- Email: [email protected]
Luteinizing hormone-releasing hormone as a potent stimulator of the thyroidal axis in ranid frogs
GENERAL
AND
COMPARATIVE
Luteinizing
G.F.M.
ENDOCRINOLOGY
Hormone-Releasing Hormone as a Potent Stimulator the Thyroidal Axis in Ranid Frogs
JACOBS, M.P.
Laboratory
70, 274-283 (1988)
of Comparative
GOYVAERTS,G.VANDORPE, E.R. ICOHN Endocrinology, Naamsestraat
A.M.L.
Zoological Institute, Catholic 61, B-3000 Leuven, Belgium
of
QUAGHEBEUR,AND
University
of Leuven,
Accepted December 9, 1987 Plasma concentrations of T4, measured by radioimmunoassay, were raised significantly 2 and 4 hr after intravenous injection of synthetic luteinizing hormone-releasing hormone (LHRH) in Rana ridibunda (1 and 10 kg on 2 consecutive days) and in Rana esculenta (10 kg). A dose of 1 pg LHRH was not so effective as 50 pg synthetic thyrotropin-releasing hormone (TRH) when injected in Rana ridibunda in November. However 10 pg LHRH was equipotent to 50 kg TRH. In February somewhat less than half of the Rana temporaria group was responsive to LHRH. There is no clear indication that fluctuating plasma T, concentrations were caused by LHRH or TRH. Preinjection levels of T, and T, were higher during the breeding season (April) in R. esculenta (resp. 35.4 + 1.4 pg/ml; 744 + 134 pglml; II = 22) compared to the basal concentrations in the very closely related Rana ridibunda (November) (resp. 15.2 _+ 1.1; 162 * 24 pg/mi; n = 28). Four days after removal of the pars distalis plasma T4 concentrations were significantly decreased in Rana esculenta, whereas T, could stay longer in circulation. T, and T4 content of the thyroids was not altered by the short-term hypophysectomy. Injection of 10 pg LHRH had no influence on plasma T4 nor testosterone concentrations in these frogs, contrary to the sham-ectomized animals in which plasma testosterone remained elevated longer than T,. The results suggest that the stimulatory effect of intravenous injected LHRH on thyroid (and gonadal) activity in the frog is primarily mediated through the hypophysis. They also point to a possible correlation between the gonadal and thyroidal axis. o 1988 Academic Press, Inc.
In teleostean fishes, a thyroid-gonadal interrelationship is well evidenced by the presence of a maturational effect of thyroxine upon the gonads and the existence of cycles of thyroid activity correlated with reproduction (Sage, 1973; Hurlburt, 1977; Chakraborti and Bhattacharya, 1984). Also, the occurrence of such a relationship has been suggested in amphibians (Dickhoff and Darling, 1983; Kuhn et al., 1987). In a recent experiment performed on Rana ridibunda, a sharp increase in the plasma T4 concentration associated with a first elevation of plasma testosterone was observed at the end of hibernation, and a maximal 5 ‘-monodeiodinating activity, as measured in kidney homogenates, was reached during the breeding season (April-May-June)
(Vandorpe et al., 1987). Suzuki and Suzuki (1981) detected the highest T4 concentration in two urodeles (Hynobius and Onychoductylus) also just before the breeding period. It is generally believed that the mammalian LHRH (luteinizing hormone-releasing hormone), which is the major form of GnRH (gonadotropin-releasing hormone) in the adult amphibian brain (Sherwood et al., 1986), stimulates the release of gonadotropins (FSH and LH) from the pituitary in vivo and in vitro (Thornton and Geschwind, 1974; Daniels and Licht, 1980; Licht et al., 1984; Porter and Licht, 1986), while TRH (thyrotropin-releasing hormone), which is also abundantly present in the frog hypothalamus and brain (Jackson and Reichlin, 274
0016~6480/88 $1 so Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
LHRH
STIMULATES
1974; Jackson and Bolaffi, 1983) has been proposed as a possible thyrotropinreleasing factor in the frog Rana ridibunda (Dar-r-as and Kuhn, 1982) and in some cases also in the metamorphosed axolotl Ambystoma mexicanum (Jacobs et al., 1988). Other positive effects obtained with the tripeptide (published mostly in abstract form) concerning acceleration of metamorphosis in tadpoles (Shiomi et al., 1974; Schultheiss, 1978), enhanced thyroid hormone secretion in thyroid-hypophysis cocultures (Etkin and Kim, 1971; Peyrot et al., 1974), and increased thyroid iodine release in adult Bufo bufo (Rosenkilde, 1979) also conflict, however, with numerous negative in vivo and in vitro reports on TRH (Etkin and Gona, 1968; Gona and Gona, 1974; Taurog et al., 1974; Vandesande and Aspeslagh, 1974). The hypothesis proposed by Sage (1973) that the control of TSH evolved from the control of an original gonadotropin (which may have influenced thyroid activity), together with the observation that injection of a LHRH analog raised plasma thyroxine concentration in female sea lamprey (Sower et al., 1985), prompted us to examine whether LHRH would be able to stimulate the pituitary-thyroid axis in anuran Amphibia. MATERIAL
AND METHODS
The experiments were performed on male frogs obtained from R. Stein (Lauingen, GFR) (Rana ridibunda and Rana temporaria) or from the Centre d’elevage d’animaux de laboratoire d’Ardenay (Le Breil sur Merize, France) (Rana esculenta). Prior to the experimental period, the animals were kept in plastic tanks in a nonheated room with open windows and outside photoperiod for at least 1 month, and they were fed on mealworms once a week. The mean maximum and minimum temperature (_+standard deviation) to which the animals had been exposed since their arrival in the laboratory was respectively 19 f 2” and 17 + 3” for R. ridibunda, 13 * 3” and 9 -+ 3” for R. temporaria, and 16 + 2” and 12.5 + 2” for R. esculenta. In the morning before the experiment, the frogs were anesthetized in a 0.16% MS 222 solution (tricaine methane sulfonate) and their abdominal vein was can-
FROG
THYROID
275
