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a Department of Obstetrics and Gynecology, Brigham&Women's Hospital,
b Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Parathyroid hormone-related protein (PTHrP) is a growth factor that was first isolated in 1987 from tumors of patients with humoral hypercalcemia [7–9]. PTHrP and parathyroid hormone (PTH) share structural homology at their respective amino termini, enabling PTHrP to interact with classical PTH receptors [10, 11]. However, while PTH is secreted predominantly if not exclusively in parathyroid cells, the PTHrP gene is widely expressed in a number of endocrine and nonendocrine tissues. These sites include skin [10, 12], lactating mammary tissue [13], the chicken oviduct shell gland [14], the endocrine pancreas [15], uterine myometrium and endometrium [16, 17], ovary [18], and placenta [19, 20].
TGF-ß has been reported to increase PTHrP production by cultured endometrial stromal and myometrial cells [16] and also by various squamous carcinoma cell lines [21, 22]. PTHrP has also been reported to have biological effects similar to those of TGF-ß. PTHrP has been shown to increase fibronectin production by human dermal fibroblasts [23] and osteoblasts [24] in culture, and collagen synthesis in fetal rat long bones [25]. PTHrP can also stimulate epidermal growth factor-dependent colony formation in soft agar suspension by normal rat kidney (NRK)-49F cells [23]. These findings raise the intriguing possibility that TGF-ß exerts its biological effects via PTHrP.
The goals of this study were 1) to compare the effects of TGF-ß and PTHrP on mouse blastocyst attachment and outgrowth in vitro, 2) to determine whether TGF-ß acts through a mechanism involving PTHrP, 3) to examine effects of PTHrP on preimplantation mouse embryo development, and 4) to determine the pattern of expression of PTHrP protein in the uterus of the mouse during early gestation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals used in this work were maintained in accordance with the guidelines of the Committee on Animals of the Harvard Medical School and of the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (DHSS publication [NIH] 85–23, rev. 1985).
All embryos used were produced by mating outbred CF1 females (Crl:CF1 BR, 6–8 wk old; Charles River, Wilmington, MA) with hybrid BDF males (B6D2F1/CrlBR; Charles River). Mice were maintained on a 14L:10D cycle (lights-on at 0500 h). Female mice were superovulated by an injection of five IU eCG (Sigma, St. Louis, MO) i.p. at 1330 h, followed by an injection of five IU of hCG (Sigma) 48 h later. Females were placed in cages with males after the hCG injection and were examined the following morning (Day 1) for the presence of a vaginal plug, indicating that mating had occurred.
Embryo Culture
For culture of pronuclear zygotes, female mice were killed between 1330 h and 1430 h on Day 1. Pronuclear zygotes were flushed from each excised oviduct with 0.1 ml HEPES-buffered KSOM (High Potassium Simplex Optimized Medium) [26]. Zygotes from several mice were pooled and washed through one droplet of flushing medium containing 0.65 mg/ml hyaluronidase (Sigma) to remove cumulus cells; they were then washed through three further droplets of HEPES-buffered KSOM.
Zygotes were cultured in dishes (Costar, Cambridge, MA) in KSOM [26, 27] containing various concentrations of PTHrP 1–34 (Peninsula Labs, Belmont, CA) or PTHrP 1–141 (gift from Armen Tashjian, Jr., Harvard School of Public Health). Lyophilized PTHrP was reconstituted in KSOM at the following concentrations: 0, 10, 33, 100, 333, 1000, 3300, and 10 000 pg/ml. Four 50-µl drops of medium were placed in each dish under 5 ml mineral oil (Sigma). The dishes were made up the day before the embryos were harvested and were equilibrated overnight in an atmosphere of 6% CO2, 5% O2, and 89% N2 in a plastic culture chamber (Billups-Rothenberg, Del Mar, CA) inside a 37°C incubator. Zygotes, five at a time, were taken randomly from the collection pool and transferred to a culture dish through two serial washes of culture drops, then to a third culture medium drop of one of eight treatments. The culture dishes were returned to the culture chamber at 37°C, which was gas-equilibrated and then sealed.
