STIMULATION OF PROTEIN SYNTHESIS IN VIVO IN IMMATURE MOUSE TESTIS BY FSH A. G. DAVIES, W. E. DAVIES
and
CHRISTINE SUMNER
Departments of Physiology and Neurocommunications, Medical School, Birmingham PI5 2TJ (Received 25th April 1974) Human pituitary FSH was found to increase the inof tritiated lysine into testicular protein in prepubertal mice in vivo. Radioactivity was measured in washed trichloracetic acid precipitates prepared from crude testicular homogenates. The time of maximum response was 8 to 16 hr after subcutaneous injection of the hormone. This was considerably later than the maximum response in vitro reported by other workers. Neither HCG nor dibutyryl cyclic 3\m='\,5\m='\-adenosinemonophosphate had a significant effect on the incorporation of lysine.
Summary. corporation
INTRODUCTION
Means & Hall (1967) found that FSH increased the incorporation of amino acids into testicular protein in vitro. This paper reports experiments designed to find out whether a similar effect occurs in vivo, whether HCG has this action and whether the action can be reproduced by dibutyryl cyclic 3',5'-adenosine monophosphate (dbcAMP), as it has been shown that FSH stimulates adenyl cyclase activity in the testis (Murad, Strauch & Vaughn, 1969; Kuehl, Patanelli, Tarnoff & Humes, 1970; Dorrington, Vernon & Fritz, 1972). MATERIALS AND METHODS
Animals and experimental design The mice used were of the CFW strain. All male young born on the same day were fostered in groups of between ten and sixteen by pairs of lactating females. Each experiment was designed as a randomized-block with replication. The young of most uniform body weight in a foster group composed a randomized block.
Gonadotrophins Human pituitary FSH was extracted and assayed as described by Butt, Crooke & Cunningham (1961). In Exps 1 and 2 and in two replicates of Exp. 5, the FSH used was not highly purified. The potency of this preparation was 700 i.u. FSH and 140 i.u. LH activity/mg. In the remaining two replicates of Exp. 5 and in Exps 3 and 4, a more highly purified preparation was used. This contained 2000 i.u. (250 ^g) FSH and less than 120 i.u. LH/mg. The potency of ß
415
A. G. Davies et al.
416
the HCG (Paines and Byrne Ltd) was greater than 1500 i.u. LH and less than 15 i.u. FSH/mg. The hormones were injected subcutaneously in 0-1 ml saline. This route was used because neither intravenous nor intraperitoneal injections were practicable in the younger mice.
Radioisotope
In every experiment, each mouse was given a single subcutaneous injection of 5 µ L-[4,5-3H]lysine monohydrochloride (sp. act. 250 mCi/mmol) in 0-15 ml saline 2 hr before autopsy.
Extraction procedure The mice were killed between 10.00 and 12.30 hours by cervical dislocation. After removal of the tunicae albugineae, both testes were weighed and then homogenized together in 1 ml distilled water. Duplicate protein estimations were made on 0-1 ml samples of the crude homogenate (Lowry, Rosebrough, Farr & Randall, 1951). A 0-5-ml sample of the homogenate was mixed with 0-5 ml of 12-5% TCA, allowed to stand for 1 hr at 4°C, and centrifuged at 600 g for 10 min. The precipitate was resuspended and washed twice in 5% TCA, once in ethanol and once in an ether : ethanol mixture (1:1, / ), and was then taken up in 0-5 ml Soluene (Packard Instrument Co.). Two 0-2-ml aliquots of the solubilized extract were placed in a toluene-ethanol PPO/POPOP mixture and the radioactivity was measured by liquid scintillation spectrometry. In all but the first experiment, a portion of liver from each mouse was treated in an identical manner. In all experiments, the results were expressed either as specific radioactivity or as ratios of testicular specific activity to liver specific
activity.
Experiment
1
Five groups consisting of four mice in each group were used. Each of the four animals was allocated at random to treatment with saline, 5 i.u. FSH, 1 mg dbcAMP or 5 i.u. FSH plus 1 mg dbcAMP. These substances were given subcutaneously 22 hr and 14 hr before autopsy. The mice were killed by cervical dislocation on the 9th day after birth.
Experiment 2
Mice were killed when 6, 9, 12, 15, 18 or 21 days old. An injection of saline or 7·5 i.u. FSH, was given 16 hr before autopsy. Each age group consisted of four experimental and four control animals.
Experiment 3 Using thirty animals, five for each of the different time periods, a single dose of 5 i.u. FSH was injected at 4, 8, 12, 16, 24 or 32 hr before autopsy on the 9th day. In addition to supplying one animal for each time period, each of the five foster groups provided two or three control mice, making a total of twelve control animals. These were given an injection of saline at one of the above times.
Mouse
testis, protein synthesis in vivo and FSH
Experiment 4 Nine-day-old mice received FSH, or an injection of saline, given to three animals.
417
either 10-0, 5-0, 2-5, 1-25, 0-63 or 0-31 i.u. 16 hr before autopsy. Each dose of FSH was
Experiment 5 Single injections containing various ratios of FSH, HCG and saline were given to mice 16 hr before autopsy on the 9th day. Thirty-six animals were used. Four animals were given one of the treatments and each one received either 0-5 or 0 i.u. FSH and either block design.
5-0,
10, 1
or
0 i.u. HCG in
a
3x3 randomized-
Experiment 6
Each mouse received a single injection of saline, or 2 mg dbcAMP or 0-5 mg theophylline, or 2 mg dbcAMP plus 0-5 mg theophylline, \, \\, 4£ or 13^ hr before autopsy on the 9th day. Each of these sixteen treatments was given to two animals. RESULTS
Experiment 1 Comparison of the results from mice treated with FSH alone and the controls treated with saline showed that FSH increased the mean testicular weight by 43%, the total weight of testicular protein by 28% and the specific radio¬ activity of testicular protein by 46% (Text-fig. 1). Analyses of variance showed that the overall effects of FSH were statistically significant (P< 0-001 for testicular weight and protein sp. act.; P
and
CHRISTINE SUMNER
Departments of Physiology and Neurocommunications, Medical School, Birmingham PI5 2TJ (Received 25th April 1974) Human pituitary FSH was found to increase the inof tritiated lysine into testicular protein in prepubertal mice in vivo. Radioactivity was measured in washed trichloracetic acid precipitates prepared from crude testicular homogenates. The time of maximum response was 8 to 16 hr after subcutaneous injection of the hormone. This was considerably later than the maximum response in vitro reported by other workers. Neither HCG nor dibutyryl cyclic 3\m='\,5\m='\-adenosinemonophosphate had a significant effect on the incorporation of lysine.
Summary. corporation
INTRODUCTION
Means & Hall (1967) found that FSH increased the incorporation of amino acids into testicular protein in vitro. This paper reports experiments designed to find out whether a similar effect occurs in vivo, whether HCG has this action and whether the action can be reproduced by dibutyryl cyclic 3',5'-adenosine monophosphate (dbcAMP), as it has been shown that FSH stimulates adenyl cyclase activity in the testis (Murad, Strauch & Vaughn, 1969; Kuehl, Patanelli, Tarnoff & Humes, 1970; Dorrington, Vernon & Fritz, 1972). MATERIALS AND METHODS
Animals and experimental design The mice used were of the CFW strain. All male young born on the same day were fostered in groups of between ten and sixteen by pairs of lactating females. Each experiment was designed as a randomized-block with replication. The young of most uniform body weight in a foster group composed a randomized block.
