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BOR - Papers in Press, published online ahead of print October 23, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.006122
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BIOLOGY OF REPRODUCTION 68, 472–477 (2003)
DOI: 10.1095/biolreprod.102.006122
© 2003 by the Society for the Study of Reproduction, Inc.

Pregnancy

Effect of Leptin on Progesterone, Human Chorionic Gonadotropin, and Interleukin-6 Secretion by Human Term Trophoblast Cells in Culture1

Paula Cameo2,a, Paul Bischofc, and Juan Carlos Calvoa,b

a Instituto de Biología y Medicina Experimental b Department of Biological Chemistry, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina c Department of Obstetrics and Gynaecology, University of Geneva, Geneva, Switzerland


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leptin, the 16-kDa protein product of the obese gene, was originally seen as an adipocyte-derived signaling molecule. Recently, it has been suggested to be involved in some functions during pregnancy, particularly in the placenta. In the present study, we investigated the role of leptin in the secretion of hCG, progesterone, and interleukin-6 (IL-6) by human term trophoblast cells in culture. Placentae were obtained from cesarean sections following uncomplicated pregnancies and used immediately after delivery. Leptin, hCG, progesterone, and IL-6 were measured by ELISA, RIA, and immunoradiometric assay in the cultured media of trophoblast cells cultured for 48 and 96 h. Leptin mRNA expression in these cultures was determined by reverse transcription-polymerase chain reaction. Recombinant human leptin added to primary cultures of human term placental trophoblast cells showed a stimulatory effect on hCG and IL-6 secretion and an inhibitory effect on progesterone secretion. Primary cultures of term trophoblast cells expressed leptin mRNA. All these findings suggest a role for leptin in human placental endocrine function.

cytokines, human chorionic gonadotropin, leptin, progesterone, trophoblast


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leptin, a 16-kDa (167 amino acids) protein transcribed from the obese (OB) gene, was originally cloned in the mouse [1]. Mutant ob/ob mice are obese and infertile [1], and administration of recombinant leptin to female ob/ob mice corrects obesity and restores full fertility [2].

At first, this protein was seen as an exclusively adipocyte-derived signaling molecule, which limits food intake and increases energy expenditure. Although its principal site of synthesis is the white adipose tissue, during the last 5 yr other sites of expression, such as the placenta [3–9], the gastric epithelium [10], and the brain [11], among others, have been shown. Several observations suggest a possible role of leptin in reproduction, particularly in the fetoplacental physiology: 1) circulating leptin levels are elevated during pregnancy, reaching a peak during the second trimester [12]; 2) at the end of pregnancy, within 24 h of delivery, maternal plasma leptin levels decline to normal values [3, 12]; 3) leptin is produced by the human placenta [3–9]; 4) maternal leptin levels are significantly elevated in hydatiform mole, decreasing to normal concentrations after surgery [3]; 5) human first-trimester cytotrophoblast cells (CTBs) secrete leptin, and in these cultures, recombinant leptin stimulates, in a dose-dependent manner, the secretion of hCG [8]; and 6) a high expression of the leptin receptor in human placenta occurs during the third trimester of pregnancy [4, 13–15].

Little is known about the physiological role of leptin during human pregnancy, but many observations suggest that this small polypeptide could be a key player in its maintenance. Assuming that during this period leptin plasma levels are elevated, and considering that metabolic effects of high leptin levels (e.g., decreased food intake, decreased metabolic efficiency) are neither observed nor desired, it could be hypothesized that this protein has an another important function in such an important tissue like the placenta during pregnancy.

Structural prediction indicates that leptin might fold into a cytokine-like structure [16]. The leptin receptor belongs to the class I cytokine-receptor superfamily [17], so it could be anticipated that leptin may act as a paracrine/autocrine regulator of placental function, like other cytokines do. Because a delicate regulatory interplay of steroid and polypeptide hormones produced by the trophoblast is basic to the maintenance of pregnancy, we decided to study if leptin had any effect on the secretion of some placental hormones.

