0013-7227/90/1273-1404$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 127, No. 3 Printed in U.S.A.
Ontogeny and Pituitary Regulation of Testicular Growth Hormone-Releasing Hormone-Like Messenger Ribonucleic Acid* SUSAN A. BERRYt AND ORA HIRSCH PESCOVITZ Department of Pediatrics, Variety Club Childrens Hospital and the Institute of Human Genetics, University of Minnesota (S.A.B.), Minneapolis, Minnesota 55455; and the Departments of Pediatrics and Physiology/ Biophysics, Indiana University (O.H.P.), Indianapolis, Indiana 46223
diminution of t-GHRH mRNA (19 ± 5% and 9 ± 2% of agematched controls, respectively). In contrast, in animals hypophysectomized on day 65 and killed on either day 80 or 90, there was a much smaller difference in levels of t-GHRH mRNA compared to values in control animals (73 ± 20%). This was unlike the effect of hypophysectomy on testicular IGF-I mRNA, where uniform diminution was seen in all three groups. Because GH is important in the regulation of hypothalamic GHRH mRNA, we examined the effects of administration of recombinant human GH on the reinduction of t-GHRH mRNA after hypophysectomy and compared this to the reinduction of IGF-I mRNA. Neither t-GHRH mRNA nor testicular IGF-I mRNA increased in hypophysectomized animals treated with GH. Our results indicate that t-GHRH mRNA is developmentally regulated, and that the hypothalamic-pituitary axis is important in its expression. The influence of the pituitary may be less important in the postpubertal than in the pre- or peripubertal animal. Expression of t-GHRH mRNA is not correlated with expression of either IGF-I or IGF-II in testis. Daily administration of GH alone for 2 weeks is insufficient to alter the diminished expression of this RNA or testicular IGF-I mRNA after prepubertal hypophysectomy. The mechanisms regulating tGHRH mRNA are complex and may involve pituitary as well as testicular autocrine and paracrine factors. (Endocrinology 127: 1404-1411, 1990)
ABSTRACT. The testis is rich in central nervous system-type neuropeptides, including a GH-releasing hormone (GHRH)-like substance. We examined the ontogeny and pituitary regulation of testicular GHRH-like mRNA (t-GHRH mRNA) and compared this to expression of insulin-like growth factor-I (IGF-I) and IGF-II mRNA in developing testis. t-GHRH mRNA was measured by dot blot hybridization and quantitated using a hypothalamic GHRH cRNA standard. t-GHRH mRNA was not detectable in Northern blots in fetal testis on day 19 of gestation, but was present in low but detectable amounts in testicular dot blots on day 2 of life (0.44 pg/jug total RNA). Levels of the RNA increased beginning on day 21 (1.72 ± 0.23 pg/Mg total RNA) and reached adult levels by day 30 (4.96 ± 0.84 pg/^g total RNA). The GHRH species on Northern analysis was about 1750 nucleotides at all ages examined; there was a larger species of about 3350 nucleotides seen on days 65 and 90. There was no correlation between the ontogeny of t-GHRH mRNA and either IGF-I or IGF-II mRNAs, which were maximally expressed in the testes of day 2 animals and decreased with age. To examine the influence of the pituitary gland on t-GHRH mRNA, levels of the mRNA were measured in the testes of hypophysectomized animals and age-matched controls. In animals hypophysectomized on day 21 and killed on day 42 and in animals hypophysectomized on day 42 and killed on day 63, there was marked
T
HE TESTIS is a critical organ during all stages of male development. Its influence begins during embryonic life when male sexual differentiation is initiated. Although hypothalamic-pituitary influences on testicular function are well defined, local factors controlling the growth and differentiation of the testis during development are largely unknown. The adult testis is a source of hypothalamic neuropeptides, including a LHRH-like Received March 5, 1990. Address all correspondence and requests for reprints to: Susan A. Berry, Department of Pediatrics, University of Minnesota, 420 Delaware Street SE, Box 75 UMHC, Minneapolis, Minnesota 55455. * This work was supported in part by the Graduate School, University of Minnesota, Minnesota Medical Foundation, the Vikings Childrens Fund, Biomedical Research Grant SO7-RR-5371J, and the James Whitcomb Riley Memorial Association. t Variety Club Scholar, University of Minnesota.
