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Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel
Address all correspondence and requests for reprints to: Dr. Y. Koch, Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail: y.koch{at}weizmann.ac.il
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Abstract |
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Introduction |
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The GT1 cell line, an immortalized hypothalamic neuronal cell line (23), has been extensively used for studies concerning the regulation of expression and secretion of GnRH-I. The goal of this study was to find out whether GT1 and other neuronal cell lines express the GnRH-II gene. Our results demonstrate that extracts of GT1, the immortalized mouse hypothalamic neuronal cells, contain two distinct immunoreactive peptides that correspond to the GnRH-I and GnRH-II isoforms, and that two of the seven human neuronal cell lines evaluated express messenger RNA (mRNA) encoding for GnRH-II and GnRH-I.
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Materials and Methods |
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Tissue processing for GnRH determination
Twenty-five confluent culture dishes (100 mm) of GT1–7, TE-671,
or LAN-1 cells were immersed in ice-cold 0.1 N HCl and
homogenized by a Polytron homogenizer (Brinkmann Instruments, Inc., Westbury, NY). After centrifugation (12,000 x
g, 30 min at 4 C) the supernatant was pumped onto columns of
Sep-Pak C18 cartridges (Waters Corp., Milford, MA), washed by 0.1% trifluoroacetic acid (TFA),
eluted by methanol, and evaporated by nitrogen. After reconstitution in
0.1% TFA (1 ml), the extracts were processed through reverse phase
(RP) HPLC using C18 columns and eluted using the
following conditions: eluent A, 0.1% TFA in water; and eluent B, 75%
CH3CN in 0.1% TFA. The gradient program
consisted of a linear gradient of 20–30% eluent B for 5 min at a flow
rate of 1 ml/min, followed by an isocratic elution of 30% eluent B for
35 min, and 100% of eluent B for an additional 20 min. All fractions
were evaporated to a volume of 0.1 ml and reconstituted with 0.1
M phosphate buffer (PB; pH 7.4) containing 0.1%
of bovine 
-globulin, and the concentrations of GnRH-I and GnRH-II
were determined by RIA using the appropriate antisera. The elution
positions of the synthetic peptides were determined by application of 1
µg GnRH-I and GnRH-II. After thorough washing, a blank run was
monitored by RIA to ensure that the column was not contaminated.
Radioiodination and RIA
Iodination of synthetic GnRH-I or GnRH-II was carried out using
the chloramine-T method (31). Free iodine was removed on a
Sep-Pak C18 cartridge (Waters Corp.), and the 125I-labeled peptides were
separated from the unlabeled peptides by HPLC using the elution program
described above. GnRH concentrations in samples of brain extracts were
determined by RIA as previously described (32).
Antibodies
The following antisera were used throughout this study. A
polyclonal antibody against GnRH-I, prepared and characterized in our
laboratory, was used for RIA (32). GnRH-II or salmon GnRH
did not displace any of the bound [125I]GnRH-I
even at a concentration that exceeded by 1000 times the GnRH-I
concentration needed for displacing 50% of the tracer (20 ng
vs. 20 pg). A monoclonal antibody against GnRH-I, provided
by Dr. H. F. Urbansky, was used at dilutions ranging from 1:4,000
to 1:10,000 for the immunofluorescence studies; the specificity of this
antibody (HU4H) was reported previously (33). Two
polyclonal antibodies against GnRH-II were used. One antibody, aCII6,
was provided by Dr. K. Okuzawa, and its specificity was previously
defined (5, 34). Additional specificity tests in our
laboratory demonstrated that GnRH-I did not displace any of the bound
[125I]GnRH-II even at a concentration that
exceeded 2,500 times the GnRH-II concentration needed to displace 50%
of the tracer (20 ng vs. 8 pg). We used dilutions ranging
from 1:4,000 to 1:10,000 of this antibody for the immunohistochemical
studies. The second antiserum, KLII-2, was prepared and characterized
in our laboratory. Specificity tests of this antibody have demonstrated
that GnRH-I did not displace any of the bound
[125I]GnRH-II, even at a concentration that
exceeded 1,000 times the GnRH-II concentration needed to displace 50%
of the tracer (30 ng vs. 30 pg). Salmon GnRH cross-reacted
with this antisera by 0.003% and with antibody aCII6 by 0.013%. We
used dilutions ranging from 1:4,000 to 1:10,000 of the GnRH-II
antibodies for the immunohistochemical studies.
