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Center for Animal Biotechnology and Genomics,4 Departments of Animal Science,5 and Veterinary Integrative Biosciences,6 Texas A&M University, College Station, Texas 77843
Department of Biological Resources and Technology,7 Yonsei University, Wonju 220-710, South Korea
ABSTRACT
Fibroblast growth factor 7 (FGF7) stimulates cell proliferation, differentiation, migration and angiogenesis. The consensus is that FGF7, expressed by mesenchymal cells, binds FGF receptor 2IIIb (FGFR2) on epithelia, thereby mediating epithelial-mesenchymal interactions. The pig uterus is unique in that FGF7 is expressed by the luminal epithelium (LE) and FGFR2 is expressed by the LE, glandular epithelium (GE), and trophectoderm to effect proliferation and differentiated cell functions during conceptus development and implantation. FGF7 expression by the uterine LE of pigs increases between Days 9 and 12 of the estrus cycle and pregnancy, as circulating concentrations of progesterone increase, progesterone receptors (PGR) in the uterine epithelia decrease, and the conceptuses secrete estradiol-17beta (E2), for pregnancy recognition. Furthermore, E2 increases the expression of FGF7 in pig uterine explants. The present study investigates the relationships between progesterone, E2, and their receptors and the expression of FGF7 in the pig uterus in vivo. Pigs were ovariectomized on Day 4 of the estrus cycle and injected i.m. daily from Day 4 to Day 12 with either corn oil (CO), progesterone (P4), P4 and ZK317,316 (PZK), E2, P4 and E2 (PE), or P4 and ZK and E2 (PZKE). All gilts (n = 5/treatment) were hysterectomized on Day 12. The results suggest that: 1) P4 is permissive to FGF7 expression by down-regulating PGR in LE; 2) P4 stimulates PGR-positive uterine stromal cells to release an unidentified progestamedin that induces FGF7 expression by LE; 3) E2 and P4 can induce FGF7 when PGR are rendered nonfunctional by ZK; and 4) E2 from conceptuses interacts via estrogen receptor alpha, but not estrogen receptor beta in LE to induce maximal expression of FGF7 in LE on Day 12 of pregnancy in pigs.
endometrium, estradiol receptor, progesterone receptor
Fibroblast growth factor 7 (FGF7), also called keratinocyte growth factor, is a member of the FGF superfamily, which has been reported to stimulate cell proliferation, differentiation, migration, and vascular angiogenesis [1]. The preponderance of evidence indicates that FGF7 is expressed in cells of mesenchymal (stromal) origin. However, FGF7 receptors (FGF receptor 2IIIb, FGFR2) are present only on epithelial cells [2]. Therefore, the prevailing opinion is that mesenchymal-derived FGF7 binds receptors on epithelia to mediate epithelial-mesenchymal interactions in various organs, including the reproductive tract [2, 3]. In sharp contrast to this consensus opinion, the results of our studies have demonstrated that FGF7 mRNA is expressed in the endometrial epithelia of pigs, that FGFR2 mRNA is present in both the endometrial epithelia and conceptus trophectoderm, and that FGF7 stimulates the trophectoderm, but not endometrial epithelial cells, to undergo proliferation and differentiation, which suggest that FGF7 is a paracrine mediator of interactions between the uterus and conceptus [4–6].
Several factors, including cytokines, growth factors, and hormones, are known to affect FGF7 expression in various tissues. Interleukin (IL) 1, IL6, platelet-derived growth factor-ß, and transforming growth factor-
increase FGF7 mRNA expression [7, 8]. Parrott and coworkers have shown that FGF7 and hepatocyte growth factor (HGF) stimulate FGF7 expression in ovarian surface epithelia via a positive autocrine feedback mechanism [9]. Steroid hormones also regulate FGF7 expression in reproductive organs. In the uterine endometria of rhesus monkeys, progesterone (P4) increases stromal expression of FGF7, which may mediate P4-induced increases in epithelial cell proliferation and spiral artery development [10]. In mice, estrogen increases mammary gland expression of FGF7 and may play a role in gland development [11]. Androgens also stimulate FGF7 expression in stromal cells of the prostate [12]. It is significant that the promoter region of the FGF7 gene contains various regulatory factor binding sites, including steroid response elements for estrogen and glucocorticoids in humans [13, 14] and for androgens in rats [15].
