GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
32, 72-77 (1977)
The Effects of Prolactin on the Gonadotropin Induced Rise in Serum Estradiol and Progesterone of the Laying Turkey1 P. M. CAMPER
AND W. H. BURKE
Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55108 Accepted December 23, 1976 Mammalian LH (250 pg of NIH-LH-B9) and FSH (500 pg of FSH-P, Armour-Baldwin) elicit increases in serum estradiol (E) and progesterone (P) of laying turkeys within 30 min after a single iv injection. Prolactin (NIH-P-S1 1) at doses of 250 and 500 Kg had no effect on the E or P levels when given in a single iv injection. One thousand micrograms of prolactin caused a significant decrease in serum E within 30 min of injection. Prolactin (500 and 1000 pg), given 30 min before LH, blocked the LH induced rise in P and at doses of 250, 500 and 1000 pg it blocked the LH induced rise in E. Five hundred and 1000 pg of prolactin blocked the FSH induced rise in P and doses of 250, 500 and 1000 pg of prolactin blocked the FSH induced rise in E. It is concluded that prolactin’s antigonadotropic effect is exerted, at least in part, by interfering with gonadotropin induced increases in ovarian steroid levels in the blood.
The pituitary prolactin content of incubating (broody) chickens (Burrows and Byerly, 1936; Saeki and Tanabe, 1954; 1956), turkeys Nakajo and Tanaka, (Cherms et al., 1962; Cogger and Burke, unpublished data), pheasants (Brietenbach and Meyer, 1959) and other avian species (Nicoll, 1974) has been found to be higher than that of actively laying individuals. Injection of prolactin has induced broodiness in several avian species (Riddle et al., 1935; Nalbandov and Card, 1945; Crispens, 1957) and has caused gonadal regression in female chickens (Bates et al., 1935). Prolactin injection has failed to cause gonadal regression in some experiments with female Coturnix quail (Alexander and Wolfson, 1970) but has done so in others (Camper and Burke, unpublished data). Likewise in early experiments with male chickens pro-
lactin caused testicular regression (Bates et al., 1935; Brenemen, 1942; Nalbandov et al., 1945), an effect which could be blocked by simultaneous FSH treatment (Nalbandov ef al., 1945). Recent work has failed to show an antigonadal effect of prolactin in roosters (Shani et al., 1973). The mechanism or site of action by which prolactin causes gonadal regression, in those species in which it does, remains obscure. One possible site of action would be at the gonad, blocking the effects of gonadotropins on their target tissues. In fact, Tanaka et al. (1971) have reported that prolactin injection will inhibit LH induced premature ovulation. It has recently been demonstrated that LH or FSH injection elicits rapid rises in serum estradiol (E) and progesterone (P) in laying turkeys (Camper and Burke, 1977). These findings provide a means to test whether prolactin inhibits the acute rise in serum E and P elicited by LH and FSH.
’ Scientific Journal series paper No. 97 IO of the Minnesota Agricultural Experiment Station. 72 Copyright All rights
@ 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN 00164480
PROLACTIN.
MATERIALS
AND
GONADOTROPIN
AND
METHODS
Experiment I was conducted to determine if prolactin injection itself would alter serum E or P levels. To do this hens were bled and then injected with I ml of saline or 250, 500 or 1000 pg of prolactin (NIH-PSl 1) in 1 ml of pH 9 saline. They were bled again 5 and 30 min after injection. Serum was obtained and analyzed for E and P. Experiments 2 and 3 were designed to determine if pretreatment of turkeys with prolactin would affect the steroidogenic response elicited by LH (NIHLH-B9) or FSH (FSH-P, Armour-Baldwin Laboratories). The general test procedure was as follows: 4 or 5 hr after oviposition turkey hens were bled from a brachial vein, then injected with prolactin solution or saline, bled 30 min later, then injected with 250 pg of LH or 500 pg of FSH or saline and bled again 5 and 30 min later. All injections were given into the brachial vein in a 1 ml volume. Five to 8 ml of blood were taken at each bleeding and allowed to clot over ice. It was stored overnight at 4” and then centrifuged. Serum was collected and stored frozen at -20” until used for E and P assay. The E and P assays used throughout and the turkeys used for experiments I and 2 were described by Camper and Burke (1977). The turkeys used in experiment 3 of this study were Nicholas small white female line birds (Nicholas Turkey Breeding Farms, Sonoma, Calif.). The birds were reared under natural photoperiod conditions from May to September. At 6 months of age they were placed on a photoperiod of 6L: 18D to terminate photo refractoriness and after 3 weeks of this regimen the photoperiod was switched to 15L:9D to stimulate reproductive activity. Within 3 weeks of this switch the hens were laying eggs and they were used for these experiments during the following months. In each experiment, 4 hens were used per treatment group. Estradiol and P levels of individuals after hormone treatment were compared to their own pretreatment E and P levels. Results were analyzed by a TABLE SERUM
ESTRADIOL
(pg/ml)
IN THE
loo0
TURKEY
73
STEROIDS
split-plot analysis of variance and if a significant F value was obtained, mean differences were determined using Dunnett’s test for comparisons involving a control mean. The statistical analyses were done on absolute quantities of hormone (i.e., pg or rig/ml), but for purposes of presentation the data in the figures has been converted to percentage change from 0 time as follows: hormone 30 min level hormone
% change
level at 5 or - hormone at 0 time level at 0 time
x 100.
