0021-972x/92/7501-0281$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society
Vol. 75, No. 1 Printed in U.S.A.
Growth Hormone-Releasing Hormone-Like Messenger Ribonucleic Acid and Immunoreactive Peptide Are Present in Human Testis and Placenta* SUSAN A. BERRY, CAROLYN H. SRIVASTAVA, LESLIE WILLIAM R. PHIPPS, AND ORA HIRSCH PESCOVITZ
R. RUBIN,
Departments of Pediatrics and Obstetrics and Gynecology and the Institute of Human Genetics, University of Minnesota, Minneapolis, Minnesota 55455; and the Departments of Pediatrics, Physiology/Biophysics, and Obstetrics and Gynecology, Indiana University, Indianapolis, Indiana 46202 ABSTRACT
protein). The origin of these peptides was confirmed by extracting total RNA from human testis and placenta, with analysis on Northern blots probed with riboprobes for rat hypothalamic GHRH cDNA and human pancreatic tumor GHRH cDNA. Human testis and placenta total RNA both contain an approximately 790-nucleotide RNA species similar in size to that reported in ectopic GHRH-producing human tumors. In addition, two larger hybridization signals were seen at 3000 and 4900 nucleotides. These data suggest that testis and placenta are extrahypothalamic sites of expression of the human GHRH gene. Normal expression of the GHRH gene in extrahypothalamic sites may include transcription of larger mRNA species than those observed in ectopic pathological sources of GHRH expression. (J Clin Endocrinol Metab 75: 281-284, 1992)
Although the sequence of human GH-releasing hormone (GHRH) has been determined, all of the information concerning gene expression has been based on pathological sources of ectopic GHRH, since the only established physiological source of GHRH in humans is the hypothalamus. We recently reported the presence of extrahypothalamic GHRH-like mRNA and immunoreactive material in rat testis and placenta. To determine if human testis and placenta also contain immunoreactive GHRH-like peptides, tissue extracts were analyzed using enzyme-linked immunosorbent assay methodology. Both tissues had detectable quantities of immunoreactive peptide recognized by a monoclonal antibody to synthetic human GHRH-(l-44) (testis, 2.4 ng/ g tissue, 0.68 ng/mg protein; placenta, 2.6 rig/g tissue, 0.36 ng/mg
G
H-RELEASING hormone (GHRH) is a hypothalamic neuropeptide that plays an important role in GH regulation. When produced in excess ectopically by tumors, acromegaly may result. To date, all studies examining mRNA expression for human (h) GHRH have been based on evaluation of RNA extracted from these GHRH-producing tumors (l-3), and little is known about physiological GHRH gene expression or normal extrahypothalamic sources of GHRH in humans. Using assays for GHRH peptide, the evidence supporting the presence of GHRH-like peptide in testis and placenta is confusing and contradictory. In humans, the presence of immunoreactive GHRH-like peptide in placenta was noted in one study (4), although this finding was not confirmed in an additional study that used nearly identical methodology (5). Only one study has suggestedthe presence of immunoreactive GHRH-like peptide in human testis (6), and previous measurementsof GHRH-like peptide in human seminal plasma were inconclusive, but suggested that measurable quantities of GHRH-like immunoreactivity were artifactual(7). In contrast, recent studiesin other species suggest that other organs do contain both GHRH-like imReceived August 15, 1991. Address all correspondence and requests for reprints to: Susan A. Berry, M.D. University of Minnesota Hospital, 420 Delaware Street SE, Minneapolis, Minnesota 55455. * This work was supported by the Vikings Children’s Fund and the Minnesota Medical Foundation (to S.A.B.), the James Whitcomb Riley Memorial Association (to O.H.P.), and NIH Grant DK-41899 (to S.A.B. and O.H.P.).
munoreactivity and mRNA. We recently reported the presence of GHRH-like immunoreactivity and mRNA in rat testis (8) and placenta (9), and others have found GHRH-like mRNA in mouse placenta and testis (10). To clarify this controversy regarding physiological extrahypothalamic GHRH-like peptides in human tissues,we assayedtesticular and placental extracts using enzyme-linked immunosorbent assay (ELISA) methodology and examined human testicular and placental RNA to determine whether a GHRH-like mRNA was present. Materials and Methods Samples Human testes were obtained from adult male subjects undergoing orchidectomy for prostatic cancer. These individuals had no known abnormality in testicular function. Samples of human placentas were obtained after delivery of term infants to healthy adult women. Rat testes were harvested from a normal 65.day-old male Sprague-Dawley rat, and rat placenta was harvested from a timed pregnant female at 19 days gestation. Animals were from Taconic Farms (Germantown, NY). Tissues were rapidly aliquoted into 0.5- to l-g fragments and flashfrozen in liquid nitrogen. They remained frozen at -80 C until peptide and RNA extraction was performed.
