Leptin Serves as an Upstream Activator of an Obligatory Signaling Cascade in the Embryo-Implantation Process
http://www.100md.com
内分泌学杂志 2005年第2期
Boston Biomedical Research Institute (M.P.R., P.C.L., R.R.G.), Watertown, Massachusetts 02472; Vincent Center for Reproductive Biology (M.P.R., B.R.R., R.R.G.), Massachusetts General Hospital, Boston, Massachusetts 02114; Harvard Medical School (B.R.R.), Boston, Massachusetts 02115; and Department of Physiology (P.C.L.), Tufts University School of Medicine, Boston, Massachusetts 02111
Address all correspondence and requests for reprints to: Dr. R. R. Gonzalez, Boston Biomedical Research Institute, 64 Grove Street, Watertown, Massachusetts 02472. E-mail: gonzalezr@bbri.org.
Abstract
Leptin is essential for mouse reproduction, but the exact roles it serves are yet to be determined. Treatment of cultured endometrial cells with leptin increases the level of ?3-integrin, IL-1, leukemia inhibitory factor, and their corresponding receptors. These leptin-induced effects are eliminated by inhibitors of leptin receptor (OB-R) signaling. Herein the impact of blocking leptin/OB-R signaling in the mouse endometrium was assessed. Intrauterine injection of either leptin peptide antagonists (LPA-1 or -2) or OB-R antibody on d 3 of pregnancy impaired mouse implantation in comparison to intrauterine injection of scrambled peptides (LPA-Sc) or species-matched IgGs. Significant reduction in the number of implantation sites and uterine horns with implanted embryos was found after intrauterine injection of LPA-1 (1 of 22) vs. LPA-1Sc (11 of 15) and LPA-2 (3 of 17) vs. LPA-2Sc (14 of 16). The impact of disruption of leptin signaling on the endometrial expression of several molecules in pregnant mice was assessed by Western blot, immunohistochemistry, and confocal microscopy. Disruption of leptin signaling resulted in a significant reduction of IL-1 receptor type I, leukemia inhibitory factor, vascular endothelial growth factor receptor 2, and ?3-integrin levels. The levels of colony stimulating factor-1 receptor and OB-R were unaltered after treatment with LPAs compared with controls. Expression of OB-R protein was pregnancy dependent and found only in glandular epithelium after implantation occurred. Our findings support previous observations that leptin signaling is critical to the implantation process and suggest that molecules downstream of leptin-activated receptor may serve obligatory roles in endometrial receptivity and successful implantation.
Introduction
LEPTIN, THE PRODUCT of the ob gene, is mainly secreted by white adipocytes and plays a key role in the regulation of body weight and food intake. Since the discovery of leptin 10 yr ago (1), its significance in other physiological processes has been realized. There is now strong supportive evidence that leptin is critical for reproduction (for review see Refs.2, 3).
The leptin sequence is highly conserved, whereas there are sequence differences in the leptin receptor (OB-R, the product of the db gene) among various species. In the mouse and the human, OB-R has several splice variants. The full-length and functional OB-R (OB-Rb) is expressed by the hypothalamus (4, 5) and other tissues (2). OB-R isoforms with a shorter cytoplasmic tail (OB-Ra) are expressed in many peripheral tissues (6) and are expressed at lower levels in endometrial cancer tissue (7).
A soluble active OB-R has been described in humans (8), and its levels are enhanced in serum from pregnant compared with nonpregnant nonhuman primates (9). Binding of leptin to OB-Rb allows the binding of Janus kinase 2 (JAK2) to the intracytoplasmic tail of OB-R. JAK2 phosphorylates OB-R followed by phosphorylation of signal transducer and activator of transcription 3 (STAT3) that activates several signaling pathways. In addition to the JAK/STAT signaling pathway, the MAPK, protein kinase C, and phosphoinositol 3-kinase pathways are also activated by leptin in several cell types (10, 11). Both OB-Rb and OB-Ra can phosphorylate erbB2 upon binding and enhance MAPK activity (12). However, OB-Ra does not activate the JAK2/STAT3 pathway (13). Leptin and OB-Rb are expressed by human female reproductive tissues, including the ovary (12, 13, 14), endometrium (15, 16), and placenta (17, 18). Leptin has been found in human and mouse oocytes and preimplantation embryos (12, 19, 20, 21). In addition, leptin can promote the development of mouse preimplantation embryos through OB-R signaling (20). In vitro, the endometrial secretion of leptin is regulated by human preimplantation embryos (15). Leptin treatment increases the levels of ?3-integrin (a marker of endometrial receptivity) in human endometrial epithelial cells (22). Leptin increases p-STAT3, leukemia inhibitory factor (LIF), IL-1 (ligand and antagonist), and their cognate receptors in rabbit (23) and human endometrial cells (24) and induces the acquisition of the invasive phenotype of human trophoblast cells (25, 26). Mouse mutants deficient in leptin (ob/ob) (27) or OB-R (db/db) are obese and infertile. Fertility can be restored in ob/ob by administration of exogenous leptin (28). The withdrawal of leptin infusion in ob/ob females shortly after fertilization impairs implantation (29). Leptin injection into starved mice restores fertility (30). A postovulatory increase in serum leptin concentration appears to be associated with implantation potential (12), and low expression of OB-R has been found in endometrium from women with unexplained infertility (31). These data suggest that in vivo leptin could act in an autocrine or paracrine manner to regulate biological functions that may mediate the implantation process.
The leptin peptide antagonist LPA-2 is able to inhibit leptin binding to OB-R in vitro (23) and leptin signaling pathways that induce an increase in levels of IL-1, LIF, and ?3-integrin by endometrial cells (23, 24). In vitro studies and the phenotype of ob/ob and db/db mice would suggest that disruption of leptin signaling can have a significant impact on embryo preimplantation development and/or the implantation process. Therefore, it was hypothesized that the inhibition of leptin/OB-R signaling in endometrium by leptin peptide antagonists (LPAs) or antimouse OB-R antibodies would impair mouse embryo implantation and affect the levels of several downstream molecules with important roles in implantation.
Materials and Methods
Chemicals
Armenian hamster antibody anti-?3-integrin (N-20, mouse origin), mouse anti-Armenian hamster IgGs, goat polyclonal antibody anti-actin, rabbit polyclonal antibodies against the carboxy terminus of LIF receptor (LIF-R) (C-19, human or mouse origin), IL-1 receptor type I (IL-1R tI, antibody M-20), vascular endothelial growth factor receptor 2 (VEGF-R2), colony-stimulating factor-1 receptor (CSF-1R), and their respective blocking peptides for competition studies and mouse macrophage lysate (RAW 264.7) for Western blot, positive controls were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Goat polyclonal antimouse OB-R (AF497, anti-NH2-terminal end of mouse OB-R) and nonspecific rabbit and goat IgGs were from R&D Systems Inc. (Minneapolis, MN). Mouse normal serum was obtained from Biomeda (Foster City, CA). Normal goat and rabbit sera and biotinylated horse antimouse and rabbit antigoat IgG antibodies were from Vector Laboratories (Burlingame, CA). Biotinylated goat antirabbit IgG antibodies (ALI3409, mouse IgG adsorbed) were obtained from BioSource International (Camarillo, CA). Alexa Fluor 594 goat antirabbit IgG, Alexa Fluor 488 goat antimouse IgG, and 4',6-diamino-2 phenylindole dihydrochloride were from Molecular Probes Inc. (Eugene, OR). Other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO).
OB-R inhibitors
LPA-1 (32 amino acid residues, IQKVQDDTKTLIKTIVTRINDISHTQSVSSKQ) and LPA-2 (23) corresponding to helices I and III of leptin, respectively, and their scrambled peptides LPA-1Sc (TDKVITQSTINVKHTSSQSVQIKDIKIRQTLD) and LPA-2Sc for negative controls were synthesized as described elsewhere (23). The peptides were dissolved in sterile filtered vehicle solution composed of 0.04% dimethylsulfoxide (DMSO)-PBS. Goat antimouse OB-R antibodies and normal goat IgGs (negative control) diluted in PBS were sterile filtered and also used to assess the impact of blockade of OB-R signaling on mouse implantation.
Animals
Virgin, 8- to 10-wk-old, female C57BL6 and CD1 mice (Charles River Laboratories, Wilmington, MA) were housed in the animal facilities at the Massachusetts General Hospital or Boston Biomedical Research Institute in accordance with National Institutes of Health standards for the care and use of experimental animals. The rooms were provided with a controlled temperature range (22–24 C) on a 14-h light/10-h dark cycle. Mice were given water and food ad libitum. Ovulation was induced in female mice (n = 120) by an ip injection of 5 IU pregnant mare serum gonadotropin (Sigma) followed by 10 IU human chorionic gonadotropin (Sigma) 48 h later. Female mice were mated with fertile males of the same strain to induce pregnancy. The following morning, the females exhibiting vaginal copulatory plugs were separated for the proposed experiments. The day of vaginal plug was recorded as d 1 of pregnancy. Mice were weighed before treatment and again before they were euthanized.
Surgical procedures and intrauterine blocking treatments
On d 3 of pregnancy, mice were anesthetized by ip injection of Avertin (2,2,2 tribromoethanol, 200 mg/kg body weight; Sigma). A surgical incision was made on the dorsal midline through the skin, and each uterine horn was exposed using small forceps. Under a dissecting microscope, different compounds were delivered into the uterine lumen distal to the uterotubal junction using a Hamilton syringe (model PC010; Hamilton Co., Reno, NV) holding a pulled glass needle (<27 gauge, capillary pipette 20 μl; Unopette, Becton Dickinson, Bedford, MA). The effectiveness of the method was verified in preliminary experiments by evaluating the administration of dye. The treatments included 10 μl of LPA-1 or -2 or LPA-1Sc or -2Sc (3.3 μM) delivered into the lumen of the right horn of each mouse. Each animal served as its own internal control, with the left uterine horn receiving the vehicle. A group of mice received 10 μl goat anti-OB-R antibodies at a concentration of 1.5 or 7.5 μg in the right horn and similar concentrations of nonspecific goat IgG solutions in the left horn. Concentrations of OB-R inhibitors used in vivo were calculated from results of previous experiments in endometrial cell cultures (23). The effects of surgical treatment, intrauterine injection, and toxicity of vehicle solutions on implantation were also assessed by comparing the number of implantation sites in mice treated with intrauterine injections of PBS and PBS-DMSO and in nontreated mice. After administration of the compound(s) or sham surgery, the incision was closed with metal auto clips (9 mm; Becton Dickinson). Mice were placed in warmed cages (cages were placed on a slide warmer; Fisher, Pittsburgh, PA) until full recovery from the anesthetic.
To analyze the effects of OB-R inhibitors, the mice were euthanized on d 10 of pregnancy. The uteri were extracted, and the implantation sites were counted in each horn. The percentage of uterine horns with implanted embryos (pregnancy rate) was calculated in relation to the total number of uterine horns injected with each inhibitor of OB-R or controls. The percentage of embryos that implanted in the uterine horns receiving OB-R inhibitors with respect to control uterine horns was determined (implantation rate). To investigate the impact of inhibition of leptin signaling on the expression of several molecules (cytokine receptors and ?3-integrin), groups of mice were euthanized at d 1–6 of pregnancy. The endometrial tissue was isolated to produce cell lysates for Western blot analysis, and a small portion of each uterus was dissected and fixed in 4% paraformaldehyde in PBS or prepared for cryostat sections for qualitative analysis.
Cell lysates
Mouse endometrial tissues were homogenized on ice with lysis buffer [20 nM Tris (pH 7.4), containing 137 nM NaCl, 2 mM EDTA, 10% glycerol, 50 mM ?-glycerophosphate, 1% Nonidet P-40, and a mixture of proteases and phosphatase inhibitors composed of 100 μM antipain, 0.1 mg/ml trypsin inhibitor, protease inhibitor cocktail 1:50 (Sigma), 50 nM NaF, 2 mM phenylmethylsulfonyl fluoride, and 2 mM sodium orthovanadate]. Cellular lysates were centrifuged at 2400 x g at 4 C for 10 min. Protein concentrations were determined using the Bradford protein assay (Bio-Rad Laboratories Inc., Hercules, CA) and BSA as standard (Sigma).
Western blot
Cellular lysates were diluted 1:1 with Laemmli buffer and incubated at 95 C for 5 min. Denatured protein preparations (20 μg) from mouse endometrial samples were loaded on 7.5% SDS-PAGE gels. Electrophoresis was performed at 220 V for 5 min followed by 165 V for 20 min (Bio-Rad, electrophoresis apparatus) in Tris-glycine buffer (pH 8.4) (Bio-Rad). Electroblotting onto 0.2-μm nitrocellulose membranes was performed at 22 V overnight at 4 C in 48 nM Tris-39 nM glycine buffer containing 0.037% SDS and 20% methanol. Membranes were washed with 20 mM Tris, 137 mM NaCl (pH 7.4) buffer containing 0.15% Tween 20 (vol/vol) (wash buffer) and incubated for 1 h at room temperature in blocking buffer containing Amersham blocking product (5%, wt/vol; Amersham Biosciences Corp., Piscataway, NJ) in wash buffer. The membranes were subsequently incubated at room temperature for 1 h with 1 μg/ml of antibody to OB-R, IL-1R tI, LIF-R, VEGF-R2, CSF-1R, or ?3-integrin in wash buffer. After washing (four times for 5 min each), the membranes were incubated for 1 h at room temperature with biotinylated secondary antibody specific for anti-primary antibody species in wash buffer containing 2.5% normal horse, goat, or mouse serum. Immune detection was performed by incubation for 30 min at room temperature with streptavidin-horseradish peroxidase conjugate diluted 1:2000 in wash buffer (Amersham). Specific bands in the blots were visualized using an ECL-chemiluminescent assay (Amersham) and Imagetek-B film (American X-Ray & Medical Supply, Rancho Cordoba, CA). Nonspecific mouse, rabbit, and goat IgGs or preincubation of primary antibodies with specific blocking peptides (Santa Cruz Biotechnology) were used instead of primary antibodies to produce negative control blots. Quantitative analyses of Western blot results were performed with the program TotalLab (version 2003.02, NonLinear Dynamics Ltd., Durham, NC). Twenty micrograms of mouse macrophage lysate (RAW 264.7) were loaded in each gel as a control for identification of specific bands and to perform statistic comparison of relative levels of antigens. The data were calculated from results of three or more Western blot determinations using different endometrial protein preparations (n = 5). Actin was determined for loading control in the gels (data not shown).
Immunohistochemical determinations
To assess the potential effects of blockade of endometrial OB-R function in vivo on the expression of various cytokine receptors, immunohistochemistry in cryostat and paraffin block sections (4 μm) was performed. Unmasking of antigen epitopes (?3-integrin, LIF-R, and IL-1R tI) in paraffin sections was performed. Briefly, samples were boiled in 10 mM sodium citrate/1 mM EDTA (pH 6) solution for 10 min. After quenching endogenous peroxidase activity with H2O2 (3% water solution) and blocking (2.5% horse or rabbit normal serum), tissue sections were incubated for 1 h at room temperature with the following primary antibodies diluted in PBS-0.1% BSA: anti-?3-integrin, IL-1R tI, LIF-R, VEGF-R, CSF-1R, and OB-R antibodies (all at 1 μg/ml). Biotinylated secondary antibodies were used. The tissues were incubated with a streptavidin-biotin-peroxidase system according to the manufacturer’s directions (Vectastain, ABC-AP kit, Vector), counterstained with hematoxylin (Dako Corp., Carpinteria, CA), and mounted with VectaMount (Vector). Negative controls included tissue preparations in which the primary antibodies were substituted by irrelevant species-matched IgGs. Negative controls for competitive studies with anti-IL-1R tI, LIF-R, VEFG-R, and CSF-1R antibodies were generated by preincubation with their respective blocking peptides (20 μg/ml; Santa Cruz Biotechnology). All washing steps were performed by immersion of the preparations three times in PBS for 5 min at room temperature.
Confocal laser scanning microscopy
Confocal laser scanning microscopy was performed in serial sections from paraffin blocks to determine the expression and colocalization of receptors (OB-R, IL-1R tI, and LIF-R) and ?3-integrin within endometrial tissue from nonpregnant and pregnant mice after anti-OB-R intrauterine treatments. The protocols for immunostaining used were similar to those already described for regular color-based immunohistochemistry, but the secondary antibodies and counterstaining agent were changed. Alexa Fluor 594 goat antirabbit IgG (red fluorescence) and Alexa Fluor 488 goat antimouse IgG (green fluorescence) conjugates were used to detect LIF-R, IL-1R tI, VEGF-R, CSF-1R, and ?3-integrin, respectively. In addition, a second fluorescein isothiocyanate-conjugated antibody (rabbit antigoat IgG-fluorescein isothiocyanate) was used to detect OB-R. The double-fluorescent-stained specimens were analyzed with a confocal laser scanning microscope equipped with an external argon laser (Bio-Rad). To avoid photobleaching of fluorochromes during fluorescence microscopy, the slides were embedded in antifade solution (Dako). For nuclear and chromosome counterstaining, 300 nM 4',6-diamidino-2-phenylindole in PBS-dimethylformamide solution was used.
Statistical analysis
A one-way ANOVA test with Dunnett error protection and a confidence interval of 95% was used from the Analyze-it for Microsoft Excel (Leeds, UK, htpp://www.analyze-it.com) for data analysis. Data are expressed as mean ± SEM. Values for P < 0.05 were considered statistically significant.
Results
Impact of disruption of leptin signaling in the endometrium
Mouse embryo implantation.
The surgical process and the injection of vehicle solution (0.04% DMSO-PBS) had no effect on the number of implanted embryos (implantation rate) in any of the mouse strains used in this study. Similarly it was assessed that postsurgery trauma did not significantly affect implantation in these mice (data not shown). However, a single injection of LPA-1 or -2 (3 μM/10 μl) at d 3 of pregnancy resulted in significantly impaired implantation in both C57BL6 and CD1 mice. The number of uterine horns with implanted embryos (pregnancy rate) was negatively affected by LPA-1 or -2. Only 4.5% (1 of 22) and 17.6% (3 of 17) of the uterine horns treated with LPA-1 and LPA-2, respectively, exhibited implantation sites (Fig. 1A). In contrast, 73.3% (11 of 15) and 90% (14 of 16) of the horns injected with negative controls LPA-1Sc and LPA-2Sc, respectively, showed implanted embryos (see Fig. 1A). No significant differences were found in pregnancy rate between LPA-1Sc, LPA-2Sc, vehicle, or IgG. Intrauterine injection of anti-OB-R-specific antibodies at a concentration of 1.5 and 7.5 μg/horn showed no reduction of pregnancy rate (see Fig. 1A). The number of implanted embryos in the uterine horns treated with OB-R inhibitors was significantly reduced compared with the controls. From the total number of embryos that implanted in the uterine horns (treated with OB-R inhibitors and their controls), only 15.5, 10.7, and 28% were found in those horns injected with LPA-1, LPA-2, and anti-OB-R antibody, respectively (Fig. 1B). The embryos that implanted in LPA-2Sc-, vehicle-, or IgG-treated mice were found uniformly distributed throughout the uterine horn (Fig. 1C). In contrast, the few embryos implanted in LPA- or anti-OB-R-antibody-treated horns were mainly found in the distal portion of the uterine horn, furthest from the OB-R inhibitor injection site (Fig. 1C).
FIG. 1. Impairment of mouse embryo implantation by intrauterine injection of OB-R inhibitors in CD1 mice. A and B, Impact of OB-R inhibitors on pregnancy rate (percentage of horns with implanted embryos) (A) and on implantation rate (percentage of implantation sites related to the total number of implanted embryos found after treatment with each inhibitor of OB-R plus its control) (B). C, Representative pictures from mouse uterine horns treated with LPA-Sc vs. vehicle and LPAs vs. vehicle. Notice the reduction of pregnancy and number of implantation sites by LPA-1 and -2 intrauterine injections (10 μl/30 μM). Implantation sites (if any) in the horns treated with LPAs were found distal from the utero-tubal junction. Anti-OB-R antibodies (7.5 μg) also negatively affected the number of implantation sites significantly. The results presented are the mean ± SE; *, P value 0.05 when comparing levels in response to treatments to that of the LPA-Sc- or IgG-treated controls. Similar results were found in C57BL6 mice (data not shown).
Levels of cytokine receptors and ?3-integrin during early pregnancy in mouse endometrium
Western blot analysis revealed that levels of LIF-R, VEGF-R2, and ?3-integrin dramatically increased in mouse endometrium during early pregnancy (d 1–6; see Fig. 2, A and B). The levels of these molecules increased at preimplantation time (d 3) and after implantation occurred (d 6). CSF-1R and IL-1R tI levels did not show significant change from d 1–6. (Fig. 2, A and B). The antibody against the extracellular domain of OB-R identified several bands corresponding to long and short OB-R isoforms. However, no significant variation in the levels of the OB-R isoforms was found from d 1–6 of pregnancy. Figure 2 shows the data corresponding to the full-length OB-R (see Fig. 2, A and B).
FIG. 2. Protein levels of ?3-integrin and VEGF-R2, LIF-R, CSF-1R, IL-1 R tI, and OB-R in mouse endometrium during early pregnancy. A, Representative Western blots of cytokine receptors and ?3-integrin obtained after d 1, 3, and 6 of pregnancy (d 1 copulatory plug) from endometrial extracts of control mice without any treatment. B, Quantitative analysis of molecules normalized against positive control from mouse macrophages (RAW 264.7; see Materials and Methods). An antibody against actin was used as a loading control (data not shown). Notice that protein levels of VEGF-R2, ?3-integrin, and LIF-R increased dramatically after fertilization and particularly after implantation occurred. In contrast, the levels of CSF-1R, IL-1R, and OB-R did not show significant changes. Quantitative analyses of Western blot results were performed with the program TotalLab. All data were derived from a minimum of three independent experiments using different mouse endometrium preparations (n = 5).
