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Changes in plasma thyroid hormones following administration of exogenous pituitary hormones and steroid hormones to rainbow trout (Salmo gairdneri)
0300-9629,80;0801-0679102.00:0
CHANGES IN PLASMA THYROID HORMONES FOLLOWING ADMINISTRATION OF EXOGENOUS PITUITARY HORMONES AND STEROID HORMONES TO RAINBOW TROUT (SALMO GAIRDNERI) ROBERTS. MILNEand JOHN F. LEATHERLAND Department of Zoology, College of Biological Science, University of Guelph, Guelph, Ontario NIG 2W1, Canada (Received 5 November 19791 Abstract-l. Porcine FSH and ovine LH (10 1g.g body weight) elicited significant increases (P c: 0.01) in plasma T4 levels within 24 hr of the single injection compared with saline-injected controls whereas HCG was without effect; plasma T3 levels did not change. 2. Ovine TSH and prolactin when given together significantly elevated (P < 0.01) plasma T3 levels 24 hr after the injection compared with all other groups (662.0 + 47.8 ng’dl-’ compared with 133.3 & 30.8, 165.4 + 30.7 and 154.7 ng’dl- ’ in fish given saline, prolactin alone and TSH alone respectively). 3. TSH, given alone or together with prolactin significantly elevated (P < 0.01) plasma T4 levels 8 hr after administration. 4. Single injections, or a series of seven daily injections of /I-estradiol-3-17, dipropionate, 17-a-methyl testosterone or cortisol were without effect on either thyroid appearance or plasma thyroid hormone levels. 5. But after seven daily injections of cortisol, plasma T4 levels were significantly higher (P -C 0.01) and the T3/T4 ratio significantly lower (P i 0.01) than in the sesame oil-injected control fish.
INTRODUCTION Several studies have shown an apparent increase in thyroid activity in some salmonid fishes during the early stages of their anadromous migration, and others an apparent increase in thyroid activity associated with gonadal maturation (see review by Sonstegard & Leatherland, 1976). At these times in their life cycle the fish are known to have elevated steroid hormone levels (Fagerlund & Donaldson, 1970; Idler & Truscott, 1972) presumably in response to ACTH and gonadotropin stimulation. Also, anadromous species may well also be changing the secretory rates of other hormones such as prolactin (for purposes of freshwater osmo-(inono)regulation (Johnson, 1973)) and growth hormone (for mobilization of stored food reserves) (Higgs et al., 1975; Kayes, 1977). There is evidence to suggest that growth hormone and gonadotropins, like TSH, have thyrotropic potency in some fish (Fontaine, 1969; Donaldson & McBride, 1974; Chan & Eales, 1976; Grau & Stetson. 1977a;b; Milne & Leatherland, 1978). In addition, prolactin appears to inhibit the thyrotropic effect of TSH and lower plasma T4 levels in Fundulus heteroclitus (Grau & Stetson, 1977a), although prolactin was without affect on plasma T4 levels in Salmo gairdneri (Mime & Leatherland, 1978). Still other studies suggest that steroid hormones have a stimulatory effect on thyroid activity in some teleosts as evidenced by histological changes (Singh, 1969; Sage & Bromage, 1970; Van Overbeeke & McBride, 1971). It is thus apparent that the complex endocrine changes associated with anadromous migration and sexual maturation of salmonids may influence thyroid 679
function. The aim of this study was to evaluate the thyrotropic effects of pituitary hormones other than TSH, and of some steroid hormones in the hope of furthering the understanding of endocrine interaction in teleosts. Commercial preparations of teleost pituitary hormones are currently not available and mammalian hormone preparations were employed. Although these are undoubtedly structurally different from the teleostean hormones they appear to share some of the physiological functions of the teleostean hormones (Ahsan & Hoar, 1963; Sundararaj & Anaud, 1972; Sundararaj & Goswami, 1974; Higgs et al., 1975; Hirose, 1976; Clarke et al., 1977).
MATERIALSAND
METHODS
Source and maintenance of fish
Rainbow trout (Salmo gairdneri) (60-12Og body weight) of both sexes were obtained from Goossen’s Hatchery, Otterville, Ontario, Canada and maintained in 4001 stock aquaria in continuously running and aerated well water (hardness 316mg.kg; pH 8.1) at 11 + 1°C under a 12 hr light: 12 hr dark photoperiod for at least 28 days prior to their use in experiments. They were fed four or five times a week with a commercial pelleted trout diet (Martin’s Feed Mills, Elmira). Fish were transferred from the stock aquaria into 1001. experimental aquaria, maintained in conditions identical to those of the stock tank 7 days before the start of the experiment; the fish were last fed 24 hr before the first injection. Hormone preparations
All pituitary hormone preparations were dissolved in 0.9% NaCl (saline) and given as intraperitoneal injections
ROBERT S. MILNE and JOHN F. LEATHERLAND
680
in 0.2 ml; steroid hormones were suspended in 0.2 ml of sesame oil. The doses of hormones used (expressed as per gram body weight) were as follows: human chorionic gonadotropin (HCG) (Sigma Chemical Co.), 10 pg; ovine luteinizing hormone (LH) (NIH-LH-Sl9), IOpg; porcine follicle stimulating hormone (FSH) (NIH-FSH-PI), IOpg; ovine thyrotropin (TSH) (NIH-TSH-S8), 2 pg; ovine prolactin (either NIH-P-S10 or Ferring, Malmo, batch No. BI 1108) 1Opg; synthetic ACTH (Ferring, Malmo, batch No. YK5527), 1OOmIU; /I estradiol-3-17 dipropionate, 0.05 mg; 17-a-methyl testosterone, 0.05 mg; cortisol, 0.05 mg. ~~peri~ie~tu~ procedures ~~~er~rne~~rJa. Four groups of fish were given a single injection of LH, FSH, HCG or saline alone at 08.30 hr and samples of six or seven fish from each group were killed 3, 6, 9 or 24 hr after the injection, Experiment lb. Two groups of fish were given a single injection of synthetic ACTH or saline alone at 08.30 hr and samples of five fish from each group were killed 1, 3, 6, 9 or 24 hr after the injections. Experimenr 2~. The procedure was essentially similar to that used in experiment 1 except that fish were given prolactin alone, TSH alone, a mixture of TSH and prolactin (NIH) or saline alone at 08.30 hr and samples of six lish from each group were killed 8 or 24 hr after the injection. Experiment 2b. The procedure was identical to that of experiment I and 2a. Groups of five fish were given a single injection of either prolactin (Ferring) and TSH or saline at 08.30 hr and killed 24 hr later. Experiment 3~. Two groups of 20 fish were given a single injection of fi estradiol-3-17-dipropionate in sesame oil or sesame oil alone. Samples of five fish from each group were killed 3, 6, 9 or 24 hr after the injections. Two further groups of six fish were given seven daily injections of either estradiol or sesame oil alone and killed 18 hr after the last injection.
4
14 2
Exprrimenr 3b. Three groups of 20 fish were given a single injection of 17-z-methyl testosterone or cortisol in sesame oil or sesame oil alone. Samples of five fish were killed 3, 6, 9 or 24 hr after the injection. Three further groups of IO fish were given seven daily injections of either testosterone or cortisol in sesame oil or sesame oil alone and killed 18 hr after the last injection, Blood sampling nnd hormone assay
Blood samples were taken from each fish by caudal severance and the blood collected in heparinized Vacutamer tubes. The plasma was separated by ~ntrifugation and stored at -25°C until hormone assays were made (in all cases within 7 days of collection). Plasma T4 and T3 levels were measured using the Ames Tetralute” and Seralute@ methods as previously described (Milne & Leatherland, 1978). Histology
The lower jaw was removed from each fish, fixed in Bouin’s fluid, decalcified and embedded in Paraplast. Seven micrometer-thick sections were stained with hematoxylin and eosin. Measurements of epithelial cell height were made using an ocular micrometer; measurements were made at four locations of each of seven follicles chosen at random, at the four points crossed by the micrometer scale in the horizontal and vertical planes in the field of view. A mean of the 28 measurements from each fish was used as the epithelial cell measurement. In addition the degree of vesiculation of the colloid was estimated in the same seven follicles. An arbitrary 0 to 10 scale was used, 0 representing no vesiculation and 10 a condition where SOo/,of the colloid was occupied by vesicles. The mean value for the seven follicles was used as the ‘*thyroid droplet index”. Statisfical analysis Data of plasma T4 and T3 concentration, plasma T4/r3 ratio, thyroid epithelial cell height, and thyroid droplet index from each experiment were separately compared by one-way analysis of variance. Where F values indicated significant differences (P < 0.05) between experimental groups, individual means were compared by StudentNewman-Kneuls test.
306
RESULTS
T3
Effect ofLH,
0:
not significantly different from the saline-injected animals at any time period. T4 levels in FSH-injected
0
7 '
3 1
31 2
TDI ,
0
and HCG
The data from experiment la are summarized in Fig. 1. Plasma T4 levels in HCG-injected fish were
E 0.2
TEH
FSH
3
6
9
24
lwws afterin~ecfton
Fig. 1.Temporal effect of a single injection of follicle stimulating hormone (FSH), luteinizing hormone (LH), or human chorionic gonadotropin (HCG) on plasma thyroxine (T4) and triiodothyronine (TJ concentration, plasma T3/r4 ratio, thyroid epithelial cell height and thyroid droplet index in rainbow trout, Salmo gairdneri. Plasma T1 and Ts concentrations are expressed as pg’dl and ng.dl respectively, and epithelial cell height as pm. n = 6 unless otherwise indicated.
trout were signi~cantiy higher than corresponding saline-injected fish at 9 hr and 24 hr after the injection (P < 0.05 and P < 0.01 respectively); T4 levels were significantly (P < 0.01) higher in LH-injected ftsh than the controls 24 hr after the injection. There were no significant differences between plasma T3 levels in hormone-injected and salineinjected fish at any time period. The plasma T3/I’4 ratio in HCG-injected fish was significantly (P < 0.01) lower than the saline-injected animals 3 hr after the injection. There were no significant differences between thyroid epithelial cell height or thyroid droplet index in hormone-injected and saline-injected fish at any time period. Effect of ACTH
The data from experiment lb are summarized in Fig. 2. Plasma T4 levels in ACTH-injected fish were
Plasma
thyroid
hormones
in S&W guirdneri
681
salme ACTH Oli
estradlol
7
6 TEH
4 2
TEH
2
2 TDI
1 0
1
3 6 9 hours afterqectton
TO1
Fig. 2. Temporal effect of a single injection of corticotropin (ACYTH) or /J’ estradiol-3-17-dipropionate on plasma thyroxine (T4) and triiodothyronine (T,) concentration, plasma T,/T, ratio. thyroid epithelial cell height and thyroid droplet index in rainbow trout, Salrno gairdneri. Plasma T, and T, concentrations are expressed as ug’dl and ng’dl respectively. and epithelial cell height as pm. II = 5 unless otherwise indicated. significantly lower (P < 0.05) than in saline-injected fish 9 hr after injection. There were no significant differences between plasma T3 levels, plasma T3/r4 ratios, thyroid epithelial cell height or thyroid droplet index in ACTH-injected and saline-injected fish at any time period.
Effect of TSH and prolactin The data from experiment 2a are summarized in Table 1. Plasma T4 levels in TSH-injected trout were significantly (P < 0.01) higher than in comparable saline-injected fish 8 hr after the injection but were not significantly different after 24 hr. T4 levels in prolactin-injected animals were not significantly different from the saline-injected fish at either time period. In fish given TSH and prolactin together T4 levels were significantly (P < 0.01) higher than in saline-injected animals at both 8 hr and 24 hr after the injection. Plasma T3 levels in TSH- or prolactin-injected fish were not significantly different from saline-injected fish 8 hr and 24 hr after the injection. However, T3 levels in fish given TSH and prolactin were significantly (P < 0.01) higher than comparable salineinjected animals 24 hr after the injection, The T3/T4 ratios in hormone-injected fish were not sigmficantly different from the saline-injected animals at either time period. In the repeat experiment (experiment 2a), using an ovine
prolactin
6 I,
preparation
from
Ferring
(Malmo,
Sweden) instead of the NIH preparation, the results were essentially similar. Plasma T4 and T3 levels in fish injected with TSH and prolactin were significantly higher (P < 0.05 and P < 0.01 respectively) than in the saline-injected animals 24 hr after the injection, whereas there was no significant difference between T3,T4 ratios in the two groups.
2
,
0
3
6 9 24 hours afterqectlon
Fig. 3. Temporal effect of a single injection of cortisol or 17-methyl testosterone on plasma thyroxine (T4) and triiodothyronine (T3) concentration, plasma TJT, ratio, thyroid epithelial cell height and thyroid droplet index in rainbow trout, Salmo gairdneri. Plasma T, and T3 concentration are expressed as pcg’dl and ng’dl respectively, and epithehal cell height as pm. n = 5 unless otherwise indicated.
Effect of steroid hormones The data from experiments 3a and 3b are summarized in Figs 2 and 3 and Tables 2 and 3. In fish given a single injection or seven daily injections there was no significant differences between estradiol- and sesame oil-injected fish for plasma T4 and T3 concentration, plasma T3/r4 ratios, thyroid epithelial cell height or thyroid droplet index (Fig. 2 and Table 2). There were no significant differences in plasma T4 and T3 concentration, plasma T3/T4 ratio, thyroid epithelial cell height or thyroid droplet index between sesame oil-injected fish and testosteroneor cortisolinjected fish at any time after a single injection (Fig. 3). In fish given seven daily injections of cortisol the plasma T4 concentration was significantly higher (P < 0.01) and the T3/T4 ratio significantly lower (P < 0.01) than in sesame oil-injected animals (Table 3). Plasma 7’4 concentrations and T3/T4 ratios in testosterone-injected fish were not significantly different from the sesame oil-injected animals. There were no significant differences between any of the three groups of fish for plasma T3 concentration, thyroid epithelial cell height or thyroid droplet index (Table 3).
DISCUSSION
The data shown in Fig. 1 suggest that porcine FSH and ovine LH have thyrotropic activity in rainbow trout as evidenced by the significant increases in
ROBERT S. MILNE and JOHN F. LEATHERLAND
682
Table I. Effect of a single injection of ovine prolactin. ovine thyrotropin (TSH), or prolactin + TSH on plasma thyrowme (T4) and triiodothyronine (T3) concentrations and T3,TJ ratios in rainbow trout, SUIWI guirdwri
Treatment
Hours after injection
Hormone
R
24
-__ saline I
T4'
prolactina
T4
TSH
T4
TSH + prolactina
T4
0.88 +
o.093
0.70 _ +
0.07
0.74 +
0.07
0.52 +
0.06
1.91 +
0.30**
1.32 + _
0.33
1.97 +
0.13""
1.62 +
_~._____ saline II
T
TSH + prolactinb
4
0.25 ~_-..___
1.11 _ +
0.09
1.79 +
0.29*
T4
______ slille I
,T3)
prolactina
T3
TSH
T
TSH + prolactina
T
saline II
3 3
1!0.9 _ +
16.6
133.3
+
30.8
145.8 +
33.7
165.4
+
30.7
237.9 + 118.4
154.7
t _
37.5
280.0 +
662.0
+
47.8**
236.1
t
22.6
558.6
+ 108.4""
42.3
T3
TSH + prolactina ______~~~__ saline I
'T3 ._~___._~.__..___..~ T3/T4
prolactina
T3/T4
TSH
T3'T4
TSH t prolactinb
T3/T4
0.13 +
0.01
0.18 +
0.01
0.20 +
0.01
0.21 + _
0.02
0.08 +
0.05
0.16 t
0.01
0.13 +
0.01
0.40 _ t
0.01
0.20 _ +
0.01
0.27 _ +
0.01
--___ saline II
T3'T4
TSH t pmlactinb
T /T 3 4 ____~___~________---_-_----__~._._-.~____~____~__~_~
1Expressed as pg. dl. * Expressed as ng. dl. 3 Mean i SEM; * prolactin preparation NIH-P-SIO: h prolactin preparation Ferring MalmG, batch No. Bl I IOX. Saline I is the control group for the TSH, prolactin” and TSH + prolactin” groups (II = 6). saline 11 is the control group for the TSH + prolactinb group (0 = 5). *, **, Significantly different (P < 0.05 and P < 0.01 respectively) from comparable saline-injected group. T4 9 and 24 hr after the injection. Grau & Stetson (1977b) reported a similar thyrotropic potency of LH and FSH in F. hrteroclitus. Fontaine (1969) showed that both purified LH and FSH stimulated radioiodide uptake by the thyroid of hypophysectomized Angltilla anguilla and Ahsan & Hoar (1963) showed that several mammalian gonadotropin preparations, including LH and FSH. increased thyroid epithelium cell height in Gasterostrus uculeat~~s leiurus; a similar significant (P <: 0.01) increase in thyroid epithelial cell height was found after LH treatment of G. aculeatus trachurus (Lam 8~ Leatherland, unpublished data). Donaidson & McBride (i974) showed that salmon gonadotropin increased thyroid plasma
in gonadectomized 0. rwrka. There was no significant effect of HCG on plasma T4 or T3 levels in S. gairdneri in the conditions and doses used here although the significantly (P < 0.01) decreased T3/T4 ratio in HCG-treated fish killed 3 hr after the injection suggests a temporary lowering of T3 without concomitant change in T4 levels. Grau & Steston (1977b) report an elevation in T4 levels in HCGtreated F. heteroclitus and Ahsan & Hoar (1963) indicate that HCG appeared to stimulate thyroid activity in G. uculrutus Ieiurus. The differences between the findings of Grau & Stetson (1977b), Ahsan & Hoar (1963) and those reported here possibly represent species differences but equally may be due to the fact activity
Plasma thyroid hormones in Sulmo yairdneri
683
Table 2. Effect of seven daily injections of estradiol dipropionate on plasma thyroxine (T4) and triiodothyronine (T3) concentrations, T,/T, ratios and histological appearance of the thyroid in rainbow trout, Salmo gairdneri
Treatment
Sesame Oil
T4'
1.05 + o.155
E&radio1
1.11 _ +
(6)"
T32
187.9
+ 5.1 (6)
T3'T4
0.19 + 0.02 (6)
TD13
1.20 _ + 0.21 (4)
TEH'
3.44 f 0.54 (4)
0.08
(6)
207.2
+ 46.2 _ (5)
0.18 +
0.02
(5)
0.90 _ +
0.20
(6)
4.32 _ +
0.35
(6)
'Expressed as pg.dl. * Expressed as ng.dl. 3 Thyroid droplet index. 4 Thyroid epithelial cell height in micrometers. ’ Mean k SEM. 6 Number of samples.
that single hormone injections were used in these experiments whereas multiple injections were used by other authors. The thyrotropic effects of HCG in mammals is well documented (Nisula & Ketelsleger, 1974; Nisula et al., 1974; Amir et al., 1977), as is the permissive role of thyroid hormones in LH function (Aranda et al., 1976). It is possible that some of the thyrotropic effect of the gonadotropin preparations is caused by contamination of the preparations by TSH. However, since the thyrotropic potency of LH and FSH, as evidenced by the elevated plasma T4 levels 24 hr after the injection (Fig. 1) was comparable to that of TSH alone (Table l), either the contamination is considerable, or at least some of the thyrotropic potency of the LH and FSH preparations is not TSH-dependent. The reported TSH contamination of the LH and FSH preparations is 0.11 and 0.036 USP units.mg respectively. whereas the reported potency of the TSH preparation is 1.70 USP units’mg. Thus the LH- and FSH-injected fish received 0.0011 and 0.0036 USP units of TSH.g body weight respectively compared with 0.0034 USP units of TSH.g body weight given to TSH-injected animals. In earlier experiments (Milne & Leatherland, 19783, TSH doses of 1.1 pg.g body weight were found to
elevate plasma T4 levels after 24 hr. Thus, TSH doses of 0.0017 USP units.mg were above the minimal level required for stimulating thyroid activity and this may account for the effect of LH, but it is unlikely to explain the response to FSH. The TSH estimations were obtained using a thyroidal 32P-uptake procedure and the method does not differentiate between TSH contaminants per se and thyrotropic activity of LH and FSH. Nevertheless in light of the low thyrotropic “contamination” particularly of the FSH preparation, the TCstimulating effects of these hormones is probably due at least in part, to the thyrotropic activity of the gonadotropin preparation and not simply to contamination with TSH, although synergism between the gonadotropin and traces of TSH may have been involved. Thyrotropic potency of gonadotropins is not surprising in light of the similar chemical composition of gonadotropin and thyrotropin hormones and their probable parallel evolution (Fontaine & Burzawa-Gerard, 1977). The findings suggest that at least some of the increased thyroid activity found in sexually maturing and mature salmonids may be due to an increase in gonadotropin secretion and/or an increased secretion of growth hormone, associated with the mobilization of lipids; growth hormone has been shown to have a
ROBERT S. MILNE and JOHN F. LEATHERLAND
684
Table 3. Effect of seven daily injections of either 17-r-methyl testosterone or cortisol on plasma thyroxine (T4) and triiodothyronine (T3) concentrations, TX/T4 ratios and histological appearance of the thyroid in rainbow trout. S&IO grtirrlwri
Treatment
Sesame
Testosterone
Cortisol
oil
I_-_____ T4’
0.90
+
0.04:
+ 1.60 _
229.8
+ 20.9
162.7
T3’T4
0.26
_+
0.04
+ 0.10 _
(10)
TD13
0.65
_+
0.24
0.53 +
3.51 +
4.35 + _
(8)
0.04
(10)
1.50 _ +
0.20
0.46
(6)
3.15 +
0.73
0.39
(6)
(6)
-
_-
-
0.18 +
0.01""
(6)
0.18
+ 30.6 (10)
(10)
(8)
TEH'
199.8
+ 17.3 (10)
(10)
0.21
(10)
(10)
(lo)6
Tj2
1.25 +
0.18**
-_
-
’ Expressed
as pg. dl. ’ Expressed as ng’ dl. 3 Thyroid droplet index. 4 Thyroid epithelial cell height in micrometers 5 Mean f SEM. b Number of samples. ** Significantly different from sesame oil-injected
marked thyrotropic effect in trout (Milne & Leatherland, 1978). TSH, given either alone or together with prolactin, stimulates T4 secretion (Table 1). Conversely, a single injection of prolactin has no significant effect on plasma thyroid levels nor does it inhibit the TCstimulating activity of TSH. These findings confirm the observations in S. gairdneri using TSH and prolactin alone (Milne & Leatherland, 1978) but contradict those reported in F. heteroclitus (Grau & Stetson, 1977a). The latter showed that prolactin lowered plasma T4 levels and inhibited the T4-stimulating effect of TSH. In S. gairdneri not only was there no inhibition of the thyrotropic potency of TSH by prolactin, but also plasma T3 levels were significantly elevated 24 hr after the injection (662.0 + 47.8 ,ug/ 100 ml in fish given prolactin and TSH as compared with 154.7 k 37.4 pg/lOO ml in fish given TSH alone). A similar potentiation of T3 levels were found in preliminary experiments in which S. gairdneri were given a single injection of bovine growth hormone (which also has thyrotropic potency (Milne & Leatherland, 1978) alone or combined with prolactin (66.2 k 12.4 pg of T3’dl in saline-injected fish; 145.2 f 29.0 Pg of T3’dl in growth hormone-injected fish; 257.5 t_ 58.2 pg of T3’dl in ovine prolactin + growth hormone-injected fish) (unpublished data).
group
(P < 0.01).
The response to prolactin is known to vary depending on the time of injection of exogenous hormone (Leatherland & Holub, 1979). Thus the different observations of Grau & Stetson (1977a) and those reported here may be caused by circadian variation in the nature of receptors. The reason for the T3 elevation in prolactin + TSH-injected trout is not known. There is good evidence to suggest that, in mammals, most circulating T3 is derived from the peripheral monodeiodination of T4 (Bernal & Refetoff, 1977; Fisher et al., 1972). Similar T4 deiodination appears to occur in salmonids (Higgs & Eales. 1977; Milne & Leatherland, 1978). Thus the increase in T3 levels in TSH + prolactin-injected fish may be caused by a potentiation of the T4 to T3 conversion. In a similar manner, the apparent lowering of T3 in HCG-injected fish may be caused by inhibition of the T4 to T3 conversion. Conversely, the changes in T3 may be caused by alterations in the peripheral metabolism of T3. Some of the temporal changes in T3 found after injections of gonadotropin preparations may be explained in a similar manner. Single, rather than multiple injections of pituitary hormone preparations were used in these experiments in order to overcome the problems of reduced thyroid response noted in multiple injection situations (Chan
Plasma thyroid hormones in S&no gairdneri & Eales,
1976; Milne & Leatherland, 1978). An advantage of the single injection procedure is that responses found within the 24 hr following the injection are more likely to be the result of direct action on the thyroid rather than indirect actions mediated via other endocrine glands. The absence of an apparent change in thyroid activity (as evidenced by changes in plasma thyroid hormone levels or changed in thyroid histology) after estradiol or testosterone treatment is surprising in light of the reports of gonadal steroids stimulating thyroid activity in teleosts (Singh, 1969; Sage & Bromage, 1970; Van Overbeeke 8~ McBride, 1971) and mammals (e.g. Gross et al., 1971; Hunt & Eales, 1979). Singh (1969) and Sage & Bromage (1970) using Mystus vittatus and Poeciiia reticulata respectively showed an apparent stimulation of thyroid activity after treatment with estrogen or testosterone using in vitro culture methodology and radioiodide-uptake rates. Van Overbeeke & McBride (1971) reported an apparent activation of the thyroid in gonadectomized male sockeye salmon treated with 1I-ketotestosterone or 17-z-methyl-testosterone; estradiol or estradiol cypionate were without effect in gonadectomized female sockeye salmon. Hunt & Eales (1979) showed an increased T4 degradation and monodeiodination rate accompanied by elevated plasma T3 and T4 levels in rainbow trout injected with testosterone propionate. The difference between the findings reported here and those of Hunt & Eales (1979) cannot be explained. Hunt & Eales (1979) reported that the responses of plasma T4 levels to testosterone was inconsistent; the differences in the two experimental series mav be due to this inconsistency. eortisol elevated plasma T4 levels in fish given seven daily injections of the steroid whereas a single ACTH-injection tended to decrease plasma T4 levels. In both cases there was no concomitant change in the histological appearance of the thyroid in hormoneinjected fish compared with controls. Thus, although there is evidence for a thyrotropic role of cortisol in trout, supporting the observations of an increased radloiodide uptake by the thyroid of M. vittatus treated with cortisone (Singh, 1969), some increase in epithelial cell height might have been expected if the increase in plasma hormone represented an elevated secretion of hormone. An alternate explanation may be that the increased plasma hormone level is a compensatory response to changes in the binding capacity of the blood proteins. Conversely, the elevated T4 levels in cortisol-treated trout may be due to a depressed plasma ACTH titer in these fish. Olivereau (1968a;b) showed a clear correlation between the pituitary-thyroid and pituitary-interrenal axes in eels. Radlothyroidectomy induced a histological picture of reduced activity of both pituitary corticotrop cells and interrenal cells while T, treatment causes an apparent stimulation of the pituitary-interrenal axis. However, Van Overbeeke & McBride (1971) reported a reduction in thyroid epithelial cell height after cortisol administration to gonadectomized sockeye salmon. This study, like many similar ones, was limited by the fact that mammalian pituitary hormones were used, since comparable hormones of teleostean origin are not readily available. In addition, it is difficult to
685
evaluate the physiological relevance of the dosages of hormones used although they all were within levels used by other authors (Ahsan & Hoar, 1963; Hawkins & Ball, 1970; Sundararaj & Anaud, 1972; McLeay, 1973; Sundararaj & Goswami, 1974; Higgs et al., 1975; Sundararaj & Keshavanath, 1976; Hirose, 1976; Clarke et al., 1977). Nevertheless, the study provides
evidence that pituitary factors other than TSH and also cortisol can influence thyroid activity in salmonid fishes and may account for some of the changes reported in salmonid thyroid activity during migration, when hormones such as growth hormones and prolactin are secreted in response to changed metabolic and osmotic requirements. Although no effects of sex steroids on thyroid activity were evident in these studies it is possible that under different conditions of temperature, these hormones may exert effects on thyroid gland function in some teleostean species. Acknowledgements-We wish to thank Lucy Lin and Kim Counsel1 for their excellent technical assistance. The hormone preparations were donated by the National Institute of Health, Bethesda, Maryland, U.S.A. The work was supported by a grant-in-aid of research from the Natural Science and Engineering Research Council of Canada to J.F.L.
REFERENCES
AHSANS. N. & HOARW. S. (1963) Some effects of gonadotropic hormones on the threespine stickleback, Gasterosteus aculeatus. Can. J. 2001. 41, 10451053. AMIR S. M., UCHIMURA H. & INGBAR S. H. (1977) Interactions of bovine thyrotropin and preparation of human gonadotropin with bovine thyroid membranes. J. clin. Endocr. Metah. 45, 280-292.
ARANDAA., HERVASF., MORREALEDE ESCOBARG. & ESCOBARDEL RAY F. (1976) Effects of small doses of L-thyroxine and triiodo-thyronine on pituitary LH content of thyroidectomized rats. Acta Endocr. 83, 72&736. BERNALJ. & REFETOFF S. (1977) The action of thyroid hormones. C/in. Endocr. 6, 227-249. CHAN H. H. & EALESJ. G. (1976) Influence of bovine TSH on plasma thyroxine levels and thyroid function in brook trout, Sahelinus fontinalis (Mitchill). Gen. camp. Endocr. 28, 461-472.
CLARKEW. C., FARMERS. W. & HARTWELLK. M. (1977) EtTect of teleost pituitary growth hormone on growth of Tilapia mossambica and on growth and sea-water adaptation of sockeye salmon (Oncorhynchus nerka). Gen. camp. Endocr. 33, 174-I 78.
DONALDSON E. M. & MCBRIDEJ. R. (1974) Effect of ACTH and salmon gonadotropin on interrenal and thyroid activity of gonadectomized adult sockeye salmon (Oncorhynchus nerka). J.jish. Res. Bd. Can. 31, 1211P1214. FAGERLUNDU. H. M. & DONALDSONE. M. (1970) Dynamics of cortisone secretion in sockeye salmon (Oncorhynchus nerka) during sexual maturation and after gonadectomy. J.fish. Rex Bd. Can. 27, 2323-2331. FISHER D. A., CHOPRA I. J. & DUSSAULTJ. H. (1972) Extrathyroidal conversion of thyroxine to triiodothyronine in sheep. Endyrinology 91, 1141-l 144. FONTAINEY. A. (1969) Studies on the heterothyrotropic activity of preparations of mammalian gonadotiopins in teleost fish. Gen. camp. Endocr. 2, 417424. FONTAINEY. A. & BUR~AWA-GERARD E. (1977) Esquisse de l’evolution des hormones gonadotropes et thyrCotropes des vCrt&bres. Gen. camp. Endocr. 32. 341-347.
ROBERT S.
686
MILNEand JOHN F. LEATHERLAND
CRAW
E. G. & STETSON M. H. (1977a) The effects of prolactin and TSH on thyroid function in Fundulus hrtero-
clitus. Gen. camp. Endocr.
33, 329-335.
GRAU E. G. & STESTONJ. H. (1977b) Thyroidal responses to exogenous mammalian hormones in Fundufus heteroclitus. GROSS
Am. Zool.
17. 857.
MILNER. S. & LEATHERLAND J. F. (1978) Effect of ovine TSH. thiourea, ovine prolactin and bovine growth hormone on plasma thyroxine and tri-iodothyronine levels in rainbow trout, Salmo gairdneri. J. camp. Physiol. 124, 105-l 10. NISULA B. C. & KETELSLEGERS J. M. (1974) Thyroidstimulating activity and chorionic gonadotropin. J. c/in.