nulated (in both directions) for injection of synthetic TRH, s-LHRH (both from UCB, Bioproducts), or frog Ringer solution (in a 100 ~1 volume) as well as for several blood samplings of about 0.35 ml, the (2) following day(s). Each time after centrifugation, the blood cells were suspended again in frog Ringer solution (also taking the volume of the canula into account) and reinjected. Three experiments were performed, all at room temperature (19-20”). 1. The first experiment was performed in the beginning of November and Rana ridibunda with a mean body weight of 61.9 + 1.9 g (n = 28) were used. The frogs were injected on 2 consecutive days with either frog Ringer solution (controls, n = lo), 50 p,g s-TRH (n = 8), or two different doses of s-LHRH (1 and 10 kg, n = 10). Blood samples were taken before and at 2, 4, and 7 hr after each injection. 2. The second experiment was performed at the end of February and Rana temporaria weighing 30.1 + 1.8 g (n = 23) were used. Because the body weight of these frogs was 50% lower, a dose of 0.5 and 5 Fg of LHRH was administered consecutively (n = 13). Blood was sampled before and at 2, 4, 8, and 24 hr after the first injection, and at 2 and 4 hr after the second one. 3. A third series of experiments using Rana esculenta with a mean body weight of 50.2 + 1.Og (n = 46) was performed in the first half of April. Hypophysectomy (pars distalis) and sham operation were carried out, while working under binocular, 4 days before injection. At the level of the pituitary, the palate bone was ground until it became thin and could be loosened by 75%. After the pars distalis was removed, the little palate piece was returned for covering the hole and the skin was sutured. Hypophysectomized and sham frogs received 10 kg LHRH (resp. n = 13; 13) or 100 ~1 Ringer (resp. n = 11; 9) and blood was collected before and at 2, 4, and 7.5 hr after injection. The plasma samples were extracted with acetone (according to Darras and Kuhn, 1982) prior to RIA of T, and T,. Hormone concentrations were assayed by using tracer obtained from Amersham International (UK), rabbit T, antiserum from Mallinckrodt (GFR) and a laboratory-raised rabbit T, antiserum (final titer: l/8250). T, and T, antisera did not show any crossreactivity for respectively T4 concentrations till 5 ng/ ml and T, concentrations up to 2 nglml frog plasma. Radioimmunoassay of testosterone as described previously (Vandorpe et al., 1987), was only performed in the samples of Rana escuienta. At the end of the experiments, the frogs were killed and the thyroid glands were carefully excised under binocular. After homogenizing the glands with a Teflon hand-homogenizer in a 0.01 M sodium phosphate buffer (pH 7.6), they were treated as described by Darras and Kuhn (1982). Subsequently T3 and T4 content was measured by RIA. Cross-reactivity of the T, antiserum (Mallinckrodt, GFR) for T, was 0.3% and final
276
JACOBS
T, data were corrected. Because of the small T,/T4 ratio in the thyroid, the cross-reactivity of the T, antiserum (commercial kit from Abbott Laboratories) for T, was not considered as being significant. Statistical analysis of data was made by one-way analysis of variance, ANOVA (for comparison between two groups) or paired t test (for comparison within the same group of animals).
RESULTS 1. Influence of Intravenous TRH and LHRH Injections on Plasma Concentrations of T4 and T3 a. Rana ridibunda. Following injection of 50 pg TRH circulating control levels of T4 (212 k 53 pg/ml; n = 8) were raised about 6.5 times (up to 1420 + 277 pg/ml) within 2 hr (P < 0.005 compared to Ringerinjected frogs) and concentrations remained significantly elevated during 4 hr. Basal values were obtained again after 24 hr and at that time a new injection of 50 kg TRH caused a similar increase of T4 (Fig. 1). In a second group of frogs (n = 10) 1 pg LHRH was responsible for a fourfold elevation of the plasma T4 concentration 2 hr after injection (P < 0.005 compared to the Ringer controls). Moreover, the effect of 10 pg LHRH the next day was equally pronounced as that caused by 50 pg TRH. Values of T4 rose from 137 + 62 to 1273 k 311 pg/ml 2 hr after injection (P < 0.005) and also stayed significantly elevated after 4 hr. While a higher T4 level (P < 0.05) was still present at the end of the TRH experiment (7 hr), this was not the case after LHRH injection. Concerning the T3 plasma concentrations, which were highly variable during the whole period studied, no difference could be found between the controls and the TRH and LHRH injected animals (ANOVA). Notwithstanding the fact that a significant increase in the T, plasma level was noted 4 hr after administration of 1 pg LHRH (P < 0.025), the same effect was seen in the Ringer-injected frogs (P < 0.05 paired t test) (Fig. 1).
ET AL.
b. Rana temporaria. Control animals showed fairly constant T, plasma concentrations (90-100 pg/ml) during the whole experiment . Injection of 0.5 kg LHRH did not influence T, levels whereas 5 kg LHRH was able to induce T, release in four of the nine frogs studied the second day. Because of the considerable high standard errors found 4 hr following injection, comparison with the controls could only reveal a significant increase after 2 hr (P < 0.01). It must also be noted that T, plasma levels were slightly elevated (P < 0.05) before the second injection (Fig. 2). As in the first experiment basal T, concentrations in R. temporaria (18.6 f 1.2 pg/ ml; n = 23) were not effected by the LHRH injections. c. Rana esculenta. Circulating levels of T, (744 k 134 pg/ml; n = 22) were significantly decreased (P < 0.005) 4 days after removal of the pars distalis (195 + 9 pg/ml; n = 24). An injection of 10 pg LHRH increased T, concentrations to about 2000 pg/ ml in sham-ectomized frogs after 2 hr (Fig. 3), whereas basal values were reached again after 7.5 hr. Plasma T, levels remained unchanged in the hypophysectomized animals when compared to their controls. Comparing preinjection T, values of all hypophysectomized frogs (31.2 + 0.8 pg/ ml; n = 24) to those of sham animals (35.4 + 1.4 pg/ml; n = 22) a significant difference (caused by the hypophysectomized frogs going to receive LHRH) was noted (P < 0.025). Two hours after injection of 10 pg LHRH plasma T, concentrations were significantly raised in sham and hypox frogs as evaluated by paired t test (P < 0.005) but compared to their controls no differences were present (Fig. 3). 2. Influence of Intravenous LHRH Injections on Plasma Concentrations of Testosterone
This hormone was only measured in the samples of the third experiment (Fig. 3).
