Dysregulation of EGF Family of Growth Factors and COX-2 in the Uterus during the Preattachment and Attachment Reactions of the Blastocyst with the Luminal Epithelium Correlates with Implantation Failure in LIF- Deficient Mice

Haengseok Song, Hyunjung Lim, Sanjoy K. Das, Bibhash C. Paria and Sudhansu K. Dey

Departments of Molecular and Integrative Physiology (H.S., H.L., S.K.Dey) Obstetrics and Gynecology (S.K.Das), and Pediatrics (B.C.P.) Ralph L. Smith Research Center University of Kansas Medical Center Kansas City, Kansas 66160-7338


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Various mediators, including cytokines, growth factors, homeotic gene products, and prostaglandins (PGs), participate in the implantation process in an autocrine, paracrine, or juxtacrine manner. However, interactions among these factors that result in successful implantation are not clearly understood. Leukemia inhibitory factor (LIF), a pleiotropic cytokine, was shown to be expressed in uterine glands on day 4 morning before implantation and is critical to this process in mice. However, the mechanism by which LIF executes its effects in implantation remains unknown. Moreover, interactions of LIF with other implantation-specific molecules have not yet been defined. Using normal and delayed implantation models, we herein show that LIF is not only expressed in progesterone (P4)-primed uterine glands before implantation in response to nidatory estrogen, it is also induced in stromal cells surrounding the active blastocyst at the time of the attachment reaction. This suggests that LIF has biphasic effects: first in the preparation of the receptive uterus and subsequently in the attachment reaction. The mechanism by which LIF participates in these events was addressed using LIF-deficient mice. We observed that while uterine cell-specific proliferation, steroid hormone responsiveness, and expression patterns of several genes are normal, specific members of the EGF family of growth factors, such as amphiregulin (Ar), heparin-binding EGF-like growth factor (HB-EGF), and epiregulin, are not expressed in LIF(-/-) uteri before and during the anticipated time of implantation, although EGF receptor family members (erbBs) are expressed correctly. Furthermore, cyclooxygenase-2 (COX-2), an inducible rate-limiting enzyme for PG synthesis and essential for implantation, is aberrantly expressed in the uterus surrounding the blastocyst in LIF(-/-) mice. These results suggest that dysregulation of specific EGF-like growth factors and COX-2 in the uterus contributes, at least partially, to implantation failure in LIF(-/-) mice. Since estrogen is essential for uterine receptivity, LIF induction, and blastocyst activation, it is possible that the nidatory estrogen effects in the P4-primed uterus for implantation are mediated via LIF signaling. However, we observed that LIF can only partially resume implantation in P4-primed, delayed implanting mice in the absence of estrogen, suggesting LIF induction is one of many functions that are executed by estrogen for implantation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A cross-talk between the active blastocyst and receptive uterus is essential to implantation (1, 2, 3). Coordinated effects of ovarian progesterone (P4) and estrogen play critical roles in establishing uterine receptivity for implantation. The initiation of implantation is first marked by an increased endometrial vascular permeability at the sites of blastocyst apposition, and this coincides with the attachment reaction (2, 4). The uterine preparation for implantation is sequentially programmed into three stages termed prereceptive, receptive, and nonreceptive phases (1, 5). In the mouse, the uterus becomes prereceptive on day 3 of pregnancy or pseudopregnancy by rising ovarian P4 levels. On day 4, the P4-primed uterus becomes receptive when complemented with preimplantation ovarian estrogen secretion. Blastocysts implant only in the receptive uterus, which spontaneously enters into the nonreceptive state (2). Similar uterine phases can be produced by inducing delayed implantation by ovariectomy on the morning of day 4 of pregnancy or pseudopregnancy before the preimplantation estrogen secretion. Under this condition, blastocysts in the pregnant uterus or transferred blastocysts into the pseudopregnant uterus undergo dormancy and failed to initiate the attachment reaction. This condition can be maintained by continued P4 treatment when the uterus remains in the neutral (prereceptive phase) state, but is terminated by an estrogen injection with blastocyst activation and attainment of uterine receptivity. Again, the limited period of uterine receptivity is followed by the nonreceptive phase in the absence of implantation-competent blastocysts (1, 2, 6). The mechanisms by which estrogen renders the uterus receptive, activates dormant blastocysts, and initiates implantation are not clearly understood.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that regulates various cellular functions including proliferation and/or differentiation (7, 8, 9, 10). LIF is a member of the interleukin-6 (IL-6) family, which includes IL-6 itself, oncostatin M, ciliary neurotrophic factor, IL-11, and cardiotrophin-1. LIF functions through heterodimerization of LIF receptor (LIFR) and gp130. The IL-6 family shares gp130 as a common receptor for signal transduction, resulting in considerable functional overlap among the cytokines (11). LIF signaling pathway is suggested to involve activation of mitogen-activated protein (MAP) and/or Janus kinases, triggering phosphorylation of signal transducer and activator of transcription (STAT) factors for translocation in the nucleus and gene activation. However, the mechanism(s) by which LIF transmits cellular signaling in vivo is not clearly understood.

The epidermal growth factor (EGF) family includes EGF, transforming growth factor-{alpha} (TGF-{alpha}), heparin binding-EGF (HB-EGF), amphiregulin (Ar), betacellulin, epiregulin, and heregulins/neu differentiation factors (NDFs) (12, 13, 14, 15, 16, 17, 18). They are synthesized as transmembrane proteins that are proteolytically processed to release the mature forms (19). These ligands interact with the receptor subtypes of the erbB gene family, which is comprised of four receptor tyrosine kinases: ErbB1 (EGF-R), ErbB2, ErbB3, and ErbB4. They share a common structural feature, but differ in their ligand specificity and kinase activity (20, 21, 22). In general, the EGF-like ligands can interact with ErbB family members via homodimerization or heterodimerization (23, 24, 25). Thus, cross-talk between the receptor subtypes with various ligands can serve as a potential signaling mechanism (26, 27). The expression patterns of the EGF family of growth factors and their receptors in the periimplantation mouse uterus have been examined (4, 25, 28, 29, 30). Overall, the results show that the expression of Ar, HB-EGF, and epiregulin in the periimplantation uterus is unique. Ar is expressed in a P4-dependent manner on day 4 (the day of implantation) (28), while HB-EGF is expressed solely in the luminal epithelium at the sites of blastocyst apposition several hours before the attachment reaction, and this expression requires the presence of active blastocysts (4). In contrast, epiregulin is expressed in the luminal epithelium and underlying stroma surrounding the blastocysts during the attachment reaction (29). This expression pattern of Ar, HB-EGF, and epiregulin suggests their roles in uterine preparation and subsequent attachment reaction. Further, HB-EGF influences blastocyst activities for implantation in paracrine and juxtacrine manners (31).

Cyclooxygenase-2 (COX-2), the rate-limiting enzyme in PG biosynthesis, is expressed in luminal epithelial and stromal cells surrounding the active blastocyst during the attachment reaction (32). We have recently established that COX-2-derived prostacyclin (PGI2) is essential for implantation and decidualization (33, 34). The molecular mechanisms by which EGF-like growth factor and COX-2 genes are induced locally by blastocysts are poorly understood. Cytokines are implicated to participate in various events of implantation in an autocrine/paracrine manner (reviewed in Ref. 35). Among the cytokines, LIF is a potential candidate since it was reported to be transiently expressed in mouse uterine glands on day 4 of pregnancy or pseudopregnancy, and this expression precedes blastocyst implantation (36, 37). In addition, LIF-deficient mice exhibit implantation and decidualization failures (38). Reciprocal embryo transfer experiments have shown that LIF(-/-) blastocysts can implant after transfer into wild-type pseudopregnant uteri, although wild-type blastocysts fail to implant in LIF(-/-) uteri. This suggests that maternal LIF is essential for implantation. Uterine LIF appears to be also important for implantation in other species including humans (39, 40, 41, 42). Indeed, LIF deficiency is noted in infertile women and in those with unexplained recurrent abortions (43, 44). Blastocysts express both LIFR and gp130, and LIF influences their development (45, 46, 47, 48, 49). These results suggest that both the embryo and uterus are potential targets for LIF signaling.

The molecular mechanism by which LIF regulates the implantation process and its interactions with other implantation-specific molecules still remains unexplained. This study is the first attempt to define the mechanism of LIF action in implantation. Using multiple approaches and LIF(-/-) mice, we herein demonstrate that the expression of several members of the EGF family is down-regulated in LIF-deficient uteri during implantation, although their receptors (erbBs) are correctly expressed. Furthermore, COX-2, which is expressed in an implantation-specific manner and essential for implantation (32, 33), is aberrantly expressed in the LIF(-/-) uteri during implantation. The deficiency of EGF-like growth factors and abnormal expression of COX-2 may, in part, be responsible for implantation failure in LIF(-/-) mice.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
LIF Is Spatiotemporally Expressed in the Uterus during the Periimplantation Period
LIF was reported to be expressed transiently in endometrial glands on day 4 of pregnancy before implantation, and LIF-deficient mice show implantation failures (36, 38). However, the mechanism by which LIF executes its effects on implantation is not yet known. To better understand its role in implantation, we examined the spatiotemporal expression of LIF throughout the periimplantation period by Northern blot and in situ hybridization. As reported previously (36, 37), Northern blot hybridization detected a single transcript (4.2 kb), and the levels of this mRNA were higher on days 1 and 4 of pregnancy (data not shown). However, analysis of in situ hybridization experiments showed unique expression pattern of LIF mRNA. Distinct LIF mRNA accumulation was noted in the luminal epithelium on day 1 and in the glandular epithelium on day 4 of pregnancy (Fig. 1Go, a and d). However, contrary to the previous report of transient expression before implantation (36), LIF mRNA was clearly detected in the glandular epithelium and also in subluminal stromal cells surrounding the implanting blastocyst on day 5, but not thereafter (Fig. 1Go, e and f). To determine whether the presence of an active blastocyst is necessary for the induction of LIF, delayed implantation model was employed. As expected, LIF was not induced in the P4-primed uterus during delayed implantation (Fig. 2aGo). However, LIF was rapidly expressed in the glandular epithelium within 12 h of an estrogen injection that terminated the delayed implantation with blastocyst activation (Fig. 2bGo). Furthermore, as observed during normal implantation on day 5, LIF mRNA accumulation occurred in stromal cells surrounding the blastocyst at 24 h of an estrogen injection (Fig. 2Go, c and d). These results add new information that LIF is not only expressed in the uterus before the initiation of implantation, but the expression persists in the glands and appears in the stromal cells surrounding the implanting blastocyst. This expression pattern suggests that LIF could be closely associated with other implantation-specific genes, such as EGF-like growth factors and COX-2, that are also induced by active blastocysts before or around the time of the attachment reaction.



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Figure 1. Spatiotemporal Expression of LIF during the Periimplantation Period

In situ hybridization of LIF mRNA in uterine sections on days 1 (a), 2 (b), 3 (c), 4 (d), 5 (e), and 6 (f) are shown under darkfield at 40x. le, Luminal epithelium; s, stroma; myo, myometrium; am, antimesometrial side; m, mesometrial side. Arrowheads and arrows indicate the location of glands and blastocysts, respectively. These experiments were repeated using three mice for each day of pregnancy with similar results.

 


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Figure 2. In Situ Hybridization of LIF mRNA in Uteri of P4-Primed, Delayed Implanting Mice before and after E2 Treatment

Darkfield photomicrographs of representative longitudinal uterine sections are shown at 40x (a–c) or 100x (d). a, Uterine section from a delayed implanting mouse treated with P4 from days 5–7 and killed 24 h after the last injection. b and c, Sections of uteri from P4-treated, delayed implanting mice killed 12 and 24 h after an E2 injection, respectively. d, Higher magnification of c. Arrowheads and arrows indicate the location of glands and blastocysts, respectively. le, Luminal epithelium; s, stroma; myo, myometrium. These experiments were repeated with three mice in each group with similar results.

 
Uterine Cell Proliferation in LIF(-/-) Mice Is Normal
The uterus is comprised of heterogeneous cell types that respond differentially to estrogen and P4. In the mouse, the establishment of a receptive uterus for supporting implantation is regulated by coordinated effects of P4 and estrogen (50). Preovulatory estrogen directs proliferation of epithelial cells on days 1 and 2 of pregnancy. In contrast, rising levels of P4 from newly formed corpora lutea from day 3 onward direct stromal cell proliferation, which is further potentiated by ovarian estrogen secretion on the morning of day 4. At this time, the luminal epithelium ceases proliferating and becomes differentiated for its interactions with the blastocyst for the attachment reaction.

We suspected that the failure of implantation in LIF(-/-) mice could arise from uterine delinquency to attain receptivity resulting from abnormal proliferation and/or differentiation. Thus, we compared uterine cell proliferation profiles between the wild-type and LIF(-/-) mice on day 4 of pregnancy by proliferating cell nuclear antigen (PCNA) immunostaining (data not shown) and nuclear [3H]thymidine incorporation (Fig. 3AGo). As previously observed (50), proliferation was restricted to stromal cells, and no difference in proliferation pattern was noted between the wild-type and LIF(-/-) mice. These data suggest that uterine LIF expression on days 1 or 4 is not essential for uterine cell proliferation and differentiation in response to steroid hormones during the preimplantation period.



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Figure 3. Uterine Responsiveness to Ovarian Steroids and Expression of Angiogenic Factors in Wild-Type and LIF(-/-) Mice

A, Nuclear [3H]thymidine (3H-thy) incorporation in day 4 pregnant uteri of wild-type and LIF(-/-) mice. B, In situ hybridization of VEGF and Flk-1 mRNAs in day 4 pregnant uteri of wild-type and LIF(-/-) mice. C, In situ hybridization of P4-responsive (Ar and Hoxa-10) or estrogen-responsive (PR and LF) genes in ovariectomized wild-type and LIF(-/-) mice after E2 or P4 treatment. Mice were injected with E2 (100 ng/mouse) or P4 (2 mg/mouse) 2 weeks after ovariectomy. E2-treated mice were killed at 24 h, while those treated with P4 were killed at 8 h. Patterns of nuclear [3H]thymidine incorporation and gene expression are shown under darkfield at 40x. ge, Glandular epithelium; le, luminal epithelium; s, stroma; myo, myometrium. These experiments were repeated in three mice with similar results.

 
Angiogenic Factors Are Normally Expressed in LIF(-/-) Uteri
In the adult, angiogenesis occurs primarily in the ovary and uterus during cycles and pregnancy. Increased uterine vascular permeability and angiogenesis are critical to the process of implantation and decidualization (51). Vascular endothelial growth factor (VEGF) is an inducer of vascular permeability and a potent angiogenic factor. VEGF mediates these effects by activating its receptors, Flt-1 and Flk-1 (reviewed in Ref. 52). While activation of Flk-1 is involved in vascular permeability changes, both Flt-1 and Flk-1 are involved in angiogenesis. We surmised that implantation failure in LIF(-/-) mice could be due to defective uterine vascular permeability and/or angiogenesis. However, uterine expression patterns of VEGF and Flk-1 were normal in LIF(-/-) mice on day 4 of pregnancy (Fig. 3BGo), suggesting that the failure of uterine response to implantation was not the result of abnormal uterine vascular permeability or angiogenesis before the attachment reaction.

Steroid-Responsive Genes Are Normally Expressed in LIF(-/-) Mice
We speculated that the implantation defect in LIF(-/-) mice could result from altered responsiveness of the uterus to steroids. Thus, we compared the expression of several steroid-responsive genes in uteri of ovariectomized LIF(-/-) mice with that of wild-type mice. Lactoferrin (LF) and progesterone receptor (PR) are estrogen-responsive genes, while Ar and Hoxa-10 are P4-responsive genes (28, 53, 54). The expression pattern of LF to estrogen and of Ar to P4 in the uterine epithelium of both wild-type and LIF(-/-) mice was similar, although the level of expression of Ar in the ovariectomized uteri was little lower in response to P4 than that of the day 4 pregnant wild-type uterus (see Fig. 3CGo vs. Fig. 5Go). This reduced response to P4 could be due to lower uterine PR levels in ovariectomized mice devoid of estrogen which is required for PR induction. However, PR expression pattern in both the epithelium and stroma in response to estrogen was also normal in LIF(-/-) uteri, and so is the stromal cell expression of Hoxa-10 to P4 (Fig. 3CGo). These results show that ovariectomized LIF(-/-) uteri respond to steroid hormones normally with respect to specific marker genes. Although these genes are normally expressed in the ovariectomized LIF(-/-) uteri by estrogen and P4, we sought to know whether these and other implantation-related genes are correctly expressed in the LIF(-/-) uteri during the preimplantation period. The results are described below.



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Figure 5. Altered Uterine Expression of EGF-Like Growth Factors around the Time of Implantation in Wild-Type and LIF(-/-) Mice

Photomicrographs of representative uterine sections showing in situ hybridization of Ar, HB-EGF, and epiregulin mRNAs on day 4 or 5 of pregnancy in wild-type and LIF(-/-) mice are shown at 40x (a–d) or 100x (e–p). Note the absence of hybridization signals for Ar, HB-EGF, and epiregulin in uteri of LIF(-/-) mice. Arrows indicate the location of blastocysts. le, Luminal epithelium; ge, glandular epithelium; s, stroma; myo, myometrium. These experiments were repeated using three mice in each group with similar results.

 
Expression of Estrogen Receptor-{alpha} (ER-{alpha}), PR, and Hoxa-10 Is Normal in LIF(-/-) Uterus
ER-{alpha} and PR are expressed in the mouse uterus in a cell-specific manner during the periimplantation period (55), and these receptors are essential for normal fertility in mice, since female mice deficient for either ER-{alpha} or PR are infertile (56, 57). To examine whether these two nuclear receptors are correctly expressed in the uterus of LIF(-/-) mice, we compared their localization patterns in LIF(-/-) uteri with those of wild-type uteri on day 4 of pregnancy. No significant differences were noted between the two groups (Fig. 4Go). Hoxa-10 is a P4-responsive gene and expressed in stromal cells on day 4 of pregnancy. The deletion of this gene by null mutation results in female infertility in mice resulting from defective decidualization (58). Uterine expression of Hoxa-10 was normal in LIF(-/-) mice on day 4 of pregnancy (Fig. 4Go). These results suggest that the primary steroid responsive machinery is apparently normal and that any aberration in uterine gene expression in LIF-deficient mice should not result from the delinquency of the uterus to respond to steroid hormones. With this data in hand, we examined the expression of EGF family of growth factors and receptors in uteri of LIF(-/-) mice.



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Figure 4. Expression of Steroid Receptors and Hoxa-10 in Pregnant Wild-Type and LIF(-/-) Mice

Immunolocalization of ER-{alpha} and PR and in situ hybridization of Hoxa-10 mRNA in day 4 pregnant wild-type and LIF(-/-) uteri are shown under brightfield (200x) and darkfield (40x), respectively. Red deposits indicate positive nuclear immunostaining for ER-{alpha} or PR. le, Luminal epithelium; ge, glandular epithelium; s, stroma; myo, myometrium. These experiments were repeated in four mice with similar results.

 
Uterine Expression of EGF Family of Growth Factors in the Uterus Is Down-Regulated in LIF (-/-) Mice
We have previously suggested that EGF-like growth factors act as local mediators for uterine preparation and embryo-uterine interactions during implantation (4, 25, 28, 29, 30). Thus, Ar, which is expressed in the entire epithelium on day 4, is considered to be important for uterine preparation, while HB-EGF, because of its unique spatiotemporal localization and effects on blastocyst functions, is considered to be involved in embryo-uterine interactions during the preattachment and attachment periods (4, 31, 59). In contrast, epiregulin, because of its expression during the attachment reaction, is also likely to be involved in the attachment reaction (29). Thus, we investigated the expression of Ar, HB-EGF, and epiregulin in LIF(-/-) mice. Ar was expressed in the luminal and glandular epithelia of wild-type uteri, but its expression was not detectable in LIF(-/-) uteri on the morning of day 4 of pregnancy (Fig. 5Go, a and b vs. c and d). It should, however, be recalled that Ar was induced in ovariectomized LIF(-/-) uteri after P4 treatment, albeit at lower levels. It is possible that plasma levels of P4 are different in LIF(-/-) mice than those in wild-type mice, resulting in the down-regulation of P4-responsive Ar expression. However, the plasma levels of P4 were comparable between the two groups (data not shown), suggesting that the repression of Ar expression on day 4 of pregnancy is not the result of altered P4 levels, but may result from lack or presence of other factors in LIF(-/-) pregnant mice.

HB-EGF is the first known molecular marker that is induced in the uterine luminal epithelium exclusively at the site of blastocyst apposition several hours before the attachment reaction (4) and is known to interact with EGF receptors expressed in the blastocyst in paracrine and juxtacrine manners (4, 31, 59). This gene was not induced in the luminal epithelium at the site of blastocyst apposition in LIF(-/-) mice at the anticipated time (1800 h) before the attachment reaction on day 4 (compare Fig. 5Go, e and f vs. g and h) or even on the morning (0900 h) of day 5 of pregnancy (compare Fig. 5Go, i and j vs. k and l). However, HB-EGF was expressed in the luminal epithelium on day 1 of pregnancy or in the brain of LIF(-/-) mice in a similar manner as observed in wild-type mice (data not shown). Epiregulin, which is expressed exclusively in the luminal epithelium and underlying stroma adjacent to the implanting blastocyst at the time of the attachment reaction (2200–2300 h) on day 4 and thereafter on day 5 (27), was also not induced on day 5 of pregnancy in LIF(-/-) mice (compare Fig. 5Go, m and n vs. o and p). In contrast, TGF-{alpha} (another member of the EGF family), which is expressed in the uterus during the entire periimplantation period, showed similar pattern of expression in both the wild-type and LIF(-/-) mice on day 4 (data not shown). Collectively, the results suggest that implantation failure in LIF(-/-) mice could result from sequential deficits of specific EGF-like growth factors in the uterus around the time of implantation. This finding led us to examine whether EGF receptor family members (erbBs) are correctly expressed in LIF(-/-) uteri during early pregnancy.

Uterine erbBs Are Correctly Expressed and Responsive to EGF-Like Ligands in LIF(-/-) Mice
We examined the expression patterns of erbBs and phosphorylation status of ErbB1 in response to ligands in LIF(-/-) mice on day 4 of pregnancy. To our surprise, the expression profiles of erbBs in LIF(-/-) uteri were similar to those in wild-type uteri (Fig. 6AGo). As noted in wild-type mice, erbB1 mRNA was normally expressed in major uterine cell types of LIF(-/-) mice. Similarly, erbB2 and erbB3 mRNAs, which are expressed predominantly in the epithelium (25, 30), were correctly expressed in LIF(-/-) uteri (Fig. 6AGo). Normally, erbB4 mRNA is primarily expressed in the submyometrial stroma and myometrial connective tissues with low levels of expression in epithelial cells and other areas of the stroma (30). Again, no aberrant expression of erbB4 was noted in LIF-deficient uteri (Fig. 6AGo).



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Figure 6. Expression and Phosphorylation of erbBs in LIF(-/-) Mice

A, In situ hybridization of the erbBs (erbB1, erbB2, erbB3, and erbB4) in uteri of day 4 pregnant wild-type and LIF(-/-) mice. Photomicrographs of representative uterine sections are shown under darkfield at 40x. le, Luminal epithelium; ge, glandular epithelium; s, stroma; myo, myometrium. These experiments were repeated two times using independent samples with similar results. B, Phosphorylation of uterine ErbB1 by EGF-like ligands. Phosphorylation of ErbB1 was determined using day 4 uterine membranes of wild-type or LIF(-/-) mice after preincubation with 100 ng/ml of EGF, HB-EGF, or betacellulin, or without these ligands (control). The labeling reaction was initiated by the addition of [{gamma}32P] ATP. After labeling, immunoprecipitation was performed with antibodies to ErbB1. Immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose membrane, and detected by autoradiography. These experiments were repeated two times with similar results.

 
This finding led us to determine whether these receptors undergo phosphorylation in response to ligands in LIF(-/-) uteri (Fig. 6BGo). As a prototypic response, day 4 pregnant uterine membranes were used to determine phosphorylation of ErbB1 in response to EGF, HB-EGF, and betacellulin. All of these ligands induced phosphorylation of ErbB1 in uterine membranes of either wild-type or LIF(-/-) mice, suggesting that ErbB1 and perhaps other members are biologically active.

COX-2, but not COX-1, Is Aberrantly Expressed in Uteri of LIF (-/-) Mice
PGs are considered important for mediating increased localized endometrial vascular permeability during implantation and decidualization (reviewed in Ref. 32). COXs, which exist in two isoforms, COX-1 and COX-2, are the rate-limiting enzymes in PG biosynthesis. We have shown that COX-2(-/-), but not COX-1(-/-), female mice show multiple reproductive defects including ovulation, fertilization, implantation, and decidualization (33). COX-1 and COX-2 genes are differentially expressed in the periimplantation mouse uterus. COX-1 is expressed in the luminal epithelium irrespective of the location of blastocysts on the morning of day 4 of pregnancy, but this expression disappears during the attachment reaction. In contrast, COX-2 is exclusively induced in the uterine luminal epithelium and subluminal stromal cells surrounding the blastocyst during the attachment reaction but is not induced in the presence of dormant blastocysts during delayed implantation (32). We examined COX-1 and COX-2 expression in LIF(-/-) mice on days 4 and 5 of pregnancy, respectively, to assess whether implantation failures in LIF(-/-) mice could result from abnormal expression of COX isoforms. While COX-1 expression on day 4 was normal in LIF(-/-) mice (compare Fig. 7Go, a vs. b), COX-2 expression on day 5 was aberrant, i.e. the expression in the luminal epithelium was present, but the expression in underlying stromal cells surrounding a blastocyst was absent in LIF(-/-) mice (compare Fig. 7Go, c and e vs. d and f). These results suggest that aberrant expression of COX-2 contributes to the failure of implantation and decidualization in LIF-deficient mice.



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Figure 7. Uterine Expression of COX-1 and COX-2 in Wild-Type and LIF(-/-) Mice

In situ hybridization of COX-1 and COX-2 on days 4 and 5 of pregnancy, respectively, is shown in wild-type and LIF(-/-) uterine sections. Photomicrographs of representative uterine sections are shown at 40x (a and b) or 100x (c–f). Arrows indicate the location of blastocysts. Note the absence of hybridization signals for COX-2 in stroma adjacent to the blastocyst in LIF(-/-) mice. le, Luminal epithelium; s, stroma; myo, myometrium. These experiments were repeated two times with similar results.

 
LIF Partially Replaces Preimplantation Ovarian Estrogen Required for Implantation
Our results show that LIF is not induced in P4-treated, delayed implantation mouse uterus, but is readily induced by an injection of E2. Thus, it can be assumed that implantation-inducing effects of estrogen are mediated via uterine induction of LIF. However, our results using a delayed implantation model show that LIF can partially replace estrogen required for implantation. While LIF given systemically by constant infusion (5 ng/h or 10 ng/h, sc) by miniosmotic pumps for a period of 48 h or 72 h (n = 8) failed to induce implantation in P4-treated, delayed-implanting wild-type mice in the absence of estrogen, a bolus ip injection of LIF (2 µg/mouse) showed few implantations (2.7 ± 0.7) in only 3 of 12 similarly treated mice. Moreover, two of these three mice each had implantation sites in only one uterine horn. A lower dose of LIF (1 µ/mouse) showed no effects on implantation (n = 5). Unimplanted embryos with morphological appearance of dormant blastocysts were recovered from uterine horns that did not show any sign of implantation. In contrast, a sc injection of E2 (3 ng or 10 ng/mouse) induced expected number of implantation sites (3 ng: 6.0 ± 0.8; 10 ng: 7.4 ± 0.7) in all of the P4-treated, delayed-implantation mice (n = 5 in each group). These results suggest that although LIF can partially replace estrogen required for implantation, uterine LIF induction is not the only function of estrogen in implantation.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
It has recently been recognized that there are close interactions between steroid hormones and local factors during implantation. These local factors are generated by the uterus or embryo independently or cooperatively under the guidance of steroid hormones. Recent progress in molecular biology and genetics has significantly contributed to our understanding on the roles of locally derived peptide growth factors and cytokines in embryo-uterine interactions during implantation and decidualization (4, 25, 28, 29, 30, 31, 38, 59, 60, 61, 62). Expression and gene targeting studies have established LIF as one of the uterine cytokines that is critical to the implantation process (36, 38). However, the molecular mechanism by which LIF executes its effects on implantation is not yet known. Our present results show that before and during implantation LIF is expressed in a biphasic manner; first in uterine glands on day 4 morning and then again in stromal cells surrounding the implanting blastocyst on day 5. This suggests that LIF participates in both uterine preparation for implantation and attachment reaction. However, normal cell type-specific proliferation/differentiation and expression of angiogenic factors and several other implantation-related genes in LIF(-/-) uteri on the morning of day 4 of pregnancy suggest that the first phase of LIF expression in uterine glands for preparation of the uterine receptivity is not as critical as the second phase of stromal LIF expression during implantation. In contrast, aberrant expression of Ar, HB-EGF, epiregulin, and COX-2 during the preattachment and attachment phases in LIF(-/-) uteri suggests that the second phase of stromal LIF expression is more critical for implantation. This is supported by our observation of stromal cell induction of LIF surrounding activated blastocysts within 24 h of an estrogen injection in P4-primed, delayed-implanting mice.

The expression of Ar in the epithelium on day 4 followed by HB-EGF expression in the luminal epithelium surrounding the blastocyst during the preattachment and attachment periods, and of epiregulin in the luminal epithelium and underlying stromal cells at the sites of blastocysts during the attachment period, suggests their distinct and overlapping functions in implantation. The down-regulation of these genes in LIF(-/-) uteri during these periods raises interesting questions. Is this down-regulation due to direct effects of LIF deficiency or the consequence of implantation failure from LIF deficiency or down-regulation of one member in the absence of LIF leads to repression of other members? Answers to these questions will require experiments to rescue implantation in LIF-deficient mice by sequential replacement of these growth factors singly or in combination.

PGs have long been considered important for implantation. Recently, expression and gene deletion studies have established that COX-2-derived PGI2 is essential for implantation and decidualization (33, 34). COX-2 induction in the luminal epithelium and underlying stroma at the site of the attachment reaction requires the presence of an active blastocyst, since this expression is absent at the sites of dormant blastocysts during delayed implantation, but reappears with blastocyst activation after termination of the delay by estrogen (32). Implantation failure in LIF(-/-) mice reassembles delayed implantation with morphologically appearing dormant blastocysts (38). Our present observation of COX-2 expression exclusively in the luminal epithelium, but not in the underlying stroma, at the sites of blastocysts in LIF(-/-) mice on day 5 suggests that the conditions of delayed implantation in the absence of LIF are different from those produced experimentally in wild-type mice by ovariectomy and P4 treatment. This is an intriguing observation and suggests that uterine and blastocyst quiescence produced by estrogen deficiency is different from that executed by LIF deficiency.

PGs can mediate their effects via G protein-coupled cell surface receptors or peroxisome proliferator-activated receptors (PPARs), members of the steroid hormone nuclear receptor superfamily (63, 64). Gene targeting experiments have shown that three of the four PGE2 cell surface receptor subtypes (EP1, EP2, and EP3) are not critical for implantation (65, 66), while EP4 deficiency results in embryonic lethality and thus its role in implantation remains unknown (67). Nonetheless, EP4 is normally expressed in LIF(-/-) uteri on day 4 of pregnancy (data not shown). Furthermore, mice deficient in PGI2 cell surface receptor (IP) or PGF2{alpha} receptor (FP) do not exhibit overt implantation defects (68, 69). In contrast, we have recently shown that COX-2-derived PGI2 can induce implantation via activation of PPAR{delta} in COX-2(-/-) mice (34), suggesting that PGI2 mediates its effects on implantation via activation of a nuclear receptor. Normally, PPAR{delta} is expressed in stromal cells and becomes more localized surrounding the blastocyst with the initiation of the attachment reaction (34). However, the expression of PPAR{delta} remains diffuse in the stroma surrounding a blastocyst in the LIF(-/-) uterus on day 5 of pregnancy (data not shown). Collectively, these results suggest that COX-2-coupled nuclear PG signaling is defective in the LIF-deficient uteri during early pregnancy. Again, the cause and effect relationship between aberrant COX-2 expression and LIF deficiency is not clear. Future experiments to rescue implantation in LIF(-/-) mice with PGI2 or PPAR{delta} agonists will be required to address this question.

