0013-7227/92/1303-1097$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine Society
Vol. 130, No. 3 Printed
in U.S.A.
Expression of the Growth Hormone-Releasing Hormone Gene and Its Peptide Product in the Rat Ovary* ANNA
BAGNATOt, COSTANZO J. CATT
MORETTI,
JUNJI
OHNISHI,
GAETANO
FRAJESE,
AND KEVIN
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; and Clinica Medica V, University Rome La Sapienza (G.F.), 00161 Rome, Italy
ABSTRACT. GH-releasing hormone (GHRH) is a potent CAMP-mediated agonist in the rat ovary, where it binds to a common vasoactive intestinal peptide/GHRH receptor and enhances the actions of FSH on granulosa cell maturation. A GHRH-like peptide has been detected by immunocytochemistry in the human ovary and by RIA in follicular fluid, suggesting local synthesis of the peptide. In rat ovarian poly(A)+ RNA, Northern blot hybridization analysis with a 32P-labeled 48-nucleotide (nt) rat GHRH oligonucleotide probe revealed the presence of one major and two minor mRNA species. The major ovarian GHRH mRNA (1750 nt) was much larger than that present in hypothalamus and placenta (750 nt), but was similar to that observed in the rat testis. Two well defined higher mol wt forms of 3.2 and 3.6 kilobases were also present and probably represent unprocessed precursors of the 1750-nt mRNA. Further evidence of GHRH gene expression in the ovary and testis was provided by reverse transcription polymerase chain reaction of ovarian mRNA and restriction enzyme analysis of the amplified
R
AT GH -releasing hormone (GHRH) is a 43-amino acid hypothalamic peptide which regulates the synthesis and secretion of GH in the anterior pituitary gland (1). The rat GHRH gene is 10 kilobases (kb) in length and contains 5 exons that encode a 104-amino acid precursor to the 5.2-kilodalton (kDa) rat GHRH peptide (2). The intron-exon organization of the GHRH gene is similar to those of the vasoactive intestinal peptide and glucagon genes, each of which encodes large precursors that generate several biologically active and structurally related petides by proteolytic processing (3). In the rat, GHRH-like immunoreactivity is largely localized to cell bodies in the arcuate nucleus and the medial perifornical region of the lateral hypothalamus, with fiber projections to the median eminence to form a dense
of
product. In addition, immunoreactive (ir) GHRH was detected in ovarian extracts and in the incubation medium of cultured rat granulosa cells. Ovaries from PMSG-treated female rats, aged 22-27 days, contained 400 + 25 pg/g ir-GHRH. The GHRH content of the hypothalamus of the same animals was 2.9 + 0.1 rig/g. Cultured rat granulosa cells released 20 f 0.1 pg ir-GHRH/ 4 X 10’ cells.3 h into the incubation medium. The GHRH immunoreactivity detected in ovarian extracts coeluted on gel filtration chromatography with authentic rGHRH (5.2 kilodaltons). A larger form of ir-GHRH (-16.5 kilodaltons) was also present. These data demonstrate that the rat ovary contains a 1750-nt transcript that could arise from the GHRH gene by tissue-specific initiation, alternative splicing, or transcript termination. The translation product of this mRNA is the same similar size as the rat hypothalamic neuropeptide and may promote follicular maturation by autocrine or paracrine modulation of the stimulatory action of FSH on granulosa cell function. (Endocrinology 130: 1097-1102,1992)
accumulation of GHRH-containing processes and terminals (4). Like several other neuropeptides, GHRH is also present in extrahypothalamic tissues (5), including the gastrointestinal tract (6), tumors of neuroendocrine origin (7), placenta (a), and testis (9). However, the characterization and sequence analysis of the cDNA encoding the precursor peptide have been reported only in hypothalamus (2) and placenta (10). The placental and hypothalamic GHRH transcripts are similar in size [-750 nucleotides (nt)], as are their peptide products (5.2 kDa) (11). Despite these similarities, it has been suggested that GHRH gene expression may be differentially regulated in the two tissues, reflecting different sites of transcription initiation and raising the possibility of common evolutionary mechanisms for tissue-specific regulation of neuropeptide production in these organs (12). In addition, a larger mRNA species (1750) that hybridizes to GHRH cDNA has been observed in the testis of postpubertal rats, where immunoreactive (ir-) GHRH is detectable in amounts of about 1.6 rig/g tissue (13). A GHRH-like peptide has also been demonstrated
Received July 26,199l. Address requests for reprints to: Dr. Kevin J. Catt, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, Building 10, Room Bl-L400, Bethesda, Maryland 20892. * This work was supported in part by a grant from Industria Pharmaceutica Serono (Rome, Italy). t Supported by a grant from Sigma-Tau (Rome, Italy). 1097
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 15:00 For personal use only. No other uses without permission. . All rights reserved.
EXPRESSION
1098
OF GHRH
in human ovarian tissue by immunoperoxidase staining (14), and ir-GHRH has been detected in human follicular fluid (15). GHRH acts through vasoactive intestinal peptide/GHRH receptors in granulosa cells to promote maturation by amplifying the stimulatory action of FSH on CAMP production and granulosa cell differentiation (16). In addition, the expression of GHRH receptors is increased by the CAMP-mediated actions of FSH in granulosa cells, suggesting a positive autoregulatory action of this peptide on FSH-induced follicular maturation (17). The present study was performed to characterize the expression of GHRH in rat ovaries and to analyze its molecular heterogeneity. Materials
and Methods
Animals
Twenty-one-day-old Sprague-Dawleyrats obtained from Taconic Farm (Germantown, NY) were injected SCon day 23 with 10IU PMSG (Sigma, St. Louis, MO) in 0.9% NaCl. Forty-eight hours later (day 25), the rate were killed by decapitation to minimize stressand potential changesin hypothalamic function associatedwith anesthesia,and their ovarieswere removed. Studieswerealsoperformed in immature femalerata implanted scon day 21 with lo-mm diethylstilbestrol (DES)-filled Silastic capsulesto stimulate granulosa cell proliferation. These animalswere alsotreated on day 25 with 10 IU PMSG and killed 24,48, and 72 h later. Testes for RNA extraction were removed from postpuberal male rats (-90 days). Hypothalamic tissue was removed to a depth of 3-4 mm from the optic chiasm anteriorly to the mammillary bodies posteriorly, along the hypothalamic sulci laterally. Placentas (mean weight, 1.1 g) wereobtained from pregnant rats killed on day 20 of gestation. The individual tissueswere immediately weighed, then frozen in liquid nitrogen, and stored at -70 C. Preparation
and culture of granulosa
cells
Granulosacells were prepared from ovaries of PMSG- and DES- and PMSG-treated immature female rats, as previously described(16). Cell viability, determined by trypan blue exclusion, was usually greater than 80%. Approximately 4 X 10’ viable cells were added to 24-well plastic culture plates in a total volume of 1 ml McCoy’s 5a medium supplementedwith 10mM HEPES (pH 7.4), 4 mM L-glutamine, 100U/ml penicillin, 100 rg/ml streptomycin sulfate, and 0.1% BSA. Cell cultures were incubated at 37 C in a humidified 95% air-5% COZ incubator for 3 h; the media were then removed and stored at -70 C for subsequentextraction. Extraction
of GHRH from tissue and medium
Tissueswere homogenizedin ice-cold 2 M acetic acid. The homogenatewas placed in a boiling water bath for 10 min to destroy peptidaseactivity and then centrifuged at 10,000 X g for 10 min. The supernatant was lyophilized and subsequently dissolvedin chromatography solvent (1% trifluoracetic acid), then centrifuged to remove any insoluble residue. Tissue extracts were slowly passedthrough Sep-Pak Cl8 cartridges