factor (1), TRH (2), GnRH (3), POMC (4), CRF (5, 6), and somatostatin (7). The role of these neuropeptides as paracrine regulators of gonadal function in the testis is unknown, although both CRF and POMC levels increase during periods of testicular development, suggesting that they could participate in testicular differentiation (5, 8, 9). Testis is also a site of growth factor synthesis (1012). Testicular insulin-like growth factor-I (IGF-I) may be regulated by GH (13), although this has not been firmly established (14). Recently, we observed that an immunoreactive GHreleasing hormone (GHRH)-like substance is present in testis, and that hybridization of testicular total RNA with a hypothalamic cDNA for GHRH results in a RNA species larger than that seen in hypothalamic total RNA (15). To our knowledge, GH has not been reported in
1404
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REGULATION OF TESTICULAR GHRH mRNA
testis. However, GH receptor (16) and both IGF-I and IGF-II (13) mRNAs are present in testis. Thus, in analogy to the hypothalamic-pituitary-growth factor axis, we speculated that testicular GHRH (t-GHRH) might function locally to mediate testicular IGFs. To investigate this, we examined the ontogeny of t-GHRH mRNA and compared it with the ontogeny of testicular IGF-I and IGF-II mRNAs. In addition, because of the influence of the pituitary and, specifically, GH in the expression of hypothalamic GHRH mRNA (17, 18), we studied the effects of hypophysectomy with and without GH replacement on expression of t-GHRH mRNA.
Materials and Methods Animals For examination of the ontogeny of GHRH, IGF-I, and IGFII mRNAs, Sprague-Dawley rats were obtained from Taconic Farms (Germantown, NY). For day 20 fetuses, timed pregnant dams were killed on day 20 of gestation. Fetuses were inspected for anogenital distance, and testes were isolated from male fetuses. For animals 2-42 days of age, pups were raised in litters adjusted to seven to nine pups per litter. Animals 65 and 90 days of age were obtained directly from the vendor. Testes were removed, decapsulated, and frozen at each age, except at day 20 of gestation and day 2, when testes were frozen without removal of the capsule. For examination of the effects of hypophysectomy, male Sprague-Dawley rats were hypophysectomized by the vendor (Harlan, Indianapolis, IN) at 21,42, and 60 days of age. Animals were given 10% glucose solution to drink, but received no other hormonal replacement. Weights were recorded on arrival and weekly. Animals that did not grow were considered hypophysectomized. Animals were killed approximately 3 weeks after their procedures (days 42, 63, and 80). Some day 60 animals were killed on day 90. As there was no difference between values from animals killed on either day 80 or day 90 these groups were pooled in the analysis of data. For evaluation of GH treatment after hypophysectomy, two experiments were performed using Sprague-Dawley rats from Hormone Assay Laboratory (Chicago, IL). In the first, animals were hypophysectomized at 21-22 of age, and GH treatment was initiated on day 42. Animals were treated with saline or 10 or 150 ng recombinant human (h) GH (Genentech, San Francisco, CA) by single daily injection for 14 days. Animals were killed approximately 1 h after their last daily dose. In the second experiment, animals were treated for 14 days with saline or 100 Mg hGH/100 g BW. Analysis of RNA Total RNA was extracted using guanidine HC1, as previously described (19), with volumes adjusted to extract 100 mg tissue. This necessitated pooling of testes for day 2 animals (16 pairs), the use of pairs of testes for day 10 and hypophysectomized animals, and the use of fragments of testes for all adults. Constant amounts of tissue were used so that samples could be extracted under uniform conditions, with the exception of fetal
1405