Gene deletion experiments have demonstrated that LIF, COX-2, and Hoxa-10 are each essential for implantation and/or decidualization. The hierarchical arrangement of these molecules in directing uterine functions during implantation is not clearly understood. However, it is interesting to note that while COX-2 expression is aberrant in LIF(-/-) uteri, uterine LIF expression is normal in COX-2(-/-) mice (33). This suggests that LIF functions upstream of COX-2 in implantation. Interestingly, LIF expression is normal in Hoxa-10(-/-) uteri (58) and Hoxa-10 expression is normal in LIF(-/-) uteri during the preimplantation period (Fig. 4Go), insinuating that the regulation of these genes is independent of each other. However, COX-2 expression is down-regulated in Hoxa-10(-/-) uteri. Collectively, these results raise the following issues. First, it is possible that the information sequentially originating from the LIF, EGF-like growth factor, and Hoxa-10 signaling pathways finally converges to the COX-2 pathway for implantation. Second, each pathway may stand on its own right and has distinctive functions in implantation. Thus, disruption of one pathway will lead to implantation failure, and apparent dysregulation of other pathways could be the consequence of implantation failure. Finally, two of the three pathways may be interconnected, and interruption of either pathway will lead to implantation failure. These complex relationships may hinder rescue of implantation defects by supplementation of specific factors.

LIF is synthesized in soluble and matrix-associated forms and was shown to be transiently expressed in the glandular epithelium on day 4 of pregnancy in mice (36, 37). As we show herein, LIF is also expressed in the stroma at the sites of blastocysts during the attachment reaction. The targets for LIF action during the preattachment and attachment periods are not fully understood. However, the results of reciprocal embryo transfer experiments and failure to induce deciduoma in LIF(-/-) mice suggest that LIF targets its own creator for implantation, but the exact site of action is not known. Spatiotemporal expression of LIFR and gp130 in the uterus during implantation is required to address this issue. Previous observations (36, 37) and our present finding with delayed implantation show that estrogen is an absolute requirement for LIF induction in the mouse uterus. Is it then possible that LIF replaces estrogen as a down stream effector for implantation? Our present results show that LIF can only partially replace nidatory estrogen for implantation, suggesting that LIF induction is one of many functions estrogen exerts during implantation. However, it is possible that systemic delivery of LIF did not reach the target in appropriate time and concentration for exerting its full effects. In conclusion, the present investigation provides evidence for relative importance of biphasic expression of uterine LIF in implantation and its interactions with other implantation-specific genes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mice
Adult CD-1 wild-type (Charles River Laboratories, Inc., Wilmington, MA) and LIF(+/-) mice on a mixed background were housed in the Animal Care Facility at the University of Kansas Medical Center according to NIH and institutional guidelines for laboratory animals. LIF(+/-) mice were kindly provided by Story Landis (NINDS/NIH). These mice were originally generated by Philipe Brulet (Pasteur Institute, Paris, France). The disruption of the LIF gene was achieved in (129/Sv) ES cells by homologous recombination as described (70). PCR analysis of tail genomic DNA was used for genotyping of mutant mice. CD-1 and LIF(-/-) mice were mated with fertile or vasectomized males of the same strain to induce pregnancy or pseudopregnancy, respectively. The morning of finding a vaginal plug was designated day 1 of pregnancy. Implantation sites on day 5 were visualized by iv injection (0.1 ml/mouse) of Chicago Blue dye solution in 0.1% saline. To induce delayed implantation, mice were ovariectomized on the morning (0900 h) of day 4 of pregnancy and maintained with daily injections of progesterone (P4, 2 mg/mouse) from days 5–7. To initiate implantation, P4-primed, delayed-implanting pregnant mice were injected with 17ß-estradiol (E2, 25 ng/mouse) (71). All steroids were dissolved in sesame oil and injected subcutaneously (0.1 ml/mouse). Uterine tissues were processed for various analyses.

Systemic Administration of LIF in Delayed-Implanting Wild-Type Mice
Miniosmotic pumps (Alzet Corp., Palo Alto, CA) containing LIF (Life Technologies, Inc., Gaithersburg, MD) at a constant infusion rate of 5 ng/h or 10 ng/h were placed under the back skin on the third day of P4-treated delayed implantation (day 7) as described above and continued for 48 h or 72 h. Another batch of P4-treated, delayed-implanting mice were given a single injection of E2 (3 ng or 10 ng/mouse) or LIF (1 µg or 2 µg/mouse) on the second day (day 6) of the delay and killed 48 h later. P4 (2 mg/mouse) treatment was continued throughout the experimental period. At the termination of the experiments, implantation sites were examined by the blue dye method (71). If implantation sites (blue bands) were absent, uterine horns were flushed with saline to recover unimplanted blastocysts. Mice without implantation sites or blastocysts were excluded from the experiments.

Uterine Responsiveness to E2 and P4 in LIF(-/-) Mice
To determine whether the LIF(-/-) uteri respond to E2 and/or P4, wild-type and LIF(-/-) mice were ovariectomized irrespective of the stage of the estrous cycle and rested for 2 weeks. They were given an injection of E2 (100 ng/mouse) or P4 (2 mg/mouse). The control mice received oil (0.1 ml/mouse) alone. They were killed at different times, and uteri were collected for in situ hybridization to study specific gene expression.

To examine uterine cell-specific proliferation on day 4 of pregnancy under the influence of P4 and estrogen, day 4 pregnant wild-type or LIF(-/-) mice received an injection (ip) of [methyl-3H]thymidine (25 µCi/0.1 ml, NEN Life Science Products, Boston, MA) at 0900 h on day 4 and were killed 2 h later. Nuclear uptake of [3H]thymidine was detected in uterine sections by autoradiography (33).

Antibodies
The affinity-purified rabbit polyclonal antibody, C1355 for rat ER-{alpha}, was kindly provided by Margaret Shupnik (University of Virginia Medical Center, Charlottesville, VA). This antibody was raised against a peptide for the last 14 amino acids in the C-terminal end of the rat ER-{alpha} (72). Mouse monoclonal antihuman PR was purchased from Zymed Laboratories, Inc. (South San Francisco, CA). This antibody was developed against a peptide in the N-terminal proline-rich region of human PR. Both ER-{alpha} and PR antibodies cross-react with corresponding mouse proteins. Mouse monoclonal antibody for PCNA was purchased from Sigma (St. Louis, MO). These antibodies were used for immunohistochemistry.

