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BOR - Papers in Press, published online ahead of print August 11, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.031864
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BIOLOGY OF REPRODUCTION 71, 2003–2011 (2004)
DOI: 10.1095/biolreprod.104.031864
© 2004 by the Society for the Study of Reproduction, Inc.

Female Reproductive Tract

Embryo-Uterine Interactions via the Neuregulin Family of Growth Factors During Implantation in the Mouse1

Naoko Brown, Kaushik Deb, Bibhash C. Paria, Sanjoy K. Das, and Jeff Reese2

Division of Reproductive and Developmental Biology, Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2370


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neuregulins (NRGs) are cell-signaling molecules with recognized roles in cancer and development, but little is known about their role in embryo implantation. Among representative NRG-1 isoforms, neu differentiation factor (NDF, type I) is expressed in the female reproductive tract and is localized to the implantation site. Here, we show that sensory and motor neuron-derived factor (SMDF, type III) is expressed in the uterine subepithelial stroma around the blastocyst and is only upregulated at the time of implantation. The cellular distribution of SMDF is similar to that of NDF and requires an implantation-competent blastocyst. The glial growth factor (GGF, type II) isoform of NRG-1 and the NRG-2 and NRG-3 genes were not expressed in the peri-implantation uterus, as determined by reverse transcription-polymerase chain reaction or in situ hybridization. In contrast to the cellular expression pattern of NDF and SMDF, NRG-4 was present in the luminal and glandular epithelium throughout the uterus during the preimplantation period. Expression of NRG-4 declined in the uterine luminal epithelium during implantation but persisted in the glandular epithelium through Day 8 of pregnancy. Studies in ovariectomized mice showed that NRG-4 is a progesterone-regulated gene, with partial augmentation by estrogen. We also observed upregulation of the erbB2 and erbB3 receptors at the blastocyst stage of embryo development. Together, these findings suggest that a distinct subset of NRGs participates in the signaling network that directs embryo implantation. Upregulation of embryonic erbB2/ erbB3 in the blastocyst trophectoderm and induction of certain NRG-1 isoforms with blastocyst activation help to define additional aspects of the embryo-uterine cross-talk that underlies the implantation process.

growth factors, implantation, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Successful implantation requires precise coordination between the embryo and uterus under the influence of ovarian steroids. After fertilization, differentiation and proliferation take place in specific uterine cell types to provide a suitable environment for embryo implantation and development, and the zygote undergoes a series of cell divisions, culminating in formation of the blastocyst. Generalized uterine swelling and progressive closure of the uterine lumen positions the blastocyst immediately adjacent to the luminal epithelium [1, 2], with eventual attachment of the embryo to the uterus during the evening (2200–2300 h) of Day 4 of pregnancy in mice. Estrogen and progesterone (P4) play major roles in this process. Preovulatory ovarian estrogen directs epithelial cell proliferation on Days 1 and 2 of pregnancy, whereas on Day 3, P4 from newly formed corpora lutea directs stromal cell proliferation that is further stimulated by a brief pulse of preimplantation estrogen secretion early on Day 4. Estrogen secretion on Day 4 is required, because ovariectomy before the rise in estrogen levels results in failure of implantation. Under this condition, the blastocysts become dormant and can remain in the uterus for many days if continuous P4 supplementation is provided [3]. In this state, which is termed delayed implantation, estrogen replacement can activate dormant blastocysts and reinitiate uterine receptivity and implantation. Despite recognition of a growing number of factors that contribute to implantation [4, 5], significant gaps remain in our understanding of the molecular mechanisms that direct this process.

Several members of the epidermal growth factor (EGF) family of mitogens and their receptors are expressed in the uterus and embryo in a temporal and cell type-specific manner during the critical window for implantation [6–8]. The EGF family of ligands interacts with the receptor subtypes of the erbB gene family, which is composed of four receptor tyrosine kinases: erbB1 (EGF receptor), erbB2, erbB3, and erbB4. Dimerization between coexpressed receptors on ligand binding establishes the classical mechanism of action of EGF-like ligands, and interactions between receptor subtypes with various ligands initiate intercellular communication pathways [9].