LHRH
STIMULATES
FROG
277
THYROID
hours T
1
after
injection
**tlr
0.6 -
FIG . 1. Plasma concentrations of T, and T, (means * SEM) in Rana ridibundu following injection of frog Ringer solution (0; n = 10 unless indicated otherwise on the figure), 50 pg TRH (A; n = 8), or 1 and 10 kg LHRH (0; n = 10) on 2 consecutive days. *P < 0.05; ** P < 0.01; *** P < 0.005 (ANOVA).
Hypophysectomy resulted in a slight decrease (P < 0.05) of the morning testosterone concentrations 4 days after the operation (3.16 _+ 0.41; n = 20 vs. 2.08 + 0.28; n = 21). Two hours following injection of 10 bg LHRH a threefold increase in plasma concentrations of testosterone was observed in sham-ectomized frogs (up to 10.3 2 1.2 rig/ml). This high value was main-
tained during 2 hr, and was still significantly elevated from controls 7.5 hr after injection (P < 0.005). No influence on testosterone levels was found in hypox animals following injection of LHRH. 3. T3 and T4 content of the thyroid gland A survey of the thyroid
hormone
con-
278
JACOBS
1
0
2
ET AL.
I
4
8
I,
II
I
I
I
h204u/fs etter21n,ectlo:
FIG . 2. Plasma concentrations of T4 (means + SEM) in Rana temporaria following injection of frog Ringer solution (0; n = 10 unless indicated otherwise on the figure) or 0.5 and 5 pg LHRH (0; n = 13) on 2 consecutive days. * P < 0.05; ** P < 0.01 (ANOVA).
tents, measured 7 hr (first experiment), 4 hr (second experiment), and 7.5 hr (third experiment) after injection, is presented in Table 1, together with the respective body weights . The amount of glandular T, was not influenced by any of the experimental treatments used. T, content assayed in the glands of the control R. ridibundu frogs was significantly higher (P < 0.05) compared to the LHRH-(but not TRH-) injected animals. However this effect was no longer seen in R. esculenta nor R. temporaria. DISCUSSION
The present study indicates that intravenously injected mammalian LHRH is able to raise circulating levels of T, (but not of T3) in three frog species. As expected, plasma testosterone concentrations were elevated as well (in R. esculenta) (McCreery et al., 1982; Moore et al., 1982; Licht et al., 1984). Synthetic TRH also caused an increase in plasma T4 when injected in R. ridibunda, confirming a previous study (Dar-r-as and Kuhn, 1982) and in R. temporaria (Darras and Kuhn, unpublished observation). Since only two different doses of LHRH (1 and 10 pg) and one dose of TRH (50 pg) were tested (first experiment), no definite
conclusions regarding the relative potencies of LHRH and TRH can be drawn. The fact that 8.5 nmol LHRH (10 pg) was as effective as 138 nmol TRH (50 pg) in R. ridibunda in November may be an important observation. However, one has to consider the possibility that 50 pg TRH may also have acted in an inhibitory way regarding lower effective doses. Seasonal variations in the response caused by LHRH and TRH may occur as well, but our study does not permit any conclusions in this regard. On the other hand, our results indicate that in R. ridibunda 1 pg LHRH was also sufficient for stimulating the release of T4 (fourfold elevation of the preinjection value after 2 hr), and since in a previous study (Darras and Kuhn, 1982) it was found that the minimal effective dose of TRH in the same species was also around 1 kg (3.3-fold elevation of the preinjection value), one may conclude that LHRH is at least as potent as TRH to increase plasma T, concentrations in this species. Immunological and chromatographic evidence suggests that the mammalian form of GnRH is present in the anuran brain (Alpert et al., 1976; Eiden and Eskay, 1980; King and Millar, 1980; Sherwood, 1986; Sherwood et al., 1986). It is known that 1 pg of LHRH will cause ovulation in Xenopus (Thornton and Geschwind, 1974). In R. es-
LHRH
STIMULATES
FROG
279
THYROID
7.5
4
hours
Testosterone nglml
after
InJectlo~
T
10 -
8-
6-
4-
0.2
-i
-sc=pm-=$--==== lo t b ’ 0
2
4
0 hours
after
lnjectk.%
2
4
hours
after
InjectI::
FIG. 3. Plasma concentrations of T,, T4, and testosterone (means rt SEM) in pars distalis (- - -) and sham-ectomized (-) frogs (Rana esculenta) following injection of 10 kg LHRH (0; n = resp. 13, 13) or frog Ringer solution (0; II = resp. 11, 9, unless indicated otherwise on the figure). * P < 0.05; *’ P < 0.025; *** P < 0.005 (ANOVA).
