0013-7227/90/1275-2336$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society

Vol. 127, No. 5 Printed in U.S.A.

Localization and Growth Hormone (GH)-Releasing Activity of Rat Testicular GH-Releasing Hormone-Like Peptide* ORA HIRSCH PESCOVITZ, SUSAN A. BERRY, MOSHE LAUDON, NIRA BENJONATHAN, ANN MARTIN-MYERS, SU-MING HSU, THEODORE J. LAMBROS, AND ARTHUR M. FELIX Departments of Pediatrics (O.H.P., A.M.-M.) and Physiology/Biophysics (O.H.P., M.L., N.B.-JJ, Indiana University, Indianapolis, Indiana 46223; the Department of Pediatrics, Variety Club Childrens Hospital (S.A.BJ, and the Institute of Human Genetics, University of Minnesota (S.A.B.), Minneapolis, Minnesota 55455; the Department of Pathology, University of Arkansas for Medical Sciences (S.-M.H.), Little Rock, Arkansas, 72201; and Peptide Research Department, Hoffman-La Roche, Inc. (T.J.L., A.M.F.), Nutley, New Jersey 07110

ABSTRACT. The testis contains many peptides originally described as originating in the central nervous system. The physiological function of these factors in the testis is generally unknown. We previously reported that the rat testis contains both a peptide with GH-releasing hormone-like immunoactivity (tGHRH-LI) and a mRNA species that cross-hybridizes with a hypothalamic cDNA for rat GHRH (rGHRH). The current study was designed to further characterize tGHRH-LI by determining its location within rat testis, and to evaluate whether tGHRHLI and hypothalamic GHRH share similar biological and electrophoretic properties. Partially purified tGHRH is capable of stimulating GH secretion from cultured anterior pituitary cells in a dose-dependent manner. Testicular GHRH and rGHRH have different HPLC

T

HE TESTIS is a source of numerous neuropeptides and growth factors. These include LHRH (1, 2), TRH (3), POMC (4), CRH (5, 6), oxytocin (7), vasopressin (7), somatostatin (8), epidermal growth factor (9), insulin-like growth factors (10, 11), nerve growth factor (12), and transforming growth factor-a and -/3 (13, 14). The role of these factors in testicular physiology is largely unknown. The testis contains a barrier consisting of junctional complexes between Sertoli cells that restricts the movement of substances into and out of the seminiferous tubules (15). Some factors enter the tubules rapidly, Received June 8, 1990. Address all correspondence and requests for reprints to: Ora Hirsch Pescovitz, Department of Pediatrics, Indiana University Medical Center, 702 Barnhill Drive, Riley A593, Indianapolis, Indiana 46202. * This work was supported in part by NIH RO-1DK-41899, Biomedical Research Grant SO7-RR-5371J, the James Whitcomb Riley Memorial Association, the Graduate School, University of Minnesota, Minnesota Medical Foundation, and the Vikings Children's Fund.

retention times and significantly different electrophoretic properties by Western gel analysis. The estimated size of tGHRHLI is approximately 3.7 times that of synthetic rGHRH. Using immunohistochemistry, tGHRH-LI is localized to mature sperm forms in rat testis. We conclude that rat tGHRH-LI and rGHRH share some structural and functional properties and are probably related peptides. However, the difference in electrophoretic mobility and HPLC retention time indicates that they are not identical. The presence of tGHRH-LI in rat sperm, within the confines of the blood-testis barrier, which is generally impermeable to peptides, leads us to speculate that tGHRH serves a paracrine or autocrine role in testicular physiology. {Endocrinology 127: 2336-2342,1990)

whereas others are almost completely excluded. For example, testosterone and glucose have accelerated entry rates, while dyes and peptide hormones have restricted entry to the tubular lumen (16). One of the functions of the barrier is to ensure that proper conditions for meiosis exist within the tubules. In addition, the barrier prevents the recognition of haploid germ cells as foreign antigens (17). In contrast to the lipophilic steroid hormones that have free mobility, peptide hormones produced or secreted into the tubular lumina are retained by the barrier and may have limited function as endocrine factors outside the testis (18). Because entry of circulating factors into the tubular fluid is restricted, the testis must produce many of its own regulatory substances. Thus, the testis functions in some ways as an autonomous unit, and the local production of neuropeptides may serve a paracrine or autocrine function to modulate testicular activity and development. We previously reported that rat testis contains an

2336

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 06:13 For personal use only. No other uses without permission. . All rights reserved.

