Molecular Human Reproduction, Vol. 7, No. 6, 567-572,
June 2001
© 2001 European Society of Human Reproduction and Embryology
Uterine physiology |
Progesterone inhibition of functional leptin receptor mRNA expression in human endometrium
Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566, Japan
Abstract
Leptin is secreted by adipocytes and regulates appetite through interaction with hypothalamic leptin receptors (OB-R). Leptin is involved in the stimulation of reproductive functions, and local expression of leptin and OB-R in the ovary, oocyte, embryo, and placenta might play a role in early development. The mRNA and protein of the long form leptin receptor (OB-RL) but not of leptin are expressed in the human endometrium and the abundance of OB-R mRNA expression varies during the menstrual cycle with a peak in the early secretory phase. We examined the steroidal regulation of OB-RL mRNA expression. Northern blot analyses showed that in organ-cultured proliferative endometrial specimens, oestradiol (10–9 and 10–8 mol/l) had no acute effect on the OB-RL mRNA expression, whereas oestradiol plus progesterone (10–8, 10–7 and 10–6 mol/l) or medroxyprogesterone acetate (10–8 and 10–7 mol/l) suppressed the expression by ~50%. This progestin-induced suppression was blocked by a concomitant addition of mifepristone. Additionally, incubation of endometrial specimens in the presence of leptin resulted in the phosphorylation of its intracellular target, STAT3 (signal transducer and activator of transcription 3). These results indicate that, in the human endometrium, progestins act via the progesterone receptors to suppress functional OB-RL mRNA expression, and may thereby alter the sensitivity of the endometrium to leptin.
endometrium/intracellular signalling/leptin receptor/oestrogen/progestin
Introduction
Leptin, the obese gene (Ob) product, is synthesized and secreted by adipose tissue (Zhang et al., 1994
) and regulates the amount of food intake through interaction with hypothalamic leptin receptors (Tartaglia et al., 1995). Leptin binds to its receptors on the cell membrane and is involved in the ob/ob activation of STAT3, a member of the signal transducer and activator of transcription family of proteins (Vaisse et al., 1996). At least four types of splice variants of OB-R mRNA have been identified to encode proteins which differ in the length of their cytoplasmic domains. The long form (OB-RL) is the full-length variant with an ability to activate the STAT pathway, while the three types of short form lack several sequences that are responsible for intracellular signalling (Baumann et al., 1996; Cioffi et al., 1996; Bjørbæk et al., 1997).
In addition to the action on energy metabolism, leptin influences various reproductive functions. Injecting leptin into ob/ob mice that are infertile with lack of leptin increases the weight of the uterus and ovaries and the number of follicles (Barash et al., 1996), resulting in restoration of fertility (Chehab et al., 1996). Administering leptin to normal female mice accelerates puberty (Ahima et al., 1997), and in humans higher leptin levels have been shown to relate to the earlier onset of menarche (Matkovic et al., 1997). These actions of leptin are considered to be mediated mainly through brain OB-R. In contrast, the leptin mRNA and protein and OB-R mRNA are expressed in peripheral reproductive tissue including granulosa cells and cumulus cells of human pre-ovulatory follicles (Antczak et al., 1997; Cioffi et al., 1997; Karlsson et al., 1997), oocytes and embryos (Antczak and Van Blerkom, 1997, 1999), and human placental trophoblasts (Luoh et al., 1997; Masuzaki et al., 1997; Señarís et al., 1997; Henson et al., 1998). Leptin directly affects oestrogen-producing activity in granulosa-luteal cells (Kitawaki et al., 1999a). Furthermore, the mRNA and protein of OB-R but not of leptin, are expressed in human endometrium (Alfer et al., 2000; Kitawaki et al., 2000a), with OB-RL mRNA expression low in the early proliferative phase, increasing gradually during the proliferative phase, greatest in the early secretory phase, and declining toward menstruation (Kitawaki et al., 2000a). These findings suggest that leptin plays a physiological role in implantation in human endometrium and led us to investigate the effect of oestrogen and progestins on OB-RL mRNA expression and to determine whether leptin activates intracellular signalling in the endometrium.
