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Exogenous interferon- alters murine inner cell mass and trophoblast development. Effect on the expression of ErbB1, ErbB4 and heparan sulfate proteoglycan (perlecan)

Laboratorio Biología de la Reproducción, Ecuador 1465 2°B (1425), ¹ Instituto de Oncología A.H. Roffo (UBA), ² Instituto de Biología y Medicina Experimental and ³ Department of Biological Chemistry, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires (UBA), Buenos Aires, Argentina

Correspondence should be addressed to M Cameo; Email: monicameo{at}fibertel.com.ar

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Implantation is a crucial event in human pregnancy. The participation of cytokines in the implantation process has been widely documented, although the role of many of these molecules is still a matter of controversy. In a previous report from our laboratory, we demonstrated that addition of interferon- to the culture medium produces deleterious effects on mouse embryo development. In this study we investigated the effect of this cytokine on outgrowing embryo morphology and on the expression of epidermal growth factor receptors (ErbBs) and heparan sulfate proteoglycan (perlecan) in mouse embryos cultured in vitro. Morphological assessment of inner cell mass and trophoblast development was carried on in-situ fixed and stained outgrowths. Localization of ErbB1, ErbB4 and perlecan on pre- and peri-implantation embryos was investigated by immunocytochemistry. Addition of interferon- produced a deleterious effect on both inner cell mass and trophoblast morphology. Immunostaining demonstrated that ErbB1, ErbB4 and perlecan are present on pre-implantation embryos and blasto-cysts; interferon- altered the expression of ErbB4 and Perlecan at the blastocyst stage. We propose that the effects produced by this cytokine could be related to the altered acquisition of adhesion competence and low implantation rates observed in certain reproductive immunological disorders.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Crosstalk between blastocyst and uterus is essential to implantation and requires interactions at the molecular and cellular levels in a temporally and spatially co-ordinated manner (Kimber 2000). The invasive phase of implantation poses the problem of antigenic differences between the mother and the foetus and implies the necessity of a very particular immunological environment (Heyborne & Silver 1996).

In the last few years, important progress has been made in the understanding of the mechanisms through which the genetically dissimilar foetus survives without immunological rejection. Such mechanisms include the preponderance of certain cytokines, the selective expression of antigens and the local production of specific immunosuppressive agents (Thellin et al. 2000). Since 1993, the consensus seems to be that a Th-2-enriched state promotes healthy gestation while Th-1 dominance is detrimental for successful pregnancy (Hill et al. 1992, Wegmann et al. 1993, Hill et al. 1995, Raghupathy et al. 1999). In fact, women with multiple implantation failures in in vitro IUF/embryo transfer (ET) cycles have been shown to upregulate Th1-type cytokines and down-regulate Th2-type molecules (Chung-Bang et al. 2000). However, it is now clear that this paradigm is not sufficient to explain implantation failures given the "complexity of the cytokine network at the materno-fetal interface" (Chaouat et al. 2003). While a deleterious effect of inflammatory molecules is certainly true for a number of pathologies, the presence of certain inflammatory cytokines has been shown to be critical for successful implantation by knockout experiments and other technologies (Laird et al. 1997).

Among Th1-type cytokines, interferon- (IFN-) has been shown to inhibit the secretion of colony-stimulating factor-1 that promotes blastocyst growth and differentiation (Wegmann et al. 1993). IFN- receptors have been found in mouse oocytes and pre-implantation embryos (Truchet et al. 2001) and IFN- level is increased in sera of women suffering from spontaneous miscarriage (Jenkins et al. 2000). In a previous study we demonstrated that human recombinant interferon- (hIFN-) added to the culture medium inhibited mouse blastocyst development impairing the outgrowing stage, mimicking the effect of embryo exposure to sera from women suffering from recurrent spontaneous abortions (Cameo et al. 1999). Given that normal embryo outgrowth is a prerequisite for successful implantation, these results suggest that embryos exposed to high concentrations of the cytokine could have an altered spatio-temporal expression of molecules that are crucial for the implantation process, such as growth factors and their receptors.

