film. Staining patterns were then analyzed, taking into account the localization and intensity. Staining intensity was visually estimated as strong (+++), moderate (++), weak (+), or nil (-).
Image Analysis and Quantitation
Image analysis and quantitation was performed according to a standard, established protocol [14]. Differences in the intensity of immunoreactivity of normal, dormant, and activated blastocysts were assessed quantitatively by subjecting the stained samples to image analysis using Leica Q 500 MC software with a gray scale of 0–255 (0 = black, 255 = white). The higher gray values indicate weaker immunoreactivity, and vice versa. The image analyzer captures the black-and-white image and converts the pixels into micron units, thereby quantitating the intensity. Under 5x magnification, the blastocysts were outlined in a measuring frame using a graphic "pen and tablet," and the gray values were obtained. From each blastocyst, the gray values of different areas (inner cell mass and trophectoderm, n = 10 readings per blastocyst) were pooled to obtain a "mean gray value" for each sample. Mean gray values from six samples were grouped, and their mean ± SD were considered as the final cumulative mean gray value representing the study group (normal, dormant, or activated).
Statistics
Values (mean ± SD) were statistically analyzed. Comparison among different treatment groups was done by the Kruskal-Wallis test with post hoc analysis. A P value of less than 0.05 was considered as the level for statistical significance. The statistical software SPSS 7.5 (Indian Council of Medical Research, New Delhi, India) was used for analysis of the study.
In Vivo Intrauterine Blocking Experiments
To determine whether 4 integrin plays a role in implantation, in vivo blocking of this integrin was carried out by injecting 4 monoclonal antibody (clone RI-2; isotype, rat [Fischer] IgG2ß, 
; Pharmingen) [15–17] directly in the uterine horn of pregnant mice on the morning of Day 4 between 1000 and 1200 h (time of implantation, between 1600 and 1700 h). A total of 15 mice were given intrauterine injection of 4 antibody in a volume of 10 µl in their right uterine horns at a concentration of 5 µg/ml. Each injection was infused slowly, over a period of 3 min, from the cervix toward the uterotubal junction. Each animal served as her own internal control, with the contralateral left uterine horn receiving 10 µl of 0.9% saline. A total of 10 mice were injected with an equal volume of 0.9% saline (10 µl) in both horns on the morning of Day 4 of pregnancy and served as normal controls. On Day 9 of pregnancy, the animals were killed, their uteri removed, and the number of implants counted in each horn.
Similarly, on the afternoon of Day 6 of delay, as explained earlier (see the discussion of delayed-implantation mice), a total of five mice were given intrauterine injection of 4 monoclonal antibody in their right uterine horns along with termination of delayed implantation by a single injection of estradiol in conjunction with progesterone. The mice were killed on the morning of Day 9 of pregnancy (i.e., ovariectomy performed on Day 3, progesterone injection from Day 3 to 6, estrogen-and-progesterone injection on the afternoon of Day 6 along with injection of 4 antibody in the right horn, and collection of uterus after 3 days of estrogen injection [on Day 9]). The number of implantation sites was counted in each horn.
Morphological Analysis
Hematoxylin-eosin (H-E) staining of uterine sections of mice injected with 4 antibody in the right horn and with normal saline in the left horn was performed to compare the morphology of embryo implantation in the uterine horn treated with 4 antibody to that of the contralateral, saline-treated control horn. The uteri were dissected out and immediately snap-frozen in liquid nitrogen for cryosectioning. H-E staining was performed on frozen sections (thickness, 6 µm) according to a standard protocol, and the photographs were recorded on Kodak film.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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4ß1 integrin receptor showed positive staining in both the blastocysts and the uteri of normal, dormant, and activated mice. Normal blastocysts showed intense staining for 4 integrin subunit on the inner cell mass as well as the trophectoderm (+++) (Fig. 1A). The negative control for the normal blastocyst did not show any detectable level of 4 staining (-) (Fig. 1B). Immunocytochemical pictures of delayed blastocyst showed a marked reduction in the immunoreactivity of 4 integrin subunit (+) (Fig. 1C), with less staining in both the trophectoderm and the inner cell mass. Interestingly, immunostaining for 4 was regained considerably in the activated blastocyst after estrogen treatment (+++) (Fig. 1E) and was comparable to that observed in the normal blastocysts. Immunolocalization of ß1 integrin subunit carried out on normal, dormant, and activated blastocysts, however, showed a lesser degree of modulation (Fig. 2). The expression of ß1 integrin subunit in the dormant blastocysts was approximately the same as that in the normal blastocysts (++) (Fig. 2C); however, its expression was marginally increased in the activated blastocysts (+++) (Fig. 2E).
