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BOR - Papers in Press, published online ahead of print September 15, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.032078
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BIOLOGY OF REPRODUCTION 72, 102–106 (2005)
DOI: 10.1095/biolreprod.104.032078
© 2005 by the Society for the Study of Reproduction, Inc.

Nitric Oxide Mediates Inhibitory Effect of Leptin on Insulin-Like Growth Factor I Augmentation of 17ß-Estradiol Production in Human Granulosa Cells1

He-Feng Huang3,4, Bo Wang4, Xiao-Fu Yang4, Qiong Luo4, and Jian-Zhong Sheng2,5

Department of Reproductive Endocrinology,4 Women's Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310006, China Department of Pharmacology and Therapeutics,5 Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study the authors investigated if the inhibitory effect of leptin in the ovary was mediated via nitric oxide (NO) using human granulosa cells (GCs). Human GCs were obtained from preovulatory follicles of women who underwent IVF. Reverse transcription–polymerase chain reaction (RT-PCR) demonstrated that human GCs expressed mRNA of leptin and mRNA of isoforms of leptin receptor, including one long form and two types of short forms. Exposure of human GCs to leptin at concentrations of 3–30 ng/ml for 60 min dose-dependently increased the fluorescence of 4,5-diaminofluorescein (DAF-2), an NO-sensitive dye. The effect of leptin on DAF-2 fluorescence was inhibited by pretreatment of human GCs with 100 µM nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase (NOS), indicating that the increase in DAF-2 fluorescence properly reflected the intracellular NO production. FSH (1 ng/ ml) and IGF-I (30 ng/ml) stimulated 17ß-estradiol (E2) production in human GCs, respectively. FSH plus IGF-I induced a further increase in E2 production. Leptin did not significantly alter basal or FSH-dependent E2 production, but it inhibited the effect of IGF-I on E2 production and the synergistic effect of IGF-I on FSH-stimulated E2 production. The inhibitory effect of leptin on IGF-I argumentation of E2 production was attenuated by pretreatment of human GCs with 100 µM L-NAME. In conclusion, leptin could induce NO production in human GCs. The inhibitory effect of leptin on IGF-I augmentation of E2 production in human GCs was attenuated by L-NAME, strongly suggesting that NO may mediate the action of leptin in human GCs.

estradiol, granulosa cells, leptin, leptin receptor, nitric oxide


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nitric oxide (NO) is a diffusible gas that has been implicated as a signaling molecule in a variety of cellular functions, including vasodilation, neurotransmission, and macrophage toxicity [1]. NO is synthesized via the oxidation of L-arginine to NO plus citrulline by one of three isoforms of NO synthase (NOS), including neural (nNOS), inducible (iNOS), and endothelial (eNOS). Inducible NOS and eNOS have been found to be expressed in human [2], rat [3], and mouse granulosa cells (GCs) [4]. Recently, many studies suggest that NO may play important roles in ovary, influencing processes such as ovulation, follicular development, oocyte maturation, apoptosis, and steroidogenesis [2, 3, 5–7].

Leptin, a product of the obesity (ob) gene [8], is a 16-kDa hormone that is best known as a regulator of food intake and energy expenditure via hypothalamic-mediated effect [9]. It is now appreciated that this hormone has many additional effects, often as a consequence of direct peripheral actions [10]. Adipocytes are the major source of leptin synthesis and secretion [10]. Leptin acts via transmembrane receptors (obR), which show structural similarity to those of the cytokine family [11]. There are different isoforms of leptin receptor existing in human and other species [12]. Both long and short forms of the leptin receptor were demonstrated in the human ovary [13]. There is accumulating evidence suggesting that leptin may play important roles in regulating functions of the reproductive system. A homozygous mutation in the leptin (ob) gene was responsible for the obesity syndrome and infertility in the obese (ob/ob) mouse [8]. Correction of leptin deficiency in ob/ob mice by peripheral injections of recombinant leptin activated the reproductive axis and restored fertility [14]. It has been demonstrated that leptin could increase NO production in pituitary [15] and serum [16]. Furthermore, leptin-induced expression of LHRH and LH secretion has been reported to be mediated via nitricoxidergic mechanisms [17]. These data suggest that NO might be a messenger coupled to leptin receptor. However, although both leptin and NO were demonstrated to affect the functions of ovary, it is still unclear whether NO is involved in the action of leptin in ovary.

