Testicular Metabolism and Serum Testosterone in Aging Male Rats S. W. C. CHAN, J. H. LEATHEM, AND T. ESASHI Bureau of Biological Research, Rutgers University, New Brunsioick, New Jersey 08903 ABSTRACT. Male Long-Evans rats, 4 and 18 months old, were studied for age differences in competence of androgen production. Testes from these animals were incubated in vitro with labeled progesterone. In the older rats, testosterone production was significantly lower than in the young rats. Aging also resulted in a sharp decline in 5a-androstane3a,17/3-diol production and a concomitant increase in a polar compound tentatively identified as 7ahydroxytestosterone, but had no effects on the formation of 17a-hydroxyprogesterone, androstenedione and 5a-dihydrotestosterone. Basal levels of serum testosterone were measured in a circadian time course in both 4 and 18 month old rats. Serum testosterone was significantly greater at all times in the younger animals with the exception of 2000 h
and of 2400 h, the latter being the nadir for both age groups. Peak levels of serum testosterone were found at 0800 h and 1600 h in the young and at 0800 h and 2000 h in the older animals. Thus, the older rats exhibited reduced testosterone levels but circadian rhythmicity was retained in a manner that mimicked that of the young rats. Testicular responsivity to human chorionic gonadotropin (hCG) was compared in the two age groups. Whereas both groups of animals responded to 50 IU of hCG with a rise in serum testosterone, a delayed responsiveness of lower magnitude was noted in aging. These results indicate an inherent loss in steroidogenic function of the rat testis in aging. (Endocrinology 101: 128, 1977)
P
REVIOUS reports on testicular incubation with progesterone have indicated that the pattern of metabolism varies with the age of the animal. In the rat, the ratio of production of testosterone to that of 5areduced C-19 steroids appeared to change depending on the stage of testicular differentiation. Thus, amounts of 5a-reduced metabolites were high in the sexually immature rat, an age at which testosterone production was minimal, whereas the converse was found for the adult male rat (1-4). However, little information is available relative to testicular metabolism of steroid hormones in senescence. Collins et at. (5) examined the interstitial tissue dissected from testes of rats 4, 6 and 15 months of age. In vitro incubations using pregnenolone and progesterone as substrates revealed a reduced conversion to androstenedione and testosterone in aging. Furthermore, the enzyme A5-3/3-hydroxysteroid dehydrogenase was found to decline between 12 and 18 months of age in the Long-Evans rat (6). Serum testosterone concentrations are Received October 13, 1976. Supported by the Charles and Johanna Busch Memorial Fund and U.S.P.H.S. research grant AG 00468.
greater in mature than in senescent rats (7) and guinea pigs (8) but no change was present in aging DBA and C57BL/6J (9) mice unless pathology was also present (10). Serum testosterone levels reflect pulsatile release of the steroid and diurnal variations. In man, peak levels of testosterone occur in the morning and the nadir is at midnight (11,12), but the converse has been reported for the, rhesus monkey (13) and the lemur (14). Kinson and Liu (15) observed a circadian rhythm in the rat in which serum testosterone was maximal at 0300-0600 h and a nadir occurred at midnight. In the immature rat, however, the peak level occurred at 0800 h and the nadir at 16002000 h (16). The present study examines the changes with age in testicular steroid metabolism, in serum testosterone levels and in the response of the latter to human chorionic gonadotropin (hCG) stimulation. Additionally, a malfunction in the hypothalamuspituitary axis has been implicated in aging, and since neural centers are involved in monitoring diurnal changes in environmental stimuli, the question of retention or modification of the circadian rhythm in testosterone secretion was also studied.
128
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TESTICULAR STEROIDOGENESIS IN AGING RATS
129
Materials and Methods
cubated for 4 h at 34 C in an atmosphere of 95% O 2 -5% CO2 in a Dubnoff Metabolic Shaking Incubator.
Male rats of Long-Evans strain from our inbred colony were used. Animals were kept at 78 F and controlled light from 0600 to 1800 h. All animals were of known birth date and had never been used as breeders. They were fed Purina laboratory chow supplemented weekly with cod liver oil on bread. Animals 4 and 18 months of age were used, and on autopsy, they were examined for gross pathology; animals bearing tumors of the reproductive system were discarded.