Tissue extraction
and ELISA
The extraction procedure involved minor modifications of previously described methodology (5, 11). The tissues were lyophilized overnight, homogenized in ice-cold 50% acidified methanol (0.1 N HCl-CH30H) including 10 Fg/mL phenylmethylsulfonylfluoride, 20 pg/mL aprotinin,
281
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BERRY ET AL. and 10 &mL pepstatin. Supernatants were fractionated over C-18 SepPak cartridges (Waters Associates, Milford, MA), as described. Extracts were pooled and lyophilized overnight. A direct-bindine ELISA usine a monoclonal antibodv to hGHRH generated against “hGHRH-(I-4>)-NH2 (Bachem, Torrence, CA) was used to measure GHRH immunoreactivity, as previously described (12). hGHRH-(1-44) from the same source was used for standard curves.
JCE & M .1992 Vol75.Nol
HP
HT
RP
RT St
Northern gel analysis Total RNA was extracted from tissues by guanidine hydrochloride or acid guanidininum thiocyanate (13, 14). Poly(A)+ RNA was isolated using oligo(dT)-cellulose. Ten to 20 rg total RNA or 5 pg poly(A)+ RNA were electrophoresed in a 1.5% agarose gel in 1% formaldehyde, 20 mM 4-morpholinepropanesulfonic acid, 5 mM sodium acetate, 1 mM EDTA, and 9 mM sodium hydroxide (15) and transferred to a nylon-based membrane. Filters were hybridized either to [32P]UTP-labeled GHRH cRNA riboprobe generated from a rat hypothalamic cDNA (R. Evans, The Salk Institute, La Jolla, CA), as previously described (16), or to a GHRH cRNA riboprobe generated from subcloning of the human cDNA (phGRF-54, R. Evans, The Salk Institute) (17) into EcoRI-BamHI-digested pGEM7z (Promega, Madison, WI), using SP6 RNA polymerase to generate this riboprobe. The specific activities of the labeled cRNAs were on the order of 0.5-l X 10’ cpm/pg DNA. Blots were prehybridized and hybridized as previously described (9). Filters were washed in 1 x SSPE [180 mM NaCl, 10 mM sodium phosphate (pH 7.5), and1 mM EDTA]0.1% SDS for 15 min four times at room temperature and twice in 0.1 X SSPE-0.1% SDS at 65 C for 20 min. The hybridized washed blots were exposed to Kodak XAR-5 film (Eastman Kodak, Rochester, NY) at -80 C.
Results ELISA ELISAs were performed on tissue extracts from human testis and placenta to determine if these normal tissues contain a peptide recognized by a monoclonal antibody against hGHRH-(1-44). This antibody is directed against the carboxy-terminus of hGHRH (12). The testicular sample had 2.4 ng GHRH/g wet weight of testis (0.68 ng/mg protein), and placenta had 2.6 ng GHRH/g wet wt placenta (0.36 ng/ mg protein). The least detectable dose of GHRH was 40 pg. Northern analysis To determine if the testicular peptide recognized by the human monoclonal antibody to hGHRH-(1-44) was represented in testicular and placental RNA by a mRNA species with homology to rat hypothalamic GHRH cDNA, a Northern blot was prepared. Total RNA extracted from human testis and placenta was examined and compared to total RNA extracted from adult rat testis and term rat placenta. Using a rat hypothalamic GHRH riboprobe, the RNA species seen in rat placenta in this gel was a broad band of about 650-850 nucleotides, and that in rat testis was about 1750 nucleotides, as have been described previously (9, 16). Human testis and placenta both have a single RNA transcript on these gels of about 790 nucleotides (Fig. 1). To further confirm that the signal demonstrated using the rat hypothalamic GHRH riboprobe hybridized to a molecular species that is like hGHRH, poly(A)+ RNA from additional human testis and placenta samples was prepared and probed with a riboprobe for human pancreatic tumor-derived GHRH (17). A hybridizing signal of the same size as that in rat
FIG. 1. Northern gel analysis of GHRH-like mRNA in human and rat tissues, using a rat hypothalamic GHRH riboprobe. Ten micrograms of total RNA were subjected to electrophoresis for each sample. HP, Human placenta; HT, human testis; RP, rat placenta; RT, rat testis. Sixty picograms of cold sense-strand hypothalamic cRNA were also placed on the gel (St). Size markers are 18s and 28s ribosomal RNAs. Exposure was for 3 days at -80 C.
GHRH riboprobe hybridizations was seen on blots hybridized with the human probe. In addition, two larger bands of about 4900 and 3000 nucleotides were seen in poly(A)’ RNA blots from these tissues (Fig. 2). Discussion In this report we demonstrate the presence of GHRH-like mRNAs in extrahypothalamic human tissues. Evolutionary conservation of this organ-specific expression suggests that the GHRH-like peptide in other tissues may be biologically important. As yet, the physiological role(s) of GHRH-like
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GHRH IN HUMAN
TESTIS AND PLACENTA
HP HI- RH
28s
18s -.*
FIG. 2. Northern gel analysis of GHRH-like mRNA in human and rat tissues, using a human pancreatic tumor GHRH riboprobe. Five micrograms of poly(A)+ RNA were subjected to electrophoresis for each sample. HP, Human placenta; HT, human testis; RH, rat hypothalamus. Size markers are 18s and 28s ribosomal RNAs. Exposure was for 2 days at -80 C.
peptide in nonhypothalamic tissues remains unknown. In rat testis, GHRH-like mRNA is developmentally regulated and dependent on the pituitary for expression in young animals. Expression in postnatal life increases beginning on about day 21 of life and reaches adult levels in the late prepubertal period (day 30) (16). Our current observation demonstrates that GHRH-like mRNA is present in adult testis, but expression in prepubertal testis has not been investigated. GHRH mRNA is also developmentally regulated in rat placenta, as it is most abundant in the third trimester of gestation (9).
283
Alterations in human placental expression with gestation are not known. In both rat (8) and mouse (10) testis, the predominant GHRH-like RNA species is larger than that in the hypothalamus. The reason for the difference in size is unknown. The predominant RNA species in rat placenta is similar in size to that in rat hypothalamus (9, 18). The sizes of human GHRH mRNAs from ectopic GHRH-secreting tumors have been variously reported as ranging between 750-820 nucleotides in length (l-3), although in 1 case (3) a larger species of 1600 nucleotides was also present. The smallest RNA species seen in human placenta and adult testis was similar in size to that found in GHRH-secreting tumors. The size of the RNA in human hypothalamus has not been reported. The observation of multiple, less intense, larger GHRH mRNA species was also noted in both rat and murine testis and placenta (9, 10, 16). Immunoreactive GHRH-like material in human testis was reported by Moretti et al. (6). They noted intense staining of the interstitial compartment of the testis with localization to Leydig cells (6). In contrast, in the rat we found that GHRHlike immunoreactivity was localized to spermatogenic cells (19). Because of this discrepancy in localization, the site of origin of GHRH-like peptide in the testis remains uncertain, although there may be species-dependent differences in localization. Losa et al. (7) examined human seminal plasma for GHRH-like immunoreactivity. In unextracted seminal plasma, they found 3.8 pg/L GHRH-like immunoreactivity using RIA, but concluded that this measurement was artifactual, because after extraction by absorption on hydrophobic Cl8 Sep-Pak cartridges, no further GHRH-like immunoreactivity could be measured (7). In contrast, the present ELISA uses a similar absorption onto Cl8 Sep-Pak cartridges with retention of immunoreactivity in whole testicular and placental extracts. We also were unable to measure GHRH-like immunoreactive material from fresh human semen (Rubin, L. R., and 0. H. Pescovitz, unpublished results). It is possible that if testicular GHRH-like peptide is present in seminal plasma, it is easily degraded into an inactive or nonassayable form, resulting in the loss of detectable activity in the extraction process. Our observation of a GHRH-like immunoreactive peptide and mRNA in human placenta is the first substantiation of the presence of both this neuropeptide and its mRNA in this human tissue, although rat placenta has been shown to have significant immunoreactive peptide and RNA present from days 13-14 of gestation (5, 9). One previous study looking for GHRH-like immunoreactivity in human placenta using RIA with a polyclonal antiserum against synthetic hGHRH(l-44) could not detect evidence of a GHRH-like peptide (5), although another group found results similar to those noted in this study (4). Several researchers have commented that the GHRH peptide is highly labile (4, 20), so that the brief delay due to separation and subsequent delivery of the placenta may result in degradation of the peptide. Our experience also suggests that very fresh or fresh-frozen tissue is required for detection of the peptide by ELISA. The localization of GHRH in human placenta is still unknown, al-
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BERRY
284
though GHRH mRNA has been localized to the cytotrophoblast in mouse and rat placenta by in situ hybridization (10, 18). The demonstration of both GHRH-like immunoreactive peptide and RNA in human testis and placenta considerably extends the evolutionary distance during which this tissuespecific gene expression has been observed and suggests additional roles for this neuropeptide beyond the regulation of GH releasefrom the pituitary. Despite abundant production of GHRH-like peptide in these tissues,they do not seem to contribute substantially to circulating GHRH (21); thus, extrahypothalamic GHRH-like peptides are more likely to act in an autocrine or paracrine fashion. The potential functions of these peptides are unknown. The presence of other growth axis genes, such as insulin-like growth factor-I and II (9, 16), in testis and placenta invites consideration that GHRH-like peptides may participate in the regulation of these gene products locally. Further studies to define a role for GHRH-like peptide in extrahypothalamic sites are needed. Acknowledgments We thank R. Evans for the rat and human GHRH cDNAs, and Mary Bundy and Ann-Margaret Myers for expert technical assistance. We appreciate the efforts of Dr. Richard Foster in obtaining testicular samples.