testicular RNA extraction, where 10 pairs of testes were extracted by the method of Chomczynski and Sacchi (20). All RNA used in these experiments had OD 260/280 ratios of 2.00 ± 0.15, and quantitation of concentration was based on OD 260. In addition, random samples from all extractions were run on denaturing gels to screen for intact 18S and 28S ribosomal RNA bands and for the relative visual intensity of ethidium bromide staining when equivalent quantities were loaded. Dot blots were prepared as previously described, using concentrations of 4, 2,1, and 0.5 ng total RNA (19). For standards, 0.5-32 pg cold sense GHRH cRNA were also applied to dot blots. To control for nonspecific hybridization, 0.5- to 4-^ig dots of yeast tRNA were placed on each filter. For Northern analysis of ontogeny of testicular total RNA, 10 ng total RNA from an individual sample at each age were subjected to electrophoresis in a 1.8% agarose gel (2.2 M formaldehyde-20 mM phosphate). The RNA was transferred to Zetaprobe membrane (Bio-Rad, Chicago, IL) by electrotransfer in 7.5 mM Tris (pH 7.8), 3.75 mM sodium acetate, and 37.5 fiM EDTA at 80 V for 6 h. For hybridization of GHRH mRNA, rat hypothalamic GHRH cDNA [gift of R. Evans (21)] was subcloned into the bifunctional plasmid vector pSP-72 (Promega, Madison, WI). RNA was generated using either T7- or SP6-RNA polymerases in a reaction mix of 40 mM Tris-HCl (pH 8.0) and 5 mM dithiothreitol with 1 mM ATP, CTP, and GTP in the presence of 1 U//xl RNAs in for 30 min at 37 C (22). Antisense [32P]labeled GHRH RNA was prepared by the addition of 40 nM UTP and 50 MCi [32P]UTP (3000 MCi/mM; New England Nuclear-DuPont, Boston, MA). Unlabeled sense strand RNA was prepared for standards by the addition of 1 mM UTP. DNA templates were removed from the RNA generated in the reaction by digestion with RNAse-free DNAse-I, followed by phenol extraction. The specific activity of the labeled probe was 1-8 x 108 cpm//ig cDNA. Dot blots were prehybridized and hybridized in 50% formamide, 2.5 X Denhardt's solution, 50 mM sodium phosphate (pH 7.5), 1 mM EDTA, 0.8 M NaCl, 10 /xg/ml poly(A), 200 Mg/ml sheared salmon sperm DNA, 100 Mg/ml yeast t-RNA. Northern blots were prehybridized in 50% formamide, 5x Denhardt's solution, 0.5% sodium dodecyl sulfate (SDS), 5 X SSPE [1 x SSPE = 180 mM NaCl, 10 mM sodium phosphate (pH 7.5), 1 mM EDTA], and 200 Mg/ml sheared salmon sperm DNA. The concentration of Denhardt's solution was decreased to 2.5fold for hybridization. Prehybridization was carried out for 412 h, and hybridization was performed for 18-24 h at 60 C. Filters were washed in 1 X SSPE-0.1% SDS for 15 min at room temperature, 1 X SSPE-0.1% SDS at 65 C for 20 min three times, and 0.1 x SSPE-0.1% SDS at 60 C for 50 min. For hybridization of IGF-I (23) and IGF-II [gift of M. M. Rechler (24)] cDNA inserts were excised by digestion with restriction endonucleases (EcoRl for IGF-I; Pstl for IGF-II) and labeled by random primer extension (25, 26) with 50 /uCi [32P]dCTP (3000 ^Ci/mM; New England Nuclear-DuPont) using a kit from Pharmacia (Piscataway, NJ). The specific activities of probes ranged from 7-10 X 108. Prehybridization and hybridization were performed in 50% formamide, 5 X Denhardt's solution, 50 mM sodium phosphate (pH 7.5), 0.2% SDS, 200 ixg/voX sheared salmon sperm DNA, and 100 Mg/ml yeast tRNA. Times of prehybridization and hybridization were the
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REGULATION OF TESTICULAR GHRH mRNA
1406
same as those for GHRH cRNA, but these procedures were carried out at 42 C. Filters were washed four times for 10 min each at room temperature in 2 X SSC (1 x SSC = 0.15 M NaCl, 0.015 M NaCitrate)-0.1% SDS and two or three times in 0.2 x SSC-0.2% SDS at 60 C. All filters were exposed to Kodak XAR5 film (Eastman Kodak, Rochester, NY) for varying times. Quantitation and statistical analysis Dot blots were quantitated by videodensitometry using a modification of the method of Mariash et al (27). Quantitation was based on comparison to fixed amounts of pooled RNA from the testes of day 30 and 65 animals consistently applied to all filters. Data were analyzed for significance by analysis of variance (ANOVA). Post-hoc testing by one-way ANOVA was performed using Fisher least mean differences at 95% significance. Analysis was performed using the program Statview 512+ (Abacus Concepts, Calabasas, CA).