Members of the epidermal growth factor (EGF) family and heparan sulfate proteoglycans (HSPGs) have been considered to be possible regulators of blastocyst implantation. Heparin binding-EGF (HB-EGF), encoded by the Hegfl gene, plays important roles in implantation in several species, including humans (Yoo et al. 1997, Paria et al. 2001a, 2001b). In the mouse its expression is upregulated solely in the uterine luminal epithelium at the site of blastocyst apposition before initial attachment (Das et al. 1994, 1997). It promotes embryonic growth via EGF receptors, also termed the ErbB family (ErbBs), expressed on the blastocyst cell surface (Wang et al. 2000). It is also expressed in the receptive human uterus and stimulates growth of in vitro-fertilized embryos (Carson et al. 2000). The presence of HSPG has been observed (Smith et al. 1997) on the surface of implantation-competent mouse blastocysts, HSPG being a candidate to interact with HB-EGF expressed in the uterine luminal epithelium.

The aim of this study was to test the hypothesis that an abnormal concentration of IFN- may result in a decreased ability to form trophoblastic outgrowths, failure to form and maintain a compact aggregate inner cell mass (ICM) and alteration of the expression of ErbB receptors and HSPGs on the surface of pre-implantation blastocysts. As the effects of Th1 cytokines on the expression of cell-surface molecules involved in uterine epithelium–blastocyst interactions remain poorly understood, the information obtained would be of value to help further understand the complex process of embryo implantation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Superovulation and embryo recovery
B6CF1 hybrid female mice (age 45–60 days, weight 19–21 g) were superovulated by subcutaneous injection of 5 iu pregnant mare serum gonadotopin (Sigma) followed 45 h later by 5 iu human chorionic gonadotropin (Sigma). Two-cell stage embryos were collected from oviducts of mated female mice 46–47 h after human chorionic gonadotropin injection. The embryos were washed three times with pre-warmed PBS. All of the two-cell embryos recovered were pooled in pre-equilibrated human tubal fluid (HTF) medium (Irvine Scientific, Santa Ana, CA, USA) and kept at 37 °C in an atmosphere of 5% CO₂ until seeding.

Mice were obtained from our Animal Care Division and treated in compliance with regulations for the protection of animal rights. Animals were handled in accordance with National Institutes of Health’s Guide for the Care and Use of Laboratory Animals.

Embryo culture
Two-cell embryos were randomly placed into groups of 10 and placed in 20 µl drops of pre-equilibrated HTF, incubated in vitro for 72 h (day 3) and the number of blastocysts recorded. Blastocysts were cultured for 24 h (day 4) in CMRL-1066 medium (Gibco, Carlsbad, CA, USA) supplemented with BSA (Sigma; 4 mg/ml) to allow complete zona hatching. Hatched blastocysts were transferred to 20 µl drops of CMRL-1066 medium supplemented with 10% (v/v) fetal bovine serum (Gibco) on fibronectin (Gibco; 50 µg/ml)-coated Petri dishes and cultured for an additional 72 h. Each assay was carried out in duplicate. Cultures were maintained at 37 °C in 5% CO₂ in air.

Effect of IFN-
To examine the effect of IFN-, a group of embryos (experimental group) was cultured at the lowest concentration of cytokine producing a significant inhibition of outgrowth (Cameo et al. 1999). Human recombinant IFN- (3 x 10⁵ iu/ml; Boehringer Ingelheim, Vienna, Austria) was added to the medium at different culture times: day 0, 3 or 4 of culture. Cultures were maintained at 37 °C in 5% CO₂ in air, and embryos inspected when appropriate under an Olympus CH2 microscope. The control group included embryos not exposed to IFN-.

Outgrowing blastocyst morphology
At the end of the culture period (day 7), embryos were fixed in 2% (w/v) paraformaldehyde in PBS for 45 min and washed with PBS. Harris’s hematoxylin conventional staining technique was adapted in order to colour embryos attached to a polystyrene culture dish. ICM and trophoblasts (TBs) of each outgrowth were evaluated and a morphological classification developed. Evaluation of the effect of IFN- on embryo morphology was performed as a blinded study.