Extraction, isolation and identificattion of radiometabolites
Animals
Steroids Testosterone, 5a-dihydrotestosterone, androsterone, androstanediol (5a-androstane-3a,17/3diol), androstenedione, progesterone, 17ahydroxyprogesterone and dehydroepiandrosterone were purchased from Sigma Co., U.S.A. Radioactive steroids, [7a-3H]testosterone (SA 17.5 Ci/mmol), [1,2,6,7-3H]testosterone (SA 106 Ci/mmol), [4-I4C]testosterone (SA 55 Ci/ mmol), [7a-3H]progesterone (SA 10 Ci/mmol), [4-14C]progesterone (SA 52.8 mCi/mmol) and [4- 14 C]androstenedione (SA 50 mCi/mmol), were purchased from New England Nuclear Corp., U.S.A. Radiochemical purity was checked, and the steroids were purified by thin-layer chromatography prior to use. Liquid scintillation counting Biofluor, purchased from New England Nuclear Corp., was used for radioimmunoassay counting. For the counting of nonaqueous samples, PPO/POPOP (4 g and 0.1 g per liter) in toluene was used as a phosphor. Samples were counted with a Packard Tricarb liquid scintillation counter, Model 3320. Incubation At autopsy, the animals were killed by decapitation; the testes were removed, and trimmed of adhering tissue and weighed. The tunica albuginea was dissected and removed, and the tissue lightly blotted on filter paper. Approximately 250 mg of tissue, accurately weighed, was transferred to a 25 ml flask containing 2 fxCi [7a-3H]progesterone, previously dried, and 10 ml of Krebs-Hensleit bicarbonate buffer was added. The tissue was teased with fine forceps, and in-
Following incubation, the incubates were filtered through glass wool, and the residual tissue was counted for radioactivity after digestion with hydroxide of hyamine 10-X. Small amounts of purified [4- l4 C]progesterone, [4- 14 C]androstenedione and [4-14C]testosterone were added to the filtrates as internal standards to correct for loss. The incubates were extracted twice with four volumes of methylene chloride. The organic phase was evaporated to dryness. Prior to chromatography, non-radioactive steroids, 100 fxg each of progesterone, androstenedione, 17a-hydroxyprogesterone and testosterone were added as carriers. The following paper (PPC) and thin-layer chromatographic (TLC) systems were used. PPC 1. Light petroleum (60-80 C):methanol: water (100:70:30) 2. Propylene glycol/cyclohexane:water (1:1) TLC 1. Acetone:benzene (2:8, 2 developments) 2. Benzene:light petroleum (60-80 C): ethyl acetate (1:1:4) 3. Chloroform:methanol (94:6) 4. Benzene:ethanol (9:1) 5. n-Heptane:ethyl acetate (5:2) The radiometabolites were tentatively identified by identical mobilities with reference steroids, and were further characterized by recrystallization to constant specific activities (17). Circadian rhythm of serum testosterone Two groups of six animals, 4 and 18 months of age, were used. Blood samples (0.3-0.5 ml) were obtained by cardiac puncture with animals under light ether anesthesia. The animals were then immediately injected ip with an equal volume of 0.9% saline. Consecutive bleedings on the same animals were carried out no more frequently than 24 h apart so that six different bleeding times were obtained. Triplicate aliquots of 0.05 ml of sera were used for each determination of testosterone by radioimmunoassay. Briefly, the procedure included ether extraction, separation by Sephadex LH-20 column chroma-
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TABLE 1. Identification of radiometabolites from testicular incubations with [7a-3H]progesterone by recrystallization to constant specific activity (dpni/mg)1 Recrystallization Sample
1st
2nd
3rd
Mean
173 181 185
175 183 195
Testosterone2 1 2 3
177 181 190
176 186 210
Androstenedione2 1 2 3
4. 99 3. 72 2. 77
4.86 3.77 2.72
4.91 3.72 2.74
4.88 3.68 2.74
0.1 ml of 0.9% saline. Control animals were injected with vehicle only. At 15, 30 and 45 min after injection, animals were bled. They were lightly anesthetized with ether, and blood samples (0.3-0.5 ml) were withdrawn by cardiac puncture; an equal volume of 0.9% saline was then immediately injected ip. Blood was allowed to clot and serum was obtained following centrifugation. Triplicate aliquots of 0.05 ml sera were used for each determination of testosterone by radioimmunoassay. Statistical analysis Results were analyzed by ANOVA, "Studentized Range Test" and Student's t test.