Endo • 1990 Vol 127 • No 3
least significant difference, 95%). The actual mass of the RNA is probably underestimated by this method, as the testicular mRNA species is larger than that found in the hypothalamus (15). To determine if the mRNA detected in dot blot hybridizations was uniformly of the larger size seen in adult animals, the same RNA was subjected to electrophoresis in a 2.2 M formaldehyde denaturing gel (Northern analysis; Fig. 2). In addition, this permitted the examination of total RNA isolated from fetal testis. The mRNA species detected in testis was 1750 basepairs (bp) at all •2 2
10 15 17 19 21 23 25 30 42 65 90
28S
Results Ontogeny of GHRH-like mRNA in rat testis By dot blot hybridization, GHRH mRNA was present by day 2 of life in low, but detectable, levels in testicular total RNA (0.44 pg//ug total RNA; n = 2). The quantity of GHRH mRNA was significantly increased beginning on day 21 [1.72 ± 0.23 pg//ig total RNA (mean ± SD); n = 4], and maximal levels were reached by day 30 (4.96 ± 0.84 pg//ig total RNA; n = 4). These remained similar at subsequent ages tested (day 60, 4.53 ± 0.72; day 90, 4.93 ± 0.53 pg/^g total RNA; n = 4 for both; Fig. 1). There was a 10-fold change in detectable t-GHRH mRNA over the period tested. There was a significant effect of age on expression of t-GHRH mRNA by one-way ANOVA (df = 11,45; F = 41.63; P < 0.001). Differences from 2, 10, and 15 day levels were significant on day 21 (Fisher
18S 1750 nt
3350 nt
90
•2
< oc 5o O)
I
cc E x oc X (3
20
40
60
80
100
Days of Age
FIG. 1. Ontogeny of testicular GHRH-like mRNA. Testes from 4 separate animals were analyzed at each time point, except at 2 days, where 32 individuals were pooled into 2 individual samples (16/sample). The mean ± SD are shown.
FIG. 2. Northern blot of total RNA from testes of rats of varying ages. Each lane contains 10 ng total testicular RNA from a representative individual. Days of age are indicated above. - 2 , RNA isolated from pooled testes of 10 fetuses at 20 days gestation. Sizes of 18S and 28S ribosomal RNA bands are indicated. The middle inset shows the overexposed area of the gel where a larger, approximately 3350-nucleotide GHRH-like mRNA species is evident in 65- and 90-day-old adult male testicular total RNA. The lower inset shows the ethidium-stained gel from which this RNA was transferred. The relatively uniform density of the ribosomal bands indicates that similar quantities of total RNA were applied in each lane.
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REGULATION OF TESTICULAR GHRH mRNA
ages examined. On prolonged exposures, a larger 3350bp RNA species was seen in day 65 and 90 testes, but no evidence of the smaller hypothalamic-size species was present in any of the samples, and there was no detectable t-GHRH mRNA on Northern blots of fetal testicular total RNA. Ontogeny of IGF-I and II mRNAs in rat testis IGF-I and IGF-II mRNAs were also measured in total RNA from testes of postnatal animals to determine if their developmental expression correlated with that of tGHRH mRNA (Fig. 3). IGF-I was maximal on day 2, but fell to an intermediate value by day 19 [61 ± 16% of day 2 value (mean ± SD; n = 4 at all ages]. A further decrease was noted after day 60 (28 ± 6% of day 2 value). IGF-II was present in abundant quantities on day 2, but fell rapidly by day 10 to amounts only 37 ± 28% of day 2 values, and decreased further by day 15 (27 ± 12% of day 2 value). This level of expression was maintained until day 30, after which levels decreased further. Each of these incremental changes was significant (95%) by post-hoc testing after one-way ANOVA. Changes in both IGF-I and IGF-II mRNAs with age were significant (IGF-I: df = 9,37; F = 9.33; P < 0.001; IGF-II: df = 9,37; F = 33.96; P < 0.001). Neither IGF-I nor IGF-II mRNA was increased when expression of t-GHRH mRNA was maximal, and expression of these RNAs was highest in neonatal animals.
1407
measured by dot blot analysis in the testes of hypophysectomized rats and age-matched controls. Because of the distinctive ontogeny of t-GHRH mRNA, animals were hypophysectomized before t-GHRH mRNA levels rise, at the time of rising t-GHRH levels, and after tGHRH mRNA levels were stable. In all cases hypophysectomy resulted in marked decreases in testis weight. In animals hypophysectomized on day 21 and killed on day 42, t-GHRH mRNA was 19 ± 5% (mean ± SD; n = 5) of that observed in age-matched controls. Similarly, in animals hypophysectomized on day 42 and examined on day 63, t-GHRH mRNA levels were 9 ± 2% (n = 6) of age-matched control values. In contrast, in the testes of animals hypophysectomized on day 60 and examined on either day 80 or day 90 there was only a small difference in t-GHRH mRNA compared to those in agematched controls (73 ± 20%; n = 10 for hypophysectomized and 9 for controls; Fig. 4A). To determine if this
(6)
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Effect of hypophysectomy and response after GH administration To determine the effects of pituitary factors on the expression of t-GHRH mRNA, t-GHRH mRNA was
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Vol. 127, No. 3 Printed in U.S.A.