Northern blot and in situ hybridization analyses of FGF7 in the pig endometrium indicate expression during the estrus cycle and peri-implantation period. FGF7 expression was first detected in LE between Days 9 and 12 of both the estrus cycle and pregnancy, peaked on Day 12 of pregnancy, and remained high through Day 20 [4]. Since the increase in FGF7 expression on Day 12 of pregnancy was significantly higher than that on Day 12 of the estrus cycle, subsequent studies using an endometrial explant culture system were performed to examine the hormonal and cytokine regulation of FGF7. In these studies, estradiol-17ß (E2), but not P4, increased the level of FGF7 mRNA [5]. However, the peri-implantation period of pregnancy is highly complex and cannot be duplicated in in vitro culture systems. In pigs, the uterine environment is influenced by the overlapping events of E2 release by conceptuses for maternal recognition of pregnancy and by extended exposure to P4, which is the hormone of pregnancy, to mediate developmental changes in the uterus that are conducive to successful implantation and placentation [16, 17]. It is known that estrogen receptor (ESR1) is present in uterine endometrial epithelial cells between Days 12 and 15 of the estrus cycle and pregnancy in the pig, whereas P4 receptors (PGR) are absent from epithelial cells [18, 19]. Given that E2 is secreted by pig conceptuses into the uterine lumen [16, 17], it is likely that FGF7 expression in the LE is up-regulated by E2 via ESR1 that is present in the endometrial epithelia. However, since P4 is the dominant hormone during diestrus of the estrus cycle and during pregnancy and since FGF7 is expressed by LE between Day 12 and Day 15 of both the estrus cycle and pregnancy, it is possible that P4 is required as a permissive hormone to allow E2 to stimulate FGF7 expression by endometrial LE. Therefore, we hypothesized that P4 is required as a permissive hormone for E2-induced FGF7 expression in the LE of pig endometrium, and we conducted a study to investigate the relationship between P4 and E2 and their receptors on FGF7 expression in vivo.
All experimental and surgical procedures complied with the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Agricultural Animal Care and Use Committee of Texas A&M University. In all, 26 sexually mature gilts of similar age, weight, and genetic background were ovariectomized on Day 4 after the onset of estrus (Day 0) and were assigned randomly to be treated daily at 0700 h from Day 4 through Day 12 as follows: 1) 200 mg P4 in corn oil vehicle (CO; Sigma Chemical Co., St. Louis, MO) (5 gilts); 2) P4 plus 75 mg ZK317,316 (ZK, which was generously provided by Dr. Kristoff Chwalisz, Schering AG, Berlin, Germany) (5 gilts); 3) P4 plus E2 (100 µg; Sigma) (5 gilts); 4) P4 plus ZK plus E2 (5 gilts); 5) estradiol benzoate (E2, 5 mg in 5 ml of CO/day) (3 gilts); and 6) CO alone (3 gilts). The doses of hormones used are those that have been shown to maintain pregnancy in ovariectomized gilts [20, 21] or to induce pseudopregnancy in pigs [22].
All the gilts were hysterectomized on Day 12, and uterine flushings were obtained by introducing and recovering 20 ml of sterile saline per uterine horn at hysterectomy. The flushings were clarified by centrifugation (3000 x g for 10 min at 4°C), aliquoted, and frozen at –80°C until analyzed. Several tissue sections (
0.5 cm) from the middle of each uterine horn were fixed in 4% paraformaldehyde in PBS (pH 7.2) and embedded in Paraplast-Plus (Oxford Laboratory, St. Louis, MO). The remaining endometrium was physically dissected from the myometrium, frozen in liquid nitrogen, and stored at –80 C for RNA extraction.
