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Original Studies |
Research Centre for Endocrinology and Metabolism, Departments of Internal Medicine (C.K., K.L., L.M.S.C., B.C.) and Department of Obstetrics and Gynecology (E.S., C.B., H.B.), Sahlgrenska University Hospital; and the Department of Physiology and Pharmacology (E.S., H.B.), Goteborg University, Goteborg; and the Department of Molecular Biology, Pharmacia and Upjohn (P.L.), Stockholm, Sweden
Address all correspondence and requests for reprints to: Dr. Björn Carlsson, Research Center for Endocrinology and Metabolism, Department of Internal Medicine, Gröna Stråket 8, Sahlgrenska University Hospital, S-413 45 Goteborg, Sweden. E-mail: bjorn.carlsson{at}ss.gu.se
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Abstract |
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In conclusion, this study has demonstrated that the leptin receptor is expressed in the human ovary, that leptin is present in follicular fluid, and that leptin can induce a biological response in ovarian cells. These results suggest that leptin may have a direct effect on the human ovary.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Leptin is an adipocyte-derived protein (7) that acts in an endocrine fashion by reporting the size of the adipose tissue mass to hypothalamic leptin receptors, and serum leptin levels correlate with the amount of body fat (8, 9). Administration of recombinant leptin to mice reduces their body weight by increasing energy consumption and decreasing food intake (10, 11, 12). Leptin is also a candidate for mediation of the signal between fat stores and the reproductive system (13, 14, 15, 16, 17, 18). Genetically obese, ob/ob mice, are infertile (19). Thinning of ob/ob female mice by diet restriction failed to correct their sterility; however, administration of recombinant leptin restored their fertility (14). The mechanism by which leptin modulates fertility is not clear. In rodents, there are indications that leptin modulates the reproductive endocrine system in the hypothalamus, pituitary, and ovary (20, 21, 22, 23).
Leptin exerts its effects via the leptin receptor, which is a member of the cytokine receptor superfamily (24). Several isoforms of the leptin receptor are produced by alternative splicing, resulting in receptors with different intracellular domains (24, 25, 26). Only the isoform with a long intracellular domain is capable of activating the JAK-STAT signaling pathway (27, 28), whereas the function of the short isoforms is unknown. The leptin receptor is expressed in the human ovary (25); however, it is unclear which of the isoforms of the receptor is present in the ovary.
The aim of our study was to determine whether there are prerequisites for a direct action of leptin on the human ovary.
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Subjects and Methods |
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Ovarian and adipose tissue were obtained from women with
regular menstrual cycles undergoing laparotomy for reasons unrelated to
ovarian pathology. Serum, follicular fluid, and granulosa cells were
obtained from four women undergoing oocyte retrieval in stimulated
cycles in the course of in vitro fertilization (IVF)/embryo
transfer. The patients, numbered 1–4 (Fig. 4
) had body mass indexes
(BMI) of 27.4, 23.1, 21.6, and 29.1 kg/m2, respectively.
Informed consent was obtained from all women, and the study was
approved by the local ethical committee.
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Total RNA was isolated as described by Chomczynski and Sacchi (29). First strand cDNA was generated as described previously (30).
Ribonuclease (RNase) protection assay
A leptin receptor probe that specifically allows analysis of
expression of the long and short isoforms of the receptor was generated
by reverse transcription-PCR (RT-PCR; Fig. 1A). The primers,
5'-CCAGTTCAGTCTTTACCC-3' (nucleotides 2421–2438) and 5'-CTCAGCCTCAGA
GAAGTT-3' (nucleotides 2904–2887), were based on the human leptin
receptor cDNA sequence (24). cDNA was amplified using a stepdown
procedure, with annealing temperatures of 60, 57, 54, and 51 C (3
cycles each) and finally 57 C (30 cycles). The amplified 483-bp
fragment was subcloned into pBluescript SK (Stratagene, La Jolla, CA),
generating pCK6:95. Plasmids were purified (Qiagen, Chatsworth, CA),
and the subcloned fragment was sequenced using a Terminator
Double-Stranded DNA Sequencing Kit (Applied Biosystems Division,
Perkin-Elmer, Foster City, CA).
