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Endocrinological Oncology |
Department of Laboratory Medicine and Pathology (N.S., L.J., X.Q., B.S., R.V.L.), Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905; Department of Pathology (N.S., R.Y.O.), Tokai University School of Medicine, Isehara-City, Kanagawa, 259–11, Japan; and Department of Pathology (K.K.), St. Michael’s Hospital, Toronto, Ontario M5B 1W8, Canada
Address all correspondence and requests for reprints to: Ricardo V. Lloyd, M.D., Department of Laboratory Medicine and Pathology, Mayo Clinic and Mayo Foundation, 200 First Street SW, Rochester, Minnesota 55905.
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Abstract |
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This is the first report of the localization of GnRH and GnRH-R mRNAs in individual pituitary adenoma cells using in situ RT-PCR. The frequent expression of GnRH and GnRH-R mRNAs in pituitary cells suggests that GnRH has autocrine/paracrine functions in nontumorous and neoplastic pituitary tissues.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The presence of GnRH-producing cells (11) and of GnRH messenger RNA (mRNA) (12) has been reported in rat anterior pituitary, which suggests that endogenously synthesized GnRH may be involved in local regulatory mechanisms. Alexander et al. (13) detected GnRH-R mRNA in human pituitary adenomas in vitro. In addition, using PCR techniques, Miller et al. (14) reported both GnRH and GnRH-R mRNAs in gonadotroph tumors, as well as in the normal human pituitary. Little is known, however, about GnRH and GnRH-R gene expression in other types of pituitary adenomas.
In situ hybridization is a technique useful in demonstrating gene expression in individual cells, but it is limited in its ability to detect low copy numbers of mRNAs. Recently, combined in situ hybridization and reverse transcription PCR (in situ RT-PCR) methods were applied to pituitary cells (15). In the present report, we used this new technique to examine the expression of GnRH mRNA and GnRH-R mRNA in nontumorous human pituitaries and in various types of pituitary adenomas.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three normal autopsy pituitaries, each obtained within 8 h of death from adult patients without endocrine abnormalities, and 33 pituitary adenomas were used in this study. Of patients with pituitary adenomas, 5 patients had GH-secreting tumors and acromegaly and elevated serum levels of GH and/or insulin-like growth factor, 4 had PRL-secreting adenomas with elevated serum PRL levels, three had ACTH-secreting tumors and Cushing’s disease, and 21 had clinically nonfunctioning adenomas with no evidence of hormone hypersecretion and serum PRL levels less than 100 ug/L. Tumors, in which 25% or more of adenoma cells immunostained for FSH or LH ß-subunits, were classified as gonadotropic adenomas. The remaining 12 tumors, which did not show hormone immunoreactivity or in which less than 25% of cells stained for gonadotropin ß-subunits, were classified as null cell adenomas.
Tumor tissue fragments were frozen immediately at -70 C for the
purpose of RNA extraction, immunohistochemistry, in situ
hybridization, and in situ RT-PCR studies. As controls for
RT-PCR, in situ hybridization, and in situ RT-PCR
studies, two experimental cell lines were used: 1) the GH3
rat PRL and GH-producing cell line (American Type Culture Collection,
Rockwille, MD); and 2) the 
T3-1 mouse pituitary
gonadotroph cell line that produces -subunit (Dr. P. Mellon,
University of California, San Diego, CA). Both cell lines were
maintained in DMEM (Life Technologies, Grand Island, NY) with 15%
horse serum, 2.5% FBS, 5 µg/mL insulin and 1% antibiotics. Cells
were harvested for RNA extraction, and cell aliquots were affixed to
slides by cytocentrifugation, fixed for 20 min in 4% paraformaldehyde
at pH7.2, as previously reported (15), and used for in situ
studies.
Frozen sections of pituitary adenomas and nonneoplastic autopsy pituitaries were cut at 8 microns, fixed in 4% paraformaldehyde, washed in 2x standard saline citrate (SSC), dehydrated in alcohol, and stored at -70 C. Mounted on silane-coated glass slides, these tissues were used for immunohistochemistry, in situ hybridization, and in situ RT-PCR experiments.