H. A., APPLEMANM. D. & NICOLOFFJ. T. (1971) Invest. 54, 49&501_ Effect of biologically active steroids on thyroid function NISULA B. C., MORGAN F. J. & CANFIELDR. E. (1974) in man. J. clin. Endocr. 33, 242-248. Evidence that chorionic gonadotropin has intrinsic thyrHAWKINSE. F. & BALLJ. N. (1970) Physiological studies otropic activity. Biochem. hiophys. Res. Commun. 59, on the hypothalam@hypophysial-interrenal axis in Poe86-90. cilia [atipinna (Teleostei). J. Endocr. 48, xxvii-xxviii. HIGGS D. A., DONALDSON E. M., DYE H. M. & MCBRIDE OLIVEREAU M. (1968a) RCsponse hypophysocorticosurrenalienne de I’anguille a un apport de thyroxine. J. R. (1975) A preliminary investigation of the effect of Arch. Anclt. Hist. Emb: Norm. Exp. 5,.510-516. bovine growth hormone on growth and muscle compoOLIVEREAIJM. (1968b) Modifications des ccllules corticosition of coho salmon (Oncorhynchus kisutch). Gen. camp. tropes de l’h‘ypopiyse chez l’anguille radiothyroidecEndocr. 27, 24g-253. tomiske. Z. Zellforsch. Mikros. Anat. 90, 289-295. HIGGS D. A. & EALESJ. G. (1977) Influence of food depriN. R. (1970) Interactions of the TSH vation or radioiodothyronine and radioiodide kinetics in SAGE M. & BROMAGE and thyroid cells with the gonadotropic cells and gonads yearling brook trout, Salaelinusfontinalis (Mitchill), with in Poecilid fishes. Gen. camp. Ettdocr. 14, 137-140. a consideration of the extent of L-thyroxine conversion SINCH T. P. (1969) Observations on the effect of gonadal to 3,5,3’-triiodo-L-thyronine. Gen. camp. Endocr. 32, and adrenal cortical steroids upon thyroid gland in 29-40. hypophysectomized catfish, Mrstus virtatus (Bloch). Get!. HIROSE K. (1976) Endocrine control of ovulation in camp. Endocr. 12, 556-560. medaka (Oryzias latipes) and ayu (Plecoglossus altivelis). SONSTEGARI) R. A. 81 LEATHERLAND J. F. (1976) The epiJ. fish. Res. Bd. Can. 33, 989-994. zootiology and pathogenesis of thyroid hyperplasia in HUNT D. W. C. & EALESJ. G. (1979) The influence of coho salmon (Oncorhpnchus kisutch) in Lake Ontario. testosterone propionate on thyroid function of immature Cancer Res. 36, 4467-4475. rainbow trouf Salmo gairdneri Richardson. Gen. camp. SUNDARARAJ B. I. & ANAUUT. C. (1972) Effects of piscine Endocr. 37, 115-121. and mammalian gonadotropins on gametogenesis in the IDLERD. R. & TRUSCOTTB. (1972) Corticosteroids in fish. catfish, Heteroprleustes ,fossilis (Bloch). Gen. camp. In Steroids in Non-mammalian Vertebrates (Edited by IDLERD. R.). Academic Press, New York. Endocr. Suppf. 3, 688-702. SUNDARARAJ B. I. & GOSWAMIS. V. (1974) Effects of ovine JOHNSOND. W. (1973) Endocrine control of hydromineral balance in teleosts. Am. Zool. 13, 799-818. luteinizing hormone and porcine adrenocorticotropin on maturation of oocytes of the catfish, Heteropneustes fosKAYEST. (1977) ElTects of temperature on hypophyseal silis (Bloch), in ovary-interrenal co-culture. Gen. camp. (growth hormone) regulation of length, weight and alloEndocr. 23, 276-281. metric growth and total lipid and water concentrations in the black bullhead (Ictalurus melas). Gen. camp. SUNDARARAJ, B. I. & KESHAVANATH, P. (1976) Effects of Endocr. 33, 382-393. melatonin and prolactin treatment on the hypophysialovarian system in the catfish, Heteropneustes ,fossilis LEATHERLAND J. F. & HOLUBB. J. (1979) Circadian effects (Bloch). Gen. camp. Endocr. 29, 8496. of prolactin on lipid metabolism and hydromineral reguVAN OVERBEEKE A. P. & MCBRIDEJ. R. (1971) Histological lation in intact and hypophysectomized goldfish, Carassius auratus L. J. interdisc. Cycle Res. 9, 12s-136. effects of 1 I-ketotestosterone. I7-r-methyltestosterone, estradiol, estradiol cypionate, and cortisol on the interMCLEAY D. J. (1973) Effects of ACTH on the pituitaryrenal tissue, thyroid gland, and pituitary gland of gonainterrenal axis and abundance of white blood cell types dectomized sockeye salmon (Oncorkynchus nerka). J. jish. in juvenile coho salmon, Oncorhynchus kisutch. Gen. camp.
Endocr.
21, 431-440.
Res. Bd. Can.
28, 477-484.
CHANGES IN PLASMA THYROID HORMONES FOLLOWING ADMINISTRATION OF EXOGENOUS PITUITARY HORMONES AND STEROID HORMONES TO RAINBOW TROUT (SALMO GAIRDNERI) ROBERTS. MILNEand JOHN F. LEATHERLAND Department of Zoology, College of Biological Science, University of Guelph, Guelph, Ontario NIG 2W1, Canada (Received 5 November 19791 Abstract-l. Porcine FSH and ovine LH (10 1g.g body weight) elicited significant increases (P c: 0.01) in plasma T4 levels within 24 hr of the single injection compared with saline-injected controls whereas HCG was without effect; plasma T3 levels did not change. 2. Ovine TSH and prolactin when given together significantly elevated (P < 0.01) plasma T3 levels 24 hr after the injection compared with all other groups (662.0 + 47.8 ng’dl-’ compared with 133.3 & 30.8, 165.4 + 30.7 and 154.7 ng’dl- ’ in fish given saline, prolactin alone and TSH alone respectively). 3. TSH, given alone or together with prolactin significantly elevated (P < 0.01) plasma T4 levels 8 hr after administration. 4. Single injections, or a series of seven daily injections of /I-estradiol-3-17, dipropionate, 17-a-methyl testosterone or cortisol were without effect on either thyroid appearance or plasma thyroid hormone levels. 5. But after seven daily injections of cortisol, plasma T4 levels were significantly higher (P -C 0.01) and the T3/T4 ratio significantly lower (P i 0.01) than in the sesame oil-injected control fish.
INTRODUCTION Several studies have shown an apparent increase in thyroid activity in some salmonid fishes during the early stages of their anadromous migration, and others an apparent increase in thyroid activity associated with gonadal maturation (see review by Sonstegard & Leatherland, 1976). At these times in their life cycle the fish are known to have elevated steroid hormone levels (Fagerlund & Donaldson, 1970; Idler & Truscott, 1972) presumably in response to ACTH and gonadotropin stimulation. Also, anadromous species may well also be changing the secretory rates of other hormones such as prolactin (for purposes of freshwater osmo-(inono)regulation (Johnson, 1973)) and growth hormone (for mobilization of stored food reserves) (Higgs et al., 1975; Kayes, 1977). There is evidence to suggest that growth hormone and gonadotropins, like TSH, have thyrotropic potency in some fish (Fontaine, 1969; Donaldson & McBride, 1974; Chan & Eales, 1976; Grau & Stetson. 1977a;b; Milne & Leatherland, 1978). In addition, prolactin appears to inhibit the thyrotropic effect of TSH and lower plasma T4 levels in Fundulus heteroclitus (Grau & Stetson, 1977a), although prolactin was without affect on plasma T4 levels in Salmo gairdneri (Mime & Leatherland, 1978). Still other studies suggest that steroid hormones have a stimulatory effect on thyroid activity in some teleosts as evidenced by histological changes (Singh, 1969; Sage & Bromage, 1970; Van Overbeeke & McBride, 1971). It is thus apparent that the complex endocrine changes associated with anadromous migration and sexual maturation of salmonids may influence thyroid 679
function. The aim of this study was to evaluate the thyrotropic effects of pituitary hormones other than TSH, and of some steroid hormones in the hope of furthering the understanding of endocrine interaction in teleosts. Commercial preparations of teleost pituitary hormones are currently not available and mammalian hormone preparations were employed. Although these are undoubtedly structurally different from the teleostean hormones they appear to share some of the physiological functions of the teleostean hormones (Ahsan & Hoar, 1963; Sundararaj & Anaud, 1972; Sundararaj & Goswami, 1974; Higgs et al., 1975; Hirose, 1976; Clarke et al., 1977).