GENE IN RAT OVARY
Endo Vol130
l l
1992 No 3
(Waters Associates, Milford, MA), followed by elution with methanol. The eluates were evaporated under vacuum and resuspendedin 0.1 M NaCl, 0.05 M Na2P0,, 0.025 M EDTA, 0.1% Na azide, 0.1% BSA, and 0.1% Triton, pH 7.4 (RIA buffer). Media from cultured granulosa cells were similarly extracted after acidification by passagethrough Sep-Pak cartridges and methanol elution. RIA of GHRH
GHRH was measuredby the RIA procedure of Vale et al. (18) with minor modifications. Sampleswere equilibrated for 24 h at 4 C with specific GHRH antibody (Peninsula Laboratories, Belmont, CA); then, about 18,000 cpm [lz61]rat (r) GHRH (Peninsula) were added,and incubation was continued for an additional 24 h. Free tracer wasseparatedfrom antibodybound tracer by the addition of sheep antiserum to rabbit immunoglobulin and continuation of incubation for an additional 24 h. The sensitivity of the assaywas8 pg/tube, and the standard curve waslinear between 8-120 pg/tube. Gel filtration
chromatography
Lyophilized ovarian extracts were resuspendedin 0.5 ml 0.2 acetic acid-0.1% BSA and centrifuged at 10,000X g for 10 min. The supernatants were applied to a 1.2 x 50-cm column packed with Sephadex G-50 gel (Pharmacia, Piscataway, NJ) previously equilibrated in the samesolution. The columnswas eluted at a flow rate of 8.2 ml/h at 4 C, and l-ml fractions were collected, lyophilized, and reconstituted for RIA. Markers used for mol wt calibration of the column were: blue dextran, cytochrome-c, rGHRH, and [3H]inositol. M
Northern
blot analysis
Isolation of total RNA from frozen tissue wasperformed by the guanidinium thiocyanate-cesiumchloride method (19). All RNA used in these experiments had OD 260/280 ratios of 2.0 -C0.2, and quantitation was basedon measurementof OD 260. In addition, samplesfrom all extractions were run on denaturing gelsto screenfor intact 18s and 28s ribosomalRNA bands. Poly(A)+ RNA wasenriched by oligo(dT)-cellulosechromatography (20). Aliquots of 20 ~1 containing 20 rg poly(A)+ RNA were subjectedto electrophoresison 1.5% agarosegelscontaining 2.2 M formaldehyde and transferred to Nytran membranes (Schleicher and Schuell, Keene, NH). Size markers were included as a RNA ladder (Bethesda Research Laboratories, Gaithersburg, MD). Prehybridization was performed for 3 h at 42 C in 1% sodium dodecyl sulfate (SDS), 10 X Denhardt’s solution, 50 Fg/ml tRNA, 50 rg/ml shearedsalmonspermDNA (5 Prime-3 Prime, Inc., West Chester,PA), 6 X SSPE [l X SSPE = 180mM NaCl, 10 mM sodiumphosphate (pH 7.4), and 1 mM EDTA]. A synthetic 48-mer oligonucleotide of rGHRH mRNA (generouslyprovided by Dr. A. W. Margioris) wasend labeled by incubation with T4 polynucleotide kinase (New England BioLabs, Beverly, MA) and [T-~*P]ATP (4500 Ci/ mmol; ICN, Costa Mesa, CA) to a specific activity of l-5 x 10’ cpm/pg DNA. Hybridization with the probe wasperformed for 18 h at 55 C (2.9 x lo6 cpm/ml) in 6 X SSPE-1% SDS. The nylon membraneswere washedthree times at room temperature and once at 55 C in 6 x SSPE (pH 7.4)-l% SDS. The
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 15:00 For personal use only. No other uses without permission. . All rights reserved.
EXPRESSION
OF GHRH
filters were exposed at -70 C to Kodak XAR-S film (Eastman Kodak, Rochester, NY) for varying times. Polymerase chain reaction (PCR) amplification First strand cDNA synthesis was carried out on 1 pg poly(A)+ RNA using the following components: 200 U Moloney murine leukemia viral reverse transcriptase (Bethesda Research Laboratories), 1 U RNase inhibitor (Promega, Madison, WI), 1 mM dNTPs, and 100 pmol random hexamer primers (Boehringer Mannhein, Indianapolis, IN) in a final volume of 20 ~1 50 mM Tris-Cl (pH 8.3), 75 mM KCl, 10 mM dithiothreitol, and 3 mM MgC12. The mixture was incubated for 10 min at room temperature, followed by 120 min at 37 C, and the reaction was terminated by heating at 94 C for 10 min. The cDNA mixture was supplemented with 2.5 U Thermus aquaticus polymerase (Boehringer), 50 pmol of each primer, 7.5 ~1 10 mM Tris-Cl (pH 8.3), 50 mM KCl, 1.5 M~C&, and 0.01% gelatin and water to a final volume of 100 ~1.The sequencesof the PCR primers (Lofstrand Laboratories, Gaithersburg, MD) usedwere GHRHspecific senseprimer (5’-GCACGAAATCATGAACAGGC-3’) from the region of the third exon and antisenseprimer (5’CTCTCCAGGGCCATCTGCTT-3) from the region of the fourth exon. Forty cycles of amplification were carried out under the following conditions: denaturation at 94 C for 1 min, annealing at 55 C for 2.5 min, and extension at 72 C for 3 min. MboI-digested and uncut PCR products were size-fractionated by electrophoresisin a 3% agarosegel. PCR products were subclonedinto pGEM-3Z (Promega) using blunt-ended ligation. The DNA sequencewas analyzed by the dideoxy chain termination method (SequenaseII, U.S. Biochemical Corp., Cleveland, OH).
Results ir-GHRH was present in the rat ovary (Fig. 1) at a mean concentration of 400 + 25 pg/g. No GHRH was
GENE IN RAT OVARY
1099
detected in extracts from muscle, which served as a negative control tissue. The GHRH content of the hypothalamus, measured as a positive control, was 2.9 + 0.1 rig/g. Serial dilution of hypothalamic extracts purified on Sep-Pak Cl8 cartridges gave a binding curve parallel to that of standard GHRH. The same result was obtained with ovarian extracts, indicating that the ovarian GHRH-like peptide is immunologically indistinguishable from authentic GHRH. Cultured rat granulosa cells were found to release immunoreactive GHRH-like material (20 f 0.1 pg/4 x 10’ cells- 3 h) into the incubation medium. The authenticity of ir-GHRH was indicated by its parallel binding inhibition with that of the standard rGHRH in the RIA (Fig. 2). The Sephadex G-50 gel filtration chromatography protile of the ir-GHRH in an extract of ovarian tissue is shown in Fig. 3. The major peak of ir-GHRH was eluted with a Kd of 0.50, coincident with the elution position of synthetic rat GHRH chromatographed on an identical column. In addition, a second peak of immunoreactivity was eluted earlier at a Kd of 0.30. The calculated relative molecular size of the early eluting peak was approximately 16,500, and that of the major peak was 5,000. Northern blot hybridization analyses of poly(A)+ RNA isolated from testes of postpuberal rats (90 days), placentas (20 days gestation), and ovaries of PMSG-treated immature rats are shown in Fig. 4. The hybridization signal of poly(A)+ ovarian RNA revealed that the major GHRH mRNA was a 1750-nt species, much larger than that extracted from rat placenta (750 nt), but similar to that present in the rat testis. Two well defined larger mRNAs of 3.2 and 3.6 kb were also observed in the ovary pl Samples
Tissue I 0.12
r
Equivalent/Tube
I 0.25
I 0.5
I
12.5
2.5
I
I
50
100
50
100
I 1.0
SO-
E60 x0 ? m 40-
8 60x0 em 40-
I
I 10
I 20 rGHRH (pg/tube)
I 50
I 100
I
FIG. 1. Inhibition of [l%I]rGHRH binding to specific antiserum by standard rGHRH (O), hypothalamic rGHRH (W), and ovarian rGHRH (A). The recovery of rGHRH through the tissue extraction procedure was 65-70%, as monitored by the addition of unlabeled rGHRH before homogenization. B/B.,, Bound/free ratio.
10
20 rGHRH (pghube)
FIG. 2. Parallel inhibition of [‘261]rGHRH binding to specific antiserum by standard rGHRH (0) and rGHRH released from granulosa cells (4 x 10’ cells/ml) cultured for 3 h (W). The recovery of rGHRH through the medium extraction procedure was 65-90%, as monitored by the addition of unlabeled rGHRH before extraction. B/B,, Bound/ free ratio.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 15:00 For personal use only. No other uses without permission. . All rights reserved.