RAT TESTICULAR GHRH

2337

immunoreactive GH-releasing hormone-like substance (tGHRH-LI) and mRNA (19). The objectives of this study were 1) to initiate purification of tGHRH-LI by HPLC, 2) to determine whether tGHRH-LI has biological activity similar to that of rat GHRH (rGHRH), 3) to compare the electrophoretic properties of tGHRH-LI with those of rGHRH by Western gel analysis, and 4) to localize endogenous tGHRH-LI within rat testis by immunohistochemistry.

Materials and Methods Tissue extraction Adult male Sprague-Dawley rats (350 g) were obtained from Harlan Laboratories (Indianapolis, IN). Animals were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Seventy-two testes were removed, decapsulated, and flash-frozen in liquid nitrogen until later extraction. The extraction procedure involved modifications of previously described methodology (20, 21). In brief, the testes were lyophilized overnight, homogenized in ice-cold 50% acidified methanol (0.1 N HC1-CH3OH), including 10 ixg/m\ phenylmethylsulfonylfluoride, 20 fig/ml aprotinin, and 10 ixg/ml pepstatin. The homogenate was spun at 10,000 X g for 30 min at 4 C. The supernatant was fractionated over C-18 Sep-Pak cartridges (Waters Associates, Milford, MA) that had been treated by successive 4-ml washes of 0.01 M trifluoroacetic acid (TFA), 80% acetonitrile-20% 0.01 M TFA (vol/vol), and 0.01 M TFA. Acidified samples were applied to the cartridges, followed by 3 ml 0.1 M TFA. The samples were eluted with 2 ml 80% acetonitrile-20% 0.01 M TFA. HPLC Extracts from 72 testes, after passage over Sep-Pak columns, were pooled and evaporated to 60 ml (0.5% TFA in H2O). The sample was applied onto a DuPont C-8 reverse phase column (2.5 X 25 cm), which had previously been standardized with rGHRH (Bachem, Inc., Torrance, CA) and was eluted at 15 ml/min, with a linear gradient of 0.5% TFA in H2O (A) and 0.25% TFA in CH3CN (B) going from 20% (B) to 45% (B) over 60 min (Fig. 1). Fractions were collected at 1-min intervals (15 ml/fraction), and 1.5 ml (10%) of each fraction were removed and lyophilized. The lyophilized samples were reconstituted in PBS and assayed for the presence of GHRH immunoactivity by enyzme-linked immunosorbant assay (ELISA). The remaining 90% of the three samples immunoactive for GHRH (see Results and Fig. 1) were pooled and the total residue (9.5 mg) was applied to a Nucleosil C-18 300 A column (2.5 X 25 cm). The column was eluted with a linear gradient consisting of 0.1% TFA in H2O (A) and 0.1% TFA in CH3CN (B) going from 20% (B) to 40% (B) in 60 min at a flow rate of 15 ml/min. Fractions were collected at 1-min intervals, and 1.5 ml (10%) of each fraction were removed and lyophilized until reconstitution for GHRH determination by ELISA.

O

10

20

30

40

50

60

70

80

90

100

FRACTION NUMBER

FIG. 1. Preparative HPLC purification of crude testicular extract using a DuPont C-8 reverse phase column with a linear gradient of 0.5% TFA in H2O (A) and 0.25% TFA in CH3CN (B), 20% (B) to 45% (B) over 60 min. Detection was at 280 nm. The sensitivity was 2.0 absorbance units full scale. Lower curve, GHRH immunoreactivity, determined by ELISA, of individual fractions from preparative HPLC. The retention time for synthetic rGHRH was determined from a separate run using the same column and column conditions. Biological activity

Anterior pituitary cells were cultured as previously described (22, 23). In brief, pituitary glands were removed from adult male Wistar rats (Harlan), and the posterior lobes were discarded. The anterior pituitaries were cut into small pieces and incubated with 0.2% trypsin (Worthington Biochemical Corp., Freehold, NJ) for 30 min. After adding lima bean trypsin inhibitor (Worthington) and DNAse (Sigma Chemical Co., St. Louis, MO), the cells were filtered and dispersed by gentle trituration. Cells (50,000/well) were cultured in 96-well plates (Nunc, Copenhagen, Denmark) with Dulbecco's Modified Eagle's Medium (Gibco, Grand Island, NY) containing 10% horse serum (Gibco), 2.5% fetal calf serum, and antibiotics. After 4 days in culture, the cells were washed three times with serumfree medium 199 (Gibco) containing 0.1% BSA and then incubated for 3 h with 0.1 ml of the same medium containing different dilutions of fractions of the Nucleosil HPLC or rGHRH (Bachem, Torrance, CA). Each treatment was tested in quadruplicate. At the end of the incubation period, the media were removed, diluted with PBS containing 0.1% BSA, and stored at —20 C until analyzed for GH. Medium GH concentrations were determined in triplicate using a NIDDK RIA kit, with rat GH RP-2 as a reference preparation. Western gel analysis Reconstituted samples from the Nucleosil HPLC fractions immunoreactive for GHRH were used for Western gel analysis, using modifications of the method of Towbin et al. (24). Syn-

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 06:13 For personal use only. No other uses without permission. . All rights reserved.

RAT TESTICULAR GHRH

2338

thetic rGHRH was used for comparison with tGHRH-LI. Myoglobin low mol wt markers ranging from 2,510-16,950 Da (Sigma) were also run. In brief, HPLC fractions and 20 ng synthetic rGHRH were subjected to electrophoresis on a 12.5% sodium dodecyl sulfate-polyaerylamide gel electrophoresis (Hoefer Scientific, San Fransisco, CA). The gel was run for 18 h at 20 mamp at 4 C and was then transferred to nitrocellulose paper (0.2 ^m; Bio-Rad, Richmond, CA). The transfer buffer consisted of 25 mM Tris, 192 mM glycine, 20% methanol, and 0.15 M NaCl. The nitrocellulose paper was blocked with 1% BSA and PBS-0.1% Tween for 1 h at room temperature under gentle shaking. The paper was washed three times, 5 min each, in 0.05 M Tris-0.15 M NaCl, pH 7.6, and then incubated with anti-GHRH polyclonal serum (1:400) in PBS-0.1% Tween and 0.1% BSA for 2 h at room temperature with gentle shaking. After three washes, as stated above, the paper was incubated in a solution containing peroxidase-conjugated goat antirabbit immunoglobulin G (Ig; Cappel, Warrington, PA) at a dilution of 1:4,000 (in PBS-0.1% Tween and 0.1% BSA). After three additional washes, the blots were developed with 50 mM TrisHC1, pH 7.6, containing 50 mg/100 ml diaminobenzidine (Sigma) and 0.01 ml H2O2 until the appearance of bands. The paper was then rinsed five times in deionized water to stop the reaction and dried between sheets of filter paper. Antiserum generation Six 3-month-old female New Zealand White rabbits were immunized with rGHRH (Bachem). Each animal received 300 Hg unconjugated rGHRH in a solution containing equal volumes of complete Freund's adjuvant and 0.15 M NaCl. They also received an im injection of 0.5 ml diphtheria-tetanus vaccine (2 limiting flocculating units diphtheria toxoid and 5 limiting flocculating units tetanus toxoid) at the time of immunization. Initial immunization was performed by 20 intradermal injections of the immunogen. One set of booster injections with half the original dose of immunogen was given after 3 weeks. After the booster, the animals were bled weekly, and the serum was analyzed for antibody formation by ELISA. All animals developed antibodies with a titer of more than 1:10,000, and no cross-reactivity was demonstrated against somatostatin, glucagon, LH, hCG, FSH, TSH, vasoactive intestinal peptide, or pancreatic polypeptide.

Endo • 1990 Vol 127 • No 5

25 ml citrate buffer and 6 nl 30% H2O2), were added per well. The reaction was stopped after 11 min with 50 /nl H2SO4. The recovery of exogenous GHRH after extraction averaged 80%. The least detectable dose was 100 pg/well. The intra- and interassay coefficients of variation at the ED50 were 8.3% and 14.9%, respectively. Immunohistochemistry The avidin-biotin-peroxidase technique of Hsu et al. (25) was used to examine the localization of tGHRH-LI. Testes from adult male rats were removed, fixed in 10% buffered formalin for 4 h at room temperature, transferred to 70% ethanol, and embedded in paraffin. The sections were deparaffinized and rehydrated. After being washed in 0.05 M Trisbuffered saline, pH 7.6, the sections were incubated with rabbit anti-GHRH serum at a dilution of 1:3000 for 30 min at room temperature, followed by sequential 30-min incubations with biotin-labeled goat antirabbit Ig (1:200) and avidin-biotin-peroxidase complex (Vector Laboratory, Burlingame, CA). Each step was followed by a 5-min wash with Tris-buffered saline. The slides were developed in a diaminobenzidine-hydrogen peroxide-nickel chloride solution. Preimmune serum and omission of the primary antiserum were used as controls.

Results Elution profile of tGHRH-LI on HPLC GHRH immunoactivity was detected in fractions 5355, but in none of the other samples. The maximum GHRH immunoactivity was detected in fraction 54 (800 ng/ml). The retention time for tGHRH-LI was about 6 min later than that for rGHRH, using the C-8 reverse phase column (Fig. 1). The three samples positive for GHRH immunoactivity were pooled and applied to a Nucleosil C-18 column (see Materials and Methods). As illustrated in Fig. 2, fractions 42-45 contained GHRH immunoactivity, while the remaining fractions were negative. These latter fractions were tested for biological activity and used for Western gel analysis. Biological activity of tGHRH-LI

GHRH ELISA A direct binding ELISA was used to measure GHRH immunoreactivity, as previously described (19). In brief, 200-^1 samples containing rGHRH (Bachem), reconstituted tissue extract, or PBS buffer were added to wells of a 96-well immunoassay plate (Nunc) for 1 h at room temperature. The plates were washed 5 times with PBS-0.1% Tween. One hundred microliters of antirat GHRH polyclonal serum at a dilution of 1:3000 were added to each well and allowed to incubate for 1 h at room temperature. The plates were washed 5 times and 100 fA peroxidase-conjugated goat antirabbit Ig (Cappel Laboratories, Cochranville, PA) at a dilution of 1:12,000 were added for 1 h at room temperature. The plates were again washed 5 times, and 200 /x\ of the substrate, orthophenylene-diamine (10 mg/

Using dispersed anterior pituitary cells, the ability of HPLC fractions containing immunoreactive GHRH to release GH was tested. HPLC fractions that were negative for GHRH immunoactivity were used as controls; synthetic rGHRH was used as a positive control. The immunoactive tGHRH-LI fractions stimulated GH release in a dose-dependent fashion (Fig. 3). A 10-fold dilution of the immunoactive tGHRH-LI sample (equivalent to 5% of one testis/well) resulted in a 200% increase in GH release. This was comparable to the stimulation of GH release generated by 3 ng/ml (0.3 ng/well) rGHRH. Even an 80-fold dilution of the positive tGHRH-LI fractions (equivalent to 0.63% of one testis/

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 06:13 For personal use only. No other uses without permission. . All rights reserved.

RAT TESTICULAR GHRH

2339

Western gel analysis

-10000 -1000

100

0

10 20 30 40 50 60 70 80 90 100 FRACTION NUMBER

FiG. 2. Preparative HPLC purification of semipure product using a Nucleosil C-18 (300 A0) reverse phase column with a linear gradient of 0.1% TFA in H 2 0 (A) and 0.1% TFA in CH3CN (B), 20% (B) to 40% (B) in 60 min. Detection was at 206 nm. The sensitivity was 2.0 absorbance units full scale. Lower curve, GHRH immunoreactivity was determined by ELISA in 0.2 ml HPLC fractions. 300-1