culenta a total of three subcutaneous injections, each containing 300 ng of a longacting LHRH analog, raised the testicular testosterone content, an effect which was abolished after hypophysectomy (Pierantoni et al., 1984). Our hypophysectomy experiments on R. esculenta revealed that the elevation of plasma testosterone was mediated through release of a gonadotropin,
probably LH (Licht, 1979), and not via direct action of LHRH on the steroidogenesis in the testis as was demonstrated in vitro in R. pipiens (Segal and Adjuwon, 1979). In the bullfrog (5450 g) a dose of 2.5 and 5 pg LHRH was able to increase plasma gonadotropins (FSH and LH), but its duration of action was short (less than 1 hr) (Daniels and Licht, 1980). In view of these results, a
280
JACOBS
lo-pg dose of LHRH as used in R. escuZenta in order to increase T4 and testosterone must be considered high, whereas 1 pg as used in R. ridibunda would be more physiological. The results clearly indicate that LHRH stimulates the release of T, via the pituitary. There are however several possibilities for the peptide to exert its action. (a) A direct effect of testosterone upon the thyroid gland (or pituitary) should not be excluded since a stimulatory influence of sex steroids on thyroid activity has been reported in teleostean fishes (Sage, 1973; Chakraborti and Bhattacharya, 1984), the chick embryo (Hoshino et al., 1987), and in mammals (see Galton, 1971). However, the fact that in our study using R. esculenta testosterone and T4 were released simultaneously after LHRH injection and that T4 was already decreasing 4 hr after injection to reach control values when the testosterone level was still significantly elevated is not favorable for this hypothesis. (b) LHRH may act as a neurotransmitter to effect other hypothalamic hormones, e.g., TRH, leading to TSH secretion. A small amount of salmonid-like GnRH has been detected in the brain of some anuran and urodelan species (Sherwood et al., 1986). This GnRH is similar to that found in the bullfrog retina (Eiden et al., 1982) and sympathetic ganglia (Eiden and Eskay, 1980; Jan and Jan, 1982; Jones et al., 1984) and has been proposed to have a neurotransmitter function (Sherwood et al., 1986). (c) Paracrine interactions in the pituitary may lead to an LHRH-induced activation of the thyrotropes. (d) LHRH may stimulate the thyrotropes directly, apart from the gonadotropes. Since in this assumption TSH, LH, and FSH will be increased, the animal should be capable of distinguishing between endogenous thyrotropins and gonadotropins. In the bullfrog it was shown that homologous LH and FSH had relatively lit-
ET AL.
tle effect on the thyroid gland, while homologous TSH stimulated thyroxine release and accelerated metamorphosis (MacKenzie et al., 1978). In addition MacKenzie and Licht (1984) found no clear indication that the thyroid gland of the leopard frog (Rana pipiens) or the tree frog (Hyla regilla) would respond to bullfrog LH. So it could be possible that the receptors on the thyrotropes of a frog do not discriminate between TRH and LHRH (e.g., by reacting with the identical pyroglutamyl-histidyl residue), that two different kinds of receptors are present on the same cell, or that two types of TSH cells occur. The last two possibilities would give the animal the opportunity to use its thyroidal and gonadal axis simultaneously as well as independently if necessary (e.g., during metamorphosis or short periods of greater T, need). Injection of LHRH into R. temporaria did not cause the same pronounced T, increase as in the other frogs, although about the same doses, relative to the body weight, were used. Only the higher concentration (5 pg/animal) was effective in four of the nine animals tested. This may be due to species or seasonal differences in receptor sensitivity or number. While plasma T, concentrations were very significantly decreased 4 days after removal of the pars distalis in R. esculenta, the influence on plasma T, levels was much slower, probably because they had been sustained by peripheral T4 conversion. The rise of circulating T, which was noticed (paired t test) 4 hr after injection of Ringer and LHRH in R. ridibunda, and 2 hr after administration of LHRH in shamectomized R. esculenta may be caused by the existence of a circadian rhythm in the T,-S-monodeiodinating activity. Kidneys dissected in the evening from winterfrogs (adapted to room temperature) exhibited a higher T4 converting activity in vitro than when removed in the morning or at noon (P < 0.001) (Jacobs and Kuhn, unpublished observation). In addition, in the hypophy-
LHRH
STIMULATES
FROG
TABLE T4 AND T, CONTENT
Expt. 1: R. ridibunda Ringer LHRH TRH Expt. 2: R. temporaria Ringer LHRH Expt. 3: R. esculenta Sham: Ringer LHRH Hypophysectomized:
OF THE THYROID
Ringer LHRH
281
THYROID
1
GLANDS REMOVED (SEE RESULTS)
AT THE END OF EACH EXPERIMENT
Body weight (g)
T, (rig/thyroid)
T3 (&thyroid)
60.7 + 3.5” (10)’ 63.7 + 3.0 (10) 61.0 ? 3.7 (8)
1858 + 323 (9) 1357 + 160 (9) 1502 k 295 (8)
13.1 + 1.7 (9) 9.0 + 0.8 (9) 8.8 + 1.3 (8)
28.1 + 2.7 (10) 31.7 k 2.4 (13)
837 + 93 (8) 947 k 151 (9)
9.7 2 1.4 (8) 8.2 2 1.2 (9)
50.3 49.5 49.4 51.5
2 2 k +
2.5 (9) 1.6 (13) 2.0 (11) 2.3 (13)
1441 1339 1785 1438
+ 224 (9) + 130 (13) +- 195 (9) + 172 (13)
3.6 4.0 6.5 4.3
2 2 k 2
0.8 0.6 1.1 0.9
(9) (13) (9) (13)
a Means + SEM. ’ Number of animals.
sectomized frogs fluctuating T, concentrations were also observed, whereby the original slight difference between hypox and sham animals was abolished. The results of the present experiments may again raise the question of whether GnRH was the original controlling hormone of a gonadotropin which influenced both thyroid gland and gonads, while TRH evolved later to control a gonadotropindeduced thyrotropin. Until now only few data supported this hypothesis. In the female sea lamprey plasma T4 was raised after injection of a LHRH analog (Sower et al., 1985). However, other studies demonstrated that in fish, a rise in blood gonadotropin after injection of LHRH was not accompanied by a change in thyroid activity (Brown et al., 1985; MacKenzie et al., 1987). Perifused pituitaries of turtles did not secrete TSH during LHRH stimulation (Licht and Porter, 1985). It is also generally accepted that the mammalian thyrotrope is not directly stimulated by LHRH. Only after chronic LHRH treatment a slight increase in plasma T4 was observed, perhaps due to changes in ovarian estrogen secretion (Fraser, 1983). ACKNOWLEDGMENTS The skillful technical assistance of Mrs. F. Voets,
Ms. L. Noterdaeme, and Mr. W. Van Ham is gratefully acknowledged. G. Jacobs was supported by the National Fund for Scientific Research (Belgium). G. Vandorpe was supported by the Belgian IWONL (Instituut tot aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw).