Impact of disruption of leptin signaling on levels of cytokine receptors and ?3-integrin in mouse endometrium
Western blot analysis revealed that intrauterine injections of LPA-1 and LPA-2 significantly decreased the levels of VEGF-R2, IL-1R tI, and LIF-R in the mouse endometrium (Fig. 3, A–F). LPA treatment also decreased the levels of ?3-integrin, but a significant reduction was found only in mouse endometria injected with LPA-1 (see Fig. 3, G and H). No significant variation in the levels of CSF-1R after LPA treatment was found (see Fig. 3, I and J). The negative effects of inhibition of leptin/OB-R signaling by LPAs on the endometrial levels of the above molecules were corroborated qualitatively by immunohistochemistry. Intensity of staining for all of the above antigens showed a marked decrease in endometrium (d 6) from mice with documented copulatory plug treated with LPA-2 or LPA-1 compared with control endometria treated with scrambled LPAs (see Fig. 4, A–L). These changes were more evident when embryo implantation was inhibited. CSF-1R staining was not significantly changed after LPA treatment (data not-shown). OB-R was found in endometrial epithelial cells (lumen and glands) in the uteri without implanted embryos (Fig. 4N). However, in those uteri with implanted embryos, OB-R staining was detected only in some epithelial glands (Fig. 4P).
FIG. 3. Impact of intrauterine injection of LPAs on protein levels of ?3-integrin and cytokine receptors in mouse endometrium at d 5 of pregnancy. Representative Western blot and quantitative analysis of normalized levels depicting changes in VEGF-R2 (A and B), IL-1R tI (C and D), LIF-R (E and F), ?3-integrin (G and H), and CSF-1R (I and J), respectively. Quantitative analyses of Western blot results were performed with the program TotalLab. An antibody against actin was used as a loading control (data not shown). Notice the reduction of VEGF-R2, IL-1R tI, LIF-R, and ?3-integrin protein levels by LPA-1 or LPA-2 treatments. The results presented are the mean ± SE; *, P value 0.05 when comparing levels in response to nontreated controls. All data were derived from a minimum of three independent experiments using different mouse endometrium preparations (n = 5). Mouse macrophage lysate (RAW 264.7) was used in each gel as a control for identification of specific bands and to perform statistical comparison of relative levels of antigens.
FIG. 4. Representative immunohistochemical analysis for levels of expression of ?3-integrin and cytokine receptors after intrauterine injection of LPAs and scrambled peptides (LPA-Sc) in mice at d 6 of pregnancy. Uterine horns were injected on d 3 as described in Materials and Methods, and uteri were removed on d 6 for immunohistochemical analysis. A–D, ?3-Integrin expression after LPA-1Sc (A and C) and LPA-1 (B and D) treatments; E–L, IL-1R tI (E–H) and LIF-R (I–L) levels after LPA-2Sc (E, G, I, and K) and LPA-2 (F, H, J, and L) treatments; M and N, OB-R levels in nonpregnant and pregnant control mice; A, B, E, F, I, J, and M, nonpregnant mice; C, D,G, H, K, L and N, pregnant mice; O and P, negative controls from nonpregnant and pregnant mice, respectively. Notice that inhibition of leptin signaling with LPA-1 negatively affected the levels of immunoreactive ?3-integrin. LPA-2 treatment reduces the levels of IL-1R tI and LIF-R in mouse endometrium. Similar results were found with LPA-1 (data not shown). OB-R levels were not affected by treatment with LPA-1 or -2. Notice that OB-R was expressed by luminal epithelial cells only before implantation occurs. Arrows and arrowheads indicate luminal and glandular staining of the antigens, respectively. Magnification, x100 (M, O, and P); x200 (A–C and N); x400 (D, F, G, and I–K); x1000 (E, H, and L).
To enhance sensitivity in the localization of immunoreactive IL-1R tI, LIF-R, or VEGF-R2 and integrin-?3 proteins in mouse endometrium, we conducted double-fluorescent confocal laser scanning microscopy. Red immunofluorescence for IL-1R tI, LIF-R, and VEGF-R and green fluorescence for ?3 staining (Fig. 5) revealed a strong membrane-associated granular pattern of expression. Immunofluorescence analysis showed that intrauterine injection of LPA-2Sc did not affect the levels of IL-1R tI (Fig. 5A) or ?3-integrin (Fig. 5B). Colocalization of IL-1R tI and ?3-integrin in the same cells (mainly glandular and luminal epithelium) was indicated by yellow fluorescence (Fig. 5C). In contrast, LPA-2 treatment negatively impacted the immunofluorescence levels of IL-1 R tI (Fig. 5D) and ?3-integrin (Fig. 5E) and their colocalization pattern (Fig. 5F). Similar results were found for LIF-R (Fig. 5, G–L) and for VEGF-R2 (Fig. 5, M–R). LPA-1 effects were similar to those found for LPA-2.
FIG. 5. Confocal laser scanning microscopy analysis for protein levels of cytokine receptors and ?3-integrin at d 5 of pregnancy in mouse endometrium after intrauterine injections of LPA-2 and LPA-2Sc at d 3 of pregnancy. A–F, IL-1R tI, ?3-integrin, and colocalization of both antigens in endometrium from mouse treated with LPA-2Sc (A–C) and LPA-2 (D–F). G–L, LIF-R, ?3-integrin, and colocalization of both antigens in endometrium from mouse treated with LPA-2Sc (G–I) and LPA-2 (J–L). M–R, VEGF-R2, ?3-integrin, and colocalization of both antigens in endometrium from mouse treated with LPA-2Sc (M–O) and LPA-2 (P–R). Notice the negative impact of LPA-2 on immunofluorescence levels for cytokine receptors and ?3-integrin.
Discussion
During the preimplantation period, the endometrium evolves to allow embryo implantation. In the mouse, implantation is estrogen driven (32) and involves the endometrial expression of adhesion molecules and cytokines and their receptors. There is evidence to support the idea that leptin and OB-R are among these molecules and serve important roles in embryo-maternal cross talk at the time of implantation (15, 20, 31, 33). In endometrial cells from human and rabbit, leptin signaling increases ?3-integrin, LIF/LIF-R, IL-1/IL-1R tI, and IL-1Ra protein levels (23, 24, 34). Moreover, leptin signaling also induces the secretion of VEGF-A by human and mouse endometrial cells (our unpublished results).
?3-Integrin, (35, 36), LIF (37), IL-1 (38), and VEGF (39) have been implicated in the implantation process. Emerging data support an important role for ?3-integrin in mouse implantation (40). In contrast to IL-1 (41, 42), LIF/LIF-R signaling is absolutely required for mouse implantation (37). Exogenous LIF enhanced mouse embryo development, and the blockade of LIF signaling with neutralizing antibodies in mouse endometrium negatively impacted embryo implantation (43). Similarly, VEGF/VEGF-R signaling in the rat endometrium has been found essential for implantation (39).
Reproduction in the mouse does not occur without leptin (28, 29, 30, 44). Infertility of leptin-deficient mice (ob/ob) (28) could be to some extent because of impaired folliculogenesis (34). Leptin is present in glandular and luminal endometrial epithelium and oviduct of pregnant mice during the preimplantation period but not in those tissues of immature and nonpregnant mice at estrus (20). However, no data have been previously reported on the specific role of leptin endometrial signaling in the mouse embryo implantation process.
In the present studies, it was determined that the mouse endometrium expresses OB-R. Because leptin has profound effects on the expression of cytokines fundamental to endometrial receptivity, it is likely that leptin itself has profound effects on implantation. Therefore, we hypothesized that the blockade of OB-R signaling in mouse endometrium would disrupt the sequential activation of leptin-induced molecules and negatively affect embryo implantation. Embryo implantation in the mouse occurs on d 4 (45, 46). Thus, to block leptin/OB-R signaling in the mouse endometrium before implantation, inhibitors of leptin signaling were injected into the uterus on d 3 of pregnancy. The leptin receptor antagonists used in these studies were generated to interact with the two major regions of human leptin that mediate its interaction with its receptor (23). Leptin is highly conserved among species, and LPA-1 (our unpublished results) and LPA-2 bind with high affinity and inhibit leptin/OB-R signaling in human and rabbit endometrial cells (23, 24). LPA-1 and -2 were more effective at preventing implantation than OB-R antibody, which inhibited implantation only when used at a high concentration. Blockade of leptin/OB-R signaling with LPAs or OB-R antibody treatments did not affect the expression of OB-R isoforms in vivo by mouse endometrium. Similar findings were previously found in vitro in human (24) and rabbit endometrial cell cultures (23). However, differences found in total OB-R immunoreactivity in mouse endometrial luminal epithelial cells before and after implantation suggest that leptin/OB-R signaling plays a role in implantation. OB-R expression in uterine epithelial lining was turned off after the embryo successfully implanted. Therefore, the temporal and spatial expression of OB-R by the luminal epithelium could represent a mechanism to allow an active molecular communication between the preimplantation embryo and the endometrium.
During the preimplantation period and early pregnancy, the levels of LIF-R, VEGF-R2, IL-1R tI, and ?3-integrin significantly increased in mouse endometrium. Immunoreactive ?3-integrin was spatially and temporally associated with immunoreactive cytokine receptors in both mouse endometrial epithelial luminal and glandular cells. Disruption of leptin signaling dramatically decreased mouse embryo implantation and the levels of LIF-R, VEGF-R2, IL-1R tI, and ?3-integrin. These data correlate with previous findings in endometrial cell cultures (23, 24).
Because it has been demonstrated that leptin is fundamental to implantation in several mammalian species, leptin signaling may also be essential for human embryo implantation. OB-R is expressed by human endometrium, and both human preimplantation embryos and endometrial epithelial cells secrete leptin. Moreover, the secretion of leptin is regulated when human blastocysts are cocultured with human endometrial cells (15). In vitro studies suggest that leptin/OB-R signaling in human and rabbit endometrial cells involves JAK/STAT pathways (23, 24, 34). Other signaling pathways (i.e. MAPK, protein kinase C, and phosphoinositol 3-kinase) could be also activated by leptin (11, 47). Potentially, both OB-Rb and OB-Ra can contribute to the leptin-induced increase in cytokines and adhesion molecules in the mouse endometrium. However, the specific contribution of the different OB-R isoforms and leptin signaling pathways to endometrial receptivity has yet to be defined.
Taken together, the present data suggest that the anti-implantation effects of inhibitors of leptin/OB-R signaling in the mouse endometrium are closely related to leptin regulation of levels of ?3-integrin, IL-1, LIF, and VEGF components. However, the contribution of leptin signaling via the embryo cannot be ruled out. The anti-implantation effects found by intrauterine injections of OB-R inhibitors could also involve the blockade of leptin/OB-R signaling required by the mouse preimplantation embryo to acquire implantation capabilities, to secrete putative factors that could contribute to some extent to the endometrial biochemical changes found in this study and/or vascular recruitment during the implantation process.
The impairment of leptin-induced angiogenic effects by LPAs could have implications beyond implantation and placentation processes. It is tempting to speculate that LPAs could have potential applications for treatment of diseases characterized by neovascularization (i.e. cancer and endometriosis) by inhibiting leptin induction of VEGF-A/VEGF-R2.