200-

Western gel analysis was used to compare the electrophoretic properties of partially purified tGHRH-LI and synthetic rGHRH (Fig. 4). As expected, the band containing synthetic rGHRH corresponded with mol wt markers of 4,500 Da. Another band of lesser intensity, which might represent either a degradation product or an impurity, was also visualized with 20 ng rGHRH, but not with lesser amounts of rGHRH. The electrophoretic mobility of HPLC fractions containing tGHRH-LI resulted in a wide band that corresponded with mol wt markers of 16,000 Da. The intensity of this tGHRH-LI band increased with increasing concentrations of partially purified tGHRH-LI (20-80 /x\; Fig. 4). A band was visualized with the least amount of tGHRH-LI tested (equivalent to 15% of one testis). Partially purified tGHRH-LI showed no visible band at the site corresponding to synthetic rGHRH. Immunohistochemistry The avidin-biotin-peroxidase immunohistochemical technique was used to localize tGHRH-LI within the testis. Testicular sections treated with the anti-GHRH serum had intense staining in mature sperm cells and in early sperm forms (Fig. 5). No staining was seen in other cells, such as interstitial, Sertoli, or endothelial cells. No staining was visualized in sections treated with preimmune serum.

200-

6.5

100-

3.4

-50J

20

10

HPLC FRACTIONS (DILUTION FACTOR)

FIG. 3. Comparison of GH-releasing activity of tGHRH-LI-enriched HPLC fractions (fractions 42-45; Fig. 2) and synthetic rGHRH. A dose response of the GH-releasing activity of GHRH is shown in A. The GH-releasing activity of serial dilutions of HPLC fractions from rat testicular extract is shown in B. A dilution factor of 10 is equivalent to 5% of one testis. D, Fractions that were immunoactive for GHRH; • , fractions that lacked immunoactivity. The results are expressed as the mean ± SEM (n = 4).

well) resulted in a 30% increase in GH release. Samples that did not contain GHRH immunoactivity did not alter GH release.

20

40

80

rGHRH 20 \iq

FIG. 4. Western gel analysis of tGHRH-LI-enriched HPLC fractions (fractions 42-45; Fig. 2). A band representing synthetic rGHRH corresponds with mol wt markers of 4500 Da. A dose-dependent band corresponding to mol wt markers of about 16,000 Da is seen with increasing volumes of tGHRH-LI-enriched HPLC fractions (20, 40, and 80 nl). Twenty microliters of the tGHRH-LI-enriched HPLC fraction are equivalent to 15% of one testis.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 06:13 For personal use only. No other uses without permission. . All rights reserved.

RAT TESTICULAR GHRH

2340

FIG. 5. Immunohistochemical localization of tGHRH-LI in mature rat testis. Testicular sections treated with anti-GHRH serum are shown in panel a (note the intense staining in the sperm), and testicular sections treated with preimmune serum are shown in panel b.

Discussion These studies confirm that the mature rat testis contains a substance with GHRH immunoreactivity. Like synthetic rat GHRH, testicular GHRH stimulates GH secretion from dispersed anterior pituitary cells in a dosedependent fashion. Although they share biological similarities, rGHRH and tGHRH-LI do not appear to be identical, since they possess different HPLC elution profiles and have different electrophoretic properties. The physiological function of tGHRH-LI is yet unknown, but its localization in rat sperm leads us to speculate that it may play a role in reproductive processes. Hypothalamic GHRH is found in the arcuate nucleus in the rat (26). Its predominant action is to stimulate anterior pituitary somatotrophs to secrete GH. In contrast to the wide distribution of other neuropeptides, such as CRH, the central nervous system distribution of

Endo • 1990 Vol 127-No 5

GHRH is limited. GHRH has been found in a variety of neuroendocrine tumors and has been detected in placenta, pancreas, adrenal, and gut in both human and rat tissues (27-32). When produced in excess, extrahypothalamic GHRH is a rare cause of acromegaly (33-35). The clinical presentation of patients with ectopic GHRH production does not differ from that of classical acromegaly, which suggests that the anterior pituitary is the primary site of GHRH action. Outside of a role in stimulating GH secretion, there is no known physiological function for extrahypothalamic GHRH. The possibility that GHRH may be a paracrine or autocrine modulator of cell function in tissues outside of the hypothalamicpituitary complex has not been explored. Given that other hypothalamic neuropeptides are found in the testis (1-14), the presence of a testicular GHRH-like substance is not entirely unexpected. Ontogeny studies reveal that the tGHRH mRNA transcript increases with age, becoming maximally expressed 2-3 weeks after birth (36). Because of this developmental pattern and because neuropeptides such as CRH (6) and POMC (37, 38) have been localized to Leydig cells, we hypothesized that tGHRH-LI would be found in Leydig cells. Thus, the immunohistochemical localization of tGHRH-LI to mature rat sperm forms was not anticipated. However, other growth factors and neurotropic factors have also been localized to germ cells. Evaluation of the immunoreactivity of insulin-like growth factor-I in mature rat testis results in strong staining in spermatocytes (39). In prepubertal animals, insulin-like growth factor-I immunoactivity is seen in Sertoli cells and Leydig cells, in addition to germ cells (10,11). Nerve growth factor protein and mRNA have also been localized to rat and mouse germ cells (12). In addition, the nerve growth factor receptor mRNA is found in rat Sertoli cells, and its expression is down-regulated by testosterone (40). These data suggest that sperm-derived factors may play a role in the regulation of other testicular products by mediating an interaction between germ cells and other tissues in the reproductive tract. Further support for this concept comes from the finding that a sperm-derived factor is also important for the regulation of proenkephalin gene expression in the epididymis (41). Thus, the localization of tGHRH-LI to germ cells leads us to speculate that it participates in spermatogenesis. The demonstration that tGHRH-LI has the capacity to stimulate GH secretion from cultured anterior pituitary cells suggests that it shares some biological functions with GHRH of hypothalamic origin. It does not, however, imply that the primary function of tGHRH is to stimulate anterior pituitary GH secretion. In view of the bloodtestis barrier, the abundance of tGHRH-LI in rat testis, and the absence of detectable GHRH in the peripheral circulation (19), it seems unlikely that tGHRH is an

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 06:13 For personal use only. No other uses without permission. . All rights reserved.

RAT TESTICULAR GHRH

important regulator of the hypothalamic-pituitary axis. Rather, tGHRH may function as an autocrine or paracrine factor within the testis. GHRH is a member of the glucagon gene superfamily which includes glucagon, secretin, vasoactive intestinal peptide, and gastrin inhibitory peptide (42). The full biological activity of rat hypothalamic GHRH resides in amino acid residues 1-29 (34). There appears to be only one mammalian gene that codes for GHRH (43). The gene structure suggests that the rat prepro-GHRH gene encodes a 104-amino acid precursor in 5 exons spanning a length of approximately 9 kilobases. In contrast to the 750-basepair (bp) hypothalamic transcript, the major mRNA species we detected in testis is a 1750-bp band (19). The larger RNA species in normal rat testis may represent a physiologically significant alternative gene transcript. This larger transcript could be an unprocessed precursor to the 750-bp RNA or be due to alternative processing of the 9-kilobase GHRH gene, resulting in a much longer mRNA. Different start or polyadenylation sites could also explain discrepancies in the transcript size. On the basis of mobility in a sodium dodecyl sulfategel, tGHRH-LI is approximately 3.7 times larger than hypothalamic GHRH. The larger mRNA species in testis could encode a longer peptide that contains a region which cross-reacts with the epitope recognized by GHRH antiserum. Alternatively, posttranslational modification of the GHRH precursor peptide could result in a product of greater mol wt than the principal hypothalamic product. In summary, tGHRH-LI and rGHRH share some biological properties, but differ in structure. The localization of tGHRH-LI in sperm cells raises intriguing questions regarding the intratesticular function of this factor. We speculate that tGHRH serves a paracrine or autocrine role in the process of spermatogenesis. Studies to determine the species specificity of tGHRH-LI and further studies to characterize both the tGHRH peptide and its mRNA are necessary to substantiate a biological role for this factor in testicular physiology.