REFERENCES Alpert, L. C., Brawer, J. R., Jackson, I. M. D., and Reichlin, S. (1976). Localization of LHRH in neurons in frog brain (Rana pipiens and Rana catesbeiana). Endocrinology 98, 910-921. Brown, C. L., Grau, E. G., and Stetson, M. H. (1985). Functional specificity of gonadotropin and thyrotropin in Fundulus heteroclitus. Gen. Comp. Endocrinol.
58, 252-258.
Chakraborti, P., and Bhattacharya, S. (1984). Plasma thyroxine levels in freshwater perch: Influence of season, gonadotropins, and gonadal hormones. Gen. Comp. Endocrinol. 53, 179-186. Daniels, E., and Licht, P. (1980). Effects of gonadotropin-releasing hormone on the levels of plasma gonadotropins (FSH and LH) in the bullfrog, Rana
catesbeiana.
Gen.
Comp.
Endocrinol.
42,
455-463. Darras, V. M., and Kuhn, E. R. (1982). Increased plasma levels of thyroid hormones in a frog Rana ridibunda following intravenous administration of TRH. Gen. Comp. Endocrinol. 48, 469-475. Dickhoff, W. W., and Darling, D. S. (1983). Evolution of thyroid function and its control in lower vertebrates. Amer. Zool. 23, 697-707. Eiden, L. E., and Eskay, R. L. (1980). Characterization of LRF-like immunoreactivity in the frog sympathetic ganglia: Non-identity with LRF decapeptide. Neuropeptides 1, 29-37.
282
JACOBS
Eiden, L. E., Loumaye, E., Sherwood, N., and Eskay, R. L. (1982). Two chemically and immunologically distinct forms of luteinizing hormonereleasing hormone are differentially expressed in frog neural tissues. Peptides 3, 323-327. Etkin, W., and Gona, A. G. (1968). Failure of mammalian thyrotropin-releasing factor preparation to elicit metamorphic responses in tadpoles. Endocrinology 82, 1067-1068. Etkin, W., and Kim, Y. S. (1971). Effects of TRH on tadpole tissue. Amer. Zool. 11, 654. (Abstract 148) Fraser, H. M. (1983). Effect of treatment for 1 year with a luteinizing hormone-releasing hormone agonist on ovarian, thyroidal, and adrenal function and menstruation in the stumptailed monkey (Mucca
arctoides).
Endocrinology
112, 245-253.
Galton, V. A. (1971). Thyroxine metabolism and thyroid function in rats following administration of estrogen. Endocrinology 88, 976-982. Gona, A. G., and Gona, 0. (1974). Failure of synthetic TRF to elicit metamorphosis in frog tadpoles or red-spotted newts. Gen. Comp. Endocrinol. 24, 223-225. Hoshino, S., Huybrechts, L. M., Darras, V. M., Decuypere, E., and Kuhn, E. R. (1987). Thyrotrophic and peripheral activity of estradiol in the chick embryo. Med. Sci. Res. 15, 1409-1410. Hurlburt, M. E. (1977). Role of the thyroid gland in ovarian maturation of the goldfish, Carussius uurutus L. Cunud. J. Zoo/. 55, 1906-1913. Jackson, I. M. D., and Bolaffi, J. L. (1983). Phylogenetic distribution of TRH: Significance and function. In “Thyrotropin-releasing hormone” (E. C. Griffiths and G. W. Bennett, Eds.), p. 191-202. Raven Press, New York. Jackson, I. M. D., and Reichlin, S. (1974). Thyrotropin-releasing hormone (TRH): Distribution in hypothalamic and extrahypothalamic brain tissue of mammalian and submammalian Chordates. Endocrinology
95, 854862.
Jacobs, G. F. M., Michielsen, R. P. A., and Kuhn, E. R. (1988). Thyroxine and triiodothyronine in plasma and thyroids of the neotenic and metamorphosed axolotl Ambystomu mexicunum: Influence of TRH injections. Gen. Comp. Endocrinol 70, 145-151. Jan, L. Y., and Jan, Y. N. (1982). Peptidergic transmission in sympathetic ganglia of the frog. J. Physiol.
327, 219-246.
Jones, S. W., Adams, P. R., Brownstein, M. J., and Rivier, J. E. (1984). Teleost luteinizing hormonereleasing hormone: Action on bullfrog sympathetic ganglia is consistent with role as neurotransmitter. J. Neurosci. 4, 420-429. King, J. A., and Millar, R. P. (1980). Comparative aspects of luteinizing hormone-releasing hormone
ET AL.
structure and function in vertebrate phylogeny. Endocrinology
106, 707-7 17.
Kuhn, E. R., Gevaerts, H., Jacobs, G., and Vandorpe, G. (1987). Reproductive cycle, thyroxine and corticosterone in females of the giant swamp frog Dicroglossus occipitalis at the equator. Gen. Comp. Endocrinol. 66, 137-144. Licht, P. (1979). Reproductive endocrinology of reptiles and amphibians: Gonadotropins. Annu. Rev. Physiol.
41, 337-351.
Licht, P., Millar, R., King, J. A. McCreery, B. R., Mendonca, M. T., Bona-Gallo, A., and Lofts, B. (1984). Effects of chicken and mammalian gonadotropin-releasing hormones (GnRH) on in vivo pituitary gonadotropin release in amphibians and reptiles. Gen. Comp. Endocrinol. 54, 89-96. Licht, P., and Porter, D. A. (1985). In vivo and in vitro responses to gonadotropin releasing hormone in the turtle, Chrysemys pictu, in relation to sex and reproductive stage. Gen. Comp. Endocrinol. 60, 75-85. MacKenzie, D. S., and Licht, P. (1984). Studies on the specificity of thyroid response to pituitary glycoprotein hormones. Gen. Comp. Endocrinol. 56, 156-166. MacKenzie, D. S., Licht, P., and Papkoff, H. (1978). Thyrotropin from amphibian (Ranu catesbeiana) pituitaries and evidence for heterothyrotropic activity of bullfrog luteinizing hormone in reptiles.Gen.