Ontogeny and Pituitary Regulation of Testicular Growth Hormone-Releasing Hormone-Like Messenger Ribonucleic Acid* SUSAN A. BERRYt AND ORA HIRSCH PESCOVITZ Department of Pediatrics, Variety Club Childrens Hospital and the Institute of Human Genetics, University of Minnesota (S.A.B.), Minneapolis, Minnesota 55455; and the Departments of Pediatrics and Physiology/ Biophysics, Indiana University (O.H.P.), Indianapolis, Indiana 46223
diminution of t-GHRH mRNA (19 ± 5% and 9 ± 2% of agematched controls, respectively). In contrast, in animals hypophysectomized on day 65 and killed on either day 80 or 90, there was a much smaller difference in levels of t-GHRH mRNA compared to values in control animals (73 ± 20%). This was unlike the effect of hypophysectomy on testicular IGF-I mRNA, where uniform diminution was seen in all three groups. Because GH is important in the regulation of hypothalamic GHRH mRNA, we examined the effects of administration of recombinant human GH on the reinduction of t-GHRH mRNA after hypophysectomy and compared this to the reinduction of IGF-I mRNA. Neither t-GHRH mRNA nor testicular IGF-I mRNA increased in hypophysectomized animals treated with GH. Our results indicate that t-GHRH mRNA is developmentally regulated, and that the hypothalamic-pituitary axis is important in its expression. The influence of the pituitary may be less important in the postpubertal than in the pre- or peripubertal animal. Expression of t-GHRH mRNA is not correlated with expression of either IGF-I or IGF-II in testis. Daily administration of GH alone for 2 weeks is insufficient to alter the diminished expression of this RNA or testicular IGF-I mRNA after prepubertal hypophysectomy. The mechanisms regulating tGHRH mRNA are complex and may involve pituitary as well as testicular autocrine and paracrine factors. (Endocrinology 127: 1404-1411, 1990)
ABSTRACT. The testis is rich in central nervous system-type neuropeptides, including a GH-releasing hormone (GHRH)-like substance. We examined the ontogeny and pituitary regulation of testicular GHRH-like mRNA (t-GHRH mRNA) and compared this to expression of insulin-like growth factor-I (IGF-I) and IGF-II mRNA in developing testis. t-GHRH mRNA was measured by dot blot hybridization and quantitated using a hypothalamic GHRH cRNA standard. t-GHRH mRNA was not detectable in Northern blots in fetal testis on day 19 of gestation, but was present in low but detectable amounts in testicular dot blots on day 2 of life (0.44 pg/jug total RNA). Levels of the RNA increased beginning on day 21 (1.72 ± 0.23 pg/Mg total RNA) and reached adult levels by day 30 (4.96 ± 0.84 pg/^g total RNA). The GHRH species on Northern analysis was about 1750 nucleotides at all ages examined; there was a larger species of about 3350 nucleotides seen on days 65 and 90. There was no correlation between the ontogeny of t-GHRH mRNA and either IGF-I or IGF-II mRNAs, which were maximally expressed in the testes of day 2 animals and decreased with age. To examine the influence of the pituitary gland on t-GHRH mRNA, levels of the mRNA were measured in the testes of hypophysectomized animals and age-matched controls. In animals hypophysectomized on day 21 and killed on day 42 and in animals hypophysectomized on day 42 and killed on day 63, there was marked
T
HE TESTIS is a critical organ during all stages of male development. Its influence begins during embryonic life when male sexual differentiation is initiated. Although hypothalamic-pituitary influences on testicular function are well defined, local factors controlling the growth and differentiation of the testis during development are largely unknown. The adult testis is a source of hypothalamic neuropeptides, including a LHRH-like Received March 5, 1990. Address all correspondence and requests for reprints to: Susan A. Berry, Department of Pediatrics, University of Minnesota, 420 Delaware Street SE, Box 75 UMHC, Minneapolis, Minnesota 55455. * This work was supported in part by the Graduate School, University of Minnesota, Minnesota Medical Foundation, the Vikings Childrens Fund, Biomedical Research Grant SO7-RR-5371J, and the James Whitcomb Riley Memorial Association. t Variety Club Scholar, University of Minnesota.