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Quantitative analysis of the integrin expression was carried out by image analysis of the immunocytochemical expression obtained earlier by the immunocytochemical technique. The results are shown in Tables 1 and 2. The "mean gray" is a measure of the intensity of staining and is inversely related to the intensity of staining. Quantitation of the immunocytochemistry data through the image-analysis system clearly indicated approximately 81% reduction in 4 integrin expression in the dormant blastocysts as compared to the normal blastocysts (Table 1). In the activated blastocysts, the estrogen treatment increased the 4 expression, which was comparable to the 4 expression exhibited by the normal blastocysts (88% of normal). The expression of ß1 integrin subunit in the dormant blastocysts as well as in the normal blastocysts was comparable (Table 2). In the activated blastocysts, the expression of ß1 integrin showed a significant increase in comparison to normal and dormant blastocysts (P < 0.001).
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In the normal uterus, strong 4 expression was localized in the basement membrane of the lumen and the proliferating stromal cells (+++) (Fig. 3A). Whereas the expression of 4 was shown to be down-regulated in the dormant uterus (+) (Fig. 3B), estrogen induced its expression back on the basement membrane of the lumen and the proliferating stromal cells (+++) (Fig. 3C) in the activated uterus.
To evaluate the potential role of 4 integrin in the process of implantation in mice, in vivo functional blocking experiments were performed. Figure 4A shows the effect of intrauterine injection of 4 integrin antibody on Day 4 pregnant mice. The uterus was recovered on the morning of Day 9 of pregnancy. The right uterine horn, in which the 4 antibody was injected, was devoid of any embryo, whereas 3 embryos were implanted in the contralateral, control uterine horn. A total of 15 mice were given intrauterine injection of 4 antibody in their right uterine horns. The number of implantation sites in the control uterine horn of the 15 animals was 3 ± 0.54. Of the 15 mice that were given intrauterine injection of 4 integrin antibody, only one showed a single embryo implanted in the right uterine horn, where the antibody could not completely block the implantation, whereas a normal number of implantations were found in the control, left uterine horn. Normal Day 9 saline controls injected with the same volume of 0.9% saline in both horns on Day 4 showed 2 implanted embryos in the right horn and 4 in the left horn (Fig. 4B), and the embryo sizes were comparable with those observed in the saline-treated left horn (Fig. 4A).
The uteri injected with the antibody as well as the control uteri were further dissected out from the mice, cryosectioned, and stained with H-E. The control uterine horn showed a normally implanted embryo (Fig. 5A). The endometrial epithelium indicated many proliferating stromal cells, suggesting that the embryo had implanted in the lumen. In contrast, no embryo implantation was observed in the uterine horn that was treated with 4 monoclonal antibody (Fig. 5B). Although the presence of a large number of proliferating stromal cells in the antibody-treated horn was suggestive of a proliferative endometrium for embryo implantation (Fig. 5B), the endometrial thickness of the antibody-treated uterine horn (Fig. 5D) appeared to be thinner than that of the control horn (Fig. 5C).
Implantation in mice takes place on the afternoon of Day 4 of pregnancy, and a preimplantation estrogen surge is essential for blastocyst implantation in mice. To evaluate a direct role of estrogen in modulating the expression of 4 integrin during implantation in vivo, intrauterine injection of 4 monoclonal antibody was administered in delayed-implanting mice before the injection of estradiol, as explained in Materials and Methods. The right uterine horn of mice treated with 4 integrin antibody did not show any implantation site, whereas five implantation sites could be seen in the untreated left horn (Fig. 6), suggesting that, even in the presence of estrogen, the blastocysts were unable to implant if the 4 integrin is blocked in vivo. The number of implantation sites in the untreated left horn over the 5 animals was 5 ± 0.61.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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4 integrin expression by progesterone and its up-regulation by estradiol is demonstrated in the implanting mouse blastocysts as well as in the endometrium using an in vivo, delayed-implantation mouse model system. The potential role of 4 integrin in the process of implantation in mice has further been shown in the present study by functional in vivo blocking experiments.
A constantly changing repertoire of integrins is expressed on the surface of invading trophoblasts, which may help in attachment of the embryo to the uterine epithelium by reacting with specific ECM proteins. Integrins such as 5ß1 (fibronectin receptor), 6Bß1 (laminin receptor), and vß3 (vitronectin and fibronectin receptor) seem to be expressed continuously by the mouse embryo throughout early development, whereas five other ß1-associated integrin subunits, 1, 2, 3, 6A, and 7, show developmentally regulated expression [10]. The embryo thus has the potential to interact with fibronectin through 5ß1 receptor and with vitronectin via vß3 receptor. The pivotal role of vß3 integrin during the process of implantation in the mouse has recently been demonstrated by Illera et al. [18].
