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Original Studies |
Instituto de Investigaciones Materno Infantil, Facultad de Medicina (R.R.G., L.D.), Hospital San Borja Arriarán, Universidad de Chile, Santiago de Chile, Chile; and Fundación del Instituto Valenciano de Infertilidad para el estudio de la Reproducción (FIVIER) and Departamento de Pediatría, Obstetría y Ginecología (P.C.-C., M.J., A.M., A.P., C.S.), Universidad de Valencia, 46020 Valencia, Spain.
Address correspondence and requests for reprints to: Carlos Simón, Instituto Valenciano de Infertilidad, Guardia Civil 23, 46020 Valencia, Spain. E-mail: csimon{at}interbook.net
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Abstract |
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Introduction |
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In addition to adipose tissue, leptin has been found to be secreted by a variety of reproductive organs including placenta (10, 11). In vitro cultures of cytotrophoblastic cells secreted leptin, and this secretion is regulated by interleukin 1 (IL-1) and estradiol (12). Leptin protein (13) and leptin messenger RNA (mRNA) (14) have also been detected in human ovary, and follicular and serum leptin production may be influenced by the ovarian functional state (15). Leptin is coexpressed in a polarized manner with STAT3 protein in oocytes and embryos at all stages of development (16). Leptin is also expressed in mammary glands (17).
The long form of the leptin receptor (OB-R), has signaling capabilities and is primarily localized at the hypothalamus, anterior pituitary, and several other areas of the brain (18, 19, 20). OB-R mRNA has been detected in granulosa and theca cells of the ovary (13, 14), and the OB-R protein has been detected by immunofluorescence in mouse oocytes fertilized eggs and cleavage stage embryos (16). OB-R is also expressed in hepatic and adrenal cells (21, 22). A soluble form of the leptin receptor has also been reported to exist in the human (23, 24).
Although it has been suggested that leptin does not directly regulate reproduction itself, leptin could regulate the function of reproductive organs (25). However, the specific mechanism(s) whereby leptin modulates reproductive function is unknown (1, 2, 5, 6, 25). Endocrine data in in vitro fertilization (IVF) suggest that leptin production may be influenced by the ovarian functional state (15) and follicular fluid leptin concentrations could be a marker of assisted reproduction treatment success (26). However, a possible role for leptin in the endometrial-embryonic dialogue during implantation has not yet been described.
In the present study, we investigate the expression of leptin and its receptor (long form) by secretory endometrium and cultured endometrial epithelial cells (EECs) in vitro and analyze leptin secretion by the human blastocyst in the presence or absence of cultured EECs.
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Materials and Methods |
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This project was approved by the institutional review board on the use of human subjects in research at the Instituto Valenciano de Infertilidad [Spanish Law of Assisted Reproductive Technologies (35/1988)]. Embryos and granulosa cells were obtained from nine patients after ovarian hyperstimulation and insemination using routine IVF procedures. Endometrial biopsies were obtained from fertile women (age, 23–39 yr) during the midsecretory phase of the menstrual cycle. Human adipose tissue, used as positive control for leptin and leptin receptor expression, was obtained from patients undergoing gynecological surgery. Human placental tissue was obtained following routine delivery.
EEC cultures
A portion of each endometrial biopsy was dated according to the Noyes’s method (27). EECs were isolated and cultured until confluent in DMEM (Sigma, St. Louis, MO) and MCDB-105 (Sigma) (3:1) containing 10% heat inactivated fetal bovine serum (Life Technologies, Grand Island, NY), antibiotics, and 5 µg/mL insulin (Sigma), as described previously (28, 29, 30).
Embryo culture and coculture with EECs
A human in vitro model was used to study interactions between the human embryo and EECs (31, 32). Forty-eight hours after insemination, two to four cell embryos were cocultured individually on the EEC monolayer for 5 days, from day 2 until day 6 of embryonic development. Embryonic development was monitored daily, and embryos were grown in 1 mL IVF/S2 or CCM medium (1/1) (Scandinavian IVF Science, Gothenburg, Sweden) until they reached the eight-cell stage, and then were cultured in CCM medium until blastocyst stage. Conditioned media were changed every 24 h of culture, however, only the last 24 h (corresponding to 120 h at day 6 of culture) media sample was collected to measure leptin concentrations. After embryo transfer, EEC wells were classified according to embryonic status: EECs with embryos that reached the blastocyst stage and EECs with arrested embryos. In each experiment, EECs cultured under the same conditions without embryos and human embryos cultured in the absence of EECs were used as controls.