Differential Fluorescent Staining of Inner Cell Mass (ICM) and Trophectoderm
At 96 h of culture, blastocysts were stained according to the method of Ebert et al. [28] and Papaioannou and Ebert [29]. Blastocysts, three to five at a time, were transferred from culture drops to acid Tyrode's solution (8.0 g/L NaCl, 0.2 g/L CaCl2, 0.1 g/L MgCl2·6H2O, 0.05 g/L NaHPO4·H2O, 1.0 g/L glucose, 1.0 g/L NaHCO3 (pH adjusted to 2.5 with 5 N HCl), under constant observation, for 5–15 sec until the zonae pellucidae were completely dissolved. Blastocysts were then removed to three rapid, successive washes in Ham's medium F-12 (10% calf serum; Sigma) and then to an antiserum (R105: rabbit anti-mouse prepared against mouse whole spleen) diluted to 10% in Ham's F-12, for 30 min at 37°C. After 30 min, embryos were transferred through three successive 5-min washes and then to 10% guinea pig complement (Gibco Laboratories, Grand Island, NY), 10 mg/ml bisbenzimide, and 10 mg/ml propidium iodide (Sigma) in Ham's F-12 for 30 min at 37°C. To count differentially stained nuclei, embryos were removed individually from complement solution to acetone-washed microscope slides, squashed under coverslips, and viewed using a Zeiss (Thornwood, NY) epifluorescence microscope with a 365-nm band pass excitation filter and a 420-nm long pass barrier filter.
Blastocyst Outgrowth Assays
The method used for assessing effects on outgrowth was the well-characterized assay established by Armant et al. [30] and used by other investigators [31–34]. These studies have shown that when intact mouse blastocysts are placed in culture on fibronectin, there is a significant outgrowth of trophoblast cells. Immunostaining with cell markers such as cytokeratin, vimentin, and DBA (Dolichos biflorus agglutinin) indicated that the cells undergoing outgrowth were trophoblast cells since the cells did show immunoreactivity for cytokeratin but were negative for vimentin and DBA (markers for ICM-derived cells). ICM cells do undergo significant outgrowth when cultured in the absence of trophectoderm [34]. Blastocysts were obtained from superovulated mice at 83 h after hCG injection by flushing them from the uterine horns with 0.1 ml Dulbecco's PBS (DPBS; Sigma). The embryos were cultured at 37°C in a 5% CO2 incubator for 24 h to allow hatching from the zona pellucida. For the trophoblast outgrowth assays, individual wells in 96-well microtiter plates (Becton Dickinson, Rutherford, NJ) were coated with fibronectin (human serum-derived; 0.25 mg/ml; Collaborative Research, Bedford, MA) for 4 h at 37°C [25]. After 4 h, wells were washed with 0.3% BSA (Sigma) in Hanks' Balanced Salt Solution (HBSS; Gibco) three times; then wells were filled with 50 µl of 0.3% BSA in Eagle's Minimal Essential Medium (Eagle's MEM; Gibco) containing various concentrations of individual growth factors or antibodies. Hatched blastocysts were added to the wells, one blastocyst per well, and cultured at 37°C in a 5% CO2:95% air humidified incubator. The percentage of blastocysts attached to fibronectin-coated plates was recorded after a 24-h incubation as described by Sherman [35]. Briefly, attachment was scored by counting under a dissecting microscope the number of blastocysts that remained attached to the substratum when the contents of the dish were swirled in a circular motion. The surface area of outgrowth containing contiguous cells was measured at 24, 48, and 72 h using a microscope eyepiece grid [32]. Media were collected from some of the treatment groups at 72 h for assay of PTHrP levels.
