GENERAL
AND
COMPARATIVE
37, 333-342 (1979)
ENDOCRINOLOGY
Hypothalamic Control of Prolactin and Growth Hormone Secretion in Different Vertebrate Species T. R. HALL' Department
of Pure
and Applied
AND A. CHADWICK
Zoology. The University,
Leeds
LS2 9JT,
England
Accepted December 20. 1978 Pituitaries from different vertebrates representing mammals, birds, reptiles, and amphibians, were incubated in vitro with various hypothalamic extracts (HE). Prolactin and growth hormone (GH) in medium and pituitary were measured by densitometry after polyacrylamide gel electrophoretic separation (PAGE). Rat (Rattus norvegicus), chicken (Callus domesticus), terrapin (Chrysemys picta), and toad (Xenopus faevis) pituitaries were incubated with homologous HE. Rat HE inhibited prolactin release. In the other species the HE stimulated prolactin release. In all four species GH release was stimulated by HE. The effects on prolactin and GH release were proportional to the dose of HE added. Chicken pituitaries were incubated with chicken HE together with rat HE. The rat HE inhibited the chicken HE-stimulated release of prolactin, as measured by radioimmunoassay. Heterologous incubations were used to test HE for prolactin releasing and inhibiting factors and for GH releasing and inhibiting factors. Chicken pituitaries were incubated with HE from the eel (Anguilla an&la), the cod (Gudus gadus) and the flounder (Pleuronectes Jesus) as well as from the other species listed. Both cod and flounder HE marginally inhibited autonomous chicken prolactin release. HE from these species dose-responsively inhibited chicken HEstimulated prolactin release. Cod HE also inhibited chicken HE-stimulated GH release. HE from the eel, the terrapin and the toad stimulated chicken prolactin release. Hormone release from terrapin and toad pituitaries incubated with heterologous HE was consistent with hypothalamic control via releasing factors in these species.
The mammalian hypothalamus contains a factor which inhibits prolactin secretion (prolactin-inhibiting factor, PIF). Pituitaries incubated in vitro have been found to release large amounts of prolactin, and this release was inhibited following the addition of hypothalamic extract (HE) (Pasteels, 1961; Talwalker et al., 1963; Clemens and Meites, 1974) or hypothalamic fragments (Quijada et ui., 1973/74; Hall et al., 1976a) to the medium. Mammalian HE stimulates growth hormone (GH) release in vitro (Franz et al., 1962; Deuben and Meites, 1964; Schally et al., 1965). In addition to the GH-releasing factor (GRF) a GH-inhibiting factor (GIF) has been detected and iden’ Present address: Department of Biology, Marquette University, Milwaukee, Wis.
tified (Krulich et al., 1968; Coy et al., 1973; Brazeau et ui., 1973). Prolactin secretion in birds is controlled by a hypothalamic prolactin-releasing factor (PRF) (Kragt and Meites, 1965). HE stimulated prolactin release both in viva (Knight and Chadwick, 1975; Hall and Chadwick, submitted-a) and in vitro (Nicoll, 1965; Tixier-Vidal and Gourdji, 1972; Hall et al., 1975: Hall and Chadwick, submitted-b). Miiller et al. (1967) found that pigeon HE stimulates GH release in rats irz viva. We have shown that avian HE stimulates GH release in birds both in vitro and in vivo (Hall and Chadwick, 1975b, 1976a, submitted-a). Little is known about the control of prolactin secretion in reptiles. Reptile pituitaries have been found to release large
333 0016~6480/79/030333-10$01.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
334
HALL
AND
