EFFECTS OF TESTOSTERONE AND PROLACTIN OR GROWTH HORMONE ON THE ACCESSORY SEX ORGANS OF CASTRATED MICE Department
E. J. KEENAN AND J. A. THOMAS of Pharmacology, West Virginia University Medical Center, West Virginia 26506, U.S.A.
Morgantown,
(Received 23 April 1974) SUMMARY
In 5-day experiments, neither bovine prolactin (300 or 600 i.u./kg) nor ovine growth hormone (25 i.u./kg) alone significantly enhanced accessory sex organ weights in the castrated mouse. Seminal vesicle weights, and to a lesser extent anterior prostate gland weights, were augmented by the simultaneous injection of testosterone (1\m=.\5mg/kg) daily plus prolactin or growth hormone. The effect was greater than that produced by testosterone alone. The levels of fructose in accessory sex organs used to indicate androgenic activity were similar in castrated mice receiving testosterone alone or in combination with prolactin or growth hormone. Prolactin alone did not influence uptake of [3H]testosterone by the seminal vesicles or anterior prostate gland over a 5 min period in vivo. INTRODUCTION
growth hormone influence the action of testosterone upon male organs. Grayhack, Bunce, Kearns & Scott (1955) initially observed accessory that the simultaneous administration of testosterone and prolactin to castratedhypophysectomized rats produced a greater augmentation of prostate gland weights than did testosterone alone. The seminal vesicles of castrated-hypophysectomized rats (Chase, Geschwind & Bern, 1957) and guinea-pigs (Antliff, Prasad & Meyer, 1960) showed similar responses. Later studies by Grayhack (1963) showed that the dorsolateral lobe was more responsive than the other lobes of the castrated-hypo¬ physectomized rat prostate gland to the interaction of testosterone and prolactin. Further studies (Grayhack & Lebowitz, 1967) established that prolactin augmented the androgen-mediated increase in citric acid levels in the dorsolateral and particu¬ larly in the lateral lobes of the prostate gland of hypophysectomized-castrated rats. In the castrated Indian palm squirrel, Reddi (1969) demonstrated that prolactin Prolactin and sex
*
USPHS Pre-doctoral fellow supported by grant TOI GM00076. Some of the data contained herein submitted for a doctoral dissertation in the Department of Pharmacology at West Virginia
were
University. t Reprint requests
should be sent to Dr J. A. Thomas at above address.
enhanced testosterone stimulation of weight and citric acid content in the prostate gland; levels of fructose in the seminal vesicles were also increased. The action of growth hormone upon male accessory sex organs has received less attention than has the action of prolactin. Lostroh (1962) observed that growth hormone enhanced testosterone stimulation of the rat ventral prostate gland and seminal vesicle weights as well as both RNA and protein synthesis. The present studies were undertaken to examine the relationship between testo¬ sterone and prolactin or growth hormone in the accessory sex organs of castrated mice. MATERIALS AND METHODS
Mature, male albino mice (Swiss-Webster strain, 30-40 g) were used in these studies and maintained on a standard diet of laboratory chow and water ad libitum. Animals were castrated by the abdominal route under ether anaesthesia. On day 7 after castration, the animals were randomly divided into groups and each animal received daily injections (for 5 days) of testosterone (1-5 mg/kg, s.c), bovine pro¬ lactin, NIH-P-B3 (300 i.u./kg or 600 i.u./kg, i.p.) or ovine growth hormone, NIHGH-S9 (25 i.u./kg, i.p.). Other groups of animals received simultaneous injections of testosterone (1-5 mg/kg, s.c.) and prolactin (300 or 600 i.u./kg) or testosterone (1-5 mg/kg, s.c.) and growth hormone (25 i.u./kg, i.p.). Control animals were injected with corn oil and saline. Testosterone (Sigma Chemical Co., St. Louis) was dissolved in corn oil and the pituitary hormone preparations were dissolved in isotonic saline adjusted to pH 9-0 with 0-1 M-NaOH. All injections were made in a volume of 0-2 ml or less. There were no significant differences in mean body weights between the various treatment groups. Approximately 24 h after the fifth and final hormone injection, animals were killed by cervical dislocation. The anterior prostate glands and seminal vesicles (empty) were rapidly dissected, rinsed in ice-cold saline, blotted and weighed before freezing in liquid nitrogen. Accessory sex organs were homogenized in distilled water (4%, w/v), precipitated with barium hydroxide and zinc sulphate, centrifuged (1000 g), and the supernatant was used for the determination of fructose (Thomas, Mawhinney & Mason, 1968). In the studies involving the uptake in vivo of [l,2-3H]testosterone (New England Nuclear Corp., Boston; specific activity 43-5 Ci/mM) a 5 min uptake period was chosen since previous studies in this laboratory (Thomas, Smith, Mawhinney & Kynch, 1970) demonstrated that significant, yet submaximal accumulation of labelled steroid occurs 5 min after injection. Castrated mice were given daily injections (for 5 days) of bovine prolactin (600 i.u./kg, i.p.) beginning on day 7 after castration. Approximately 24 h after the final prolactin injection, animals received a single i.p. injection of [3H]testosterone (100/iCi/kg, 10/ig/kg). Five minutes after the injection of labelled testosterone the animals were killed by cervical dislocation and the accessory sex organs were rapidly removed, rinsed in ice-cold sahne, blotted and frozen in liquid nitrogen. Glands were homogenized and the radioactive steroid extracted with chloroform: ether (2:1, v/v). Aliquots of the chloroform: ether extracts were placed in glass scintillation vials and evaporated. Liquid scintillation counting fluid (2,5 diphenyl-oxazole and 2,2'-£>-phenylene-bis [5-phenyloxazole]) (PPO-
POPOP) was added to the vials and the radioactive steroid was counted in a liquid scintillation spectrophotometer. Radioactivity was corrected for background and counting efficiency and expressed as disintegrations per minute. Differences between mean values were determined by a two-tailed Student's i-test (Snedecor & Cochran, 1967). RESULTS
Prolactin (600 i.u./kg) alone did not influence anterior prostate gland or seminal vesicle wet weights in castrated mice (Table 1). As expected, testosterone increased the weights of both accessory sex organs. However, the simultaneous administration of testosterone and prolactin produced a further enhancement of anterior prostate gland wet weight and markedly increased (P < 0-05) seminal vesicle weight. A lower dose of prolactin (300 i.u./kg) when given with testosterone produced similar results
(Table 1).