References 1. Gubler U, Monahan JJ, Lomedico PT, et al. 1983 Cloning and sequence analysis of a cDNA for the precursor of human growth hormone-releasing factor, somatocrinin. Proc Nat1 Acad Sci USA. 80:4311-4. 2. Mayo KE, 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. 82:63-7. 3. Roth KA, Wilson DW, Eberwine J, et al. 1986 Acromegaly and pheochromocytoma: a multiple endocrine syndrome caused by a plurihormonal adrenal medullary tumor. J Clin Endocrinol Metab. 63:1421-6. 4. Losa M, Wolfram G, Mujto J, et al. 1990 Presence of growth hormone-releasing hormone-like immunoreactivity in human tumors: characterization of immunological and biological properties. J Clin Endocrinol Metab. 70:62-S. 5. Meigan G, Sasaki A, Yoshinaga K. 1988 Immunoreactive growth hormone-releasing hormone in rat placenta. Endocrinology.
ETAL.
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123:1098-102. 6. Moretti C, Fabbri A, Gnessi L, et al. 1990 Immunohistochemical localization of growth hormone releasing hormone in human gonads J Endocrinol Invest. 13:301-5. 7. Losa M, Wolfram G, Schopohl J, Sobieszczyk S, von Werder K. 1988 Evidence for a nonspecific factor interfering in the radioimmunoassay of somatoliberin-like immunoreactivity in human seminal plasma. J Clin Chem Clin Biochem. 26:367-71. 8. Berrv SA. Pescovitz OH. 1988 Identification of a GHRH-like substanfe and its messenger RNA in rat testis. Endocrinology. 123:6613. OH, Johnson N, Berry SA. 1990 Ontogeny of growth 9. Pescovitz hormone-releasing hormone and insulin-like growth factors I and II messenger RNAs in rat placenta. Pediatr Res. 29:510-6. 10. Suhr ST, Rahal JO, Mayo KE. 1989 Mouse growth hormonereleasing hormone: precursor structure and expression in brain and placenta. Mol Endocrinol. 3:1693-700. 11. Jansson JO, Ishikawa K, Katakami H, Frohman L. 1987 Pre- and postnatal developmental changes in hypothalamic content of rat growth hormone releasing factor. Endocrinology. 120:525-30. 12. Pescovitz OH, Gelato MC, Bundy M, Loriaux DL, Merriam CR, Pescovitz MD. 1986 Production of monoclonal antibodies against human growth hormone releasing hormone and their use h an enzvme-linked immunosorbent assav i (ELISAl. Methods. ~ I ,I Immunol 94~257-62. 13. Chomczynski P, Sacchi N. 1987 Single step method of RNA isolation bv acid guanidinium thiocvanate extraction. Anal Biochem. 162:156-9: 14. Berry SA, Seelig S. 1986 Differential regulation of alpha-2,-globulin gene products. Endocrinology. 119:600-5. RA, Siebert E. 1990 Optimization of Northern analysis by 15. Kriczek vacuum-blotting, RNA-transfer visualization, and ultraviolet fixation. Anal BiochYm. 184:90-5. 16. Berry SA, Pescovitz OH. 1990 Ontogeny and pituitary regulation of testicular growth hormone-releasing hormone-like messenger RNA. Endocrinology. 127:1404-10. 17. Mayo KE, Vale W, Rivier J, Rosenfeld MG, Evans RM. 1983 Expression-cloning and sequence of a cDNA encoding human growth hormone-Releasing factor. Nature. 306;86-8. 18. Mareioris AN, Brockmann G. Bohler HCL. Grino M. Vamvakopot&s N, Chrbusos 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. 126:151-S. 19. Pescovitz OH, Berry SA, Laudon M, et al. 1990 Localization and growth hormone releasing activitv of rat testis growth hormone yeleasing hormone-like peptide. Endocrinology. 127:2336-42. LA, Downs TR, Williams TC, Heimer El’, Pan Y-CE, 20. Frohman Felix AM. 1986 Rapid enzymatic degredation of growth hormone releasing hormone by plasma in vitro and in vim to a biologically inactive product cleaved at the NH2 terminus. J Clin Invest. 78:90613. 21. Mazlan M, Spence-Jones C, Chard T, Landon J, McLean C. 1990 Circulating levels of GH-releasing hormone and GH during human pregnancy. J Endocrinol. 125:161-7.