amounts of prolactin in vitro, suggesting inhibitory control by the hypothalamus as in mammals. However, reptile HE further stimulated prolactin release from these pituitaries (Nicoll, 1965: Nicoll and Fiorindo. 1969; Nicoll et al. , 19701. We have reported, however, that prolactin release from terrapin pituitaries, in \>itr’o, is low, although it was found to be stimulated by terrapin HE (Hall rt al., 1978). Control of prolactin release in amphibians may involve both stimulatory and inhibitory hypothalamic influences. Etkin and Lehrer (1960), Everett (19661, and Guardabassi et ul. (1969) all suggested an inhibitory control of prolactin via hypothalamic PIF. On the other hand both Hall and Chadwick (1976b) and Kuhn and Engelen ( 19761 found evidence for an amphibian hypothalamic PRF. The latter authors suggested that the amphibian hypothalamus may contain both PRF and PIF. There are conflicting reports concerning the control of prolactin release in teleosts. Sage ( 1966, 1968) reported that cultured released large Xiphophorus pituitaries amounts of prolactin, indicating hypothalamic PIF. Based on autotransplantation studies, Ball et al. (1972) concluded that prolactin cells were under inhibitory control. We have reported that in the eel Anguilfa the hypothalamus contains prolactin stimulating activity in \ftro (Hall and Chadwick, 1978). The present experiments, which were in part reported in abstract (Hall et al., 1976b) were designed to determine the effect of hypothalamic extracts on prolactin and GH secretion in tsit~o in a variety of different vertebrate species. First, homologous pituitary-HE incubations were carried in the different species and second. pituitary glands were incubated in \‘itro with heterologous HE preparations. MATERIALS Rats, clawed
AND METHODS
chickens, terrapins toads (Xenopuu lark),
(Chr~~semj~~
eels (Anguih
picta). un-
CHADWICK guillu). and flounders (Pleuronec~tc~~ Jesus) were obtained from various commercial sources and housed in appropriate conditions in the laboratory for at least 1 week before use. Heads from freshly killed chicken and cod (Gud~s ~n&ts) were obtained from packing stations and dissected as soon as possible postmortem. The live animals were killed by decapitation and pituitaries and hypothalami were quickly removed. HE was made according to the method of Follett ( 1970). Similar extracts of forebrain were used as controls. The incubation technique (Hall et ul., 1975. submitted-b) can be summarized briefly as follows. Pituitaries were incubated individually in small glass tubes containing 100 yl medium 199 (Wellcome Laboratories, Ltd.) under an atmosphere of 95%’ OdS% CO, for 120 min. This “preincubation” medium was discarded and fresh medium incorporating the various treatments with HE was added and the incubation carried out for up to 24 hr. Tetrapod pituitaries were incubated at 37”, and teleost pituitaries at 12”. Medium and pituitary samples were stored deep-frozen until assayed. The prolactin and GH content of the medium and pituitary samples were measured by densitometric evaluation of stained protein bands in the gels following polyacrylamide gel electrophoretic separation (PAGE) of the samples (Hall ct ul.. 1975). Results are expressed in arbitrary “density units” (du) per milligram wet weight pituitary before incubation (duimg). Some medium samples were assayed forprolactin using an homologous fowl prolactin radioimmunoassay (RIA) (Scanes ef al.. 1976b). Statistical analyses of differences between groups were performed using Student’s t test.
RESULTS
Rat pituitaries were incubated for 1 hr in medium containing rat HE in amounts ranging from the equivalent of l-l.5 hypothalami per tube. Prolactin released is shown in Table 1. The densitometric method showed that relatively large amounts of prolactin were released autonomously by the pituitary and that release was inhibited by the addition of rat HE, proportionally to amount of HE added. Chicken pituitaries were incubated for 5 hr and terrapin and toad pituitaries for 24 hr in medium containing cortical extract or various doses of homologous HE. Prolactin release is summarized in Table 2 (Xenopus). Prolactin release in all three
CONTROL
EFFECT
OF RAT FROM
OF
PROLACTIN
TABLE I HE ON PROLACTIN RAT
AND
RELEASE
PITUITARIES
IN VITRO Dose of HE (hypothalamic equivalents)
Prolactin release” (duimg pituitary)
0
16.9 9.7 8.0 6.0
I 2 5 IO I5 II Values
k t 2 f
5.4( I.3 I .8( 0.9(
IO) IO) IO) IO)
NS NS NS P < 0.05
5.0 + 1310) P i 0.05 P < 0.02
4.0 5 l.l(lO) are mean
? SEM.