References 1. Sharpe RM, Fraser HM, Cooper I 1982 The secretion, measurement, and function of a testicular LHRH-like factor. Ann NY Acad Sci 383:272-94 2. Bhasin S, Heber D, Peterson M, Swerdloff R 1983 Partial isolation and characterization of testicular GnRH-like factors. Endocrinology 112:1144-6 3. Morley JM, Meyer N, Pekary AE, Melmed S, Carlson HE, Briggs JE, Hershman JM 1980 A prolactin inhibitory factor with immunocharacteristics similar to thyrotropin releasing factor (TRH) is present in rat pituitary tumors (GH3 and W5), testicular tissue and a plant material, alfalfa. Biochem Biophys Res Commun 96:4753 4. Pintar JE, Schachter BS, Herman AB, Durgerian S, Kreiger DT 1984 Characterization and localization of proopiomelanocortin messenger RNA in the adult rat testis. Science 225:632-4

2341

5. Yoon DJ, Sklar C, David R 1988 Presence of immunoreactive corticotropin-releasing factor in rat testis. Endocrinology 122:75961 6. Audhya T, Hollander CS, Schlesinger DH, Hutchinson B 1989 Structural characterization and localization of corticotropin-releasing factor in testis. Biochim Biophys Acta 995:10-6 7. Nicholson HD, Swann RW, Burford GD, Wathes DC, Porter DG, Pickering BT 1984 Identification and characterization of oxytocin and vasopressin in the testis and in adrenal tissue. Regul Peptides 8:141-6 8. Pekary AE, Yameda T, Sharp B, Basin S, Swerdloff RS, Hershman J 1984 Somatostatin-14 and -28 in the male rat reproductive system. Life Sci 34:939-45 9. Tsutsumi O, Kurachi H, Oka T 1986 A physiological role of epidermal growth factor in male reproductive function. Science 233:975-7 10. D'Ercole AJ, Stiles AD, Underwood LE 1984 Tissue concentrations of somatomedin C: further evidence for multiple sites of synthesis and paracrine or autocrine mechanisms of action. Proc Natl Acad Sci USA 81:935-9 11. Casella SJ, Smith EP, Van Wyk JJ, Joseph DR, Hynes MA, Hoyt EC, Lund PK 1987 Isolation of rat testis cDNAs encoding an insulin-like growth factor-I precursor. DNA 6:325-30 12. Ayer-LeLievre C, Olson L, Ebendal T, Hallbook F, Persson H 1988 Nerve growth factor mRNA and protein in the testis and epididymis of mouse and rat. Proc Natl Acad Sci USA 85:2628-32 13. Skinner MK, Moses HL 1989 Transforming growth factor beta gene expression and action in the seminiferous tubule: peritubular cell-Sertoli cell interactions. Mol Endocrinol 3:625-34 14. Skinner MK, Takacs K, Coffey RJ 1989 Transforming growth factor-alpha gene expression and action in the seminiferous tubule: peritubular cell-Sertoli cell interactions. Endocrinology 124:84554 15. Setchell BP 1980 The functional significance of the blood-testis barrier. J Androl 1:3-10 16. Waites GMH, Gladwell RT 1982 Physiological significance of fluid secretion in the testis and blood-testis barrier. Physiol Rev 62:62471 17. Setchell BP, Waites GMH 1975 The blood-testis barrier. In: Hamilton DW, Greep RO (eds) Handbook of Physiology. American Physiological Society, Washington, vol 5:143-72 18. Tindall DJ, Vitale R, Means AR 1975 Androgen binding protein as a biochemical marker of formation of the blood testis barrier. Endocrinology 97:636-48 19. Berry SA, Pescovitz OH 1988 Identification of a GHRH-like substance and its messenger RNA in rat testis. Endocrinology 123:6613 20. 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 21. Meigan G, Sasaki A, Yoshinaga K 1988 Immunoreactive growth hormone-releasing hormone in rat placenta. Endocrinology 123:1098-102 22. Hoefer MT, Heiman ML, Ben-Jonathan N 1984 Prolactin secretion by cultured anterior pituitary cells: influence of culture conditions and endocrine status of the pituitary donor. Mol Cell Endocrinol 35:229-35 23. Laudon M, Hyde JF, Ben-Jonathan N 1989 Ontogeny of prolactin releasing and inhibiting activities in the posterior pituitary of male rats. Neuroendocrinology 50:644-9 24. Towbin H, Staehelin T, Gordon J 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:3116-20 25. Hsu S-M, Raine L, Fanger H 1981 Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577-80 26. Bloch B, Ling N, Wehrenberg WB, Benoit R, Guillemin R 1984 Specific depletion of immunoreactive growth hormone-releasing factor by monosodium glutamate in rat median eminence. Nature 307:272-3

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 06:13 For personal use only. No other uses without permission. . All rights reserved.

2342

RAT TESTICULAR GHRH

27. 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: extra-hypothalamic localization and possible functional significance. Acta Endocrinol [Suppl] (Copenh) 276:34-40 28. Christofides ND, Stephanou A, Suzuki H, Yiangou Y, Bloom SR 1984 Distribution of immunoreactive growth hormone releasing hormone in the human brain and intestine and its production by tumors. J Clin Endocrinol Metab 59:747-51 29. Baird A, Wehrenberg WB, Bohlen D, Ling N 1985 Immunoreactive and biologically active growth hormone-releasing factor in the rat placenta. Endocrinology 117:1598-601 30. Shibisaki T, Kiyosawa Y, Masuda A, Nakahara M, Imaki T, Wakabayashi I, Demura H, Shizume K, Ling N 1984 Distribution of growth hormone-releasing-factor-like immunoreactivity in human tissues. J Clin Endocrinol Metab 59:263-8 31. Bruhn TO, Mason RT, Vale W 1985 Presence of growth hormonereleasing factor-like immunoreactivity in rat duodenum. Endocrinology 117:1710-2 32. Bosman FT, Van Assche C, Nieuwenhuyzen Krusman AC, Jackson S, Lowry PJ 1984 Growth hormone releasing factor immunoreactivity in human and rat gastrointestinal tract and pancreas. J Histochem Cytochem 32:1139-44 33. Guillemin R, Brazeau P, Bohlen P, Esch F, Ling N, Wehrenberg WB 1982 Growth hormone releasing factor from a human pancreatic tumor that caused acromegaly. Science 218:585-7 34. Rivier J, Spiess J, Thorner M, Vale W 1982 Characterization of a growth hormone releasing factor from a human pancreatic islet

Endo•1990 Vol 127 • No 5

tumour. Nature 300:276-8 35. Melmed S 1990 Acromegaly. N Engl J Med 322:966-77 36. Berry S, Pescovitz OH 1990 The ontogeny of testicular growth hormone releasing hormone-like messenger RNA. Endocrinology 127:1404-11 37. Bardin CW, Chen C-LC, Morris PL, Gerendai I, Boitani C, Liotta AS, Margioris A, Krieger DT 1987 Proopiomelanocortin-derived peptides in testis, ovary and tissues of reproduction. Recent Prog Horm Res 43:1-28 38. Ginzang-Ginsberg E, Wolgemuth DJ 1985 Localization of mRNAs in mouse testes by in situ hybridization: distribution of a-tubulin and developmental stage specificity of proopiomelanocortin transcripts. Dev Biol 111:293-305 39. Hansson HA, Billig H, Isgaard J 1989 Insulin-like growth factor I in the developing and mature rat testis: immunohistochemical aspects. Biol Reprod 40:1321-8 40. Persson H, Ayer-LeLievre C, Soder O, Villar MJ, Metsis M, Olson L, Ritzen M, Hokfelt T 1990 Expression of /3-nerve growth factor receptor mRNA in Sertoli cells downregulated by testosterone. Science 247:704-7 41. Garrett JE, Garrett SH, Douglass J 1990 A spermatozoa-associated factor regulates proenkephalin gene expression in the rat epididymis. Mol Endocrinol 4:108-18 42. Bell GI 1986 The glucagon superfamily: precursor structure and gene organization. Peptides [Suppl 1] 7:27-36 43. 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 314:464-7

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 06:13 For personal use only. No other uses without permission. . All rights reserved.

Localization and growth hormone (GH)-releasing activity of rat testicular GH-releasing hormone-like peptide.

The testis contains many peptides originally described as originating in the central nervous system. The physiological function of these factors in th...
1MB Sizes 0 Downloads 0 Views