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Vol. 75, No. 1 Printed in U.S.A.
Growth Hormone-Releasing Hormone-Like Messenger Ribonucleic Acid and Immunoreactive Peptide Are Present in Human Testis and Placenta* SUSAN A. BERRY, CAROLYN H. SRIVASTAVA, LESLIE WILLIAM R. PHIPPS, AND ORA HIRSCH PESCOVITZ
R. RUBIN,
Departments of Pediatrics and Obstetrics and Gynecology and the Institute of Human Genetics, University of Minnesota, Minneapolis, Minnesota 55455; and the Departments of Pediatrics, Physiology/Biophysics, and Obstetrics and Gynecology, Indiana University, Indianapolis, Indiana 46202 ABSTRACT
protein). The origin of these peptides was confirmed by extracting total RNA from human testis and placenta, with analysis on Northern blots probed with riboprobes for rat hypothalamic GHRH cDNA and human pancreatic tumor GHRH cDNA. Human testis and placenta total RNA both contain an approximately 790-nucleotide RNA species similar in size to that reported in ectopic GHRH-producing human tumors. In addition, two larger hybridization signals were seen at 3000 and 4900 nucleotides. These data suggest that testis and placenta are extrahypothalamic sites of expression of the human GHRH gene. Normal expression of the GHRH gene in extrahypothalamic sites may include transcription of larger mRNA species than those observed in ectopic pathological sources of GHRH expression. (J Clin Endocrinol Metab 75: 281-284, 1992)
Although the sequence of human GH-releasing hormone (GHRH) has been determined, all of the information concerning gene expression has been based on pathological sources of ectopic GHRH, since the only established physiological source of GHRH in humans is the hypothalamus. We recently reported the presence of extrahypothalamic GHRH-like mRNA and immunoreactive material in rat testis and placenta. To determine if human testis and placenta also contain immunoreactive GHRH-like peptides, tissue extracts were analyzed using enzyme-linked immunosorbent assay methodology. Both tissues had detectable quantities of immunoreactive peptide recognized by a monoclonal antibody to synthetic human GHRH-(l-44) (testis, 2.4 ng/ g tissue, 0.68 ng/mg protein; placenta, 2.6 rig/g tissue, 0.36 ng/mg
G
H-RELEASING hormone (GHRH) is a hypothalamic neuropeptide that plays an important role in GH regulation. When produced in excess ectopically by tumors, acromegaly may result. To date, all studies examining mRNA expression for human (h) GHRH have been based on evaluation of RNA extracted from these GHRH-producing tumors (l-3), and little is known about physiological GHRH gene expression or normal extrahypothalamic sources of GHRH in humans. Using assays for GHRH peptide, the evidence supporting the presence of GHRH-like peptide in testis and placenta is confusing and contradictory. In humans, the presence of immunoreactive GHRH-like peptide in placenta was noted in one study (4), although this finding was not confirmed in an additional study that used nearly identical methodology (5). Only one study has suggestedthe presence of immunoreactive GHRH-like peptide in human testis (6), and previous measurementsof GHRH-like peptide in human seminal plasma were inconclusive, but suggested that measurable quantities of GHRH-like immunoreactivity were artifactual(7). In contrast, recent studiesin other species suggest that other organs do contain both GHRH-like imReceived August 15, 1991. Address all correspondence and requests for reprints to: Susan A. Berry, M.D. University of Minnesota Hospital, 420 Delaware Street SE, Minneapolis, Minnesota 55455. * This work was supported by the Vikings Children’s Fund and the Minnesota Medical Foundation (to S.A.B.), the James Whitcomb Riley Memorial Association (to O.H.P.), and NIH Grant DK-41899 (to S.A.B. and O.H.P.).
munoreactivity and mRNA. We recently reported the presence of GHRH-like immunoreactivity and mRNA in rat testis (8) and placenta (9), and others have found GHRH-like mRNA in mouse placenta and testis (10). To clarify this controversy regarding physiological extrahypothalamic GHRH-like peptides in human tissues,we assayedtesticular and placental extracts using enzyme-linked immunosorbent assay (ELISA) methodology and examined human testicular and placental RNA to determine whether a GHRH-like mRNA was present. Materials and Methods Samples Human testes were obtained from adult male subjects undergoing orchidectomy for prostatic cancer. These individuals had no known abnormality in testicular function. Samples of human placentas were obtained after delivery of term infants to healthy adult women. Rat testes were harvested from a normal 65.day-old male Sprague-Dawley rat, and rat placenta was harvested from a timed pregnant female at 19 days gestation. Animals were from Taconic Farms (Germantown, NY). Tissues were rapidly aliquoted into 0.5- to l-g fragments and flashfrozen in liquid nitrogen. They remained frozen at -80 C until peptide and RNA extraction was performed.