NS. not significant
species was stimulated by HE in a doseresponsive manner. Chicken HE had relatively less effect on prolactin release than terrapin and toad HE in homologous incubation, probably due to the shorter duration of the chicken pituitary incubations. GH release was also measured and the results are shown in Table 3 (Xewop~s). HE
GROWTH
from all three species contained GHreleasing activity, shown by the increased amounts of GH released into the medium following incubation. Release of GH was proportional to the amount of HE added to the medium, over the range of 0.3 hypothalamic equivalents to 3.0 hypothalamic equivalents. Chicken pituitaries were incubated for 5 hr in medium containing either chicken HE, rat HE, or both at a concentration equivalent to 3.0 hypothalami per pituitary. Prolactin released into the medium was measured by RIA, and the results are shown in Table 4. Rat HE inhibited prolactin release from the unstimulated pituitary, though the inhibition was not significant. Chicken HE significantly stimulated prolactin release from chicken pituitaries. and rat HE significantly inhibited this stimulation, though rat HE did not completely block the pituitary response to chicken HE. Chicken pituitaries were incubated for 5
TABLE EFFECT
OF HOMOLOGOUS
2
HE ON PROLACTIN RELEASE FROM AND TOAD PITUITARIES IN VITRO
Prolactin Species Chicken Terrapin Toad (1 Mean 2 SEM. + Significantly different
‘I Mean ? SEM. * Significantly different
(duimg) I.0 HE
3.0 HE
32 t 311 38 2 6 120 i 33
35 t 4 62 2 9 130 + I9
45 2 5” 96 t II” 421 2 43”
70 2 6* 142 f 2l* 547 + 34*
from
control
P < 0.05: II = 4 in each case. 3
HE
OF HOMOLOGOUS
ON GH RELEASE FROM TOAD PITUITARIES IN VITRO
GH
Chicken Terrapin Toad
TERRAPIN,
0.3 HE
AND
Species
CHICKEN,
Control
TABLE EFFECT
335
HORMONE
Control 50 2 5” 49 f I 389 t 27
from
control
release
CHICKEN,
TERRAPIN,
(duimg)
0.3 HE
I .O HE
3.0 HE
52 + 6 59 c II 436 + 32
682 I2 100 2 5* 615 t 66*
85 k 8* 165 t 25* 720 f l7*
P < 0.05: n = 4 in each case
336
HALL
AND
CHADWICK
lease from chicken pituitaries and also stimulated an increase in total prolactin. Rat HE, like the HE of cod and flounder, marginally inhibited chicken prolactin reProlactin release” lease without affecting total prolactin Treatment levels. Control 1.26 2 0.11 GH in these incubations is illustrated in NS (medium 199) Fig. 2. HE from flounder, eel, toad, terraRat HE 0.89 + 0.18 pin, and rat, as well as from chicken, all (3.0 hypothalami) 4.43 + 0.97 Chicken HE stimulated GH release and stimulated an (3.0 hypothalami) P < 0.05 increase in total GH. Only cod HE inhibited Chicken HE 2.25 + 0.07 1 GH release from chicken pituitary glands + rat HE incubatedin vitro and this response was only marginal. The cod HE did not affect total B Measured by radioimmunoassay; pg/mg wet wt of pituitary. Values represent mean + SEM; II = 6 in GH content. each case. Further incubations were carried out to test the inhibitory actions of cod and flounhr in medium containing HE equivalent to 1 der HE. Chicken pituitaries were incubated hypothalamus derived from chicken, cod, with various amounts of cod HE or flounder flounder, eel, toad, terrapin, and rat. Pro- HE (equivalent to 0.03, 0.3, and 3.0 lactin and GH in the medium and pituitary hypothalami) either alone or with chicken samples were measured by densitometry. HE equivalent to 3.0 hypothalami. Figure 3 Prolactin results are shown in Fig. 1. shows that cod HE inhibited the chicken Chicken HE stimulated prolactin release and HE-stimulated release of both chicken prosynthesis thus increasing total prolactin lactin and chicken GH. The results in Table (prolactin in medium + prolactin in pitui5 show that cod HE not only inhibited retary after incubation) compared with the lease of prolactin and GH but also blocked medium-only control incubation. Both cod hormone synthesis. Flounder HE (Fig. 4) and flounder HE marginally inhibited pro- inhibited the autonomous release of chicken lactin release without affecting total prolacprolactin but appeared to stimulate the tin. On the other hand, eel HE, toad HE, release of chicken GH. Flounder HE also and terrapin HE all stimulated prolactin re- inhibited the chicken HE-stimulated release EFFECT
TABLE 4 OF CHICKEN AND RAT HE ON PROLACTIN RELEASE FROM CHICKEN PITUITARIES IN VITRO
1
NO HE
Chii
Cod
Flolndl?r
Eel
Toad
FIG. 1. Effect of HE from different vertebrate species on prolactin secretion by chicken pituitary glands incubated in virro. Pairs of histograms show, on left, hormone released into medium (open columns). remaining in pituitary (cross-hatched) and, on right, total hormone after incubation. Vertical bars represent SEM; n = 6. Hormone release significantly different from control (0)P < 0.05; (*)P < 0.01.
