Original Paper Neuroendocrinology 1992;56:264-270
Departments of Pediatrics and Pharmacology, University of Virginia Health Sciences Center, Charlottesville, Va.; Department of Anatomy, Medical College of Virginia, Richmond, Va., USA
Key Words Proopiomelanocortin Messenger ribonucleic acid Androgen action In situ hybridization histochemistry
Exogenous Androgen Does Not Alter Hypothalamic Proopiomelanocortin Gene Transcript Levels in the Sexually Immature Male Rat
Abstract To investigate possible mechanisms whereby the augmentation of hypotha lamic proopiomelanocortin (POMC) messenger ribonucleic acid (mRNA) levels occurs with pubertal development, we employed the techniques of tes tosterone administration and in situ hybridization histochemistry in sexually immature male rats. Six animals from each of the following groups were stud ied: (1) untreated controls (CTRL); (2) empty capsule (SHAM); (3) testoster one capsule (TEST), and (4) untreated adults (ADLT). Capsules were im planted at 21 days of age. Groups 1-3 were sacrificed at 35 days of life; group 4 at 55 days. Ventral prostate and seminal vesicle weights were obtained to assess the biologic effect of testosterone. Hybridizations were performed on coronal brain slices through the region of the arcuate nucleus using a 35Slabeled oligonucleotide probe complementary to a 30-base sequence within POMC mRNA. Anatomically matched tissue sections (1 1 per animal, from the retrochiasmatic region rostrally to the premammillary nucleus caudally) were exposed to x-ray film, followed by densitometric analysis. The mean se rum testosterone concentration of the TEST group was significantly greater than that of the ADLT animals; values for the CTRL and SHAM rats were undetectable. The accessory sex organ weights of the ADLT animals were greater than those of the TEST rats; both values were greater than those of the CTRL and SHAM groups which were indistinguishable. Increased levels of hypothalamic POMC mRNA were observed in the male rat after pubertal development. However, no effect of exogenous androgen administration was demonstrable on hypothalamic POMC gene transcript levels in sexually im mature rats despite an observed biologic effect of increased size of the acces sory sex organs. These findings suggest that gonadal steroid hormones alone are insufficient, at least in the prepubertal state, to stimulate the expression of the hypothalamic POMC gene. Additional factors, such as those responsible for the initiation of puberty and maturational processes of puberty, may be important in the regulation of POMC gene expression.
Received: July 18, 1991 Accepted after revision January 2. 1992
James R. Kerrigan. MD Department of Pediatries MR-4 Building/Room 3037 University o f Virginia Health Sciences Center Charlottesville. VA 22908 (USA)
Downloaded by: Stockholm University Library 130.237.165.40 - 11/14/2018 5:00:42 PM
James R. Kerrigan“ Richard J. Krieg, Jr.c Alan D. Rogola h
Materials and Methods Animals Male King x Holtzman rats (Stanley-Gumbreck line, Introgene, Oklahoma City, Okla., USA), were housed under a 14:10 h light-dark cycle with lights on at 05:00 h; the temperature was main tained at 21-23 °C. All animals were obtained from a colony kept in the Medical College of Virginia facilities. The rats were allowed free access to standard laboratory chow and water. All animals were weaned at 21 days oflife. Protocol Six animals from each group were included. Untreated controls (CTRL) received no intervention, whereas empty Silastic capsules were implanted subcutaneously in the placebo-treated group (SHAM). Testosterone-filled Silastic capsules were implanted in the third group (TEST), while the fourth group, untreated adults (ADLT), received no treatment. Silastic capsules were implanted at
2 1 days of age. Groups 1-3 were sacrificed at 35 days of age; group 4 at 55 days. Prostate and seminal vesicle weights were obtained to assess the biologic effect of testosterone. The study was approved and conducted according to guidelines set forth by the University of Virginia Department of Comparative Medicine and the Medical College of Virginia Division of Animal Resources which are fully accredited by the American Association for Accreditation of Labo ratory Animal Care. Experimental Treatment At 21 days of life, animals from the SHAM and TEST groups were anesthetized with ether prior to surgical implantation of Silas tic capsules (1.58 mm ID and 3.18 mm OD; Dow Corning, Mid land, Mich., USA). The capsules (all 