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Endocrinology Vol. 142, No. 2 830-837
Copyright © 2001 by The Endocrine Society

ARTICLES

Two Isoforms of Gonadotropin-Releasing Hormone Are Coexpressed in Neuronal Cell Lines1

Alon Chen, Dror Yahalom, Orly Laskar-Levy, Shai Rahimipour, Nurit Ben-Aroya and Yitzhak Koch

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GnRH-I serves as the neuropeptide that regulates mammalian reproduction. Recently, several groups have identified in the brain of rodents, monkeys, and humans a second isoform of GnRH (GnRH-II) whose structure is 70% identical to that of GnRH-I. In this study we demonstrate for the first time human and mouse neuronal cell lines that express both GnRH-I and GnRH-II. Following the screening of several human neuronal cell lines by RT-PCR and Southern hybridization, we demonstrated that two cell lines, TE-671 medulloblastoma and LAN-1 neuroblastoma cells, coexpress messenger RNA encoding the two isoforms of GnRH. Nucleotide sequencing indicated that the complementary DNA fragments are identical to those of the known human GnRH-I and GnRH-II sequences. Extracts obtained from the TE-671 and LAN-1 cell lines as well as from the immortalized mouse hypothalamic GT1–7 neuronal cell line were found to contain the two isoforms of GnRH, which exhibited identical chromatographic properties as synthetic GnRH-I and GnRH-II, in HPLC followed by specific RIAs. Furthermore, double immunofluorescence studies demonstrated the two GnRH isoforms in LAN-1, TE-671, and GT1–7 cells. The identification of neuronal cell lines expressing both GnRH-I and GnRH-II provides tools for studying the differential regulation of gene expression and secretion and for studying the interaction between the two isoforms. Such studies may contribute to elucidation of the physiological functions of GnRH-II, which are still unknown.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GnRH-I (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2), originally isolated from the mammalian hypothalamus, plays a pivotal role as the physiological regulator of reproduction (1, 2). This peptide is synthesized and released by hypothalamic neurosecretory cells and reaches the pituitary gland by way of a specialized portal system to induce the synthesis and secretion of the gonadotropic hormones, which regulate gonadal function (3). Recently, several groups have identified a second isoform of GnRH (GnRH-II; His5,Trp7,Tyr8-GnRH-I), in the brain of mammalian species (4, 5, 6). The GnRH-II gene was cloned from human (7) and monkey (8) brains. Originally, GnRH-II was isolated as a second form of GnRH from the chicken brain (9) and was termed chicken GnRH-II. Since then, it was found to be expressed in cartilaginous and bony fish (10, 11, 12, 13), amphibians (14, 15, 16), reptiles (17, 18), birds (9, 19), and metatherian mammals (20, 21) and recently in rodents (5, 6), monkeys (4, 8), and humans (5, 7). The wide distribution of this neuropeptide over all vertebrate classes demonstrates its conservation over the years of evolution and may imply that its physiological functions are very important. In the vertebrate brain the localization of GnRH-II neurons is restricted mainly to the brainstem and hypothalamic structures. The cells are scattered in the periaqueductal and central regions of the midbrain and in the paraventricular, supraoptic, and medial-basal nuclei of the hypothalamus (4, 5, 6, 8). Recently, a third isoform of GnRH in the human, calf, and mouse brain has been demonstrated (22).

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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Neuronal cell lines
The following neuronal cell lines were used: human medulloblastoma cell lines Daoy (24) and TE-671 (25); human neuroblastoma cell lines SK-N-MC (26), SHSY5Y (27), and LAN-1 (28); human cortical neuronal cell line HCN (29); human peripheral neuroepithelioma cell line A673 (30); and an immortalized mouse hypothalamic neuronal cell line, GT1–7 (23). The GT1–7, HCN, TE-671, SK-N-MC, SHSY5Y, and A673 cell lines were maintained in DMEM (Sigma, St. Louis, MO), whereas the LAN-1 and Daoy cell lines were maintained in RPMI 1640 medium (Sigma). The media were supplemented with 10% FCS (Biological Industries, Beth Haemek, Israel), penicillin (100 IU/ml), and streptomycin (100 µg/ml; Life Technologies, Inc., Paisley, Scotland, UK). The cells were cultured at 37 C in a humidified atmosphere of 5% CO2-95% air.

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 {gamma}-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).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
mRNA for GnRH-I and GnRH-II in human cell lines
Total RNA preparations derived from the seven human neuronal cell lines were reverse transcribed to generate cDNA pools. The cDNA products were used as templates to the PCR by using specific primers for GnRH-I, GnRH-II, and the ribosomal protein S14 that served as an internal control. Sense and antisense primers were selected to be located on different exons for both for GnRH-I and GnRH-II to avoid false positive results caused by DNA contamination (Fig. 1AGo). 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. 1BGo, upper panel) and GnRH-II (Fig. 1BGo, 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. 1BGo, 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. 1CGo).



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  		Figure 1. Expression of GnRH-I and GnRH-II genes in human neuronal cell lines. A, Schematic representation of the GnRH-I and GnRH-II transcripts. GnRH-I and GnRH-II cDNA are shown with the introns (lines), exons (square), polyadenylase tail (wavy line), and location of the PCR fragments (shaded square). The lengths in base pairs of the introns, exons, and each of the PCR fragment are indicated. B, Southern blot hybridization of amplified GnRH-I, GnRH-II, and the ribosomal protein S14 cDNA fragments. Amplified GnRH-I, GnRH-II, and S14 cDNA fragments from human neuronal cell lines, LAN-1 and TE-671, were hybridized to a human GnRH-I (upper panel), GnRH-II (middle panel), and S14 (lower panel) 32P-labeled oligonucleotide probes. The hybridizations were performed sequentially on the same membrane. The predicted sizes of GnRH-I, GnRH-II, and S14 fragments are 248, 197, and 143 bp, respectively. Lanes 1 and 3, PCR containing GnRH-I and S14 primers; lanes 2 and 4, PCR containing GnRH-II and S14 primers; lane 5, PCR without cDNA that served as negative control. C, The nucleotide sequence of the amplified GnRH-I and GnRH-II cDNA fragments. The 248-bp product is identical to nucleotides 1134–1192 (exon 1) and 2063–3766 (exon 2 and 3) of human GnRH-I (38 ). The 197-bp product is identical to nucleotides 1312–1355 (exon 1) and 2098–2250 (exon 2) of human GnRH-II (7 ). The locations of the primers used in the PCR are underlined, and the locations of the primers used as probes for hybridization are marked by squares.

 
Identification of GnRH-I and GnRH-II peptides in human cell lines
Acid extracts of 25 confluent tissue culture dishes (100 mm) of the TE-671 and LAN-1 cells were eluted through RP C18 columns on HPLC using an isocratic elution program that separates synthetic GnRH-I (Fig. 2Go, open arrow) from GnRH-II (Fig. 2Go, black arrow). The concentrations of GnRH-I or GnRH-II in the eluate of TE-671 (Fig. 2AGo) and LAN-1 (Fig. 2BGo) cell extracts were determined using specific RIA systems for GnRH-I or GnRH-II. Figure 2Go 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 3Go demonstrates GnRH-I and GnRH-II immunoreactivity in TE-671 (Fig. 3Go, A–D) and LAN-1 (Fig. 3Go, 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. 3Go) due to different exposure times and variance in the chromophore intensity and the film sensitivity. Figure 3Go, 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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  		Figure 2. Elution profiles of GnRH-I and GnRH-II extracted from TE-671 (A) and LAN-1 (B) cells and eluted through RP HPLC. Fractions (1 ml) of the eluate were collected, evaporated, and reconstituted with phosphate buffer. All fractions were assayed for GnRH-I and GnRH-II by RIA, using specific antibodies for either GnRH-I ({square}) or GnRH-II ({blacksquare}). 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.

 


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  		Figure 3. Double fluorescence microscopy of the TE-671 (A–D), LAN-1 (E–H), and GT1–7 (I–L) cell lines. The cells were incubated with a mixture of a monoclonal antibody against GnRH-I and a polyclonal anti-GnRH-II serum. A mixture of secondary antibodies, goat antimouse (Cy2), and goat antirabbit (rhodamine) was used to label the appropriate antibody. Immunoreactive cells were observed with the green filter (antibody against GnRH-I; A, C, E, G, I, and K) and with the red filter (antibody against GnRH-II; B, D, F, H, J, and L). The arrows point to immunoreactive clusters in the cell bodies. M and N, TE-671 cells reacted with antibody against vasopressin followed by secondary antibodies. Scale bar, 10 µm.

 
Identification of GnRH-II peptide in the mouse GT1–7 cell line
We also looked into the possibility that GnRH-II is produced in GT1 cells, the well established GnRH-I cell line model. Acid extracts of 25 confluent tissue culture dishes (100 mm) of the GT1–7 cells, a subclone of mouse hypothalamus immortalized neurons, were eluted through RP C18 columns on HPLC using an isocratic elution program that separates synthetic GnRH-I (Fig. 4Go, open arrows) from GnRH-II (Fig. 4Go, 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 4Go shows that the elution profiles of the immunoreactive GnRH-I (Fig. 4AGo) and GnRH-II (Fig. 4BGo) 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. 3Go, I and K) and GnRH-II (Fig. 3Go, J and L) was observed in this mouse hypothalamic neuronal cell line.



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  		Figure 4. Elution profile of GnRH-I and GnRH-II extracted from GT1–7 cells and eluted through RP HPLC. Fractions (1 ml) of the eluate were collected, evaporated, and reconstituted. All fractions were assayed for GnRH-I (Fig. 3AGo) and GnRH-II (Fig. 3BGo) by RIA, using specific antibodies for either GnRH-I ({square}) or GnRH-II ({blacksquare}). 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 lysate calculated as picograms of peptide per milligram of protein are 8530 and 80 pg for GnRH-I and GnRH-II, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
By using GnRH-I- and GnRH-II-specific oligonucleotide primers for the RT-PCR and specific radioactive probes for Southern hybridization, we demonstrated that GnRH-I and GnRH-II are coexpressed by the human medulloblastoma cell line TE-671 and by the human neuroblastoma cell line LAN-1. The PCR fragments from TE-671 were isolated and cloned into pGEM-T vector, and the nucleotide sequences were identical to the known GnRH-I and GnRH-II sequences. The presence of the neuropeptides GnRH-I and GnRH-II in the TE-671 cells was demonstrated by HPLC followed by RIA. The presence of the two neuropeptides in LAN-1 and TE-671 was also demonstrated by double immunofluorescence staining.

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.


   Acknowledgments
 
The authors thank Drs. K. Okuzawa (Japan) and H. F. Urbansky (U.S.) for supplying antibodies for GnRH-II and GnRH-I, respectively. The authors also thank Dr. P. Mellon for permission to use the GT1 cells.


   Footnotes
 
1 This work was supported by the Israel Science Foundation, administered by the Israel Academy of Sciences and Humanities. Back

Received June 5, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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