In conclusion, the present findings show that leptin signaling in the mouse endometrium is essential for embryo implantation. Leptin effects in the endometrium are related to endometrial receptivity and angiogenesis. We hypothesize that the mechanism by which endometrial leptin signaling acts during implantation includes the induction of LIF, IL-1, VEGF components, and ?3-integrin expression. Therefore, a putative mechanism for LPA anti-implantation effects could involve the inhibition of leptin/OB-R signaling required for the expression of these molecules in the endometrium. The regulation of leptin signaling in the endometrium could impact the control of fertility. Overall, our results suggest that leptin could be one of the primary factors that initiates and regulates the cascade system of molecules that promote the development of endometrial receptivity and successful implantation.
Acknowledgments
We thank Dr. Maureen P. Lynch for her critical review of the manuscript. We thank Ms. E. Gowell for helping in the purification and characterization of peptides.
References
Zhang F, Basinski MB, Beals JM, Briggs SL, Churgay LM, Clawson DK, DiMarchi RD, Furman TC, Hale JE, Hsiung HM, Schoner BE, Smith DP, Zhang XY, Wery JP, Schevitz RW 1997 Crystal structure of the obese protein leptin-E100. Nature 387:206–209
Gonzalez RR, Simon C, Caballero-Campo P, Norman R, Chardonnens D, Devoto L, Bischof P 2000 Leptin and reproduction. Hum Reprod Update 6:290–300
Castracane VD, Henson MC 2002 When did leptin become a reproductive hormone? Semin Reprod Med 20:89–92
Tartaglia LA, Dembski M, Weng X, Deng N, Culpepper J, Devos R, Richards GJ, Campfield LA, Clark FT, Deeds J, Muir C, Sanker S, Moriarty A, Smutko JS, Mays GG, Wool EA, Monroe CA, Tepper RI 1995 Identification and expression cloning of a leptin receptor, OB-R. Cell 83:1263–1271
Tartaglia LA 1997 The leptin receptor. J Biol Chem 272:6093–6096
Wang Y, Kuropatwinski KK, White DW, Hawley TS, Hawley RG, Tartaglia LA, Baumann H 1997 Leptin receptor action in hepatic cells. J Biol Chem 272:16216–16223
Yuan SS, Tsai KB, Chung YF, Chan TF, Yeh YT, Tsai LY, Su JH 2004 Aberrant expression and possible involvement of the leptin receptor in endometrial cancer. Gynecol Oncol 92:769–775
Lewandowski K, Horn R, O’Callaghan CJ, Dunlop D, Medley GF, O’Hare P, Brabant G 1999 Free leptin, bound leptin, and soluble leptin receptor in normal and diabetic pregnancies. J Clin Endocrinol Metab 84:300–306
Edwards DE, Bohm Jr RP, Purcell J, Ratterree MS, Swan KF, Castracane VD, Henson MC 2004 Two isoforms of the leptin receptor are enhanced in pregnancy-specific tissues and soluble leptin receptor is enhanced in maternal serum with advancing gestation in the baboon. Biol Reprod 71:1746–1752
Ghilardi N, Ziegler S, Wiestner A, Stoffel R, Heim MH, Skoda RC 1996 Defective STAT signaling by the leptin receptor in diabetic mice. Proc Natl Acad Sci USA 93:6231–6235
Zabeau L, Lavens D, Peelman F, Eyckerman S, Vandekerckhove J, Tavernier J 2003 The ins and outs of leptin receptor activation. FEBS Lett 546:45–50
Cioffi JA, Van Blerkom J, Antczak M, Shafer A, Wittmer S, Snodgrass HR 1997 The expression of leptin and its receptors in pre-ovulatory human follicles. Mol Hum Reprod 3:467–472
Zachow RJ, Magoffin DA 1997 Direct intraovarian effects of leptin: impairment of the synergistic action of insulin-like growth factor-I on follicle-stimulating hormone-dependent estradiol-17? production by rat ovarian granulosa cells. Endocrinology 138:847–850
Agarwal SK, Vogel K, Weitsman SR, Magoffin DA 1999 Leptin antagonizes the insulin-like growth factor-I augmentation of steroidogenesis in granulosa and theca cells of the human ovary. J Clin Endocrinol Metab 84:1072–1076
Gonzalez RR, Caballero-Campo P, Jasper M, Mercader A, Devoto L, Pellicer A, Simon C 2000 Leptin and leptin receptor are expressed in the human endometrium and endometrial leptin secretion is regulated by the human blastocyst. J Clin Endocrinol Metab 85:4883–4888
Wu MH, Chuang PC, Chen HM, Lin CC, Tsai SJ 2002 Increased leptin expression in endometriosis cells is associated with endometrial stromal cell proliferation and leptin gene up-regulation. Mol Hum Reprod 8:456–464
Masuzaki H, Ogawa Y, Sagawa N, Hosoda K, Matsumoto T, Mise H, Nishimura H, Yoshimasa Y, Tanaka I, Mori T, Nakao K 1997 Nonadipose tissue production of leptin: leptin as a novel placenta-derived hormone in humans. Nat Med 3:1029–1033
Senaris R, Garcia-Caballero T, Casabiell X, Gallego R, Castro R, Considine RV, Dieguez C, Casanueva FF 1997 Synthesis of leptin in human placenta. Endocrinology 138:4501–4504
Antczak M, Van Blerkom J 1997 Oocyte influences on early development: the regulatory proteins leptin and STAT3 are polarized in mouse and human oocytes and differentially distributed within the cells of the preimplantation stage embryo. Mol Hum Reprod 3:1067–1086
Kawamura K, Sato N, Fukuda J, Kodama H, Kumagai J, Tanikawa H, Nakamura A, Tanaka T 2002 Leptin promotes the development of mouse preimplantation embryos in vitro. Endocrinology 143:1922–1931
Matsuoka T, Tahara M, Yokoi T, Masumoto N, Takeda T, Yamaguchi M, Tasaka K, Kurachi H, Murata Y 1999 Tyrosine phosphorylation of STAT3 by leptin through leptin receptor in mouse metaphase 2 stage oocyte. Biochem Biophys Res Commun 256:480–484
Gonzalez RR, Leavis P 2001 Leptin upregulates ?3-integrin expression and interleukin-1?, upregulates leptin and leptin receptor expression in human endometrial epithelial cell cultures. Endocrine 16:21–28
Gonzalez RR, Leavis PC 2003 A peptide derived from the human leptin molecule is a potent inhibitor of the leptin receptor function in rabbit endometrial cells. Endocrine 21:185–195
Gonzalez RR, Rueda BR, Ramos MP, Littell RD, Glasser S, Leavis PC 2004 Leptin-induced increase in leukemia inhibitory factor and its receptor by human endometrium is partially mediated by interleukin 1 receptor signaling. Endocrinology 145:3850–3857
Castellucci M, De Matteis R, Meisser A, Cancello R, Monsurro V, Islami D, Sarzani R, Marzioni D, Cinti S, Bischof P 2000 Leptin modulates extracellular matrix molecules and metalloproteinases: possible implications for trophoblast invasion. Mol Hum Reprod 6:951–958
Gonzalez RR, Devoto L, Campana A, Bischof P 2001 Effects of leptin, interleukin-1, interleukin-6, and transforming growth factor-? on markers of trophoblast invasive phenotype: integrins and metalloproteinases. Endocrine 15:157–164
Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM 1994 Positional cloning of the mouse obese gene and its human homologue. Nature 372:425–432
Chehab FF, Lim ME, Lu R 1996 Correction of the sterility defect in homozygous obese female mice by treatment with the human recombinant leptin. Nat Genet 12:318–320
Malik NM, Carter ND, Murray JF, Scaramuzzi RJ, Wilson CA, Stock MJ 2001 Leptin requirement for conception, implantation, and gestation in the mouse. Endocrinology 142:5198–5202
Ahima RS, Prabakaran D, Mantzoros C, Qu D, Lowell B, Maratos-Flier E, Flier JS 1996 Role of leptin in the neuroendocrine response to fasting. Nature 382:250–252
Alfer J, Muller-Schottle F, Classen-Linke I, von Rango U, Happel L, Beier-Hellwig K, Rath W, Beier HM 2000 The endometrium as a novel target for leptin: differences in fertility and subfertility. Mol Hum Reprod 6:595–601
Curtis Hewitt S, Goulding EH, Eddy EM, Korach KS 2002 Studies using the estrogen receptor knockout uterus demonstrate that implantation but not decidualization-associated signaling is estrogen dependent. Biol Reprod 67:1268–1277
Kitawaki J, Koshiba H, Ishihara H, Kusuki I, Tsukamoto K, Honjo H 2000 Expression of leptin receptor in human endometrium and fluctuation during the menstrual cycle. J Clin Endocrinol Metab 85:1946–1950
Gonzalez RR, Leary K, Petrozza JC, Leavis PC 2003 Leptin regulation of the interleukin-1 system in human endometrial cells. Mol Hum Reprod 9:151–158
Lessey BA, Damjanovich L, Coutifaris C, Castelbaum A, Albelda SM, Buck CA 1992 Integrin adhesion molecules in the human endometrium. Correlation with the normal and abnormal menstrual cycle. J Clin Invest 90:188–195
Gonzalez RR, Palomino A, Boric A, Vega M, Devoto L 1999 A quantitative evaluation of 1, 4, V and ?3 endometrial integrins of fertile and unexplained infertile women during the menstrual cycle. A flow cytometric appraisal. Hum Reprod 14:2485–2492
Stewart CL, Kaspar P, Brunet LJ, Bhatt H, Gadi I, Kontgen F, Abbondanzo SJ 1992 Blastocyst implantation depends on maternal expression of leukaemia inhibitory factor. Nature 359:76–79
Simon C, Frances A, Piquette GN, el Danasouri I, Zurawski G, Dang W, Polan ML 1994 Embryonic implantation in mice is blocked by interleukin-1 receptor antagonist. Endocrinology 134:521–528
Rockwell LC, Pillai S, Olson CE, Koos RD 2002 Inhibition of vascular endothelial growth factor/vascular permeability factor action blocks estrogen-induced uterine edema and implantation in rodents. Biol Reprod 67:1804–1810
Illera MJ, Cullinan E, Gui Y, Yuan L, Beyler SA, Lessey BA 2000 Blockade of the (v)?(3) integrin adversely affects implantation in the mouse. Biol Reprod 62:1285–1290
Abbondanzo SJ, Cullinan EB, McIntyre K, Labow MA, Stewart CL 1996 Reproduction in mice lacking a functional type 1 IL-1 receptor. Endocrinology 137:3598–3601
Stewart CL, Cullinan EB 1997 Preimplantation development of the mammalian embryo and its regulation by growth factors. Dev Genet 21:91–101
Mitchell MH, Swanson RJ, Oehninger S 2002 In vivo effect of leukemia inhibitory factor (LIF) and an anti-LIF polyclonal antibody on murine embryo and fetal development following exposure at the time of transcervical blastocyst transfer. Biol Reprod 67:460–464
Takeda K, Noguchi K, Shi W, Tanaka T, Matsumoto M, Yoshida N, Kishimoto T, Akira S 1997 Targeted disruption of the mouse Stat3 gene leads to early embryonic lethality. Proc Natl Acad Sci USA 94:3801–3804
Paria BC, Huet-Hudson YM, Dey SK 1993 Blastocyst’s state of activity determines the "window" of implantation in the receptive mouse uterus. Proc Natl Acad Sci USA 90:10159–10162
Psychoyos A 1973 Endocrine control of egg implantation. Washington, DC: American Physiological Society
Takahashi Y, Okimura Y, Mizuno I, Iida K, Takahashi T, Kaji H, Abe H, Chihara K 1997 Leptin induces mitogen-activated protein kinase-dependent proliferation of C3H10T1/2 cells. J Biol Chem 272:12897–12900(M. P. Ramos, B. R. Rueda,)
Address all correspondence and requests for reprints to: Dr. R. R. Gonzalez, Boston Biomedical Research Institute, 64 Grove Street, Watertown, Massachusetts 02472. E-mail: gonzalezr@bbri.org.
Abstract
Leptin is essential for mouse reproduction, but the exact roles it serves are yet to be determined. Treatment of cultured endometrial cells with leptin increases the level of ?3-integrin, IL-1, leukemia inhibitory factor, and their corresponding receptors. These leptin-induced effects are eliminated by inhibitors of leptin receptor (OB-R) signaling. Herein the impact of blocking leptin/OB-R signaling in the mouse endometrium was assessed. Intrauterine injection of either leptin peptide antagonists (LPA-1 or -2) or OB-R antibody on d 3 of pregnancy impaired mouse implantation in comparison to intrauterine injection of scrambled peptides (LPA-Sc) or species-matched IgGs. Significant reduction in the number of implantation sites and uterine horns with implanted embryos was found after intrauterine injection of LPA-1 (1 of 22) vs. LPA-1Sc (11 of 15) and LPA-2 (3 of 17) vs. LPA-2Sc (14 of 16). The impact of disruption of leptin signaling on the endometrial expression of several molecules in pregnant mice was assessed by Western blot, immunohistochemistry, and confocal microscopy. Disruption of leptin signaling resulted in a significant reduction of IL-1 receptor type I, leukemia inhibitory factor, vascular endothelial growth factor receptor 2, and ?3-integrin levels. The levels of colony stimulating factor-1 receptor and OB-R were unaltered after treatment with LPAs compared with controls. Expression of OB-R protein was pregnancy dependent and found only in glandular epithelium after implantation occurred. Our findings support previous observations that leptin signaling is critical to the implantation process and suggest that molecules downstream of leptin-activated receptor may serve obligatory roles in endometrial receptivity and successful implantation.
Introduction
LEPTIN, THE PRODUCT of the ob gene, is mainly secreted by white adipocytes and plays a key role in the regulation of body weight and food intake. Since the discovery of leptin 10 yr ago (1), its significance in other physiological processes has been realized. There is now strong supportive evidence that leptin is critical for reproduction (for review see Refs.2, 3).
The leptin sequence is highly conserved, whereas there are sequence differences in the leptin receptor (OB-R, the product of the db gene) among various species. In the mouse and the human, OB-R has several splice variants. The full-length and functional OB-R (OB-Rb) is expressed by the hypothalamus (4, 5) and other tissues (2). OB-R isoforms with a shorter cytoplasmic tail (OB-Ra) are expressed in many peripheral tissues (6) and are expressed at lower levels in endometrial cancer tissue (7).
A soluble active OB-R has been described in humans (8), and its levels are enhanced in serum from pregnant compared with nonpregnant nonhuman primates (9). Binding of leptin to OB-Rb allows the binding of Janus kinase 2 (JAK2) to the intracytoplasmic tail of OB-R. JAK2 phosphorylates OB-R followed by phosphorylation of signal transducer and activator of transcription 3 (STAT3) that activates several signaling pathways. In addition to the JAK/STAT signaling pathway, the MAPK, protein kinase C, and phosphoinositol 3-kinase pathways are also activated by leptin in several cell types (10, 11). Both OB-Rb and OB-Ra can phosphorylate erbB2 upon binding and enhance MAPK activity (12). However, OB-Ra does not activate the JAK2/STAT3 pathway (13). Leptin and OB-Rb are expressed by human female reproductive tissues, including the ovary (12, 13, 14), endometrium (15, 16), and placenta (17, 18). Leptin has been found in human and mouse oocytes and preimplantation embryos (12, 19, 20, 21). In addition, leptin can promote the development of mouse preimplantation embryos through OB-R signaling (20). In vitro, the endometrial secretion of leptin is regulated by human preimplantation embryos (15). Leptin treatment increases the levels of ?3-integrin (a marker of endometrial receptivity) in human endometrial epithelial cells (22). Leptin increases p-STAT3, leukemia inhibitory factor (LIF), IL-1 (ligand and antagonist), and their cognate receptors in rabbit (23) and human endometrial cells (24) and induces the acquisition of the invasive phenotype of human trophoblast cells (25, 26). Mouse mutants deficient in leptin (ob/ob) (27) or OB-R (db/db) are obese and infertile. Fertility can be restored in ob/ob by administration of exogenous leptin (28). The withdrawal of leptin infusion in ob/ob females shortly after fertilization impairs implantation (29). Leptin injection into starved mice restores fertility (30). A postovulatory increase in serum leptin concentration appears to be associated with implantation potential (12), and low expression of OB-R has been found in endometrium from women with unexplained infertility (31). These data suggest that in vivo leptin could act in an autocrine or paracrine manner to regulate biological functions that may mediate the implantation process.
The leptin peptide antagonist LPA-2 is able to inhibit leptin binding to OB-R in vitro (23) and leptin signaling pathways that induce an increase in levels of IL-1, LIF, and ?3-integrin by endometrial cells (23, 24). In vitro studies and the phenotype of ob/ob and db/db mice would suggest that disruption of leptin signaling can have a significant impact on embryo preimplantation development and/or the implantation process. Therefore, it was hypothesized that the inhibition of leptin/OB-R signaling in endometrium by leptin peptide antagonists (LPAs) or antimouse OB-R antibodies would impair mouse embryo implantation and affect the levels of several downstream molecules with important roles in implantation.
Materials and Methods
Chemicals
Armenian hamster antibody anti-?3-integrin (N-20, mouse origin), mouse anti-Armenian hamster IgGs, goat polyclonal antibody anti-actin, rabbit polyclonal antibodies against the carboxy terminus of LIF receptor (LIF-R) (C-19, human or mouse origin), IL-1 receptor type I (IL-1R tI, antibody M-20), vascular endothelial growth factor receptor 2 (VEGF-R2), colony-stimulating factor-1 receptor (CSF-1R), and their respective blocking peptides for competition studies and mouse macrophage lysate (RAW 264.7) for Western blot, positive controls were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Goat polyclonal antimouse OB-R (AF497, anti-NH2-terminal end of mouse OB-R) and nonspecific rabbit and goat IgGs were from R&D Systems Inc. (Minneapolis, MN). Mouse normal serum was obtained from Biomeda (Foster City, CA). Normal goat and rabbit sera and biotinylated horse antimouse and rabbit antigoat IgG antibodies were from Vector Laboratories (Burlingame, CA). Biotinylated goat antirabbit IgG antibodies (ALI3409, mouse IgG adsorbed) were obtained from BioSource International (Camarillo, CA). Alexa Fluor 594 goat antirabbit IgG, Alexa Fluor 488 goat antimouse IgG, and 4',6-diamino-2 phenylindole dihydrochloride were from Molecular Probes Inc. (Eugene, OR). Other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO).
OB-R inhibitors
LPA-1 (32 amino acid residues, IQKVQDDTKTLIKTIVTRINDISHTQSVSSKQ) and LPA-2 (23) corresponding to helices I and III of leptin, respectively, and their scrambled peptides LPA-1Sc (TDKVITQSTINVKHTSSQSVQIKDIKIRQTLD) and LPA-2Sc for negative controls were synthesized as described elsewhere (23). The peptides were dissolved in sterile filtered vehicle solution composed of 0.04% dimethylsulfoxide (DMSO)-PBS. Goat antimouse OB-R antibodies and normal goat IgGs (negative control) diluted in PBS were sterile filtered and also used to assess the impact of blockade of OB-R signaling on mouse implantation.
Animals
Virgin, 8- to 10-wk-old, female C57BL6 and CD1 mice (Charles River Laboratories, Wilmington, MA) were housed in the animal facilities at the Massachusetts General Hospital or Boston Biomedical Research Institute in accordance with National Institutes of Health standards for the care and use of experimental animals. The rooms were provided with a controlled temperature range (22–24 C) on a 14-h light/10-h dark cycle. Mice were given water and food ad libitum. Ovulation was induced in female mice (n = 120) by an ip injection of 5 IU pregnant mare serum gonadotropin (Sigma) followed by 10 IU human chorionic gonadotropin (Sigma) 48 h later. Female mice were mated with fertile males of the same strain to induce pregnancy. The following morning, the females exhibiting vaginal copulatory plugs were separated for the proposed experiments. The day of vaginal plug was recorded as d 1 of pregnancy. Mice were weighed before treatment and again before they were euthanized.
Surgical procedures and intrauterine blocking treatments
On d 3 of pregnancy, mice were anesthetized by ip injection of Avertin (2,2,2 tribromoethanol, 200 mg/kg body weight; Sigma). A surgical incision was made on the dorsal midline through the skin, and each uterine horn was exposed using small forceps. Under a dissecting microscope, different compounds were delivered into the uterine lumen distal to the uterotubal junction using a Hamilton syringe (model PC010; Hamilton Co., Reno, NV) holding a pulled glass needle (<27 gauge, capillary pipette 20 μl; Unopette, Becton Dickinson, Bedford, MA). The effectiveness of the method was verified in preliminary experiments by evaluating the administration of dye. The treatments included 10 μl of LPA-1 or -2 or LPA-1Sc or -2Sc (3.3 μM) delivered into the lumen of the right horn of each mouse. Each animal served as its own internal control, with the left uterine horn receiving the vehicle. A group of mice received 10 μl goat anti-OB-R antibodies at a concentration of 1.5 or 7.5 μg in the right horn and similar concentrations of nonspecific goat IgG solutions in the left horn. Concentrations of OB-R inhibitors used in vivo were calculated from results of previous experiments in endometrial cell cultures (23). The effects of surgical treatment, intrauterine injection, and toxicity of vehicle solutions on implantation were also assessed by comparing the number of implantation sites in mice treated with intrauterine injections of PBS and PBS-DMSO and in nontreated mice. After administration of the compound(s) or sham surgery, the incision was closed with metal auto clips (9 mm; Becton Dickinson). Mice were placed in warmed cages (cages were placed on a slide warmer; Fisher, Pittsburgh, PA) until full recovery from the anesthetic.
To analyze the effects of OB-R inhibitors, the mice were euthanized on d 10 of pregnancy. The uteri were extracted, and the implantation sites were counted in each horn. The percentage of uterine horns with implanted embryos (pregnancy rate) was calculated in relation to the total number of uterine horns injected with each inhibitor of OB-R or controls. The percentage of embryos that implanted in the uterine horns receiving OB-R inhibitors with respect to control uterine horns was determined (implantation rate). To investigate the impact of inhibition of leptin signaling on the expression of several molecules (cytokine receptors and ?3-integrin), groups of mice were euthanized at d 1–6 of pregnancy. The endometrial tissue was isolated to produce cell lysates for Western blot analysis, and a small portion of each uterus was dissected and fixed in 4% paraformaldehyde in PBS or prepared for cryostat sections for qualitative analysis.
Cell lysates
Mouse endometrial tissues were homogenized on ice with lysis buffer [20 nM Tris (pH 7.4), containing 137 nM NaCl, 2 mM EDTA, 10% glycerol, 50 mM ?-glycerophosphate, 1% Nonidet P-40, and a mixture of proteases and phosphatase inhibitors composed of 100 μM antipain, 0.1 mg/ml trypsin inhibitor, protease inhibitor cocktail 1:50 (Sigma), 50 nM NaF, 2 mM phenylmethylsulfonyl fluoride, and 2 mM sodium orthovanadate]. Cellular lysates were centrifuged at 2400 x g at 4 C for 10 min. Protein concentrations were determined using the Bradford protein assay (Bio-Rad Laboratories Inc., Hercules, CA) and BSA as standard (Sigma).
Western blot
Cellular lysates were diluted 1:1 with Laemmli buffer and incubated at 95 C for 5 min. Denatured protein preparations (20 μg) from mouse endometrial samples were loaded on 7.5% SDS-PAGE gels. Electrophoresis was performed at 220 V for 5 min followed by 165 V for 20 min (Bio-Rad, electrophoresis apparatus) in Tris-glycine buffer (pH 8.4) (Bio-Rad). Electroblotting onto 0.2-μm nitrocellulose membranes was performed at 22 V overnight at 4 C in 48 nM Tris-39 nM glycine buffer containing 0.037% SDS and 20% methanol. Membranes were washed with 20 mM Tris, 137 mM NaCl (pH 7.4) buffer containing 0.15% Tween 20 (vol/vol) (wash buffer) and incubated for 1 h at room temperature in blocking buffer containing Amersham blocking product (5%, wt/vol; Amersham Biosciences Corp., Piscataway, NJ) in wash buffer. The membranes were subsequently incubated at room temperature for 1 h with 1 μg/ml of antibody to OB-R, IL-1R tI, LIF-R, VEGF-R2, CSF-1R, or ?3-integrin in wash buffer. After washing (four times for 5 min each), the membranes were incubated for 1 h at room temperature with biotinylated secondary antibody specific for anti-primary antibody species in wash buffer containing 2.5% normal horse, goat, or mouse serum. Immune detection was performed by incubation for 30 min at room temperature with streptavidin-horseradish peroxidase conjugate diluted 1:2000 in wash buffer (Amersham). Specific bands in the blots were visualized using an ECL-chemiluminescent assay (Amersham) and Imagetek-B film (American X-Ray & Medical Supply, Rancho Cordoba, CA). Nonspecific mouse, rabbit, and goat IgGs or preincubation of primary antibodies with specific blocking peptides (Santa Cruz Biotechnology) were used instead of primary antibodies to produce negative control blots. Quantitative analyses of Western blot results were performed with the program TotalLab (version 2003.02, NonLinear Dynamics Ltd., Durham, NC). Twenty micrograms of mouse macrophage lysate (RAW 264.7) were loaded in each gel as a control for identification of specific bands and to perform statistic comparison of relative levels of antigens. The data were calculated from results of three or more Western blot determinations using different endometrial protein preparations (n = 5). Actin was determined for loading control in the gels (data not shown).
Immunohistochemical determinations
To assess the potential effects of blockade of endometrial OB-R function in vivo on the expression of various cytokine receptors, immunohistochemistry in cryostat and paraffin block sections (4 μm) was performed. Unmasking of antigen epitopes (?3-integrin, LIF-R, and IL-1R tI) in paraffin sections was performed. Briefly, samples were boiled in 10 mM sodium citrate/1 mM EDTA (pH 6) solution for 10 min. After quenching endogenous peroxidase activity with H2O2 (3% water solution) and blocking (2.5% horse or rabbit normal serum), tissue sections were incubated for 1 h at room temperature with the following primary antibodies diluted in PBS-0.1% BSA: anti-?3-integrin, IL-1R tI, LIF-R, VEGF-R, CSF-1R, and OB-R antibodies (all at 1 μg/ml). Biotinylated secondary antibodies were used. The tissues were incubated with a streptavidin-biotin-peroxidase system according to the manufacturer’s directions (Vectastain, ABC-AP kit, Vector), counterstained with hematoxylin (Dako Corp., Carpinteria, CA), and mounted with VectaMount (Vector). Negative controls included tissue preparations in which the primary antibodies were substituted by irrelevant species-matched IgGs. Negative controls for competitive studies with anti-IL-1R tI, LIF-R, VEFG-R, and CSF-1R antibodies were generated by preincubation with their respective blocking peptides (20 μg/ml; Santa Cruz Biotechnology). All washing steps were performed by immersion of the preparations three times in PBS for 5 min at room temperature.
Confocal laser scanning microscopy
Confocal laser scanning microscopy was performed in serial sections from paraffin blocks to determine the expression and colocalization of receptors (OB-R, IL-1R tI, and LIF-R) and ?3-integrin within endometrial tissue from nonpregnant and pregnant mice after anti-OB-R intrauterine treatments. The protocols for immunostaining used were similar to those already described for regular color-based immunohistochemistry, but the secondary antibodies and counterstaining agent were changed. Alexa Fluor 594 goat antirabbit IgG (red fluorescence) and Alexa Fluor 488 goat antimouse IgG (green fluorescence) conjugates were used to detect LIF-R, IL-1R tI, VEGF-R, CSF-1R, and ?3-integrin, respectively. In addition, a second fluorescein isothiocyanate-conjugated antibody (rabbit antigoat IgG-fluorescein isothiocyanate) was used to detect OB-R. The double-fluorescent-stained specimens were analyzed with a confocal laser scanning microscope equipped with an external argon laser (Bio-Rad). To avoid photobleaching of fluorochromes during fluorescence microscopy, the slides were embedded in antifade solution (Dako). For nuclear and chromosome counterstaining, 300 nM 4',6-diamidino-2-phenylindole in PBS-dimethylformamide solution was used.
Statistical analysis
A one-way ANOVA test with Dunnett error protection and a confidence interval of 95% was used from the Analyze-it for Microsoft Excel (Leeds, UK, htpp://www.analyze-it.com) for data analysis. Data are expressed as mean ± SEM. Values for P < 0.05 were considered statistically significant.
Results
Impact of disruption of leptin signaling in the endometrium
Mouse embryo implantation.
The surgical process and the injection of vehicle solution (0.04% DMSO-PBS) had no effect on the number of implanted embryos (implantation rate) in any of the mouse strains used in this study. Similarly it was assessed that postsurgery trauma did not significantly affect implantation in these mice (data not shown). However, a single injection of LPA-1 or -2 (3 μM/10 μl) at d 3 of pregnancy resulted in significantly impaired implantation in both C57BL6 and CD1 mice. The number of uterine horns with implanted embryos (pregnancy rate) was negatively affected by LPA-1 or -2. Only 4.5% (1 of 22) and 17.6% (3 of 17) of the uterine horns treated with LPA-1 and LPA-2, respectively, exhibited implantation sites (Fig. 1A). In contrast, 73.3% (11 of 15) and 90% (14 of 16) of the horns injected with negative controls LPA-1Sc and LPA-2Sc, respectively, showed implanted embryos (see Fig. 1A). No significant differences were found in pregnancy rate between LPA-1Sc, LPA-2Sc, vehicle, or IgG. Intrauterine injection of anti-OB-R-specific antibodies at a concentration of 1.5 and 7.5 μg/horn showed no reduction of pregnancy rate (see Fig. 1A). The number of implanted embryos in the uterine horns treated with OB-R inhibitors was significantly reduced compared with the controls. From the total number of embryos that implanted in the uterine horns (treated with OB-R inhibitors and their controls), only 15.5, 10.7, and 28% were found in those horns injected with LPA-1, LPA-2, and anti-OB-R antibody, respectively (Fig. 1B). The embryos that implanted in LPA-2Sc-, vehicle-, or IgG-treated mice were found uniformly distributed throughout the uterine horn (Fig. 1C). In contrast, the few embryos implanted in LPA- or anti-OB-R-antibody-treated horns were mainly found in the distal portion of the uterine horn, furthest from the OB-R inhibitor injection site (Fig. 1C).
FIG. 1. Impairment of mouse embryo implantation by intrauterine injection of OB-R inhibitors in CD1 mice. A and B, Impact of OB-R inhibitors on pregnancy rate (percentage of horns with implanted embryos) (A) and on implantation rate (percentage of implantation sites related to the total number of implanted embryos found after treatment with each inhibitor of OB-R plus its control) (B). C, Representative pictures from mouse uterine horns treated with LPA-Sc vs. vehicle and LPAs vs. vehicle. Notice the reduction of pregnancy and number of implantation sites by LPA-1 and -2 intrauterine injections (10 μl/30 μM). Implantation sites (if any) in the horns treated with LPAs were found distal from the utero-tubal junction. Anti-OB-R antibodies (7.5 μg) also negatively affected the number of implantation sites significantly. The results presented are the mean ± SE; *, P value 0.05 when comparing levels in response to treatments to that of the LPA-Sc- or IgG-treated controls. Similar results were found in C57BL6 mice (data not shown).
Levels of cytokine receptors and ?3-integrin during early pregnancy in mouse endometrium
Western blot analysis revealed that levels of LIF-R, VEGF-R2, and ?3-integrin dramatically increased in mouse endometrium during early pregnancy (d 1–6; see Fig. 2, A and B). The levels of these molecules increased at preimplantation time (d 3) and after implantation occurred (d 6). CSF-1R and IL-1R tI levels did not show significant change from d 1–6. (Fig. 2, A and B). The antibody against the extracellular domain of OB-R identified several bands corresponding to long and short OB-R isoforms. However, no significant variation in the levels of the OB-R isoforms was found from d 1–6 of pregnancy. Figure 2 shows the data corresponding to the full-length OB-R (see Fig. 2, A and B).
FIG. 2. Protein levels of ?3-integrin and VEGF-R2, LIF-R, CSF-1R, IL-1 R tI, and OB-R in mouse endometrium during early pregnancy. A, Representative Western blots of cytokine receptors and ?3-integrin obtained after d 1, 3, and 6 of pregnancy (d 1 copulatory plug) from endometrial extracts of control mice without any treatment. B, Quantitative analysis of molecules normalized against positive control from mouse macrophages (RAW 264.7; see Materials and Methods). An antibody against actin was used as a loading control (data not shown). Notice that protein levels of VEGF-R2, ?3-integrin, and LIF-R increased dramatically after fertilization and particularly after implantation occurred. In contrast, the levels of CSF-1R, IL-1R, and OB-R did not show significant changes. Quantitative analyses of Western blot results were performed with the program TotalLab. All data were derived from a minimum of three independent experiments using different mouse endometrium preparations (n = 5).