Oligonucleotide primers and probes
Oligonucleotide primers and hybridization probes were produced
on a DNA oligonucleotide synthesizer (Applied Biosystems, Foster City,
CA) (Table 1
). Both primers and probes for human GnRH
(GenEMBL: X15215) and human GnRH-R (GenEMBL: L07949) were synthesized
on the basis of published sequences (14, 16, 17). Mouse GnRH (GenEMBL:
M14872) and GnRH-R (GenEMBL: M93108) primers also were synthesized (18, 19).
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First-strand complementary DNA (cDNA) was prepared from total RNA by using a first-strand synthesis kit (Stratagene, La Jolla, CA). The RT reaction was performed at 37 C for 60 min in a final vol of 50 µL with 5 µg total RNA, 300 ng antisense primer, 1x RT buffer, 1.0 mmol/L of each deoxyribonucleotide (dATP, dCTP, dTTP and dGTP), 40 U ribonuclease (RNase) inhibitor, and 50 U Moloney murine leukemia virus RT. The reaction product was then heated at 95 C for 5 min and immediately placed on ice.
The PCR was performed in 100-µL final reaction volumes containing 5 µL RT reaction product as template DNA, corresponding to cDNA synthesized from 500 ng total RNA, 1x PCR buffer (Promega, Madison, WI), 1.5 mmol/L MgCl2, 0.2 mmol/L of each deoxynucleotide (Boehringer Mannheim, Indianapolis, IN), 300 ng of each sense and antisense primer for GnRH and GnRH-R, and 2.5 U Taq DNA polymerase (Promega). Programmable temperature cycling (Perkin-Elmer/Cetus 480, Norwalk, CT) was performed with the following cycle profile: 95 C for 5 min, followed by 94 C for 1 min, 60 C for 1 min, and 72 C for 2 min (30 cycles) for GnRH; and 94 C for 1 min, 62 C for 1 min, and 72 C for 2 min (35 cycles) for GnRH-R, respectively. After the last cycle, the elongation step was extended at 72 C for 10 min.
A 20-µL aliquot of PCR product was analyzed by gel electrophoresis, using a 2% agarose gel, and was stained with ethidium bromide. PHx174 DNA/HaeIII digest (Boehringer Mannheim) was used as the standard. The separated PCR products were transferred to nylon membrane filters. Southern hybridization, with a single internal probe that hybridized to regions within the amplified sequences, was performed. Hybridization was performed with 1 x 106 cpm/mL [33P] deoxyadenosine diphosphate-labeled probe at 42 C for 18 h. After washing with 6x SSC-0.1% SDS at 23 C for 20 min and at 42 C for 20 min, autoradiography was performed at -70 C with Kodak Omat-AR film (Eastman Kodak, Rochester, NY) with intensifying screens. In RT-PCR experiments, total RNAs from the aT3-1 and GH3 cell lines were included as respective positive and negative controls for GnRH and GnRH-R.
Immunohistochemistry.
Immunostaining for anterior pituitary
hormones used the avidin-biotin peroxidase complex method (Vector
Laboratories, Burlingame, CA). Primary antibodies were directed against
the full spectrum of anterior pituitary hormones, including GH (1:1000
dilution), PRL (1:1000), LHß (1:500), FSHß (1:500), TSHß (1:1000)
(all rabbit polyclonal and obtained from the National Pituitary Agency,
Bethesda, MD), and rabbit polyclonal ACTH (1:1000) (Dako Corp., Santa
Barbara, CA). The monoclonal antibody to -subunit of glycoprotein
hormones (1:250) was purchased from Biogenex (San Ramon, CA).
Chromogranin A antibody (LK2H 10, 1:1000) was produced in our
laboratory, as previously described (22). The reaction products were
visualized by 3,3'-diaminobenzidine tetrahydrochloride.
In situ hybridization
A cocktail of four oligonucleotide probes for GnRH and for GnRH-R, internal to the amplified products from the primers, were labeled with digoxigenin-deoxyuridine 5-triphosphate (Boehringer Mannheim) by terminal deoxyribonucleotidyl transferase reaction, as previously reported (23). The ISH procedure was performed as previously described (23, 24). In brief, after deparaffinization, the sections were treated with 1 µg/mL proteinase K (Boehringer Mannheim) at 23 C for 15 min, followed by heat treatment, hydrochloride treatment, acetylation, and then prehybridization. Thereafter, the sections were hybridized with 1 ng/µL of the cocktail probe at 50 C for 18 h. After hybridization, immunodetection was performed using antidigoxigenin at a 1:500 dilution (Boehringer Mannheim). The reaction product was visualized by nitroblue tetrazolium salt and 5-bromo-4 chloro-3 indolyl phosphate (NBT-BCIP, Life Technologies). Control experiments were carried out using sense probes that had complementary sequences to one of the antisense probes.
In situ RT-PCR
The in situ RT-PCR technique was performed according to a three-step protocol previously described (15). The same GnRH and GnRH-R primers and probes used for RT-PCR were employed for in situ RT-PCR. Briefly, tissue sections or aT3-1 cytospin cells were digested with 1 µg/mL proteinase K at 23 C for 5 min and the enzyme was subsequently inactivated by heating to 80 C in PBS for 10 min. Then, after rinsing with H2O, the RT reaction was performed on the slides. The RT reaction mixture included: 10 mmol/L Tris-HCl, 50 mmol/L KCl, 1.5 mmol/L MgCl2, 10 mmol/L dithiothreitol, 0.2 mmol/L each of deoxyribonucleotide (Boehringer Mannheim), antisense oligonucleotide primer (1 µg/100 µL), RNasin (75U/100 µL), and Superscript II RT (1000 U/100 µL; Life Technologies). The reaction solution (50 µL) was applied to each slide and was covered by glass coverslips. After a 2-h incubation at 42 C, the slides were washed in 2x SSC, 1xSSC, 0.5xSSC, and water. For PCR, a total vol of 100 mL PCR solution with 10 mmol/L Tris-HCl, 50 mmol/L KCl, 2.5 mmol/L MgCl2, 1 mmol/L dithiothreitol, 0.2 mmol/L of each deoxynucleotide, sense and antisense primers (1 µg each/100 µL), and Taq DNA polymerase (10 U/100 µL; Promega) was applied to each slide. The solution was sealed, using mineral oil (Sigma, St. Louis, MO), and then placed on the block of the thermocycler (OmniSlide; Hybaid, Middlesex, UK). After initial denaturation at 95 C for 5 min, PCR amplification was performed using a programmable cycle profile of 20 cycles for GnRH mRNA and 30 cycles for GnRH-R mRNA of amplification, with denaturing at 94 C for 2 min, annealing at 60 C for 1.5 min, extension at 72 C for 1.5 min, and final extension at 72 C for 10 min. After the PCR reaction, tissues were fixed in 4% paraformaldehyde for 5 min, followed by incubation in ethanol and a rinse in 2x SSC. Slides were incubated with prehybridization solution at 23 C for 20 min and then hybridized overnight with labeled probes at 42 C. Immunodetection of the hybridization signals was performed as above. Control experiments performed included: 1) omission of RT or Taq polymerase; 2) omission of PCR primers; 3) pretreatment with RNase (Sigma), 100 µg/mL in PBS at 37 C for 2 h before reverse transcription; and 4) analyzing the in situ RT-PCR amplified products from the supernatant by gel electrophoresis and Southern hybridization blotting (15).
Grading of the in situ hybridization and in situ RT-PCR reactions was based on signal intensity as follows: -, negative; 1+, weak; 2+, moderate; 3+, strong.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunohistochemical analyses showed 4 adenomas positive only for
PRL; all 5 GH adenomas were positive for GH, with 4 of these cases also
positive for PRL and for -subunit of glycoprotein hormones (-SU).
There were 3 ACTH adenomas that were positive only for ACTH; 9
gonadotroph (GTH) adenomas, all of which were positive for FSHß and
-SU, 4 of which also expressed LHß. The 12 null cell adenomas were
all diffusely positive for chromogranin A but had less than 25% of
cells in the adenomas positive for FSHß or LHß.