EXPRESSION
1100
OF GHRH
GENE
IN RAT
OVARY
Endo * 1992
Vol130*No3
Fraction I 0
Number I
I 1.0
0.5 Kd
FIG. 3. Distribution profile of ovarian rGHRH (B) on a Sephadex G50 (fine) column eluted with 0.2 N acetic acid-0.1% BSA. The column dimensions were 50 x 1.2 cm, and the flow rate was 8.2 ml/h. The positions of the void volume (Vo) and the elution volumes of cytochrome-c (mol wt, 12,500), rGHRH (mol wt, 5,200), and inositol ([3H] Ins.; mol wt, 108) are also shown. Identical results were obtained in two such experiments performed on separate ovarian extracts. A
6 E p
r-i”‘+ ---
p --
.g
SZ$;g%l2 -
-Origin
=ggg -1:75
kb
-0.75
kb
2
DES + PMSG i:L i i’ -
-
f
-Origin
=3% :I: -1.75 kb -0.75
WlObp 43 bp r47bp
FIG. 5. Ethidium bromide-stained gel of GHRH mRNA amplified from rat ovary and testis by reverse transcription PCR and subjected to restriction enzyme analysis. The first lane contains size markers from f?laeIII-digested $X174 DNA. The expected sire of the uncut rat GHRH PCR product was 110 nt, and those of the Mb01 fragments were 63 and 47 nt.
Ovary P, g
;
234 194= 11872-
kb
FIG. 4. Northern blot analysis of GHRH mRNA. A, Twenty micrograms of poly(A)+ RNA isolated from testis, placenta, and ovary. B, Twenty micrograms of RNA isolated from rat muscle (negative control) and 10 rg poly(A)+ RNA isolated from rat testis and ovary. Other lanes show autoradiographs of ovarian GHRH mRNA during follicular development. DES-implanted rats were treated with 10 IU PMSG and killed after 24, 48, and 72 h. The deduced sizes of mRNA species are indicated on the right.
and may represent unprocessed precursors of the 1750 nt mRNA (Fig. 4A). No GHRH hybridization signal was detected with muscle RNA (negative control). Northern analysis was also performed on 10 pg poly(A)+ RNA isolated from ovaries of normal and DES-implanted rats after treatment with PMSG (10 IU) at specific times. The three major bands (1.75, 3.2, and 3.6 kb) were present during follicular development. After PMSG treatment of DES-implanted rats, the levels of GHRH mRNA measured 24 h after treatment with PMSG were similar in normal and DES-treated rats and declined at 48 and 72 h (Fig. 4B). Further evidence of GHRH gene expression in the ovary and testis was provided by the use of reverse transcription PCR. As shown in Fig. 5, PCR products of 110 nt, the predicted size based on the primers employed
and the sequence of the corresponding rat GHRH-(l43) segment, were amplified from ovarian and testicular cDNA. These PCR products were digested by MboI, yielding two fragments of the expected sizes (63 and 47 nt). DNA sequence analysis of these products revealed 100% homology with regions of exons 3 and 4 of rat hypothalamic GHRH cDNA (data not shown). Discussion These observations demonstrate that the rat ovary, in addition to being a target of GHRH action, is a site of GHRH-like mRNA and peptide synthesis. Our data indicate that the GHRH gene is expressed in the rat ovary and that its product is translated into a peptide similar to GHRH. Northern blot analysis showed that the size of rat ovarian GHRH transcript was about 1750 basepairs, similar to the mRNA encoding testicular GHRH. The rat prepro-GHRH gene encodes a 104-amino acid precursor in five exons, which, like the human GHRH gene, span more than 9 kb. The human and rat preproGHRHs are not highly conserved, and the mature rat hypothalamic peptide contains only 43 amino acids and lacks a COOH-terminal amide. The GHRH peptide sequence encoded by cloned cDNA is followed by a 30amino acid peptide of unknown function (2). The nucleotide sequences of the rat and human GHRH genes differ in the 3’-domain because of changes in the
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 15:00 For personal use only. No other uses without permission. . All rights reserved.
EXPRESSION
OF GHRH
splicing of intron D (21), which is the site of evolutionary divergence for other members of the glucagon gene superfamily (3). This suggests that alterations in mRNA transcripts from the GHRH gene could be more likely in the 3’regions of the ovarian transcript. The 1750-nt mRNA species in rat ovary and testis may represent a physiologically significant alternative gene transcript derived from an unprocessed precursor of the 750-nt RNA, or arising during tissue-specific initiation, alternative splicing, or transcription termination of the GHRH gene. The presence of a larger GHRH mRNA and a GHRHlike peptide in the rat gonads raises several questions, including the possible existence of a GHRH-like gene containing additional coding regions, the heterogeneity of the 3’nontranslated sequence, and the presence of different transcriptional start sites. The ir-GHRH present in ovarian extracts was shown by gel filtration chromatography to be similar in size to authentic rat GHRH. A higher mol wt form (-16,000 kDa) was also detected by gel filtration and may represent pro-GHRH, as predicted by the larger mRNA species observed on Northern blots. Our data also show that cultured rat granulosa cells secrete a GHRH-like substance, suggesting a role for this peptide as a mediator in the transfer of intercellular information at the ovarian level. The presence of a GHRH mRNA species larger than that found in the hypothalamus and of a GHRH-like substance has been described in the rat testis (9). Although rGHRH and testicular GHRH share biological similarities, they do not appear to be identical, since they exhibit different HPLC elutions profiles and molecular sizes. In fact, on the basis of SDS-gel analysis, testicular GHRH was reported to be almost 4 times larger than hypothalamic GHRH (22). In the testis, the larger mRNA species might encode a longer peptide, which contains a region that cross-reacts with the epitope recognized by the GHRH antiserum or results from postranslational modifications, leading to a product of larger molecular size. In the endocrine and nervous systems, several gene products that serve multiple and complex roles are generated by tissue-specific RNA and precursor processing (23). Recently, utilization of the transgenic technique (24) has provided a valuable approach to the problem of molecular heterogeneity and tissue distribution of neuropeptides. In transgenic mice expressing the human (h) GHRH gene, the highest concentration of GHRH was found in the pituitary gland, followed by the pancreas (25). The finding that pituitary tissue of transgenic mice contains high levels of GHRH confirms the recent observation related to the capacity of perifused human hypophyseal fragments to release GHRH (26). In transgenie mice, intermediate levels of ir-GHRH were present in hypothalamus and liver and lower levels in visceral organs, heart, and gonads. Gel filtration and assay of ir-
GENE
IN RAT
OVARY
1101
hGHRH in extracts of pituitary, brain, pancreas, and gut revealed the mature hormone and a more hydrophobic form, which probably represents the hGHRH precursor (pro-GHRH) on the basis of its estimated molecular size (9 kDa) (25). The gonadal expression of hGHRH-(1-44) in transgenic mice demonstrates the presence of tissuespecific enzymes that are capable of processing the preproGHRH peptide. In this study we observed that the ovarian levels of GHRH mRNA in PMSG-treated normal and DES-implanted rats were similar. The mRNA levels measured in DES-treated rats declined 48 and 72 h after PMSG treatment, suggesting a regulatory action of gonadotropins on GHRH gene expression. It has been recently reported that FSH treatment of hypophysectomized rats or whole ovarian dispersates for up to 48 h caused timeand dose-dependent decrements in insulin-like growth factor gene expression (27). Whether the decrease in GHRH mRNA observed after PMSG treatment reflects a decline after transient stimulation of GHRH gene expression or an inhibitory action of the gonadotropin has yet to be determined. In the hypothalamus, GH exerts a negative feedback action on the expression of GHRH mRNA, which increases significantly after hypophysectomy (28). In hypophysectomized rats and in the lit/lit mouse, a model of isolated GH deficiency due to abnormal GHRH receptor expression in somatotrophs, the increase in hypothalamic GHRH mRNA is not accompanied by a change in placental GHRH mRNA (29). In the testis, where GHRH mRNA appears to be developmentally regulated, the GHRH trancript decreases after hypophysectomy, probably as a consequence of gonadotropin deficiency. These observations suggest that in the ovary, as in other peripheral tissues, the transcript would not be regulated by GH. Furthermore, structural and physiological studies have demonstrated differences between hypothalamic and placental regulation of mouse and rat GHRH gene expression. Despite the presence of mRNAs of similar size, the existence of alternative 5’-untranslated regions has revealed differencies in the sites of initiation of transcription in the two tissues (10, 11). This is also evident for the rat insulin-like growth factor-I gene, which is expressed and regulated by GH in a tissuespecific manner, since its transcripts contain at least three alternative 5’-untranslated regions associated with a common sequence encoding the mature peptide (30). Also, the CRF gene is differentially regulated by glucocorticoids in specific tissues, being suppressed at the hypothalamic level and increased in the placenta (31, 32). These examples demonstrate that tissue-specific expression of a gene can generate the same biologically active mature peptide, as predicted by its exons, through different routes of transcription and precursor biosyn-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 15:00 For personal use only. No other uses without permission. . All rights reserved.