Total cellular RNA was isolated from endometrial tissues using Trizol reagent (Invitrogen, Carlsbad, CA) according to manufacturer's recommendations. Total endometrial RNA (20 µg) was loaded onto a 1.2% (wt/vol) agarose gel, electrophoresed, and transferred to a 0.2-µm pore size nylon membrane, as described previously [23]. The blot was then hybridized with [32P]-radiolabeled antisense cRNA probes generated from a linearized porcine FGF7 partial cDNA [4]. Autoradiographs of the Northern blots were prepared using Kodak X-OMAT x-ray film (Eastman Kodak Co., Rochester, NY).
The steady-state levels of FGF7 were assessed by slot blot hybridization, as described previously [4]. Denatured total endometrial RNA (20 µg) from each pig was analyzed using a [32P]-radiolabeled antisense pig cRNA probe [4]. To correct for variations in total RNA loading, a duplicate RNA slot membrane was hybridized with radiolabeled antisense 18S rRNA (pTRI 18S; Ambion, Austin, TX). Following washing, nonspecific hybridization was removed by RNase A digestion. The radioactivity associated with each slot was quantitated by electronic autoradiography using an Instant Imager (Packard Instrument Company, Meridian, CT) and is expressed as total counts.
Total endometrial cDNAs were produced using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen) with 1 µl (100 ng) of total RNA. PCR was performed with Platinum Taq DNA polymerase (Invitrogen) in 50-µl reactions that contained 2 µl of cDNA, 5 µl of 10x PCR buffer, 1 µl of 10 mM dNTP mixture, 1.5 µl of 50 mM MgCl2, 0.2 µl of Platinum Taq DNA polymerase, and 1 µl of either 10 µM ERS1 (5'-AGGGAAGCTCCTATTTGCTCC-3') or 10 µM ERß (5'-GCTTCGTGGAGCTCAGCCTG-3') sense primer and 1 µl of either 10 µM ERS1 (5'-CGGTGGATGTGGTCCTTCTCT-3') or 10 µM ERß (5'-AGGATCATGGCCTTGACACAGA3') antisense primer [24, 25]. Amplification was performed as previously described [20], with initial denaturation at 94°C for 10 min, followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, extension at 72°C for 2 min, and a final extension step at 72°C for 5 min. The PCR products (5 µl) were analyzed on a 2% (wt/vol) agarose gel. The resulting gels were scanned using the BioRad ChemiDoc XRS scanner and analyzed with the Quantity One software (Bio-Rad).
The location of FGF7 mRNA in uterine tissue sections was determined by in situ hybridization analysis, as described previously [26]. Briefly, deparaffinized, rehydrated, and deproteinated uterine tissue sections (5 µm) were hybridized with radiolabeled antisense or sense porcine FGF7 cRNA probes that were generated from linearized plasmid templates by in vitro transcription with [35S]-UTP (Perkin Elmer Life Sciences). The plasmid templates were partial cDNAs for porcine FGF7 [4]. After hybridization, washing, and digestion with RNase A, autoradiography was performed using Kodak NTB-2 liquid photographic emulsion (Eastman Kodak). The slides were exposed at 4°C for 2 wk, developed in Kodak D-19 developer, counterstained with Harris modified hematoxylin (Fisher Scientific, Fairlawn, NJ), dehydrated through a graded series of alcohol to Citrisolv (Decon Laboratories Inc., King of Prussia, PA), and protected with coverslips.
FGF7 was purified from 1 mg of total protein from each uterine flushing by incubating with 200 µl heparin-beaded agarose (Sigma) at 4°C overnight and washing three times with Hanks balanced salt solution (Sigma) and centrifugation at 10 000 x g for 5 min. Proteins that bound to the heparin-beaded agarose were denatured in Laemmli buffer and separated by SDS-PAGE, transferred to nitrocellulose, and blocked with 5% (wt/vol) nonfat milk-TBST (Tris-buffered saline with 0.1% Tween-20), as described previously [4]. Blots were incubated with a goat polyclonal antibody against human FGF7 synthetic peptide (2 µg/ml; Santa Cruz Biotechnology, Santa Cruz, CA) or normal goat IgG (Sigma) as the primary antibody in 2% (wt/vol) milk-TBST overnight at 4°C, rinsed for 30 min at room temperature with TBST, incubated with peroxidase-conjugated rabbit anti-goat IgG (1:10 000; Zymed Laboratories Inc., San Francisco, CA) as the secondary antibody for 1 h at room temperature, and then rinsed again for 30 min at room temperature with TBST. Immunoreactive proteins were detected using enhanced chemiluminescence (Amersham/Pharmacia, Arlington Heights, NY) according to the manufacturer's recommendations.