Tissue extraction
and ELISA
The extraction procedure involved minor modifications of previously described methodology (5, 11). The tissues were lyophilized overnight, homogenized in ice-cold 50% acidified methanol (0.1 N HCl-CH30H) including 10 Fg/mL phenylmethylsulfonylfluoride, 20 pg/mL aprotinin,
281
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BERRY ET AL. and 10 &mL pepstatin. Supernatants were fractionated over C-18 SepPak cartridges (Waters Associates, Milford, MA), as described. Extracts were pooled and lyophilized overnight. A direct-bindine ELISA usine a monoclonal antibodv to hGHRH generated against “hGHRH-(I-4>)-NH2 (Bachem, Torrence, CA) was used to measure GHRH immunoreactivity, as previously described (12). hGHRH-(1-44) from the same source was used for standard curves.
JCE & M .1992 Vol75.Nol
HP
HT
RP
RT St
Northern gel analysis Total RNA was extracted from tissues by guanidine hydrochloride or acid guanidininum thiocyanate (13, 14). Poly(A)+ RNA was isolated using oligo(dT)-cellulose. Ten to 20 rg total RNA or 5 pg poly(A)+ RNA were electrophoresed in a 1.5% agarose gel in 1% formaldehyde, 20 mM 4-morpholinepropanesulfonic acid, 5 mM sodium acetate, 1 mM EDTA, and 9 mM sodium hydroxide (15) and transferred to a nylon-based membrane. Filters were hybridized either to [32P]UTP-labeled GHRH cRNA riboprobe generated from a rat hypothalamic cDNA (R. Evans, The Salk Institute, La Jolla, CA), as previously described (16), or to a GHRH cRNA riboprobe generated from subcloning of the human cDNA (phGRF-54, R. Evans, The Salk Institute) (17) into EcoRI-BamHI-digested pGEM7z (Promega, Madison, WI), using SP6 RNA polymerase to generate this riboprobe. The specific activities of the labeled cRNAs were on the order of 0.5-l X 10’ cpm/pg DNA. Blots were prehybridized and hybridized as previously described (9). Filters were washed in 1 x SSPE [180 mM NaCl, 10 mM sodium phosphate (pH 7.5), and1 mM EDTA]0.1% SDS for 15 min four times at room temperature and twice in 0.1 X SSPE-0.1% SDS at 65 C for 20 min. The hybridized washed blots were exposed to Kodak XAR-5 film (Eastman Kodak, Rochester, NY) at -80 C.
Results ELISA ELISAs were performed on tissue extracts from human testis and placenta to determine if these normal tissues contain a peptide recognized by a monoclonal antibody against hGHRH-(1-44). This antibody is directed against the carboxy-terminus of hGHRH (12). The testicular sample had 2.4 ng GHRH/g wet weight of testis (0.68 ng/mg protein), and placenta had 2.6 ng GHRH/g wet wt placenta (0.36 ng/ mg protein). The least detectable dose of GHRH was 40 pg. Northern analysis To determine if the testicular peptide recognized by the human monoclonal antibody to hGHRH-(1-44) was represented in testicular and placental RNA by a mRNA species with homology to rat hypothalamic GHRH cDNA, a Northern blot was prepared. Total RNA extracted from human testis and placenta was examined and compared to total RNA extracted from adult rat testis and term rat placenta. Using a rat hypothalamic GHRH riboprobe, the RNA species seen in rat placenta in this gel was a broad band of about 650-850 nucleotides, and that in rat testis was about 1750 nucleotides, as have been described previously (9, 16). Human testis and placenta both have a single RNA transcript on these gels of about 790 nucleotides (Fig. 1). To further confirm that the signal demonstrated using the rat hypothalamic GHRH riboprobe hybridized to a molecular species that is like hGHRH, poly(A)+ RNA from additional human testis and placenta samples was prepared and probed with a riboprobe for human pancreatic tumor-derived GHRH (17). A hybridizing signal of the same size as that in rat
FIG. 1. Northern gel analysis of GHRH-like mRNA in human and rat tissues, using a rat hypothalamic GHRH riboprobe. Ten micrograms of total RNA were subjected to electrophoresis for each sample. HP, Human placenta; HT, human testis; RP, rat placenta; RT, rat testis. Sixty picograms of cold sense-strand hypothalamic cRNA were also placed on the gel (St). Size markers are 18s and 28s ribosomal RNAs. Exposure was for 3 days at -80 C.
GHRH riboprobe hybridizations was seen on blots hybridized with the human probe. In addition, two larger bands of about 4900 and 3000 nucleotides were seen in poly(A)’ RNA blots from these tissues (Fig. 2). Discussion In this report we demonstrate the presence of GHRH-like mRNAs in extrahypothalamic human tissues. Evolutionary conservation of this organ-specific expression suggests that the GHRH-like peptide in other tissues may be biologically important. As yet, the physiological role(s) of GHRH-like
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GHRH IN HUMAN
TESTIS AND PLACENTA
HP HI- RH
28s
18s -.*
FIG. 2. Northern gel analysis of GHRH-like mRNA in human and rat tissues, using a human pancreatic tumor GHRH riboprobe. Five micrograms of poly(A)+ RNA were subjected to electrophoresis for each sample. HP, Human placenta; HT, human testis; RH, rat hypothalamus. Size markers are 18s and 28s ribosomal RNAs. Exposure was for 2 days at -80 C.
peptide in nonhypothalamic tissues remains unknown. In rat testis, GHRH-like mRNA is developmentally regulated and dependent on the pituitary for expression in young animals. Expression in postnatal life increases beginning on about day 21 of life and reaches adult levels in the late prepubertal period (day 30) (16). Our current observation demonstrates that GHRH-like mRNA is present in adult testis, but expression in prepubertal testis has not been investigated. GHRH mRNA is also developmentally regulated in rat placenta, as it is most abundant in the third trimester of gestation (9).
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Alterations in human placental expression with gestation are not known. In both rat (8) and mouse (10) testis, the predominant GHRH-like RNA species is larger than that in the hypothalamus. The reason for the difference in size is unknown. The predominant RNA species in rat placenta is similar in size to that in rat hypothalamus (9, 18). The sizes of human GHRH mRNAs from ectopic GHRH-secreting tumors have been variously reported as ranging between 750-820 nucleotides in length (l-3), although in 1 case (3) a larger species of 1600 nucleotides was also present. The smallest RNA species seen in human placenta and adult testis was similar in size to that found in GHRH-secreting tumors. The size of the RNA in human hypothalamus has not been reported. The observation of multiple, less intense, larger GHRH mRNA species was also noted in both rat and murine testis and placenta (9, 10, 16). Immunoreactive GHRH-like material in human testis was reported by Moretti et al. (6). They noted intense staining of the interstitial compartment of the testis with localization to Leydig cells (6). In contrast, in the rat we found that GHRHlike immunoreactivity was localized to spermatogenic cells (19). Because of this discrepancy in localization, the site of origin of GHRH-like peptide in the testis remains uncertain, although there may be species-dependent differences in localization. Losa et al. (7) examined human seminal plasma for GHRH-like immunoreactivity. In unextracted seminal plasma, they found 3.8 pg/L GHRH-like immunoreactivity using RIA, but concluded that this measurement was artifactual, because after extraction by absorption on hydrophobic Cl8 Sep-Pak cartridges, no further GHRH-like immunoreactivity could be measured (7). In contrast, the present ELISA uses a similar absorption onto Cl8 Sep-Pak cartridges with retention of immunoreactivity in whole testicular and placental extracts. We also were unable to measure GHRH-like immunoreactive material from fresh human semen (Rubin, L. R., and 0. H. Pescovitz, unpublished results). It is possible that if testicular GHRH-like peptide is present in seminal plasma, it is easily degraded into an inactive or nonassayable form, resulting in the loss of detectable activity in the extraction process. Our observation of a GHRH-like immunoreactive peptide and mRNA in human placenta is the first substantiation of the presence of both this neuropeptide and its mRNA in this human tissue, although rat placenta has been shown to have significant immunoreactive peptide and RNA present from days 13-14 of gestation (5, 9). One previous study looking for GHRH-like immunoreactivity in human placenta using RIA with a polyclonal antiserum against synthetic hGHRH(l-44) could not detect evidence of a GHRH-like peptide (5), although another group found results similar to those noted in this study (4). Several researchers have commented that the GHRH peptide is highly labile (4, 20), so that the brief delay due to separation and subsequent delivery of the placenta may result in degradation of the peptide. Our experience also suggests that very fresh or fresh-frozen tissue is required for detection of the peptide by ELISA. The localization of GHRH in human placenta is still unknown, al-
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though GHRH mRNA has been localized to the cytotrophoblast in mouse and rat placenta by in situ hybridization (10, 18). The demonstration of both GHRH-like immunoreactive peptide and RNA in human testis and placenta considerably extends the evolutionary distance during which this tissuespecific gene expression has been observed and suggests additional roles for this neuropeptide beyond the regulation of GH releasefrom the pituitary. Despite abundant production of GHRH-like peptide in these tissues,they do not seem to contribute substantially to circulating GHRH (21); thus, extrahypothalamic GHRH-like peptides are more likely to act in an autocrine or paracrine fashion. The potential functions of these peptides are unknown. The presence of other growth axis genes, such as insulin-like growth factor-I and II (9, 16), in testis and placenta invites consideration that GHRH-like peptides may participate in the regulation of these gene products locally. Further studies to define a role for GHRH-like peptide in extrahypothalamic sites are needed. Acknowledgments We thank R. Evans for the rat and human GHRH cDNAs, and Mary Bundy and Ann-Margaret Myers for expert technical assistance. We appreciate the efforts of Dr. Richard Foster in obtaining testicular samples.