EXPRESSION
1102
OF GHRH
thesis. In conclusion, these findings demonstrate that expression of the GHRH gene in the rat ovary leads to the production of a GHRH-like peptide of similar size to the rat hypothalamic neuropeptide. These data and our previous observations indicate that this peptide can promote follicular maturation by autocrine modulation of the stimulatory action of FSH on granulosa cell function. The granulosa cell serves as a site of production, receptors, and actions of GHRH, which may, thus, exert a positive autoregulatory action on ovarian follicular maturation. References 1. Frohman LA, Jansson JO 1986 Growth hormone releasing hormone. Endocr Rev 7:223-253 2. Mayo KE, Cerelli GM, Rosenfeld MG, Evans RM 1985 Characterization of cDNA and genomic clones encoding the precursor to rat hypothalamic growth hormone-releasing factor. Nature 314:464467 3. Bell GI 1986 The glucagon superfamily: precursor structure and gene organization. Peptides [Suppl l] 7:27 4. Merchenthaler I. Vigh S, Schallv AV, Petrusz P 1984 Immunocytochemical localization of growth hormone-releasing factor in the rat hypothalamus. Endocrinology 114:1082-1085 5. Thorner MO, Vance ML, Evans WS, Ho K, Rogol AD, Blizzard RM, Furlanetto R, Rivier J, Vale W 1986 Growth hormone releasing factor and somatomedin C production: extrahypothalamic localization and possible functional significance. Acta Endocrinol [Suppl] (Copenh) 276:34-40 6. Bruhn TO, Mason RT, Vale W 1985 Presence of growth hormone releasing factor-like immunoreactivity in rat duodenum. Endocrinology 117:1710-1712 7. Rivier J, Spiess J, Thorner M, Vale W 1982 Characterization of a growth hormone-releasing factor from a human pancreatic islet tumour. Nature 300:276-278 8. Meigan G, Sasaki A, Yoshinaga K 1988 Immunoreactive growth hormone-releasing hormone in rat placenta. Endocrinology 123:1098-1102 9. Berry SA, Pescovitz OH 1988 Identification of a rat GHRH-like substance and its messenger RNA in rat testis. Endocrinology 122661-663 10. Surh ST, Rahal JO, Mayo KE 1989 Mouse growth hormonereleasing hormone: precursor structure and expression in brain and placenta. Mol Endocrinol3:1693-1700 11. Margioris AN, Brockmann G, Bohler HCL, Grino M, Vamvakopoulos N, Chrousos GP 1990 Expression and localization of growth hormone releasing hormone messenger ribonucleic acid in rat placenta: in vitro secretion and regulation of its peptide product. Endocrinology 126151-158 12. Mizobuchi M, Frohman MA, Downs TR, Frohman LA 1991 Tissue specific transcription initiation and effects of growth hormone (GH) deficiencv on the regulation of mouse and rat GH-releasing hormone gene-in hypothilamus and placenta. Mol Endocrinoi 5:476-484 13. Berry SA, Pescovitz OH 1988 Ontogeny and pituitary regulation of testicular growth-hormone releasing hormone-like messenger ribonucleic acid. Endocrinology 127~1404-1401 14. Moretti C, Fabhri A, Gnessi L, Bonifacio V, Bolotti M, Arizzi M, Nazzicone Q, Spera G 1990 Immunohistochemical localization of growth hormone releasing hormone in human gonads. J Endocrinol Invest. 13:301-305 15. Moretti C, Fahhri A, Gnessi L, Forni L, Fraioli F, Frajese G 1989
GENE
16. 17. 18. 19. 20. 21.
22.
23. 24.
25.
26.
27.
28. 29.
30.
31. 32.
IN RAT
OVARY
Endo. Voll30.
1992 No 3
GHRH stimulates follicular growth and amplified FSH-induced ovarian folliculogenesis in women with anovulatory infertility. In: Frajese G, Steinberg F, Rodriguez-Rigau LJ (eds) Reproductive Medicine: Medical Therapy. Proceeding of the Second International Symposium on Reproductive Medicine. Elsevier, New York, no. 875:103-112 Moretti C, Bagnato A, Solan N, Frajese G, Catt KJ 1990 Receptor mediated actions of growth hormone-releasing factor on granulosa cell differentiation. Endocrinoloav 127:2117-2126 Bagnato A, Moretti C, Frajese-G, Catt KJ 1991 Gonadotropininduced expression of receptors for growth hormone releasing factor in cultured granulosa cells. Endocrinology 1282889-2894 Vale W, Vaughan J, Jolley D, Yamamoto G, Bruhn T, Seifert H, Perrin M, Thorner M, Rivier J 1986 Assay of growth hormonereleasing factor. Methods Enzymol 124:389-401 Maniatis T, Frisch EF, Samhrook J 1982 Molecular Cloning-A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor Aviv H, Leder P 1972 Purification of biologically active globin messenger RNA by chromatography on oligothymidilic acid cellulose. Proc Nat1 Acad Sci USA 69:1408-1414 Mayo EK, Cerelli GM, Lebo RV, Bruce BD, Rosenfeld MG, Evans RM 1985 Gene encoding human growth hormone-releasing factor precursor: structure, sequence, and chromosome assignment. Proc Nat1 Acad Sci USA 82:63-67 Pescovitz OH, Berry SA, Laudon M, Benjonathan N, MartinMvers A. Hsu S. Lamhros TJ. Felix AM 1990 Localization and growth hormone. (GH)-releasing activity of rat testicular GHreleasing hormone-like peptide. Endocrinology 127~2336-2342 Mayo EK, Evans RM, Rosenfeld MG 1986 Genes encoding mammalian neuroendocrine peptides. Annu Rev Physiol48:431-446 Palmiter RD, Brinster RL, Hammer RE, Trumhauer ME, Rosenfeld MG 1982 Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature 300~611-615 Frohman LA, Downs TR, Kashio Y, Brinster RL 1990 Tissue distribution and molecular heterogeneity of human growth hormone-releasing factor in the transgenic mouse. Endocrinology 127:2149-2156 Joubert-Bression D, Benlot C, Lagoguey A, Garnier P, Brandi AM, Gautron JP, Legrand JC, Peillon F 1989 Normal and growth hormone (GH)-secreting adenomatous human pituitaries release somatostatin and GH-releasing hormone. J Clin Endocrinol Metab 68572-577 Hernandez ER, Botero L. Ricciarelli E, Follicle-stimulating hormone (FSH) inhibits ovarian granulosa cell IGF-I gene expr&sion. 73rd Annual Meeting of The Endocrine Society, Washington DC, 1991 (Abstract 334:114) De Gennaro Colonna V, Cattaneo E, Cocchi D, Muller EE, Maggi A 1988 Growth hormone regulation of growth hormone-releasing hormone gene expression. Peptides 9:985-988 Frohman MA, Downs TR, Chomazynski P, Frohman LA 1989 Cloning and characterization of mouse growth hormone-releasing hormone (GRH) cDNA: increased GRH mRNA levels in the growth hormone deficient lit/lit mouse. Mol Endocrinol 3:15291536 Lowe WL, Roberts CT, Lasky SR, Le Roith D 1987 Differential expression of alternative 5’ untranslated regions in mRNAs encoding rat insulin-like growth factor I. Proc Nat1 Acad Sci USA 848946-8950 Jones SA, Brooks AN, Challis JRG 1989 Steroids modulate corticotropin-releasing hormone production in human fetal membranes and placenta. J Clin Endocrinol Metab 68825-830 Jingami H, Matsukura S, Numa S, Imura H 1985 Effects of adrenalectomy and dexamethazone administration on the level of preprocorticotropin releasing factor messenger ribonucleic acid (mRNA) in the hypothalamus and adrenocorticotropin/@lipotropin precursor in the pituitary in rats. Endocrinology 117:13141320
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 15:00 For personal use only. No other uses without permission. . All rights reserved.