Effect of simultaneously injected prolactin or growth hormone and testosterone (1·5 mg¡kg daily for 5 days) on wet weights of accessory sex organs of castrated male mice (means + s.e.m., seven observations¡group) Table 1.
Anterior prostate
Seminal vesicle
(mg)
(mg)
12·8±0·5
29-3 ±1-0 29-2 + 1-7
Treatment
A. Prolactin Castrated Castrated + prolactin (600 i.u./kg) Castrated + testosterone Castrated + testosterone + prolactin (300 Castrated + testosterone + prolactin (600
11-1 + 0-6
i.u./kg) i.u./kg)
20-3 ±0-9 22-3 ±1-7 23-4+1-1
49·4±1·9
ll-4±l-0 13-3 + 0-7
27-7 ±1-4 25-6 + 2-1
19-9±l-2 23-5±l-4
42·0±1·6 54-9 ±2-2*
30-7 + 2-8
84-2 + 3-5
60-9 + 1-9* 57-5 + 2-2*
. Growth hormone
Castrated Castrated + growth hormone (25 i.u./kg) Castrated + testosterone Castrated-)-testosterone + growth hormone Intact, untreated *
(25 i.u./kg)
Significantly different from the testosterone-treated control mice:
E. J. KEENAN AND J. A. THOMAS of Pharmacology, West Virginia University Medical Center, West Virginia 26506, U.S.A.
Morgantown,
(Received 23 April 1974) SUMMARY
In 5-day experiments, neither bovine prolactin (300 or 600 i.u./kg) nor ovine growth hormone (25 i.u./kg) alone significantly enhanced accessory sex organ weights in the castrated mouse. Seminal vesicle weights, and to a lesser extent anterior prostate gland weights, were augmented by the simultaneous injection of testosterone (1\m=.\5mg/kg) daily plus prolactin or growth hormone. The effect was greater than that produced by testosterone alone. The levels of fructose in accessory sex organs used to indicate androgenic activity were similar in castrated mice receiving testosterone alone or in combination with prolactin or growth hormone. Prolactin alone did not influence uptake of [3H]testosterone by the seminal vesicles or anterior prostate gland over a 5 min period in vivo. INTRODUCTION
growth hormone influence the action of testosterone upon male organs. Grayhack, Bunce, Kearns & Scott (1955) initially observed accessory that the simultaneous administration of testosterone and prolactin to castratedhypophysectomized rats produced a greater augmentation of prostate gland weights than did testosterone alone. The seminal vesicles of castrated-hypophysectomized rats (Chase, Geschwind & Bern, 1957) and guinea-pigs (Antliff, Prasad & Meyer, 1960) showed similar responses. Later studies by Grayhack (1963) showed that the dorsolateral lobe was more responsive than the other lobes of the castrated-hypo¬ physectomized rat prostate gland to the interaction of testosterone and prolactin. Further studies (Grayhack & Lebowitz, 1967) established that prolactin augmented the androgen-mediated increase in citric acid levels in the dorsolateral and particu¬ larly in the lateral lobes of the prostate gland of hypophysectomized-castrated rats. In the castrated Indian palm squirrel, Reddi (1969) demonstrated that prolactin Prolactin and sex
*
USPHS Pre-doctoral fellow supported by grant TOI GM00076. Some of the data contained herein submitted for a doctoral dissertation in the Department of Pharmacology at West Virginia
were
University. t Reprint requests
should be sent to Dr J. A. Thomas at above address.