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-33P]UTP (DuPont, Dreieich, Germany),
and 10 U T3 polymerase (Promega). After synthesis for 15 min at room
temperature, 10 U RNase-free deoxyribonuclease were added (37 C for 15
min). Finally, the probe was purified using NICK columns, Sephadex G-50
DNA grade (Pharmacia Biotech, Uppsala, Sweden). Hybridization was
performed overnight at 45 C in 1 x hybridization buffer (RPA II
Kit, Ambion) using 100 µg RNA. Subsequently, the samples were treated
with 25 U RNase A and 1000 U RNase T1 for 30 min at 37 C. Protected
fragments were precipitated and analyzed on a denaturing polyacrylamide
gel. The gel was exposed on a PhosphorImager screen (Molecular
Dynamics, Sunnyvale, CA) for 10 days and then analyzed. RT-PCR
Leptin receptor cDNA. Amplification of leptin receptor cDNA
was performed in 1 x Taq extender reaction buffer
(Stratagene) containing cDNA, 0.8 mmol/L deoxy-NTP
(Boehringer-Mannheim, Mannheim, Germany), and 1 µmol/L each of sense
and antisense primers, 5'-TAGAATTCCCTCGAATGTTAA-3' (nucleotides
2358–2378) and 5'-CGTGATTTTCTTCAGGAA-3' (nucleotides 3217–3199; Fig. 1A
). The forward primer was based on the mouse leptin receptor cDNA
sequence, and the reverse primer was based on the rat leptin receptor
cDNA sequence. After denaturation (4 min at 94 C), 5 U each of
Taq DNA polymerase (B/M) and Taq extender PCR
additive (Stratagene) were added, resulting in a final volume of 50
µL. cDNA was amplified using a stepdown procedure, with annealing
temperatures of 56, 53, and 50 C (3 cycles each) and finally 48 C (30
cycles). The identity of the PCR product (Fig. 3A) was verified by DNA
sequencing using the Prism Dye Primer Cycle Sequencing Kit (Applied
Biosystems Division, Perkin-Elmer) and an ABI 373A automatic sequencer
(Applied Biosystems).
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).
Cyclophilin cDNA. Amplification of cyclophilin cDNA using
the same cDNA templates as those described above was included as a
positive control and was performed with 1 µmol/L each of sense and
antisense primers, 5'-biotin-GCAGACAAGGTCCCAAAGA-3' (nucleotides
76–94) and 5'-GCAGCGAGAGCACAAAGA-3' (nucleotides 231–209; Fig. 1C)
and an annealing temperature of 57 C (30 cycles). The primers were
based on the human cyclophilin gene sequence (33) and were designed to
span 3 introns (Fig. 1C).
The PCR products were separated on a 0.8% agarose gel containing
ethidium bromide and visualized by UV light (Fig. 3, A–C). Controls
for possible PCR contamination were included in all experiments and
were always negative (Fig. 3, A–C, lane 6).
Leptin RIA
Serum and follicular fluid leptin concentrations were determined in duplicate by a human leptin RIA (Linco Research, St. Charles, MO). The limit of sensitivity for the assay was 0.5 µg/L. The intraassay coefficient of variation was 6.3% at a leptin concentration of 15.6 µg/L. All samples were analyzed in the same assay.
Leptin
Recombinant human leptin was produced essentially as described by Halaas et al. (11). In brief, human leptin was expressed as a hexahistidine tag leptin fusion protein and purified with Q-Sepharose (Pharmacia Biotech). The hexahistidine tag was removed by thrombin cleavage, and leptin was further purified by gel filtration (Superdex 75, Pharmacia Biotech) to near homogeneity (>99%). The endotoxin concentration, determined by Limulus amoebocyte endotoxin assay, was 0.00065 U/µg leptin.