MATERIALSAND
METHODS
Source and maintenance of fish
Rainbow trout (Salmo gairdneri) (60-12Og body weight) of both sexes were obtained from Goossen’s Hatchery, Otterville, Ontario, Canada and maintained in 4001 stock aquaria in continuously running and aerated well water (hardness 316mg.kg; pH 8.1) at 11 + 1°C under a 12 hr light: 12 hr dark photoperiod for at least 28 days prior to their use in experiments. They were fed four or five times a week with a commercial pelleted trout diet (Martin’s Feed Mills, Elmira). Fish were transferred from the stock aquaria into 1001. experimental aquaria, maintained in conditions identical to those of the stock tank 7 days before the start of the experiment; the fish were last fed 24 hr before the first injection. Hormone preparations
All pituitary hormone preparations were dissolved in 0.9% NaCl (saline) and given as intraperitoneal injections
ROBERT S. MILNE and JOHN F. LEATHERLAND
680
in 0.2 ml; steroid hormones were suspended in 0.2 ml of sesame oil. The doses of hormones used (expressed as per gram body weight) were as follows: human chorionic gonadotropin (HCG) (Sigma Chemical Co.), 10 pg; ovine luteinizing hormone (LH) (NIH-LH-Sl9), IOpg; porcine follicle stimulating hormone (FSH) (NIH-FSH-PI), IOpg; ovine thyrotropin (TSH) (NIH-TSH-S8), 2 pg; ovine prolactin (either NIH-P-S10 or Ferring, Malmo, batch No. BI 1108) 1Opg; synthetic ACTH (Ferring, Malmo, batch No. YK5527), 1OOmIU; /I estradiol-3-17 dipropionate, 0.05 mg; 17-a-methyl testosterone, 0.05 mg; cortisol, 0.05 mg. ~~peri~ie~tu~ procedures ~~~er~rne~~rJa. Four groups of fish were given a single injection of LH, FSH, HCG or saline alone at 08.30 hr and samples of six or seven fish from each group were killed 3, 6, 9 or 24 hr after the injection, Experiment lb. Two groups of fish were given a single injection of synthetic ACTH or saline alone at 08.30 hr and samples of five fish from each group were killed 1, 3, 6, 9 or 24 hr after the injections. Experimenr 2~. The procedure was essentially similar to that used in experiment 1 except that fish were given prolactin alone, TSH alone, a mixture of TSH and prolactin (NIH) or saline alone at 08.30 hr and samples of six lish from each group were killed 8 or 24 hr after the injection. Experiment 2b. The procedure was identical to that of experiment I and 2a. Groups of five fish were given a single injection of either prolactin (Ferring) and TSH or saline at 08.30 hr and killed 24 hr later. Experiment 3~. Two groups of 20 fish were given a single injection of fi estradiol-3-17-dipropionate in sesame oil or sesame oil alone. Samples of five fish from each group were killed 3, 6, 9 or 24 hr after the injections. Two further groups of six fish were given seven daily injections of either estradiol or sesame oil alone and killed 18 hr after the last injection.
4
14 2
Exprrimenr 3b. Three groups of 20 fish were given a single injection of 17-z-methyl testosterone or cortisol in sesame oil or sesame oil alone. Samples of five fish were killed 3, 6, 9 or 24 hr after the injection. Three further groups of IO fish were given seven daily injections of either testosterone or cortisol in sesame oil or sesame oil alone and killed 18 hr after the last injection, Blood sampling nnd hormone assay
Blood samples were taken from each fish by caudal severance and the blood collected in heparinized Vacutamer tubes. The plasma was separated by ~ntrifugation and stored at -25°C until hormone assays were made (in all cases within 7 days of collection). Plasma T4 and T3 levels were measured using the Ames Tetralute” and Seralute@ methods as previously described (Milne & Leatherland, 1978). Histology
The lower jaw was removed from each fish, fixed in Bouin’s fluid, decalcified and embedded in Paraplast. Seven micrometer-thick sections were stained with hematoxylin and eosin. Measurements of epithelial cell height were made using an ocular micrometer; measurements were made at four locations of each of seven follicles chosen at random, at the four points crossed by the micrometer scale in the horizontal and vertical planes in the field of view. A mean of the 28 measurements from each fish was used as the epithelial cell measurement. In addition the degree of vesiculation of the colloid was estimated in the same seven follicles. An arbitrary 0 to 10 scale was used, 0 representing no vesiculation and 10 a condition where SOo/,of the colloid was occupied by vesicles. The mean value for the seven follicles was used as the ‘*thyroid droplet index”. Statisfical analysis Data of plasma T4 and T3 concentration, plasma T4/r3 ratio, thyroid epithelial cell height, and thyroid droplet index from each experiment were separately compared by one-way analysis of variance. Where F values indicated significant differences (P < 0.05) between experimental groups, individual means were compared by StudentNewman-Kneuls test.
306
RESULTS
T3
Effect ofLH,
0:
not significantly different from the saline-injected animals at any time period. T4 levels in FSH-injected
0
7 '
3 1
31 2
TDI ,
0
and HCG
The data from experiment la are summarized in Fig. 1. Plasma T4 levels in HCG-injected fish were
E 0.2
TEH
FSH
3
6
9
24
lwws afterin~ecfton
Fig. 1.Temporal effect of a single injection of follicle stimulating hormone (FSH), luteinizing hormone (LH), or human chorionic gonadotropin (HCG) on plasma thyroxine (T4) and triiodothyronine (TJ concentration, plasma T3/r4 ratio, thyroid epithelial cell height and thyroid droplet index in rainbow trout, Salmo gairdneri. Plasma T1 and Ts concentrations are expressed as pg’dl and ng.dl respectively, and epithelial cell height as pm. n = 6 unless otherwise indicated.
trout were signi~cantiy higher than corresponding saline-injected fish at 9 hr and 24 hr after the injection (P < 0.05 and P < 0.01 respectively); T4 levels were significantly (P < 0.01) higher in LH-injected ftsh than the controls 24 hr after the injection. There were no significant differences between plasma T3 levels in hormone-injected and salineinjected fish at any time period. The plasma T3/I’4 ratio in HCG-injected fish was significantly (P < 0.01) lower than the saline-injected animals 3 hr after the injection. There were no significant differences between thyroid epithelial cell height or thyroid droplet index in hormone-injected and saline-injected fish at any time period. Effect of ACTH
The data from experiment lb are summarized in Fig. 2. Plasma T4 levels in ACTH-injected fish were
Plasma
thyroid
hormones
in S&W guirdneri
681
salme ACTH Oli
estradlol
7
6 TEH
4 2
TEH
2
2 TDI
1 0
1
3 6 9 hours afterqectton
TO1
Fig. 2. Temporal effect of a single injection of corticotropin (ACYTH) or /J’ estradiol-3-17-dipropionate on plasma thyroxine (T4) and triiodothyronine (T,) concentration, plasma T,/T, ratio. thyroid epithelial cell height and thyroid droplet index in rainbow trout, Salrno gairdneri. Plasma T, and T, concentrations are expressed as ug’dl and ng’dl respectively. and epithelial cell height as pm. II = 5 unless otherwise indicated. significantly lower (P < 0.05) than in saline-injected fish 9 hr after injection. There were no significant differences between plasma T3 levels, plasma T3/r4 ratios, thyroid epithelial cell height or thyroid droplet index in ACTH-injected and saline-injected fish at any time period.
Effect of TSH and prolactin The data from experiment 2a are summarized in Table 1. Plasma T4 levels in TSH-injected trout were significantly (P < 0.01) higher than in comparable saline-injected fish 8 hr after the injection but were not significantly different after 24 hr. T4 levels in prolactin-injected animals were not significantly different from the saline-injected fish at either time period. In fish given TSH and prolactin together T4 levels were significantly (P < 0.01) higher than in saline-injected animals at both 8 hr and 24 hr after the injection. Plasma T3 levels in TSH- or prolactin-injected fish were not significantly different from saline-injected fish 8 hr and 24 hr after the injection. However, T3 levels in fish given TSH and prolactin were significantly (P < 0.01) higher than comparable salineinjected animals 24 hr after the injection, The T3/T4 ratios in hormone-injected fish were not sigmficantly different from the saline-injected animals at either time period. In the repeat experiment (experiment 2a), using an ovine
prolactin
6 I,
preparation
from
Ferring
(Malmo,
Sweden) instead of the NIH preparation, the results were essentially similar. Plasma T4 and T3 levels in fish injected with TSH and prolactin were significantly higher (P < 0.05 and P < 0.01 respectively) than in the saline-injected animals 24 hr after the injection, whereas there was no significant difference between T3,T4 ratios in the two groups.