enhanced testosterone stimulation of weight and citric acid content in the prostate gland; levels of fructose in the seminal vesicles were also increased. The action of growth hormone upon male accessory sex organs has received less attention than has the action of prolactin. Lostroh (1962) observed that growth hormone enhanced testosterone stimulation of the rat ventral prostate gland and seminal vesicle weights as well as both RNA and protein synthesis. The present studies were undertaken to examine the relationship between testo¬ sterone and prolactin or growth hormone in the accessory sex organs of castrated mice. MATERIALS AND METHODS
Mature, male albino mice (Swiss-Webster strain, 30-40 g) were used in these studies and maintained on a standard diet of laboratory chow and water ad libitum. Animals were castrated by the abdominal route under ether anaesthesia. On day 7 after castration, the animals were randomly divided into groups and each animal received daily injections (for 5 days) of testosterone (1-5 mg/kg, s.c), bovine pro¬ lactin, NIH-P-B3 (300 i.u./kg or 600 i.u./kg, i.p.) or ovine growth hormone, NIHGH-S9 (25 i.u./kg, i.p.). Other groups of animals received simultaneous injections of testosterone (1-5 mg/kg, s.c.) and prolactin (300 or 600 i.u./kg) or testosterone (1-5 mg/kg, s.c.) and growth hormone (25 i.u./kg, i.p.). Control animals were injected with corn oil and saline. Testosterone (Sigma Chemical Co., St. Louis) was dissolved in corn oil and the pituitary hormone preparations were dissolved in isotonic saline adjusted to pH 9-0 with 0-1 M-NaOH. All injections were made in a volume of 0-2 ml or less. There were no significant differences in mean body weights between the various treatment groups. Approximately 24 h after the fifth and final hormone injection, animals were killed by cervical dislocation. The anterior prostate glands and seminal vesicles (empty) were rapidly dissected, rinsed in ice-cold saline, blotted and weighed before freezing in liquid nitrogen. Accessory sex organs were homogenized in distilled water (4%, w/v), precipitated with barium hydroxide and zinc sulphate, centrifuged (1000 g), and the supernatant was used for the determination of fructose (Thomas, Mawhinney & Mason, 1968). In the studies involving the uptake in vivo of [l,2-3H]testosterone (New England Nuclear Corp., Boston; specific activity 43-5 Ci/mM) a 5 min uptake period was chosen since previous studies in this laboratory (Thomas, Smith, Mawhinney & Kynch, 1970) demonstrated that significant, yet submaximal accumulation of labelled steroid occurs 5 min after injection. Castrated mice were given daily injections (for 5 days) of bovine prolactin (600 i.u./kg, i.p.) beginning on day 7 after castration. Approximately 24 h after the final prolactin injection, animals received a single i.p. injection of [3H]testosterone (100/iCi/kg, 10/ig/kg). Five minutes after the injection of labelled testosterone the animals were killed by cervical dislocation and the accessory sex organs were rapidly removed, rinsed in ice-cold sahne, blotted and frozen in liquid nitrogen. Glands were homogenized and the radioactive steroid extracted with chloroform: ether (2:1, v/v). Aliquots of the chloroform: ether extracts were placed in glass scintillation vials and evaporated. Liquid scintillation counting fluid (2,5 diphenyl-oxazole and 2,2'-£>-phenylene-bis [5-phenyloxazole]) (PPO-
POPOP) was added to the vials and the radioactive steroid was counted in a liquid scintillation spectrophotometer. Radioactivity was corrected for background and counting efficiency and expressed as disintegrations per minute. Differences between mean values were determined by a two-tailed Student's i-test (Snedecor & Cochran, 1967). RESULTS
Prolactin (600 i.u./kg) alone did not influence anterior prostate gland or seminal vesicle wet weights in castrated mice (Table 1). As expected, testosterone increased the weights of both accessory sex organs. However, the simultaneous administration of testosterone and prolactin produced a further enhancement of anterior prostate gland wet weight and markedly increased (P < 0-05) seminal vesicle weight. A lower dose of prolactin (300 i.u./kg) when given with testosterone produced similar results
(Table 1).
Effect of simultaneously injected prolactin or growth hormone and testosterone (1·5 mg¡kg daily for 5 days) on wet weights of accessory sex organs of castrated male mice (means + s.e.m., seven observations¡group) Table 1.
Anterior prostate
Seminal vesicle
(mg)
(mg)
12·8±0·5
29-3 ±1-0 29-2 + 1-7
Treatment
A. Prolactin Castrated Castrated + prolactin (600 i.u./kg) Castrated + testosterone Castrated + testosterone + prolactin (300 Castrated + testosterone + prolactin (600
11-1 + 0-6
i.u./kg) i.u./kg)
20-3 ±0-9 22-3 ±1-7 23-4+1-1
49·4±1·9
ll-4±l-0 13-3 + 0-7
27-7 ±1-4 25-6 + 2-1
19-9±l-2 23-5±l-4
42·0±1·6 54-9 ±2-2*
30-7 + 2-8
84-2 + 3-5
60-9 + 1-9* 57-5 + 2-2*
. Growth hormone
Castrated Castrated + growth hormone (25 i.u./kg) Castrated + testosterone Castrated-)-testosterone + growth hormone Intact, untreated *
(25 i.u./kg)
Significantly different from the testosterone-treated control mice:
