GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
27, 28-33 (1975)
Effect of Prolactin on Thyroid Function in Japanese Quail (Coturnix co&mix japonica) MASARU WADA,’ YASUYOSHI ARIMATSU,’ AND HIDESHI KOBAYASHI Misaki
Marine Misaki,
Biological Station, University of Tokyo, Kanagawa-ken, 238-02, Japan
Accepted April 11, 1975 Ovine prolactin administration in doses of 10 and 50 IU given over periods of 10 or 20 days induced hypertrophy of the epithehal cells of the thyroid gland in Japanese quail. A decrease of serum thyroxine was observed in the birds that received prolactin (10 or 50 IU) for 10 days, but not in the birds injected with the same doses for 20 days. Prolactin seems to act at the thyroid level to inhibit thyroxine secretion, resulting in hypertrophy of the gland presumably through negative feedback. Prolactin also induced fat deposition.
The diverse actions of prolactin in different vertebrates have aroused much interest during the past several years. One of the most interesting of these is the antithyroidal and antimetamorphic effect of mammalian prolactin in anuran tadpoles (see Etkin, 1968; Frye et al., 1972). As an explanation of these phenomena, the following two hypotheses have been proposed: (1) prolactin acts as a goitrogen, preventing the formation of physiologically active hormone by an “active” thyroid gland (Etkin and Gona, 1967a, b; Gona, 1967, 1968), or (2) prolactin blocks some thyroxine actions at the peripheral level (Bern et al., 1967; Gona, 1967; Brown and Frye, 1969; Blatt et al., 1969; Medda and Frieden, 1970; Jaffe and Geschwind, 1974). In the lizard, A&is carolinensis, prolactin induces an increase of the epitheha1 cell height of the thyroid during the winter (Licht and Jones, 1967). In the eel, Olivereau (1966) has reported that injec-
tion of ovine prolactin produces a histological picture of thyroid stimulation, including marked hypertrophy and hyperplasia. Osewold and Fiedler (1968) reported similar results in the teleost, Pterophyhn seafare. They considered prolactin to be a kind of “thyrotropinreleasing factor.” However, there is a possibility that prolactin might have evoked a hypothyroid state and, by lowering feedback, increased the production of endogenous thyroid stimulating hormone, resulting in the histological picture of an activated thyroid (Gona, 1967). Thus, the mechanism of action of prolactin on thyroid function in various vertebrates has not been fully elucidated. Effects of prolactin on thyroid function have not been studied in avian species. This paper presents our study of the effects of prolactin on the thyroid of Japanese quail. MATERIALS
Male Japanese quail (Coturnix coturnir japonica) were obtained from a commercial source at the age of 3 wk. They were kept on a short daily photoperiod of 8L: 16D (light on from 0800 to 1600) for 2 wk. Thereafter, they were transferred to a long daily photoperiod (long day) of 16L: SD (light on from 0800 to 2400). During photostimulation body weight was
’ Present address: Department of Zoology, University of Washington, Seattle, Washington 98 195. z Present address: Laboratory of Neurochemistry, Mitsubishi Kasei Institute of Life Sciences, Minamiotani, Machida-shi, Tokyo, Japan. 28 Copyright All rights
0 1975by AcademicPress,Inc. of reproduction
in any form reserved
AND METHODS
PROLACTIN
ON
THYROID
FUNCTION
measured to the nearest 0.1 g daily at about 1300. Powdered quail food (Nihon Haigo Shiryo Co. Ltd., Tokyo) and water were given ad lib. The room temperature was 22-28” during the experimental period. Ovine prolactin (50 IU/mg) was a gift from the Teikoku Zoki Co. Ltd., Tokyo. It was dissolved in 0.9% NaCl solution alkalinized with 0.1 N sodium hydroxide and neutralized just before the injection. After 2 wk of long days, 10 and 50 IU of prolactin were injected daily over period of 10 or 20 days. All injections were intramuscular in a volume of 0.1 ml at 1400. Quail injected with saline served as controls. One day after the last injection, the birds were anesthetized lightly with Nembutal and blood was collected by heart puncture. The sera were obtained after centrifugation for 30 min at 3000 rpm and stored at -20” until thyroxine measurement. After collecting blood the birds were killed by decapitation. Thyroids, adrenals, and adenohypophyses were weighed with a torsion balance whose smallest scale division was 0.1 mg. Testes and cervical adipose tissue were weighed to the nearest 0.01 g with a Mettler-type direct reading balance whose smallest scale division was 0.01 g. Differences in tissue weights between groups were determined by Student’s f-test. The thyroids were fixed in Bouin’s solution, embedded in paraffin, sectioned serially at 6 pm, and stained with hematoxylin and eosin. Thirty epithelial cells of the thyroid were randomly selected from each bird and the cell height was measured with a micrometer. Differences in epithelial cell height among the groups were tested by one-way analysis of variance. Total serum thyroxine was measured with the Res-
O-Mat T., kit (Mallinckrodt Nuclear, delivered through Daiichi Radioisotope Laboratories, Ltd., Tokyo); this is a clinical diagnostic kit for Tq measurement in serum using the competitive protein binding method (Murphy and Pattee, 1964; Murphy, 1965; Murphy et al., 1966). Differences in serum thyroxine between the groups were determined by Student’s t-test.