Impact of disruption of leptin signaling on levels of cytokine receptors and ?3-integrin in mouse endometrium
Western blot analysis revealed that intrauterine injections of LPA-1 and LPA-2 significantly decreased the levels of VEGF-R2, IL-1R tI, and LIF-R in the mouse endometrium (Fig. 3, A–F). LPA treatment also decreased the levels of ?3-integrin, but a significant reduction was found only in mouse endometria injected with LPA-1 (see Fig. 3, G and H). No significant variation in the levels of CSF-1R after LPA treatment was found (see Fig. 3, I and J). The negative effects of inhibition of leptin/OB-R signaling by LPAs on the endometrial levels of the above molecules were corroborated qualitatively by immunohistochemistry. Intensity of staining for all of the above antigens showed a marked decrease in endometrium (d 6) from mice with documented copulatory plug treated with LPA-2 or LPA-1 compared with control endometria treated with scrambled LPAs (see Fig. 4, A–L). These changes were more evident when embryo implantation was inhibited. CSF-1R staining was not significantly changed after LPA treatment (data not-shown). OB-R was found in endometrial epithelial cells (lumen and glands) in the uteri without implanted embryos (Fig. 4N). However, in those uteri with implanted embryos, OB-R staining was detected only in some epithelial glands (Fig. 4P).
FIG. 3. Impact of intrauterine injection of LPAs on protein levels of ?3-integrin and cytokine receptors in mouse endometrium at d 5 of pregnancy. Representative Western blot and quantitative analysis of normalized levels depicting changes in VEGF-R2 (A and B), IL-1R tI (C and D), LIF-R (E and F), ?3-integrin (G and H), and CSF-1R (I and J), respectively. Quantitative analyses of Western blot results were performed with the program TotalLab. An antibody against actin was used as a loading control (data not shown). Notice the reduction of VEGF-R2, IL-1R tI, LIF-R, and ?3-integrin protein levels by LPA-1 or LPA-2 treatments. The results presented are the mean ± SE; *, P value 0.05 when comparing levels in response to nontreated controls. All data were derived from a minimum of three independent experiments using different mouse endometrium preparations (n = 5). Mouse macrophage lysate (RAW 264.7) was used in each gel as a control for identification of specific bands and to perform statistical comparison of relative levels of antigens.
FIG. 4. Representative immunohistochemical analysis for levels of expression of ?3-integrin and cytokine receptors after intrauterine injection of LPAs and scrambled peptides (LPA-Sc) in mice at d 6 of pregnancy. Uterine horns were injected on d 3 as described in Materials and Methods, and uteri were removed on d 6 for immunohistochemical analysis. A–D, ?3-Integrin expression after LPA-1Sc (A and C) and LPA-1 (B and D) treatments; E–L, IL-1R tI (E–H) and LIF-R (I–L) levels after LPA-2Sc (E, G, I, and K) and LPA-2 (F, H, J, and L) treatments; M and N, OB-R levels in nonpregnant and pregnant control mice; A, B, E, F, I, J, and M, nonpregnant mice; C, D,G, H, K, L and N, pregnant mice; O and P, negative controls from nonpregnant and pregnant mice, respectively. Notice that inhibition of leptin signaling with LPA-1 negatively affected the levels of immunoreactive ?3-integrin. LPA-2 treatment reduces the levels of IL-1R tI and LIF-R in mouse endometrium. Similar results were found with LPA-1 (data not shown). OB-R levels were not affected by treatment with LPA-1 or -2. Notice that OB-R was expressed by luminal epithelial cells only before implantation occurs. Arrows and arrowheads indicate luminal and glandular staining of the antigens, respectively. Magnification, x100 (M, O, and P); x200 (A–C and N); x400 (D, F, G, and I–K); x1000 (E, H, and L).
To enhance sensitivity in the localization of immunoreactive IL-1R tI, LIF-R, or VEGF-R2 and integrin-?3 proteins in mouse endometrium, we conducted double-fluorescent confocal laser scanning microscopy. Red immunofluorescence for IL-1R tI, LIF-R, and VEGF-R and green fluorescence for ?3 staining (Fig. 5) revealed a strong membrane-associated granular pattern of expression. Immunofluorescence analysis showed that intrauterine injection of LPA-2Sc did not affect the levels of IL-1R tI (Fig. 5A) or ?3-integrin (Fig. 5B). Colocalization of IL-1R tI and ?3-integrin in the same cells (mainly glandular and luminal epithelium) was indicated by yellow fluorescence (Fig. 5C). In contrast, LPA-2 treatment negatively impacted the immunofluorescence levels of IL-1 R tI (Fig. 5D) and ?3-integrin (Fig. 5E) and their colocalization pattern (Fig. 5F). Similar results were found for LIF-R (Fig. 5, G–L) and for VEGF-R2 (Fig. 5, M–R). LPA-1 effects were similar to those found for LPA-2.
FIG. 5. Confocal laser scanning microscopy analysis for protein levels of cytokine receptors and ?3-integrin at d 5 of pregnancy in mouse endometrium after intrauterine injections of LPA-2 and LPA-2Sc at d 3 of pregnancy. A–F, IL-1R tI, ?3-integrin, and colocalization of both antigens in endometrium from mouse treated with LPA-2Sc (A–C) and LPA-2 (D–F). G–L, LIF-R, ?3-integrin, and colocalization of both antigens in endometrium from mouse treated with LPA-2Sc (G–I) and LPA-2 (J–L). M–R, VEGF-R2, ?3-integrin, and colocalization of both antigens in endometrium from mouse treated with LPA-2Sc (M–O) and LPA-2 (P–R). Notice the negative impact of LPA-2 on immunofluorescence levels for cytokine receptors and ?3-integrin.
Discussion
During the preimplantation period, the endometrium evolves to allow embryo implantation. In the mouse, implantation is estrogen driven (32) and involves the endometrial expression of adhesion molecules and cytokines and their receptors. There is evidence to support the idea that leptin and OB-R are among these molecules and serve important roles in embryo-maternal cross talk at the time of implantation (15, 20, 31, 33). In endometrial cells from human and rabbit, leptin signaling increases ?3-integrin, LIF/LIF-R, IL-1/IL-1R tI, and IL-1Ra protein levels (23, 24, 34). Moreover, leptin signaling also induces the secretion of VEGF-A by human and mouse endometrial cells (our unpublished results).
?3-Integrin, (35, 36), LIF (37), IL-1 (38), and VEGF (39) have been implicated in the implantation process. Emerging data support an important role for ?3-integrin in mouse implantation (40). In contrast to IL-1 (41, 42), LIF/LIF-R signaling is absolutely required for mouse implantation (37). Exogenous LIF enhanced mouse embryo development, and the blockade of LIF signaling with neutralizing antibodies in mouse endometrium negatively impacted embryo implantation (43). Similarly, VEGF/VEGF-R signaling in the rat endometrium has been found essential for implantation (39).
Reproduction in the mouse does not occur without leptin (28, 29, 30, 44). Infertility of leptin-deficient mice (ob/ob) (28) could be to some extent because of impaired folliculogenesis (34). Leptin is present in glandular and luminal endometrial epithelium and oviduct of pregnant mice during the preimplantation period but not in those tissues of immature and nonpregnant mice at estrus (20). However, no data have been previously reported on the specific role of leptin endometrial signaling in the mouse embryo implantation process.
In the present studies, it was determined that the mouse endometrium expresses OB-R. Because leptin has profound effects on the expression of cytokines fundamental to endometrial receptivity, it is likely that leptin itself has profound effects on implantation. Therefore, we hypothesized that the blockade of OB-R signaling in mouse endometrium would disrupt the sequential activation of leptin-induced molecules and negatively affect embryo implantation. Embryo implantation in the mouse occurs on d 4 (45, 46). Thus, to block leptin/OB-R signaling in the mouse endometrium before implantation, inhibitors of leptin signaling were injected into the uterus on d 3 of pregnancy. The leptin receptor antagonists used in these studies were generated to interact with the two major regions of human leptin that mediate its interaction with its receptor (23). Leptin is highly conserved among species, and LPA-1 (our unpublished results) and LPA-2 bind with high affinity and inhibit leptin/OB-R signaling in human and rabbit endometrial cells (23, 24). LPA-1 and -2 were more effective at preventing implantation than OB-R antibody, which inhibited implantation only when used at a high concentration. Blockade of leptin/OB-R signaling with LPAs or OB-R antibody treatments did not affect the expression of OB-R isoforms in vivo by mouse endometrium. Similar findings were previously found in vitro in human (24) and rabbit endometrial cell cultures (23). However, differences found in total OB-R immunoreactivity in mouse endometrial luminal epithelial cells before and after implantation suggest that leptin/OB-R signaling plays a role in implantation. OB-R expression in uterine epithelial lining was turned off after the embryo successfully implanted. Therefore, the temporal and spatial expression of OB-R by the luminal epithelium could represent a mechanism to allow an active molecular communication between the preimplantation embryo and the endometrium.
During the preimplantation period and early pregnancy, the levels of LIF-R, VEGF-R2, IL-1R tI, and ?3-integrin significantly increased in mouse endometrium. Immunoreactive ?3-integrin was spatially and temporally associated with immunoreactive cytokine receptors in both mouse endometrial epithelial luminal and glandular cells. Disruption of leptin signaling dramatically decreased mouse embryo implantation and the levels of LIF-R, VEGF-R2, IL-1R tI, and ?3-integrin. These data correlate with previous findings in endometrial cell cultures (23, 24).
Because it has been demonstrated that leptin is fundamental to implantation in several mammalian species, leptin signaling may also be essential for human embryo implantation. OB-R is expressed by human endometrium, and both human preimplantation embryos and endometrial epithelial cells secrete leptin. Moreover, the secretion of leptin is regulated when human blastocysts are cocultured with human endometrial cells (15). In vitro studies suggest that leptin/OB-R signaling in human and rabbit endometrial cells involves JAK/STAT pathways (23, 24, 34). Other signaling pathways (i.e. MAPK, protein kinase C, and phosphoinositol 3-kinase) could be also activated by leptin (11, 47). Potentially, both OB-Rb and OB-Ra can contribute to the leptin-induced increase in cytokines and adhesion molecules in the mouse endometrium. However, the specific contribution of the different OB-R isoforms and leptin signaling pathways to endometrial receptivity has yet to be defined.
Taken together, the present data suggest that the anti-implantation effects of inhibitors of leptin/OB-R signaling in the mouse endometrium are closely related to leptin regulation of levels of ?3-integrin, IL-1, LIF, and VEGF components. However, the contribution of leptin signaling via the embryo cannot be ruled out. The anti-implantation effects found by intrauterine injections of OB-R inhibitors could also involve the blockade of leptin/OB-R signaling required by the mouse preimplantation embryo to acquire implantation capabilities, to secrete putative factors that could contribute to some extent to the endometrial biochemical changes found in this study and/or vascular recruitment during the implantation process.
The impairment of leptin-induced angiogenic effects by LPAs could have implications beyond implantation and placentation processes. It is tempting to speculate that LPAs could have potential applications for treatment of diseases characterized by neovascularization (i.e. cancer and endometriosis) by inhibiting leptin induction of VEGF-A/VEGF-R2.
In conclusion, the present findings show that leptin signaling in the mouse endometrium is essential for embryo implantation. Leptin effects in the endometrium are related to endometrial receptivity and angiogenesis. We hypothesize that the mechanism by which endometrial leptin signaling acts during implantation includes the induction of LIF, IL-1, VEGF components, and ?3-integrin expression. Therefore, a putative mechanism for LPA anti-implantation effects could involve the inhibition of leptin/OB-R signaling required for the expression of these molecules in the endometrium. The regulation of leptin signaling in the endometrium could impact the control of fertility. Overall, our results suggest that leptin could be one of the primary factors that initiates and regulates the cascade system of molecules that promote the development of endometrial receptivity and successful implantation.
Acknowledgments
We thank Dr. Maureen P. Lynch for her critical review of the manuscript. We thank Ms. E. Gowell for helping in the purification and characterization of peptides.
References
Zhang F, Basinski MB, Beals JM, Briggs SL, Churgay LM, Clawson DK, DiMarchi RD, Furman TC, Hale JE, Hsiung HM, Schoner BE, Smith DP, Zhang XY, Wery JP, Schevitz RW 1997 Crystal structure of the obese protein leptin-E100. Nature 387:206–209
Gonzalez RR, Simon C, Caballero-Campo P, Norman R, Chardonnens D, Devoto L, Bischof P 2000 Leptin and reproduction. Hum Reprod Update 6:290–300
Castracane VD, Henson MC 2002 When did leptin become a reproductive hormone? Semin Reprod Med 20:89–92
Tartaglia LA, Dembski M, Weng X, Deng N, Culpepper J, Devos R, Richards GJ, Campfield LA, Clark FT, Deeds J, Muir C, Sanker S, Moriarty A, Smutko JS, Mays GG, Wool EA, Monroe CA, Tepper RI 1995 Identification and expression cloning of a leptin receptor, OB-R. Cell 83:1263–1271
Tartaglia LA 1997 The leptin receptor. J Biol Chem 272:6093–6096
Wang Y, Kuropatwinski KK, White DW, Hawley TS, Hawley RG, Tartaglia LA, Baumann H 1997 Leptin receptor action in hepatic cells. J Biol Chem 272:16216–16223
Yuan SS, Tsai KB, Chung YF, Chan TF, Yeh YT, Tsai LY, Su JH 2004 Aberrant expression and possible involvement of the leptin receptor in endometrial cancer. Gynecol Oncol 92:769–775
Lewandowski K, Horn R, O’Callaghan CJ, Dunlop D, Medley GF, O’Hare P, Brabant G 1999 Free leptin, bound leptin, and soluble leptin receptor in normal and diabetic pregnancies. J Clin Endocrinol Metab 84:300–306
Edwards DE, Bohm Jr RP, Purcell J, Ratterree MS, Swan KF, Castracane VD, Henson MC 2004 Two isoforms of the leptin receptor are enhanced in pregnancy-specific tissues and soluble leptin receptor is enhanced in maternal serum with advancing gestation in the baboon. Biol Reprod 71:1746–1752
Ghilardi N, Ziegler S, Wiestner A, Stoffel R, Heim MH, Skoda RC 1996 Defective STAT signaling by the leptin receptor in diabetic mice. Proc Natl Acad Sci USA 93:6231–6235
Zabeau L, Lavens D, Peelman F, Eyckerman S, Vandekerckhove J, Tavernier J 2003 The ins and outs of leptin receptor activation. FEBS Lett 546:45–50
Cioffi JA, Van Blerkom J, Antczak M, Shafer A, Wittmer S, Snodgrass HR 1997 The expression of leptin and its receptors in pre-ovulatory human follicles. Mol Hum Reprod 3:467–472
Zachow RJ, Magoffin DA 1997 Direct intraovarian effects of leptin: impairment of the synergistic action of insulin-like growth factor-I on follicle-stimulating hormone-dependent estradiol-17? production by rat ovarian granulosa cells. Endocrinology 138:847–850
Agarwal SK, Vogel K, Weitsman SR, Magoffin DA 1999 Leptin antagonizes the insulin-like growth factor-I augmentation of steroidogenesis in granulosa and theca cells of the human ovary. J Clin Endocrinol Metab 84:1072–1076
Gonzalez RR, Caballero-Campo P, Jasper M, Mercader A, Devoto L, Pellicer A, Simon C 2000 Leptin and leptin receptor are expressed in the human endometrium and endometrial leptin secretion is regulated by the human blastocyst. J Clin Endocrinol Metab 85:4883–4888
Wu MH, Chuang PC, Chen HM, Lin CC, Tsai SJ 2002 Increased leptin expression in endometriosis cells is associated with endometrial stromal cell proliferation and leptin gene up-regulation. Mol Hum Reprod 8:456–464
Masuzaki H, Ogawa Y, Sagawa N, Hosoda K, Matsumoto T, Mise H, Nishimura H, Yoshimasa Y, Tanaka I, Mori T, Nakao K 1997 Nonadipose tissue production of leptin: leptin as a novel placenta-derived hormone in humans. Nat Med 3:1029–1033
Senaris R, Garcia-Caballero T, Casabiell X, Gallego R, Castro R, Considine RV, Dieguez C, Casanueva FF 1997 Synthesis of leptin in human placenta. Endocrinology 138:4501–4504
Antczak M, Van Blerkom J 1997 Oocyte influences on early development: the regulatory proteins leptin and STAT3 are polarized in mouse and human oocytes and differentially distributed within the cells of the preimplantation stage embryo. Mol Hum Reprod 3:1067–1086
Kawamura K, Sato N, Fukuda J, Kodama H, Kumagai J, Tanikawa H, Nakamura A, Tanaka T 2002 Leptin promotes the development of mouse preimplantation embryos in vitro. Endocrinology 143:1922–1931
Matsuoka T, Tahara M, Yokoi T, Masumoto N, Takeda T, Yamaguchi M, Tasaka K, Kurachi H, Murata Y 1999 Tyrosine phosphorylation of STAT3 by leptin through leptin receptor in mouse metaphase 2 stage oocyte. Biochem Biophys Res Commun 256:480–484
Gonzalez RR, Leavis P 2001 Leptin upregulates ?3-integrin expression and interleukin-1?, upregulates leptin and leptin receptor expression in human endometrial epithelial cell cultures. Endocrine 16:21–28
Gonzalez RR, Leavis PC 2003 A peptide derived from the human leptin molecule is a potent inhibitor of the leptin receptor function in rabbit endometrial cells. Endocrine 21:185–195
Gonzalez RR, Rueda BR, Ramos MP, Littell RD, Glasser S, Leavis PC 2004 Leptin-induced increase in leukemia inhibitory factor and its receptor by human endometrium is partially mediated by interleukin 1 receptor signaling. Endocrinology 145:3850–3857
Castellucci M, De Matteis R, Meisser A, Cancello R, Monsurro V, Islami D, Sarzani R, Marzioni D, Cinti S, Bischof P 2000 Leptin modulates extracellular matrix molecules and metalloproteinases: possible implications for trophoblast invasion. Mol Hum Reprod 6:951–958
Gonzalez RR, Devoto L, Campana A, Bischof P 2001 Effects of leptin, interleukin-1, interleukin-6, and transforming growth factor-? on markers of trophoblast invasive phenotype: integrins and metalloproteinases. Endocrine 15:157–164
Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM 1994 Positional cloning of the mouse obese gene and its human homologue. Nature 372:425–432
Chehab FF, Lim ME, Lu R 1996 Correction of the sterility defect in homozygous obese female mice by treatment with the human recombinant leptin. Nat Genet 12:318–320
Malik NM, Carter ND, Murray JF, Scaramuzzi RJ, Wilson CA, Stock MJ 2001 Leptin requirement for conception, implantation, and gestation in the mouse. Endocrinology 142:5198–5202
Ahima RS, Prabakaran D, Mantzoros C, Qu D, Lowell B, Maratos-Flier E, Flier JS 1996 Role of leptin in the neuroendocrine response to fasting. Nature 382:250–252
Alfer J, Muller-Schottle F, Classen-Linke I, von Rango U, Happel L, Beier-Hellwig K, Rath W, Beier HM 2000 The endometrium as a novel target for leptin: differences in fertility and subfertility. Mol Hum Reprod 6:595–601
Curtis Hewitt S, Goulding EH, Eddy EM, Korach KS 2002 Studies using the estrogen receptor knockout uterus demonstrate that implantation but not decidualization-associated signaling is estrogen dependent. Biol Reprod 67:1268–1277
Kitawaki J, Koshiba H, Ishihara H, Kusuki I, Tsukamoto K, Honjo H 2000 Expression of leptin receptor in human endometrium and fluctuation during the menstrual cycle. J Clin Endocrinol Metab 85:1946–1950
Gonzalez RR, Leary K, Petrozza JC, Leavis PC 2003 Leptin regulation of the interleukin-1 system in human endometrial cells. Mol Hum Reprod 9:151–158
Lessey BA, Damjanovich L, Coutifaris C, Castelbaum A, Albelda SM, Buck CA 1992 Integrin adhesion molecules in the human endometrium. Correlation with the normal and abnormal menstrual cycle. J Clin Invest 90:188–195
Gonzalez RR, Palomino A, Boric A, Vega M, Devoto L 1999 A quantitative evaluation of 1, 4, V and ?3 endometrial integrins of fertile and unexplained infertile women during the menstrual cycle. A flow cytometric appraisal. Hum Reprod 14:2485–2492
Stewart CL, Kaspar P, Brunet LJ, Bhatt H, Gadi I, Kontgen F, Abbondanzo SJ 1992 Blastocyst implantation depends on maternal expression of leukaemia inhibitory factor. Nature 359:76–79
Simon C, Frances A, Piquette GN, el Danasouri I, Zurawski G, Dang W, Polan ML 1994 Embryonic implantation in mice is blocked by interleukin-1 receptor antagonist. Endocrinology 134:521–528
Rockwell LC, Pillai S, Olson CE, Koos RD 2002 Inhibition of vascular endothelial growth factor/vascular permeability factor action blocks estrogen-induced uterine edema and implantation in rodents. Biol Reprod 67:1804–1810
Illera MJ, Cullinan E, Gui Y, Yuan L, Beyler SA, Lessey BA 2000 Blockade of the (v)?(3) integrin adversely affects implantation in the mouse. Biol Reprod 62:1285–1290
Abbondanzo SJ, Cullinan EB, McIntyre K, Labow MA, Stewart CL 1996 Reproduction in mice lacking a functional type 1 IL-1 receptor. Endocrinology 137:3598–3601
Stewart CL, Cullinan EB 1997 Preimplantation development of the mammalian embryo and its regulation by growth factors. Dev Genet 21:91–101
Mitchell MH, Swanson RJ, Oehninger S 2002 In vivo effect of leukemia inhibitory factor (LIF) and an anti-LIF polyclonal antibody on murine embryo and fetal development following exposure at the time of transcervical blastocyst transfer. Biol Reprod 67:460–464
Takeda K, Noguchi K, Shi W, Tanaka T, Matsumoto M, Yoshida N, Kishimoto T, Akira S 1997 Targeted disruption of the mouse Stat3 gene leads to early embryonic lethality. Proc Natl Acad Sci USA 94:3801–3804
Paria BC, Huet-Hudson YM, Dey SK 1993 Blastocyst’s state of activity determines the "window" of implantation in the receptive mouse uterus. Proc Natl Acad Sci USA 90:10159–10162
Psychoyos A 1973 Endocrine control of egg implantation. Washington, DC: American Physiological Society
Takahashi Y, Okimura Y, Mizuno I, Iida K, Takahashi T, Kaji H, Abe H, Chihara K 1997 Leptin induces mitogen-activated protein kinase-dependent proliferation of C3H10T1/2 cells. J Biol Chem 272:12897–12900(M. P. Ramos, B. R. Rueda,)