Vol. 130, No. 3 Printed
in U.S.A.
Expression of the Growth Hormone-Releasing Hormone Gene and Its Peptide Product in the Rat Ovary* ANNA
BAGNATOt, COSTANZO J. CATT
MORETTI,
JUNJI
OHNISHI,
GAETANO
FRAJESE,
AND KEVIN
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; and Clinica Medica V, University Rome La Sapienza (G.F.), 00161 Rome, Italy
ABSTRACT. GH-releasing hormone (GHRH) is a potent CAMP-mediated agonist in the rat ovary, where it binds to a common vasoactive intestinal peptide/GHRH receptor and enhances the actions of FSH on granulosa cell maturation. A GHRH-like peptide has been detected by immunocytochemistry in the human ovary and by RIA in follicular fluid, suggesting local synthesis of the peptide. In rat ovarian poly(A)+ RNA, Northern blot hybridization analysis with a 32P-labeled 48-nucleotide (nt) rat GHRH oligonucleotide probe revealed the presence of one major and two minor mRNA species. The major ovarian GHRH mRNA (1750 nt) was much larger than that present in hypothalamus and placenta (750 nt), but was similar to that observed in the rat testis. Two well defined higher mol wt forms of 3.2 and 3.6 kilobases were also present and probably represent unprocessed precursors of the 1750-nt mRNA. Further evidence of GHRH gene expression in the ovary and testis was provided by reverse transcription polymerase chain reaction of ovarian mRNA and restriction enzyme analysis of the amplified
R
AT GH -releasing hormone (GHRH) is a 43-amino acid hypothalamic peptide which regulates the synthesis and secretion of GH in the anterior pituitary gland (1). The rat GHRH gene is 10 kilobases (kb) in length and contains 5 exons that encode a 104-amino acid precursor to the 5.2-kilodalton (kDa) rat GHRH peptide (2). The intron-exon organization of the GHRH gene is similar to those of the vasoactive intestinal peptide and glucagon genes, each of which encodes large precursors that generate several biologically active and structurally related petides by proteolytic processing (3). In the rat, GHRH-like immunoreactivity is largely localized to cell bodies in the arcuate nucleus and the medial perifornical region of the lateral hypothalamus, with fiber projections to the median eminence to form a dense
of
product. In addition, immunoreactive (ir) GHRH was detected in ovarian extracts and in the incubation medium of cultured rat granulosa cells. Ovaries from PMSG-treated female rats, aged 22-27 days, contained 400 + 25 pg/g ir-GHRH. The GHRH content of the hypothalamus of the same animals was 2.9 + 0.1 rig/g. Cultured rat granulosa cells released 20 f 0.1 pg ir-GHRH/ 4 X 10’ cells.3 h into the incubation medium. The GHRH immunoreactivity detected in ovarian extracts coeluted on gel filtration chromatography with authentic rGHRH (5.2 kilodaltons). A larger form of ir-GHRH (-16.5 kilodaltons) was also present. These data demonstrate that the rat ovary contains a 1750-nt transcript that could arise from the GHRH gene by tissue-specific initiation, alternative splicing, or transcript termination. The translation product of this mRNA is the same similar size as the rat hypothalamic neuropeptide and may promote follicular maturation by autocrine or paracrine modulation of the stimulatory action of FSH on granulosa cell function. (Endocrinology 130: 1097-1102,1992)
accumulation of GHRH-containing processes and terminals (4). Like several other neuropeptides, GHRH is also present in extrahypothalamic tissues (5), including the gastrointestinal tract (6), tumors of neuroendocrine origin (7), placenta (a), and testis (9). However, the characterization and sequence analysis of the cDNA encoding the precursor peptide have been reported only in hypothalamus (2) and placenta (10). The placental and hypothalamic GHRH transcripts are similar in size [-750 nucleotides (nt)], as are their peptide products (5.2 kDa) (11). Despite these similarities, it has been suggested that GHRH gene expression may be differentially regulated in the two tissues, reflecting different sites of transcription initiation and raising the possibility of common evolutionary mechanisms for tissue-specific regulation of neuropeptide production in these organs (12). In addition, a larger mRNA species (1750) that hybridizes to GHRH cDNA has been observed in the testis of postpubertal rats, where immunoreactive (ir-) GHRH is detectable in amounts of about 1.6 rig/g tissue (13). A GHRH-like peptide has also been demonstrated
Received July 26,199l. Address requests for reprints to: Dr. Kevin J. Catt, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, Building 10, Room Bl-L400, Bethesda, Maryland 20892. * This work was supported in part by a grant from Industria Pharmaceutica Serono (Rome, Italy). t Supported by a grant from Sigma-Tau (Rome, Italy). 1097
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 15:00 For personal use only. No other uses without permission. . All rights reserved.
EXPRESSION
1098
OF GHRH
in human ovarian tissue by immunoperoxidase staining (14), and ir-GHRH has been detected in human follicular fluid (15). GHRH acts through vasoactive intestinal peptide/GHRH receptors in granulosa cells to promote maturation by amplifying the stimulatory action of FSH on CAMP production and granulosa cell differentiation (16). In addition, the expression of GHRH receptors is increased by the CAMP-mediated actions of FSH in granulosa cells, suggesting a positive autoregulatory action of this peptide on FSH-induced follicular maturation (17). The present study was performed to characterize the expression of GHRH in rat ovaries and to analyze its molecular heterogeneity. Materials
and Methods
Animals
Twenty-one-day-old Sprague-Dawleyrats obtained from Taconic Farm (Germantown, NY) were injected SCon day 23 with 10IU PMSG (Sigma, St. Louis, MO) in 0.9% NaCl. Forty-eight hours later (day 25), the rate were killed by decapitation to minimize stressand potential changesin hypothalamic function associatedwith anesthesia,and their ovarieswere removed. Studieswerealsoperformed in immature femalerata implanted scon day 21 with lo-mm diethylstilbestrol (DES)-filled Silastic capsulesto stimulate granulosa cell proliferation. These animalswere alsotreated on day 25 with 10 IU PMSG and killed 24,48, and 72 h later. Testes for RNA extraction were removed from postpuberal male rats (-90 days). Hypothalamic tissue was removed to a depth of 3-4 mm from the optic chiasm anteriorly to the mammillary bodies posteriorly, along the hypothalamic sulci laterally. Placentas (mean weight, 1.1 g) wereobtained from pregnant rats killed on day 20 of gestation. The individual tissueswere immediately weighed, then frozen in liquid nitrogen, and stored at -70 C. Preparation
and culture of granulosa
cells
Granulosacells were prepared from ovaries of PMSG- and DES- and PMSG-treated immature female rats, as previously described(16). Cell viability, determined by trypan blue exclusion, was usually greater than 80%. Approximately 4 X 10’ viable cells were added to 24-well plastic culture plates in a total volume of 1 ml McCoy’s 5a medium supplementedwith 10mM HEPES (pH 7.4), 4 mM L-glutamine, 100U/ml penicillin, 100 rg/ml streptomycin sulfate, and 0.1% BSA. Cell cultures were incubated at 37 C in a humidified 95% air-5% COZ incubator for 3 h; the media were then removed and stored at -70 C for subsequentextraction. Extraction
of GHRH from tissue and medium
Tissueswere homogenizedin ice-cold 2 M acetic acid. The homogenatewas placed in a boiling water bath for 10 min to destroy peptidaseactivity and then centrifuged at 10,000 X g for 10 min. The supernatant was lyophilized and subsequently dissolvedin chromatography solvent (1% trifluoracetic acid), then centrifuged to remove any insoluble residue. Tissue extracts were slowly passedthrough Sep-Pak Cl8 cartridges