17 mm in length) were inserted through a midline incision between the scapulae of the rats. Empty implants were used for the SHAM group while testosterone-filled capsules were implanted in the TEST animals. The dose of testoster one was chosen based on previous experimental work in our labora tory (data not shown) and was intended to provide circulating levels of the hormone within the normal adult male range. The 14-day period of treatment was used to approximate the duration of an drogen exposure to which the 55-day-old adult animals would have been exposed; serum testosterone levels rise by 40 days oflife in normal male rats [10]. Tissue Preparation At the appropriate age, animals were weighed prior to decapita tion. All rats were sacrificed between 13:00 and 15:00 h. The brains were removed immediately following sacrifice, frozen on dry ice, wrapped in aluminum foil and plastic wrap and stored at -80 °C. Trunk blood was obtained from each animal, and the serum sepa rated and frozen at -20 ”C prior to measurement of gonadal steroid hormone concentrations. Accessory sex organs were dissected, blotted on absorbent paper, and weighed. Fluid was expressed with positive pressure prior to weighing. For in situ hybridization histochemical analysis, the frozen brains were allowed to equilibrate in a cryostat at -16 °C. Coronal sections were cut at 16 pm and then thaw-mounted onto gelatincoated slides. With the aid of an anatomical atlas [II], brain sections were collected beginning at the retrochiasmatic area rostrally and continuing caudally to the premammillary area (plates No. 25 through 33). After drying at room temperature for 2-3 h, the tissue sections were stored at -20 °C prior to in situ hybridization histo chemical analysis. hi situ Hybridization Histochemistry The technique was performed as described previously [9] with several modifications. All tissue sections from every animal were included in a single run to eliminate interassay variability. Tissue sections were fixed in 4% paraformaldehyde for 5 min, rinsed in 0.1 M phosphate-buffered saline (PBS) for 5-10 min and then trans ferred to 2 x SSC (I x SSC = 0.15 M NaCI/0.015 M Na citrate) for 30 min at room temperature. While not allowing the tissue sec tions to dry, the radiolabeled POMC oligonucleotide probe (No. NEP-504, Dupont NEN Research Products, Wilmington, Del., USA) contained in 300 pi of hybridization buffer [50% (vol/vol) formamide/10% dextran sulfate/1 x Denhardt’s solution/0.25 mg of yeast tRNA per ml/1.5 mg dithiothreitol per ml/0.5 mg salmon sperm DNA per ml in 4 x SSC] was applied to each slide (4 tissue sections per slide). The oligonucleotide probe was 3’-end labeled
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Downloaded by: Stockholm University Library 130.237.165.40 - 11/14/2018 5:00:42 PM
Hypothalamic opioid peptides play an important role in modulating the feedback action of gonadal steroid hormones on gonadotropin secretion [1, 2], Indeed, the functional activity of the endogenous opiates has been demonstrated to vary with circulating levels of the sex hormones [3]. In animal studies, an association has been demonstrated between changes of gonadal steroid hor mone levels and alterations of hypothalamic proopiome lanocortin (POMC, the precursor molecule of |)-endorphin and related peptides) gene transcript levels [4-7]. In adult male rats, surgical castration results acutely in de creased levels of POMC messenger ribonucleic acid (mRNA). The replacement in such gonadectomized ani mals with physiologic doses of testosterone results in nor malization of the quantity of gene transcript [5, 6]. In the chronically (2-4 weeks) orchidectomized adult animals, increased levels of POMC mRNA have been detected [7]. A rise in hypothalamic POMC gene transcript levels has been observed across pubertal development in the male rat, a biologic process associated with increasing cir culating sex hormone concentrations [8, 9]. However, the precise role of the gonadal steroid hormones in this phe nomenon is presently undetermined. Additional critical factors associated with sexual maturation may be of equal, or greater, importance in the regulation of the hy pothalamic POMC gene. To investigate whether gonadal steroid hormone ac tion alone, in the absence of pubertal development, can alter steady state levels of hypothalamic POMC mRNA, we have employed the techniques of exogenous androgen administration and in situ hybridization histochemistry in the normal prepubertal male rat.