In the present study, we examined the expression of leptin mRNA and leptin receptor mRNA in human GCs and carried out fluorometric analysis by use of 4,5-diaminofluorescein (DAF-2), an NO-sensitive fluorescent dye [18], to investigate the effect of leptin on NO production in human GCs. Leptin has been shown to have an inhibitory effect on IGF-I augmentation of steroidogenesis in human GCs [19]. For this reason we also examined the effect of leptin on IGF-I augmentation of 17ß-estradiol (E2) production in human GCs in the absence or the presence of nitro-L-arginine methyl ester (L-NAME), a specific NOS inhibitor [1], to determine whether this inhibitory effect of leptin was mediated via NO.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Human GC Culture

Human GCs were obtained from preovulatory follicles of women undergoing in vitro fertilization/embryo transfer (IVF-ET) in the program at Woman's Hospital, Zhejiang University School of Medicine, China. The GCs were a by product of the IVF-ET procedure. This study was approved by the Clinical Research Ethics Committee at the Zhejiang University School of Medicine, and informed consent was obtained from each patient. The infertility of the patients was due to tubal or male factors. The menstrual cycle, ovulation and endocrine function of the patients were normal. All women underwent a long-cycle ovarian stimulation protocol. Pituitary desensitization began from Day 7 after an increase in the basal body temperature in the previous menstrual cycle with daily subcutaneous administration of 0.1 mg GnRHa (Decapeptyl, Ferring, Germany). FSH 300 IU (Metrodin, Serono, Switzerland) was intramuscularly administered daily from the third day of the IVF-ET program and 150 IU daily from the fifth day. FSH was discontinued when the dominant follicle reached a diameter of 18 mm or at least three follicles reached an average diameter of 16 mm. After the final injection of FSH, 10 000 IU hCG (Serono, Switzerland) was intramuscularly administrated. Transvaginal oocyte retrieval was scheduled 34–36 h after hCG injection.

At the time of retrieval, aspirates from different follicles of each patient were pooled and centrifuged. The pellets were resuspended in Dulbecco modified Eagle medium (DMEM; Gibco, Grand Island, NY). After washing, Percoll gradient centrifugation (2500 rpm for 30 min) was used to collect GCs. The GCs were resuspended in DMEM containing 1 µg/ml insulin, 0.4% BSA, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 µg/ ml low-density lipoprotein (LDL), and 10% FBS. The GC viability and number were determined by the method of trypan blue exclusion. The range of GC viability was 60%–85%. From one patient, 2 to 8 x 106 GCs could be collected. GCs were incubated at a density of 40 000/well on a 96-well culture plate (approximately 2 x 105 cells/ml). The 96-well plates were pretreated with serum cellulose (1 µg/l; Sigma) to improve the adhesion and growth of cells [20]. The culture plates were kept in a humidified atmosphere containing 5% CO2 at 37°C. Each experiment was based on cultures of GCs derived from all preovulatory follicles from one patient. The experiment was performed at least three times with GCs from three different women.

Leptin mRNA and Leptin Receptor Isoform mRNA Expression

Total RNA from the GCs was extracted using the Trizol Reagent method according to the manufacturer's protocols (Sangon, China). The messenger RNA was reverse transcribed into cDNA, then amplified by PCR. Total RNA of 1 µg was taken to synthesize cDNA in a reaction volume of 10 µl. PCR was carried out in 50 µl reactions containing 2 µl of the cDNA product as template and 0.5 µmol/L of each primer. The primers were leptin (sense, 5'-CCTCACTGAATGCCTCAATG-3'; antisense, 5'-CCAGCTCTTGCTCAGATGAA-3', which yielded a 352 bp PCR product); isoforms of leptin receptors (each primer pair shared a common 5' primer: 5'-ATCCCCATTGAGAAGTACCAG-3'; antisense, 5'-GAAGTTGGCACATTGGGTTC-3', which yielded a 332 bp PCR product; 5'-AATAGTGGAGGGAGGGTCAG-3', which yielded a 425 bp product; 5'-TGTCCTGGAGAACTCTGATG-3', which yielded a 952 bp product, representing the full-length signal transducing isoform).

The thermal cycling protocol for leptin began with a denaturing step of 94°C for 5 min, then 35 cycles at 94°C for 45 sec, 54°C for 45 sec, 72°C for 45 sec, and finished at 72°C for 10 min. The thermal cycling protocol for the leptin receptor isoform began with a denaturing step of 95°C for 5 min, then 35 cycles of 94°C for 1 min, 56°C for 1 min, 72°C for 1 min, and finished at 72°C for 10 min. The amplified products were separated on a 1% agarose gel and visualized with ethidium bromide staining.

Measurement of NO Production in Human GCs

Intracellular NO was monitored with DAF-2, a fluorescence indicator of NO that emits fluorescence in response to a reaction with NO. DAF-2 is a weakly fluorescent compound that was recently developed to measure NO production [18]. DAF-2 is converted to triazolofluorescein (DAF-2-T) by NO via triazolo ring formation with NO concentration-dependent enhancement of fluorescence. The detection limit of NO has been reported to be as low as 5 nM. The DAF-2 has a visible excitation wavelength (excitation maximum ~495 nm, emission maximum ~515 nm) [18], which is much less damaging to the cells than UV excitation and does not interfere with autofluorescence from the tissue [18]. To measure intracellular NO, the human GCs were placed on coverslips and incubated with 5 µM 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-2-FM diacetate; Molecular Probe, Eugene, OR) in DMEM at room temperature for 30 min. After loading, cells were rinsed three times with DMEM, kept in the dark, and maintained at 37°C with a warming stage on a Nikon inverted microscope equipped with a SFX-2 micro fluorometer (Soamere Tech, Salt Lake City, UT). In some experiments, 100 µM L-NAME (Sigma, St. Louis, MO), an inhibitor of NOS, was added 20 min before loading with DAF-2-FM diacetate. NOS leptin-induced vasorelaxation was inhibited with 100 µM L-NAME [21, 22]. As a control, some experiments were performed using 100 µM nitro-D-arginine methyl ester (D-NAME; Sigma), an inactive isomer of L-NAME. Typically, fluorescence from two to five human GCs in a field was measured in one experiment. The experiment was repeatedly performed with GCs from different women. NO production was detected at an excitation wavelength of 488 nm and emission wavelength of 515 nm. During the experiments, cells were maintained in a chamber at 37°C containing HEPES buffer (135 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES, pH 7.4). Because the DAF-2 dye undergoes significant photobleaching, cells loaded with DAF-2 were briefly (5 sec) and repeatedly exposed with excitation light at 30-sec time intervals. For measurement of DAF-2 fluorescence, all solutions used in the present study contained 3 mM L-arginine (Sigma), except for the experiments using L-NAME. Fluorescence signals were acquired on a PC computer using Axonscope software and a DigiData 1200 AD/DA interface (Axon Instruments, Inc.).

Measurement of E2 in Human GCs

For measurement of E2 production, GC culture media were replaced by serum-free DMEM containing the desired agonists including leptin (Sigma), FSH (Sigma), and IGF-I (Gibco). GCs in the control group were incubated without agonists. In some experiments, 100 µM L-NAME or 100 µM D-NAME was added 20 min before addition of agonists. Culture was terminated at 24 h after addition of agonists, and the media were collected and frozen at –20°C pending the radioimmunoassays (RIAs) to measure the E2 level. RIAs were conducted according to the manufacturer's protocol (Depu, Tianjin, China). The level of sensitivity of the RIA is 1.4 pg/ml, and the interassay variation is 4.9% (n = 3); intraassay variation is 4.2% (n = 3).

Statistical Analyses

The data are expressed as mean ± SEM for triplicate culture wells from three or more experiments. Multiple comparisons among different groups were performed using one-way analysis of variance (ANOVA) for repeated measurements, and values from independent experiments were analyzed by Student t-test. Values were determined to be significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Culture and Calculation of Human GCs

Most human GCs were attached to the bottom of culture plates after incubation for 48 h. After treatment of human GCs with FSH, IGF-1, and leptin for 24 h, the cellular morphology did not show drastic change when observed directly through a phase contrast microscope. The number of human GCs underwent no significant change after digestion with trypsin. Leptin, FSH, and IGF-I did not affect the number of cultured human GCs.

Leptin and Leptin Receptor Isoform mRNA Expression in Human GCs

We examined the expression of leptin and leptin receptor isoforms in human GCs. Human GC expressed leptin mRNA (353 bp; Fig. 1A, lane 1) and mRNA of isoforms of leptin receptor, including one long form (952 bp) and two types of short forms (332 and 425 bp; Fig. 1B).



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  		FIG. 1. A) Leptin mRNA expression in human GC. M, Marker. Numbers indicate the following: 1, GCs; 2, lipocytes, used as a positive control; 3, negative control. Complementary DNA for the leptin was amplified by RT-PCR. The amplified bands (352 bp) were detected on an ethidium bromide-stained agarose gel. B) Leptin receptor isoforms of mRNA expression in human GCs. Complementary DNA for the long form of the human leptin receptor and two short isoforms were amplified by RT-PCR. M, Marker. Numbers indicate the following: 1, short receptor I; 3, short receptor II; 5, long receptor; 2, 4, and 6, negative control; 7, ß-actin mRNA (224 bp) as control for expression quality and procedural variables. The results shown in this figure are representative of data observed in two additional experiments

Effects of Leptin on NO Production in Human GCs

After exposure of human GCs to leptin at concentrations of 3–30 ng/ml for 5–10 min, an increase in DAF-2 fluorescence was detected (Fig. 2A). After 50–60 min, the net increment in DAF-2 fluorescence was up 20%–40% of the basal level (Fig. 2, A and B). The effect of leptin on DAF-2 fluorescence in human GCs was inhibited by pretreatment of human GCs with 100 µM L-NAME (Fig. 2, A and B), but not by pretreatment of human GCs with 100 µM D-NAME (Fig. 2B). The results indicated that the increase in DAF-2 fluorescence properly reflected the intracellular NO production in our hands. In human GCs, leptin induced a dose-dependent and time-dependent NO production (Fig. 2, A and B).



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  		FIG. 2. A) Effect of leptin on NO production. Fluorescence response of DAF-2 to leptin at 3–30 ng/ml in human GCs. B) Net increment of relative DAF-2 fluorescence at 60 min after administration of leptin at different concentrations in the presence or absence of 100 µM L-NAME or 100 µM D-NAME in human GCs. Data represent the mean ± SEM. n, number of experiments. **P < 0.01 vs. corresponding values in the group without L-NAME

Effect of Leptin on E2 Production in Human GCs

FSH (1 ng/ml) and IGF-I (30 ng/ml) alone each stimulated E2 production in human GCs (Fig. 3). FSH plus IGF-I induced a further increase in E2 production. Leptin at 10 ng/ml did not significantly alter basal or FSH-dependent E2 production, but it attenuated the effect of IGF-I on E2 production and the synergistic effect of IGF-I on FSH-stimulated E2 production (Fig. 3). The inhibitory effect of leptin on IGF-I argumentation of E2 production was significantly attenuated by pretreatment of human GCs with 100 µM L-NAME, but not by pretreatment with 100 µM D-NAME (Fig. 3). The results suggested that NO might mediate this inhibitory action of leptin in human GCs.



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  		FIG. 3. The effect of leptin on E2 production stimulated by FSH, IGF-I, or FSH plus IGF-I in human GCs. GCs (2 x 108/L) were incubated with FSH (1 ng/ml), or IGF-1 (30 ng/ml), or FSH (1 ng/ml) plus IGF-1 (30 ng/ ml) in the presence or absence of leptin (10 ng/ml). Controls were incubated without FSH and IGF-I. All cultures were terminated at 24 h, and the E2 level in media was measured by RIA. Data represent the mean ± SEM from three independent experiments. a, P < 0.01 vs. corresponding value in the group of FSH. b, P < 0.01 vs. corresponding value in the group of IGF-I. c and d, P < 0.05 or P < 0.01 vs. corresponding values in the group without leptin. e, P < 0.01 vs. corresponding values in the group with L-NAME


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we found that leptin dose- and time-dependently induced NO production in human GCs. Leptin inhibited IGF-I augmentation of E2 production in human GCs, and this inhibitory action of leptin was attenuated by L-NAME, an inhibitor of NOS. Our results showed for the first time that NO mediated the action of leptin in human GCs.

It has been shown that mRNA coding for leptin receptors was expressed in human ovary, including GCs [13, 23, 24]. The present study demonstrated the expression of leptin mRNA in human GCs. At the same time, our data also confirmed the expression of one long form and two alternatively spliced short forms of the leptin receptor in human GCs, sharing a similar result with other researchers [13, 23, 24]. The expression of both leptin and leptin receptor in human GCs suggested that leptin might regulate the physiological functions of GCs by autocrine and/or paracrine.

DAF-2 is a highly sensitive fluorescent probe for the real-time detection of NO. The detection limit of NO has been reported to be as low as 5 nM. Many groups have examined NO production in endothelial cells using DAF-2 as a probe. However, no previous studies have directly shown NO production in GCs. In the present study we examined intracellular NO production induced by leptin using the DAF-2 fluorescent method. The study showed the direct recordings of the intracellular NO production in GCs.

NO interacts with soluble guanylate cyclase to evoke a surprisingly large array of physiological responses [25]. The widespread implications of NO have become even more pronounced with the realization that NO can also interact with molecular oxygen and superoxide radicals to produce reactive nitrogen species, which can then modify proteins, lipids, and nucleic acids [26]. Three types of NOS, including nNOS, iNOS, and eNOS, have been identified. Expression of iNOS and eNOS was demonstrated in GCs of human and other species [2–4]. It is noteworthy that eNOS and iNOS activity and production change during the cycle in the rat ovary [27]. Cyclic changes in the expression of NOS subtypes in ovary lend support to the role of NO in directly affecting the ovarian function. An increase in NOS activity in the ovary corresponding to the time of the preovulatory surge suggests that NO may be assisting the process of ovulation [27]. It has been shown that NO has an inhibitory effect on E2 production in GCs [28]. NO plays important roles in ovulation, follicular development, oocyte maturation, and apoptosis in ovary [29, 30]. Jablonka-Shariff and Olson[27] reported a 5-fold higher concentration of E2 in eNOS knockout mice compared with wild type mice, whereas no change in progesterone concentration was noticed [27]. On the other hand, nNOS was also shown to be important in regulating reproductive functions, because nNOS knockout in mice resulted in hypogonadism and infertility [31]. There is evidence suggesting that leptin increased NO production. Intravenous administration of leptin to rats increased serum NO concentration, an effect not observed in obese (fa/fa) rats [32]. It was subsequently found that preincubation of endothelial cells with leptin enhanced NO production as measured using DAF-2 and measurement of nitrate and nitrite concentration [33]. In the present study we observed that leptin dose- and time-dependently increased NO production in human GCs. NO levels in human GCs began to increase 5–10 min after administration of leptin and gradually increased to a high level in 60 min. Because of the limitation of one excitation wavelength, it is difficult to calculate the concentration of NO in the present study. The results not only confirmed that leptin could stimulate NO production, but also showed direct evidence for the first time that leptin increased NO levels in GCs.

In the present study, leptin was shown to attenuate IGF-I augmentation of E2 synthesis in human GCs. The data were consistent with the results of previous studies [19, 34]. The mechanisms involved in the inhibitory effect of leptin on E2 production stimulated by IGF-I are unclear. Both leptin and NO were found to have an inhibitory effect on the activity of aromatase, an enzyme in the synthesis of E2 [28, 34]. In the present study, we observed that the inhibitory effect of leptin on IGF-I augmentation of E2 production was attenuated by NOS inhibitor, L-NAME, suggesting that NO may be involved in this inhibitory action of leptin in human GCs. It is possible that NO induced by leptin might inhibit the activity of aromatase, resulting in an attenuated E2 production stimulated by IGF-I. Additional studies are needed to clarify this possibility.

In conclusion, we have demonstrated that leptin induced NO production in human GCs. The inhibitory effect of leptin on IGF-I augmentation of E2 production in human GCs was attenuated by L-NAME, strongly suggesting that NO may mediate the action of leptin in human GCs.


   FOOTNOTES
 
1 Supported by Zhejiang Natural Science Foundation of China and the Pao Yu-Kong and Pao Zhao-long Scholarship. Back

2 Correspondence: Jian-Zhong Sheng, Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, AB T2N 4N1, Canada. FAX: 403 270 2211; jzsheng{at}ucalgary.ca Back

3 Correspondence: He-Feng Huang, Department of Reproductive Endocrinology, Women's Hospital, Zhejiang University School of Medicine, 2 Xue Shi Road, Hangzhou, Zhejiang 310006, China. FAX: 86 571 8721 8744; huanghefg{at}hotmail.com Back

Received: 13 May 2004.

First decision: 8 June 2004.

Accepted: 23 August 2004.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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A G Ricci, M P Di Yorio, and A G Faletti
Inhibitory effect of leptin on the rat ovary during the ovulatory process.
Reproduction, November 1, 2006; 132(5): 771 - 780.
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