GENE IN RAT OVARY
Endo Vol130
l l
1992 No 3
(Waters Associates, Milford, MA), followed by elution with methanol. The eluates were evaporated under vacuum and resuspendedin 0.1 M NaCl, 0.05 M Na2P0,, 0.025 M EDTA, 0.1% Na azide, 0.1% BSA, and 0.1% Triton, pH 7.4 (RIA buffer). Media from cultured granulosa cells were similarly extracted after acidification by passagethrough Sep-Pak cartridges and methanol elution. RIA of GHRH
GHRH was measuredby the RIA procedure of Vale et al. (18) with minor modifications. Sampleswere equilibrated for 24 h at 4 C with specific GHRH antibody (Peninsula Laboratories, Belmont, CA); then, about 18,000 cpm [lz61]rat (r) GHRH (Peninsula) were added,and incubation was continued for an additional 24 h. Free tracer wasseparatedfrom antibodybound tracer by the addition of sheep antiserum to rabbit immunoglobulin and continuation of incubation for an additional 24 h. The sensitivity of the assaywas8 pg/tube, and the standard curve waslinear between 8-120 pg/tube. Gel filtration
chromatography
Lyophilized ovarian extracts were resuspendedin 0.5 ml 0.2 acetic acid-0.1% BSA and centrifuged at 10,000X g for 10 min. The supernatants were applied to a 1.2 x 50-cm column packed with Sephadex G-50 gel (Pharmacia, Piscataway, NJ) previously equilibrated in the samesolution. The columnswas eluted at a flow rate of 8.2 ml/h at 4 C, and l-ml fractions were collected, lyophilized, and reconstituted for RIA. Markers used for mol wt calibration of the column were: blue dextran, cytochrome-c, rGHRH, and [3H]inositol. M
Northern
blot analysis
Isolation of total RNA from frozen tissue wasperformed by the guanidinium thiocyanate-cesiumchloride method (19). All RNA used in these experiments had OD 260/280 ratios of 2.0 -C0.2, and quantitation was basedon measurementof OD 260. In addition, samplesfrom all extractions were run on denaturing gelsto screenfor intact 18s and 28s ribosomalRNA bands. Poly(A)+ RNA wasenriched by oligo(dT)-cellulosechromatography (20). Aliquots of 20 ~1 containing 20 rg poly(A)+ RNA were subjectedto electrophoresison 1.5% agarosegelscontaining 2.2 M formaldehyde and transferred to Nytran membranes (Schleicher and Schuell, Keene, NH). Size markers were included as a RNA ladder (Bethesda Research Laboratories, Gaithersburg, MD). Prehybridization was performed for 3 h at 42 C in 1% sodium dodecyl sulfate (SDS), 10 X Denhardt’s solution, 50 Fg/ml tRNA, 50 rg/ml shearedsalmonspermDNA (5 Prime-3 Prime, Inc., West Chester,PA), 6 X SSPE [l X SSPE = 180mM NaCl, 10 mM sodiumphosphate (pH 7.4), and 1 mM EDTA]. A synthetic 48-mer oligonucleotide of rGHRH mRNA (generouslyprovided by Dr. A. W. Margioris) wasend labeled by incubation with T4 polynucleotide kinase (New England BioLabs, Beverly, MA) and [T-~*P]ATP (4500 Ci/ mmol; ICN, Costa Mesa, CA) to a specific activity of l-5 x 10’ cpm/pg DNA. Hybridization with the probe wasperformed for 18 h at 55 C (2.9 x lo6 cpm/ml) in 6 X SSPE-1% SDS. The nylon membraneswere washedthree times at room temperature and once at 55 C in 6 x SSPE (pH 7.4)-l% SDS. The
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 15:00 For personal use only. No other uses without permission. . All rights reserved.
EXPRESSION
OF GHRH
filters were exposed at -70 C to Kodak XAR-S film (Eastman Kodak, Rochester, NY) for varying times. Polymerase chain reaction (PCR) amplification First strand cDNA synthesis was carried out on 1 pg poly(A)+ RNA using the following components: 200 U Moloney murine leukemia viral reverse transcriptase (Bethesda Research Laboratories), 1 U RNase inhibitor (Promega, Madison, WI), 1 mM dNTPs, and 100 pmol random hexamer primers (Boehringer Mannhein, Indianapolis, IN) in a final volume of 20 ~1 50 mM Tris-Cl (pH 8.3), 75 mM KCl, 10 mM dithiothreitol, and 3 mM MgC12. The mixture was incubated for 10 min at room temperature, followed by 120 min at 37 C, and the reaction was terminated by heating at 94 C for 10 min. The cDNA mixture was supplemented with 2.5 U Thermus aquaticus polymerase (Boehringer), 50 pmol of each primer, 7.5 ~1 10 mM Tris-Cl (pH 8.3), 50 mM KCl, 1.5 M~C&, and 0.01% gelatin and water to a final volume of 100 ~1.The sequencesof the PCR primers (Lofstrand Laboratories, Gaithersburg, MD) usedwere GHRHspecific senseprimer (5’-GCACGAAATCATGAACAGGC-3’) from the region of the third exon and antisenseprimer (5’CTCTCCAGGGCCATCTGCTT-3) from the region of the fourth exon. Forty cycles of amplification were carried out under the following conditions: denaturation at 94 C for 1 min, annealing at 55 C for 2.5 min, and extension at 72 C for 3 min. MboI-digested and uncut PCR products were size-fractionated by electrophoresisin a 3% agarosegel. PCR products were subclonedinto pGEM-3Z (Promega) using blunt-ended ligation. The DNA sequencewas analyzed by the dideoxy chain termination method (SequenaseII, U.S. Biochemical Corp., Cleveland, OH).
Results ir-GHRH was present in the rat ovary (Fig. 1) at a mean concentration of 400 + 25 pg/g. No GHRH was
GENE IN RAT OVARY
1099
detected in extracts from muscle, which served as a negative control tissue. The GHRH content of the hypothalamus, measured as a positive control, was 2.9 + 0.1 rig/g. Serial dilution of hypothalamic extracts purified on Sep-Pak Cl8 cartridges gave a binding curve parallel to that of standard GHRH. The same result was obtained with ovarian extracts, indicating that the ovarian GHRH-like peptide is immunologically indistinguishable from authentic GHRH. Cultured rat granulosa cells were found to release immunoreactive GHRH-like material (20 f 0.1 pg/4 x 10’ cells- 3 h) into the incubation medium. The authenticity of ir-GHRH was indicated by its parallel binding inhibition with that of the standard rGHRH in the RIA (Fig. 2). The Sephadex G-50 gel filtration chromatography protile of the ir-GHRH in an extract of ovarian tissue is shown in Fig. 3. The major peak of ir-GHRH was eluted with a Kd of 0.50, coincident with the elution position of synthetic rat GHRH chromatographed on an identical column. In addition, a second peak of immunoreactivity was eluted earlier at a Kd of 0.30. The calculated relative molecular size of the early eluting peak was approximately 16,500, and that of the major peak was 5,000. Northern blot hybridization analyses of poly(A)+ RNA isolated from testes of postpuberal rats (90 days), placentas (20 days gestation), and ovaries of PMSG-treated immature rats are shown in Fig. 4. The hybridization signal of poly(A)+ ovarian RNA revealed that the major GHRH mRNA was a 1750-nt species, much larger than that extracted from rat placenta (750 nt), but similar to that present in the rat testis. Two well defined larger mRNAs of 3.2 and 3.6 kb were also observed in the ovary pl Samples
Tissue I 0.12
r
Equivalent/Tube
I 0.25
I 0.5
I
12.5
2.5
I
I
50
100
50
100
I 1.0
SO-
E60 x0 ? m 40-
8 60x0 em 40-
I
I 10
I 20 rGHRH (pg/tube)
I 50
I 100
I
FIG. 1. Inhibition of [l%I]rGHRH binding to specific antiserum by standard rGHRH (O), hypothalamic rGHRH (W), and ovarian rGHRH (A). The recovery of rGHRH through the tissue extraction procedure was 65-70%, as monitored by the addition of unlabeled rGHRH before homogenization. B/B.,, Bound/free ratio.
10
20 rGHRH (pghube)
FIG. 2. Parallel inhibition of [‘261]rGHRH binding to specific antiserum by standard rGHRH (0) and rGHRH released from granulosa cells (4 x 10’ cells/ml) cultured for 3 h (W). The recovery of rGHRH through the medium extraction procedure was 65-90%, as monitored by the addition of unlabeled rGHRH before extraction. B/B,, Bound/ free ratio.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 15:00 For personal use only. No other uses without permission. . All rights reserved.