The biological actions of leptin are carried out through interaction with its specific surface receptor Ob-R, which exists in different spliced isoforms that share the extracellular domain but differ in the transmembrane/cytoplasmic region [17, 18]. At least four types of splice variants of Ob-R mRNA have been identified; among them, the variant with the longest cytoplasmic domain (Ob-Rb) appears as the functional, signal-transducing isoform in the hypothalamus [19]. The ability of the Ob-Ra isoform to perform signal transduction has been reported [20]. In addition, a role for the OB-Re isoform as a soluble leptin-binding protein has been proposed [17]. In this scenario, regulation of leptin action on target tissues likely depends, at least partially, on balanced expression of the different Ob-R isoforms. Both the short and the long Ob-R isoforms are present in the human placenta [4, 14]. Leptin binding to the Ob-Rb isoform results in activation of divergent signal transduction pathways, such as the Janus kinase-signal transducer and activator of transcription (JAK-STAT) and the mitogen-activated protein kinase (MAPK), which eventually induce expression of several genes involved in transcriptional regulation and signaling as well as cellular physiology. On the other hand, the short isoforms contain only box1 JAK-binding sites for JAK and MAPK activation [21]. Leptin receptor signaling appears to be initiated by ligand-induced receptor homodimerization [22].

The CTBs of the human term placenta produce hCG and steroid hormones [23, 24]. These mononuclear cells ultimately fuse to form syncytial structures and synthesize larger quantities of hCG and other polypeptide and steroid hormones. The CTBs isolated from term human placentae can be stimulated to augment production of hCG and progesterone by analogues of cAMP without altering cell aggregation and, thus, formation of syncytiotrophoblast cells (STBs) [25].

Knowing that leptin has a stimulatory effect on hCG secretion by first-trimester trophoblast cells [8] and that interleukin-6 (IL-6) is involved in the regulation of hCG secretion by term trophoblast [26, 27] tissue, we decided to study the effect of the obesity protein on hCG and IL-6 secretion by term CTBs. On the other hand, because one of the two most important steroid hormones produced by the human placenta is progesterone, we aimed to elucidate the role of leptin on placental progesterone secretion.

In the present study, we demonstrate that recombinant human (rh) leptin added to primary cultures of human term placental trophoblast cells has a stimulatory effect on hCG and IL-6 secretion and an inhibitory effect on progesterone secretion.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Preparation

The CTBs were isolated and purified according to the technique of Kliman et al. [24] with slightly modifications. Briefly, 18 normal human term (36–42 wk of gestation) placentae were obtained from cesarean sections following uncomplicated pregnancies and used within 1 h after delivery. Villous tissue from the maternal surface was dissected from connective tissue, separated from blood cells, and cut into small pieces. Tissue (35–40 g wet weight) was digested with trypsin and filtered through a cell strainer (100 µm). The CTBs were isolated on a discontinuous Percoll gradient (5–70%) and immunopurified with antibody-coated magnetic particles (anti-CD45) to eliminate all contaminating leukocytes, according to a previous report [28]. Viability of the CTBs was assessed by eosin exclusion, and cells were diluted to 106 cells/ml.

Culture Conditions

The CTBs were plated (6 x 105 to 1 x 106 cells/ml) in 24-well (16-mm) or 6-well (35-mm) culture plates containing Dulbecco modified Eagle medium (DMEM) with 25 mM glucose, 25 mM Hepes, 100 U/ml of penicillin, 100 µg/ml of streptomycin, 0.25 µg/ml of amphotericin B, and 4 mM L-glutamine and incubated in a humidified atmosphere (5% CO2 and 95% air) at 37°C. Medium was changed after the first 24 h to let the cells stabilize. After that moment, medium was collected every 48 h, and fresh medium was added. Aliquots of the supernatants were kept at -20°C until measured. The CTBs were incubated in duplicates in the presence of different concentrations of rh-leptin (1, 10, 100, 1000, and 2000 ng/ml). The CTBs were cultured in the presence of 10% (v/v) fetal bovine serum (FBS) when STBs were desired, because CTBs differentiate to STBs in serum-supplemented medium. At the end of the cultures (4 days of treatment), cells were washed with PBS and stored at -20°C for protein determination or resuspended in guanidinium thiocyanate for RNA isolation. At least three independent experiments (including the appropriate controls) were run for each culture condition using different cell preparations.

Hormone and Protein Assays

Progesterone, hCG, IL-6, and leptin released by trophoblast cells into the culture medium were measured in the supernatants in duplicates using commercial kits (COAT-A-COUNT Progesterone RIA, Coat-A-Count hCG IRMA [Diagnostics Products Corp., Los Angeles, CA], Human Leptin IRMA [Diagnostic System Laboratories Inc. Webster, TX], and Quantikine Human IL-6 ELISA [R&D Systems, Minneapolis, MN]). The limit of sensitivity was 0.02 ng/ml for progesterone measurements, 0.3 mIU/ml for hCG, 0.1 ng/ml for leptin, and 0.7 pg/ml for IL-6. Proteins were determined by the Lowry assay, slightly modified according to Oyama et al. [29], using bovine serum albumin as standard.

RNA Isolation and Reverse Transcription-Polymerase Chain Reaction

Total RNA was extracted using the method described by Chomczynski and Sacchi [30]. Reverse transcription-polymerase chain reaction (RT-PCR) was performed with Access RT-PCR System (Promega, Madison, WI) using the following two pairs of primers: 5'-CCAAAAAGTCCAAGATGACACC-3' (sense) and 5'-TCTGTGGAGTAGCCTGAAGC-3' (antisense) and 5'-GGATCAATGACATTTCACACACG-3' (sense) and 5'-AGCCCAGGAATGAAGTCCAA-3' (antisense) for human leptin. The RT-PCR mixture contained 1 µg of total RNA, 1 µmol/L of each of the primers mentioned above, 0.2 mmol/L of dNTP, 0.1 U/µl of Taq DNA polymerase, 1.5 mmol/L of MgSO4, and 0.1 U/µl AMV Reverse Transcriptase in a total volume of 50 µl. After 45 min at 48°C for RT and a denaturation step at 94°C for 2 min, PCR was carried out at 94°C for 30 sec, 60°C for 60 sec, and 68°C for 2 sec. The PCR reactions were run for 45 cycles.

Statistics

Released hormones and cytokines were measured at the end of each experiment. Values were normalized per milligram of recovered cells and per their respective controls and then pooled and analyzed. Statistical analysis was performed by ANOVA using the GraphPad Instat computer program (San Diego, CA). Every experiment was performed at least three times in duplicates or triplicates.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Leptin Secretion and mRNA Expression in Human Trophoblast Cells in Culture

Human term trophoblast cells cultured in DMEM expressed leptin mRNA (two fragments of the expected sizes [357 and 149 bp] were obtained by RT-PCR) (Fig. 1). The same fragments were obtained when CTBs were cultured in the presence of 10% FBS (data not shown). Moreover, leptin protein was released to the culture media after 2 and 4 days of culture in the presence (2 days, 0.296 ± 0.1 ng/ml; 4 days, 0.029 ± 0.007 ng/ml) or absence of 10% FBS (2 days, 0.223 ± 0.034 ng/ml; 4 days, 0.037 ± 0.013 ng/ml).



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  		FIG. 1. RT-PCR for human leptin mRNA expression in human term trophoblast cells. A) First pair of primers for leptin mRNA (fragment expected size, 357 bp). Mx, Molecular weight markers. B) Second pair of primers for leptin mRNA (fragment expected size, 129 bp)

Effect of rh-Leptin on hCG Secretion

The effect of rh-leptin on hCG secretion by cultured trophoblast cells is shown in Figure 2. Addition of rh-leptin (1 or 2 µg/ml) led to a significant stimulation of hCG release after 2 days (control, 100%; 1 µg/ml, 172 ± 9.6 treatment/control, P < 0.01; 2 µg/ml, 200 ± 17 treatment/control, P < 0.001) (Fig. 2A) or 4 days (control, 100; 1 µg/ml, 276.25 ± 69.12 treatment/control; 2 µg/ml, 259.75 ± 48.4 treatment/control; P < 0.05) of culture (Fig. 2B). Lower concentrations of leptin did not produce a significant effect. Basal hCG secretion varied between experiments, probably because of biological variations, yet leptin was able to significantly increase hCG release to the medium in all the experiments.