CONTROL
OF
PROLACTIN
AND
./1/
GROWTH
HORMONE
n
1’
Fknmder
Eel
Toad
TWPWl
Rat
FIG. 2. Effect of HE from different vertebrate species on GH secretion by chicken pituitary glands incubated in vitro. Pairs of histograms show, on left, hormone released in medium (open columns), remaining in pituitary (cross-hatched) and. on right. total hormone after incubation. Vertical bars represent SEM; n = 6. Hormone release significantly different from control (0)P < 0.05; (*)P < 0.01.
of prolactin, the highest concentration of flounder HE almost completely counteracting the stimulatory effect of homologous HE. However, flounder HE enhanced the chicken HE-stimulated release of GH. The response to both cod and flounder HE was proportional to the amount of HE added.
FIG. 3. Effect of cod HE on prolactin and GH release from chicken pituitaries incubated in rho. Histograms show prolactin (open) and GH (crosshatched). Results in the incubation medium expressed as percentage change compared to control (medium only) incubation. Vertical bars represent SEM. Hormone release significantly different from medium 199 control. (+) P < 0.01; from 3.0 chicken HE, (0)P < 0.05; (*) P < 0.01.
Terrapin pituitaries were incubated for 5 hr either alone or with homologous HE, rat HE, chicken HE, or eel HE, all equivalent to one hypothalamus per pituitary. Figure 5 shows that small but detectable amounts of prolactin were released without any HE and that the addition of homologous HE stimulated both synthesis and release. Chicken HE had a powerful stimulatory effect, as did eel HE, but rat HE had no stimulatory effect. Figure 6 shows the results of similar experiments on Xenopus. After 5 hr incubation at 37” both prolactin and GH were stimulated by homologous HE, chicken HE, and terrapin HE. Rat HE appeared to inhibit prolactin release but stimulated GH release. Possible inhibitory influence of rat HE on prolactin stimulation by homologous HE could not be tested in the terrapin and the toad due to shortage of animals. DISCUSSION
The prolactin and GH bands visible after PAGE have been identified in numerous vertebrates by both bioassay and RIA. Nicoll and Nichols ( 197 1) and Nicoll and Licht (1971) identified prolactin and GH
338
HALL
AND TABLE
EFFECT
CHADWICK 5
OF COD HE ON PROLACTIN AND CHICKEN PITUITARIES INCUBATED
GH
SECRETION IN VITRO
FROM
Prolactin (duimg)
Growth
Treatment
Medium
Control (no HE) Chicken HE ( = 3 .O hypothalami) Chicken HE + cod HE (0.03 hypothalami) Chicken HE + cod HE (0.3 hypothalami) Chicken HE + cod HE (3.0 hypothalami)
27 2 4
56 f
62 2 3":
Total”
hormone (duimg)
Medium
Total R
66 2 4
141 2 13
98 k 8"
88 t- 4"
179 2 8#:
61 t 3”
95 2 6*
85 k 3"
171 + 6':
50 + 2*
gg + 4°C
76 + 4
150 + 5
31+3
62 + 3*
51 + 3
130 2 5
10
* Significantly different from control P < 0.05; n = 6 in each case. ” Total = hormone in medium plus hormone in pituitary at end of incubation.
bands in pituitary electrophoretograms in a wide variety of tetrapods. In the chicken the prolactin band identification has been confirmed by RIA (Scanes et cd., 1976a). Bolton ef al. (1976) showed that in chicken pituitary incubations there was a linear relationship between prolactin concentrations measured by PAGE and densitometry and
Values
represent
mean t SEM.
by radioimmunoassay. Knight et czf. (1970) and Hall and Chadwick (1978) identified the prolactin band in eel electrophoretograms, and Ingleton and Stribley (1975), using an immunoassay technique, demonstrated the position of the GH bands in eel pituitary electrophoretograms. The mammalian hypothalamus contains
FIG. 4. Effect of flounder HE on prolactin and GH release from rfitro. Histograms show prolactin (open) and GH (cross-hatched) in expressed as percentage change compared to control (medium only) sent SEM. Hormone release significantly different from medium 199 ,O.Ol; from 3.0 chicken HE, (*) P < 0.01.