Immunohistochemistry
Uteri were collected from wild-type and LIF(-/-) mice on day 4 of pregnancy, fixed in 10% neutral formalin, and embedded in paraffin. After dehydration, nuclear antigens were retrieved by microwave processing with 10 mM Na-citrate (pH 6.0) for 8 min followed by washings (5 min each x 2) in PBS. Sections were processed for immunohistochemical staining for ER-{alpha}, PR, and PCNA using Histostain-SP Kits (Zymed Laboratories, Inc., South San Francisco, CA) appropriate for the respective antibodies (55). After immunostaining, sections were lightly counterstained with Fast Green. Red deposits indicated the sites of immunoreactive proteins.

Hybridization Probes
Sense or antisense 35S-labeled cRNA probes were generated using appropriate polymerases from cDNAs to LIF, Ar, HB-EGF, epiregulin, erbBs (erbB1, erbB2, erbB3, erbB4), Hoxa-10, COX-1, COX-2, Flk-1, VEGF, LF, PR, PGE2 receptor subtype (EP4), and PPAR{delta} for in situ hybridization as described previously (4, 25, 29, 30, 33, 34). For Northern hybridization, antisense 32P-labeled cRNA probe for LIF was generated. The probes had specific activities of {approx}2 x 109 dpm/µg.

In Situ Hybridization
In situ hybridization was performed as described previously (4, 33). Small pieces of tissues were flash-frozen in liquid Histo-Freeze (Fisher Scientific, Pittsburgh, PA). Frozen sections (11 µm) were mounted onto poly-L-lysine-coated slides, fixed in cold 4% paraformaldehyde solution in PBS, acetylated, and hybridized at 45 C for 4 h in hybridization buffer containing the 35S-labeled antisense cRNA probes. After hybridization, the sections were incubated with ribonuclease A (RNase A, 20 µg/ml) at 37 C for 20 min. RNase A-resistant hybrids were detected by autoradiography using NTB-2 liquid emulsion (Eastman Kodak Co., Rochester, NY). Sections hybridized with sense probes served as negative controls.

Northern Blot Hybridization
Total RNA were extracted from whole uteri pooled from 7–10 mice on the indicated days of pregnancy by a modified guanidine thiocyanate procedure. Polyadenylated [poly (A+)] RNAs were isolated from total RNAs by oligo(dT)-cellulose column chromatography as previously described (55). Poly (A+) RNA (2.0 µg) was denatured, separated by formaldehyde-agarose gel electrophoresis, and transferred to nylon membranes. RNA was cross-linked to the membranes by UV irradiation (Spectrolinker, XL-1500; Spectronics Corp., Westbury, NY), and the blots were prehybridized, hybridized, and washed as previously described by us (4). The hybrids were detected by autoradiography.

Autophosphorylation of Uterine ErbB1 in LIF(-/-) Mice
The autophosphorylation of ErbB1 was studied in day 4 pregnant uterine membranes of wild-type or LIF(-/-) mice by the protocol as previously described (25, 30). Membranes (150 µg protein) were suspended in 50 ml phosphorylation reaction buffer containing 0.1 mM Na-vanadate and preincubated with or without a specific ligand (100 ng/ml) for 10 min at 4 C. The labeling reaction was performed for 2 min at 4 C after addition of 5 µCi[{gamma}32P]ATP (1 mM). The reaction was terminated by the addition of 15 µl of an ice-cold mixture of 1 mM ATP and 0.1% BSA followed by an equal volume (wt/vol) of 10% trichloroacetic acid. After incubation on ice for 1 h, the mixture was centrifuged. The precipitate was washed with a mixture of diethylether and ethanol (1:1) and suspended in 50 µl of 50 mM Tris buffer (pH 7.5). An equal volume of protein A/Sepharose-antibody conjugate (3 mg: 0.5 mg) was added to this mixture and incubated for 90 min at 4 C. The protein A/Sepharose-antibody conjugates were washed sequentially with buffer A (50 mM HEPES, 0.1% Triton X-100, 0.1% SDS, 5 mM EGTA, pH 8.0), buffer B (50 mM HEPES, 0.1% Triton X-100, 0.1% SDS, 150 mM NaCl, pH 8.0), and buffer C (10 mM Tris-HCl, pH 8.0). The pellets were boiled in 1x SDS sample buffer for 3 min and centrifuged. The supernatants were subjected to 7.5% SDS-PAGE. The gel was transferred to nitrocellulose membrane, and the products were visualized by autoradiography.


    ACKNOWLEDGMENTS
 
We thank Y. Zhu for her help in genotyping of LIF knock-out mice.


    FOOTNOTES
 
Address requests for reprints to: Dr. Sudhansu K. Dey, Department of Molecular and Integrative Physiology, Kansas University Medical Center, 3901 Rainbow Boulevard, MRRC 37/3017, Kansas City, Kansas 66160-7338. E-mail: sdey{at}kumc.edu

This work was supported by NIH Grants HD-12304 and HD-29968, as part of the National Cooperative Program on Markers of Uterine Receptivity for Blastocyst Implantation (S. K. Dey), ES 07814 (S. K. Das), and HD-37394 (B. C. Paria). H. Lim was a Kansas Health Foundation predoctoral fellow. Center grant in Reproductive Biology (HD-33994) and Mental Retardation (HD-02528) provided access to various core facilities. S. K. Dey is a MERIT Awardee of the NICHD/NIH.

Received for publication February 29, 2000. Revision received April 14, 2000. Accepted for publication April 18, 2000.


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