Table 1. Body weights, serum testoster one concentrations and accessory sex organ weights
Experimental group
Body weight g
Testosterone nmol/l
Paired seminal vesicle weight mg
Ventral prostate weight mg
CTRL SHAM TEST ADLT
99 ±3* 100 ± 2* 101 ±2* 230 ±7**
Departments of Pediatrics and Pharmacology, University of Virginia Health Sciences Center, Charlottesville, Va.; Department of Anatomy, Medical College of Virginia, Richmond, Va., USA
Key Words Proopiomelanocortin Messenger ribonucleic acid Androgen action In situ hybridization histochemistry
Exogenous Androgen Does Not Alter Hypothalamic Proopiomelanocortin Gene Transcript Levels in the Sexually Immature Male Rat
Abstract To investigate possible mechanisms whereby the augmentation of hypotha lamic proopiomelanocortin (POMC) messenger ribonucleic acid (mRNA) levels occurs with pubertal development, we employed the techniques of tes tosterone administration and in situ hybridization histochemistry in sexually immature male rats. Six animals from each of the following groups were stud ied: (1) untreated controls (CTRL); (2) empty capsule (SHAM); (3) testoster one capsule (TEST), and (4) untreated adults (ADLT). Capsules were im planted at 21 days of age. Groups 1-3 were sacrificed at 35 days of life; group 4 at 55 days. Ventral prostate and seminal vesicle weights were obtained to assess the biologic effect of testosterone. Hybridizations were performed on coronal brain slices through the region of the arcuate nucleus using a 35Slabeled oligonucleotide probe complementary to a 30-base sequence within POMC mRNA. Anatomically matched tissue sections (1 1 per animal, from the retrochiasmatic region rostrally to the premammillary nucleus caudally) were exposed to x-ray film, followed by densitometric analysis. The mean se rum testosterone concentration of the TEST group was significantly greater than that of the ADLT animals; values for the CTRL and SHAM rats were undetectable. The accessory sex organ weights of the ADLT animals were greater than those of the TEST rats; both values were greater than those of the CTRL and SHAM groups which were indistinguishable. Increased levels of hypothalamic POMC mRNA were observed in the male rat after pubertal development. However, no effect of exogenous androgen administration was demonstrable on hypothalamic POMC gene transcript levels in sexually im mature rats despite an observed biologic effect of increased size of the acces sory sex organs. These findings suggest that gonadal steroid hormones alone are insufficient, at least in the prepubertal state, to stimulate the expression of the hypothalamic POMC gene. Additional factors, such as those responsible for the initiation of puberty and maturational processes of puberty, may be important in the regulation of POMC gene expression.
Received: July 18, 1991 Accepted after revision January 2. 1992
James R. Kerrigan. MD Department of Pediatries MR-4 Building/Room 3037 University o f Virginia Health Sciences Center Charlottesville. VA 22908 (USA)
Downloaded by: Stockholm University Library 130.237.165.40 - 11/14/2018 5:00:42 PM
James R. Kerrigan“ Richard J. Krieg, Jr.c Alan D. Rogola h
Materials and Methods Animals Male King x Holtzman rats (Stanley-Gumbreck line, Introgene, Oklahoma City, Okla., USA), were housed under a 14:10 h light-dark cycle with lights on at 05:00 h; the temperature was main tained at 21-23 °C. All animals were obtained from a colony kept in the Medical College of Virginia facilities. The rats were allowed free access to standard laboratory chow and water. All animals were weaned at 21 days oflife. Protocol Six animals from each group were included. Untreated controls (CTRL) received no intervention, whereas empty Silastic capsules were implanted subcutaneously in the placebo-treated group (SHAM). Testosterone-filled Silastic capsules were implanted in the third group (TEST), while the fourth group, untreated adults (ADLT), received no treatment. Silastic capsules were implanted at
2 1 days of age. Groups 1-3 were sacrificed at 35 days of age; group 4 at 55 days. Prostate and seminal vesicle weights were obtained to assess the biologic effect of testosterone. The study was approved and conducted according to guidelines set forth by the University of Virginia Department of Comparative Medicine and the Medical College of Virginia Division of Animal Resources which are fully accredited by the American Association for Accreditation of Labo ratory Animal Care. Experimental Treatment At 21 days of life, animals from the SHAM and TEST groups were anesthetized with ether prior to surgical implantation of Silas tic capsules (1.58 mm ID and 3.18 mm OD; Dow Corning, Mid land, Mich., USA). The capsules (all 