RT-PCR
Total cellular RNA was extracted, purified, and reverse transcribed from secretory phase endometrial biopsies, EECs, adipose tissue, placenta, and granulosa cells using Trizol reagent (Life Technologies, Paisley, Scotland) according to the manufacturer’s instructions. First-strand cDNA was reverse-transcribed from 1 µg RNA using a MMLV Reverse transcriptase and Advantage RT-for-PCR kit (60 min/42 C) (Clontech, Palo Alto, CA). Contradictory data about leptin mRNA expression in ovary has been reported (13, 14) by RT-PCR using different primers. Therefore, we assayed two pairs of primers previously reported as successful in detecting the leptin mRNA expression in ovary (14) and placenta (11) to be more confident about the RT-PCR in the assessment of leptin mRNA expression in endometrium and EEC. Primers specific for leptin were, 5'-ATGCATTGGGAACCCTGTGCGG-3' (forward) and 5'-TGAGGTCCAGCTGCCACAGCATG-3' (reverse) (11), and 5'-CCAAAACCCTCATAAGAC-3' (forward) and 5'-CACCTCTGGAGTAG-3' (reverse) (14) were used to amplify products of 490 bp and 341 bp, respectively. Primers specific for the long form of leptin receptor were 5'-GCTATTTTGGGAAGATGT-3' (forward) and 5'-TGCCTGGGCCTCTATCTC-3' (reverse) (14). All primers were synthesized by Life Technologies. PCR conditions specific for each primer pair were determined following optimization with adipose tissue, placenta, and granulosa cells. The PCR amplification was performed in a final reaction volume of 25 µL consisting of 67 mM Tris-HCl (pH 8.8), 16 mM (NH4)2 SO4, 0.01% Tween 20, 2.5–3.25 mM MgCl2, 0.2 mM dNTPs (Sigma), 0.2 µM forward and reverse primers, and 2.5 µL cDNA, overlayed with 50 µL mineral oil (Sigma). Taq DNA polymerase (0.5 U, BIOTAQ; Bioline, London, UK) was added at 94 C. PCR amplification was as 5–10 min at 94 C, followed by a 1-min cycle of denaturation at 94 C, annealing at 58–59 C, and extension at 72 C, followed by a final extension for 10 min at 72 C; repeated for 30 cycles. The negative control included in each reaction consisted of H2O substituted for complementary DNA (cDNA). In addition, in some experiences we performed PCR using total RNA without RT treatment as additional negative controls. Also, total RNA extracted from samples was previously digested with DNAase I, and RT-PCR experiences were performed. Therefore, we were sure that genomic DNA was not present in RNA extracts. The integrity of each cDNA preparation was analyzed by RT-PCR of ß-actin mRNA (33). The primers for ß-actin amplification used were those reported by Ponte et al. (33). Concentrations of cDNA used for PCR experiments were similar and adjusted for ß-actin mRNA expression. PCR reaction products were analyzed by gel electrophoresis in 2% agarose gel containing 0.5 µg/mL ethidium bromide and parallel to a molecular weight marker of 100 bp (Life Technologies). The PCR assay was repeated at least three times on each cDNA sample.
Western blot analysis
Adipose, endometrial, and placental tissue (100 mg) were disrupted by homogenization in RIPA buffer on ice and subsequently by ultrasonic treatment. Total protein was determined using the Bradford protein assay (BioRad Laboratories, Inc., Munich, Germany). Five micrograms (adipose tissue) and 10 µg (for rest of samples) proteins were loaded per lane for leptin and 10 µg proteins (all samples) for leptin receptor, and electrophoresis was performed at 125 V for 1.5 h and 2.5 h (NOVEX Electrophoresis GmbH, Frankfurt, Germany) for leptin and leptin receptor, respectively, in 8% SDS and 14% polyacrylamide gel (SDS-PAGE). Electroblotting (NOVEX apparatus) onto nitrocellulose membranes of 0.2 µm and 0.45 µm pore size (BioRad) for leptin and leptin receptor, respectively, was performed at 35 mA for 1.5 h. Membranes were blocked with a TRIS-Glycine (TBS) solution consisting of 3% BSA and 0.05% Tween 20 overnight at 4 C and washed three times with TBS plus 0.05% Tween 20 for 5 min each. The membranes were subsequently incubated at room temperature for 2 h with 0.5 µg/mL rabbit antihuman leptin antibody or 2 µg/mL goat antihuman leptin receptor antibody (Ob Y-20 and Ob R Sc-20, respectively; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in TBS containing 1% BSA and 0.05% Tween 20. Following an incubation with biotinylated antirabbit (Sigma) and antigoat (DAKO Diagnosticos, S.A, Barcelona, Spain) biotinylated antibodies and peroxidase-labeled streptavidin (Sigma) for 1 h at room temperature. Positive specific antigen-antibody reactions were analyzed with a chemiluminescent assay (Amersham, Germany). Western blot experiments were also performed with human recombinant leptin at 2 µg (R&D Systems, Bühlmann Laboratories, Basel, Switzerland) and to demonstrate that the antileptin antibody specifically detects the peptide (a band about 16 kDa). To analyze nonspecific binding in the blot, PAGE-SDS gel electrophoresis and membrane electroblotting were run in parallel experiments and irrelevant rabbit or goat immunoglobulin preparations were substituted for primary specific antibodies. Afterward, nonspecific bands in the blot for leptin and leptin receptor were identified. A broad range biotinylated marker (Bio-Rad) was used to calculate protein weight.
Immunohistochemistry
Formalin-fixed endometrial tissues were sectioned and deparaffinized. Unmasking of tissue antigens was performed by heat treatment in sodium citrate buffer (pH 6, 10 mM) at 95 C for 15 min and partial digestion at 37 C by 10 min with protease (Sigma) (16). Endometrial tissue sections were blocked with PBS containing 5% BSA and 0.1% Tween 20 for 2 h at room temperature or overnight at 4 C. Samples were incubated with 0.5 µg/mL rabbit antihuman leptin antibody (Ob Y-20) or 2 mg/mL goat antihuman leptin receptor (Ob.R Sc-20) in 1% PBS-BSA/0.05% Tween 20, for 1.5 h at 37 C. Immunofluorescent labeling was performed with fluorescein isothiocianate-antirabbit or antigoat IgG for 1 h at 37 C.
Leptin measurements in conditioned media from EEC cultures
Leptin concentrations were measured by enzyme-linked immunosorbent assay (ELISA) (DRG Diagnostic International, Inc., USA and Instruments GmbH, Germany) in conditioned media from the last 24 h (5 days) of EECs cultured alone (n = 7), with arrested embryos (n = 10) or transferred blastocyst (n = 10), as well as arrested embryos (n = 7) and transferred blastocysts (n = 3) cultured alone. Leptin was also determined in fresh IVF media (CCM) and in control samples in the kit. Samples (0.8–1.0 mL) and fresh conditioned medium were lyophilized and resuspended in 120 µL deionized water or of deionized water at basic pH, to compare total solubility of leptin after lyophilization. Recovery experiments were made by the addition of human recombinant leptin (R&D System) to fresh conditioned media, following lyophilization and resuspension of the powder. Total recovery of leptin in these samples ranged between 87% and 90%. Measured concentrations of leptin were within the dynamic range of the standard curve of the ELISA kit. Leptin concentrations were divided by the lyophilization concentration factor and expressed in pg/mL. According to the manufacturer, the performance characteristics of leptin-ELISA were as: sensitivity, 200 pg/mL; 100% specificity for human leptin; less than 0.2% cross-reactivity with rat and mouse leptin; and no detectable cross-reactivity with human insulin, human proinsulin, rat insulin, human C-peptide, glucagon, or insulin-like growth factor I. Standards, controls, and samples were assayed in duplicate. The intra-assay CV were between 0.75–5.32%.
Statistical analysis
Results are expressed as mean ± SE. Statistics were performed using SPSS computer software program (SPSS Inc., Chicago, IL). Differences in leptin protein concentrations were evaluated by ANOVA using the Scheffe test for multiple comparison; P less than 0.05 was considered to be statistically significant.
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Results |
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Analysis of PCR products with the two different sets of primers
demonstrated that leptin mRNA is expressed in cultured EECs (Fig. 1
, A and B). In addition, leptin mRNA was
also shown to be expressed in secretory endometrium, placenta,
granulosa cells, and adipose tissue. Each cDNA generated a single
product of the expected size, with no evidence of product generated
from contaminating genomic DNA showed the same cDNA product for leptin
(see Fig. 1, A and B). A PCR product of 501 bp encoding a region of
mRNA for the full-length leptin receptor (OB-R) was detected in all EEC
cultures and secretory endometrial biopsies (Fig. 1C). RT-PCR product
specific for leptin receptor was identified in granulosa cell
preparations at the same size. Analysis of the ß-actin PCR product by
gel electrophoresis (Fig. 1D) confirmed the initial amount of cDNA to
be approximately equivalent in all samples. However, from the
calculations of the yields of cDNA amplification using the ratio leptin
or leptin receptor/ß-actin, we were not able to demonstrate whether
leptin mRNA or leptin receptor mRNA were significantly increased in
EECs cocultured with blastocyst, with arrested embryos, or without
embryos.
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Using immunohistochemical technique, we identified a positive
signal for leptin and leptin receptor in secretory endometrium.