The expression of PGR and ESR1 proteins was evaluated in paraformaldehyde-fixed, paraffin-embedded, uterine tissue cross-sections (4 µm) using procedures described previously [23]. Briefly, boiling citrate buffer was used for retrieval of PGR, and pronase E (0.5 mg/ml in PBS) was used to retrieve the ESR1 antigen. Proteins were detected with mouse anti-human PGR IgG (2 µg/ml 2C5 PGR; Zymed Labs) and rat anti-human-ESR1 IgG (2 µg/ml H222; Abbott Laboratories, Chicago, IL) and visualized with the Super ABC Mouse/Rat IgG Kit (Biomeda, Foster City, CA). For negative controls, primary antibodies were substituted with mouse (for PGR) or rat (for ESR1) IgG at the same concentrations as the primary antibodies.
Digital photomicrographs of representative bright-field (immunohistochemistry) or bright-field and dark-field illumination (in situ) images were evaluated with a Zeiss Axioplan2 microscope (Carl Zeiss, Thornwood, NY) fitted with an Axiocam HR digital camera. Digital images for immunohistochemistry and in situ hybridization were recorded using the Axiovision 4.3 software. All immunohistochemistry and in situ hybridization figures were assembled using Adobe Photoshop 8.0 (Adobe Systems Inc., San Jose, CA).
All quantitative data were subjected to least-squares ANOVA using the general linear models procedures of the Statistical Analysis System (SAS Institute, Cary, NC). Slot blot hybridization data were corrected for differences in sample loading using the 18S rRNA data as a covariate. Orthogonal contrasts were used to determine the effects of treatment. Preplanned contrasts were CO vs. E2, E2 vs. P4, P4 vs. P4 + E2, P4 + ZK vs. P4 + ZK + E2, and P4 vs. P4 + ZK. All tests of significance were performed using the appropriate error terms according to the expectation of the mean squares for error. A P value equal to or less than 0.05 was considered statistically significant. Data are presented as least-square means (LSM) with standard errors (SEM).
Steady-State Levels of FGF7 mRNA in the Pig Endometrium and of FGF7 Protein in the Uterine Lumen
The FGF7 cRNA detected a single transcript of 2.4 kb in Northern blot analysis of pig endometrial total RNA (Fig. 1A). The steady-state levels of endometrial FGF7 mRNA were not different between pigs that received CO or E2 (P > 0.10), or between pigs that received P4 or P4ZK (P > 0.10). However FGF7 mRNA increased in pigs injected with P4 compared to those injected with E2 (P < 0.05). The combination of P4 and E2 increased endometrial FGF7 mRNA compared to P4 alone (P < 0.01), and the addition of E2 (PZKE2) to the P4ZK treatment increased the level of FGF7 mRNA significantly (P > 0.01). The results of the Western blotting analysis to detect FGF7 protein purified from uterine flushings using heparin-coated agarose beads are shown in Figure 2. An immunoreactive FGF7 protein of 17-kDa was detected in the flushings from pigs treated with P4 and E2, and two molecular mass variants of the FGF7 protein, 17 kDa and 25 kDa, were detected in the flushings from pigs treated with the combination of P4, E2, and ZK, which indicates the secretion of FGF7 into the uterine lumen of pigs (Fig. 2).