Double fluorescence immunocytochemical analysis
GT1–7, TE-671, and LAN-1 cells were analyzed by double
fluorescence immunocytochemistry using fluorescence microscopy,
according to the procedure described by Kim et al.
(35). The neuronal cells were plated on round glass
coverslips (13 mm) coated with poly-L-lysine (15
µg/ml) in 24-well culture plates. Two days later the cells were fixed
by the addition of 4% paraformaldehyde and 4% sucrose in 0.1
M PB, pH 7.4 (30 min), washed (three times, 5 min
each time) with PB, and permeabilized for 3 min with 0.5% Triton
X-100. After washing (three times) the cells were incubated for 2
h at room temperature in a blocking medium (PBS containing 10% normal
goat serum, 2% BSA, 1% glycine, and 0.5% Triton X-100) to saturate
nonspecific binding sites for IgG. The primary antibodies were added
for 12–15 h at 4 C, and the cells were washed (three times, 5 min each
time) with 0.1 M PB. The cells were than
incubated for 2 h at room temperature with either fluorescein- or
rhodamine-conjugated secondary antibody (goat antimouse conjugated
to Cy2 and goat antirabbit conjugated to rhodamine,
Jackson Immuno-Research Laboratories, Inc., West
Grove, PA), or with both conjugated secondary antibodies. Fluorescence
was visualized by fluorescence microscopy using green and red filters
for GnRH-I and GnRH-II, respectively. To determine the specificity of
the signals we included several control groups in which the antibodies
were preabsorbed with excess (10–100 µg) GnRH-I or GnRH-II for
24 h. Additional controls were incubated without the first
antibody or with an antibody for vasopressin (INCSTAR Corp., Stillwater, MN).
RT-PCR and Southern analysis
Total RNA was extracted by using TRIzol RNA isolation reagent
(Molecular Research Center, Inc., Cincinnati, OH) based on
the acid guanidinium thiocyanate-phenol-chloroform extraction method
according to the manufacturer’s recommendations. We used RT-PCR
(36) to amplify the levels of endogenous GnRH-II and
GnRH-I mRNA that may be present in the human neuronal cell lines
samples. The expression of the ribosomal protein, S-14
(37), served as an internal control. Each reaction
contained four oligonucleotide primers, two for the GnRH isoform
(GnRH-I or GnRH-II) and two for the S-14 internal control.
Complementary DNA (cDNA) equivalent to 0.5 µg RNA was amplified by
PCR for 35 cycles; the annealing temperatures were 62 and 60 C for
GnRH-II and GnRH-I reactions, respectively. The final
MgCl2 concentration was 2.5 mM. The
PCR products were transferred onto a nylon membrane (Nytran 0.45,
Schleicher & Schuell, Inc., Dassel, Germany) by overnight
capillary blotting in 20x SSC (standard saline citrate) solution, and
the nylon was baked in a vacuum oven at 80 C for 2 h.
Prehybridization was performed in the presence of 6x SSC, 5x
Denhardt’s solution, 5 mM EDTA, and 0.2 mg/ml salmon sperm
DNA for 3 h at 60 or 64 C for GnRH-I or GnRH-II, respectively.
Overnight hybridizations were performed, sequentially on the same
membrane, in the presence of a 32P-labeled probe
specific to the GnRH-I, GnRH-II, or S-14 cDNA. Hybridizations were
performed at 64 C for the GnRH-II and S-14 probes and at 60 C for the
GnRH-I probe. The corresponding bands could be seen after exposure of
the membranes to PhosphorImager plates (445 SI, Molecular Dynamics, Inc., Jersey City, NJ). Gels were also exposed to
x-ray film (Fuji Photo Film Co., Ltd., Tokyo, Japan) for
2–16 h at -80 C and were developed in CURIX 60 processor (AGFA, Koln,
Germany).