Surface area of the outgrowths
Expansion of the outgrowths was evaluated after fixation and staining. Surface area was calculated using the formula S = •1/2•d₁•1/2•d₂, where d₁ and d₂ are the major and minor diameters of the outgrowth in micrometers (Barlow & Sherman 1972).

Cell proliferation and differentiation
Nuclei were counted and nuclear area measured on TB outgrowths from control and experimental groups (IFN- day 3 and day 4) using Scion Image for Windows. Data obtained were registered and analyzed.

Immunostaining of ErbB1 and ErbB4 in mouse embryos
The presence and localization of ErbBs was analyzed in embryos at different stages of development. Embryos were fixed in 2% (w/v) paraformaldehyde in PBS for 30 min at 4 °C followed by washing in cold PBS. Zona pellucida was mechanically removed and non-specific binding of IgG was blocked by incubating the embryos in 10% normal donkey serum in PBS for 20 min followed by a wash with PBS. Embryos were then incubated with 2 µg/ml polyclonal antibody against EGF receptor or with 2 µg/ml polyclonal antibody against ErbB4 receptor (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 60 min at room temperature. After being rinsed carefully in PBS, embryos were incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibody (0.5 µg/ml; Santa Cruz Biotechnology) for 45 min at room temperature in the dark. Embryos were washed three times with PBS, mounted in 90% (v/v) glycerol in PBS and then examined using a fluorescence microscope (Olympus CH2) equipped with a reflected-light fluorescence attachment (U-LH50HG).

The following negative controls were included: (1) FITC-conjugated secondary antibody alone; (2) primary antibody replaced by the same concentration of normal goat IgG (Santa Cruz Biotechnology) and (3) primary antibody pre-incubated with a specific blocking peptide.

Immunostaining of HSPG (perlecan) in mouse embryos
The presence and localization of perlecan was analyzed in embryos at the blastocyst stage. Embryos were fixed in 2% paraformaldehyde as described above and, following zona pellucida removal, incubated with rat anti-perlecan monoclonal antibody (1/100 in 0.15 M NaCl; Chemicon International) for 30 min at 37 °C. After careful rinsing in NaCl, embryos were incubated with rhodamine isothiocyanate (RITC)-conjugated secondary antibody (5 µg/ml; Santa Cruz Biotechnology) in 0.15 M NaCl for 40 min at 37 °C in the dark. Embryos were washed three times with 0.15 M NaCl, mounted in 90% (v/v) glycerol in PBS and then examined using an Olympus CH2 fluorescence microscope equipped with a filter for rhodamine (U-LH50HG). For the negative control the primary antibody was omitted.

Statistical analysis
Statistical analysis was performed using Instat Mathpad. Statistical differences between control and experimental distribution patterns were tested by ² analysis. Impact of IFN- on surface area of the outgrowths was determined by one-way ANOVA followed by Dunn’s multiple comparisons test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Morphological classification of outgrowing blastocysts
The adapted staining methodology used in this study permitted a more detailed observation of embryo morphology, which in turn allowed the development of a classification system for ICM and TB on fixed and stained embryos.

Blastocyst outgrowths developed in control and study groups were evaluated at the end of the culture period (day 7). An outgrowth consists of a monolayer of spreading trophoblastic cells surrounding a protruding aggregate of inner cell mass and reflects the capacity of peri-implantation blastocysts to implant in utero (Enders et al. 1981). After fixation and staining, ICM and TB of each outgrowth were characterized as type A or B.