RESULTS
Injection of laying turkeys with 250 or 500 Fg of prolactin had no effect on serum E levels (Table l), but 1000 pg caused a significant depression in E within 30 min. Three of the 4 birds given the 1000 pg injection had markedly lower E levels 30 min after prolactin treatment. In 1 bird the E level was depressed to a nearly nondetectable level and in 1 bird the E level was nondetectable 30 min after prolactin treatment. The fourth bird in the group had no change in its E level over the course of the experiment. Progesterone levels of these same birds remained unchanged by the prolactin treatment (Table 2). The effect of prolactin on the LH induced rise in P and E is shown in Fig. la and b, respectively. Saline injection had no significant effect on P or E levels. Injection of 250 pg of LH caused a highly significant increase in serum P (131.58 + 24.94Yo) and I HEN
AFTER
A SINGLE
INJECTION
OF PROLACTIN”
After prolactin (min)
Prolactin w 0 250 500
LAYING
SERUM
0 15.11 37.96 18.33 26.35
ir f. 2 ?I
5 2.42b 5.87 3.62 2.34
a Prolactin (NIH P-S-II) given at time 0. b Mean +- standard error; n = 4 in all groups. c Estradiol decreased from time 0; P < 0.05.
13.25 32.81 20.85 23.74
? k 2 k
30 1.61 4.42 3.48 1.38
15.81 30.10 16.07 8.99
tf 2 f
1.95 7.18 2.83 3.95”
CAMPER
74
AND BURKE
TABLE 2 SERUM PROGESTERONE (r&ml) IN THE LAYING TURKEY HEN AFTER A SINGLE INJECTION OF PROLACTIN" After prolactin (min)
Prolactin (M)
0 1.32 1.80 2.29 2.20
0
250 500 1000
? 2 + +
5 0.31b 0.22 0.30 0.22
1.18 2.06 2.01 1.95
+ f k *
30 0.33 0.23 0.44 0.13
1.48 1.86 2.42 2.10
” 0.23 -c 0.22 f 0.59 f 0.17
a Prolactin (NIH P-S-11) injected at time 0. b Mean f standard error; n = 4 in all groups.
a significant increase in E (231.68 2 74.40%). Treatment of hens with 500 or 1000 pg of prolactin 30 min prior to treatment with LH completely blocked the rise in steroids. The birds receiving 250 Fg of prolactin prior to LH treatment showed a highly significant increase in P (93.53 + 6.20%) but no significant change in E. The E levels were higher 30 min after LH treatment in 3 of the 4 hens receiving pretreatment with 250 pg of prolactin, but statistical tests failed to show a significant rise. As in the preliminary experiment, 1000 ,ug of prolactin significantly depressed serum E within 30 min of its injection. UC!
FSH treatment caused a significant rise in serum P (143.58 r 42.39%, Fig. 2a). Five hundred and 1000 pg of prolactin significantly reduced the response to FSH. Estradiol levels were increased by the injection of 500 pg of FSH (236.38 f 84.76%). There was no significant change in E levels in FSH treated hens that were pretreated with 250,500 or 1000 pg of prolactin (Fig. 2b). DISCUSSION
It is generally assumed that prolactin induces incubation behavior in Gallinaceous birds and is responsible for the ovarian re-
r
Minutes
after
LH
Miies
after
LH
FIG. 1. Changes in serum progesterone (a) levels or estradiol levels (b) following the injection of bovine LH (NIH-LH-B9) preceded by the injection of saline or various doses of ovine prolactin (NIH-P-Sll). Lines followed by asterisks indicate that the hormone level is significantly different (P G 0.01) than at time 0 in the same animals.