chicken pituitaries incubated in the incubation media. Results incubation. Vertical bars reprecontrol: (0) P < 0.05; (+) P
AND
COMPARATIVE
37, 333-342 (1979)
ENDOCRINOLOGY
Hypothalamic Control of Prolactin and Growth Hormone Secretion in Different Vertebrate Species T. R. HALL' Department
of Pure
and Applied
AND A. CHADWICK
Zoology. The University,
Leeds
LS2 9JT,
England
Accepted December 20. 1978 Pituitaries from different vertebrates representing mammals, birds, reptiles, and amphibians, were incubated in vitro with various hypothalamic extracts (HE). Prolactin and growth hormone (GH) in medium and pituitary were measured by densitometry after polyacrylamide gel electrophoretic separation (PAGE). Rat (Rattus norvegicus), chicken (Callus domesticus), terrapin (Chrysemys picta), and toad (Xenopus faevis) pituitaries were incubated with homologous HE. Rat HE inhibited prolactin release. In the other species the HE stimulated prolactin release. In all four species GH release was stimulated by HE. The effects on prolactin and GH release were proportional to the dose of HE added. Chicken pituitaries were incubated with chicken HE together with rat HE. The rat HE inhibited the chicken HE-stimulated release of prolactin, as measured by radioimmunoassay. Heterologous incubations were used to test HE for prolactin releasing and inhibiting factors and for GH releasing and inhibiting factors. Chicken pituitaries were incubated with HE from the eel (Anguilla an&la), the cod (Gudus gadus) and the flounder (Pleuronectes Jesus) as well as from the other species listed. Both cod and flounder HE marginally inhibited autonomous chicken prolactin release. HE from these species dose-responsively inhibited chicken HEstimulated prolactin release. Cod HE also inhibited chicken HE-stimulated GH release. HE from the eel, the terrapin and the toad stimulated chicken prolactin release. Hormone release from terrapin and toad pituitaries incubated with heterologous HE was consistent with hypothalamic control via releasing factors in these species.
The mammalian hypothalamus contains a factor which inhibits prolactin secretion (prolactin-inhibiting factor, PIF). Pituitaries incubated in vitro have been found to release large amounts of prolactin, and this release was inhibited following the addition of hypothalamic extract (HE) (Pasteels, 1961; Talwalker et al., 1963; Clemens and Meites, 1974) or hypothalamic fragments (Quijada et ui., 1973/74; Hall et al., 1976a) to the medium. Mammalian HE stimulates growth hormone (GH) release in vitro (Franz et al., 1962; Deuben and Meites, 1964; Schally et al., 1965). In addition to the GH-releasing factor (GRF) a GH-inhibiting factor (GIF) has been detected and iden’ Present address: Department of Biology, Marquette University, Milwaukee, Wis.
tified (Krulich et al., 1968; Coy et al., 1973; Brazeau et ui., 1973). Prolactin secretion in birds is controlled by a hypothalamic prolactin-releasing factor (PRF) (Kragt and Meites, 1965). HE stimulated prolactin release both in viva (Knight and Chadwick, 1975; Hall and Chadwick, submitted-a) and in vitro (Nicoll, 1965; Tixier-Vidal and Gourdji, 1972; Hall et al., 1975: Hall and Chadwick, submitted-b). Miiller et al. (1967) found that pigeon HE stimulates GH release in rats irz viva. We have shown that avian HE stimulates GH release in birds both in vitro and in vivo (Hall and Chadwick, 1975b, 1976a, submitted-a). Little is known about the control of prolactin secretion in reptiles. Reptile pituitaries have been found to release large
333 0016~6480/79/030333-10$01.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
334
HALL
AND