2
,
0
3
6 9 24 hours afterqectlon
Fig. 3. Temporal effect of a single injection of cortisol or 17-methyl testosterone on plasma thyroxine (T4) and triiodothyronine (T3) concentration, plasma TJT, ratio, thyroid epithelial cell height and thyroid droplet index in rainbow trout, Salmo gairdneri. Plasma T, and T3 concentration are expressed as pcg’dl and ng’dl respectively, and epithehal cell height as pm. n = 5 unless otherwise indicated.
Effect of steroid hormones The data from experiments 3a and 3b are summarized in Figs 2 and 3 and Tables 2 and 3. In fish given a single injection or seven daily injections there was no significant differences between estradiol- and sesame oil-injected fish for plasma T4 and T3 concentration, plasma T3/r4 ratios, thyroid epithelial cell height or thyroid droplet index (Fig. 2 and Table 2). There were no significant differences in plasma T4 and T3 concentration, plasma T3/T4 ratio, thyroid epithelial cell height or thyroid droplet index between sesame oil-injected fish and testosteroneor cortisolinjected fish at any time after a single injection (Fig. 3). In fish given seven daily injections of cortisol the plasma T4 concentration was significantly higher (P < 0.01) and the T3/T4 ratio significantly lower (P < 0.01) than in sesame oil-injected animals (Table 3). Plasma 7’4 concentrations and T3/T4 ratios in testosterone-injected fish were not significantly different from the sesame oil-injected animals. There were no significant differences between any of the three groups of fish for plasma T3 concentration, thyroid epithelial cell height or thyroid droplet index (Table 3).
DISCUSSION
The data shown in Fig. 1 suggest that porcine FSH and ovine LH have thyrotropic activity in rainbow trout as evidenced by the significant increases in
ROBERT S. MILNE and JOHN F. LEATHERLAND
682
Table I. Effect of a single injection of ovine prolactin. ovine thyrotropin (TSH), or prolactin + TSH on plasma thyrowme (T4) and triiodothyronine (T3) concentrations and T3,TJ ratios in rainbow trout, SUIWI guirdwri
Treatment
Hours after injection
Hormone
R
24
-__ saline I
T4'
prolactina
T4
TSH
T4
TSH + prolactina
T4
0.88 +
o.093
0.70 _ +
0.07
0.74 +
0.07
0.52 +
0.06
1.91 +
0.30**
1.32 + _
0.33
1.97 +
0.13""
1.62 +
_~._____ saline II
T
TSH + prolactinb
4
0.25 ~_-..___
1.11 _ +
0.09
1.79 +
0.29*
T4
______ slille I
,T3)
prolactina
T3
TSH
T
TSH + prolactina
T
saline II
3 3
1!0.9 _ +
16.6
133.3
+
30.8
145.8 +
33.7
165.4
+
30.7
237.9 + 118.4
154.7
t _
37.5
280.0 +
662.0
+
47.8**
236.1
t
22.6
558.6
+ 108.4""
42.3
T3
TSH + prolactina ______~~~__ saline I
'T3 ._~___._~.__..___..~ T3/T4
prolactina
T3/T4
TSH
T3'T4
TSH t prolactinb
T3/T4
0.13 +
0.01
0.18 +
0.01
0.20 +
0.01
0.21 + _
0.02
0.08 +
0.05
0.16 t
0.01
0.13 +
0.01
0.40 _ t
0.01
0.20 _ +
0.01
0.27 _ +
0.01
--___ saline II
T3'T4
TSH t pmlactinb
T /T 3 4 ____~___~________---_-_----__~._._-.~____~____~__~_~
1Expressed as pg. dl. * Expressed as ng. dl. 3 Mean i SEM; * prolactin preparation NIH-P-SIO: h prolactin preparation Ferring MalmG, batch No. Bl I IOX. Saline I is the control group for the TSH, prolactin” and TSH + prolactin” groups (II = 6). saline 11 is the control group for the TSH + prolactinb group (0 = 5). *, **, Significantly different (P < 0.05 and P < 0.01 respectively) from comparable saline-injected group. T4 9 and 24 hr after the injection. Grau & Stetson (1977b) reported a similar thyrotropic potency of LH and FSH in F. hrteroclitus. Fontaine (1969) showed that both purified LH and FSH stimulated radioiodide uptake by the thyroid of hypophysectomized Angltilla anguilla and Ahsan & Hoar (1963) showed that several mammalian gonadotropin preparations, including LH and FSH. increased thyroid epithelium cell height in Gasterostrus uculeat~~s leiurus; a similar significant (P <: 0.01) increase in thyroid epithelial cell height was found after LH treatment of G. aculeatus trachurus (Lam 8~ Leatherland, unpublished data). Donaidson & McBride (i974) showed that salmon gonadotropin increased thyroid plasma
in gonadectomized 0. rwrka. There was no significant effect of HCG on plasma T4 or T3 levels in S. gairdneri in the conditions and doses used here although the significantly (P < 0.01) decreased T3/T4 ratio in HCG-treated fish killed 3 hr after the injection suggests a temporary lowering of T3 without concomitant change in T4 levels. Grau & Steston (1977b) report an elevation in T4 levels in HCGtreated F. heteroclitus and Ahsan & Hoar (1963) indicate that HCG appeared to stimulate thyroid activity in G. uculrutus Ieiurus. The differences between the findings of Grau & Stetson (1977b), Ahsan & Hoar (1963) and those reported here possibly represent species differences but equally may be due to the fact activity
Plasma thyroid hormones in Sulmo yairdneri
683
Table 2. Effect of seven daily injections of estradiol dipropionate on plasma thyroxine (T4) and triiodothyronine (T3) concentrations, T,/T, ratios and histological appearance of the thyroid in rainbow trout, Salmo gairdneri
Treatment
Sesame Oil
T4'
1.05 + o.155
E&radio1
1.11 _ +
(6)"
T32
187.9
+ 5.1 (6)
T3'T4
0.19 + 0.02 (6)
TD13
1.20 _ + 0.21 (4)
TEH'
3.44 f 0.54 (4)
0.08
(6)
207.2
+ 46.2 _ (5)
0.18 +
0.02
(5)
0.90 _ +
0.20
(6)
4.32 _ +
0.35
(6)
'Expressed as pg.dl. * Expressed as ng.dl. 3 Thyroid droplet index. 4 Thyroid epithelial cell height in micrometers. ’ Mean k SEM. 6 Number of samples.