PROLACTIN,
GONADOTROPIN
ug PRL J@FSH
0
75
STEROIDS
pg PRL
P after
SERUM
a
5 Minutes
AND
FSH
0
5
w FSH
b
30 Minutes
after
FSH
2. Changes in serum progesterone (a) or estradiol levels (b) following the injection of porcine FSH (Armour FSH-P) preceded by the injection of saline or various doses of ovine prolactin (NIH-P-Sll). Lines followed by asterisks indicate that the hormone level is significantly different (P G 0.05) than at time 0 in the same animals. FIG.
gression that accompanies broodiness. Prolactin often has been said to have an antigonadotropic effect in birds. Bates et al. (1937) concluded that prolactin acted upon the pituitary to decrease FSH secretion, a conclusion shared by Nalbandov et al. (1945). The latter authors also stated that “Prolactin may prevent FSH from exerting its normal action.” Lofts and Marshall (1956) suggested that the gonad suppressing action of prolactin might be due to a direct effect on the gonad. The work of Tanaka et al. (1971) demonstrated that prolactin can act at the ovarian level to block the ovulation inducing effects of LH. Meier (1969) showed that prolactin could inhibit the ovarian and oviducal response to FSH and LH in the white-throated sparrow. The latter effect was attributed to an interference with steroid production. Stetson and Erickson (1970) failed to show that prolactin would block gonadotropin induced testicular 32P uptake in young chicks. The recent finding of Shahabi et al. (1975) showing that LH injection into laying hens causes a rapid increase in follicular E, P
and testosterone and an increase in serum P and testosterone. This provided a means to test one possible method by which prolactin could exert an antigonadotropic effect. Camper and Burke (1977) extended these findings and showed both FSH and LH caused rapid increases in serum E and P in the turkey. While this paper clearly shows that LH and FSH induced rises in serum E and P are blocked by prolactin, it provides no information on the site of action. A variety of sites or mechanisms of action are possible. If prolactin acts on the gonad to reduce steroid output, it might do so by blocking the binding of gonadotropins to receptor sites or by blocking one or more of the steps between binding of the gonadotropin and synthesis of the steroids or by acting to increase enzymes which could result in decreased E or P synthesis. A completely different site of action is also possible. Prolactin might act upon the liver to increase steroid degradation. Bates et al. (1937) demonstrated a hepatotropic effect of prolactin in pigeons and Breneman (1942) showed that prolactin injection in-
76
CAMPER
AND BURKE
Bates, R. W., Lahr, E. L.. and Riddle, 0. (1935). The creased the weight of liver and other gross action of prolactin and follicle stimulating viscera in the cockerel. More acute inhormone on the mature ovary and sex accestracellular effects of prolactin on the liver sories of fowl. Amer. J. Physiol. 111, 361-368. seem possible. Bates, R. W., Riddle, O., and Lahr. E. L. (1937). The The fact that high levels of prolactin (i.e., mechanism of the antigonadal action of prolactin in adult pigeons. Amer. J. Physiol. 119,610-614. 1000 pg) reduce endogenous serum E levels, but have no effect on P levels, Breitenbach, R. P., and Meyer, R. K. (1959). Pituitary prolactin levels in laying, incubating, and suggests that prolactin is not simply inbrooding pheasants (Phasianus colchicus). Proc. creasing total steroid degradation. Sot. Exp. Biol. Med. 101, 16-19. This study certainly does not support the Breneman, W. R. (1942). Action of prolactin and esidea that prolactin’s antigonadotropic actrone on weights of reproductive organs and viscera of the cockerel. Endocrinology 30, 60!9tion is at the pituitary level, but it does not 615. exclude this as one possible site of action. Burrows, W. H., and Byerly, T. C. (1936). Studies of While previous workers (Bates et al., 1937; prolactin in the fowl pituitary. I. Broody hens Nalbandov et al., 1945) drew this conclucompared with laying hens and males. Proc. Sot. Exp. Biol. Med. 34, 841-844. sion from their data, it is doubtful whether Camper, P. M., and Burke, W. H. (1977). Serum esit can be supported. tradiol and progesterone levels of the laying turIncreased prolactin secretion might act to key hen following acute treatment with mammainterfere with ovarian activity by reducing lian luteinizing or follicle stimulating hormone. serum steroids and disrupting the normal Gen. Comp. Endocrinol. 31, 224-232. Cherms, F. L., Jr., Herrick, R. B., McShan, W. H., ovarian-hypothalamic-pituitary relationand Hymer, W. C. (1962). Prolactin content of ships which are responsible for ovum the anterior pituitary gland of turkey hens in difgrowth and ovulation. Progesterone injecferent reproductive stages. Endocrinology 71, tion will release LH in the hen (Wilson and 288-292. Sharp, 1975) and induce ovulation. InjecCrispens, C. G., Jr. (1957). Use of prolactin to induce broodiness in two wild turkeys. J. Wildlife tion of antiprogesterone or antitestosterone Mgmt. 21, 462. antisera will block ovulation in the hen B. J. A., and Smith, G. K. (1975). Effects of (Furr and Smith, 1975). Thus there is a cer- Furr, antisera against gonadal steroids on ovulation in tainly delicate balance between serum stethe hen (Gallus domesticus). J. Endocrinol. 66, roids and gonadotropin release. 303-304. Identification of the precise site of pro- Lofts, B., and Marshall, A. J. (1956). The effects of prolactin on the internal rhythm of reproduction lactin’s “antigonadotropic” action in in male birds. J. Endocrinol. 13, 101-106. female birds awaits further work.
ACKNOWLEDGMENTS The authors are indebted to Mr. M. J. Wineland and Dr. B. C. Wentworth for the gift of antisera for the estradiol assay. This work was supported in part by grant No. HD07242 from the National Institutes of Health and by grants from the Minnesota Turkey Growers Association. The LH and prolactin used in these studies were a gift from the National Institute of Arthritis, Metabolism and Digestive Diseases.
REFERENCES Alexander, B., and Wolfson, A. (1970). Prolactin and sexual maturation in the Japanese quail, Cotrurnix coturnix japonica. Poult. Sci. 49, 632-640.
Meier, A. H. (1969). Diurnal variations of metabolic responses to prolactin in lower vertebrates. Gen. Camp. Endocrinol. Suppl. 2, 55-62. Nakajo, S., and Tanaka, K. (1956). Prolactin potency of the cephalic and the caudal lobe of the anterior pituitary in relation to broodiness in the domestic fowl. Poult. sci. 55, 990-994. Nalbandov, A. V., and Card, L. E. (1945). Endocrine identification of the broody genotype of cocks. J. Hered. 36, 34-39. Nalbandov, A. V.. Hochauser, M., and Dugas, M. (1945). A study of the effect of prolactin on broodiness and cock testes. Endocrinology 36, 251-258. Nicoll, C. S. (1974). Physiological actions of prolactin. In “Handbook of Physiology-Endocrinology” (E. Knobil and W. H. Sawyer, eds.), Vol. 4, Part
PROLACTIN,
GONADOTROPIN
2, pp. 253-292. American Physiological Society, Washington, DC. Riddle, O., Bates, R. W., and Lahr, E. L. (1935). Prolactin induces broodiness in fowl. Amer. J. Physiol. 111, 352-360. Saeki, Y., and Tanabe, Y. (1954). Changes in prolactin content of fowl pituitary during broody periods and some experiments on the induction of broodiness. Poult. Sci. 34, 909-919. Shahabi, N. A., Bahr, J. M., and Nalbandov, A. V. (1975). Effect of LH injection on plasma and follicular steroids in the chicken. Endocrinology 96, 969-972. Shani, J., Furr, B. J. A., and Forsyth, I. A. (1973). A reexamination of the antigonadal effect of prolac-
AND SERUM STEROIDS
77
tin in the male fowl, Gatlus domesticus. J. Reprod. Fert. 34, 167-170. Stetson, M. H., and Erickson, J. E. (1970). The antigonadal properties of prolactin in birds: failure of prolactin to inhibit the uptake of szPby testes of cockerels in vivo. Gen. Comp. Endocrinol. 15, 484-487. Tanaka, K., Kamiyoshi, M., and Tanabe, Y. (1971). Inhibition of premature ovulation by prolactin in the hen. Poult. Sci. SO, 63-66. Wilson, S. C., and Sharp, P. J. (1975). Effects of progesterone and synthetic luteinizing hormone releasing hormone on the release of luteinizing hormone during sexual maturation in the hen (Gallus domesticus). .I. Endocrinol. 67, 359-369.
AND
COMPARATIVE
ENDOCRINOLOGY
32, 72-77 (1977)
The Effects of Prolactin on the Gonadotropin Induced Rise in Serum Estradiol and Progesterone of the Laying Turkey1 P. M. CAMPER
AND W. H. BURKE
Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55108 Accepted December 23, 1976 Mammalian LH (250 pg of NIH-LH-B9) and FSH (500 pg of FSH-P, Armour-Baldwin) elicit increases in serum estradiol (E) and progesterone (P) of laying turkeys within 30 min after a single iv injection. Prolactin (NIH-P-S1 1) at doses of 250 and 500 Kg had no effect on the E or P levels when given in a single iv injection. One thousand micrograms of prolactin caused a significant decrease in serum E within 30 min of injection. Prolactin (500 and 1000 pg), given 30 min before LH, blocked the LH induced rise in P and at doses of 250, 500 and 1000 pg it blocked the LH induced rise in E. Five hundred and 1000 pg of prolactin blocked the FSH induced rise in P and doses of 250, 500 and 1000 pg of prolactin blocked the FSH induced rise in E. It is concluded that prolactin’s antigonadotropic effect is exerted, at least in part, by interfering with gonadotropin induced increases in ovarian steroid levels in the blood.
The pituitary prolactin content of incubating (broody) chickens (Burrows and Byerly, 1936; Saeki and Tanabe, 1954; 1956), turkeys Nakajo and Tanaka, (Cherms et al., 1962; Cogger and Burke, unpublished data), pheasants (Brietenbach and Meyer, 1959) and other avian species (Nicoll, 1974) has been found to be higher than that of actively laying individuals. Injection of prolactin has induced broodiness in several avian species (Riddle et al., 1935; Nalbandov and Card, 1945; Crispens, 1957) and has caused gonadal regression in female chickens (Bates et al., 1935). Prolactin injection has failed to cause gonadal regression in some experiments with female Coturnix quail (Alexander and Wolfson, 1970) but has done so in others (Camper and Burke, unpublished data). Likewise in early experiments with male chickens pro-
lactin caused testicular regression (Bates et al., 1935; Brenemen, 1942; Nalbandov et al., 1945), an effect which could be blocked by simultaneous FSH treatment (Nalbandov ef al., 1945). Recent work has failed to show an antigonadal effect of prolactin in roosters (Shani et al., 1973). The mechanism or site of action by which prolactin causes gonadal regression, in those species in which it does, remains obscure. One possible site of action would be at the gonad, blocking the effects of gonadotropins on their target tissues. In fact, Tanaka et al. (1971) have reported that prolactin injection will inhibit LH induced premature ovulation. It has recently been demonstrated that LH or FSH injection elicits rapid rises in serum estradiol (E) and progesterone (P) in laying turkeys (Camper and Burke, 1977). These findings provide a means to test whether prolactin inhibits the acute rise in serum E and P elicited by LH and FSH.
’ Scientific Journal series paper No. 97 IO of the Minnesota Agricultural Experiment Station. 72 Copyright All rights
@ 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN 00164480
PROLACTIN.
MATERIALS
AND
GONADOTROPIN
AND
METHODS
Experiment I was conducted to determine if prolactin injection itself would alter serum E or P levels. To do this hens were bled and then injected with I ml of saline or 250, 500 or 1000 pg of prolactin (NIH-PSl 1) in 1 ml of pH 9 saline. They were bled again 5 and 30 min after injection. Serum was obtained and analyzed for E and P. Experiments 2 and 3 were designed to determine if pretreatment of turkeys with prolactin would affect the steroidogenic response elicited by LH (NIHLH-B9) or FSH (FSH-P, Armour-Baldwin Laboratories). The general test procedure was as follows: 4 or 5 hr after oviposition turkey hens were bled from a brachial vein, then injected with prolactin solution or saline, bled 30 min later, then injected with 250 pg of LH or 500 pg of FSH or saline and bled again 5 and 30 min later. All injections were given into the brachial vein in a 1 ml volume. Five to 8 ml of blood were taken at each bleeding and allowed to clot over ice. It was stored overnight at 4” and then centrifuged. Serum was collected and stored frozen at -20” until used for E and P assay. The E and P assays used throughout and the turkeys used for experiments I and 2 were described by Camper and Burke (1977). The turkeys used in experiment 3 of this study were Nicholas small white female line birds (Nicholas Turkey Breeding Farms, Sonoma, Calif.). The birds were reared under natural photoperiod conditions from May to September. At 6 months of age they were placed on a photoperiod of 6L: 18D to terminate photo refractoriness and after 3 weeks of this regimen the photoperiod was switched to 15L:9D to stimulate reproductive activity. Within 3 weeks of this switch the hens were laying eggs and they were used for these experiments during the following months. In each experiment, 4 hens were used per treatment group. Estradiol and P levels of individuals after hormone treatment were compared to their own pretreatment E and P levels. Results were analyzed by a TABLE SERUM
ESTRADIOL
(pg/ml)
IN THE
loo0
TURKEY
73
STEROIDS
split-plot analysis of variance and if a significant F value was obtained, mean differences were determined using Dunnett’s test for comparisons involving a control mean. The statistical analyses were done on absolute quantities of hormone (i.e., pg or rig/ml), but for purposes of presentation the data in the figures has been converted to percentage change from 0 time as follows: hormone 30 min level hormone
% change
level at 5 or - hormone at 0 time level at 0 time
x 100.
RESULTS
Injection of laying turkeys with 250 or 500 Fg of prolactin had no effect on serum E levels (Table l), but 1000 pg caused a significant depression in E within 30 min. Three of the 4 birds given the 1000 pg injection had markedly lower E levels 30 min after prolactin treatment. In 1 bird the E level was depressed to a nearly nondetectable level and in 1 bird the E level was nondetectable 30 min after prolactin treatment. The fourth bird in the group had no change in its E level over the course of the experiment. Progesterone levels of these same birds remained unchanged by the prolactin treatment (Table 2). The effect of prolactin on the LH induced rise in P and E is shown in Fig. la and b, respectively. Saline injection had no significant effect on P or E levels. Injection of 250 pg of LH caused a highly significant increase in serum P (131.58 + 24.94Yo) and I HEN
AFTER
A SINGLE
INJECTION
OF PROLACTIN”
After prolactin (min)
Prolactin w 0 250 500
LAYING
SERUM
0 15.11 37.96 18.33 26.35
ir f. 2 ?I
5 2.42b 5.87 3.62 2.34
a Prolactin (NIH P-S-II) given at time 0. b Mean +- standard error; n = 4 in all groups. c Estradiol decreased from time 0; P < 0.05.
13.25 32.81 20.85 23.74
? k 2 k
30 1.61 4.42 3.48 1.38
15.81 30.10 16.07 8.99
tf 2 f
1.95 7.18 2.83 3.95”
CAMPER
74
AND BURKE
TABLE 2 SERUM PROGESTERONE (r&ml) IN THE LAYING TURKEY HEN AFTER A SINGLE INJECTION OF PROLACTIN" After prolactin (min)
Prolactin (M)
0 1.32 1.80 2.29 2.20
0
250 500 1000
? 2 + +
5 0.31b 0.22 0.30 0.22
1.18 2.06 2.01 1.95
+ f k *
30 0.33 0.23 0.44 0.13
1.48 1.86 2.42 2.10
” 0.23 -c 0.22 f 0.59 f 0.17
a Prolactin (NIH P-S-11) injected at time 0. b Mean f standard error; n = 4 in all groups.
a significant increase in E (231.68 2 74.40%). Treatment of hens with 500 or 1000 pg of prolactin 30 min prior to treatment with LH completely blocked the rise in steroids. The birds receiving 250 Fg of prolactin prior to LH treatment showed a highly significant increase in P (93.53 + 6.20%) but no significant change in E. The E levels were higher 30 min after LH treatment in 3 of the 4 hens receiving pretreatment with 250 pg of prolactin, but statistical tests failed to show a significant rise. As in the preliminary experiment, 1000 ,ug of prolactin significantly depressed serum E within 30 min of its injection. UC!
FSH treatment caused a significant rise in serum P (143.58 r 42.39%, Fig. 2a). Five hundred and 1000 pg of prolactin significantly reduced the response to FSH. Estradiol levels were increased by the injection of 500 pg of FSH (236.38 f 84.76%). There was no significant change in E levels in FSH treated hens that were pretreated with 250,500 or 1000 pg of prolactin (Fig. 2b). DISCUSSION
It is generally assumed that prolactin induces incubation behavior in Gallinaceous birds and is responsible for the ovarian re-
r
Minutes
after
LH
Miies
after
LH
FIG. 1. Changes in serum progesterone (a) levels or estradiol levels (b) following the injection of bovine LH (NIH-LH-B9) preceded by the injection of saline or various doses of ovine prolactin (NIH-P-Sll). Lines followed by asterisks indicate that the hormone level is significantly different (P G 0.01) than at time 0 in the same animals.
PROLACTIN,
GONADOTROPIN
ug PRL J@FSH
0
75
STEROIDS
pg PRL
P after
SERUM
a
5 Minutes
AND
FSH
0
5
w FSH
b
30 Minutes
after
FSH
2. Changes in serum progesterone (a) or estradiol levels (b) following the injection of porcine FSH (Armour FSH-P) preceded by the injection of saline or various doses of ovine prolactin (NIH-P-Sll). Lines followed by asterisks indicate that the hormone level is significantly different (P G 0.05) than at time 0 in the same animals. FIG.
gression that accompanies broodiness. Prolactin often has been said to have an antigonadotropic effect in birds. Bates et al. (1937) concluded that prolactin acted upon the pituitary to decrease FSH secretion, a conclusion shared by Nalbandov et al. (1945). The latter authors also stated that “Prolactin may prevent FSH from exerting its normal action.” Lofts and Marshall (1956) suggested that the gonad suppressing action of prolactin might be due to a direct effect on the gonad. The work of Tanaka et al. (1971) demonstrated that prolactin can act at the ovarian level to block the ovulation inducing effects of LH. Meier (1969) showed that prolactin could inhibit the ovarian and oviducal response to FSH and LH in the white-throated sparrow. The latter effect was attributed to an interference with steroid production. Stetson and Erickson (1970) failed to show that prolactin would block gonadotropin induced testicular 32P uptake in young chicks. The recent finding of Shahabi et al. (1975) showing that LH injection into laying hens causes a rapid increase in follicular E, P
and testosterone and an increase in serum P and testosterone. This provided a means to test one possible method by which prolactin could exert an antigonadotropic effect. Camper and Burke (1977) extended these findings and showed both FSH and LH caused rapid increases in serum E and P in the turkey. While this paper clearly shows that LH and FSH induced rises in serum E and P are blocked by prolactin, it provides no information on the site of action. A variety of sites or mechanisms of action are possible. If prolactin acts on the gonad to reduce steroid output, it might do so by blocking the binding of gonadotropins to receptor sites or by blocking one or more of the steps between binding of the gonadotropin and synthesis of the steroids or by acting to increase enzymes which could result in decreased E or P synthesis. A completely different site of action is also possible. Prolactin might act upon the liver to increase steroid degradation. Bates et al. (1937) demonstrated a hepatotropic effect of prolactin in pigeons and Breneman (1942) showed that prolactin injection in-
76
CAMPER
AND BURKE
Bates, R. W., Lahr, E. L.. and Riddle, 0. (1935). The creased the weight of liver and other gross action of prolactin and follicle stimulating viscera in the cockerel. More acute inhormone on the mature ovary and sex accestracellular effects of prolactin on the liver sories of fowl. Amer. J. Physiol. 111, 361-368. seem possible. Bates, R. W., Riddle, O., and Lahr. E. L. (1937). The The fact that high levels of prolactin (i.e., mechanism of the antigonadal action of prolactin in adult pigeons. Amer. J. Physiol. 119,610-614. 1000 pg) reduce endogenous serum E levels, but have no effect on P levels, Breitenbach, R. P., and Meyer, R. K. (1959). Pituitary prolactin levels in laying, incubating, and suggests that prolactin is not simply inbrooding pheasants (Phasianus colchicus). Proc. creasing total steroid degradation. Sot. Exp. Biol. Med. 101, 16-19. This study certainly does not support the Breneman, W. R. (1942). Action of prolactin and esidea that prolactin’s antigonadotropic actrone on weights of reproductive organs and viscera of the cockerel. Endocrinology 30, 60!9tion is at the pituitary level, but it does not 615. exclude this as one possible site of action. Burrows, W. H., and Byerly, T. C. (1936). Studies of While previous workers (Bates et al., 1937; prolactin in the fowl pituitary. I. Broody hens Nalbandov et al., 1945) drew this conclucompared with laying hens and males. Proc. Sot. Exp. Biol. Med. 34, 841-844. sion from their data, it is doubtful whether Camper, P. M., and Burke, W. H. (1977). Serum esit can be supported. tradiol and progesterone levels of the laying turIncreased prolactin secretion might act to key hen following acute treatment with mammainterfere with ovarian activity by reducing lian luteinizing or follicle stimulating hormone. serum steroids and disrupting the normal Gen. Comp. Endocrinol. 31, 224-232. Cherms, F. L., Jr., Herrick, R. B., McShan, W. H., ovarian-hypothalamic-pituitary relationand Hymer, W. C. (1962). Prolactin content of ships which are responsible for ovum the anterior pituitary gland of turkey hens in difgrowth and ovulation. Progesterone injecferent reproductive stages. Endocrinology 71, tion will release LH in the hen (Wilson and 288-292. Sharp, 1975) and induce ovulation. InjecCrispens, C. G., Jr. (1957). Use of prolactin to induce broodiness in two wild turkeys. J. Wildlife tion of antiprogesterone or antitestosterone Mgmt. 21, 462. antisera will block ovulation in the hen B. J. A., and Smith, G. K. (1975). Effects of (Furr and Smith, 1975). Thus there is a cer- Furr, antisera against gonadal steroids on ovulation in tainly delicate balance between serum stethe hen (Gallus domesticus). J. Endocrinol. 66, roids and gonadotropin release. 303-304. Identification of the precise site of pro- Lofts, B., and Marshall, A. J. (1956). The effects of prolactin on the internal rhythm of reproduction lactin’s “antigonadotropic” action in in male birds. J. Endocrinol. 13, 101-106. female birds awaits further work.
ACKNOWLEDGMENTS The authors are indebted to Mr. M. J. Wineland and Dr. B. C. Wentworth for the gift of antisera for the estradiol assay. This work was supported in part by grant No. HD07242 from the National Institutes of Health and by grants from the Minnesota Turkey Growers Association. The LH and prolactin used in these studies were a gift from the National Institute of Arthritis, Metabolism and Digestive Diseases.
REFERENCES Alexander, B., and Wolfson, A. (1970). Prolactin and sexual maturation in the Japanese quail, Cotrurnix coturnix japonica. Poult. Sci. 49, 632-640.
Meier, A. H. (1969). Diurnal variations of metabolic responses to prolactin in lower vertebrates. Gen. Camp. Endocrinol. Suppl. 2, 55-62. Nakajo, S., and Tanaka, K. (1956). Prolactin potency of the cephalic and the caudal lobe of the anterior pituitary in relation to broodiness in the domestic fowl. Poult. sci. 55, 990-994. Nalbandov, A. V., and Card, L. E. (1945). Endocrine identification of the broody genotype of cocks. J. Hered. 36, 34-39. Nalbandov, A. V.. Hochauser, M., and Dugas, M. (1945). A study of the effect of prolactin on broodiness and cock testes. Endocrinology 36, 251-258. Nicoll, C. S. (1974). Physiological actions of prolactin. In “Handbook of Physiology-Endocrinology” (E. Knobil and W. H. Sawyer, eds.), Vol. 4, Part
PROLACTIN,
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2, pp. 253-292. American Physiological Society, Washington, DC. Riddle, O., Bates, R. W., and Lahr, E. L. (1935). Prolactin induces broodiness in fowl. Amer. J. Physiol. 111, 352-360. Saeki, Y., and Tanabe, Y. (1954). Changes in prolactin content of fowl pituitary during broody periods and some experiments on the induction of broodiness. Poult. Sci. 34, 909-919. Shahabi, N. A., Bahr, J. M., and Nalbandov, A. V. (1975). Effect of LH injection on plasma and follicular steroids in the chicken. Endocrinology 96, 969-972. Shani, J., Furr, B. J. A., and Forsyth, I. A. (1973). A reexamination of the antigonadal effect of prolac-
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tin in the male fowl, Gatlus domesticus. J. Reprod. Fert. 34, 167-170. Stetson, M. H., and Erickson, J. E. (1970). The antigonadal properties of prolactin in birds: failure of prolactin to inhibit the uptake of szPby testes of cockerels in vivo. Gen. Comp. Endocrinol. 15, 484-487. Tanaka, K., Kamiyoshi, M., and Tanabe, Y. (1971). Inhibition of premature ovulation by prolactin in the hen. Poult. Sci. SO, 63-66. Wilson, S. C., and Sharp, P. J. (1975). Effects of progesterone and synthetic luteinizing hormone releasing hormone on the release of luteinizing hormone during sexual maturation in the hen (Gallus domesticus). .I. Endocrinol. 67, 359-369.