Oligonucleotide primers
For the PCR reactions the following specific human GnRH-I,
GnRH-II, and S-14 oligonucleotide primers were used: GnRH-I,
5'-AGTACTCAACCTACTTCAAG-3' and 5'-CATTCAAAGCGTTGGGTTTCT-3',
corresponding to nucleotides 1134–1153 (sense) and 3746–3766
(antisense), respectively (38) (the predicted size of the
band is 248 bp); GnRH-II, 5'-CTGCAGCTGCCTGAAGGAG-3' and
5'-CTAAGGGCATTCTGGGGAT-3', corresponding to nucleotides 1312–1330
(sense) and 2232–2250 (antisense), respectively (7) (the
predicted size of the band is 197 bp); and S-14,
5'-GGCAGACCGAGATGAATCCTCA-3' and 5'-CAGGTCCAGGGGTCTTGGTCC-3',
corresponding to nucleotides 2941–2962 (sense) and 4166–4186
(antisense), respectively (39) (the predicted size of the
band is 143 bp). The oligonucleotide probes for hybridization were:
GnRH-I, 5'-CCAAGTCAGTAGAATAAGGCC-3', corresponding to nucleotides
2091–2111; GnRH-II,
5'-GCAGGAGGCCTC-GCCTGGAGCTGGCCATGGCTGCT-3'
corresponding to nucleotides 2098–2132; and S-14,
5'-ATATGCTGCTATGTTGGCTGC-3' corresponding to nucleotides
2965–2985.
DNA sequencing
The appropriate cDNA fragments of GnRH-I and GnRH-II from the
human medulloblastoma cell line, TE-671, were extracted from the gels
by using the QIAquick Gel Extraction Kit (QIAGEN, Hilden,
Germany) and subcloned into pGEM-T vector using the pGEM-T Easy Vector
System I (Promega Corp., Madison, WI). Nucleotide
sequencing of the specific PCR bands was obtained by automated direct
DNA sequencing, according to the manufacturer’s recommendations (model
377, PE Applied Biosystems, Perkin-Elmer Corp., Foster City, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Each PCR tube contained four
oligonucleotide primers, two for a GnRH isoform (GnRH-I or GnRH-II) and
two for the internal control (S14). Southern hybridizations were
performed sequentially on the same membrane using
32P- labeled oligonucleotide probes specific
to S14, GnRH-I, or GnRH-II. The RT-PCR and Southern hybridization
demonstrate that GnRH-I (Fig. 1B, upper panel) and GnRH-II
(Fig. 1B, middle panel) are expressed in the medulloblastoma
cell line, TE-671, and the neuroblastoma cell line, LAN-1. The other
human cell lines studied (HCN, A673, SK-N-MC, SHSY5Y, and Daoy
neuronal cells) did not express either GnRH-I or GnRH-II mRNA under
these experimental conditions. The ribosomal protein S14, which served
as an internal control, was expressed, as expected, in all cDNA
preparations (Fig. 1B, lower panel). The appropriate TE-671
cDNA fragments of GnRH-I and GnRH-II were isolated from the agarose gel
and subcloned into pGEM-T vector, and the nucleotide sequences were
determined using the T7 primer. These sequences were compared with
those in the GenBank database and were identical to the known sequences
of GnRH-I and GnRH-II (Fig. 1C).