Type A TBs
Regular monolayer with predominantly large elongated epithelioid phenotype and absence of mitotic figures corresponding to a giant-cell transformation. Nuclei were oval or round, with many nucleoli. At a higher magnification (1000 x ; not shown) a thin network of small vacuoles could be observed in the cytoplasm. A few small picnotic bodies were identified, although their exact localization could not be established because the intercellular boundaries were not clearly seen. The outline surface of the TB outgrowth showed regions with irregular edges. These corresponded to cytoplasm projections and apical vesicles, which may represent structural traits of spreading and phagocitosis/exocytosis stages, respectively. Some small cells were observed with circular nuclei containing one nucleolus and a vacuolated cytoplasm pattern (1000 x ) similar to that of giant cells.

Type B TBs
Irregular or limited monolayer with little giant-cell reaction and prevalence of cells with no more than one nucleolus per cell. The cytoplasm was organized with a pattern of perinuclear and large vacuoles (1000 x ). Picnotic bodies were abundant, heterogeneous in size and in some cases had perinuclear localization. There was a predominance of regions with smooth edges in the outline surface of the TB, which reflected limited structural signals of the spreading stage.

Type A ICM
A protruding aggregate of compact and intensively stained cells inside a TB outgrowth.

Type B ICM
A few, disaggregated and non-compacted ICM cells inside a TB outgrowth. TB outgrowths without ICM were also observed.

Figure 1 shows, on the left-hand panel, one embryo with type A ICM and TB and, on the right, one embryo with type B ICM and TB, as observed at 400 x magnification. Embryos with hybrid phenotype were frequently observed; in such cases the ICM cells belonged to the type A group and TB cells belonged to the type B, or vice versa.

View larger version (50K): [in this window] [in a new window]   Figure 1 Outgrowth classification. Embryos were processed as described in the text. Type A outgrowth (left-hand panel): compact ICM in close and continuous cell contact with a regular monolayer of TB cells. Giant cells (black arrows) can be seen at the periphery of the outgrowth with abundant nucleoli (black arrowhead). Irregular edges on the outline surface correspond to cytoplasm projections and apical vesicles (inset: grey arrowhead). Some small cells (white arrow) with round nuclei containing one nucleolus can also be observed. Type B outgrowths (right-hand panel): ICM formed by scattered cells with light compaction (white arrowhead) inside a disorganized TB outgrowth containing a few giant cells. Abundant picnotic bodies (arrow) and predominance of smooth edges on outline surface (arrowheads). Scale bar, 27 µm.

Effect of IFN- on ICM and TB morphology

View larger version (16K): [in this window] [in a new window]   Figure 2 Impact of exogenous IFN- on developing embryos. Blastocysts from control and IFN- groups were cultured in vitro for up to 96 h. After fixation and staining, outgrowths were examined morphologically. Blastocysts were classified according to ICM and TB morphology as type A (white area) or type B (black area). The figure shows blastocyst distribution according to ICM (left-hand panel) or TB (right-hand panel) morphology. Values are expressed as percentages; *P < 0.0001 and **P < 0.002 versus control by 2 test. Numbers in parentheses correspond to total number of embryos evaluated in each experimental condition. Each experiment was repeated five times.

Impact of IFN- on surface area of TB outgrowth

View larger version (16K): [in this window] [in a new window]   Figure 3 Impact of exogenous IFN- on outgrowth area. (A) Surface area of outgrowths incubated with or without IFN- added at different developmental stages (days 3 (grey bar) and 4 (black bar)). Outgrowth area was calculated as described in the Materials and Methods section. Values are expressed as means±S.E.M. from four experiments, *P < 0.01 versus control (white bar) by one-way ANOVA followed by Dunn’s multiple comparisons test. (B) Percentage of normal outgrowth following exposure to IFN-. ‘Normal outgrowth’ was defined as described in text. Values are expressed as percentages, *P < 0.05 and **P < 0.001 versus control by 2. Numbers in parentheses correspond to total number of embryos evaluated in each experimental condition. Experiments were repeated four times.

To clarify whether the alteration observed was due to loss of organization or to a decrease in proliferation, the number of nuclei was determined and nuclear area measured on TB outgrowths in embryos from control and experimental groups. The existence of a relationship between nuclear area and DNA content in murine TB outgrowths was previously described by Barlow and Sherman (1972). There was a significant decrease (P < 0.05, by ANOVA) in nuclear area following exposure to IFN- but nuclei number was the same as in control.