Materials and methods
Tissue samples
Endometrial specimens were obtained from patients who had undergone hysterectomy at the Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine. This study protocol was approved by the Kyoto Prefectural University of Medicine institutional review board, and informed consent was obtained from each patient. All patients had normal menstrual cycles and were not receiving any endocrine therapy such as gonadotrophin-releasing hormone analogue, danazol, or steroids. Each specimen was diagnosed by histological examination using excised uteri. Since the expression pattern for OB-R is similar between the endometrial samples of patients with endometriosis, adenomyosis, and/or leiomyomas, and in disease-free endometria (Kitawaki et al., 2000a), the samples showing these diseases were included in this study. However, the cases with malignant neoplasms other than cervical cancer in situ, ovarian neoplasm, pelvic inflammation, and pregnancy were excluded from this study. Thirty-eight patients met the criteria for enrollment. The mean age and body mass index (BMI) were 45.9 ± 4.1 (mean ± SD, 34–49 years) and 21.4 ± 1.9 (19.0–25.8 kg) respectively. There was no significant difference in the mean ages or BMI between patient groups of the proliferative phase (n = 28) and the secretory phase (n = 10). Endometrial dating was performed using chronological dating, basal body temperature, and published criteria (Noyes et al., 1950).
Organ culture
Organ culture of endometrial tissues was performed as described previously (Kitawaki et al., 2000b). Briefly, the endometrial tissue was washed immediately after sampling and cut into ~1 mm cubes in ice-cold Hanks' balanced salt solution (HBSS) (Life Technologies, Inc., Grand Island, NY, USA). The tissue fragments were placed on several pieces of 1 cm cubes of Spongel (Yamanouchi Pharmaceuticals, Tokyo, Japan), which were immersed in 10 ml Dulbecco's modified Eagles's medium/Ham's F-12 medium (1:1) with 15 mmol/l HEPES buffer without Phenol Red (DMEM/F-12) (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Inc.), penicillin (100 IU/ml), streptomycin (100 µg/ml), and fungizone (0.25 µg/ml). The FBS had been treated twice with charcoal (6.25 mg/ml) and Dextran T-70 (0.625 mg/ml) and then incubated at 56°C for 30 min to remove endogenous cytokines and steroids. The tissue fragments were cultured in the presence or absence of oestradiol (Sigma, St Louis, MO, USA), progesterone (Sigma), medroxyprogesterone acetate (MPA) (Sigma), and/or mifepristone (Sigma), a progesterone receptor antagonist, in a humidified atmosphere of 5% CO2-95% air at 37°C and were subjected to the total RNA extraction.
Primary culture of epithelial cells and stromal cells
Separation and culture of endometrial epithelial and stromal cells were performed essentially as described (Satyaswaroop et al., 1979). Endometrial tissues were cleaned, trimmed, minced, and digested with 0.25% collagenase (type 1; Sigma) in serum-free DMEM/F-12 medium at 37°C in a shaking water bath for 2 h. All incubations and cultures were then performed in a humidified atmosphere of 5% CO2-95% air. The cells were passed through a 40 µm nylon mesh filter. The filtrate containing stromal cells was resuspended in DMEM/F-12 medium containing 10% FBS, plated in 25 cm2 tissue culture flasks (Becton Dickinson Labware, Franklin Lakes, NJ, USA), and incubated for 30 min. After washing away unattached cells, morphologically homogeneous stromal cell cultures were obtained. Isolated glands retained on the filter were washed free of adhering stromal cells with HBSS, transferred by backwashing with DMEM/F-12 medium containing 10% charcoal-treated FBS, and distributed in 100 mm Petri dishes (Becton Dickinson). After allowing the remaining stromal cells to adhere selectively to Petri dishes for 30 min, unattached epithelial cells were collected. This process was repeated and a purified epithelial cell preparation was obtained. Epithelial or stromal cells were cultured in DMEM/F-12 medium containing 10% charcoal-treated FBS in 25 cm2 tissue culture flasks. The purity of cell preparations was confirmed by immunohistochemical staining using anti-cytokeratin antibody (Dako, Carpinteria, CA, USA) for epithelial cells and anti-vimentin antibody (Dako) for stromal cells. After reaching confluence, the cells were further incubated with or without test compounds in serum-free DMEM/F-12 medium, and were scraped and subjected to the total RNA extraction.
RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted using Trizol reagent (Life Technologies, Inc.), and the first strand complementary DNA (cDNA) synthesis from total RNA was catalysed by Superscript II RT (Life Technologies, Inc.) using oligo(dT) 12–18, as previously described (Kitawaki et al., 1999a). The resulting first strand cDNA was used for PCR amplification with the following primers: 5'-TTGTGCCAGTAATTATTTCCTCTT-3' (forward; nucleotides 2727–2750) and 5'-CTGATC- AGCGTGGCGTATTT-3' (reverse; nucleotides 3165–3146) (Kielar et al., 1998) for OB-RL and the human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) amplimer set for G3PDH (Clontech, Palo Alto, CA, USA). The PCR mixture comprised 1 µl first strand cDNA, 0.2 µmol/l of each of the primers mentioned above, 0.2 mmol/l dNTP, and 1 µl AdvanTaq DNA polymerase (Clontech) in a total volume of 50 µl PCR buffer (Clontech). After an initial denaturation at 95°C for 1 min, PCR was carried out at 95°C for 30 s; at 60°C (for OB-RL) or 55°C (for G3PDH) for 30 s; and at 68°C for 30 s. The PCR reactions were run for 35 cycles.
Northern blot analysis
Northern blotting was performed as described previously (Kitawaki et al., 2000a). Briefly, 20 µg total RNA was subjected to electrophoresis in a 1% agarose/formaldehyde gel, transferred to a nylon membrane (Hybond N+; Amersham Pharmacia Biotech, Piscataway, NJ, USA) by capillary blotting, and UV cross-linked. Membranes were prehybridized for 1 h at 57°C in 0.5 mol/l Na2HPO4/H3PO4 buffer (pH 7.2) containing 1 mmol/l EDTA and 7% sodium dodecyl sulphate (SDS). The radiolabelled probes for OB-RL and G3PDH were derived from the amplified cDNA fragments produced in RT-PCR. DNA bands were excised from the agarose gel and extracted using a NucleoTrap DNA purification kit (Clontech). An aliquot of the DNA product was sequenced by the dye terminator method using a model 100 DNA analyser (PE Applied Biosystems, Foster City, CA, USA) and the sequence was confirmed to be equal to that reported in the GenBank databank (U43168 for OB-RL). The probes were radiolabelled with [
-32P]dCTP using a Random Primer Plus extension labelling system (New England Nuclear, Boston, MA, USA). After hybridization for 24 h at 57°C, membranes were washed three times for 5 min each at 65°C, and then washed for 15 min at 65°C in 0.04 mol/l Na2HPO4/H3PO4 buffer (pH 7.2) containing 1% SDS. The hybridized signal was analysed using a bioimaging analyser (BAS 2000; Fujix, Tokyo, Japan).
Immunoprecipitation and Western blot analysis for leptin-induced tyrosine phosphorylation of STAT3
Approximately 0.4 g wet wt endometrial tissue was cut into 1 mm cubes and incubated in serum-free DMEM/F-12 medium containing 15 ng/ml recombinant human leptin (Immugenex, Los Angeles, CA, USA) in a humidified atmosphere of 5% CO2-95% air at 37°C. The pieces were homogenized, solubilized in 0.4 ml lysis buffer [phosphate-buffered saline (PBS), pH 7.4, containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and 0.57 mmol/l phenylmethylsulphonylfluoride], and centrifuged at 15 000 g for 20 min at 4°C. The resulting supernatant was incubated with anti-STAT3 antibody (1:100; New England Biolabs, Beverly, MA, USA) for 60 min at 4°C. The immunocomplexes were adsorbed to Protein A-Agarose (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C on a rocker platform. After washing with PBS, the samples were mixed with sample buffer and electrophoresed under reducing conditions in 7.5% SDS–polyacrylamide gel. ECL protein molecular weight markers (Amersham Pharmacia Biotech) were used as standards. Proteins were electrotransferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA). After incubating with blocking buffer containing 5% bovine serum albumin to prevent non-specific protein binding, the membrane was incubated with the anti-STAT3 antibody (1:500), washed, and incubated with a second antibody conjugated with horseradish peroxidase (1:10 000; Santa Cruz Biotechnology). Immunoreactions were detected with enhanced chemiluminescence using ECL plus Western blotting detection reagents (Amersham Pharmacia Biotech). The membrane was exposed to Kodak BioMax film (Eastman Kodak Company, Rochester, NY, USA). The membrane was then stripped and reprobed with anti-phospho-STAT3 antibody (New England Biolabs) to detect tyrosine phosphorylation of STAT3.
Statistical analysis
Differences in ages and BMI between patient groups of the proliferative phase and the secretory phase were analysed with Student's t-test. Differences in OB-RL mRNA levels in the endometrial specimens cultured with or without steroids were analysed with the one-factor ANOVA and multiple comparisons were performed using Bonferroni/Dunn's procedure. P < 0.05 was considered to be significant.
Results
Progestin suppression of OB-RL mRNA
By Northern blot analyses, OB-RL mRNA was detected as a doublet of 6.4 and 4.5 kb in endometrial specimens that had been organ-cultured in the presence or absence of steroids. The amount of OB-RL mRNA expression relative to G3PDH mRNA was calculated based on the Northern blot (Figure 1). In the specimens obtained from patients in the proliferative phase, the addition of oestradiol (10–9 or 10–8 mol/l) had no acute effect on OB-RL mRNA expression, whereas the addition of progesterone (10–8, 10–7 and 10–6 mol/l) with or without oestradiol (10–9 mol/l) suppressed OB-RL mRNA expression by 48–53% of control (P < 0.01). The addition of oestradiol (10–9 mol/l) with MPA (10–8 and 10–7 mol/l) also suppressed OB-RL mRNA expression by 52% of control (P < 0.01). However, progestin suppression was blocked by a concomitant addition of mifepristone, a progesterone receptor antagonist (Figure 1).
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In the specimens obtained from patients in the secretory phase, the level of OB-RL/G3PDH mRNA expression was similar to that of the proliferative endometria that had been suppressed by progestins, and was not affected by the addition of oestradiol, progesterone, and/or mifepristone (Figure 1
). The expression of OB-RL mRNA was suppressed by progesterone at 12 h of exposure and in 24 h reached a minimum level of 45% of control (P < 0.01) (Figure 2).
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To investigate whether the steroid modulation of OB-RL mRNA expression was identical between cellular components, similar experiments were performed in primary cultured epithelial and stromal cells that were separated from the proliferative phase endometria. The level of OB-RL/G3PDH mRNA expression was comparable in epithelial and stromal cells. Furthermore, progesterone suppressed OB-RL mRNA and mifepristone blocked the suppression in both cellular components in a similar manner (Figure 3
).
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Leptin-induced tyrosine phosphorylation of STAT3
To determine whether leptin activates intracellular signalling in human endometrium, leptin-induced tyrosine phosphorylation of STAT3 was measured. Endometrial specimens obtained from patients in the proliferative phase were cut into small pieces and stimulated by leptin at 15 ng/ml, a standard concentration observed in human serum. Immunoprecipitation and Western blotting analysis of the lysed samples showed a comparable amount of a 89 kDa protein, which is identical to STAT3, for each incubation time listed. Stripping and reblotting the membrane with antibody specific for phosphorylated STAT3 showed a time-dependent increase in STAT3 phosphorylation, reaching a maximum ~11-fold increase 30 min after leptin addition (Figure 4
).
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Discussion
To the best of our knowledge, this is the first report demonstrating the transcriptional regulation of OB-RL expression in the human tissue. The present study indicates that in the human endometrium progestins suppresses OB-RL mRNA expression via the progesterone receptors. We also demonstrated leptin-induced phosphorylation of STAT3, indicating the presence of a functional OB-R and intracellular signalling in the human endometrium.
During the menstrual cycle, the abundance of total OB-R and OB-RL mRNA increases from the early to late proliferative phase, reaches a peak in the early secretory phase, and decreases from the mid to late secretory phase (Kitawaki et al., 2000a). However, the present in-vitro study showed that oestradiol had no acute effect on OB-RL mRNA expression, indicating that oestrogens are not a factor that directly increase endometrial OB-R expression. The OB-R expression may be increased by another unknown factor that is expressed in the proliferative phase endometrium, probably in the presence of oestrogens. Leptin has been shown to be involved in angiogenesis and blood vessel growth. The stimulation of endothelial cells by leptin leads to a marked enhancement of angiogenesis (Bouloumié et al., 1998). Therefore leptin may be involved in the proliferation of the endometrium, and its receptor may be regulated by any proliferative factor. In addition, the oestrogen metabolism, namely the enzyme regulation of aromatase responsible for oestrogen biosynthesis and of 17ß-hydroxysteroid dehydrogenase type 2 responsible for inactivation of oestradiol to oestrone, is remarkably different between disease-free endometria and those of patients with endometriosis, adenomyosis, and/or leiomyomas (Kitawaki et al., 1999b, 2000b), whereas the incidence and abundance of OB-RL mRNA expression are comparable among diseased and disease-free endometria (Kitawaki et al., 2000a). These findings are supportive of the view that oestrogens are not a direct factor regulating the OB-RL expression.
In contrast with the proliferative endometrium, the abundance of OB-RL mRNA expression in the secretory endometrium was decreased by ~50%. The decrease was reproduced by progesterone exposure to the proliferative endometrium but not to the secretory endometrium. This suggests that OB-RL mRNA in the secretory endometrium had been fully suppressed by progesterone. The organ culture technique has been previously validated; using the same method, we have shown progestin induction of 17ß-hydroxysteroid dehydrogenase type 2 (Kitawaki et al., 2000b). The results suggest that the decrease in the OB-RL expression is mainly due to progesterone that is secreted from corpus luteum. Moreover, the present study showed that the progesterone suppressed the OB-RL mRNA to a similar extent in both epithelial and stromal cells, indicating that the decrease was not caused by a change in conformation ratio of epithelial and stromal cells in the secretory phase. Under our conditions, the progesterone suppression of OB-RL mRNA was obtained more efficiently by the monolayer culture technique (~80%) than by the organ culture technique (~50%). We consider that the discrepancy might be caused by the difference of experimental conditions. Although at the transcriptional level, progesterone suppression was obtained quickly in 12–24 h, the molecular mechanism has not been clarified. Since a progesterone-binding site has not been identified in the promoter region of the OB-R gene, the decrease in OB-RL mRNA expression might be mediated indirectly by other factor(s).
Although the abundance of OB-RL expression decreases during the secretory phase, it should be noted that OB-RL is substantially expressed in the midluteal phase endometrium. While the abundance further decreases in the late secretory phase, the expression is maintained in the decidua of early pregnancy (Kitawaki et al., 2000a). Since leptin and OB-R are expressed in the preimplantation stage embryo (Antczak and Van Blerkom, 1997, 1999), leptin may participate in the implantation process and in early development by a local cross-talk between the embryo and endometrium.
The present study was performed using only the endometrial specimens that expressed OB-RL mRNA. We did not examine the difference in OB-R expression of patients with fertility and infertility. Since the incidence of OB-RL mRNA expression was 84% (42 of 50) in regularly cycling women (Kitawaki et al., 2000a), the remaining 16% might lack expression for an unknown reason. Alfer et al. reported a certain group of normally ovulating subfertile patients who lacked the OB-R and postulated that this lack might adversely affect endometrial receptivity (Alfer et al., 2000). Additionally, although the abundance of endometrial OB-RL mRNA expression is not related to the BMI within the normal range (Kitawaki et al., 2000a), the expression in the women who are excessively slender or obese remains to be examined.
In conclusion, the present study has demonstrated steroidal regulation of the functional OB-R expression in the human endometrium. The serum leptin level rises during the follicular phase and peaks during the luteal phase of the spontaneous menstrual cycle (Hardie et al., 1997; Shimizu et al., 1997; Mannucci et al., 1998; Messinis et al., 1998; Riad-Gabriel et al., 1998); however, the fluctuation is within double that of the basal level. It is interesting that progesterone stimulates leptin secretion in vivo (Messinis et al., 2000) on the one hand and suppresses OB-R expression in the endometrium on the other hand. The present study suggests that the sensitivity to the endometrium fluctuates to a great extent based on the change of abundance of OB-RL expression. Further studies are needed to clarify the physiological role of leptin in the human endometrium.
Notes
1 To whom correspondence should be addressed. E-mail: kitawaki{at}koto.kpu-m.ac.jp 
References
Ahima, R.S., Dushay, J., Flier, S.N. et al. (1997) Leptin accelerates the onset of puberty in normal female mice. J. Clin. Invest., 99, 391–395.[Web of Science][Medline]
Alfer, J., Müller-Schöttle, F., I. Classen-Linke et al. (2000) The endometrium as a novel target for leptin: differences in fertility and subfertility. Mol. Hum. Reprod., 6, 594–600.
Antczak, M. and 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.
Antczak, M. and Van Blerkom, J. (1999) Temporal and spatial aspects of fragmentation in early human embryos: possible effects on developmental competence and association with the differential elimination of regulatory proteins from polarized domains. Hum. Reprod., 14, 429–447.
Antczak, M., Van Blerkom, J. and Clark, A. (1997) A novel mechanism of vascular endothelial growth factor, leptin and transforming growth factor-ß2 sequestration in a subpopulation of human ovarian follicle cells. Hum. Reprod., 12, 2226–2234.
Barash, I.A., Cheung, C.C., Weigle, D.S. et al. (1996) Leptin is a metabolic signal to the reproductive system. Endocrinology, 137, 3144–3147.[Abstract]
Baumann, H., Morella, K.K., White, D.W. et al. (1996) The full-length leptin receptor has signaling capabilities of interleukin 6-type cytokine receptors. Proc. Natl Acad. Sci. USA, 93, 8374–8378.
Bjørbæk, C., Uotani, S., da Silva, B. et al. (1997) Divergent signaling capacities of the long and short isoforms of the leptin receptor. J. Biol. Chem., 272, 32686–32695.
Bouloumié, A., Drexler, H.C.A., Lafontan, M. et al. (1998) Leptin, the product of ob gene, promotes angiogenesis. Circ. Res., 83, 1059–1066.
Chehab, F.F., Lim, M.E. and Lu, R. (1996) Correction of the sterility defect in homozygous obese female mice by treatment with the human recombinant leptin. Nature Genet., 12, 318–320.[Web of Science][Medline]
Cioffi, J.A., Shafer, A.W., Zupancic, T.J. et al. (1996) Novel B219/OB receptor isoforms; possible role of leptin in hematopoiesis and reproduction. Nature Med., 2, 585–589.[Web of Science][Medline]
Cioffi, J.A., Van Blerkom, J., Antczak, M. et al. (1997) The expression of leptin and its receptors in pre-ovulatory human follicles. Mol. Hum. Reprod., 3, 467–472.
Hardie, L., Trayhurn, P., Abramovich, D. et al. (1997) Circulating leptin in women: a longitudinal study in the menstrual cycle and during pregnancy. Clin. Endocrinol. (Oxf.), 47, 101–106.[Medline]
Henson, M.C., Swan, K.F. and O'Neil, J.S. (1998) Expression of placental leptin and leptin receptor transcripts in early pregnancy and at term. Obstet. Gynecol., 92, 1020–1028.[Web of Science][Medline]
Karlsson, C., Lindell, K., Svensson, E. et al. (1997) Expression of functional leptin receptors in the human ovary. J. Clin. Endocrinol. Metab., 82, 4144–4148.
Kielar, D., Clark, J.S.C., Ciechanowicz, A. et al. (1998) Leptin receptor isoforms expressed in human adipose tissue. Metabolism, 47, 844–847.[Web of Science][Medline]
Kitawaki, J., Kusuki, I., Koshiba, H. et al. (1999a) Leptin directly stimulates aromatase activity in human luteinized granulosa cells. Mol. Hum. Reprod., 5, 708–713.
Kitawaki, J., Kusuki, I., Koshiba, H. et al. (1999b) Detection of aromatase cytochrome P450 in endometrial biopsy specimens as a diagnostic test for endometriosis. Fertil. Steril., 72, 1100–1106.[Web of Science][Medline]
Kitawaki, J., Koshiba, H., Ishihara H. et al. (2000a) Expression of leptin receptor in human endometrium and fluctuation during the menstrual cycle. J. Clin. Endocrinol. Metab., 85, 1946–1950.
Kitawaki, J., Koshiba, H., Ishihara H. et al. (2000b) Progesterone induction of 17ß-hydroxysteroid dehydrogenase type 2 during the secretory phase occurs in the endometrium of estrogen-dependent benign diseases but not in normal endometrium. J. Clin. Endocrinol. Metab., 85, 3292–3296.
Luoh, S.M., Di Marco, F., Levin, N. et al. (1997) Cloning and characterization of a human leptin receptor using a biologically active leptin immunoadhesion. J. Mol. Endocrinol., 18, 77–85.
Mannucci, E., Ognibene, A., Becorpi, A. et al. (1998) Relationship between leptin and oestrogens in healthy women. Eur. J. Endocrinol., 139, 198–201.[Abstract]
Masuzaki, H., Ogawa, Y., Sagawa, N. et al. (1997) Nonadipose tissue production of leptin: leptin as a novel placenta-derived hormone in humans. Nature Med., 3, 1029–1033.[Web of Science][Medline]
Matkovic, V., Ilich, J.Z., Skugor, M. et al. (1997) Leptin is inversely related to age at menarche in human females. J. Clin. Endocrinol. Metab., 82, 3239–3245.
Messinis, I.E., Milingos, S., Zikopoulos, K. et al. (1998) Leptin concentrations in the follicular phase of spontaneous cycles and cycles superovulated with follicle stimulating hormone. Hum. Reprod., 13, 1152–1156.
Messinis, I.E., Kariotis, I., Milingos, S. et al. (2000) Treatment of normal women with oestradiol plus progesterone prevents the decrease of leptin concentrations induced by ovariectomy. Hum. Reprod., 15, 2383–2387.
Noyes, R.W., Hertig, A.T. and Rock, J. (1950) Dating the endometrial biopsy. Fertil. Steril., 1, 3–25.
Riad-Gabriel, M.G., Jinagouda, S.D., Sharma, A. et al. (1998) Changes in plasma leptin during the menstrual cycle. Eur. J. Endocrinol., 139, 528–531.[Abstract]
Satyaswaroop, P.G., Bressler, R.S., de la Pena, M.M. et al. (1979) Isolation and culture of human endometrial glands. J. Clin. Endocrinol. Metab., 48, 639–641.
Señarís, R., Garcia-Caballero, T., Casabiell, X. et al. (1997) Synthesis of leptin in human placenta. Endocrinology, 138, 4501–4504.
Shimizu, H., Shimomura, Y., Nakanishi, Y. et al. (1997) Estrogen increases in vivo leptin production in rats and human subjects. J. Endocrinol., 154, 285–292.
Tartaglia, L.A., Dembski, M., Weng, X. et al. (1995) Identification and expression cloning of a leptin receptor, OB-R. Cell, 83, 1263–1271.[Web of Science][Medline]
Vaisse, C., Halaas, J., Horvath, C. et al. (1996) Leptin activation of Stat3 in the hypothalamus of wild-type and ob/ob mice but not db/db mice. Nature Genet., 14, 95–97.[Web of Science][Medline]
Zhang, Y., Proenca, R., Maffei, M. et al. (1994) Positional cloning of the mouse obese gene and its human homologue. Nature, 372, 425–432.[Medline]
Submitted on January 3, 2001; accepted on April 2, 2001.
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