17a-Hydroxyprogesterone 1 2
761 718
719 685
Results
730 661
736 688
5a-Dihydrotestosterone 1 2 3
2,370 2,970 2,380
2,380 2,910 2,470
2,370 2,910 2,410
2,370 2,930 2,420
5a-Androstane-3a ,17/3-diol 1 2 3
Endo i 1977 Vol 101 i No 1
CHAN, LEATHEM AND ESASHI
130
16,200 23,500 17,900
16,500 25,200 18,900
16,300 25,600 18,300
16,300 24,800 18,400
1
Radiochemical purity is established when deviation from mean is
and of 2400 h, the latter being the nadir for both age groups. Peak levels of serum testosterone were found at 0800 h and 1600 h in the young and at 0800 h and 2000 h in the older animals. Thus, the older rats exhibited reduced testosterone levels but circadian rhythmicity was retained in a manner that mimicked that of the young rats. Testicular responsivity to human chorionic gonadotropin (hCG) was compared in the two age groups. Whereas both groups of animals responded to 50 IU of hCG with a rise in serum testosterone, a delayed responsiveness of lower magnitude was noted in aging. These results indicate an inherent loss in steroidogenic function of the rat testis in aging. (Endocrinology 101: 128, 1977)
P
REVIOUS reports on testicular incubation with progesterone have indicated that the pattern of metabolism varies with the age of the animal. In the rat, the ratio of production of testosterone to that of 5areduced C-19 steroids appeared to change depending on the stage of testicular differentiation. Thus, amounts of 5a-reduced metabolites were high in the sexually immature rat, an age at which testosterone production was minimal, whereas the converse was found for the adult male rat (1-4). However, little information is available relative to testicular metabolism of steroid hormones in senescence. Collins et at. (5) examined the interstitial tissue dissected from testes of rats 4, 6 and 15 months of age. In vitro incubations using pregnenolone and progesterone as substrates revealed a reduced conversion to androstenedione and testosterone in aging. Furthermore, the enzyme A5-3/3-hydroxysteroid dehydrogenase was found to decline between 12 and 18 months of age in the Long-Evans rat (6). Serum testosterone concentrations are Received October 13, 1976. Supported by the Charles and Johanna Busch Memorial Fund and U.S.P.H.S. research grant AG 00468.
greater in mature than in senescent rats (7) and guinea pigs (8) but no change was present in aging DBA and C57BL/6J (9) mice unless pathology was also present (10). Serum testosterone levels reflect pulsatile release of the steroid and diurnal variations. In man, peak levels of testosterone occur in the morning and the nadir is at midnight (11,12), but the converse has been reported for the, rhesus monkey (13) and the lemur (14). Kinson and Liu (15) observed a circadian rhythm in the rat in which serum testosterone was maximal at 0300-0600 h and a nadir occurred at midnight. In the immature rat, however, the peak level occurred at 0800 h and the nadir at 16002000 h (16). The present study examines the changes with age in testicular steroid metabolism, in serum testosterone levels and in the response of the latter to human chorionic gonadotropin (hCG) stimulation. Additionally, a malfunction in the hypothalamuspituitary axis has been implicated in aging, and since neural centers are involved in monitoring diurnal changes in environmental stimuli, the question of retention or modification of the circadian rhythm in testosterone secretion was also studied.
128
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TESTICULAR STEROIDOGENESIS IN AGING RATS
129
Materials and Methods
cubated for 4 h at 34 C in an atmosphere of 95% O 2 -5% CO2 in a Dubnoff Metabolic Shaking Incubator.
Male rats of Long-Evans strain from our inbred colony were used. Animals were kept at 78 F and controlled light from 0600 to 1800 h. All animals were of known birth date and had never been used as breeders. They were fed Purina laboratory chow supplemented weekly with cod liver oil on bread. Animals 4 and 18 months of age were used, and on autopsy, they were examined for gross pathology; animals bearing tumors of the reproductive system were discarded.