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Relationships Between FGF7 mRNA, PGR, and ESR1 in Pig Endometria
Previous studies have reported the presence of ESR2 mRNA and ESR2 protein in the pig uterus [27–29]. A specific PCR product was detected in the pig endometrium using primers for ESR2. Sequence analysis indicated that the product was pig ESR2. However, although ESR1 was readily detected by RT-PCR, ESR2 was barely detected above background in total endometrium from pigs from all the treatment groups (Fig. 3). In all the endometrial samples, the level of ESR1 was 8-fold higher than that of ESR2. In addition, ESR2 was not detected above the level of the sense cRNA probe or irrelevant IgG background using in situ hybridization or immunohistochemistry, respectively (data not shown). In situ hybridization analysis of pig endometrium localized FGF7 mRNA to the LE of ovariectomized pigs treated with P4, P4 and E2 or P4, E2, and ZK. However, FGF7 mRNA was not detected in the endometrial LE of pigs treated with CO vehicle, P4 and ZK or E2 alone (Fig. 4). Immunohistochemistry for PGR revealed expression in the endometrial LE of pigs treated with CO vehicle or E2, whereas PGR expression was absent in the LE of pigs treated with P4 alone or P4 in combination with ZK and/or E2. In contrast, PGR was expressed in the endometrial stromal cells of pigs in all the treatment groups (Fig. 4). Immunohistochemistry for ESR1 revealed expression in the LE, stromal cells, and GE of all pigs in all treatment groups. In the following section, the relationships between treatment, PGR and ESR1 status, and FGF7 mRNA expression are summarized by treatment group (Fig. 4).
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Ovariectomized pigs injected with corn oil. In the absence of ovarian steroid hormones following ovariectomy, the absence of P4 and E2 resulted in default expression of both PGR and ESR1 in the endometrial LE, stromal cells, and GE (Fig. 4). The presence of PGR in the LE is considered to preclude the expression of FGF7 mRNA in CO-treated pigs.
Ovariectomized pigs injected with progesterone. Continuous 9-day exposure of pigs to P4 resulted in the down-regulation of PGR in endometrial LE but not in stromal cells, and did not affect the expression of ESR1 in LE, GE or stromal cells (Fig. 4). The combined effects of P4-induced down-regulation of PGR in LE and the maintenance of PGR in stromal cells is considered to enable P4 to interact with PGR in stromal cells to induce the synthesis and secretion of a putative progestamedin that induces FGF7 in the LE.
Ovariectomized pigs treated with progesterone and ZK137,316. It appears that 75 mg ZK daily does not recapitulate the previous results obtained for sheep [30] to block completely the effects of P4 on the pig endometrium. PGR in the LE and stromal cells are differentially sensitive to the effects of ZK. Although ZK did not inhibit P4-induced down-regulation of PGR in endometrial LE in 5/5 pigs, ZK bound PGR in the endometrial stromal cells, thereby rendering the stromal cells unresponsive to P4 (Fig. 4). Therefore, it seems likely that a progestamedin was not synthesized or released by stromal cells in response to P4 so as to mediate the induction of FGF7 mRNA in the LE of the 3/5 pigs that received both P4 and ZK.
Ovariectomized pigs treated with estradiol benzoate. The absence of P4 to down-regulate PGR and the effect of E2 in inducing PGR resulted in the expression of PGR in the LE, which is considered to have prevented the induction of FGF7 mRNA in the LE by E2 (Fig. 4).
Ovariectomized pigs treated with progesterone and E2. Treatment of pigs with both P4 and E2 allowed P4 to down-regulate the PGR in the LE and allowed E2 to induce FGF7 mRNA expression in the endometrial LE (Fig. 3). Furthermore, the interaction of P4 with PGR in stromal cells probably allows the induction of a progestamedin that acts on the LE to induce the expression of FGF7 mRNA. Alternatively, the expression of FGF7 mRNA may be induced in this treatment group through a combination of these two mechanisms.
Ovariectomized pigs treated with progesterone, ZK, and E2. The action of ZK on PGR in stromal cells inhibits the production of the putative progestamedin that is necessary for the induction of FGF7 expression in the LE (Fig. 4). Therefore, in the absence of functional PGR, E2 action via ESR1 is considered to be sufficient for the induction of FGF7 mRNA expression in pig endometrial LE.
The results of the present study of pig endometria indicate that: 1) P4 is permissive for FGF7 expression through its action in down-regulating PGR in LE; 2) P4 stimulates PGR-positive uterine stromal cells to release an unidentified progestamedin, which induces FGF7 expression by the LE; 3) the combined effects of E2 and P4 or E2, P4, and ZK can induce FGF7 due to the effect of P4 in down-regulating PGR or the effect of ZK in blocking PGR function and allowing E2 to act on PGR-negative LE; and 4) E2 from conceptuses interacts via ESR1 in LE to induce maximal expression of FGF7 in PGR-negative LE on Day 12 of pregnancy in pigs. The group that received both P4 and E2 is the most physiologically relevant, with P4 from the CL being permissive for the actions of E2 from pig conceptuses during the peri-implantation period. A model that summarizes the present working hypothesis for hormonal regulation and the role of uterine FGF7 during early pregnancy in pigs is presented in Figure 5. The mechanism responsible for the lack of expression of FGF7 by endometrial GE is not known.
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P4, which is the hormone of pregnancy in all mammals, is critical for the control of the temporal and spatial (cell-specific) changes in gene expression within the uterus that ensure synchrony between uterine and conceptus (embryo/fetus and associated membranes) development [31]. Indeed, treatment with exogenous P4 significantly alters the expression of a number of genes in rodent, primate, and sheep uteri, as measured in microarray analyses [32–34]. Although similar studies have not been performed in pigs, P4 increases the expression of calbindin-D9k [35], vascular endothelial growth factor [36], FGF2 and two of its receptors, FGFR1 and FGFR2 [37], and the 4, 5, and ß1 integrin receptor subunits [38], as well as suppressing the expression of MUC1 [38]. Importantly, P4 increases the expression levels of various uterine secretory proteins, components of the histotroph, which is hypothesized to support conceptus development in pigs [39–41]. Previous studies with endometrial explant cultures and in vivo steroid replacement experiments have failed to demonstrate FGF7 regulation by P4 in the pig uterus, since the investigators assumed that increases in FGF7 expression during the estrus cycle and pregnancy were most likely influenced by P4 [5, 42]. The results of the present study clearly indicate that FGF7 is induced in the uterine LE of ovariectomized pigs treated with P4, and that the expression of FGF7 is blocked by ZK, a PGR antagonist.
The permissive effects of P4 on FGF7 expression are mediated by PGR in the pig endometrium [43, 44]. In most mammalian uteri, PGR are expressed in endometrial epithelia and stromal cells during the early to midluteal phase, allowing direct regulation of genes by P4. However, in sheep, the presence of PGR in LE precludes the expression of most P4-regulated genes in the LE until continuous exposure of the endometrium to P4 down-regulates PGR expression exclusively in the LE and GE [31]. This paradigm of loss of PGR from uterine epithelia prior to implantation appears to be common among mammals [45] and predicates that the endocrine effects of ovarian P4 on endometrial epithelia during the peri-implantation period are mediated indirectly by either P4-induced paracrine-acting factors (progestamedins) produced by PGR-positive stromal cells or by the induction of factors in the LE that simultaneously down-regulate PGR and either allow or stimulate the expression of endometrial genes [30, 46]. In pigs, the expression of PGR in endometrial LE and GE is down-regulated by Day 10 of the estrus cycle and pregnancy, whereas the expression of PGR is maintained in stromal cells and the myometrium [19]. Removal of PGR from LE correlates with loss of MUC1 and expression of secreted phosphoprotein 1 (SPP1; also known as osteopontin) on the apical surface of the LE, which exposes integrins to extracellular matrix proteins for trophoblast attachment to the uterus [38, 47]. This is also the period during which the endometrium releases many cytokines and growth factors into the uterine lumen of the pig to support conceptus development and trophoblast elongation [48]. Although the loss of PGR from the endometrial epithelia of pigs is well established [19], the present results are the first to dissect effectively P4 regulation of gene expression in the LE during the estrus cycle and early pregnancy.