EXPRESSION
1100
OF GHRH
GENE
IN RAT
OVARY
Endo * 1992
Vol130*No3
Fraction I 0
Number I
I 1.0
0.5 Kd
FIG. 3. Distribution profile of ovarian rGHRH (B) on a Sephadex G50 (fine) column eluted with 0.2 N acetic acid-0.1% BSA. The column dimensions were 50 x 1.2 cm, and the flow rate was 8.2 ml/h. The positions of the void volume (Vo) and the elution volumes of cytochrome-c (mol wt, 12,500), rGHRH (mol wt, 5,200), and inositol ([3H] Ins.; mol wt, 108) are also shown. Identical results were obtained in two such experiments performed on separate ovarian extracts. A
6 E p
r-i”‘+ ---
p --
.g
SZ$;g%l2 -
-Origin
=ggg -1:75
kb
-0.75
kb
2
DES + PMSG i:L i i’ -
-
f
-Origin
=3% :I: -1.75 kb -0.75
WlObp 43 bp r47bp
FIG. 5. Ethidium bromide-stained gel of GHRH mRNA amplified from rat ovary and testis by reverse transcription PCR and subjected to restriction enzyme analysis. The first lane contains size markers from f?laeIII-digested $X174 DNA. The expected sire of the uncut rat GHRH PCR product was 110 nt, and those of the Mb01 fragments were 63 and 47 nt.
Ovary P, g
;
234 194= 11872-
kb
FIG. 4. Northern blot analysis of GHRH mRNA. A, Twenty micrograms of poly(A)+ RNA isolated from testis, placenta, and ovary. B, Twenty micrograms of RNA isolated from rat muscle (negative control) and 10 rg poly(A)+ RNA isolated from rat testis and ovary. Other lanes show autoradiographs of ovarian GHRH mRNA during follicular development. DES-implanted rats were treated with 10 IU PMSG and killed after 24, 48, and 72 h. The deduced sizes of mRNA species are indicated on the right.
and may represent unprocessed precursors of the 1750 nt mRNA (Fig. 4A). No GHRH hybridization signal was detected with muscle RNA (negative control). Northern analysis was also performed on 10 pg poly(A)+ RNA isolated from ovaries of normal and DES-implanted rats after treatment with PMSG (10 IU) at specific times. The three major bands (1.75, 3.2, and 3.6 kb) were present during follicular development. After PMSG treatment of DES-implanted rats, the levels of GHRH mRNA measured 24 h after treatment with PMSG were similar in normal and DES-treated rats and declined at 48 and 72 h (Fig. 4B). Further evidence of GHRH gene expression in the ovary and testis was provided by the use of reverse transcription PCR. As shown in Fig. 5, PCR products of 110 nt, the predicted size based on the primers employed
and the sequence of the corresponding rat GHRH-(l43) segment, were amplified from ovarian and testicular cDNA. These PCR products were digested by MboI, yielding two fragments of the expected sizes (63 and 47 nt). DNA sequence analysis of these products revealed 100% homology with regions of exons 3 and 4 of rat hypothalamic GHRH cDNA (data not shown). Discussion These observations demonstrate that the rat ovary, in addition to being a target of GHRH action, is a site of GHRH-like mRNA and peptide synthesis. Our data indicate that the GHRH gene is expressed in the rat ovary and that its product is translated into a peptide similar to GHRH. Northern blot analysis showed that the size of rat ovarian GHRH transcript was about 1750 basepairs, similar to the mRNA encoding testicular GHRH. The rat prepro-GHRH gene encodes a 104-amino acid precursor in five exons, which, like the human GHRH gene, span more than 9 kb. The human and rat preproGHRHs are not highly conserved, and the mature rat hypothalamic peptide contains only 43 amino acids and lacks a COOH-terminal amide. The GHRH peptide sequence encoded by cloned cDNA is followed by a 30amino acid peptide of unknown function (2). The nucleotide sequences of the rat and human GHRH genes differ in the 3’-domain because of changes in the
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 15:00 For personal use only. No other uses without permission. . All rights reserved.
EXPRESSION
OF GHRH
splicing of intron D (21), which is the site of evolutionary divergence for other members of the glucagon gene superfamily (3). This suggests that alterations in mRNA transcripts from the GHRH gene could be more likely in the 3’regions of the ovarian transcript. The 1750-nt mRNA species in rat ovary and testis may represent a physiologically significant alternative gene transcript derived from an unprocessed precursor of the 750-nt RNA, or arising during tissue-specific initiation, alternative splicing, or transcription termination of the GHRH gene. The presence of a larger GHRH mRNA and a GHRHlike peptide in the rat gonads raises several questions, including the possible existence of a GHRH-like gene containing additional coding regions, the heterogeneity of the 3’nontranslated sequence, and the presence of different transcriptional start sites. The ir-GHRH present in ovarian extracts was shown by gel filtration chromatography to be similar in size to authentic rat GHRH. A higher mol wt form (-16,000 kDa) was also detected by gel filtration and may represent pro-GHRH, as predicted by the larger mRNA species observed on Northern blots. Our data also show that cultured rat granulosa cells secrete a GHRH-like substance, suggesting a role for this peptide as a mediator in the transfer of intercellular information at the ovarian level. The presence of a GHRH mRNA species larger than that found in the hypothalamus and of a GHRH-like substance has been described in the rat testis (9). Although rGHRH and testicular GHRH share biological similarities, they do not appear to be identical, since they exhibit different HPLC elutions profiles and molecular sizes. In fact, on the basis of SDS-gel analysis, testicular GHRH was reported to be almost 4 times larger than hypothalamic GHRH (22). In the testis, the larger mRNA species might encode a longer peptide, which contains a region that cross-reacts with the epitope recognized by the GHRH antiserum or results from postranslational modifications, leading to a product of larger molecular size. In the endocrine and nervous systems, several gene products that serve multiple and complex roles are generated by tissue-specific RNA and precursor processing (23). Recently, utilization of the transgenic technique (24) has provided a valuable approach to the problem of molecular heterogeneity and tissue distribution of neuropeptides. In transgenic mice expressing the human (h) GHRH gene, the highest concentration of GHRH was found in the pituitary gland, followed by the pancreas (25). The finding that pituitary tissue of transgenic mice contains high levels of GHRH confirms the recent observation related to the capacity of perifused human hypophyseal fragments to release GHRH (26). In transgenie mice, intermediate levels of ir-GHRH were present in hypothalamus and liver and lower levels in visceral organs, heart, and gonads. Gel filtration and assay of ir-
GENE
IN RAT
OVARY
1101
hGHRH in extracts of pituitary, brain, pancreas, and gut revealed the mature hormone and a more hydrophobic form, which probably represents the hGHRH precursor (pro-GHRH) on the basis of its estimated molecular size (9 kDa) (25). The gonadal expression of hGHRH-(1-44) in transgenic mice demonstrates the presence of tissuespecific enzymes that are capable of processing the preproGHRH peptide. In this study we observed that the ovarian levels of GHRH mRNA in PMSG-treated normal and DES-implanted rats were similar. The mRNA levels measured in DES-treated rats declined 48 and 72 h after PMSG treatment, suggesting a regulatory action of gonadotropins on GHRH gene expression. It has been recently reported that FSH treatment of hypophysectomized rats or whole ovarian dispersates for up to 48 h caused timeand dose-dependent decrements in insulin-like growth factor gene expression (27). Whether the decrease in GHRH mRNA observed after PMSG treatment reflects a decline after transient stimulation of GHRH gene expression or an inhibitory action of the gonadotropin has yet to be determined. In the hypothalamus, GH exerts a negative feedback action on the expression of GHRH mRNA, which increases significantly after hypophysectomy (28). In hypophysectomized rats and in the lit/lit mouse, a model of isolated GH deficiency due to abnormal GHRH receptor expression in somatotrophs, the increase in hypothalamic GHRH mRNA is not accompanied by a change in placental GHRH mRNA (29). In the testis, where GHRH mRNA appears to be developmentally regulated, the GHRH trancript decreases after hypophysectomy, probably as a consequence of gonadotropin deficiency. These observations suggest that in the ovary, as in other peripheral tissues, the transcript would not be regulated by GH. Furthermore, structural and physiological studies have demonstrated differences between hypothalamic and placental regulation of mouse and rat GHRH gene expression. Despite the presence of mRNAs of similar size, the existence of alternative 5’-untranslated regions has revealed differencies in the sites of initiation of transcription in the two tissues (10, 11). This is also evident for the rat insulin-like growth factor-I gene, which is expressed and regulated by GH in a tissuespecific manner, since its transcripts contain at least three alternative 5’-untranslated regions associated with a common sequence encoding the mature peptide (30). Also, the CRF gene is differentially regulated by glucocorticoids in specific tissues, being suppressed at the hypothalamic level and increased in the placenta (31, 32). These examples demonstrate that tissue-specific expression of a gene can generate the same biologically active mature peptide, as predicted by its exons, through different routes of transcription and precursor biosyn-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 15:00 For personal use only. No other uses without permission. . All rights reserved.