factor (1), TRH (2), GnRH (3), POMC (4), CRF (5, 6), and somatostatin (7). The role of these neuropeptides as paracrine regulators of gonadal function in the testis is unknown, although both CRF and POMC levels increase during periods of testicular development, suggesting that they could participate in testicular differentiation (5, 8, 9). Testis is also a site of growth factor synthesis (1012). Testicular insulin-like growth factor-I (IGF-I) may be regulated by GH (13), although this has not been firmly established (14). Recently, we observed that an immunoreactive GHreleasing hormone (GHRH)-like substance is present in testis, and that hybridization of testicular total RNA with a hypothalamic cDNA for GHRH results in a RNA species larger than that seen in hypothalamic total RNA (15). To our knowledge, GH has not been reported in
1404
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REGULATION OF TESTICULAR GHRH mRNA
testis. However, GH receptor (16) and both IGF-I and IGF-II (13) mRNAs are present in testis. Thus, in analogy to the hypothalamic-pituitary-growth factor axis, we speculated that testicular GHRH (t-GHRH) might function locally to mediate testicular IGFs. To investigate this, we examined the ontogeny of t-GHRH mRNA and compared it with the ontogeny of testicular IGF-I and IGF-II mRNAs. In addition, because of the influence of the pituitary and, specifically, GH in the expression of hypothalamic GHRH mRNA (17, 18), we studied the effects of hypophysectomy with and without GH replacement on expression of t-GHRH mRNA.
Materials and Methods Animals For examination of the ontogeny of GHRH, IGF-I, and IGFII mRNAs, Sprague-Dawley rats were obtained from Taconic Farms (Germantown, NY). For day 20 fetuses, timed pregnant dams were killed on day 20 of gestation. Fetuses were inspected for anogenital distance, and testes were isolated from male fetuses. For animals 2-42 days of age, pups were raised in litters adjusted to seven to nine pups per litter. Animals 65 and 90 days of age were obtained directly from the vendor. Testes were removed, decapsulated, and frozen at each age, except at day 20 of gestation and day 2, when testes were frozen without removal of the capsule. For examination of the effects of hypophysectomy, male Sprague-Dawley rats were hypophysectomized by the vendor (Harlan, Indianapolis, IN) at 21,42, and 60 days of age. Animals were given 10% glucose solution to drink, but received no other hormonal replacement. Weights were recorded on arrival and weekly. Animals that did not grow were considered hypophysectomized. Animals were killed approximately 3 weeks after their procedures (days 42, 63, and 80). Some day 60 animals were killed on day 90. As there was no difference between values from animals killed on either day 80 or day 90 these groups were pooled in the analysis of data. For evaluation of GH treatment after hypophysectomy, two experiments were performed using Sprague-Dawley rats from Hormone Assay Laboratory (Chicago, IL). In the first, animals were hypophysectomized at 21-22 of age, and GH treatment was initiated on day 42. Animals were treated with saline or 10 or 150 ng recombinant human (h) GH (Genentech, San Francisco, CA) by single daily injection for 14 days. Animals were killed approximately 1 h after their last daily dose. In the second experiment, animals were treated for 14 days with saline or 100 Mg hGH/100 g BW. Analysis of RNA Total RNA was extracted using guanidine HC1, as previously described (19), with volumes adjusted to extract 100 mg tissue. This necessitated pooling of testes for day 2 animals (16 pairs), the use of pairs of testes for day 10 and hypophysectomized animals, and the use of fragments of testes for all adults. Constant amounts of tissue were used so that samples could be extracted under uniform conditions, with the exception of fetal
1405