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  		FIG. 2. Effect of leptin on hCG (mIU/ml) release after 2 days (A) or 4 days (B) of culture. Data are expressed as a percentage of respective controls. #P < 0.01, ##P < 0.001, ###P < 0.05 versus vehicle-treated cells

Effect of rh-Leptin on IL-6 Secretion

The effect of rh-leptin on IL-6 secretion is shown in Figure 3. After incubating term trophoblast cells with leptin (0.1 and 1 µg/ml) for 4 days, a significant stimulatory effect on IL-6 release into the culture medium was observed (control, 100; 0.1 µg/ml, 72.19 ± 32 treatment/control; 1 µg/ml, 1328.8 ± 547 treatment/control; P < 0.05). The same result was observed after 2 days of culture, but differences were not statistically significant. Lower concentrations of leptin did not significantly increase IL-6 release.



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  		FIG. 3. Effect of leptin on IL-6 secretion after 4 days of culture. Data are expressed as a percentage of respective controls. #P < 0.05 versus vehicle-treated cells

Effect of rh-Leptin on Progesterone Secretion

Treatment with rh-leptin inhibited progesterone secretion by trophoblast cells cultured in vitro. When compared to controls, progesterone release into the medium was significantly lower after 2 days of incubation with 2 µg/ml of rh-leptin (control, 100; 1 µg/ml, 88 ± 2.4 treatment/control; 2 µg/ml, 75.3 ± 3.6 treatment/control; P < 0.001) (Fig. 4A) and after 4 days of culture with 1 and 2 µg/ml of rh-leptin (control, 100; 1 µg/ml, 69.3 ± 4.6 treatment/control, P < 0.01; 2 µg/ml, 63.6 ± 5.5 treatment/control, P < 0.001) (Fig. 4B). As generally accepted, we found variability in hormonal basal release between different placentas, but leptin always led to an inhibitory effect on progesterone secretion.



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  		FIG. 4. Leptin inhibits progesterone secretion after 2 days (A) and 4 days (B) of culture. Data are expressed as a percentage of respective controls. #P < 0.001, ##P < 0.01 versus vehicle-treated cells

Correlation Between hCG and Progesterone Secretion by Trophoblast Cells Treated with rh-Leptin

As shown in Figure 5, a negative correlation exists between progesterone and hCG secretion by these cells in culture when treated with 2 µg/ml of leptin during 2 days (correlation coefficient, -0.8; P < 0.05) and 4 days of culture (correlation coefficient, -0.81; P < 0.049).



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  		FIG. 5. Correlation between hCG and progesterone secretion by trophoblast cells treated with rh-leptin (2 µg/ml) after 2 days (A) and 4 days (B) of culture (correlation coefficients, -0.8 and -0.81, respectively)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Successful pregnancy depends on a well-developed and functional placenta regulated by growth factors, cytokines, and steroid and polypeptide hormones acting in an autocrine/paracrine fashion. Using cultures of human term trophoblast cells, we found an effect of leptin on some placental secretions. We also demonstrated that leptin could be one important participant of such regulation of the human fetoplacental unit.

Previous studies on this subject were performed using first-trimester trophoblast. In the present study, we provide evidence for an effect of leptin on hCG, IL-6, and progesterone secretion by human term trophoblast cells. The molecular mechanisms of such effects remain unknown.

Our results indicate that leptin stimulates hCG and IL-6 secretion, which is consistent with previous results showing a stimulation of hCG secretion by leptin in first-trimester trophoblasts [8] and with others showing a stimulation on IL-6 production by choriocarcinoma JAR cell line [31]. Trophoblast secretion of hCG is regulated by many paracrine/autocrine factors, among which are IL-6 [28, 32], IL-1 [28, 32], LIF (leukemia inhibitory factor), and EGF (epidermal growth factor) [33]. One of the cytokines expressed at the fetomaternal interface, IL-6 plays an important role in regulating some placental functions [26, 27, 32, 34]. Considering that IL-6 and IL-1 are recognized as promoters of hCG secretion by term human trophoblasts and that our results show lower doses of leptin are required to stimulate IL-6 secretion compared to hCG, we suggest that leptin's stimulating effect on hCG secretion could be mediated, at least partially, by IL-6. Clearly, our current data do not exclude the possibility of leptin's action changing the proportion of the different spliced isoforms of Ob-R, thus modifying the amount of signal-transducing receptor and a leptin-binding activity [35]. As discussed previously, the different spliced isoforms of Ob-R have distinct signaling capabilities. It remains to be clarified which isoforms and which signaling pathways are involved in the effects shown in the present study.