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, open arrow) from GnRH-II
(Fig. 2, black arrow). The concentrations of
GnRH-I or GnRH-II in the eluate of TE-671 (Fig. 2A) and LAN-1 (Fig. 2B)
cell extracts were determined using specific RIA systems for GnRH-I or
GnRH-II. Figure 2 demonstrates that the elution profiles of the
immunoreactive neuropeptides that were extracted from TE-671 and LAN-1
cells were identical to those of the synthetic peptides. These results
demonstrate the existence of the two neuropeptides, GnRH-I and GnRH-II,
in TE-671 and LAN-1 cell lines. Further support for the translation of
the mRNA of these two neuropeptides came from immunofluorescence
staining for GnRH-I and GnRH-II. Figure 3
demonstrates GnRH-I and GnRH-II immunoreactivity in TE-671 (Fig. 3, A–D) and LAN-1 (Fig. 3, E–H) cells. GnRH-I monoclonal antibody was
reacted with goat antimouse Cy2 (green fluorescence), whereas GnRH-II
polyclonal antibody was reacted with goat antirabbit rhodamine (red
fluorescence). We could not observe heterogeneity among the cells
within a cell line. The cells were immunoreactive for both isoforms,
but different levels of intensity could be observed in the same cell
line. The immunocytochemistry study did not attempt to compare
quantitatively the two isoforms, but, rather, verified their presence
in the examined cells. Therefore, the differences in the amount of the
endogenous GnRH isoform, as demonstrated by HPLC and RIA, is not
reflected by the immunocytochemistry results (Fig. 3) due to different
exposure times and variance in the chromophore intensity and the film
sensitivity. Figure 3, M and N, demonstrates the specificity of the
GnRH staining. Thus, TE-671 cells reacted with antibody against
vasopressin followed by secondary antibodies did not result in any
staining. In addition, preabsorption of GnRH-I antibody with excess
synthetic GnRH-I abolished the immunoreactive staining, whereas
preabsorption with excess synthetic GnRH-II did not affect the signal
(data not shown). Likewise, a similar procedure carried out with the
antibody against GnRH-II exhibited the expected specificity of the
antibody.
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, open
arrows) from GnRH-II (Fig. 4, black arrows).
In this elution program dogfish GnRH eluted in fraction 35, whereas
salmon GnRH emerged only after the concentration of acetonitrile was
increased. The concentration of GnRH-I or GnRH-II in the eluate of the
cell line extracts was determined by RIA using specific antibodies to
either GnRH-I or GnRH-II. Figure 4 shows that the elution profiles of
the immunoreactive GnRH-I (Fig. 4A) and GnRH-II (Fig. 4B) that were
extracted from the GT1–7 cells were identical to those of the
synthetic peptides. Thus, these results demonstrate the existence of
GnRH-II, in addition to GnRH-I, in the GT1–7 cell line. However, the
total amount of GnRH-II was less than 1% that of GnRH-I. Double
immunofluorescence labeling for GT1–7 cells was performed as described
above. Indeed, immunoreactivity for both GnRH-I (Fig. 3
, I and K) and
GnRH-II (Fig. 3, J and L) was observed in this mouse hypothalamic
neuronal cell line.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The establishment of the mouse immortalized hypothalamic neuronal cell line, GT1 cells, by Mellon et al. (23) provided a powerful tool for studying the regulation of GnRH-I expression, secretion, and function (40, 41, 42, 43, 44). The recent evidence for the presence of an additional GnRH isoform in the mammalian hypothalamus (4, 5, 6, 8) led us to assume that GT1 cells may also produce an additional isoform of GnRH. As the GnRH-II gene has not been yet cloned in rodents, we used HPLC followed by specific RIA for the two isoforms as well as immunohistochemistry to demonstrate that the GT1–7 cell line also contains the GnRH-II isoform. The concentration ratio between the two isoforms in GT1–7 cells is approximately 100:1, whereas in the TE-671 cells the ratio is about 10:1 for GnRH-I and GnRH-II, respectively. Preliminary results have indicated that the two isoforms of GnRH are also present in the medium (data not shown). The coexpression of the two isoforms of GnRH by GT1–7 cells and the human cell lines could be attributed to their tumorigenic source and is not necessarily due to their normal repertoire of expression, although only two of the seven human neuronal cell lines that were studied were found to express GnRH. Indeed, it is of interest to note that Urbanski and co-workers (8) showed a largely dissimilar distribution pattern of cells containing either GnRH-I or GnRH-II mRNA in the macaque brain, and that the cells that contain GnRH-II mRNA exhibit some differences in their axonal projections compared with those that express the GnRH-I mRNA. More recently, Latimer and co-workers (45) reported that they did not find any colocalization of the two isoforms in the hypothalamic neurons of the rhesus macaque. Previous studies carried out in amphibia are inconsistent regarding the distribution of the two isoforms in the hypothalamus. Although Collin et al. (46) had demonstrated by immunostaining the coexistence of GnRH-I and GnRH-II in neurons of the septal-anterior preoptic area, the studies of Muske et al. (47) and D’Aniello et al. (48) suggested separate distribution. It is interesting to note that we have not found a cell line that expresses only one of the GnRH isoforms; all cell lines that were found to express GnRH-I also expressed the GnRH-II isoform. This observation may imply a relationship in the expression of these two isoforms. Indeed, it can be assumed that all known GnRH isoforms in the vertebrata have evolved from a single ancestral gene that underwent modifications and chromosomal segregation during evolution. Therefore, it is not unexpected to find more then one isoform of GnRH in a single neuron.