testicular RNA extraction, where 10 pairs of testes were extracted by the method of Chomczynski and Sacchi (20). All RNA used in these experiments had OD 260/280 ratios of 2.00 ± 0.15, and quantitation of concentration was based on OD 260. In addition, random samples from all extractions were run on denaturing gels to screen for intact 18S and 28S ribosomal RNA bands and for the relative visual intensity of ethidium bromide staining when equivalent quantities were loaded. Dot blots were prepared as previously described, using concentrations of 4, 2,1, and 0.5 ng total RNA (19). For standards, 0.5-32 pg cold sense GHRH cRNA were also applied to dot blots. To control for nonspecific hybridization, 0.5- to 4-^ig dots of yeast tRNA were placed on each filter. For Northern analysis of ontogeny of testicular total RNA, 10 ng total RNA from an individual sample at each age were subjected to electrophoresis in a 1.8% agarose gel (2.2 M formaldehyde-20 mM phosphate). The RNA was transferred to Zetaprobe membrane (Bio-Rad, Chicago, IL) by electrotransfer in 7.5 mM Tris (pH 7.8), 3.75 mM sodium acetate, and 37.5 fiM EDTA at 80 V for 6 h. For hybridization of GHRH mRNA, rat hypothalamic GHRH cDNA [gift of R. Evans (21)] was subcloned into the bifunctional plasmid vector pSP-72 (Promega, Madison, WI). RNA was generated using either T7- or SP6-RNA polymerases in a reaction mix of 40 mM Tris-HCl (pH 8.0) and 5 mM dithiothreitol with 1 mM ATP, CTP, and GTP in the presence of 1 U//xl RNAs in for 30 min at 37 C (22). Antisense [32P]labeled GHRH RNA was prepared by the addition of 40 nM UTP and 50 MCi [32P]UTP (3000 MCi/mM; New England Nuclear-DuPont, Boston, MA). Unlabeled sense strand RNA was prepared for standards by the addition of 1 mM UTP. DNA templates were removed from the RNA generated in the reaction by digestion with RNAse-free DNAse-I, followed by phenol extraction. The specific activity of the labeled probe was 1-8 x 108 cpm//ig cDNA. Dot blots were prehybridized and hybridized in 50% formamide, 2.5 X Denhardt's solution, 50 mM sodium phosphate (pH 7.5), 1 mM EDTA, 0.8 M NaCl, 10 /xg/ml poly(A), 200 Mg/ml sheared salmon sperm DNA, 100 Mg/ml yeast t-RNA. Northern blots were prehybridized in 50% formamide, 5x Denhardt's solution, 0.5% sodium dodecyl sulfate (SDS), 5 X SSPE [1 x SSPE = 180 mM NaCl, 10 mM sodium phosphate (pH 7.5), 1 mM EDTA], and 200 Mg/ml sheared salmon sperm DNA. The concentration of Denhardt's solution was decreased to 2.5fold for hybridization. Prehybridization was carried out for 412 h, and hybridization was performed for 18-24 h at 60 C. Filters were washed in 1 X SSPE-0.1% SDS for 15 min at room temperature, 1 X SSPE-0.1% SDS at 65 C for 20 min three times, and 0.1 x SSPE-0.1% SDS at 60 C for 50 min. For hybridization of IGF-I (23) and IGF-II [gift of M. M. Rechler (24)] cDNA inserts were excised by digestion with restriction endonucleases (EcoRl for IGF-I; Pstl for IGF-II) and labeled by random primer extension (25, 26) with 50 /uCi [32P]dCTP (3000 ^Ci/mM; New England Nuclear-DuPont) using a kit from Pharmacia (Piscataway, NJ). The specific activities of probes ranged from 7-10 X 108. Prehybridization and hybridization were performed in 50% formamide, 5 X Denhardt's solution, 50 mM sodium phosphate (pH 7.5), 0.2% SDS, 200 ixg/voX sheared salmon sperm DNA, and 100 Mg/ml yeast tRNA. Times of prehybridization and hybridization were the
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REGULATION OF TESTICULAR GHRH mRNA
1406
same as those for GHRH cRNA, but these procedures were carried out at 42 C. Filters were washed four times for 10 min each at room temperature in 2 X SSC (1 x SSC = 0.15 M NaCl, 0.015 M NaCitrate)-0.1% SDS and two or three times in 0.2 x SSC-0.2% SDS at 60 C. All filters were exposed to Kodak XAR5 film (Eastman Kodak, Rochester, NY) for varying times. Quantitation and statistical analysis Dot blots were quantitated by videodensitometry using a modification of the method of Mariash et al (27). Quantitation was based on comparison to fixed amounts of pooled RNA from the testes of day 30 and 65 animals consistently applied to all filters. Data were analyzed for significance by analysis of variance (ANOVA). Post-hoc testing by one-way ANOVA was performed using Fisher least mean differences at 95% significance. Analysis was performed using the program Statview 512+ (Abacus Concepts, Calabasas, CA).