EXPRESSION
1102
OF GHRH
thesis. In conclusion, these findings demonstrate that expression of the GHRH gene in the rat ovary leads to the production of a GHRH-like peptide of similar size to the rat hypothalamic neuropeptide. These data and our previous observations indicate that this peptide can promote follicular maturation by autocrine modulation of the stimulatory action of FSH on granulosa cell function. The granulosa cell serves as a site of production, receptors, and actions of GHRH, which may, thus, exert a positive autoregulatory action on ovarian follicular maturation. References 1. Frohman LA, Jansson JO 1986 Growth hormone releasing hormone. Endocr Rev 7:223-253 2. Mayo KE, Cerelli GM, Rosenfeld MG, Evans RM 1985 Characterization of cDNA and genomic clones encoding the precursor to rat hypothalamic growth hormone-releasing factor. Nature 314:464467 3. Bell GI 1986 The glucagon superfamily: precursor structure and gene organization. Peptides [Suppl l] 7:27 4. Merchenthaler I. Vigh S, Schallv AV, Petrusz P 1984 Immunocytochemical localization of growth hormone-releasing factor in the rat hypothalamus. Endocrinology 114:1082-1085 5. Thorner MO, Vance ML, Evans WS, Ho K, Rogol AD, Blizzard RM, Furlanetto R, Rivier J, Vale W 1986 Growth hormone releasing factor and somatomedin C production: extrahypothalamic localization and possible functional significance. Acta Endocrinol [Suppl] (Copenh) 276:34-40 6. Bruhn TO, Mason RT, Vale W 1985 Presence of growth hormone releasing factor-like immunoreactivity in rat duodenum. Endocrinology 117:1710-1712 7. Rivier J, Spiess J, Thorner M, Vale W 1982 Characterization of a growth hormone-releasing factor from a human pancreatic islet tumour. Nature 300:276-278 8. Meigan G, Sasaki A, Yoshinaga K 1988 Immunoreactive growth hormone-releasing hormone in rat placenta. Endocrinology 123:1098-1102 9. Berry SA, Pescovitz OH 1988 Identification of a rat GHRH-like substance and its messenger RNA in rat testis. Endocrinology 122661-663 10. Surh ST, Rahal JO, Mayo KE 1989 Mouse growth hormonereleasing hormone: precursor structure and expression in brain and placenta. Mol Endocrinol3:1693-1700 11. Margioris AN, Brockmann G, Bohler HCL, Grino M, Vamvakopoulos N, Chrousos GP 1990 Expression and localization of growth hormone releasing hormone messenger ribonucleic acid in rat placenta: in vitro secretion and regulation of its peptide product. Endocrinology 126151-158 12. Mizobuchi M, Frohman MA, Downs TR, Frohman LA 1991 Tissue specific transcription initiation and effects of growth hormone (GH) deficiencv on the regulation of mouse and rat GH-releasing hormone gene-in hypothilamus and placenta. Mol Endocrinoi 5:476-484 13. Berry SA, Pescovitz OH 1988 Ontogeny and pituitary regulation of testicular growth-hormone releasing hormone-like messenger ribonucleic acid. Endocrinology 127~1404-1401 14. Moretti C, Fabhri A, Gnessi L, Bonifacio V, Bolotti M, Arizzi M, Nazzicone Q, Spera G 1990 Immunohistochemical localization of growth hormone releasing hormone in human gonads. J Endocrinol Invest. 13:301-305 15. Moretti C, Fahhri A, Gnessi L, Forni L, Fraioli F, Frajese G 1989
GENE
16. 17. 18. 19. 20. 21.
22.
23. 24.
25.
26.
27.
28. 29.
30.
31. 32.
IN RAT
OVARY
Endo. Voll30.
1992 No 3
GHRH stimulates follicular growth and amplified FSH-induced ovarian folliculogenesis in women with anovulatory infertility. In: Frajese G, Steinberg F, Rodriguez-Rigau LJ (eds) Reproductive Medicine: Medical Therapy. Proceeding of the Second International Symposium on Reproductive Medicine. Elsevier, New York, no. 875:103-112 Moretti C, Bagnato A, Solan N, Frajese G, Catt KJ 1990 Receptor mediated actions of growth hormone-releasing factor on granulosa cell differentiation. Endocrinoloav 127:2117-2126 Bagnato A, Moretti C, Frajese-G, Catt KJ 1991 Gonadotropininduced expression of receptors for growth hormone releasing factor in cultured granulosa cells. Endocrinology 1282889-2894 Vale W, Vaughan J, Jolley D, Yamamoto G, Bruhn T, Seifert H, Perrin M, Thorner M, Rivier J 1986 Assay of growth hormonereleasing factor. Methods Enzymol 124:389-401 Maniatis T, Frisch EF, Samhrook J 1982 Molecular Cloning-A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor Aviv H, Leder P 1972 Purification of biologically active globin messenger RNA by chromatography on oligothymidilic acid cellulose. Proc Nat1 Acad Sci USA 69:1408-1414 Mayo EK, Cerelli GM, Lebo RV, Bruce BD, Rosenfeld MG, Evans RM 1985 Gene encoding human growth hormone-releasing factor precursor: structure, sequence, and chromosome assignment. Proc Nat1 Acad Sci USA 82:63-67 Pescovitz OH, Berry SA, Laudon M, Benjonathan N, MartinMvers A. Hsu S. Lamhros TJ. Felix AM 1990 Localization and growth hormone. (GH)-releasing activity of rat testicular GHreleasing hormone-like peptide. Endocrinology 127~2336-2342 Mayo EK, Evans RM, Rosenfeld MG 1986 Genes encoding mammalian neuroendocrine peptides. Annu Rev Physiol48:431-446 Palmiter RD, Brinster RL, Hammer RE, Trumhauer ME, Rosenfeld MG 1982 Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature 300~611-615 Frohman LA, Downs TR, Kashio Y, Brinster RL 1990 Tissue distribution and molecular heterogeneity of human growth hormone-releasing factor in the transgenic mouse. Endocrinology 127:2149-2156 Joubert-Bression D, Benlot C, Lagoguey A, Garnier P, Brandi AM, Gautron JP, Legrand JC, Peillon F 1989 Normal and growth hormone (GH)-secreting adenomatous human pituitaries release somatostatin and GH-releasing hormone. J Clin Endocrinol Metab 68572-577 Hernandez ER, Botero L. Ricciarelli E, Follicle-stimulating hormone (FSH) inhibits ovarian granulosa cell IGF-I gene expr&sion. 73rd Annual Meeting of The Endocrine Society, Washington DC, 1991 (Abstract 334:114) De Gennaro Colonna V, Cattaneo E, Cocchi D, Muller EE, Maggi A 1988 Growth hormone regulation of growth hormone-releasing hormone gene expression. Peptides 9:985-988 Frohman MA, Downs TR, Chomazynski P, Frohman LA 1989 Cloning and characterization of mouse growth hormone-releasing hormone (GRH) cDNA: increased GRH mRNA levels in the growth hormone deficient lit/lit mouse. Mol Endocrinol 3:15291536 Lowe WL, Roberts CT, Lasky SR, Le Roith D 1987 Differential expression of alternative 5’ untranslated regions in mRNAs encoding rat insulin-like growth factor I. Proc Nat1 Acad Sci USA 848946-8950 Jones SA, Brooks AN, Challis JRG 1989 Steroids modulate corticotropin-releasing hormone production in human fetal membranes and placenta. J Clin Endocrinol Metab 68825-830 Jingami H, Matsukura S, Numa S, Imura H 1985 Effects of adrenalectomy and dexamethazone administration on the level of preprocorticotropin releasing factor messenger ribonucleic acid (mRNA) in the hypothalamus and adrenocorticotropin/@lipotropin precursor in the pituitary in rats. Endocrinology 117:13141320
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 15:00 For personal use only. No other uses without permission. . All rights reserved.