The results presented herein are restricted to the cell lines that were studied, and therefore, they do not necessarily reflect the endogenous expression of the GnRH isoforms in the brain. However, the distribution of the GnRH-I- and GnRH-II-expressing neurons in the mammalian hypothalamus should be further investigated to explore the possibility of the existence of neurons that express more than one GnRH isoform. Nevertheless the TE-671, LAN-1, as well as GT1–7 cell lines are useful in studying the regulation of GnRH-I and GnRH-II expression and secretion as well as for studies of the relationship between the two GnRH isoforms in the same cell. These cell lines can be used for transfection with GnRH-I or GnRH-II promoter constructs to investigate the differential regulation of their genes. Ideally, the host cell line should express the endogenous gene of interest, as regulatory substances that affect the endogenous promoter will also control the transfected constructs.
The conserved structure of the neuropeptide GnRH-II from the primitive fish to the human suggests that this neuropeptide possess vital bioactivities. However, the biological functions of GnRH-II are practically still unknown. In the bullfrog, application of exogenous GnRH-I has been reported to inhibit a specific voltage-dependent potassium current leading to depolarization of sympathetic neurons and induction of a late, slow excitatory postsynaptic potential (49). Later, it was found that GnRH-II is at least 1000 times more potent than GnRH-I in inducing this effect (50), and that indeed GnRH-II, but not GnRH-I, is present in extracts of sympathetic ganglions (51). These findings imply that GnRH-II is the endogenous transmitter that mediates the excitatory postsynaptic potential in amphibian. Studies in mammals have indicated a role for GnRH in the process of sexual differentiation of the brain (52). Other studies demonstrated the involvement of GnRH in the induction of sexual behavior. Thus, GnRH-I administered to the midbrain central gray has been demonstrated to facilitate sexual behavior (53, 54). However, the high doses of GnRH-I (micromolar range) that were needed to induce this phenomenon and the discovery of GnRH-II in the midbrain of mammals raise the possibility that GnRH-II is the physiological regulator of mating behavior. Critical examination of the relevant literature projects discrepancies between the reported biological potencies of various GnRH-I analogs in the pituitary gland and with their potencies in the brain. Thus, analogs or fragments of GnRH-I that are unable to activate the pituitary gland could induce sexual behavior, whereas other GnRH analogs that are potent secretagogues of gonadotropins were unable to induce mating behavior (55, 56). These observations suggest that the activities of GnRH in the brain are not mediated by the known GnRH-I receptor and imply that the behavioral effects of GnRH in the brain are mediated by an as yet unknown receptor. The recent cloning of a GnRH receptor in catfish (57) and two GnRH receptors from the brain and pituitary of goldfish (58) that bind GnRH-II at a higher affinity compared with the binding affinities to other GnRH isoforms suggest that additional GnRH receptor(s) may be present in the pituitary and brain of mammals.
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Acknowledgments |
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Footnotes |
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Received June 5, 2000.
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) or GnRH-II (
). The elution positions of synthetic GnRH-I (I)
and GnRH-II (II) are indicated by open and black
arrows, respectively. A broken line indicates
the acetonitrile gradient program. The amounts of GnRH-I and GnRH-II in
cell lysates calculated as picograms of peptide per milligram of
protein are: TE-671 cells, 21 and 2 pg for GnRH-I and GnRH-II,
respectively; and LAN-1 cells, 23 and 0.5 pg for GnRH-I and GnRH-II,
respectively.