Extraction, isolation and identificattion of radiometabolites
Animals
Steroids Testosterone, 5a-dihydrotestosterone, androsterone, androstanediol (5a-androstane-3a,17/3diol), androstenedione, progesterone, 17ahydroxyprogesterone and dehydroepiandrosterone were purchased from Sigma Co., U.S.A. Radioactive steroids, [7a-3H]testosterone (SA 17.5 Ci/mmol), [1,2,6,7-3H]testosterone (SA 106 Ci/mmol), [4-I4C]testosterone (SA 55 Ci/ mmol), [7a-3H]progesterone (SA 10 Ci/mmol), [4-14C]progesterone (SA 52.8 mCi/mmol) and [4- 14 C]androstenedione (SA 50 mCi/mmol), were purchased from New England Nuclear Corp., U.S.A. Radiochemical purity was checked, and the steroids were purified by thin-layer chromatography prior to use. Liquid scintillation counting Biofluor, purchased from New England Nuclear Corp., was used for radioimmunoassay counting. For the counting of nonaqueous samples, PPO/POPOP (4 g and 0.1 g per liter) in toluene was used as a phosphor. Samples were counted with a Packard Tricarb liquid scintillation counter, Model 3320. Incubation At autopsy, the animals were killed by decapitation; the testes were removed, and trimmed of adhering tissue and weighed. The tunica albuginea was dissected and removed, and the tissue lightly blotted on filter paper. Approximately 250 mg of tissue, accurately weighed, was transferred to a 25 ml flask containing 2 fxCi [7a-3H]progesterone, previously dried, and 10 ml of Krebs-Hensleit bicarbonate buffer was added. The tissue was teased with fine forceps, and in-
Following incubation, the incubates were filtered through glass wool, and the residual tissue was counted for radioactivity after digestion with hydroxide of hyamine 10-X. Small amounts of purified [4- l4 C]progesterone, [4- 14 C]androstenedione and [4-14C]testosterone were added to the filtrates as internal standards to correct for loss. The incubates were extracted twice with four volumes of methylene chloride. The organic phase was evaporated to dryness. Prior to chromatography, non-radioactive steroids, 100 fxg each of progesterone, androstenedione, 17a-hydroxyprogesterone and testosterone were added as carriers. The following paper (PPC) and thin-layer chromatographic (TLC) systems were used. PPC 1. Light petroleum (60-80 C):methanol: water (100:70:30) 2. Propylene glycol/cyclohexane:water (1:1) TLC 1. Acetone:benzene (2:8, 2 developments) 2. Benzene:light petroleum (60-80 C): ethyl acetate (1:1:4) 3. Chloroform:methanol (94:6) 4. Benzene:ethanol (9:1) 5. n-Heptane:ethyl acetate (5:2) The radiometabolites were tentatively identified by identical mobilities with reference steroids, and were further characterized by recrystallization to constant specific activities (17). Circadian rhythm of serum testosterone Two groups of six animals, 4 and 18 months of age, were used. Blood samples (0.3-0.5 ml) were obtained by cardiac puncture with animals under light ether anesthesia. The animals were then immediately injected ip with an equal volume of 0.9% saline. Consecutive bleedings on the same animals were carried out no more frequently than 24 h apart so that six different bleeding times were obtained. Triplicate aliquots of 0.05 ml of sera were used for each determination of testosterone by radioimmunoassay. Briefly, the procedure included ether extraction, separation by Sephadex LH-20 column chroma-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 10:30 For personal use only. No other uses without permission. . All rights reserved.
TABLE 1. Identification of radiometabolites from testicular incubations with [7a-3H]progesterone by recrystallization to constant specific activity (dpni/mg)1 Recrystallization Sample
1st
2nd
3rd
Mean
173 181 185
175 183 195
Testosterone2 1 2 3
177 181 190
176 186 210
Androstenedione2 1 2 3
4. 99 3. 72 2. 77
4.86 3.77 2.72
4.91 3.72 2.74
4.88 3.68 2.74
0.1 ml of 0.9% saline. Control animals were injected with vehicle only. At 15, 30 and 45 min after injection, animals were bled. They were lightly anesthetized with ether, and blood samples (0.3-0.5 ml) were withdrawn by cardiac puncture; an equal volume of 0.9% saline was then immediately injected ip. Blood was allowed to clot and serum was obtained following centrifugation. Triplicate aliquots of 0.05 ml sera were used for each determination of testosterone by radioimmunoassay. Statistical analysis Results were analyzed by ANOVA, "Studentized Range Test" and Student's t test.
17a-Hydroxyprogesterone 1 2
761 718
719 685
Results
730 661
736 688
5a-Dihydrotestosterone 1 2 3
2,370 2,970 2,380
2,380 2,910 2,470
2,370 2,910 2,410
2,370 2,930 2,420
5a-Androstane-3a ,17/3-diol 1 2 3
Endo i 1977 Vol 101 i No 1
CHAN, LEATHEM AND ESASHI
130
16,200 23,500 17,900
16,500 25,200 18,900
16,300 25,600 18,300
16,300 24,800 18,400
1
Radiochemical purity is established when deviation from mean is