Three conclusions can made based on the results of the present study. First, similar to sheep [23], P4 negatively auto-regulates PGR in the LE, but not PGR in the endometrial stromal cells of pigs. The expression of PGR in the LE and stromal cells was detected in pigs that received CO but it was down-regulated in the LE of all pigs treated with P4. Second, similar to the results for P4-regulated genes in the endometrial epithelia of sheep [23, 30], the presence of PGR in pig LE precludes the induction of FGF7. All pigs with PGR in the LE, i.e., those treated with CO alone or E2 alone, failed to express FGF7 in LE. Third, the combined effects of P4-induced down-regulation of PGR in LE and P4 interaction with PGR in stromal cells result in the expression of a progestamedin(s) from stromal cells that acts on LE to induce FGF7. Pigs treated with P4 exhibited down-regulation of PGR in LE concomitant with induction of FGF7. Furthermore, FGF7 expression requires functional stromal PGR, since ZK treatment ablated P4-induced FGF7 expression. However, in the absence of functional PGR (treatment with P4, E2, and ZK), E2 alone interacts with ESR1 to induce FGF7 expression.
The mechanisms by which P4 both down-regulates PGR and up-regulates the expression of other genes within the uterine LE of pigs are not understood. Induction of stromal-derived progestamedins is one explanation for these phenomena, although an attractive alternative hypothesis has been put forward by Geisert and colleagues [46]. They propose that P4 interacts with PGR in LE to stimulate factors that activate nuclear factor kappa B (NF-
B), which then functions to inhibit PGR expression and activate transcription of genes that are believed to be involved in implantation [46]. The results of the present study are consistent with both of these theories of endometrial gene regulation by P4.
Estrogens secreted by pig conceptuses on Day 12 of gestation comprise the maternal recognition signal that switches the secretion of endometrial prostaglandin F2 from the endocrine to exocrine direction to prevent CL regression [16]. In addition, conceptus estrogens modulate uterine gene expression to support uterine secretions and the controlled inflammatory-like events that characterize changes in conceptus morphology and uterine remodeling for implantation in pigs [49]. Indeed, secreted SPP1 is induced by estrogen in LE, and it initially localizes to the LE in close proximity to the Day-12 implanting conceptuses [47], whereas conceptus secretion of estrogens correlates with conceptus secretion of IL1ß and may modulate uterine responses to this cytokine [50]. The importance of estrogen for the early survival of pig conceptuses is underscored by pregnancy loss in response to premature exposure of the pregnant uterus to estrogen. Administration of estrogen on Days 9 and 10 of pig pregnancy is associated with altered expression of SPP1 and cyclooxygenase 1 in LE [46, 51] and degeneration of conceptuses by Day 15 [52]. In pigs, ESR1 is readily detectable in LE from Day 5 through Day 12 of the estrus cycle and pregnancy, then decreases, but remains detectable until Day 15 [18]. The presence of ESR1 in LE provides a mechanism by which estrogens secreted by the elongating pig conceptus can stimulate the changes in uterine function that are necessary for the maintenance of pregnancy [18]. Previous reports, using endometrial explant cultures and in vivo steroid replacement experiments, have strongly suggested that the induction of FGF7 in uterine LE during early pregnancy in pigs is stimulated primarily by conceptus estrogens [5, 42]. The results of the present study support these previous reports and further indicate that FGF7 is induced in the uterine LE of ovariectomized pigs injected with E2, but only when P4 has down-regulated PGR in the LE. The uterine LE of pigs treated with E2 alone does not express FGF7 mRNA, probably due to the lack of P4 to down-regulate PGR in the LE, whereas pigs treated with a combination of E2 and P4 exhibit FGF7 expression. Furthermore, functional stromal PGR, and therefore progestamedins, are not required for E2 induction of FGF7 because the addition of ZK did not alter the effects of the combination of P4 and E2.
There is a discrepancy between the results of the present study and those in the previous reports with respect to the fining that estrogen in the absence of P4 can increase endometrial expression of FGF7 [5, 37]. Ka et al. [5] used endometrial explant cultures from Day 9 of the estrus cycle, at which time-point the PGR are significantly reduced compared to Days 0–5 [19, 53]. It is likely that the PGR were already decreased to levels that were insufficient to prevent the induction of FGF7. Although the study conducted by Wollenhaupt et al. [42] employed ovariectomized, steroid-replaced pigs and detected a significant increase in endometrial FGF7 mRNA in response to estradiol benzoate compared to P4 treatment, the levels of expression of FGF7 mRNA overlapped between gilts that were treated with vehicle and estradiol benzoate. Furthermore, the effects on FGF7 expression of estradiol benzoate alone did not differ from the combined effects of P4 and estradiol benzoate, which did not differ from the results of treatment with either vehicle or P4 alone. Interestingly, increases in FGF7 protein were detected primarily in the vascular smooth muscle cells and endothelium, whereas weak expression in LE was not affected by the different treatments [42]. In contrast, the present study utilized ZK, which is a PGR antagonist, to dissect the hormonal regulation of FGF7, and in situ hybridization analyses to understand cell-specific changes in FGF7 expression. The results clearly indicate the complex and overlapping regulation of FGF7 in endometrial LE by P4 and E2.
The FGF7 synthesized and secreted by uterine epithelial cells into the uterine lumen can stimulate the proliferation and differentiation of conceptus trophectoderm [4, 5] by influencing DNA synthesis and through motility, differentiation, cytoprotective, and antiapoptotic effects on cells [54]. Western blotting detected an immunoreactive FGF7 protein of about 25 kDa only in the uterine flushings of pigs treated with E2. Interestingly, a 17-kDa FGF7 protein was also detected in uterine flushings from pigs treated with E2; this protein may be a cleavage fragment of the native 25-kDa FGF7 protein. These results indicate that E2 regulates the secretion of FGF7 from LE cells. It is known that estrogen (of conceptus origin or administered exogenously) induces pseudopregnancy, modulates the redirection of prostaglandin F2 from primarily endocrine secretion into the underlying vasculature to exocrine secretion into the uterine lumen [16], and increases the secretory activities of endometrial epithelia directly or by increasing uterine expression of prolactin receptors on uterine epithelia [55]. Therefore, we hypothesize that FGF7 secretion is regulated by conceptus estrogens during pregnancy and by ovarian estrogens during the estrus cycle. The detailed cellular mechanism by which estrogen affects epithelial FGF7 secretion remains to be determined.
In summary, the uterine environment of early pregnancy in pigs is complex and influenced by the overlapping actions of conceptus estrogens for pregnancy recognition and the permissive effects of P4 acting on uterine epithelia to down-regulate PGR and/or via PGR expressed by uterine stromal cells to stimulate the expression of a progestamedin(s) that mediates the developmental changes in uterine functions necessary for the establishment and maintenance of pregnancy. The dynamic regulation of FGF7 by estrogen and P4 reflects this complexity, and strongly suggests important roles for FGF7 in stimulating the proliferation and differentiation of the pig conceptus during elongation and implantation. The results of the present study provide new insights into the intricate interplay between the actions of estrogen and P4 to modify gene expression in the peri-implantation pig uterus. A similar interplay between pregnancy recognition signals and progesterone has been reported for sheep [56, 57]. However, the magnitude and extent of the influence that conceptus estrogens impart on the early pregnant uterus are remarkable and unique to the pig. Clearly, further studies are warranted to dissect further the endocrine and paracrine regulation of gene expression in the uterus of pregnant pigs.
FOOTNOTES
3These authors contributed equally to this work. 
1Supported by USDA-NRICGP 2000–02290 (F.W.B. and L.A.J.) and NIH P30 ES09106 (G.A.J., R.C.B., T.E.S., F.W.B.).
Correspondence: 2Fuller W. Bazer, Department of Animal Science and Center for Animal Biotechnology and Genomics, 442D Kleberg Center, Texas A&M University, College Station, TX 77843-2471. FAX: 979 845 9938; e-mail: fbazer{at}cvm.tamu.edu
Received: 7 August 2006.
First decision: 17 August 2006.
Accepted: 22 March 2007.
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