RT-PCR analysis
The results of RT-PCR are shown in Fig. 1. Analysis
of GnRH mRNA demonstrated that the expected 246-bp PCR product was
detected by ethidium bromide staining in all 3 nontumorous pituitaries
and in all 17 tumors studied. The expected 387-bp PCR product for
GnRH-R mRNA was detected in all 3 nontumorous pituitaries and in 5
gonadotropin secreting adenomas, 6 null cell tumors, 1 of 2
GH-secreting adenomas, and 1 of 2 ACTH-secreting tumors analyzed. It
was not seen, however, in 2 PRL-secreting adenomas. Southern
hybridization with GnRH and GnRH-R internal probes showed strong bands
for GnRH mRNA in all cases and for GnRH-R mRNA in 13 of the 17
adenomas. To further evaluate the absence of GnRH-R mRNA in
PRL-secreting adenomas, the film was exposed for 10 days; the PRL
adenomas remained negative like the negative control lanes (data not
shown). The control RT-PCR experiment, using RNA extracted from
T3-1 gonadotroph cells, was positive for GnRH and GnRH-R
mRNA, whereas RNA from the GH3 cell line was negative for
both (data not shown).
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The in situ hybridization signal for GnRH mRNA was
detected within the cytoplasm of both nontumorous pituitary cells and
the T3-1 cells. Compared with negative controls without
probe or with sense probe, the signals were variably positive. A
positive, but weak, signal for GnRH-R mRNA also was detected in normal
pituitaries and in T3–1 cells.
A positive hybridization signal for GnRH mRNA was detected in 12 of 33
adenomas (36.3%) by in situ hybridization (Table 2). In most cases, the signal was relatively weak (1+).
A weak cytoplasmic signal for GnRH-R mRNA also was detected in the
cytoplasm of 10 of 33 adenomas (30.3%). In situ
hybridization controls with the sense probes were always negative.
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T3-1
cells, the hybridization signal for GnRH mRNA was amplified
significantly. For the detection of GnRH-R mRNA, 30 cycles of PCR
produced a stronger signal than did 20 cycles (Fig. 2).
Various controls for in situ RT-PCR were used, including
analysis of the supernatant from the reaction. When the products in the
supernatants were electrophoresed, after 20 or 30 cycles of
amplification, bands of expected size were observed for GnRH mRNA and
GnRH-R mRNA. Using mouse primer sets, in situ RT-PCR
products of T3-1 cells resulted in a stronger band than
that seen when using human primers. Southern blot hybridization, with
internal oligonucleotide probes labeled with 33P, confirmed
the specificity of the bands for GnRH and GnRH-R from the supernatant
solutions (Fig. 3). Although some nonneoplastic
pituitary cells were positive in the slide preparation, no band was
detected after 30 cycles of in situ RT-PCR. In
situ RT-PCR products from a gonadotropin-producing adenoma showed
strong bands for both GnRH and GnRH-R mRNAs. Other controls, including
omission of PCR primers, RT, Taq polymerase, or RNase
digestion before performing in situ RT-PCR, resulted in only
background staining of tissue sections.
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and 3. GnRH and
GnRH-R mRNAs were detected in 28 (84.8%) and 25 (75.8%) of the 33
adenomas, respectively. Although GnRH mRNA was found in all types of
adenomas, GnRH-R mRNA was lacking in all 4 PRL-secreting adenomas
examined. GnRH and GnRH-R mRNAs were detected in several adenomas that
were negative by standard in situ hybridization (Table 3).
The signal intensity also was increased for GnRH-R mRNA; moderate (2+)
to strong (3+) signals were observed in tissues that expressed only
weak signal by in situ hybridization. A positive signal for
GnRH mRNA was detected in most cells of all types of adenomas (Fig. 4). A positive hybridization signal for GnRH-R was
detected in the majority of cells in gonadotropin-secreting and null
cell adenomas, whereas it was present in 10 to 50% of adenoma cells in
GH and ACTH adenomas and not in PRL adenomas.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GnRH-R mRNA has been characterized in the pituitaries of several species (25, 26). Alexander et al. (13) and Miller et al. (14) found GnRH and GnRH-R mRNAs in nontumorous and neoplastic human pituitaries by in vitro experiments and the PCR technique. These reports, however, did not investigate gene expression at the single-cell level.
In the present work, we applied in situ RT-PCR methods for
the detection of GnRH and GnRH-R mRNAs. Using this technique, low
amounts of mRNAs were detected in individual cells, and the signal
intensity was found to be increased when compared with conventional
in situ hybridization. An increase in the number of PCR
cycles (from 20 to 30 cycles) increased the percentage of cases
positive for GnRH-R mRNA, a finding consistent with the results of
solution RT-PCR. In situ RT-PCR and in situ PCR
both have been useful in the amplification and localization of RNA and
DNA when present in low copy numbers within cells (27, 28, 29).
Specifically, this technique recently has been used to detect low copy
numbers of mRNAs in endocrine cells (15, 21, 30). One advantage of
performing in situ RT-PCR with nonisotopic probes is the
excellent resolution obtained with digoxigenin or biotin labeling after
amplification of low-abundance mRNAs by the PCR technique. The approach
was successful in the present study, because more cases were found to
be positive for GnRH and GnRH-R by in situ RT-PCR than by
simple in situ hybridization. Various controls for the
in situ RT-PCR procedure were used, including analysis of
the supernatant of the reaction. After 20 or 30 cycles of amplification
and electrophoresis of the supernatant, the expected size bands for
GnRH mRNA and GnRH-R mRNA were observed. In situ RT-PCR
products from cultured T3-1 cells, using mouse primers,
resulted in stronger bands for GnRH and GnRH-R mRNA than when the human
primers were used. Performance of various controls supported the
specificity of our in situ RT-PCR studies. These consisted
of omission of PCR primers, RT, Taq polymerase, and RNase
digestion before performing in situ RT-PCR and resulted in
no staining of the tissue sections. The GnRH-R mRNA analysis of the
supernatant products from the normal pituitary was negative after 30
cycles. We have shown previously that the detection of bands by gel
electrophoresis, using the supernatants from the in situ
RT-PCR reaction, is dependent on the number of amplification cycles
(15). In the cases of normal pituitary, the supernatant was probably
diluted by PRL cells and some other cells that did not express GnRH-R
mRNA.
With the RT-PCR method, GnRH mRNA was detected in all 17 adenomas of
various types, and GnRH-R mRNA in 15 of the 17. The high incidence of
GnRH and GnRH-R gene expression in tumorous pituitaries suggests that
locally produced hypothalamic hormones may play a role in the
regulation of tumor cell growth. The presence of GnRH-R mRNA expression
in some GH and ACTH-producing adenomas could explain the abnormal
response to GnRH in some patients with acromegaly and Cushing’s
disease (9, 10). GnRH-R mRNA expression was found in all types of
adenomas, except PRL-secreting adenomas, by both RT-PCR and in
situ RT-PCR. Although our series include only a small number of
tumors of varying type, it is of note that PRL-secreting adenomas did
not express GnRH-R mRNA. In contrast, expression of GnRH-R mRNA also
has been reported in T3-1 mouse gonadotroph tumor cells
(31).
In the present study, all 5 gonadotropin-producing adenomas and 6 null-cell adenomas expressed GnRH-R mRNA. Miller et al., using the same upstream and downstream primer set, reported that 9 of 10 gonadotroph adenomas were positive for GnRH-R mRNA (14) and that only 6 of 10 adenomas were positive using another 5'GnRH primer set. One possible reason for the difference between these and our results may be the selection of adenomas studied. There also were differences in the PCR conditions. Miller et al. used a lower annealing temperature (at 53.2 C for GnRH and at 52.2 C for GnRH-R) for PCR. Despite the fact that a higher annealing temperature increases the specificity of the PCR reaction, our experiments resulted in more positive cases, even at a higher annealing temperature. Because, in the present study, the negative control using RNA from GH3 cells was consistently negative for both GnRH and GnRH-R, contamination of RNA is highly unlikely.
Genetic studies carried out by X-chromosomal inactivation analysis revealed that the majority of pituitary adenomas are monoclonal in origin (3, 4). Nonetheless, the hypothalamic hypothesis that pituitary tumors are secondary to hypothalamic dysregulation has not been completely resolved. Indeed, transgenic mouse studies, in which GHRH overproduction led to the development of pituitary adenomas in a time-dependent manner (32), do suggest that hypothalamic hormones play a role in adenoma development.