Gonadotrophins Human pituitary FSH was extracted and assayed as described by Butt, Crooke & Cunningham (1961). In Exps 1 and 2 and in two replicates of Exp. 5, the FSH used was not highly purified. The potency of this preparation was 700 i.u. FSH and 140 i.u. LH activity/mg. In the remaining two replicates of Exp. 5 and in Exps 3 and 4, a more highly purified preparation was used. This contained 2000 i.u. (250 ^g) FSH and less than 120 i.u. LH/mg. The potency of ß
415
A. G. Davies et al.
416
the HCG (Paines and Byrne Ltd) was greater than 1500 i.u. LH and less than 15 i.u. FSH/mg. The hormones were injected subcutaneously in 0-1 ml saline. This route was used because neither intravenous nor intraperitoneal injections were practicable in the younger mice.
Radioisotope
In every experiment, each mouse was given a single subcutaneous injection of 5 µ L-[4,5-3H]lysine monohydrochloride (sp. act. 250 mCi/mmol) in 0-15 ml saline 2 hr before autopsy.
Extraction procedure The mice were killed between 10.00 and 12.30 hours by cervical dislocation. After removal of the tunicae albugineae, both testes were weighed and then homogenized together in 1 ml distilled water. Duplicate protein estimations were made on 0-1 ml samples of the crude homogenate (Lowry, Rosebrough, Farr & Randall, 1951). A 0-5-ml sample of the homogenate was mixed with 0-5 ml of 12-5% TCA, allowed to stand for 1 hr at 4°C, and centrifuged at 600 g for 10 min. The precipitate was resuspended and washed twice in 5% TCA, once in ethanol and once in an ether : ethanol mixture (1:1, / ), and was then taken up in 0-5 ml Soluene (Packard Instrument Co.). Two 0-2-ml aliquots of the solubilized extract were placed in a toluene-ethanol PPO/POPOP mixture and the radioactivity was measured by liquid scintillation spectrometry. In all but the first experiment, a portion of liver from each mouse was treated in an identical manner. In all experiments, the results were expressed either as specific radioactivity or as ratios of testicular specific activity to liver specific
activity.
Experiment
1
Five groups consisting of four mice in each group were used. Each of the four animals was allocated at random to treatment with saline, 5 i.u. FSH, 1 mg dbcAMP or 5 i.u. FSH plus 1 mg dbcAMP. These substances were given subcutaneously 22 hr and 14 hr before autopsy. The mice were killed by cervical dislocation on the 9th day after birth.
Experiment 2
Mice were killed when 6, 9, 12, 15, 18 or 21 days old. An injection of saline or 7·5 i.u. FSH, was given 16 hr before autopsy. Each age group consisted of four experimental and four control animals.
Experiment 3 Using thirty animals, five for each of the different time periods, a single dose of 5 i.u. FSH was injected at 4, 8, 12, 16, 24 or 32 hr before autopsy on the 9th day. In addition to supplying one animal for each time period, each of the five foster groups provided two or three control mice, making a total of twelve control animals. These were given an injection of saline at one of the above times.
Mouse
testis, protein synthesis in vivo and FSH
Experiment 4 Nine-day-old mice received FSH, or an injection of saline, given to three animals.
417
either 10-0, 5-0, 2-5, 1-25, 0-63 or 0-31 i.u. 16 hr before autopsy. Each dose of FSH was
Experiment 5 Single injections containing various ratios of FSH, HCG and saline were given to mice 16 hr before autopsy on the 9th day. Thirty-six animals were used. Four animals were given one of the treatments and each one received either 0-5 or 0 i.u. FSH and either block design.
5-0,
10, 1
or
0 i.u. HCG in
a
3x3 randomized-
Experiment 6
Each mouse received a single injection of saline, or 2 mg dbcAMP or 0-5 mg theophylline, or 2 mg dbcAMP plus 0-5 mg theophylline, \, \\, 4£ or 13^ hr before autopsy on the 9th day. Each of these sixteen treatments was given to two animals. RESULTS
Experiment 1 Comparison of the results from mice treated with FSH alone and the controls treated with saline showed that FSH increased the mean testicular weight by 43%, the total weight of testicular protein by 28% and the specific radio¬ activity of testicular protein by 46% (Text-fig. 1). Analyses of variance showed that the overall effects of FSH were statistically significant (P< 0-001 for testicular weight and protein sp. act.; P