Matrix Binding Experiments
Ninety-six-well plates were coated with fibronectin (0.25 mg/ml) as described above. Fibronectin-coated wells were washed with HBSS/0.3% BSA three times, and growth factors were added and incubated for 2 h in 5% CO2 at 37°C. Unbound growth factors were then washed out with HBSS/0.3% BSA. Hatched blastocysts were subsequently cultured one per well in 50 µl of Eagle's MEM/0.3% BSA, and the surface area of outgrowth was scored as described above.
Growth Factors and Neutralizing Procedures
Growth factors and their antibodies were obtained from various sources. The intact form of PTHrP, PTHrP 1–141, is not commercially available so most of the studies were carried out using the biologically active fragment of PTHrP, PTHrP 1–34, which was purchased from Peninsula Laboratories (Belmont, CA). We did obtain small amounts of intact PTHrP 1–141 and PTH as gifts from Dr. Armen Tashjian Jr. at the Harvard School of Public Health. Both PTHrP preparations as well as the PTH were tested in a bone resorption-stimulation assay [36] and were found to cause significant stimulation of bone resorption, although the PTHrP 1–34 fragment was more potent than PTHrP 1–141 (Edward Voelkel, personal communication). Recombinant TGF-ß1 was obtained from R&D Systems (Minneapolis, MN). Neutralizing antibody to PTHrP 34–53 was purchased from Oncogene Science (Manhosset, NY) and that to TGF-ß1, -ß2, and -ß3 from Genzyme (Cambridge, MA). In neutralization studies, the antibody to PTHrP was diluted to 100 µg/ml, and the antibody to TGF-ß was diluted to 200 µg/ml with the appropriate growth factors for 1 h at 37°C in a 5% CO2 atmosphere. The antibody preparations were then centrifuged at 10 000 x g for 5 min to remove immune complexes, and the supernatants were subsequently added to the blastocyst outgrowth assay.
PTHrP Immunoassay
PTHrP levels were measured using an immunoradiometric assay kit from the Nichols Institute (San Juan Capistrano, CA). This kit uses a polyclonal antibody to PTHrP 60–72 to detect the larger, physiological forms of PTHrP. The kit does not measure the levels of the smaller, biologically active fragment PTHrP 1–34. Medium samples from blastocysts within a treatment group were pooled so that each sample contained the conditioned medium from 5 wells (total volume 250 µl). Each sample was then assayed in duplicate (125 µl/tube). Pooling of samples within each treatment group was necessary in order to have enough measurable PTHrP in each sample tube. The intraassay variation was 6.1% and the lower limit of sensitivity was 2.4 pg/ml.
Immunohistochemistry
Mated female mice were killed on Days 3, 4, 5, 6, and 7 of gestation (n = three/time point); uterine horns were fixed in fresh 4% paraformaldehyde for 8 h and then embedded in paraffin for histological analysis. Sections for analysis were cut at 5 µm. Deparaffinized sections were first incubated in 1.5% goat serum/PBS as a blocking step for 20 min at 25°C. Sections were then incubated with one of the following antibodies, each for 30 min at 25°C: polyclonal rabbit antibody to human PTHrP 34–53, 5 µg/ml; polyclonal rabbit antibody to chicken desmin (Chemicon, Temecula, CA), 2.5 µg/ml; monoclonal rat antibody to murine laminin (Chemicon), 1:200 dilution; or nonspecific rabbit IgG (Sigma), 5 µg/ml. Binding was visualized with use of biotinylated goat anti-rabbit or goat anti-rat secondary antibody diluted 1:200 in PBS (pH 7.4) followed by avidin/horseradish peroxidase binding (Vecta-stain kit; Vector Laboratories, Burlingame, CA). The complex was detected with diaminobenzidine (0.1%) and hydrogen peroxide (0.02%) in sections counterstained with hematoxylin (4 g/ml).
Experimental Design and Statistical Analysis
Pronuclear zygotes were cultured in various concentrations of PTHrP 1–34 (three replicates) or PTHrP 1–141 (one replicate), ten embryos per treatment in each replicate. After 96 h of culture, the numbers of ICM cells and trophoblast cells were counted. Since the growth of the preimplantation embryo is multiplicative, the counts were statistically analyzed after transformation to the logarithmic scale. Logarithms to the base 2 were used since this converts the data from counts to numbers of cell divisions. ANOVAs were done using the S-Plus 4 (Mathsoft, Seattle, WA) computer program. Confidence limits are given at the p = 0.05 level.