Evidence indicating that endogenous expression of hypothalamic hormone in the pituitary has been accumulating in recent years. A number of hypothalamic neuropeptides known to influence anterior pituitary secretion, such as GHRH, TRH, CRH, vasoactive intestinal polypeptide, neuropeptide Y, substance P, and galanin have been shown to be synthesized by pituitary cells (33, 34, 35, 36, 37, 38, 39). With regard to pituitary adenomas, it is of note that using RT-PCR methods, GHRH gene expression has been reported in all somatotroph adenomas but is not evident in either nonfunctioning or normal anterior pituitary (40). Furthermore, using RT-PCR, CRH transcript has been found in about 20% of pituitary tumors of different types (41).
The finding of various releasing hormones in pituitary adenomas sheds light on recent observations about pituitary adenoma with neuronal choristoma lesions (42, 43, 44). Such lesions, which consist of varying proportions of pituitary adenoma, usually GH cell adenoma, and neurons were long considered manifestations of ectopic hypothalamic neurons with induced adenoma (42, 43). The recent morphologic demonstration of cells transitional between adenoma cells and neurons (44) suggests a metaplastic origin of the neurons. This interpretation is supported by the present work and that of Wakabayashi et al. (40), which conclusively demonstrated releasing hormones in morphologically typical adenomas devoid of neuronal elements.
The expression of many hypothalamic hormone receptor genes by pituitary cells also has been demonstrated. For instance, the pharmacologic and biochemical characteristics of dopamine D2 receptors in PRL-secreting adenomas have been investigated extensively (45). Similarly, somatostatin (SS) receptors (SSTRs), which includes five separate subtypes, also have been detected in the pituitary; and SSTR2 and SSTR5 mRNA have been demonstrated (46). Given the therapeutic importance of bromocriptine and SS analogs binding to these receptors (47), the presence of these receptors is of clinical interest. Expression of GHRH-R mRNA and TRH-R mRNA in human pituitary and pituitary adenomas also has been reported (48).
The processes of GnRH-producing, hypothalamic neurons terminate not only in the median eminence but also on other GnRH-producing nerve cells. It is no surprise, therefore, that GnRH-producing neurons express abundant GnRH-R, an arrangement suggesting that autocrine regulation of GnRH release does occur within the brain (49). Thus, it seems that GnRH acts as a neurohormone, neurotransmitter, or neuromodulator, as well as a local hormone in the brain. It has been proposed that pituitary tumors can produce a number of substances that may have secretory, differentiating, and proliferative functions (50). Hypothalamic hormones may have just such specific roles, acting not only as classical releasing hormones, but also as neuromodulators.
Although GnRH-R has been detected in the -subunit producing
T3-1 cell line (31, 51), expression of GnRH by these
oncogenically transformed cells has not been detected previously by
in situ RT-PCR or other in situ methods. On the
other hand, our study showed that T3-1 cells express
both GnRH and GnRH-R mRNAs by RT-PCR. Similar results were obtained
using cultured cells by in situ RT-PCR. These observations
suggest that autocrine/paracrine regulatory mechanisms may be ongoing
in these cells, as well as in normal and neoplastic human pituitary
tissues.
The biological significance of GnRH expression by all pituitary adenomas and of GnRH-R expression by most tumors is uncertain. Recent studies, using endometrial carcinoma cell lines, found low levels of GnRH and GnRH-R mRNAs, but secretion of GnRH and functional receptor activity were uncommon findings in these cell lines (51). Thus, the functional significance of GnRH and GnRH-R mRNA expression by pituitary adenomas will have to be investigated further by functional studies in vitro.
In conclusion, the presence of GnRH and GnRH-R mRNA in various types of adenomas, as detected by in situ techniques, indicates that endogenous production of the hypothalamic hormone GnRH and its receptor in pituitaries may have a role in autocrine/paracrine regulation. Further investigations will be needed to clarify the mechanisms of such regulation and the possible role of hypothalamic hormones and their receptors in the genesis, growth, and differentiation of pituitary adenomas.
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Acknowledgments |
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Footnotes |
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Received January 6, 1997.
Accepted February 19, 1997.
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