amounts of prolactin in vitro, suggesting inhibitory control by the hypothalamus as in mammals. However, reptile HE further stimulated prolactin release from these pituitaries (Nicoll, 1965: Nicoll and Fiorindo. 1969; Nicoll et al. , 19701. We have reported, however, that prolactin release from terrapin pituitaries, in \>itr’o, is low, although it was found to be stimulated by terrapin HE (Hall rt al., 1978). Control of prolactin release in amphibians may involve both stimulatory and inhibitory hypothalamic influences. Etkin and Lehrer (1960), Everett (19661, and Guardabassi et ul. (1969) all suggested an inhibitory control of prolactin via hypothalamic PIF. On the other hand both Hall and Chadwick (1976b) and Kuhn and Engelen ( 19761 found evidence for an amphibian hypothalamic PRF. The latter authors suggested that the amphibian hypothalamus may contain both PRF and PIF. There are conflicting reports concerning the control of prolactin release in teleosts. Sage ( 1966, 1968) reported that cultured released large Xiphophorus pituitaries amounts of prolactin, indicating hypothalamic PIF. Based on autotransplantation studies, Ball et al. (1972) concluded that prolactin cells were under inhibitory control. We have reported that in the eel Anguilfa the hypothalamus contains prolactin stimulating activity in \ftro (Hall and Chadwick, 1978). The present experiments, which were in part reported in abstract (Hall et al., 1976b) were designed to determine the effect of hypothalamic extracts on prolactin and GH secretion in tsit~o in a variety of different vertebrate species. First, homologous pituitary-HE incubations were carried in the different species and second. pituitary glands were incubated in \‘itro with heterologous HE preparations. MATERIALS Rats, clawed
AND METHODS
chickens, terrapins toads (Xenopuu lark),
(Chr~~semj~~
eels (Anguih
picta). un-
CHADWICK guillu). and flounders (Pleuronec~tc~~ Jesus) were obtained from various commercial sources and housed in appropriate conditions in the laboratory for at least 1 week before use. Heads from freshly killed chicken and cod (Gud~s ~n&ts) were obtained from packing stations and dissected as soon as possible postmortem. The live animals were killed by decapitation and pituitaries and hypothalami were quickly removed. HE was made according to the method of Follett ( 1970). Similar extracts of forebrain were used as controls. The incubation technique (Hall et ul., 1975. submitted-b) can be summarized briefly as follows. Pituitaries were incubated individually in small glass tubes containing 100 yl medium 199 (Wellcome Laboratories, Ltd.) under an atmosphere of 95%’ OdS% CO, for 120 min. This “preincubation” medium was discarded and fresh medium incorporating the various treatments with HE was added and the incubation carried out for up to 24 hr. Tetrapod pituitaries were incubated at 37”, and teleost pituitaries at 12”. Medium and pituitary samples were stored deep-frozen until assayed. The prolactin and GH content of the medium and pituitary samples were measured by densitometric evaluation of stained protein bands in the gels following polyacrylamide gel electrophoretic separation (PAGE) of the samples (Hall ct ul.. 1975). Results are expressed in arbitrary “density units” (du) per milligram wet weight pituitary before incubation (duimg). Some medium samples were assayed forprolactin using an homologous fowl prolactin radioimmunoassay (RIA) (Scanes ef al.. 1976b). Statistical analyses of differences between groups were performed using Student’s t test.
RESULTS
Rat pituitaries were incubated for 1 hr in medium containing rat HE in amounts ranging from the equivalent of l-l.5 hypothalami per tube. Prolactin released is shown in Table 1. The densitometric method showed that relatively large amounts of prolactin were released autonomously by the pituitary and that release was inhibited by the addition of rat HE, proportionally to amount of HE added. Chicken pituitaries were incubated for 5 hr and terrapin and toad pituitaries for 24 hr in medium containing cortical extract or various doses of homologous HE. Prolactin release is summarized in Table 2 (Xenopus). Prolactin release in all three
CONTROL
EFFECT
OF RAT FROM
OF
PROLACTIN
TABLE I HE ON PROLACTIN RAT
AND
RELEASE
PITUITARIES
IN VITRO Dose of HE (hypothalamic equivalents)
Prolactin release” (duimg pituitary)
0
16.9 9.7 8.0 6.0
I 2 5 IO I5 II Values
k t 2 f
5.4( I.3 I .8( 0.9(
IO) IO) IO) IO)
NS NS NS P < 0.05
5.0 + 1310) P i 0.05 P < 0.02
4.0 5 l.l(lO) are mean
? SEM.
NS. not significant
species was stimulated by HE in a doseresponsive manner. Chicken HE had relatively less effect on prolactin release than terrapin and toad HE in homologous incubation, probably due to the shorter duration of the chicken pituitary incubations. GH release was also measured and the results are shown in Table 3 (Xewop~s). HE
GROWTH
from all three species contained GHreleasing activity, shown by the increased amounts of GH released into the medium following incubation. Release of GH was proportional to the amount of HE added to the medium, over the range of 0.3 hypothalamic equivalents to 3.0 hypothalamic equivalents. Chicken pituitaries were incubated for 5 hr in medium containing either chicken HE, rat HE, or both at a concentration equivalent to 3.0 hypothalami per pituitary. Prolactin released into the medium was measured by RIA, and the results are shown in Table 4. Rat HE inhibited prolactin release from the unstimulated pituitary, though the inhibition was not significant. Chicken HE significantly stimulated prolactin release from chicken pituitaries. and rat HE significantly inhibited this stimulation, though rat HE did not completely block the pituitary response to chicken HE. Chicken pituitaries were incubated for 5
TABLE EFFECT
OF HOMOLOGOUS
2
HE ON PROLACTIN RELEASE FROM AND TOAD PITUITARIES IN VITRO
Prolactin Species Chicken Terrapin Toad (1 Mean 2 SEM. + Significantly different
‘I Mean ? SEM. * Significantly different
(duimg) I.0 HE
3.0 HE
32 t 311 38 2 6 120 i 33
35 t 4 62 2 9 130 + I9
45 2 5” 96 t II” 421 2 43”
70 2 6* 142 f 2l* 547 + 34*
from
control
P < 0.05: II = 4 in each case. 3
HE
OF HOMOLOGOUS
ON GH RELEASE FROM TOAD PITUITARIES IN VITRO
GH
Chicken Terrapin Toad
TERRAPIN,
0.3 HE
AND
Species
CHICKEN,
Control
TABLE EFFECT
335
HORMONE
Control 50 2 5” 49 f I 389 t 27
from
control
release
CHICKEN,
TERRAPIN,
(duimg)
0.3 HE
I .O HE
3.0 HE
52 + 6 59 c II 436 + 32
682 I2 100 2 5* 615 t 66*
85 k 8* 165 t 25* 720 f l7*
P < 0.05: n = 4 in each case
336
HALL
AND
CHADWICK
lease from chicken pituitaries and also stimulated an increase in total prolactin. Rat HE, like the HE of cod and flounder, marginally inhibited chicken prolactin reProlactin release” lease without affecting total prolactin Treatment levels. Control 1.26 2 0.11 GH in these incubations is illustrated in NS (medium 199) Fig. 2. HE from flounder, eel, toad, terraRat HE 0.89 + 0.18 pin, and rat, as well as from chicken, all (3.0 hypothalami) 4.43 + 0.97 Chicken HE stimulated GH release and stimulated an (3.0 hypothalami) P < 0.05 increase in total GH. Only cod HE inhibited Chicken HE 2.25 + 0.07 1 GH release from chicken pituitary glands + rat HE incubatedin vitro and this response was only marginal. The cod HE did not affect total B Measured by radioimmunoassay; pg/mg wet wt of pituitary. Values represent mean + SEM; II = 6 in GH content. each case. Further incubations were carried out to test the inhibitory actions of cod and flounhr in medium containing HE equivalent to 1 der HE. Chicken pituitaries were incubated hypothalamus derived from chicken, cod, with various amounts of cod HE or flounder flounder, eel, toad, terrapin, and rat. Pro- HE (equivalent to 0.03, 0.3, and 3.0 lactin and GH in the medium and pituitary hypothalami) either alone or with chicken samples were measured by densitometry. HE equivalent to 3.0 hypothalami. Figure 3 Prolactin results are shown in Fig. 1. shows that cod HE inhibited the chicken Chicken HE stimulated prolactin release and HE-stimulated release of both chicken prosynthesis thus increasing total prolactin lactin and chicken GH. The results in Table (prolactin in medium + prolactin in pitui5 show that cod HE not only inhibited retary after incubation) compared with the lease of prolactin and GH but also blocked medium-only control incubation. Both cod hormone synthesis. Flounder HE (Fig. 4) and flounder HE marginally inhibited pro- inhibited the autonomous release of chicken lactin release without affecting total prolacprolactin but appeared to stimulate the tin. On the other hand, eel HE, toad HE, release of chicken GH. Flounder HE also and terrapin HE all stimulated prolactin re- inhibited the chicken HE-stimulated release EFFECT
TABLE 4 OF CHICKEN AND RAT HE ON PROLACTIN RELEASE FROM CHICKEN PITUITARIES IN VITRO
1
NO HE
Chii
Cod
Flolndl?r
Eel
Toad
FIG. 1. Effect of HE from different vertebrate species on prolactin secretion by chicken pituitary glands incubated in virro. Pairs of histograms show, on left, hormone released into medium (open columns). remaining in pituitary (cross-hatched) and, on right, total hormone after incubation. Vertical bars represent SEM; n = 6. Hormone release significantly different from control (0)P < 0.05; (*)P < 0.01.