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123:1098-102. 6. Moretti C, Fabbri A, Gnessi L, et al. 1990 Immunohistochemical localization of growth hormone releasing hormone in human gonads J Endocrinol Invest. 13:301-5. 7. Losa M, Wolfram G, Schopohl J, Sobieszczyk S, von Werder K. 1988 Evidence for a nonspecific factor interfering in the radioimmunoassay of somatoliberin-like immunoreactivity in human seminal plasma. J Clin Chem Clin Biochem. 26:367-71. 8. Berrv SA. Pescovitz OH. 1988 Identification of a GHRH-like substanfe and its messenger RNA in rat testis. Endocrinology. 123:6613. OH, Johnson N, Berry SA. 1990 Ontogeny of growth 9. Pescovitz hormone-releasing hormone and insulin-like growth factors I and II messenger RNAs in rat placenta. Pediatr Res. 29:510-6. 10. Suhr ST, Rahal JO, Mayo KE. 1989 Mouse growth hormonereleasing hormone: precursor structure and expression in brain and placenta. Mol Endocrinol. 3:1693-700. 11. Jansson JO, Ishikawa K, Katakami H, Frohman L. 1987 Pre- and postnatal developmental changes in hypothalamic content of rat growth hormone releasing factor. Endocrinology. 120:525-30. 12. Pescovitz OH, Gelato MC, Bundy M, Loriaux DL, Merriam CR, Pescovitz MD. 1986 Production of monoclonal antibodies against human growth hormone releasing hormone and their use h an enzvme-linked immunosorbent assav i (ELISAl. Methods. ~ I ,I Immunol 94~257-62. 13. Chomczynski P, Sacchi N. 1987 Single step method of RNA isolation bv acid guanidinium thiocvanate extraction. Anal Biochem. 162:156-9: 14. Berry SA, Seelig S. 1986 Differential regulation of alpha-2,-globulin gene products. Endocrinology. 119:600-5. RA, Siebert E. 1990 Optimization of Northern analysis by 15. Kriczek vacuum-blotting, RNA-transfer visualization, and ultraviolet fixation. Anal BiochYm. 184:90-5. 16. Berry SA, Pescovitz OH. 1990 Ontogeny and pituitary regulation of testicular growth hormone-releasing hormone-like messenger RNA. Endocrinology. 127:1404-10. 17. Mayo KE, Vale W, Rivier J, Rosenfeld MG, Evans RM. 1983 Expression-cloning and sequence of a cDNA encoding human growth hormone-Releasing factor. Nature. 306;86-8. 18. Mareioris AN, Brockmann G. Bohler HCL. Grino M. Vamvakopot&s N, Chrbusos 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. 126:151-S. 19. Pescovitz OH, Berry SA, Laudon M, et al. 1990 Localization and growth hormone releasing activitv of rat testis growth hormone yeleasing hormone-like peptide. Endocrinology. 127:2336-42. LA, Downs TR, Williams TC, Heimer El’, Pan Y-CE, 20. Frohman Felix AM. 1986 Rapid enzymatic degredation of growth hormone releasing hormone by plasma in vitro and in vim to a biologically inactive product cleaved at the NH2 terminus. J Clin Invest. 78:90613. 21. Mazlan M, Spence-Jones C, Chard T, Landon J, McLean C. 1990 Circulating levels of GH-releasing hormone and GH during human pregnancy. J Endocrinol. 125:161-7.
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