Comp.
MacKenzie, D. Breton, B. in goldfish ing thyroid
Endocrinol.
36, 566-574.
S., Sokolowska, M., Peter, R. E., and (1987). Increased gonadotropin levels do not result in alterations in circulathormone levels. Gen. Comp. Endocrinol. 67, 202-2 13. McCreery, B. R., Licht, P., Barnes, R., Rivier, J. E., and Vale, W. W. (1982). Actions of agonistic and antagonistic analogs of gonadotropin releasing hormone (Gn-RH) in the bullfrog Runu cutesbeiunu. Gen. Comp. Endocrinol. 46, 511-520. Moore, F. L., Miller, L. J., Spielvogel, S. P., Kubiak, T., and Folkers, K. (1982). Luteinizing hormonereleasing hormone in the reproductive behavior of a male amphibian. Neuroendocrinology 35, 212216. Peyrot, A., Pons, G., and Vellano, C. (1974). Etude du metaboiisme thyroi’dien du triton Crete par l’association in vitro de thyroide et d’hypophyse. Effet de la TRH. Gen. Comp. Endocrinol. 22, 374. (Abstract 106) Pierantoni, R., Iela, L., d’Istria, M., Fasano, S., Rastogi, R. K., and Delrio, G. (1984). Seasonal testosterone profile and testicular responsiveness to pituitary factors and gonadotrophin releasing hormone during two different phases of the sexual cycle of the frog (Ranu eculentu). J. Endocrinol. 102, 387-392.
LHRH
STIMULATES
Porter, D. A., and Licht, P. (1986). Effects of temperature and mode of delivery on responses to gonadotropin-releasing hormone by superfused frog pituitaries. Gen. Comp. Endocrinol. 63, 236-244. Rosenkilde, P. (1979). The thyroid hormones in Amphibia. In “Hormones and Evolution” (E. J. W. Barrington, Ed.), Vol. I, p. 437-491. Academic Press, New York. Sage, M. (1973). The evolution of thyroidal function in fishes. Amer. Zool. 13, 899-905. Schultheiss, H. (1978). The influence of purified amphibian pituitary hormones, pituitary grafts and TRH on thyroid activity and metamorphosis. In “Proceedings, Eighth Int. Symp. Comp. Endocrinol., Amsterdam,” Abstr. p. 75. Segal, S. J., and Adejuwon, C. A. (1979). Direct effect of LH-RH on testicular steroidogenesis in Rana pipiens. Biolog. Bull. 157, 393-394. Sherwood, N. M. (1986). Evolution of a neuropeptide family: Gonadotropin-releasing hormone. Amer. Zoo/. 26, 1041-1054. Sherwood, N. M., Zoeller, R. T., and Moore, F. L. (1986). Multiple forms of gonadotropin-releasing hormone in amphibian brains. Gen. Comp. Endocrinol. 61, 313-322. Shiomi, K., Hachiya, T., Yoshimura, M. (1974). Effect of synthetic TRH (thyrotropin releasing hormone) on tadpole metamorphosis. Excerpta Med. Sect. 3 30, 234. (Abstract 1231)
FROG
283
THYROID
Sower, S. A. Plisetskaya, E., and Gorbman A. (1985). Steroid and thyroid hormone profiles following a single injection of partly purified salmon gonadotropin or GnRH analogues in male and female sea lamprey. J. Exp. Zool. 235, 403-408. Suzuki, S., and Suzuki, M. (1981). Plasma thyroxine and triiodothyronine levels during and after metamorphosis of amphibians. In “Proceedings, Ninth Int. Symp. Comp. Endocrinol., Hong Kong,” Abstr. PlO. Taurog, A., Oliver, C., Eskay, R. L., Porter, J. C., and McKenzie, J. M. (1974). The role of TRH in the neoteny of the mexican axolotl (Ambystoma mexicunum).
Gen.
Comp.
Endocrinol.
24, 267-
279. Thornton, V. F., and Geschwind, I. I. (1974). Hypothalamic control of gonadotropin release in Amphibia: Evidence from studies of gonadotropin release in vitro and in vivo. Gen. Comp. Endocrinol. 23, 294-301. Vandesande, F., and Aspeslagh, M. R. (1974). Failure of thyrotropin-releasing hormone to increase ‘25I uptake by the thyroid in Runu temporuriu. Gen Comp.
Endocrinol.
23, 355-356.
Vandorpe, G., Jacobs, G. F. M., and Kuhn, E. R. (1987). Seasonal changes of the 5’-monodeiodination activity in kidney and skin homogenates of male Runu ridibundu: Relation to plasma thyroxine (T4) and testosterone. Gen. Comp. Endocrinol. 68, 163-169.
AND
COMPARATIVE
Luteinizing
G.F.M.
ENDOCRINOLOGY
Hormone-Releasing Hormone as a Potent Stimulator the Thyroidal Axis in Ranid Frogs
JACOBS, M.P.
Laboratory
70, 274-283 (1988)
of Comparative
GOYVAERTS,G.VANDORPE, E.R. ICOHN Endocrinology, Naamsestraat
A.M.L.
Zoological Institute, Catholic 61, B-3000 Leuven, Belgium
of
QUAGHEBEUR,AND
University
of Leuven,
Accepted December 9, 1987 Plasma concentrations of T4, measured by radioimmunoassay, were raised significantly 2 and 4 hr after intravenous injection of synthetic luteinizing hormone-releasing hormone (LHRH) in Rana ridibunda (1 and 10 kg on 2 consecutive days) and in Rana esculenta (10 kg). A dose of 1 pg LHRH was not so effective as 50 pg synthetic thyrotropin-releasing hormone (TRH) when injected in Rana ridibunda in November. However 10 pg LHRH was equipotent to 50 kg TRH. In February somewhat less than half of the Rana temporaria group was responsive to LHRH. There is no clear indication that fluctuating plasma T, concentrations were caused by LHRH or TRH. Preinjection levels of T, and T, were higher during the breeding season (April) in R. esculenta (resp. 35.4 + 1.4 pg/ml; 744 + 134 pglml; II = 22) compared to the basal concentrations in the very closely related Rana ridibunda (November) (resp. 15.2 _+ 1.1; 162 * 24 pg/mi; n = 28). Four days after removal of the pars distalis plasma T4 concentrations were significantly decreased in Rana esculenta, whereas T, could stay longer in circulation. T, and T4 content of the thyroids was not altered by the short-term hypophysectomy. Injection of 10 pg LHRH had no influence on plasma T4 nor testosterone concentrations in these frogs, contrary to the sham-ectomized animals in which plasma testosterone remained elevated longer than T,. The results suggest that the stimulatory effect of intravenous injected LHRH on thyroid (and gonadal) activity in the frog is primarily mediated through the hypophysis. They also point to a possible correlation between the gonadal and thyroidal axis. o 1988 Academic Press, Inc.
In teleostean fishes, a thyroid-gonadal interrelationship is well evidenced by the presence of a maturational effect of thyroxine upon the gonads and the existence of cycles of thyroid activity correlated with reproduction (Sage, 1973; Hurlburt, 1977; Chakraborti and Bhattacharya, 1984). Also, the occurrence of such a relationship has been suggested in amphibians (Dickhoff and Darling, 1983; Kuhn et al., 1987). In a recent experiment performed on Rana ridibunda, a sharp increase in the plasma T4 concentration associated with a first elevation of plasma testosterone was observed at the end of hibernation, and a maximal 5 ‘-monodeiodinating activity, as measured in kidney homogenates, was reached during the breeding season (April-May-June)
(Vandorpe et al., 1987). Suzuki and Suzuki (1981) detected the highest T4 concentration in two urodeles (Hynobius and Onychoductylus) also just before the breeding period. It is generally believed that the mammalian LHRH (luteinizing hormone-releasing hormone), which is the major form of GnRH (gonadotropin-releasing hormone) in the adult amphibian brain (Sherwood et al., 1986), stimulates the release of gonadotropins (FSH and LH) from the pituitary in vivo and in vitro (Thornton and Geschwind, 1974; Daniels and Licht, 1980; Licht et al., 1984; Porter and Licht, 1986), while TRH (thyrotropin-releasing hormone), which is also abundantly present in the frog hypothalamus and brain (Jackson and Reichlin, 274
0016~6480/88 $1 so Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
LHRH
STIMULATES
1974; Jackson and Bolaffi, 1983) has been proposed as a possible thyrotropinreleasing factor in the frog Rana ridibunda (Dar-r-as and Kuhn, 1982) and in some cases also in the metamorphosed axolotl Ambystoma mexicanum (Jacobs et al., 1988). Other positive effects obtained with the tripeptide (published mostly in abstract form) concerning acceleration of metamorphosis in tadpoles (Shiomi et al., 1974; Schultheiss, 1978), enhanced thyroid hormone secretion in thyroid-hypophysis cocultures (Etkin and Kim, 1971; Peyrot et al., 1974), and increased thyroid iodine release in adult Bufo bufo (Rosenkilde, 1979) also conflict, however, with numerous negative in vivo and in vitro reports on TRH (Etkin and Gona, 1968; Gona and Gona, 1974; Taurog et al., 1974; Vandesande and Aspeslagh, 1974). The hypothesis proposed by Sage (1973) that the control of TSH evolved from the control of an original gonadotropin (which may have influenced thyroid activity), together with the observation that injection of a LHRH analog raised plasma thyroxine concentration in female sea lamprey (Sower et al., 1985), prompted us to examine whether LHRH would be able to stimulate the pituitary-thyroid axis in anuran Amphibia. MATERIAL
AND METHODS
The experiments were performed on male frogs obtained from R. Stein (Lauingen, GFR) (Rana ridibunda and Rana temporaria) or from the Centre d’elevage d’animaux de laboratoire d’Ardenay (Le Breil sur Merize, France) (Rana esculenta). Prior to the experimental period, the animals were kept in plastic tanks in a nonheated room with open windows and outside photoperiod for at least 1 month, and they were fed on mealworms once a week. The mean maximum and minimum temperature (_+standard deviation) to which the animals had been exposed since their arrival in the laboratory was respectively 19 f 2” and 17 + 3” for R. ridibunda, 13 * 3” and 9 -+ 3” for R. temporaria, and 16 + 2” and 12.5 + 2” for R. esculenta. In the morning before the experiment, the frogs were anesthetized in a 0.16% MS 222 solution (tricaine methane sulfonate) and their abdominal vein was can-
FROG
THYROID
275