Effect of IFN- on the expression of ErbB1 and ErbB4 on mouse embryos
Previous results from our laboratory demonstrated that IFN- does not impair early embryo development but significantly inhibits blastocyst spreading (Cameo et al. 1999). Since it has been well stated in the literature that the adhesion between blastocyst and uterine HB-EGF may be achieved via interaction with blastocyst ErbB receptors and HSPG (Raab et al. 1996, Paria et al. 1999), we wondered whether the detrimental effect of IFN- observed on blastocyst spreading could be attributed to an alteration of ErbB1 and ErbB4 expression. Accordingly, the presence and localization of ErbB1 and ErbB4 on mouse blastocysts, cultured with or without IFN-, was analyzed by indirect immunofluorescence.

Immunostaining with anti-ErbB1 confirmed that this molecule is localized on the surface of blastomeres in pre-implantation embryos (Fig. 4A, B and C). At the blastocyst stage (Fig. 4E) the staining was mostly cytoplasmic, with good definition of cell peripheries.

View larger version (48K): [in this window] [in a new window]   Figure 4 Immunocytochemical localization of ErbB1 on pre-implantation mouse embryos developed in vitro (A, B, C, E) and effect of IFN- on the expression of ErbB1 on blastocysts (F). ErbB1 was localized on the surface of blastomeres in pre-implantation embryos (A, B, C). In blastocysts, both with (F) or without (E) IFN-, the label was uniformly distributed throughout the cytoplasm, with good definition of cell peripheries; cell nuclei remained unstained. Negative controls: embryos incubated with FITC-conjugated secondary antibody alone (D, H) or incubated with primary antibody pre-incubated with a specific blocking peptide (G). Experiments were repeated at least three times for each condition and/or developmental stage. Scale bars, 27 µm (A–D) and 22 µm (E–H).

When anti-ErbB4 was used as primary antibody (Fig. 5), blastocysts were once again uniformly labeled throughout the cytoplasm with good definition of cell peripheries (Fig. 5A). However, immunostaining was clearly different in blastocysts developed in culture medium containing IFN (Fig. 5B). ErbB4 was present in cytoplasmic and nuclear areas but there was no definition of cell peripheries (n = 16 blastocysts). The specificity of the reaction was demonstrated by absent or faint staining when the first antibody was replaced by normal goat IgG (not shown).

View larger version (68K): [in this window] [in a new window]   Figure 5 Immunocytochemical localization of ErbB4 on mouse peri-implantation embryos developed in vitro (A) and effect of IFN- on ErbB4 expression (B). The expression of ErbB4 was analyzed on blastocysts cultured with (B) or without (A) IFN-. In the absence of IFN- (A) fluorescent labeling was distributed uniformly throughout the cytoplasm with good definition of cell peripheries; cell nuclei remained unstained. When IFN- was present in the medium (B), ErbB4 was localized in the cytoplasmic and nuclear areas with no definition of cell peripheries. Negative control: embryos incubated with normal goat IgG plus FITC-conjugated secondary antibody (not shown). Experiments were repeated at least three times for each condition and/or developmental stage. Scale bars, 22 µm.

Effect of IFN- on the expression of perlecan on mouse blastocysts

As shown in Fig. 6B, after staining with anti-perlecan primary antibody, labeling was mostly cytoplasmic with good definition of cell peripheries while the nuclear area was clearly negative. This staining pattern changed following exposure to IFN- (Fig. 6C): perlecan appeared to be localized within the cytoplasm and nucleus of TB and ICM cells with no definition of cell peripheries. Some 28 4.5-day blastocysts were evaluated. The specificity of the reaction was demonstrated by absent or faint staining when embryos were incubated with secondary antibody alone (Fig. 6A).