Staining for leptin receptor and leptin was found at the luminal and
glandular endometrial epithelium (Fig. 2, A and B). Furthermore, these results were confirmed by Western blot
analysis. Human adipose and placental tissues were used as positive
controls. Figure 3 shows the Western blot
results for leptin and leptin receptor. A band of approximately 16 kDa
was found in all the samples and controls (Fig. 3A). Immunoblotting
analysis using negative control demonstrated that leptin was present in
secretory endometrium and all preparations from EEC cultured with or
without embryos (Fig. 3A). The same samples, EEC cultures, and
endometrium were positive for the leptin receptor (Fig. 3B). Western
blotting for leptin receptor identified a major band at approximately
190 kDa (34). However, other minor but specific bands were
also detected. The antileptin receptor antibody used in this
investigation was raised against amino acids 1146–1165 encoding the
carboxyl-terminus of the full-length OB-R. Therefore, minor bands found
to be lower than 190 kDa could represent different molecular size
isoforms of the leptin receptor.
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Leptin was not detected in the lyophilized fresh culture medium by
the ELISA kit. Leptin was detected in the conditioned media from both
human blastocysts and endometrial epithelial cells (Fig. 4). Interestingly, two different patterns
of secretion were found depending on whether embryos were cultured
alone or cocultured with EEC (Fig. 4). Hatched blastocysts cultured
alone secreted significantly higher concentrations of leptin
(453.3 ± 43.3 pg/mL) than arrested embryos alone (P =
0.001), blastocyst cocultured with EECs (P =
0.001) or EECs alone (P = 0.003). Nevertheless,
when blastocysts were cocultured with EECs, leptin concentrations were
lower than when arrested embryos were cocultured with EECs (247 ±
14.5 pg/mL vs. 360 ± 30 pg/mL; P =
0.012), suggesting a regulation in the secretion of leptin or
binding to leptin receptor in the cocultures of human embryos and
EECs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Higher leptin secretion was found in competent blastocyst cultures compared with arrested embryos, suggesting that this molecule may be a marker of cell viability. However, when competent embryos were cocultured with EECs, leptin concentrations in conditioned media were not different from those of EECs cultured alone. This finding suggests different possibilities: leptin secreted by a competent blastocyst may bind to EECs or the secretion of leptin is regulated in EECs and/or in the human blastocyst. In contrast to this, leptin concentration when an arrested blastocyst is cocultured with EECs is higher than leptin secreted by an arrested blastocyst cultured alone, suggesting that EECs may increase leptin production in response to embryonic death signals or bind less leptin when is cocultured with an arrested embryo. In any case, all these findings strongly suggest that this molecule is part of the embryonic-endometrial dialogue during the adhesion phase of human embryonic implantation.
The human blastocyst expresses immunoreactive leptin in a copolarized manner with STAT3 protein (16), and leptin had also been detected in mouse oocytes, but an exogenous source (maternal) has been suggested due to the absence of leptin mRNA as determined by RT-PCR (14). However, mouse embryos express leptin receptor (16). This fact reinforces our hypothesis that blastocyst and human endometrium might communicate at the time of implantation through the leptin system.
In addition, we found the expression of leptin and leptin receptor mRNA in EECs cocultured with human embryos confirmed the observations made following measurement of leptin protein secreted during culture. Expression of leptin receptor mRNA and protein within the secretory endometrium and cultured EECs indicate potential cell targets for this system. From a functional perspective, at the time of implantation the changes observed in vitro in the leptin levels in the microenvironment of the EECs (that express both ligand and receptor) close to the blastocyst (that secretes leptin) might be interpreted as a molecular communication between both partners. If a similar situation occurs in vivo, we might think that leptin is an important factor for implantation to proceed.
Leptin produced and secreted locally by epithelial cells could act in an autocrine or paracrine manner to regulate biological functions that may mediate endometrial receptivity to facilitate the implantation process. Regulation of leptin and leptin receptor expression in EECs by the human embryo could represent a coordinate molecular event to allow implantation to occur.
Further research is needed to examine the mechanism(s) through which leptin modulates reproductive function. Based on the present investigation, we propose that leptin could be an autocrine/paracrine mediator that allows the embryo and epithelial cells to communicate during implantation.
In summary, the finding of leptin and leptin receptor mRNA expression in the endometrium and in EEC cultures and the regulation of its secretion by the human embryo suggests that leptin may be involved in the molecular mechanism of the implantation process.
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Footnotes |
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Received February 1, 2000.
Revised August 14, 2000.
Accepted September 6, 2000.
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References |
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and 17ß-estradiol, and its effect on human chorionic
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