RESULTS
Weights of endocrine organs and cervical adipose tissue of each group are summarized in Table 1. The body weight was apparently heavier in the prolactin treated groups than in controls, but the difference was not statistically significant. The thyroid glands of prolactin-treated birds tended to be heavier than those of controls, especially after receiving 50 IU of prolactin. Individual differences in each group were so great, however, that a statistically significant difference in thyroid weight was not detected between control and experimental groups. Individual differences were especially pronounced in the group which received 50 IU of prolactin. The weight of the adenohypophysis decreased significantly in the birds treated with 10 IU of prolactin for 10 days, and it
TABLE WEIGHTS No.
Dose
of birds
OF BODY,
ENDOCRINE
Body weight (9)
ORGANS,
Adenohypophysis
29
IN QUAIL
AND
1 FAT
Thyroids (md100 i3 body wt)
TISSUE
AFTER
Adrenals
PROLACTIN
TREATMENT
Testes We g body wt)
Cervical adipose tissue w
10 days Saline 10 IU 50 IU
4 5 5
88.5 k 2.9” 97.6 f 3.0 96.3 k 2.1
1.71 k 0.11 1.33 2 0.096 1.40 k 0.08
6.70 -c 1.49 8.58 f 0.90 12.34 2 5.06 20
Saline
4
10 IU 50 IU
5 5
92.8 2 1.3 94.0 f 2.4 98.4 f 3.4
1.28 k 0.06 1.41 f 0.15 1.46 + 0.02a
2.47 f 0.23 2.19 -+ 0.26 1.72 2 0.20b
0.89 f 0.29 1.47 k 0.29 1.78 k 0.59
9.11 k 1.22 11.08~0.90 9.82 k 0.89
2.74 2 0.22 2.43 2 0.12 2.49 f 0.16
0.65 2 0.06 1.19 2 0.17b 1.16 f O.lSa
days
6.38 + 0.46 6.20 + 0.76 11.22 + 2.64
a Mean f standard error. * Significant (P < 0.05) compared to saline group.
10.75 + 0.65 10.32 f 0.79 10.25 + 0.91
30
WADA,
ARIMATSU
increased significantly in the birds injected with 50 IU of prolactin for 20 days. In the other groups, prolactin did not affect the adenohypophyseal weight. The weights of adrenals did not vary between control and experimental groups. The testes of the birds treated with 50 IU of prolactin for 10 days weighed significantly less than those of control birds, but among the other groups there were no differences. The cervical adipose tissue weight increased in the prolactin-treated groups, and this was es-
AND KOBAYASHI
pecially significant in groups treated for 20 days. In control birds, the thyroid follicles were large, the epithelial cells were flat, and the nuclei were ellipsoid in shape (Fig. la). The follicular colloid was not vacuolated. In contract, most of the follicles in the prolactin-treated birds were relatively small and the epithelial cells showed hypertrophy; epithelial cells became taller, nuclei became round, and vacuoles were often seen in the colloid (Fig. lb). Analyti-
FIG. 1. Light micrographs of the thyroid glands. (a) Control bird, (b) prolactin-injected days). x 460.