On the other hand, our findings provide evidence for an inhibitory effect of leptin on progesterone production by these cell cultures. These results and the negative correlation found between hCG and progesterone secretion, after leptin stimulation, are in accordance with the results of Das et al. [36] and Paul et al. [37], showing no effect of hCG on progesterone production by human placenta, unlike the known stimulatory effect of hCG on progesterone production by the corpus luteum. Our findings are in agreement with an inhibitory effect of leptin on progesterone secretion found in other tissues, such as human granulosa luteal cells [38, 39], bovine granulosa cells [40], and rat granulosa cells [41]. In these tissues, an inhibitory effect of leptin on stimulated progesterone secretion was observed. This negative correlation is an interesting finding, but we are not yet able to explain the mechanism underlying it. Experiments are in progress in our lab to further clarify this. According to the literature, nobody has so far been able to show a clear correlation between hCG and progesterone in human placenta; however, a role of hCG in progesterone synthesis seems expected. Maybe leptin is one of the missing links between these two hormones in term placenta.

Progesterone and estradiol are the two most important steroid hormones produced by the human placenta. Progesterone is indispensable for the maintenance of pregnancy and is crucial for keeping the uterus in a quiescent state to prevent premature onset of labor. Because progesterone production by the placenta depends on the mother's cholesterol and our experiments were developed in cultures of trophoblast cells with no addition of external cholesterol, a possible explanation could be related to cholesterol availability. Otherwise, leptin could also be regulating some of the cholesterol-metabolizing enzymes or the transport of cholesterol between mitochondrial membranes by the placental steroidogenic acute regulatory protein (StAR)-like protein. Experiments are in progress to evaluate this possibility. A direct inhibitory action of leptin on steroid hormone secretion has been demonstrated independently by different groups in three of the major steroidogenic tissues, namely the adrenal gland, the ovary, and the testis [42–44]. The mechanism involved for such inhibitory action has not been completely clarified: Results obtained from testis suggested that leptin-induced inhibition of steroidogenesis is, at least partially, mediated by a decrease in the expression of mRNA encoding several upstream elements in the steroidogenic route, such as P450scc and StAR [44].

Another fact to be considered is that in cultures of human granulosa cells, an inhibitory effect of IL-6 on FSH-stimulated progesterone production has been shown [45]. This result and our findings of leptin's effect on progesterone and IL-6 secretion could suggest that the leptin-induced inhibition of progesterone secretion might be mediated by IL-6.

Increased leptin secretion with 10% FBS fits with immunohistochemical studies showing more intense staining for leptin in STB compared with CTB [3, 7], because serum is a known promoter of syncytium formation.

Many other factors, such as retinoids [46], estradiol [8], insulin, glucocorticoids, and cAMP [47], are involved in the regulation of leptin secretion by human placenta, suggesting a fine regulation of the obese protein in this tissue. Differential placental leptin expression has been associated with many pregnancy-associated disorders, such as recurrent diabetic pregnancies [5, 48], abnormal pregnancies [49], implantation failures [50], and preeclampsia [51], but little is known about how this small protein could be related to these problems. In the present study, we demonstrated, to our knowledge for the first time in full-term placenta, an important role of leptin in some specific placental functions that could be related to these problems of pregnancy. More experiments should be done to understand the mechanisms involved in these processes.


   ACKNOWLEDGMENTS
 
The authors thank members of Laboratorio Biología de la Reproducción for their constant support, all the registered nurses in the delivery room from the Instituto Argentino de Diagnóstico y Tratamiento (IADT) for their collaboration, and Dr. Mónica Cameo for critical reading of the manuscript.


   FOOTNOTES
 
1 Supported by the UNDP/UNFPA/WHO World Bank Special Programme of Research, Development and Research Training in Human Reproduction, World Health Organization (no. 98166). P.C. has a postgraduate fellowship from CONICET. Back

2 Correspondence: Paula Cameo, Instituto de Biología y Medicina Experimental (IBYME), Vuelta de Obligado 2490, Capital Federal (1428), Buenos Aires, Argentina. FAX: 54 11 4783 2690; paucameo{at}fibertel.com.ar and pcameo{at}dna.uba.ar Back

Received: 2 April 2002.

First decision: 29 April 2002.

Accepted: 12 August 2002.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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