that single hormone injections were used in these experiments whereas multiple injections were used by other authors. The thyrotropic effects of HCG in mammals is well documented (Nisula & Ketelsleger, 1974; Nisula et al., 1974; Amir et al., 1977), as is the permissive role of thyroid hormones in LH function (Aranda et al., 1976). It is possible that some of the thyrotropic effect of the gonadotropin preparations is caused by contamination of the preparations by TSH. However, since the thyrotropic potency of LH and FSH, as evidenced by the elevated plasma T4 levels 24 hr after the injection (Fig. 1) was comparable to that of TSH alone (Table l), either the contamination is considerable, or at least some of the thyrotropic potency of the LH and FSH preparations is not TSH-dependent. The reported TSH contamination of the LH and FSH preparations is 0.11 and 0.036 USP units.mg respectively. whereas the reported potency of the TSH preparation is 1.70 USP units’mg. Thus the LH- and FSH-injected fish received 0.0011 and 0.0036 USP units of TSH.g body weight respectively compared with 0.0034 USP units of TSH.g body weight given to TSH-injected animals. In earlier experiments (Milne & Leatherland, 19783, TSH doses of 1.1 pg.g body weight were found to
elevate plasma T4 levels after 24 hr. Thus, TSH doses of 0.0017 USP units.mg were above the minimal level required for stimulating thyroid activity and this may account for the effect of LH, but it is unlikely to explain the response to FSH. The TSH estimations were obtained using a thyroidal 32P-uptake procedure and the method does not differentiate between TSH contaminants per se and thyrotropic activity of LH and FSH. Nevertheless in light of the low thyrotropic “contamination” particularly of the FSH preparation, the TCstimulating effects of these hormones is probably due at least in part, to the thyrotropic activity of the gonadotropin preparation and not simply to contamination with TSH, although synergism between the gonadotropin and traces of TSH may have been involved. Thyrotropic potency of gonadotropins is not surprising in light of the similar chemical composition of gonadotropin and thyrotropin hormones and their probable parallel evolution (Fontaine & Burzawa-Gerard, 1977). The findings suggest that at least some of the increased thyroid activity found in sexually maturing and mature salmonids may be due to an increase in gonadotropin secretion and/or an increased secretion of growth hormone, associated with the mobilization of lipids; growth hormone has been shown to have a
ROBERT S. MILNE and JOHN F. LEATHERLAND
684
Table 3. Effect of seven daily injections of either 17-r-methyl testosterone or cortisol on plasma thyroxine (T4) and triiodothyronine (T3) concentrations, TX/T4 ratios and histological appearance of the thyroid in rainbow trout. S&IO grtirrlwri
Treatment
Sesame
Testosterone
Cortisol
oil
I_-_____ T4’
0.90
+
0.04:
+ 1.60 _
229.8
+ 20.9
162.7
T3’T4
0.26
_+
0.04
+ 0.10 _
(10)
TD13
0.65
_+
0.24
0.53 +
3.51 +
4.35 + _
(8)
0.04
(10)
1.50 _ +
0.20
0.46
(6)
3.15 +
0.73
0.39
(6)
(6)
-
_-
-
0.18 +
0.01""
(6)
0.18
+ 30.6 (10)
(10)
(8)
TEH'
199.8
+ 17.3 (10)
(10)
0.21
(10)
(10)
(lo)6
Tj2
1.25 +
0.18**
-_
-
’ Expressed
as pg. dl. ’ Expressed as ng’ dl. 3 Thyroid droplet index. 4 Thyroid epithelial cell height in micrometers 5 Mean f SEM. b Number of samples. ** Significantly different from sesame oil-injected
marked thyrotropic effect in trout (Milne & Leatherland, 1978). TSH, given either alone or together with prolactin, stimulates T4 secretion (Table 1). Conversely, a single injection of prolactin has no significant effect on plasma thyroid levels nor does it inhibit the TCstimulating activity of TSH. These findings confirm the observations in S. gairdneri using TSH and prolactin alone (Milne & Leatherland, 1978) but contradict those reported in F. heteroclitus (Grau & Stetson, 1977a). The latter showed that prolactin lowered plasma T4 levels and inhibited the T4-stimulating effect of TSH. In S. gairdneri not only was there no inhibition of the thyrotropic potency of TSH by prolactin, but also plasma T3 levels were significantly elevated 24 hr after the injection (662.0 + 47.8 ,ug/ 100 ml in fish given prolactin and TSH as compared with 154.7 k 37.4 pg/lOO ml in fish given TSH alone). A similar potentiation of T3 levels were found in preliminary experiments in which S. gairdneri were given a single injection of bovine growth hormone (which also has thyrotropic potency (Milne & Leatherland, 1978) alone or combined with prolactin (66.2 k 12.4 pg of T3’dl in saline-injected fish; 145.2 f 29.0 Pg of T3’dl in growth hormone-injected fish; 257.5 t_ 58.2 pg of T3’dl in ovine prolactin + growth hormone-injected fish) (unpublished data).
group
(P < 0.01).
The response to prolactin is known to vary depending on the time of injection of exogenous hormone (Leatherland & Holub, 1979). Thus the different observations of Grau & Stetson (1977a) and those reported here may be caused by circadian variation in the nature of receptors. The reason for the T3 elevation in prolactin + TSH-injected trout is not known. There is good evidence to suggest that, in mammals, most circulating T3 is derived from the peripheral monodeiodination of T4 (Bernal & Refetoff, 1977; Fisher et al., 1972). Similar T4 deiodination appears to occur in salmonids (Higgs & Eales. 1977; Milne & Leatherland, 1978). Thus the increase in T3 levels in TSH + prolactin-injected fish may be caused by a potentiation of the T4 to T3 conversion. In a similar manner, the apparent lowering of T3 in HCG-injected fish may be caused by inhibition of the T4 to T3 conversion. Conversely, the changes in T3 may be caused by alterations in the peripheral metabolism of T3. Some of the temporal changes in T3 found after injections of gonadotropin preparations may be explained in a similar manner. Single, rather than multiple injections of pituitary hormone preparations were used in these experiments in order to overcome the problems of reduced thyroid response noted in multiple injection situations (Chan
Plasma thyroid hormones in S&no gairdneri & Eales,
1976; Milne & Leatherland, 1978). An advantage of the single injection procedure is that responses found within the 24 hr following the injection are more likely to be the result of direct action on the thyroid rather than indirect actions mediated via other endocrine glands. The absence of an apparent change in thyroid activity (as evidenced by changes in plasma thyroid hormone levels or changed in thyroid histology) after estradiol or testosterone treatment is surprising in light of the reports of gonadal steroids stimulating thyroid activity in teleosts (Singh, 1969; Sage & Bromage, 1970; Van Overbeeke 8~ McBride, 1971) and mammals (e.g. Gross et al., 1971; Hunt & Eales, 1979). Singh (1969) and Sage & Bromage (1970) using Mystus vittatus and Poeciiia reticulata respectively showed an apparent stimulation of thyroid activity after treatment with estrogen or testosterone using in vitro culture methodology and radioiodide-uptake rates. Van Overbeeke & McBride (1971) reported an apparent activation of the thyroid in gonadectomized male sockeye salmon treated with 1I-ketotestosterone or 17-z-methyl-testosterone; estradiol or estradiol cypionate were without effect in gonadectomized female sockeye salmon. Hunt & Eales (1979) showed an increased T4 degradation and monodeiodination rate accompanied by elevated plasma T3 and T4 levels in rainbow trout injected with testosterone propionate. The difference between the findings reported here and those of Hunt & Eales (1979) cannot be explained. Hunt & Eales (1979) reported that the responses of plasma T4 levels to testosterone was inconsistent; the differences in the two experimental series mav be due to this inconsistency. eortisol elevated plasma T4 levels in fish given seven daily injections of the steroid whereas a single ACTH-injection tended to decrease plasma T4 levels. In both cases there was no concomitant change in the histological appearance of the thyroid in hormoneinjected fish compared with controls. Thus, although there is evidence for a thyrotropic role of cortisol in trout, supporting the observations of an increased radloiodide uptake by the thyroid of M. vittatus treated with cortisone (Singh, 1969), some increase in epithelial cell height might have been expected if the increase in plasma hormone represented an elevated secretion of hormone. An alternate explanation may be that the increased plasma hormone level is a compensatory response to changes in the binding capacity of the blood proteins. Conversely, the elevated T4 levels in cortisol-treated trout may be due to a depressed plasma ACTH titer in these fish. Olivereau (1968a;b) showed a clear correlation between the pituitary-thyroid and pituitary-interrenal axes in eels. Radlothyroidectomy induced a histological picture of reduced activity of both pituitary corticotrop cells and interrenal cells while T, treatment causes an apparent stimulation of the pituitary-interrenal axis. However, Van Overbeeke & McBride (1971) reported a reduction in thyroid epithelial cell height after cortisol administration to gonadectomized sockeye salmon. This study, like many similar ones, was limited by the fact that mammalian pituitary hormones were used, since comparable hormones of teleostean origin are not readily available. In addition, it is difficult to
685
evaluate the physiological relevance of the dosages of hormones used although they all were within levels used by other authors (Ahsan & Hoar, 1963; Hawkins & Ball, 1970; Sundararaj & Anaud, 1972; McLeay, 1973; Sundararaj & Goswami, 1974; Higgs et al., 1975; Sundararaj & Keshavanath, 1976; Hirose, 1976; Clarke et al., 1977). Nevertheless, the study provides
evidence that pituitary factors other than TSH and also cortisol can influence thyroid activity in salmonid fishes and may account for some of the changes reported in salmonid thyroid activity during migration, when hormones such as growth hormones and prolactin are secreted in response to changed metabolic and osmotic requirements. Although no effects of sex steroids on thyroid activity were evident in these studies it is possible that under different conditions of temperature, these hormones may exert effects on thyroid gland function in some teleostean species. Acknowledgements-We wish to thank Lucy Lin and Kim Counsel1 for their excellent technical assistance. The hormone preparations were donated by the National Institute of Health, Bethesda, Maryland, U.S.A. The work was supported by a grant-in-aid of research from the Natural Science and Engineering Research Council of Canada to J.F.L.
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