17 mm in length) were inserted through a midline incision between the scapulae of the rats. Empty implants were used for the SHAM group while testosterone-filled capsules were implanted in the TEST animals. The dose of testoster one was chosen based on previous experimental work in our labora tory (data not shown) and was intended to provide circulating levels of the hormone within the normal adult male range. The 14-day period of treatment was used to approximate the duration of an drogen exposure to which the 55-day-old adult animals would have been exposed; serum testosterone levels rise by 40 days oflife in normal male rats [10]. Tissue Preparation At the appropriate age, animals were weighed prior to decapita tion. All rats were sacrificed between 13:00 and 15:00 h. The brains were removed immediately following sacrifice, frozen on dry ice, wrapped in aluminum foil and plastic wrap and stored at -80 °C. Trunk blood was obtained from each animal, and the serum sepa rated and frozen at -20 ”C prior to measurement of gonadal steroid hormone concentrations. Accessory sex organs were dissected, blotted on absorbent paper, and weighed. Fluid was expressed with positive pressure prior to weighing. For in situ hybridization histochemical analysis, the frozen brains were allowed to equilibrate in a cryostat at -16 °C. Coronal sections were cut at 16 pm and then thaw-mounted onto gelatincoated slides. With the aid of an anatomical atlas [II], brain sections were collected beginning at the retrochiasmatic area rostrally and continuing caudally to the premammillary area (plates No. 25 through 33). After drying at room temperature for 2-3 h, the tissue sections were stored at -20 °C prior to in situ hybridization histo chemical analysis. hi situ Hybridization Histochemistry The technique was performed as described previously [9] with several modifications. All tissue sections from every animal were included in a single run to eliminate interassay variability. Tissue sections were fixed in 4% paraformaldehyde for 5 min, rinsed in 0.1 M phosphate-buffered saline (PBS) for 5-10 min and then trans ferred to 2 x SSC (I x SSC = 0.15 M NaCI/0.015 M Na citrate) for 30 min at room temperature. While not allowing the tissue sec tions to dry, the radiolabeled POMC oligonucleotide probe (No. NEP-504, Dupont NEN Research Products, Wilmington, Del., USA) contained in 300 pi of hybridization buffer [50% (vol/vol) formamide/10% dextran sulfate/1 x Denhardt’s solution/0.25 mg of yeast tRNA per ml/1.5 mg dithiothreitol per ml/0.5 mg salmon sperm DNA per ml in 4 x SSC] was applied to each slide (4 tissue sections per slide). The oligonucleotide probe was 3’-end labeled
265
Downloaded by: Stockholm University Library 130.237.165.40 - 11/14/2018 5:00:42 PM
Hypothalamic opioid peptides play an important role in modulating the feedback action of gonadal steroid hormones on gonadotropin secretion [1, 2], Indeed, the functional activity of the endogenous opiates has been demonstrated to vary with circulating levels of the sex hormones [3]. In animal studies, an association has been demonstrated between changes of gonadal steroid hor mone levels and alterations of hypothalamic proopiome lanocortin (POMC, the precursor molecule of |)-endorphin and related peptides) gene transcript levels [4-7]. In adult male rats, surgical castration results acutely in de creased levels of POMC messenger ribonucleic acid (mRNA). The replacement in such gonadectomized ani mals with physiologic doses of testosterone results in nor malization of the quantity of gene transcript [5, 6]. In the chronically (2-4 weeks) orchidectomized adult animals, increased levels of POMC mRNA have been detected [7]. A rise in hypothalamic POMC gene transcript levels has been observed across pubertal development in the male rat, a biologic process associated with increasing cir culating sex hormone concentrations [8, 9]. However, the precise role of the gonadal steroid hormones in this phe nomenon is presently undetermined. Additional critical factors associated with sexual maturation may be of equal, or greater, importance in the regulation of the hy pothalamic POMC gene. To investigate whether gonadal steroid hormone ac tion alone, in the absence of pubertal development, can alter steady state levels of hypothalamic POMC mRNA, we have employed the techniques of exogenous androgen administration and in situ hybridization histochemistry in the normal prepubertal male rat.
Table 1. Body weights, serum testoster one concentrations and accessory sex organ weights
Experimental group
Body weight g
Testosterone nmol/l
Paired seminal vesicle weight mg
Ventral prostate weight mg
CTRL SHAM TEST ADLT
99 ±3* 100 ± 2* 101 ±2* 230 ±7**