View larger version (45K): [in this window] [in a new window]   Figure 6 Immunocytochemical localization of perlecan on mouse peri-implantation embryos developed in vitro (A, B) and effect of IFN- on perlecan expression (C). Immunolocalization of perlecan was performed on blastocysts cultured in the presence (C) or absence (B) of IFN-. In the absence of IFN- (B) fluorescent labeling was mostly cytoplasmic with good definition of cell peripheries. When blastocysts were cultured with IFN- (C) perlecan was present in both cytoplasmic and nuclear areas with no definition of cell peripheries. Negative control: embryos incubated with secondary antibody alone (A). Experiments were repeated at least three times for each condition and/or developmental stage. Scale bars, 22 µm.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

The present study provides a detailed description of the effects that exogenous IFN- exerts on both ICM and TB cells. The in-situ fixation and staining of outgrowths allowed a thorough observation of embryos and the development of a classification system for morphological characteristics of ICM and TB. This, in turn, permitted the detailed evaluation of the effect of IFN- on embryo morphology according to major structural components, growth and differentiation patterns. Attention was focused on giant-cell transformation and acquisition of spreading potential in the outgrowth stage. Preliminary results showing a significant decrease in nuclear area with no effect on TB nucleus number support the hypothesis that the effects observed are not related to cell proliferation but rather reveal a delayed morphological progression and disturbed cell differentiation.

The viability of IFN--exposed embryos was analyzed after transfer into fibronectin-coated dishes. Compared with control outgrowths, fewer IFN--treated blastocysts had the ability to maintain a compact and structured ICM at the center of the regular monolayer of flat, elongated and giant cells. Since the ability to produce an outgrowth reflects the capacity of peri-implantation blastocysts to implant in utero (Enders et al. 1981, Hogan et al. 1994) our observations may suggest that, in vivo, an aberrant IFN- production could alter blastocyst implantation capacity. There is an apparent discrepancy observed between the effect of treatment with IFN- on day 4 of culture on morphology and spreading potential. However, we must keep in mind that when IFN- is added on day 4, embryos are at a different developmental stage than when the cytokine is added on day 3. Particularly, day 3 coincides with the appearance of two-cell populations that begin to diverge at the molecular level, expressing different repertoires of genes and subject to relocalization and trafficking of molecules involved in blastocyst spreading. This may well render the day-4 blastocyst particularly sensitive to detrimental effects on its spreading capability. Additionally, morphological evaluation is a purely descriptive methodology, while spreading potential is quantified through outgrowth area measurement.

When evaluating embryo morphology, outgrowths with a hybrid phenotype were observed frequently. This could be explained on the basis of a differential sensitivity of the two cell lines. ICM cells and trophectoderm (TE) cells have many different cellular, biochemical and molecular characteristics, including their susceptibility to embryotoxic agents (Pampfer 2000). Recent observations show that the expression domains of important regulatory proteins become progressively and unequally distributed between inner and outer cells during compaction, and then between ICM and TE cells at the blastocyst stage (Antezak & Van Blerkom 1997). Early functional demarcations between ICM and TE cells, as well as their exposure to distinct microenvironments, are probably the reasons behind the differential sensitivity of the two cell lines to detrimental agents.

There are reasons to believe that morphology is not the best marker of embryo viability. Subtle changes in cytoskeletal organization in the perimplantation period could lead to failure of implantation or development (Polgár et al. 1996), although embryos may appear to be normal (Hill & Anderson 1988, Lindahl-Magnusson et al. 1971). We propose that an altered production of IFN- as a result of an inappropriate immune cell activation could affect not only the morphology of the outgrowing embryo, but also the spatio-temporal expression of surface molecules that participate in early stages of implantation.

As stated previously, members of the EGF family have been considered possible regulators of blastocyst implantation. HB-EGF, the earliest known marker of implantation in mice, which is also expressed in the receptive human uterus (Carson et al. 2000, Lim et al. 2002) exhibits dual specificity, binding both ErbB1 and ErbB4 receptors (Hynes et al. 2001) and also HSPG (Paria et al. 1999). The high-affinity binding is mediated by ErbB4 protein and by interaction with HSPG on the surface of the blastocyst. Mouse blastocysts lacking ErbB receptors exhibit altered adhesive properties and die shortly after initiation of implantation (Harvey et al. 1995). Accordingly, we evaluated the influence of the addition of IFN- to the culture medium on the expression of ErbB receptors and HSPG on the surface of mouse blastocyst cells using an immuno-staining technique.

Our results confirm ErbB1 and ErbB4 expression on mouse embryos developed in vitro; the immunoreactivity is associated with two-, four- and eight-cell embryos, morulae and blastocysts. Whereas we did not observe any difference in the distribution and/or intensity of ErbB1 immunostaining in blastocysts from control or IFN- groups, immunolocalization of ErbB4 was indeed affected by the cytokine. In experimental group blastocysts ErbB4 appeared to be localized in the cytoplasm and no staining was visible on the cell surface. Wang et al.(2000) have reported that while ErbB1, ErbB2 and ErbB3 consistently appear on the cell surface of blastocysts, ErbB4, originally localized in the cytoplasm, translocates to the apical surface of TB cells close to the time when blastocysts become responsive to HB-EGF. Our results suggest that IFN- present in the culture medium could interfere with ErbB4 translocation to the cell surface. ErbB4 is believed to be one of the most important mediators in HB-EGF activity (Elenius et al. 1997, Paria et al. 1999), so such an interference could be responsible for an altered maternal–blastocyst signaling and impaired acquisition of adhesion competence.

Perlecan has been shown to be present on the trophectodermal surface at the time of attachment competence, while absent in unhatched blastocysts even though perlecan mRNA is initially detected at this stage (Smith et al. 1997). The results presented in this paper show the presence of perlecan on trophectodermal surface of in vitro-cultured day-4.5 control blatocysts while no staining is visible on the cell surface following IFN- exposure. We propose that a delay or block in perlecan expression would be caused by the presence of the cytokine in the culture medium.

Hatching undoubtedly represents a turning point in embryo development. When the blastocyst gets rid of its zona pellucida, trophectoderm cells are exposed to the uterine cavity microenvironment and must be ready to begin successful interaction with the cells of the luminal epithelium. Diminished or delayed expression of critical surface molecules may hinder implantation, which is only just beginning. It may be worth remembering at this point that previous work from our laboratory demonstrated that sera from women suffering from recurrent spontaneous abortions significantly inhibited mouse blastocyst hatching in vitro, and that this effect could be mimicked by hIFN-.

While IFN- is certainly beneficial during implantation if secreted at the correct time, location and concentration (Ashkar et al. 2000), it is evident that its anti-differentiative action would prove extremely deleterious to the embryo.

Although the local concentration of IFN- within the uterine lumen is still unknown, enhanced IFN- synthesis has been described in spontaneous abortion in mice (Chaouat et al. 1990). Further investigation into the dysregulation of uterine IFN- production and into the influence on embryo growth would provide us with an important key to understanding the many aspects of early pregnancy failure.

In conclusion, the results presented in this paper show that, under our experimental conditions, IFN- has a deleterious effect on outgrowing blastocyst morphology and differentiation and on the expression of ErbB4 and HSPG at the protein level. It is important to note that the altered expression of ErbB4 and HSPG produced by this cytokine could be related to an altered acquisition of adhesion competence and low implantation rates observed in certain reproductive immunological disorders.

Failed implantation is a crucial factor in determining the success of both spontaneous pregnancy and assisted reproduction. The identification of molecules which affect the complex signaling mechanisms between embryo and uterus could be useful to help couples suffering from infertility problems.

	Acknowledgements

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
The authors would like to thank to Dr Paula Cameo for her comments on the manuscript and Ms Vanina Julianelli and Vanina Rodriguez for their technical support. The present work was funded by grant BID-1201-AR PICT 98, no. 05-03511 from the CONICET, Buenos Aires, Argentina.

	Footnotes

 
Received 28 May 2004
First decision 28 June 2004
Revised manuscript received 4 August 2004
Accepted 16 August 2004

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