bird (50 IU for 20
PROLACTIN
ON THYROID
FUNCTION
TABLE EFFECT
OF PROLACTIN
IN QUAIL
31
2
ON THYROID
EPITHELIAL
10 day treatment
CELL
HEIGHT
20 day treatment
Group
No. of birds
Cell height @urn) (mean f SE)
Group
No. of birds
Cell height (pm) (mean 2 SE)
A, control B, 10 IU c, 50 IU
4 5 5
3.05 -r- 0.08 4.97 iI 0.15 6.44 f 0.15
D, control E, 10 IU F, 50 IU
4 5 5
3.29 f 0.09 5.72 k 0.16 5.79 ” 0.17
Analysis of variance Sample comparison A vs B A vs C B vs C
df
1 1 1
V
F
P
1244.2 3503.7 735.6
9.63 80.43 4.93
AND
COMPARATIVE
ENDOCRINOLOGY
27, 28-33 (1975)
Effect of Prolactin on Thyroid Function in Japanese Quail (Coturnix co&mix japonica) MASARU WADA,’ YASUYOSHI ARIMATSU,’ AND HIDESHI KOBAYASHI Misaki
Marine Misaki,
Biological Station, University of Tokyo, Kanagawa-ken, 238-02, Japan
Accepted April 11, 1975 Ovine prolactin administration in doses of 10 and 50 IU given over periods of 10 or 20 days induced hypertrophy of the epithehal cells of the thyroid gland in Japanese quail. A decrease of serum thyroxine was observed in the birds that received prolactin (10 or 50 IU) for 10 days, but not in the birds injected with the same doses for 20 days. Prolactin seems to act at the thyroid level to inhibit thyroxine secretion, resulting in hypertrophy of the gland presumably through negative feedback. Prolactin also induced fat deposition.
The diverse actions of prolactin in different vertebrates have aroused much interest during the past several years. One of the most interesting of these is the antithyroidal and antimetamorphic effect of mammalian prolactin in anuran tadpoles (see Etkin, 1968; Frye et al., 1972). As an explanation of these phenomena, the following two hypotheses have been proposed: (1) prolactin acts as a goitrogen, preventing the formation of physiologically active hormone by an “active” thyroid gland (Etkin and Gona, 1967a, b; Gona, 1967, 1968), or (2) prolactin blocks some thyroxine actions at the peripheral level (Bern et al., 1967; Gona, 1967; Brown and Frye, 1969; Blatt et al., 1969; Medda and Frieden, 1970; Jaffe and Geschwind, 1974). In the lizard, A&is carolinensis, prolactin induces an increase of the epitheha1 cell height of the thyroid during the winter (Licht and Jones, 1967). In the eel, Olivereau (1966) has reported that injec-
tion of ovine prolactin produces a histological picture of thyroid stimulation, including marked hypertrophy and hyperplasia. Osewold and Fiedler (1968) reported similar results in the teleost, Pterophyhn seafare. They considered prolactin to be a kind of “thyrotropinreleasing factor.” However, there is a possibility that prolactin might have evoked a hypothyroid state and, by lowering feedback, increased the production of endogenous thyroid stimulating hormone, resulting in the histological picture of an activated thyroid (Gona, 1967). Thus, the mechanism of action of prolactin on thyroid function in various vertebrates has not been fully elucidated. Effects of prolactin on thyroid function have not been studied in avian species. This paper presents our study of the effects of prolactin on the thyroid of Japanese quail. MATERIALS
Male Japanese quail (Coturnix coturnir japonica) were obtained from a commercial source at the age of 3 wk. They were kept on a short daily photoperiod of 8L: 16D (light on from 0800 to 1600) for 2 wk. Thereafter, they were transferred to a long daily photoperiod (long day) of 16L: SD (light on from 0800 to 2400). During photostimulation body weight was
’ Present address: Department of Zoology, University of Washington, Seattle, Washington 98 195. z Present address: Laboratory of Neurochemistry, Mitsubishi Kasei Institute of Life Sciences, Minamiotani, Machida-shi, Tokyo, Japan. 28 Copyright All rights
0 1975by AcademicPress,Inc. of reproduction
in any form reserved
AND METHODS
PROLACTIN
ON
THYROID
FUNCTION
measured to the nearest 0.1 g daily at about 1300. Powdered quail food (Nihon Haigo Shiryo Co. Ltd., Tokyo) and water were given ad lib. The room temperature was 22-28” during the experimental period. Ovine prolactin (50 IU/mg) was a gift from the Teikoku Zoki Co. Ltd., Tokyo. It was dissolved in 0.9% NaCl solution alkalinized with 0.1 N sodium hydroxide and neutralized just before the injection. After 2 wk of long days, 10 and 50 IU of prolactin were injected daily over period of 10 or 20 days. All injections were intramuscular in a volume of 0.1 ml at 1400. Quail injected with saline served as controls. One day after the last injection, the birds were anesthetized lightly with Nembutal and blood was collected by heart puncture. The sera were obtained after centrifugation for 30 min at 3000 rpm and stored at -20” until thyroxine measurement. After collecting blood the birds were killed by decapitation. Thyroids, adrenals, and adenohypophyses were weighed with a torsion balance whose smallest scale division was 0.1 mg. Testes and cervical adipose tissue were weighed to the nearest 0.01 g with a Mettler-type direct reading balance whose smallest scale division was 0.01 g. Differences in tissue weights between groups were determined by Student’s f-test. The thyroids were fixed in Bouin’s solution, embedded in paraffin, sectioned serially at 6 pm, and stained with hematoxylin and eosin. Thirty epithelial cells of the thyroid were randomly selected from each bird and the cell height was measured with a micrometer. Differences in epithelial cell height among the groups were tested by one-way analysis of variance. Total serum thyroxine was measured with the Res-
O-Mat T., kit (Mallinckrodt Nuclear, delivered through Daiichi Radioisotope Laboratories, Ltd., Tokyo); this is a clinical diagnostic kit for Tq measurement in serum using the competitive protein binding method (Murphy and Pattee, 1964; Murphy, 1965; Murphy et al., 1966). Differences in serum thyroxine between the groups were determined by Student’s t-test.