The experiments employing trophoblast outgrowth assays were performed in three replicates (see Figs. 2, 3, and 5) or two replicates (see Fig. 4); in each replicate there were five observations per treatment, with the exception of one missing observation in the last experiment (see Fig. 5). The results were analyzed by two-way ANOVA. The replicate x treatment interactions were highly significant in all experiments. Consequently, the sums of squares for the replicate x treatment interactions were used to calculate pooled error means squares to test the significance of the treatment effects. Then the computations were done using the Number Crunching Statistical System 6.0 (Jerry Hintz, Kaysville, UT).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PTHrP and TGF-ß1 were tested at concentrations of 0, 0.1, 1.0, and 10.0 ng/ml. Blastocysts were collected, allowed to hatch, and then placed in fibronectin-coated wells. Cultures were assessed at 24 h for attachment and at 72 h for outgrowth. The mean percentage of blastocysts that had attached was 90% for all the treatment groups at 24 h and 99% for all treatment groups at 72 h.
Figure 1 shows the appearance of spreading trophoblast cells from a typical outgrowth assay after 72 h of treatment. The increase in outgrowth of mouse trophoblast cells in response to the addition of PTHrP 1–34 (Fig. 1, B–D) and TGF-ß1 (Fig. 1, E and F) to culture medium is evident when compared with the untreated control (Fig. 1A). Figure 2 shows the effects of various concentrations of TGF-ß1 and PTHrP 1–34 on the area of trophoblast outgrowth. At the three concentrations tested, TGF-ß1 and PTHrP 1–34 increased the surface area of trophoblast outgrowth significantly compared with medium controls. Across the three concentrations of TGF-ß1 tested, trophoblast outgrowth was linear, and greatest at the highest concentration tested. PTHrP 1–34 stimulated outgrowth maximally at 1 ng/ml. Antibodies to these growth factors abrogated their effects. The antibodies alone had no effect.
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Figure 3 shows the results of testing the addition of various concentrations of PTHrP 1–141 and PTH to blastocyst cultures on trophoblast outgrowth. PTHrP 1–141 increased outgrowth significantly at concentrations of 1.0 and 10 ng/ml compared with controls, whereas PTHrP 1–141 at 0.1 ng/ml did not. At the concentrations tested, PTH, which binds the same receptor as PTHrP [10, 11], did not significantly increase trophoblast outgrowth.
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To determine whether the increase in trophoblast outgrowth effected by TGF-ß1 was mediated by the production of PTHrP by the blastocysts themselves, experiments were carried out to examine whether the effect of TGF-ß1 was inhibited by PTHrP-neutralizing antibody. The results of these experiments are presented in Figure 4. When added in the maximally effective concentration, the PTHrP-neutralizing antibody reduced the increase in trophoblast outgrowth seen with TGF-ß1 alone. Conditioned medium from blastocysts treated with no growth factor (control), 1.0 ng/ml PTHrP 1–34, or 1.0 ng/ml TGF-ß1 was assayed for levels of intact PTHrP 1–141 using an immunoradiometric assay. The results (Table 1) showed that both PTHrP 1–34 and TGF-ß1 caused an increase in PTHrP production by cultured blastocysts.
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The possible binding of TGF-ß1 or PTHrP to the fibronectin matrix coating the culture wells of the trophoblast outgrowth assay was tested by adding growth factors to culture wells and then washing these out with medium before the addition of zona-free blastocysts for culture. The results of this experiment are shown in Figure 5. TGF-ß1 at all concentrations tested produced equivalent increases in trophoblast outgrowth that were always greater than that produced by controls. This effect was reduced to control levels by addition of anti-TGF-ß antibody and by anti-PTHrP antibody. PTHrP 1–34 and PTHrP 1–141 caused no enhancement of trophoblast outgrowth.
Effect of PTHrP on Preimplantation Development
Since PTHrP showed a significant stimulatory effect on blastocyst outgrowth, we next tested the effects of PTHrP on specific endpoints in preimplantation embryo development. Pronuclear zygotes were collected from female mice and placed in culture with a range of concentrations of PTHrP 1–34 or PTHrP 1–141 from 10 to 10 000 pg/ml. Embryos were cultured for 96 h, and the number developing to the blastocyst stage was recorded for each treatment group. The percentage of embryos developing to blastocysts ranged from 88% to 95% for the various treatment groups, indicating that PTHrP 1–34 and PTHrP 1–141 had no effect on the incidence of blastocyst formation.
The ANOVAs on the numbers of ICM cells, trophectoderm cells, and total cell divisions showed no significant differences between the cell counts in the control blastocysts and those exposed to either PTHrP 1–34 or PTHrP 1–141. The overall growth of the zygotes is summarized in Table 2. The growth of the zygotes exposed to PTHrP 1–34 is approximately the same as that described by Erbach et al. [27] for the development of control CF-1 zygotes in KSOM. The growth of zygotes exposed to PTHrP 1–141 differs slightly in that the proliferation of the trophectoderm cells was low. The reason for this effect is unknown.
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Localization of PTHrP in Mouse Uterus
The expression of PTHrP in the mouse uterus during early pregnancy was examined to see whether the protein is present in specific cells at the time of implantation. The results for the immunochemical staining are shown in Figure 6. At Days 3, 4, and 5 of gestation, uterine luminal and glandular epithelial cells stained positively for PTHrP, but stromal cells appeared negative, as can be seen particularly well in the high-power view in Figure 6C, which shows positively stained epithelial cells but little if any staining in stromal cells. Oviductal epithelial cells also showed positive immunoreactivity at this time (data not shown). Implantation in the mouse is initiated late in the evening of Day 4 of pregnancy. No positive immunoreactivity for desmin or laminin was observed in endometrial stromal cells through the morning of Day 5 (data not shown). By Days 6 and 7, decidualized stromal cells stained positively for PTHrP (see Fig. 6, H–M) and for desmin and laminin (data not shown). Positive immunoreactivity for PTHrP was restricted to decidualized stromal cells located at the site of implantation on the anti-mesometrial side of the uterine lumen (see Fig. 6J, high-power view, note arrow) and to luminal epithelial cells. Nondecidualized stromal cells in inter-implantation sites showed no immunoreactivity with PTHrP (data not shown). The myometrium also showed positive immunoreactivity on all days examined, and this confirms findings from previous investigations on human myometrium [16, 17].
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PTHrP and PTH show striking homology within the first 13 amino acids, which accounts for the ability of PTHrP to bind to the PTH receptor [10]. However, the remaining sequences of the two molecules are completely distinct. The PTHrP gene in the rat encodes a 141-amino acid peptide; while in the human, three isoforms of 139, 141, and 173 amino acids exist [8, 11]. The presence of another unique receptor for PTHrP has been suggested by studies showing that the midportion of the PTHrP molecule acts to regulate calcium transport in the sheep placenta [40].
Immunohistochemical analysis of PTHrP protein expression revealed a pattern similar to that reported for TGF-ß [5, 6]. Mouse uterine epithelial cells expressed the PTHrP protein as early as Day 3 of gestation, whereas the decidualizing stromal cells did not show expression until the morning of Day 6 of gestation. Thus, expression of PTHrP in mouse stromal cells appears to be linked with decidualization, as it is in the rat. The presence of an embryo may not be required for expression of PTHrP by decidual cells since induction of deciduomas in the rat through corn oil injections also results in the expression of PTHrP mRNA by the decidualized stromal cells [37].
The expression of PTHrP by stromal cells once they had undergone decidualization suggests that this growth factor may be involved in the process of decidualization. Decidualization of rat stromal cells is associated with an increase in expression of desmin and laminin [41]. Human endometrial stromal cells show an increase in laminin and fibronectin production when they undergo decidualization in an in vitro culture system [42]. It is possible that PTHrP is involved in the regulation of production of these ECM proteins by decidual cells. PTHrP has also been shown to be a potent smooth-muscle relaxant [43]. PTHrP produced by decidual cells in pregnancy may serve to modulate endometrial/decidual blood flow.
TGF-ß1 and PTHrP both proved to be potent stimulators of trophoblast outgrowth. Comparison of the effects of PTHrP 1–34 with those of TGF-ß1 showed that PTHrP 1–34 was a more potent stimulator of outgrowth, with the greatest stimulation occurring at 1 ng/ml, whereas the greatest stimulation for TGF-ß1 occurred at a concentration of 10 ng/ml. PTHrP 1–141 was not quite as potent as PTHrP 1–34 in stimulating outgrowth, but this was probably due to an overall reduced biological activity of the protein, since it was also less potent than PTHrP 1–34 when tested in a bone resorption-stimulating assay. Whether the increase in surface area represents an increase in both cell number and migration needs to be determined. The stimulatory effects of both PTHrP and TGF-ß1 were abrogated with concomitant treatment with an anti-PTHrP 34–53 antibody. This antibody does not recognize PTHrP 1–34. These results suggest that the stimulatory effects of exogenously added PTHrP 1–34 or TGF-ß1 are mediated indirectly through a stimulation of intact PTHrP 1–141 production by the blastocysts themselves. This was confirmed when we assayed the medium from mouse blastocysts treated with either PTHrP 1–34 or TGF-ß1. The results showed that both growth factors caused a 3- to 4-fold increase in PTHrP secretion by cultured blastocysts (Table 1). Van de Stolpe et al. [38] have reported that the trophectoderm of mouse blastocysts produces PTHrP. They also reported that ES-5 embryonic stem cells express the PTH/PTHrP receptor mRNA. We have detected the mRNA for PTH/PTHrP receptor in mouse blastocysts using polymerase chain reaction (unpublished observations). PTHrP may therefore act in an autocrine manner to stimulate further increases in PTHrP production by the mouse blastocyst. Stimulation of PTHrP production by TGF-ß1 has been reported for other cell types including epidermal squamous cancer cell lines [21] as well as human endometrial stromal cells [16]. These investigators found that TGF-ß1 not only increased PTHrP mRNA levels by stimulating increases in gene transcription but also acted to increase PTHrP mRNA stability.
Our results showing that PTHrP stimulates the outgrowth of cultured mouse blastocysts are similar to the findings reported by Behrendtsen et al. [34]. These investigators cultured isolated ICM from mouse embryos on a variety of defined ECM substrata and assessed the extent of outgrowth by the parietal endoderm cells. They found that the parietal endoderm cells showed outgrowth only when cultured on fibronectin. However, if parietal endoderm cells were cultured in combination with trophectoderm, or if parietal endoderm cells were cultured in the presence of PTHrP, then outgrowth occurred on other substrata, including laminin and vitronectin. These results suggest that PTHrP may increase blastocyst outgrowth by regulating either fibronectin production or the interaction of parietal endoderm cells with the underlying matrix protein. This could occur through regulation of specific integrins or metalloproteinases. The authors did not test the effects of PTHrP on outgrowth of intact mouse blastocysts so that effects on trophoblast cells could not be measured. Their results show that the stimulatory effect of PTHrP on blastocyst outgrowth may involve outgrowth of both primary trophoblast cells as well as parietal endoderm cells.
We observed no effect of either PTHrP 1–34 or PTHrP 1–141 on the rate of development of pronuclear zygotes to the blastocyst stage. The PTHrP peptides used had no effect on the numbers of ICM and trophoblast cells in the blastocysts that developed. One explanation for these findings may be that preimplantation mouse embryos do not express the PTHrP receptor before an expanded blastocyst is formed. Although the expression of the PTH/PTHrP receptor has been detected in both F9 embryonal carcinoma cells and ES-5 embryonic stem cells as they differentiate into primitive and parietal endoderm-like phenotypes [38] and we have detected the mRNA for PTH receptor in mouse blastocysts (unpublished observations), it has not been looked for at earlier stages of mouse embryo development. Alternatively, it may be possible that PTHrP regulates some other processes in embryo development that have not as yet been identified.
TGF-ß has been reported to stimulate trophoblast cell spreading and oncofetal fibronectin production by first-trimester human trophoblast cells [44] and by term human cytotrophoblast cells [45]. TGF-ß has also been shown to increase levels of gelatinase mRNA in both first-trimester trophoblast cells and choriocarcinoma cell lines [46]. Studies by Graham and coworkers [47, 48] have provided considerable evidence that TGF-ß modulates trophoblast differentiation by promoting trophoblast syncytial formation and limiting invasiveness in vitro. TGF-ß1 and -ß2 are expressed in a cell-specific manner during early pregnancy in the mouse uterus, suggesting a role for this growth factor in the embryo-maternal interactions that occur during implantation.
A number of studies have reported that PTHrP has biological activities similar to those of TGF-ß. PTHrP has been shown to increase fibronectin production by human dermal fibroblasts in culture [23] and collagen synthesis in fetal rat long bones [25]. These findings suggest that PTHrP and TGF-ß may stimulate blastocyst outgrowth by promoting attachment and movement of trophoblast cells on specific ECM proteins, particularly fibronectin. Both human and mouse trophoblasts undergo significant transitions in their expression of integrins and ECM components as they invade the endometrial layer. As these cells leave the basement membrane, they down-regulate expression of 
6ß4 integrin (laminin receptor) and begin to express the 5ß1 integrin, a fibronectin receptor, along with a fibronectin-rich pericellular matrix [49, 50]. PTHrP and TGF-ß may regulate these processes in normal placental development. This hypothesis is supported by a study [39] showing that PTHrP receptor (-/-) mutant mice do not develop beyond Days 10–12 of gestation.
In conclusion, the results of our studies have shown that both TGF-ß1 and PTHrP are potent stimulators of trophoblast outgrowth by mouse blastocysts cultured on fibronectin. PTH did not stimulate blastocyst outgrowth, suggesting that the actions of PTHrP occur through interaction with a receptor other than the PTH/PTHrP receptor. A neutralizing antibody to PTHrP 34–53 blocked the stimulatory effects of both TGF-ß1 and PTHrP 1–34, suggesting that these two growth factors act to stimulate trophoblast outgrowth by increasing endogenous PTHrP 1–141 production by the mouse blastocyst. This was confirmed by immunoassay of conditioned medium from blastocysts treated with either PTHrP 1–34 or TGF-ß1, which showed 3- to 4-fold higher levels of PTHrP 1–141 than did untreated controls. The mechanisms by which PTHrP increases trophoblast outgrowth may include regulation of fibronectin production or expression of specific integrins; these questions are currently being investigated in our laboratory.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Romana Nowak, Brigham&Women's Hospital, 221 Longwood Ave., Boston, MA 02115. FAX: 617 566 7980.
Accepted: August 19, 1998.
Received: May 12, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and TGFß genes in preimplantation mouse embryos. Science 1988; 242:1823–1825.This article has been cited by other articles:
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  L. Chen, M. Nakai, R. J Belton Jr, and R. A Nowak Expression of extracellular matrix metalloproteinase inducer and matrix metalloproteinases during mouse embryonic development Reproduction, February 1, 2007; 133(2): 405 - 414. [Abstract] [Full Text] [PDF]
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R. L Jones, C. Stoikos, J. K Findlay, and L. A Salamonsen TGF-{beta} superfamily expression and actions in the endometrium and placenta. Reproduction, August 1, 2006; 132(2): 217 - 232. [Abstract] [Full Text] [PDF]
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