The present study demonstrates the up-regulation of 4 expression by estrogen and its down-regulation by progesterone in the mouse blastocyst as well as in the uterus (Figs. 1 and 3). The expression of ß1 was up-regulated significantly in the activated blastocyst, but not much change was observed in the expression of ß1 in the dormant blastocyst as compared to the normal blastocyst (Fig. 2). In rodents, estrogen triggers events that allow initiation of implantation. It is likely that estrogen may, either directly or indirectly (through other paracrine molecules), induce changes in the expression of cell surface proteins around the time of implantation that are necessary for correct signaling to the uterus and for establishing proper contact of the trophoblast cells to the uterine luminal epithelial cells. Our results further support this hypothesis by showing that estrogen induces the expression of 4ß1 integrin receptor around the time of implantation (Figs. 1 and 2).
In humans, vß3, 4ß1, and 1ß1 integrins have been shown to be coexpressed during the putative window of implantation on Days 20–24 [19]. The presence of 4 integrin in the receptive endometrium and its modulation by steroids, as observed in the present study using the in vivo, delayed-implantation mouse model system, are suggestive of its crucial role during the initial attachment and implantation of the embryo in mice, and its presence may be considered as a potential marker of endometrial receptivity and implantation in this species.
Fibronectin and laminin are the two major components of uterine basement membrane at the time of implantation [20, 21]; hence, the induction of 4 (receptor for fibronectin) by estradiol signifies the necessity of the steroid for blastocyst attachment and implantation. This is further substantiated by the drastic reduction of 4 immunoreactivity observed in the dormant blastocysts that are unable to implant (Fig. 1C). An increased ratio of estrogen to progesterone during the peri-implantation period may normally be responsible for the proper expression and distribution of integrins, through which the blastocyst attaches itself to its respective ligand on the uterine epithelium. Up-regulation of 4 by estradiol and its down-regulation by progesterone, as demonstrated in the present study, further strengthen this hypothesis.
The displacement penetration model of Schlafke and Enders [22] proposes that, during the process of invasion in mouse, a number of surface epithelial cells detach from their basement membrane and from each other. These detached cells subsequently degenerate and are then phagocytosed by the invading trophoblasts. As a consequence, the trophoblasts are exposed to the bare basement membrane. The expression of 4 integrin on the basement membrane of the uterus, as observed in the present study (Fig. 3), suggests that it might help in attachment of the blastocyst to the uterine lumen. As the epithelial cells detach from the basement membrane, the 4 integrin expressed on the basement membrane gets exposed to the blastocyst and, thus, may help in its attachment. The process probably involves proteolysis with proteases being released by the trophoblast cells of the blastocyst, which disrupts the tight interepithelial junctions and, thereby, makes room for the invading blastocyst to interact with the basement membrane proteins. The expression of integrins may further facilitate the transepithelial migration, similar to diapedesis of leukocytes through the subendothelial junction during inflammation.
The modulation of 4ß1 integrin expression with blastocyst implantation using functional in vivo neutralization of the integrin has been explored in the present study as well. Yang et al. [23] previously demonstrated that 4 integrin knockout embryos failed to develop much later than implantation. In the present study, we show that in vivo blocking of the 4 integrin by monoclonal antibodies in the uterus of pregnant mice on the day of implantation results in implantation failure in both normal as well as delayed-implantation mice. This observation gains immense importance, because it may be an important lead toward understanding the molecules involved in the overall process of implantation.
Using the delayed-implantation mouse model and the in vivo blocking experiments, we have been able to delineate the differential modulation of 4ß1 integrin receptor expression by estradiol and progesterone on the blastocyst and the uterine epithelium as well as its importance in the implantation process. Because integrins are crucial for the process of implantation, targeting integrins could be one of the possible contraceptive tools that could prevent pregnancy by blocking implantation. Conversely, induction of specific integrins may prove to be beneficial in certain infertility cases.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the Indo-French Center for Promotion of Advanced Research, New Delhi, India. Fellowships provided by the Indian Council of Medical Research and Council of Scientific and Industrial Research to S.B. and R.D., respectively, are gratefully acknowledged. 
2 Correspondence. FAX: 91 11 6862663; chandana_d{at}hotmail.com
Accepted: December 12, 2001.
Received: May 15, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1, 4, V and ß3 endometrial integrins of fertile and unexplained infertile women during the menstrual cycle. A flow cytometric appraisal. Hum Reprod 1999; 14:2485-92 chain homologous to human VLA-4. Cell 1989; 56:37-46[CrossRef][Medline]4 subunit inhibit the murine contact hypersensitivity response. Eur J Immunol 1993; 23:682-688[Medline]4 integrin. J Immunol 1993; 150:1172-1182[Abstract]vß3 adversely affects implantation in the mouse. Biol Reprod 2000; 62:1285-12904 integrins are essential in placental and cardiac development. Development 1995; 121:549-560[Abstract]This article has been cited by other articles:
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  B. V. Sweet Natalizumab update Am. J. Health Syst. Pharm., April 1, 2007; 64(7): 705 - 716. [Abstract] [Full Text] [PDF]
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