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Original Studies |
Departments of Human Anatomy and Histology (T.B., G.F., M.G., G.C.B., G.B.V.) and Clinical Physiopathology, Endocrinology (S.G., R.M., E.M., M.L., C.R., M.S.) and Andrology (M.M.) Units, Institute of Internal Medicine (F.M.), University of Florence, 50134 Florence, Italy
Address all correspondence and requests for reprints to: Gabriella B. Vannelli, M.D., Department of Human Anatomy and Histology, University of Florence, Violex Morgagni 85, 50134 Florence, Italy. E-mail: vannelli{at}cesit1.unifi.it
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Abstract |
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- and
ß-subtypes of the estrogen receptor was also demonstrated by RT-PCR
and Western blot analysis. When the cells were stimulated with
increasing concentrations of 17ß-estradiol in the presence of a fixed
concentration of progesterone (10-7 mol/L), the
combination of the two steroids induced a 3- to 4-fold increase in GnRH
secretion. This stimulatory effect was completely blunted by tamoxifen.
Neither 17ß-estradiol nor progesterone was effective when tested
separately. Treatment with increasing concentrations of the odorant,
l-carvone, induced a time- and dose-dependent dramatic
increase in GnRH protein release (1000-fold increase) and gene
expression. Repeated application of the stimulus resulted in a
progressive lower responsiveness of the cells. To our knowledge, this
is the first time that primary cell cultures from human fetal olfactory
neuroepithelium have been shown to express and release GnRH. Our
results also demonstrate that these cultures, which are sensitive to
sex steroids and odorants, can be useful models in the study of the
complex array of regulatory factors that finely tune GnRH secretion in
humans. |
Introduction |
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Kallmann’s syndrome is caused by a defect in the migratory pathway for GnRH-expressing neurons as a result of Kal protein deficiency (6, 7). The disorder is clinically characterized by the association of an inability to smell (anosmia) with a defect in GnRH secretion (hypogonadotropic hypogonadism) (8).
A relationship between the olfactory and the reproductive system has been demonstrated in previous studies, and mammalian olfactory responsiveness to pheromones has been documented (9, 10, 11). It has also been recently demonstrated that pheromones may regulate ovulation in humans (12).
In a previous work, we isolated, characterized, and cloned neuroblast long term cell lines from olfactory epithelium of 8- to 12-week-old human fetuses (13). These cells showed typical neuronal and olfactory properties and were able to respond to odors. We now demonstrate that one such clone, i.e. FNC-B4, not only produces GnRH and responds to sex steroids but is sensitive to odorants in terms of GnRH production. These data represent the first evidence of a direct link between the olfactory and the neuroendocrine systems in humans.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human fetal olfactory epithelium specimens were obtained from seven 8- to 12-week-old fetuses after spontaneous or therapeutic abortion. Legal abortions were performed in authorized hospitals, and certificates of consent were obtained. The study protocols were approved by the university ethical committee. The olfactory epithelium was fixed in buffered formalin, embedded in paraffin, and used for morphological detail and immunohistochemical techniques.
Cell cultures
Primary olfactory neuroblast long term cell cultures of FNC-B4 were isolated, cloned, and propagated in vitro from the human fetal olfactory epithelium, as previously described (13). Although the cells have the some properties as those of immature neurons, they can differentiate and express both neuronal proteins and olfactory genes (13). This suggests that these cultures originate from the neuroblastic stem cell compartment and are composed at all times of cells at different stages of maturation.
These cells, grown as a monolayer, are nontumorigenic and have a normal human karyotype. FNC-B4 cells were cultured in Coon’s modified F-12 medium supplemented with 10% FBS in a 5% CO2 atmosphere at 37 C, as previously described (13, 14).
Antisera
The following polyclonal (PA) and monoclonal (MA) antibodies were used for immunocytochemistry at the indicated dilutions: rabbit PA to human GnRH (INCSTAR Corp., Stillwater, MN), dilution 1:1000; mouse MA to human estrogen receptor (ER), clone ER1D5, dilution 1:25 (BioGenex Laboratories, Inc., San Ramon, CA); and mouse MA to human progesterone (Pg) receptor (PR), clone PGR1A6, dilution 1:40 (BioGenex Laboratories, Inc.).
The MA H222 was used for Western blot analysis of the ER. It was a
gift from Prof. Geoffrey Greene (The Ben May Institute for Cancer
Research, University of Chicago, Chicago, IL).
Chemicals
[125I]GnRH (2200 Ci/mmol) and
[-35S-thio]UTP (1300 mCi/mmol) were obtained
from NEN Life Science Products (Milan, Italy). GnRH RIA
kit was obtained from Buhlmann Laboratories AG (Allschwil,
Switzerland). Odorant compounds were purchased from Aldrich Chimica
(Milan, Italy). Synthetic GnRH was obtained from INCSTAR Corp. Tamoxifen and sex steroids were purchased from Sigma Chemical Co. (St. Louis, MO).
3,4,3',4'-Tetra-aminodiphenylhydrochloride (diaminobenzidine) was
obtained from BDH Chemical Ltd. (Poole, UK). 3-Amino-9-ethyl-carbazole
was purchased from Sigma Chemical Co. Universal
immunoperoxidase staining kits were obtained from Vector Laboratories, Inc. (Burlingame, CA).
Immunohistochemistry
Immunohistochemical studies were carried out on deparaffinized and rehydrated sections or cultured cells fixed in 3.7% paraformaldehyde for 15 min, as previously described (13, 15). The specimens were subsequently exposed to 0.3% hydrogen peroxide-methanol solution to quench endogenous peroxidase activity. Slides were rinsed in tap water, then immersed in 10 mmol/L citrate buffer (pH 6) and microwaved for 40 min at 350 watts to enhance antigen exposure. The primary antibody, appropriately diluted in phosphate-buffered saline (PBS), was added to the slides and incubated overnight at 4 C. Sections were rinsed in PBS, incubated with biotinylated secondary antibodies, and finally incubated with streptavidin-biotin peroxidase complex (LSAB kit, DAKO Corp., Carpinteria, CA). The development reaction of the product was performed using diaminobenzidine tetrahydrochloride or 3-amino-9-ethyl-carbazole as chromogen. Slides were washed in running tap water and counterstained with hematoxylin followed by dehydration and coverslip mounting. Controls were performed by processing slides lacking the primary antibodies, by staining with the corresponding nonimmune serum, or by preincubating the primary antibodies with the synthetic antigen. The slides were evaluated and photographed using a Nikon Microphone-FX microscope (Nikon, Tokyo, Japan).
SDS-PAGE and Western blot analysis
FNC-B4 and MCF-7 cells grown to confluence were scraped in PBS, centrifuged, and resuspended in lysis buffer [20 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 0.25% Nonidet P-40, 1 mmol/L Na3VO4, 1 mmol/L phenylmethylsulfonylfluoride, and 1 mmol/L ethyleneglycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid].
Human uterus samples, obtained at surgery, were minced with sharp scissors, suspended in lysis buffer, and homogenized (Teflon-glass). The homogenates were centrifuged at 1500 rpm for 10 min at 4 C, and supernatants corresponding to total lysates were subjected to protein measurement (Bio-Rad Laboratories, Inc., Hercules, CA).
After protein measurement, aliquots containing 50 µg proteins
were diluted in reducing 2x SB [Laemmli’s sample buffer = 62.5
mmol/L Tris (pH 6.8), 10% glycerol, 10% SDS, 2.5% pyronin, and 100
mmol/L dithiothreitol] and loaded onto 8% SDS-PAGE. After SDS-PAGE,
proteins were transferred to nitrocellulose membranes. Membranes were
blocked overnight at 4 C in 5% BSA-TTBS (0.1% Tween-20, 20 mmol/L
Tris, and 150 mmol/L NaCl), washed in TTBS, and incubated for 2 h
with H222 antibody (16) followed by peroxidase-conjugated secondary
IgG (Sigma Chemical Co.). Finally, reacted proteins were
revealed using an enhanced chemiluminescence system (BM, Roche Molecular Biochemicals, Milan, Italy).
cAMP determination
Cells were plated in 24-well plates and grown to subconfluence in Coon’s modified Ham’s F-12 plus 10% FCS. After removing the medium, cells were incubated for 30 min at 37 C in assay buffer (0.025 mol/L Tris-acetate, 0.25 mol/L sucrose, 0.5% BSA, 5 mmol/L glucose, and 0.6 mmol/L 3-isobutyl-1-methylxanthine) containing increasing concentrations of l-carvone or the solvent. To stop the reaction equal volumes of cold (-20 C) absolute ethanol were added, and samples were stored at -20 C. After vacuum drying, samples were reconstituted with 0.05 mol/L sodium acetate, pH 6.2, and cAMP was determined by RIA as previously described (13).
Northern blot analysis and RT-PCR
Total ribonucleic acid (RNA) from FNC-B4 cells, from the
prostate and mammary cancer cell lines, DU-145 and MCF-7, and from
human testis was isolated as previously described (17) and quantified
by spectrofluorometric analysis at 260 nm. For Northern analysis, the
samples were fractionated in a 1.2% agarose gel containing 8%
formaldehyde; the RNA was then transferred onto nylon membranes
(Hybond-N, Amersham Pharmacia Biotech, Milan, Italy) and
baked at 80 C for 2 h. Membranes were prehybridized for 1 h
and hybridized overnight at 65 C with Church and Gilbert solution
containing 10 mg/mL BSA, 7% SDS, 0.25 mol/L PBS, 1 mmol/L
ethylenediaminetetraacetic acid (EDTA; pH 8), and 0.2 mg/mL
hot-denatured sonicated herring sperm DNA. The probe for the detection
of GnRH messenger RNA was derived from RT-PCR amplification of the
placental total RNA, using the Superscript One-Step RT-PCR System
(Life Technologies, Inc., Gaithersburg, MD). The sequence
of the sense primer was 5'-GCA AGC CAG CAA GTG TCT C-3' (904–924 bp);
the sequence of the antisense primer was 5'-GCA ACT TGG TGT AAG GAT-3'
(1478–1461 bp). The amplified DNA was purified using the Quiaquick PCR
Purification Kit (Qiagen S.A., Courtaboeuf, France). The probe
was labeled with 5'-[-32P]deoxy-CTP using a
random priming kit (Roche Molecular Biochemicals, Milan,
Italy) and chromatographed (Nu-Clean D25 Disposable Spun Columns,
IBI, New Haven, CT) before use. Washing was performed once
at 65 C in 5% SDS, 0.04 mol/L PBS, 1 mmol/L EDTA (pH 8), and 5 mg/mL
BSA and once at 65 C in 1% SDS, 0.04 mol/L PBS, and 1 mmol/L EDTA, pH
8. Hybridized nylon membranes were submitted to autoradiography using
Hyperfilm-MP (Amersham Pharmacia Biotech Italy, Milan,
Italy) and Kodak X-Omatic Regular intensifying screen at
-80 C for 15 days.
RT-PCR for the - and ß-subtypes of the ER was performed on 1 µg
total RNA, using the Superscript One Step RT-PCR System (Life Technologies, Inc.) according to the manufacturer’s
instructions. The ER - and ß-specific primers were, respectively:
ER-F (sense primer), 5'-GAC CCT CCA CAC CAA AGC ATC TG-3'; ER-R
(antisense primer), 5'-CTC CTC TTC GGT CTT TTC GTA TCC-3'; ERß-F
(sense primer) 5'-TAG TGG TCC ATC GCC AGT TAT-3'; and finally, ERß-R
(antisense primer) 5'-GGG AGC CAC ACT TCA CCA T-3'. The quality of the
total RNA used was assessed by performing additional RT-PCR using
primers specific for the glyceraldehyde-3-phosphate dehydrogenase
gene.
GnRH RIA
Immunoreactive GnRH was extracted from the conditioned media of the FNC-B4 cells with chilled absolute ethanol (-20 C), evaporated to dryness, and subjected to RIA using a commercial kit. The recovery of unlabeled GnRH added to the media, before extraction, was 81%. A close parallelism with the standard curve was found when serial dilution of the FNC-B4 cell-conditioned media or extracts of the rat hypothalamus were subjected to RIA. The odorants tested, including l-carvone, do not cross-react with the antibody employed in the RIA.
High performance liquid chromatography (HPLC)
Reverse phase HPLC was performed on a BIO-SIL column (250 x 4 mm; Bio-Rad Laboratories, Inc.) as previously described (18). Briefly, after extraction, samples or standards were injected and eluted with a linear gradient of acetonitrile in 0.1% trifluoroacetic acid with a flow rate of 1 mL/min. The acetonitrile gradient was from 10–70% for 40 min. Fractions (1 mL) were collected, evaporated, and subjected to GnRH RIA. The reproducibility of the elution pattern of each HPLC run was verified by adding about 3000 cpm labeled GnRH.
Statistical analysis
Data are expressed as the mean ± SE of the percent stimulation over the basal level (100%). The significance of the difference was calculated by Wilcoxon rank sum test, with P < 0.05 considered statistically significant. The computer program ALLFIT was employed to analyze the sigmoidal dose-response curves and the relative medium effective concentration (EC50) (19).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). GnRH-LI was also present in the
FNC-B4 cells (Fig. 1B) The specificity of the immunohistochemical
staining was demonstrated in the olfactory cells by the complete
absence of any staining after preabsorption of the anti-GnRH antibody
with synthetic GnRH (100 µg/mL; Fig. 1, C and D). FNC-B4 cells
secreted GnRH-LI in the spent media, as a function of time, reaching a
maximum after 24 h (Fig. 2). HPLC
analysis indicated that the GnRH-LI in the extracted medium from the
FNC-B4 cells corresponded to authentic GnRH, as it eluted in the same
fraction as the synthetic peptide GnRH (Fig. 2, inset).
Specific transcripts for GnRH were expressed in the olfactory neurons
of the FNC-B4 cells as well as in the prostatic cancer cell line
DU-145, which was used as a positive control (20) (see below).
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, A and B) and the FNC-B4 olfactory neurons (Fig. 3, C and D) were
positive for ER and PR. In the section incubated with nonimmune serum,
labeling was absent (Fig. 3, E and F). To further investigate the
nature of the ER identified in FNC-B4 cells, we studied the gene and
protein expression of the - and ß-subtypes of the ER using
specific primers and a MA (H222) that recognizes both subtypes of
the receptor. Western blot analysis (Fig. 4A) and RT-PCR (Fig. 4B) indicated that
indeed both ER and ERß were expressed by FNC-B4 cells. The effects
of sex steroid on the GnRH secretion were therefore investigated. We
incubated the FNC-B4 cells for 24 h in the presence of increasing
concentrations
(10-11–10-6 mol/L) of
Pg, estradiol (17ßE2) and a combination of the
two. Individually, neither 17ßE2 nor Pg
significantly affected GnRH secretion (data not shown; n = 3).
Conversely, in the presence of fixed concentration of Pg (100 nmol/L),
17ßE2 induced a dose-dependent 3- to 4-fold
increase in GnRH secretion, with an EC50 of 6.3
pmol/L (n = 7; Fig. 5). The
stimulatory effect of 17ßE2 started to be
significant at 10-11 mol/L (P <
0.05) and was highly significant at higher concentrations of
17ßE2
(10-10–10-8 mol/L;
P < 0.001). The specificity of the estrogen effect on
GnRH secretion was further validated by the evidence that tamoxifen
completely antagonized the stimulatory activity of
17ßE2 (n = 3; Fig. 5).
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) with a
concomitant increase in cAMP accumulation (Fig. 6B). The stimulatory
effect of l-carvone (10-11 mol/L) on
GnRH gene (Fig. 7A) and protein (Fig. 7B)
expression was time dependent, reaching a maximum after 24 h of
treatment. However, iterative stimulation with l-carvone
(10-11 mol/L, one pulse every 12 h)
resulted in a progressive reduction in FNC-B4 responsiveness,
indicating that desensitization is occurring (Fig. 7C).
l-Carvone-induced GnRH secretion was just observed in FNC-B4
cells and not in the prostatic cancer cell line DU-145, although it
expresses a specific transcript for GnRH (Fig. 7A) and secretes GnRH in
the culture medium (not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The presence of GnRH immunoreactivity in the nasal epithelia of both normal human fetuses and those with Kallmann’s syndrome at the midtrimester of gestation has been demonstrated in previous studies (23, 24). Our results show that immunopositivity for GnRH is present in the olfactory mucosa of 8- to 12-week-old human fetuses. This demonstrates the presence of GnRH-immunopositive cells in human olfactory mucosa early in development. Immunoreactivity was also present in the FNC-B4 cells that had been isolated and cloned from olfactory mucosa at the same stage of fetal maturation. The expression of the GnRH gene and the secretion of the GnRH protein in the spent medium by the FNC-B4 cells lend support to a neuroendocrine identity of these olfactory neurons.
Although several clinical and animal experimental models have shown
that gonadal steroids are among the endocrine factors that control GnRH
secretion (25, 26), there is no evidence of the presence of the steroid
receptors in prepubertal (27) or adult animals (28). In our study, the
olfactory mucosa of the 8- to 12-week-old fetuses was immunopositive
for both GnRH and sex steroid receptors. The FNC-B4 cell line is also
immunopositive for sex steroid receptors. In particular, FNC-B4 cells
express specific genes and proteins for both subtypes of ER,
i.e. the - and ß-subtypes. Therefore, we investigated
the role of sex steroids in GnRH secretion in these cells. FNC-B4 cells
were insensitive to increasing concentrations of either
17ßE2 or Pg. However, when the two steroids
were combined, a sustained increase in GnRH release was observed. Note
that the sex steroid concentrations eliciting GnRH secretion in our
in vitro system are very similar to those found in human
blood at the time of ovulation, when the physiological surge in GnRH
secretion takes place.
In mammals, the olfactory system is responsive to olfactory cues defined as pheromones; these substances are secreted by different individuals within a species. In most instances, these chemical signals provide information on reproductive status and gender and may elicit certain types of sexual behavior. They can also cause profound neuroendocrine changes (29, 30, 31). A recent report demonstrated the existence of human pheromones and hypothesized that humans have the potential to communicate pheromonally (12). In particular, the timing of ovulation can be manipulated by axillary compounds collected from the armpits of women during the different phases of their menstrual cycles.
As we had previously reported that olfactory neurons in culture were responsive to aromatic chemicals with intracellular cAMP accumulation (13), we also tested the effect of some different odorants (13, 32) on GnRH secretion. Only l-carvone induced a remarkable increase in GnRH secretion and gene expression. The selective response to l-carvone, as expressed by both GnRH protein release and gene expression, probably reflects some distinct odor specificity of the clone. It also confirms previous data, which demonstrated that a single olfactory cell expresses only a small subset of distinct receptors and responds to a restricted number of odorants (33, 34, 35).
This is the first report on GnRH production in human olfactory cells exposed to a selective odorant. The regulation of GnRH expression by sex steroids and odorants can help elucidate the complex neuroendocrine network that controls human reproductive behavior.
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Acknowledgments |
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H222. We also thank Prof.
Felice Petraglia (Department of Surgical Sciences, Division of
Obstetrics and Gynecology, University of Udine, Udine, Italy)
for his comments. We thank Dr. Clara Crescioli (Department of Clinical
Physiopathology, Endocrinology Unit, University of Florence, Florence,
Italy) and Dr. Roberto Zonefrati (Institute of Internal Medicine and
Immunoallergology, University of Florence) for their technical
assistance. |
Footnotes |
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Received February 22, 1999.
Revised August 3, 1999.
Accepted August 9, 1999.
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