CONTROL
OF
PROLACTIN
AND
./1/
GROWTH
HORMONE
n
1’
Fknmder
Eel
Toad
TWPWl
Rat
FIG. 2. Effect of HE from different vertebrate species on GH secretion by chicken pituitary glands incubated in vitro. Pairs of histograms show, on left, hormone released in medium (open columns), remaining in pituitary (cross-hatched) and. on right. total hormone after incubation. Vertical bars represent SEM; n = 6. Hormone release significantly different from control (0)P < 0.05; (*)P < 0.01.
of prolactin, the highest concentration of flounder HE almost completely counteracting the stimulatory effect of homologous HE. However, flounder HE enhanced the chicken HE-stimulated release of GH. The response to both cod and flounder HE was proportional to the amount of HE added.
FIG. 3. Effect of cod HE on prolactin and GH release from chicken pituitaries incubated in rho. Histograms show prolactin (open) and GH (crosshatched). Results in the incubation medium expressed as percentage change compared to control (medium only) incubation. Vertical bars represent SEM. Hormone release significantly different from medium 199 control. (+) P < 0.01; from 3.0 chicken HE, (0)P < 0.05; (*) P < 0.01.
Terrapin pituitaries were incubated for 5 hr either alone or with homologous HE, rat HE, chicken HE, or eel HE, all equivalent to one hypothalamus per pituitary. Figure 5 shows that small but detectable amounts of prolactin were released without any HE and that the addition of homologous HE stimulated both synthesis and release. Chicken HE had a powerful stimulatory effect, as did eel HE, but rat HE had no stimulatory effect. Figure 6 shows the results of similar experiments on Xenopus. After 5 hr incubation at 37” both prolactin and GH were stimulated by homologous HE, chicken HE, and terrapin HE. Rat HE appeared to inhibit prolactin release but stimulated GH release. Possible inhibitory influence of rat HE on prolactin stimulation by homologous HE could not be tested in the terrapin and the toad due to shortage of animals. DISCUSSION
The prolactin and GH bands visible after PAGE have been identified in numerous vertebrates by both bioassay and RIA. Nicoll and Nichols ( 197 1) and Nicoll and Licht (1971) identified prolactin and GH
338
HALL
AND TABLE
EFFECT
CHADWICK 5
OF COD HE ON PROLACTIN AND CHICKEN PITUITARIES INCUBATED
GH
SECRETION IN VITRO
FROM
Prolactin (duimg)
Growth
Treatment
Medium
Control (no HE) Chicken HE ( = 3 .O hypothalami) Chicken HE + cod HE (0.03 hypothalami) Chicken HE + cod HE (0.3 hypothalami) Chicken HE + cod HE (3.0 hypothalami)
27 2 4
56 f
62 2 3":
Total”
hormone (duimg)
Medium
Total R
66 2 4
141 2 13
98 k 8"
88 t- 4"
179 2 8#:
61 t 3”
95 2 6*
85 k 3"
171 + 6':
50 + 2*
gg + 4°C
76 + 4
150 + 5
31+3
62 + 3*
51 + 3
130 2 5
10
* Significantly different from control P < 0.05; n = 6 in each case. ” Total = hormone in medium plus hormone in pituitary at end of incubation.
bands in pituitary electrophoretograms in a wide variety of tetrapods. In the chicken the prolactin band identification has been confirmed by RIA (Scanes et cd., 1976a). Bolton ef al. (1976) showed that in chicken pituitary incubations there was a linear relationship between prolactin concentrations measured by PAGE and densitometry and
Values
represent
mean t SEM.
by radioimmunoassay. Knight et czf. (1970) and Hall and Chadwick (1978) identified the prolactin band in eel electrophoretograms, and Ingleton and Stribley (1975), using an immunoassay technique, demonstrated the position of the GH bands in eel pituitary electrophoretograms. The mammalian hypothalamus contains
FIG. 4. Effect of flounder HE on prolactin and GH release from rfitro. Histograms show prolactin (open) and GH (cross-hatched) in expressed as percentage change compared to control (medium only) sent SEM. Hormone release significantly different from medium 199 ,O.Ol; from 3.0 chicken HE, (*) P < 0.01.
chicken pituitaries incubated in the incubation media. Results incubation. Vertical bars reprecontrol: (0) P < 0.05; (+) P