RESULTS
Weights of endocrine organs and cervical adipose tissue of each group are summarized in Table 1. The body weight was apparently heavier in the prolactin treated groups than in controls, but the difference was not statistically significant. The thyroid glands of prolactin-treated birds tended to be heavier than those of controls, especially after receiving 50 IU of prolactin. Individual differences in each group were so great, however, that a statistically significant difference in thyroid weight was not detected between control and experimental groups. Individual differences were especially pronounced in the group which received 50 IU of prolactin. The weight of the adenohypophysis decreased significantly in the birds treated with 10 IU of prolactin for 10 days, and it
TABLE WEIGHTS No.
Dose
of birds
OF BODY,
ENDOCRINE
Body weight (9)
ORGANS,
Adenohypophysis
29
IN QUAIL
AND
1 FAT
Thyroids (md100 i3 body wt)
TISSUE
AFTER
Adrenals
PROLACTIN
TREATMENT
Testes We g body wt)
Cervical adipose tissue w
10 days Saline 10 IU 50 IU
4 5 5
88.5 k 2.9” 97.6 f 3.0 96.3 k 2.1
1.71 k 0.11 1.33 2 0.096 1.40 k 0.08
6.70 -c 1.49 8.58 f 0.90 12.34 2 5.06 20
Saline
4
10 IU 50 IU
5 5
92.8 2 1.3 94.0 f 2.4 98.4 f 3.4
1.28 k 0.06 1.41 f 0.15 1.46 + 0.02a
2.47 f 0.23 2.19 -+ 0.26 1.72 2 0.20b
0.89 f 0.29 1.47 k 0.29 1.78 k 0.59
9.11 k 1.22 11.08~0.90 9.82 k 0.89
2.74 2 0.22 2.43 2 0.12 2.49 f 0.16
0.65 2 0.06 1.19 2 0.17b 1.16 f O.lSa
days
6.38 + 0.46 6.20 + 0.76 11.22 + 2.64
a Mean f standard error. * Significant (P < 0.05) compared to saline group.
10.75 + 0.65 10.32 f 0.79 10.25 + 0.91
30
WADA,
ARIMATSU
increased significantly in the birds injected with 50 IU of prolactin for 20 days. In the other groups, prolactin did not affect the adenohypophyseal weight. The weights of adrenals did not vary between control and experimental groups. The testes of the birds treated with 50 IU of prolactin for 10 days weighed significantly less than those of control birds, but among the other groups there were no differences. The cervical adipose tissue weight increased in the prolactin-treated groups, and this was es-
AND KOBAYASHI
pecially significant in groups treated for 20 days. In control birds, the thyroid follicles were large, the epithelial cells were flat, and the nuclei were ellipsoid in shape (Fig. la). The follicular colloid was not vacuolated. In contract, most of the follicles in the prolactin-treated birds were relatively small and the epithelial cells showed hypertrophy; epithelial cells became taller, nuclei became round, and vacuoles were often seen in the colloid (Fig. lb). Analyti-
FIG. 1. Light micrographs of the thyroid glands. (a) Control bird, (b) prolactin-injected days). x 460.
bird (50 IU for 20
PROLACTIN
ON THYROID
FUNCTION
TABLE EFFECT
OF PROLACTIN
IN QUAIL
31
2
ON THYROID
EPITHELIAL
10 day treatment
CELL
HEIGHT
20 day treatment
Group
No. of birds
Cell height @urn) (mean f SE)
Group
No. of birds
Cell height (pm) (mean 2 SE)
A, control B, 10 IU c, 50 IU
4 5 5
3.05 -r- 0.08 4.97 iI 0.15 6.44 f 0.15
D, control E, 10 IU F, 50 IU
4 5 5
3.29 f 0.09 5.72 k 0.16 5.79 ” 0.17
Analysis of variance Sample comparison A vs B A vs C B vs C
df
1 1 1
V
F
P
1244.2 3503.7 735.6
9.63 80.43 4.93