Neuregulins (NRGs) are signaling proteins that are considered to be members of the EGF-like family of growth factors based on their binding and activation of erbB receptors. They are encoded by four distinct genes, NRG-1, NRG-2, NRG-3, and NRG-4, which mediate cell-cell interactions in the nervous system, heart, breast, and other organ systems [10–13]. NRG-1, which has been studied most extensively, has more than 15 isoforms derived from alternate splicing that are grouped into three subtypes based on their structure, function, and unique hybridization patterns [14–16]. Type I includes neu differentiation factor (NDF), heregulin, and acetylcholine receptor-inducing activity. Glial growth factor (GGF) is a prototypic type II isoform, and sensory and motor neuron-derived factor (SMDF) exemplifies type III NRG-1 isoforms. Despite some crucial differences, they all share structural and functional features, including conserved EGF domains and induction of erbB dimerization. NRG-2, NRG-3, and NRG-4 were identified as additional members of the NRG family based on their shared structural and functional features and the activation of erbB receptors [17–20].

We previously demonstrated NDF expression in the uterine subepithelial stroma around the embryo at the time of implantation [21], suggesting that some NRG-1 signals may be transduced via uterine erbBs for implantation. We also found that uterine NDF expression requires the presence of an activated blastocyst. However, to our knowledge, no information is available concerning the role of other NRGs in the implantation process, and only limited information is available regarding the potential for the blastocyst to act as a target of uterine NRG signaling. Thus, we examined the expression of representative NRG isoforms in the uterus during the peri-implantation period and during artificially induced delayed implantation. The influence of ovarian steroids on NRGs that were expressed during the preimplantation period was also examined. Finally, the ontogeny of erbB-receptor expression was evaluated at various stages of preimplantation embryo development to identify potential signaling pathways between the embryo and uterus at the time of implantation.


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

In the present study, CD-1 mice (Charles River Laboratory, Raleigh, NC) were housed in an AAALAC-approved animal care facility according to National Institutes of Health and institutional guidelines for the care of laboratory animals. Adult female CD-1 mice (weight, 20–25 g; age, 48– 60 days) were mated with fertile males of the same strain. The morning of finding a vaginal plug was designated as Day 1 of pregnancy. Mice were killed by cervical dislocation at 0830–0930 h on Days 1, 4, 5, and 8 of pregnancy and at midnight on Day 4 (n = 4–6 animals/time-point). Whole uteri were excised and divided into short (~1 cm) segments, snap-frozen, and stored at –80°C for later analysis. Pregnancy on Days 1 and 4 was confirmed by oviductal or uterine flushing to check for the presence of embryos. On Day 4.5 (2200–2300 h) and on Day 5 (0830–0930 h), mice were anesthetized by i.p. injections of 2.5% avertin (2,2,2-tribromoethanol in tert-amyl alcohol; Sigma-Aldrich, St. Louis, MO) at 0.35 ml/mouse, and implantation sites were visualized by i.v. injections of 0.1 ml of Chicago Blue B dye solution (1% in saline). The animals were killed 5 min later to identify the blue bands (implantation sites) along the uterus [2]. For reverse transcription-polymerase chain reaction (RT-PCR) and immunostaining, blastocysts were flushed out of the Day 4 uteri (0900 h) and washed with Whitten medium. An average of 70–80 blastocysts were snap-frozen with a small amount of medium in a sterile, 1.5-ml microcentrifuge tube for RNA extraction. The remaining blastocysts were freed of the zona pellucida by exposing them to warm acid-Tyrode solution for immunocytochemistry.

Isolation of Blastocyst RNA

Total RNA from blastocysts (n = 50–80 per group) was extracted as described previously [22] in the presence of Escherichia coli rRNA carrier. Recovery of a labeled tracer RNA was used to monitor yield of blastocyst RNA as follows: Ten picograms (50 000 cpm) of 32P-labeled antisense metallothionein I RNA (specific activity, 1 x 109 cpm/µg) were included in the SDS buffer, and the radioactivity in aliquots of the initial SDS buffer and of the final RNA solution was determined by liquid scintillation counting. Recovery of the tracer RNA was within 59–61%.

Delayed Implantation

To induce and maintain delayed implantation, one group of mice (n = 6) were ovariectomized in the morning (0800–0900 h) on Day 4 of pregnancy and were given daily injections of P4 (2 mg/mouse, s.c.) from Days 5 to 7 [3, 6]. To terminate delayed implantation and to induce blastocyst activation, a second group of P4-primed, delayed pregnant mice (n = 6) were given a single injection of 17ß-estradiol (E2; 25 ng/mouse) on the third day of the delay (Day 7). These steroids were reconstituted in sesame oil and injected s.c. Mice were killed at 16–24 h following E2 injection. Under these conditions, the first visually detectable implantation sites after blue dye injection became visible 16–24 h after an E2 injection. Uterine segments were excised, snap-frozen, and stored at –70°C. Representative segments from two animals per group were analyzed by in situ hybridization. Repeat experiments were performed with paired tissues from the remaining groups once in situ conditions were established.

Influence of Ovarian Steroid Hormones

To evaluate the effects of estrogen and P4 on NRG-4 gene expression, adult virgin females were ovariectomized, allowed to recover for 7 days, then treated with either P4, E2, or a combination of these. One group of ovariectomized females received oil (n = 4) or P4 (2 mg/mouse, s.c., daily; n = 6) for 3 days and were killed 6 h after the final dose. A second group of mice received P4 daily for 3 days (2 mg/mouse, s.c.) plus a single dose of estrogen (25 ng/mouse, s.c.) and were killed 1, 6, and 24 h later (n = 4 animals/time-point). A third group of mice received a single dose of estrogen (25 ng/mouse, s.c.) and were killed 1, 6, and 24 h later (n = 4 animals/time-point). Uteri were collected and stored at –80°C for later analysis.

Reverse Transcription-Polymerase Chain Reaction

Uterine total RNA was extracted using Trizol reagent (Invitrogen Corp., Carlsbad, CA) according to the manufacturer's recommendations. Reverse transcription was performed with gene-specific primers or with oligodeoxythymidine (oligo-dT) using 1 µg of total RNA from uterine tissues on Days 4 and 5 of pregnancy (Superscript II; Invitrogen). Negative-control reactions lacked reverse transcriptase, whereas adult brain (SMDF, NRG-2, NRG-3, and NRG-4) and neonatal brain (GGFII) served as positive-control tissues. Polymerase chain reaction was carried out with primers specific for each isoform (Table 1). The RT products (2 µl) were denatured at 95°C for 3 min and amplified for 35 cycles under the following general conditions: 94°C for 30 sec, 55°C for 30 sec, 72°C for 60 sec, and 72°C extension for 5 min in 20-µl reaction volumes. Blastocyst total RNA underwent oligo-dT-primed RT-PCR amplification with erbB-specific primers (Table 1) as previously described [6]. Expression of the ubiquitous housekeeping gene ribosomal protein L7 (rpL7) was used as a loading standard. The RT-PCR products were visualized in 2.5% agarose gels and blotted onto nylon membranes for subsequent Southern hybridization using 32P-labeled internal oligonucleotides.


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  		TABLE 1. Sequence of primers used in RT-PCR analysis.a

In Situ Hybridization

Templates for cRNA probes were generated from RT-PCR-derived cDNAs for NDF, GGFII, SMDF, NRG-2, NRG-3, and NRG-4. Partial cDNAs were subcloned (TopoII; Invitrogen) and sequenced to confirm correct amplification of the intended target gene and its orientation within the vector. Sense and antisense 35S-labeled cRNA probes were generated using the appropriate polymerases. Probes had specific activity of approximately 2 x 109 dpm/µg.

In situ hybridization was performed as previously described [23]. Briefly, short segments of uterine horns were snap-frozen in liquid Histo-Freeze (Fisher Scientific, St. Louis, MO). Representative sections from two to three different animals per treatment group (or time-point) were used on each hybridization slide. Tissues from different comparison groups were mounted side-by-side, along with positive-control tissues, on the same glass slide. An expression study for a single probe consisted of 5–10 slides of parallel cryostat sections of each tissue. Once the initial results and the conditions for hybridization and exposure were determined, each expression study was performed in triplicate using tissues from different representative animals in each comparison group. Frozen sections (thickness, 11 µm) were thaw-mounted onto poly-L-lysine-coated slides, fixed in cold 4% paraformaldehyde solution in PBS, and acetylated and hybridized at 45°C for 4 h in hybridization buffer containing the 35S-labeled probes. After hybridization, sections were incubated with ribonuclease A (RNase A; 20 µg/ml) at 37°C for 20 min. The RNase A-resistant hybrids were detected by autoradiography using Kodak NTB-2 liquid emulsion (Eastman Kodak, Rochester, NY). Parallel sections were hybridized with sense probes to serve as negative controls. Slides were developed after 3- to 5-wk exposure periods. Sections were briefly poststained with hematoxylin-eosin.

Immunodetection of erbB2 and erbB3 in the Blastocyst

Zona-free blastocysts (n = 3–5 per experiment) were fixed in 2% formaldehyde in PBS at room temperature (15 min), permeabilized in 0.5% Triton X-100 in PBS for 5 min, and then spun onto poly-L-lysine-coated slides for immunostaining as previously described [24, 25]. Briefly, slides were incubated overnight at 4°C with rabbit anti-human polyclonal antibodies to erbB2 or erbB3 at a concentration of 4 µg/ml (Santa Cruz Biotechnology, Santa Cruz, CA). Immunodetection experiments were performed in duplicate for each erbB antibody. For control experiments, blastocysts (n = 5) were incubated with antibodies preneutralized with a 10-fold molar excess of the respective antigenic peptides (Santa Cruz Biotechnology). Blastocysts were then washed in PBS and incubated with biotinylated goat anti-rabbit secondary antibody for 10 min. After several washings in PBS, blastocysts were exposed to streptavidin-peroxidase conjugate for 10 min. Aminoethyl carbazole enzyme substrate (Zymed Laboratories, San Francisco, CA) was used to develop the final color product. Blastocyst nuclei were stained by brief exposure to hematoxylin. Red-brown deposits indicated sites of immunolocalization.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isoform-Specific NRG Expression During Implantation

To determine which NRGs might be involved in the implantation process, RT-PCR was performed with NDF, GGFII, SMDF, NRG-2, NRG-3, and NRG-4 gene-specific primers on uteri at Days 4 and 5 of pregnancy. We previously reported NDF (NRG-1, type I) expression in the uterine subepithelial stroma on Day 5 of pregnancy [21]; thus, this primer pair served as an internal control. Of the remaining NRG-1 isoforms, SMDF (type III) was detected in the uterine tissue, but GGFII (type II) was absent. NRG-2 and NRG-3 were also not detected, but NRG-4 expression was observed in the peri-implantation uterus. Sequence analysis confirmed correct amplification of each target gene. Negative results were confirmed by Southern hybridization (results for Day 5 uteri shown in Fig. 1).



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  		FIG. 1. NRG expression in the peri-implantation mouse uterus. Representative members of the NRG-1 (NDF, GGFII, and SMDF), NRG-2, NRG-3, and NRG-4 families were analyzed by RT-PCR and Southern hybridization of whole uteri from Day 5 of pregnancy and brain (positive control). +, Presence of reverse transcriptase; –, absence of reverse transcriptase; W, water (negative control)

SMDF Is Upregulated in the Peri-Implantation Mouse Uterus

The results of RT-PCR suggested that SMDF participates in the implantation process. To examine the cell-specific expression of SMDF in a temporal manner, in situ hybridization was performed on uterine tissues on Days 1, 4 at 0900 h, 4 at 2400 h, 5, and 8 of pregnancy. Low-level accumulation of SMDF signals was noted in the uterine stroma on Day 1 (Fig. 2). Although uteri on the morning of Day 4 did not show any specific signals, tissues from Day 4 at midnight as well as from Day 5 showed increased accumulation of SMDF in the uterine stroma immediately beneath the luminal epithelium and surrounding the implanting blastocyst at the antimesometrial pole. Expression of SMDF was limited to this narrow region of subepithelial stroma around the blastocyst and did not extend into interimplantation regions. Similar results were obtained among all replicate hybridization experiments. No SMDF signals were detected in the luminal or glandular epithelium at these time-points. Hybridization with sense probes was negative at specific sites of SMDF signal accumulation (not shown).



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  		FIG. 2. SMDF localization in the mouse uterus during early pregnancy. The cell-specific expression of SMDF was examined by in situ hybridization. Representative uterine sections from different stages of pregnancy were hybridized with 35S-labeled sense or antisense cRNA probes. SMDF autoradiographic signals accumulated in the uterine stroma on Day 1 (A), but no specific signals were noted on the morning of Day 4 of pregnancy (B). At midnight on Day 4 (C) and the morning of Day 5 of pregnancy (D), SMDF signals were concentrated in the uterine stroma immediately adjacent to the luminal epithelium surrounding the implanting blastocyst (magnification, x20). Slides with sense probes were negative at specific sites of SMDF expression. bl, Blastocyst; em, embryo; le, luminal epithelium; st, stroma

Blastocyst Activation Is Required for SMDF Expression

To investigate whether the expression of SMDF requires the presence of an activated blastocyst, in situ hybridization was carried out on ovariectomized mice with P4-induced implantation day and mice with E2-induced termination of the implantation delay. Results indicate that SMDF was not expressed in any uterine cell types in the absence of the attachment reaction in P4-treated, delayed implanting uteri (n = 10 uterine segments from six females) (Fig. 3). Sections that contained dormant blastocysts (n = 4) also lacked SMDF radiographic signals. However, with the termination of delayed implantation and activation of the blastocyst, SMDF expression was again noted in subepithelial stromal cells adjacent to the implanting blastocyst (n = 12 uterine segments from six females), similar to that observed during normal implantation. Results were consistent among each replicate hybridization experiment. This pattern of SMDF expression corresponds with the previously observed distribution of NDF signals [21]. In both cases, signal accumulation occurs in the uterine stroma but not in the luminal epithelium immediately adjacent to the implanting embryo, suggesting that the activated blastocyst can induce changes in uterine gene expression across the luminal barrier.



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  		FIG. 3. Expression of SMDF in uteri of mice with delayed implantation. The expression and localization of SMDF mRNA was examined by in situ hybridization in mice with artificially induced delayed implantation. A) Ovariectomized, P4-treated mice with blastocyst dormancy and shift of the uterus into a neutral phase do not show SMDF expression. B) Mice with termination of the delayed-implanting state by estrogen treatment of P4-primed, delayed implantation uteri exhibit SMDF signals in subepithelial stromal cells adjacent to the implanting blastocysts, similar to natural implantation (magnification, x40). Slides with sense probes were negative at specific sites of SMDF expression. bl, Blastocyst

NRG-4 Expression Is P4-Responsive and Distinct from NRG-1

The expression of NRG-4 during the attachment and postattachment phases was examined by in situ hybridization on Days 1, 4, 5, and 8 of pregnancy to determine whether NRG-1 and NRG-4 might have similar roles in the implantation process. Results (from four to six uterine segments/time-point) showed no detectable NRG-4 expression on Day 1 (Fig. 4). However, NRG-4 is expressed in the luminal and glandular epithelium along the entire length of Day 4 uteri but is not specifically localized around the blastocyst. On Day 5, NRG-4 is no longer expressed in the luminal epithelium, although its expression in the glandular epithelium persists. By Day 8, NRG-4 expression is limited to the remaining glandular epithelium. Similar results were obtained among all replicate hybridization experiments. Slides with sense probes were negative at specific sites of NRG-4 signal accumulation.



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  		FIG. 4. NRG-4 expression in the peri-implantation period. The temporal and cell-specific expression of NRG-4 was examined by in situ hybridization on Days 1 (A), 4 (B), 5 (C), and 8 (D) of pregnancy with 35S-labeled sense or antisense cRNA probes. The NRG-4 signals accumulated in the luminal epithelium on Day 4 and in the glandular epithelium on Days 5 and 8 but are not specifically localized to the implanting site (magnification, x20). Slides with sense probes were negative at specific sites of NRG-4 expression. bl, Blastocyst; em, embryo; ge, glandular epithelium; le, luminal epithelium

On Day 4 of pregnancy, the uterine environment becomes receptive for implantation under the influence of ovarian steroid hormones. Because peak accumulation of uterine NRG-4 signals occurs on Day 4, the effects of estrogen and/or P4 on NRG-4 expression were evaluated. Adult ovariectomized females that received estrogen treatment alone (n = 8 uterine segments from four females/ time-point) showed minimal increase in uterine NRG-4 expression compared to vehicle-treated mice (Fig. 5). On the other hand, NRG-4 was highly expressed in the luminal and glandular epithelium with P4 treatment alone compared to vehicle (n = 8 uterine segments from six females). A similar pattern of NRG-4 signals appeared in P4-primed uteri after 1, 6 and 24 h of estrogen exposure (n = 8 uterine segments from four females/time-point). The influence of estrogen was dependent on previous exposure to P4, because animals that were treated with estrogen alone showed minimal NRG-4 expression, even after 6–24 h of exposure (Fig. 5). These patterns were consistently observed among all replicate hybridization experiments.



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  		FIG. 5. Regulation of uterine NRG-4 expression by ovarian steroids. The localization and hormonal responsiveness of NRG-4 expression was examined in uteri of nonpregnant, ovariectomized mice under the influence of exogenous ovarian steroid hormones. Ovariectomized mice received oil (vehicle), P4, or E2 alone or three daily doses of P4 plus a single dose of E2. Mice receiving oil or P4 (three daily doses) were killed 6 h after the last injection. Mice receiving a single dose of E2 or a combination of P4 and E2 were killed 1, 6, and 24 h later. In each case, NRG-4 signals were restricted to the uterine luminal and glandular epithelium. Representative longitudinal sections are shown (magnification, x40). Slides with sense probes were negative at specific sites of NRG-4 expression

Stage-Specific Expression of erbB1, erbB2, erbB3, and erbB4 in Preimplantation Embryos

Because NRGs typically exert their actions by initially binding to erbB3 or erbB4 and subsequent dimerization involves the recruitment of other erbBs, we examined the expression of embryonic erbB1 through erbB4 to determine the possibility of ligand-receptor signaling between maternal and embryonic cell types. Numerous reports are available in the literature regarding the expression of erbB1 and erbB4 in the developing embryo [6, 24–29]. Consistent with earlier observations, our analysis by RT-PCR and Southern hybridization revealed that erbB1 expression was detected at the 1-cell, 8-cell, and blastocyst stages but was at the limits of detection at the 2-cell stage (Fig. 6A). Here, we demonstrate, to our knowledge for the first time, the expression of erbB2 and erbB3 at different stages of mouse embryo development. The expression of erbB2 was less than the limits of detection at the 1-cell, 2-cell, and 8-cell stages but was strongly expressed in the blastocyst. In contrast, erbB3 was expressed at low levels at the 1-cell and 8-cell stages but was not observed at the 2-cell stage. Similar to expression of erbB1 and erbB2, expression of erbB3 was markedly upregulated at the blastocyst stage. An increase in cell number does not account for these results, because erbB2 and erbB3 expression levels were several-fold higher than the change observed in rpL7 expression. The temporal expression pattern of erbB4 was opposite that of erbB1, erbB2, and erbB3, revealing the highest levels of expression at the 1- and 2-cell stages and minimal expression at the 8-cell and blastocyst stages. Immunostaining of Day 4 blastocysts showed cytoplasmic and cell-surface localization of erbB2 protein in the blastocyst trophectoderm (Fig. 6B). Staining of erbB3 was also concentrated in the trophectoderm cells but was more consistent with a cell-surface or cell-cell contact expression pattern in the apical and lateral membranes. Staining of the inner cell mass cells did not appear to be as strong as that in the trophectoderm for either receptor.



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  		FIG. 6. Stage-specific expression of erbB receptors in the preimplantation embryo. The ontogeny of erbB-receptor expression was examined in the developing embryo. A) Southern hybridization of RT-PCR-amplified products for erbB1, erbB2, erbB3, and erbB4 at various stages of preimplantation embryo development. Lanes 1–4: 1-cell, 2-cell, 8-cell/morula, and blastocyst stages, respectively. W, Water (negative control). B) Cell type-specific localization of erbB2 and erbB3 proteins in the blastocyst trophectoderm of preimplantation embryos. Immunocytochemistry of three to five zona-free blastocysts per group showed apical and cytoplasmic localization of erbB2 compared to the predominantly apical and lateral membrane localization of erB3 in the blastocyst trophectoderm (magnification, x400). Blastocysts incubated with preneutralized antibodies were devoid of staining (right). ICM, Inner cell mass; Tr, trophectoderm


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Growth factor signaling via the EGF family of ligands and their cognate erbB receptors is a common characteristic of the mammalian implantation process [30–32]. Here, we observed differential expression and hormonal regulation of representative members of the NRG family of growth factors during the peri-implantation period in the mouse. Among NRG-1 isoforms, SMDF was restricted to the uterine subepithelial stroma around the blastocyst at the time of implantation, similar to the pattern of NDF expression, but GGF was not expressed. Like NDF, SMDF requires an activated blastocyst for its appropriate expression in the uterus. In contrast, NRG-4 was expressed throughout the uterine luminal and glandular epithelium before implantation but was not restricted to the site of blastocyst implantation. We also observed upregulation of erbB2 and erbB3 at the blastocyst stage of embryo development. These results, together with the induction of NRG expression by activated blastocysts, suggest that type I and type III NRG-1 isoforms are part of the embryo-uterine cross-talk that occurs at the time of implantation and that NRG-4 is involved in uterine preparation for implantation in the mouse.

Polypeptide growth factors not only regulate proliferation and differentiation of uterine cells during implantation, they also play important roles in mediating cellular communication between the embryo and uterus. The EGF family of ligands and their receptors are expressed in the uterus and embryo in a temporal and cell type-specific fashion that coincides with the critical window for implantation [6, 8, 25]. Of these, HB-EGF is the earliest molecular marker whose expression occurs solely at the sites of active blastocysts [23]. This marker influences blastocyst actions for implantation in a paracrine and juxtacrine manner [33], and it plays multiple roles in human embryo implantation [34– 38]. The induction of uterine HB-EGF by activated blastocysts is closely followed by the appearance of epiregulin, betacellulin, and NDF at the time of the attachment reaction [7, 21]. In contrast, amphiregulin is expressed in the uterine epithelium in a P4-dependent manner on the afternoon of Day 4 and then becomes localized to the luminal epithelium surrounding the blastocyst at the time of the attachment reaction on the evening of Day 4. Despite their localization patterns, mice deficient in amphiregulin or mice with combined deletion of EGF, transforming growth factor {alpha}, and amphiregulin do not exhibit implantation defects [39]. Normal fertility has also been reported in mice with combined deletion of HB-EGF and betacellulin, although only a few animals survive to adulthood because of severe heart defects [40]. Targeted deletions of NRG-1 cause a different spectrum of severe cardiac abnormalities [41, 42] but are uniformly lethal in midgestation, thus preventing any assessment of its role in implantation. Because NDF and SMDF display overlapping expression patterns with HB-EGF, betacellulin, epiregulin, and amphiregulin around the blastocyst at the time of attachment reaction (for review, see [7]), functional redundancy likely exists among EGF ligands in the uterus. Upregulation of one EGF member occurs after deletion of one or more members of the EGF family in other tissues [39, 43]. Thus, the presence of NDF and SMDF, together with other EGF members, may provide a compensatory mechanism to support the implantation process and maintain successful reproduction.

NRG-mediated cell-cell interactions are defined by the recruitment and dimerization of specific erbB-receptor combinations [10–12]. In general, NRGs preferentially bind to erbB3 and erbB4, with subsequent homo- or heterodimerization to other erbBs to propagate tissue- and ligand-specific signals. SMDF binds with low affinity to erbB3 and erbB4 homodimers but with higher affinity to heterodimeric erbB2/erbB3 or erbB2/erbB4 [44, 45]. For NDF, erbB3 and erbB4 are also the primary partners for receptor heterodimerization [46–49], with preference being given to erbB2/erbB3 heterodimers [50–52]. All four erbB receptors are expressed in the peri-implantation uterus: erbB1 localizes to the subepithelial stroma, erbB2 and erbB3 are concentrated in the luminal epithelium, and erbB4 is expressed in the myometrium and peripheral stroma away from the implantation site [8]. Thus, one target of NDF and SMDF likely is the contiguous cells of the uterine lumen or stroma. The addition of recombinant NDF to uterine membrane homogenates induces ligand-stimulated phosphorylation of uterine erbB receptors [8, 53], suggesting that this is an active signaling pathway during implantation. However, NDF and SMDF may have different signaling strategies despite their similar expression patterns at the time of implantation. In cultured cells where specific NRGs have been expressed by transfection, the ectodomain of NDF type I isoforms is preferentially secreted from the membrane by proteolytic processing and, therefore, generates paracrine (short-distance, diffusible) signals. On the other hand, type III isoforms, including SMDF, may remain anchored in the membrane and preferentially act by juxtacrine signaling (for review, see [10]). If true in the uterine environment, the majority of NDF might be secreted from the subepithelial stroma for potential heterodimerization with erbB2 and erbB3 in luminal epithelial cells. At the same time, SMDF likely is anchored to subepithelial stromal cell surfaces and binds to erbB2 and erbB3 in the adjacent luminal epithelial cells in a juxtacrine manner.

Another potential target for uterine NRG signaling is the implanting blastocyst. In the rodent embryo, erbB1 mRNA is initially present as a maternal transcript, is increasingly expressed from the 4-cell to the blastocyst stage, and is present as an embryo-derived protein that localizes to the apical surface of the blastocyst trophectoderm [6, 25–27, 29]. ErbB1 is localized to the inner cell mass as well as to the trophectoderm of human blastocysts [54]. Targeted deletion of erbB1 causes embryonic lethality, placental defects, or abnormalities in the skin, kidney, brain, and gastrointestinal systems, depending on strain-specific modifications [55–57]. However, erbB1-null embryos can still bind and internalize HB-EGF at the blastocyst stage [24], suggesting that embryonic erbB4 plays an active role in mouse implantation. ErbB4 translocates to the surface of the blastocyst trophectoderm during normal implantation [29] but is absent in dormant blastocysts of mice with delayed implantation [24]. ErbB4 also is localized to the apical surface of the human blastocyst [54]. Despite these findings, the highest levels of erbB4 mRNA expression occur at the 1-cell stage and are reduced to the limits of detection by the blastocyst stage in the hamster [25] and mouse (Fig. 6), consistent with high levels of maternal erbB4 transcript during the early stages of development and persistence of erbB4 protein at the blastocyst stage. The presence of erbB4 on the blastocyst surface and the predilection of NRGs for erbB4 suggest that uterine NRGs may be one of the signals involved in embryo-uterine cross-talk. Targeted deletion of erbB4 results in lethal cardiac anomalies, but mice with a conditional deletion of erbB4 that bypasses these defects have normal fertility [58]. Thus, the preferential interaction of NRGs with erbB2/erbB3 heterodimers may also be important. To our knowledge, no information is available concerning the temporal pattern of erbB2 expression in early stage embryos, and the temporal expression of erbB3 has only been documented in the bovine embryo at the 2-cell and blastocyst stage [59]. In this report, we observed a marked increase in the expression of both erbB2 and erbB3 from the 2-cell to the blastocyst stage. We also observed localization of both receptors on the surface of the blastocyst trophectoderm, which is in agreement with a previous report [29]. Our results additionally suggest erbB2 expression in the cytoplasm, although these findings may not represent the mature form of the receptor. Because NDF and SMDF preferentially exert their actions via erbB2/erbB3 heterodimers, these NRGs may play a role in the cell-cell communication between the uterus and embryo during implantation and embryo invasion. Furthermore, the presence of both erbB2 and erbB3 in the blastocyst provides additional dimerization options for potential interactions between the blastocyst and other members of the EGF family of ligands.

In contrast to the implantation site-specific expression of NRG-1 isoforms, NRG-4 expression was observed in the luminal and glandular epithelium throughout the uterus on Day 4. Its expression in the luminal epithelium diminishes during embryo implantation and invasion on Day 5 and remains only in the glandular epithelium. We also observed that NRG-4 is a P4-regulated gene, but estrogen treatment alone or the addition of estrogen to P4-primed uteri had only a minor impact. On Day 4, uterine epithelial cells switch from a proliferative to a differentiation phenotype under the influence of P4 from newly formed corpora lutea [60]. The function of NRG4 in the uterus is unknown, although its expression and hormonal regulation coincide with differentiation and proliferation of uterine epithelia in the peri-implantation period. Little is known about NRG-4 except that it contains a juxtamembrane EGF domain and a putative proteolytic cleavage site that are similar to those of other NRGs, but it does not share other common features of the NRG family [61]. The highest levels of NRG-4 expression occur in the endocrine pancreas, where it stimulates differentiation of somatostatin-producing {delta}-cells [17]. Although signaling mechanisms for NRG-4 have not been as thoroughly explored as those for NRG-1 isoforms, NRG-4 binds exclusively to the erbB4 receptor but can preferentially heterodimerize with erbB1 or erbB2 to enhance its activity in cultured cells [17, 61]. Whether endogenous NRG-4 acts in a paracrine and/or juxtacrine fashion has not been established. Thus, NRG-4 in the uterine luminal epithelium could interact with erbBs in the adjacent stromal cells in a juxtacrine manner, or it could be secreted to the underlying stroma and myometrium as a paracrine signal. The expression and hormonal regulation of NRG-4 closely matches that of amphiregulin, suggesting similar involvement of both EGF-like growth factors in uterine preparation for implantation. An alternative possibility is that NRG-4 may bind to erbB receptors in the blastocyst to act as one of the stimuli that synchronize uterine receptivity with blastocyst activation before the attachment reaction.

Overall, our results show that the NRG-1 isoforms, NDF and SMDF, are implantation-specific genes that appear in the uterus at the time of embryo-uterine attachment and require the presence of an implantation-competent blastocyst. Conversely, NRG-4 is expressed in the preimplantation period and is regulated by ovarian steroids. Their distinct and overlapping expression with other EGF family members suggests that NRGs are part of the extensive signaling complex that mediates successful female reproduction. The availability of multiple erbB-receptor combinations in the blastocyst and adjacent uterine cell types provides a diversity of potential signaling pathways to coordinate embryo-uterine communication at the time of implantation.


   ACKNOWLEDGMENTS
 
We are grateful to S.K. Dey for his research support and helpful comments.


   FOOTNOTES
 
1 Supported by NIH grants HD42636 and HD44741 to B.C.P., ES07814 and HD37830 to S.K.D., and HD37667 and HD40221 to J.R. Back

2 Correspondence: Jeff Reese, D-4106 Medical Center North, Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2370. FAX: 615 322 4704; jeff.re-ese{at}vanderbilt.edu Back

Received: 11 May 2004.

First decision: 3 June 2004.

Accepted: 23 July 2004.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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