Leptin stimulation of primary isolated human granulosa cells
Human granulosa cells were obtained in connection with IVF through follicle aspiration via transvaginal ultrasound-guided puncture. The patients were treated with a GnRH agonist for 3 weeks (Suprefact, Hoechst, Germany) before inducing follicle development with recombinant FSH (Gonal-F, Serono, Italy). The follicle aspirations were performed 36–38 h after the administration of hCG (Profasi, Serono, Italy). The granulosa cells were washed in medium 199 with Earle’s salt (Life Technologies, Paisley, UK), 25 mmol/L NaHCO3, 50 mg/L gentamicin (Life Technologies), 1% FBS (Flow Laboratories, Labkemi, Goteborg, Sweden), and 0.1 mg/mL testosterone (Sigma Chemical Co., St. Louis, MO). The cells from 4 subjects were pooled and cultured in 24-well plates (Falcon 3047, Becton Dickinson Labware, Lincoln, NJ; 96,000 cells/well) in medium 199 with the additives described above. The cells were precultured for 2–4 days and then cultured for 2 days with different combinations of leptin (100 ng/mL) and LH (0.1 ng/mL). Before adding hormones, the medium was changed. The amount of estradiol was measured in spent culture medium by Delfia assay (Wallac Oy, Finland). The experiment was performed three times, with four observations in each experiment.
Statistical analysis
Differences between groups were analyzed by Student-Newman-Keuls multiple range test. P < 0.05 was considered significant.
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Results |
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The expression of leptin receptors in granulosa cells and thecal
cells was analyzed by a RNase protection assay that differentiates
between long and short receptor isoforms. The probe was designed to
protect a 483-bp fragment for the long isoform and a 252-bp fragment
for the short isoforms (Fig. 1A). Only a
252-bp fragment was detected (Fig. 2),
indicating that the short isoforms of the leptin receptor are the most
abundant and that the long isoform is expressed at levels below the
sensitivity of our assay.
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, lanes 1–3). DNA sequencing verified
that the PCR product was identical to the cDNA sequence of the long
isoform of the leptin receptor (GenBank accession no. U43168). Genomic
DNA was included as a negative control. Analysis of immunoreactive leptin in follicular fluid
Recent studies suggest that leptin circulates in both bound and
free forms in human blood (35, 36). A binding protein may interfere
with the diffusion of leptin from blood vessels to the avascular
compartment within the follicle. We, therefore, measured immunoreactive
leptin in serum and follicular fluid obtained from four women
undergoing IVF. Serum and follicular fluid leptin concentrations ranged
from 18.3–29.7 µg/L. In all four patients, leptin concentrations in
follicular fluid were similar to those in serum (Fig. 4).
To examine whether leptin is produced in the ovary, we analyzed the
expression of the ob gene in human ovarian cells by RT-PCR.
ob gene expression was detected in adipose tissue, but not
in granulosa cells, thecal cells, or interstitial cells (Fig. 3B). To
verify the quality of the RNA and the cDNA, cyclophilin gene expression
was analyzed (Fig. 3C).
Effects of leptin on human granulosa cells
The effect of leptin (100 ng/mL) on basal and LH-stimulated (0.1
ng/mL) steroid production was investigated in granulosa cells obtained
in connection with IVF. Leptin had no effect on basal estradiol
production (Fig. 5). However, LH-induced
estradiol production in primary cultures of human granulosa cells was
suppressed by leptin (Fig. 5).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Leptin has recently been shown to be of critical importance for normal function of the female reproductive system in rodents (14, 15, 16, 17, 18). It is not known whether the effects of leptin on the reproductive system are exerted at the ovarian or pituitary/hypothalamic level or at both levels. Several leptin receptor isoforms exist that are generated by alternative splicing, and the isoforms differ in the intracellular domain. Transfection experiments have shown that only the long isoform of the leptin receptor has signaling capacity (27, 28). This is in line with studies of several other members of the cytokine receptor superfamily that have shown that two semiconserved regions, boxes 1 and 2, in the intracellular region of the receptors are required for efficient activation of protein tyrosine kinases of the JAK family (37). Leptin receptor expression was recently demonstrated in the human ovary (25); however, the analysis used detected all isoforms of the leptin receptor, including those without signaling capacity. In this study we show that the long isoform of the leptin receptor is expressed in several human ovarian cell types, although transcripts encoding the short isoforms were much more abundant. It has been proposed that the short isoform may act as a dominant negative receptor (38) as has been shown for receptors of the tyrosine kinase family (39). The biological response to leptin in granulosa cells was, therefore, unexpected because of the high ratio of the short/long receptor isoforms of the leptin receptor. However, transfection experiments have shown that the long isoform of the leptin receptor is relatively resistant to dominant negative repression by truncated receptor isoforms (40), and the effects of leptin may, therefore, be exerted through the low abundant long leptin receptor. As the long isoform of the leptin receptor was only detected by RT-PCR, which is a very sensitive method for messenger RNA (mRNA) detection, it is possible that the long receptor isoform is expressed at levels that are biologically insignificant. An alternative explanation is, therefore, that the short leptin receptor has some signaling capacity. This idea is supported by a recent study in which leptin inhibited the effects of insulin on cultured hepatocytes that only express the short isoform of the receptor (41). However, a potential problem when using recombinant proteins produced in bacteria is contamination with endotoxins that may exert biological effects. As leptin had no effect on basal estradiol production, and the endotoxin levels were low in our leptin preparation, the suppression of LH-induced estradiol production is likely to be caused by leptin itself.
In rodents, ob gene expression appears to be restricted to adipocytes (7). In contrast, in man, ob gene expression has also been detected in placenta and heart (42). However, it remains to be established which cell type within these tissues expresses the ob gene. The presence of leptin in follicular fluid and the absence of ob gene expression in the ovary indicate that leptin acts in an endocrine fashion on the ovary.
Leptin was identified based on its importance in the regulation of adipose tissue mass in an animal model of obesity (ob/ob mice) (7). Leptin seems to have similar effects in humans, as defects in the ob gene cause obesity in humans (43). However, ob gene defects are rare in human obesity (30, 43, 44, 45), and to date, there are no reports of mutations in the leptin receptor gene in man (46). Obese subjects, as a group, have elevated levels of leptin in the blood (8, 9), and it has been proposed that obese subjects are leptin resistant (9). It is not known whether the elevated levels of leptin in serum contribute to diseases secondary to obesity. It has been speculated that leptin may be of importance in obesity-associated dysfunction of the reproductive system (13, 23). One study has shown that a substantial portion of women with PCOS have leptin levels higher than expected for their BMI (47). In contrast, three independent laboratories recently provided evidence against the concept of elevated leptin levels in PCOS patients compared to those in age- and weight-matched control subjects (48, 49, 50, 51). However, as there may be subgroups of women with PCOS, it is possible that a subgroup of PCOS women exists that has higher leptin levels (48). Interestingly, leptin inhibits the production of estradiol, but not that of progesterone, in cultured rat granulosa cells (23), suggesting that it may promote a steroid microenvironment in the follicle similar to that present in PCOS (52). Based on the tissue distribution of leptin receptors (Refs. 24–26 and this study), it is possible that leptin acts on the reproductive system both at the hypothalamic-pituitary level (GnRH-FSH/LH) (20, 21, 22) and directly on the ovary (Ref. 23 and this study). The relative importance of leptin interactions at these levels is not known and could also vary during physiological and pathophysiological situations. It is possible that the major site of action of leptin may differ depending on the concentration of leptin in the blood. The direct action of leptin on the ovary may be of importance under certain conditions with elevated concentrations of leptin in the blood, such as obesity. It was recently shown that the cerebrospinal fluid/plasma leptin ratio is lower in obese compared to lean subjects, and it was suggested that this was due to a reduced efficiency of the transport of leptin from plasma to cerebrospinal fluid (53, 54). This creates a situation where peripheral tissues may be exposed to very high leptin concentrations while the central nervous system is exposed to only moderately increased levels of leptin. Conversely, leptin action at the hypothalamic level may be of relatively greater importance during conditions with low concentrations of leptin in the blood, as in women with low BMI and in ob/ob mice (20, 21, 55, 56, 57).
We conclude that both the short and long isoforms of the leptin receptor are expressed in human ovarian cells and that immunoreactive leptin is present in human follicular fluid. In addition, leptin significantly suppressed LH-induced estradiol production. These findings are consistent with an endocrine action of leptin on the human ovary, with possible implications for female reproduction in health and disease.
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Acknowledgments |
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Footnotes |
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Received October 14, 1996.
Revised March 17, 1997.
Revised September 2, 1997.
Accepted September 5, 1997.
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