Endo • 1990 Vol 127 • No 3
least significant difference, 95%). The actual mass of the RNA is probably underestimated by this method, as the testicular mRNA species is larger than that found in the hypothalamus (15). To determine if the mRNA detected in dot blot hybridizations was uniformly of the larger size seen in adult animals, the same RNA was subjected to electrophoresis in a 2.2 M formaldehyde denaturing gel (Northern analysis; Fig. 2). In addition, this permitted the examination of total RNA isolated from fetal testis. The mRNA species detected in testis was 1750 basepairs (bp) at all •2 2
10 15 17 19 21 23 25 30 42 65 90
28S
Results Ontogeny of GHRH-like mRNA in rat testis By dot blot hybridization, GHRH mRNA was present by day 2 of life in low, but detectable, levels in testicular total RNA (0.44 pg//ug total RNA; n = 2). The quantity of GHRH mRNA was significantly increased beginning on day 21 [1.72 ± 0.23 pg//ig total RNA (mean ± SD); n = 4], and maximal levels were reached by day 30 (4.96 ± 0.84 pg//ig total RNA; n = 4). These remained similar at subsequent ages tested (day 60, 4.53 ± 0.72; day 90, 4.93 ± 0.53 pg/^g total RNA; n = 4 for both; Fig. 1). There was a 10-fold change in detectable t-GHRH mRNA over the period tested. There was a significant effect of age on expression of t-GHRH mRNA by one-way ANOVA (df = 11,45; F = 41.63; P < 0.001). Differences from 2, 10, and 15 day levels were significant on day 21 (Fisher
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FIG. 1. Ontogeny of testicular GHRH-like mRNA. Testes from 4 separate animals were analyzed at each time point, except at 2 days, where 32 individuals were pooled into 2 individual samples (16/sample). The mean ± SD are shown.
FIG. 2. Northern blot of total RNA from testes of rats of varying ages. Each lane contains 10 ng total testicular RNA from a representative individual. Days of age are indicated above. - 2 , RNA isolated from pooled testes of 10 fetuses at 20 days gestation. Sizes of 18S and 28S ribosomal RNA bands are indicated. The middle inset shows the overexposed area of the gel where a larger, approximately 3350-nucleotide GHRH-like mRNA species is evident in 65- and 90-day-old adult male testicular total RNA. The lower inset shows the ethidium-stained gel from which this RNA was transferred. The relatively uniform density of the ribosomal bands indicates that similar quantities of total RNA were applied in each lane.
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REGULATION OF TESTICULAR GHRH mRNA
ages examined. On prolonged exposures, a larger 3350bp RNA species was seen in day 65 and 90 testes, but no evidence of the smaller hypothalamic-size species was present in any of the samples, and there was no detectable t-GHRH mRNA on Northern blots of fetal testicular total RNA. Ontogeny of IGF-I and II mRNAs in rat testis IGF-I and IGF-II mRNAs were also measured in total RNA from testes of postnatal animals to determine if their developmental expression correlated with that of tGHRH mRNA (Fig. 3). IGF-I was maximal on day 2, but fell to an intermediate value by day 19 [61 ± 16% of day 2 value (mean ± SD; n = 4 at all ages]. A further decrease was noted after day 60 (28 ± 6% of day 2 value). IGF-II was present in abundant quantities on day 2, but fell rapidly by day 10 to amounts only 37 ± 28% of day 2 values, and decreased further by day 15 (27 ± 12% of day 2 value). This level of expression was maintained until day 30, after which levels decreased further. Each of these incremental changes was significant (95%) by post-hoc testing after one-way ANOVA. Changes in both IGF-I and IGF-II mRNAs with age were significant (IGF-I: df = 9,37; F = 9.33; P < 0.001; IGF-II: df = 9,37; F = 33.96; P < 0.001). Neither IGF-I nor IGF-II mRNA was increased when expression of t-GHRH mRNA was maximal, and expression of these RNAs was highest in neonatal animals.
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measured by dot blot analysis in the testes of hypophysectomized rats and age-matched controls. Because of the distinctive ontogeny of t-GHRH mRNA, animals were hypophysectomized before t-GHRH mRNA levels rise, at the time of rising t-GHRH levels, and after tGHRH mRNA levels were stable. In all cases hypophysectomy resulted in marked decreases in testis weight. In animals hypophysectomized on day 21 and killed on day 42, t-GHRH mRNA was 19 ± 5% (mean ± SD; n = 5) of that observed in age-matched controls. Similarly, in animals hypophysectomized on day 42 and examined on day 63, t-GHRH mRNA levels were 9 ± 2% (n = 6) of age-matched control values. In contrast, in the testes of animals hypophysectomized on day 60 and examined on either day 80 or day 90 there was only a small difference in t-GHRH mRNA compared to those in agematched controls (73 ± 20%; n = 10 for hypophysectomized and 9 for controls; Fig. 4A). To determine if this
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Effect of hypophysectomy and response after GH administration To determine the effects of pituitary factors on the expression of t-GHRH mRNA, t-GHRH mRNA was
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