nulated (in both directions) for injection of synthetic TRH, s-LHRH (both from UCB, Bioproducts), or frog Ringer solution (in a 100 ~1 volume) as well as for several blood samplings of about 0.35 ml, the (2) following day(s). Each time after centrifugation, the blood cells were suspended again in frog Ringer solution (also taking the volume of the canula into account) and reinjected. Three experiments were performed, all at room temperature (19-20”). 1. The first experiment was performed in the beginning of November and Rana ridibunda with a mean body weight of 61.9 + 1.9 g (n = 28) were used. The frogs were injected on 2 consecutive days with either frog Ringer solution (controls, n = lo), 50 p,g s-TRH (n = 8), or two different doses of s-LHRH (1 and 10 kg, n = 10). Blood samples were taken before and at 2, 4, and 7 hr after each injection. 2. The second experiment was performed at the end of February and Rana temporaria weighing 30.1 + 1.8 g (n = 23) were used. Because the body weight of these frogs was 50% lower, a dose of 0.5 and 5 Fg of LHRH was administered consecutively (n = 13). Blood was sampled before and at 2, 4, 8, and 24 hr after the first injection, and at 2 and 4 hr after the second one. 3. A third series of experiments using Rana esculenta with a mean body weight of 50.2 + 1.Og (n = 46) was performed in the first half of April. Hypophysectomy (pars distalis) and sham operation were carried out, while working under binocular, 4 days before injection. At the level of the pituitary, the palate bone was ground until it became thin and could be loosened by 75%. After the pars distalis was removed, the little palate piece was returned for covering the hole and the skin was sutured. Hypophysectomized and sham frogs received 10 kg LHRH (resp. n = 13; 13) or 100 ~1 Ringer (resp. n = 11; 9) and blood was collected before and at 2, 4, and 7.5 hr after injection. The plasma samples were extracted with acetone (according to Darras and Kuhn, 1982) prior to RIA of T, and T,. Hormone concentrations were assayed by using tracer obtained from Amersham International (UK), rabbit T, antiserum from Mallinckrodt (GFR) and a laboratory-raised rabbit T, antiserum (final titer: l/8250). T, and T, antisera did not show any crossreactivity for respectively T4 concentrations till 5 ng/ ml and T, concentrations up to 2 nglml frog plasma. Radioimmunoassay of testosterone as described previously (Vandorpe et al., 1987), was only performed in the samples of Rana escuienta. At the end of the experiments, the frogs were killed and the thyroid glands were carefully excised under binocular. After homogenizing the glands with a Teflon hand-homogenizer in a 0.01 M sodium phosphate buffer (pH 7.6), they were treated as described by Darras and Kuhn (1982). Subsequently T3 and T4 content was measured by RIA. Cross-reactivity of the T, antiserum (Mallinckrodt, GFR) for T, was 0.3% and final
276
JACOBS
T, data were corrected. Because of the small T,/T4 ratio in the thyroid, the cross-reactivity of the T, antiserum (commercial kit from Abbott Laboratories) for T, was not considered as being significant. Statistical analysis of data was made by one-way analysis of variance, ANOVA (for comparison between two groups) or paired t test (for comparison within the same group of animals).
RESULTS 1. Influence of Intravenous TRH and LHRH Injections on Plasma Concentrations of T4 and T3 a. Rana ridibunda. Following injection of 50 pg TRH circulating control levels of T4 (212 k 53 pg/ml; n = 8) were raised about 6.5 times (up to 1420 + 277 pg/ml) within 2 hr (P < 0.005 compared to Ringerinjected frogs) and concentrations remained significantly elevated during 4 hr. Basal values were obtained again after 24 hr and at that time a new injection of 50 kg TRH caused a similar increase of T4 (Fig. 1). In a second group of frogs (n = 10) 1 pg LHRH was responsible for a fourfold elevation of the plasma T4 concentration 2 hr after injection (P < 0.005 compared to the Ringer controls). Moreover, the effect of 10 pg LHRH the next day was equally pronounced as that caused by 50 pg TRH. Values of T4 rose from 137 + 62 to 1273 k 311 pg/ml 2 hr after injection (P < 0.005) and also stayed significantly elevated after 4 hr. While a higher T4 level (P < 0.05) was still present at the end of the TRH experiment (7 hr), this was not the case after LHRH injection. Concerning the T3 plasma concentrations, which were highly variable during the whole period studied, no difference could be found between the controls and the TRH and LHRH injected animals (ANOVA). Notwithstanding the fact that a significant increase in the T, plasma level was noted 4 hr after administration of 1 pg LHRH (P < 0.025), the same effect was seen in the Ringer-injected frogs (P < 0.05 paired t test) (Fig. 1).
ET AL.
b. Rana temporaria. Control animals showed fairly constant T, plasma concentrations (90-100 pg/ml) during the whole experiment . Injection of 0.5 kg LHRH did not influence T, levels whereas 5 kg LHRH was able to induce T, release in four of the nine frogs studied the second day. Because of the considerable high standard errors found 4 hr following injection, comparison with the controls could only reveal a significant increase after 2 hr (P < 0.01). It must also be noted that T, plasma levels were slightly elevated (P < 0.05) before the second injection (Fig. 2). As in the first experiment basal T, concentrations in R. temporaria (18.6 f 1.2 pg/ ml; n = 23) were not effected by the LHRH injections. c. Rana esculenta. Circulating levels of T, (744 k 134 pg/ml; n = 22) were significantly decreased (P < 0.005) 4 days after removal of the pars distalis (195 + 9 pg/ml; n = 24). An injection of 10 pg LHRH increased T, concentrations to about 2000 pg/ ml in sham-ectomized frogs after 2 hr (Fig. 3), whereas basal values were reached again after 7.5 hr. Plasma T, levels remained unchanged in the hypophysectomized animals when compared to their controls. Comparing preinjection T, values of all hypophysectomized frogs (31.2 + 0.8 pg/ ml; n = 24) to those of sham animals (35.4 + 1.4 pg/ml; n = 22) a significant difference (caused by the hypophysectomized frogs going to receive LHRH) was noted (P < 0.025). Two hours after injection of 10 pg LHRH plasma T, concentrations were significantly raised in sham and hypox frogs as evaluated by paired t test (P < 0.005) but compared to their controls no differences were present (Fig. 3). 2. Influence of Intravenous LHRH Injections on Plasma Concentrations of Testosterone
This hormone was only measured in the samples of the third experiment (Fig. 3).
LHRH
STIMULATES
FROG
277
THYROID
hours T
1
after
injection
**tlr
0.6 -
FIG . 1. Plasma concentrations of T, and T, (means * SEM) in Rana ridibundu following injection of frog Ringer solution (0; n = 10 unless indicated otherwise on the figure), 50 pg TRH (A; n = 8), or 1 and 10 kg LHRH (0; n = 10) on 2 consecutive days. *P < 0.05; ** P < 0.01; *** P < 0.005 (ANOVA).
Hypophysectomy resulted in a slight decrease (P < 0.05) of the morning testosterone concentrations 4 days after the operation (3.16 _+ 0.41; n = 20 vs. 2.08 + 0.28; n = 21). Two hours following injection of 10 bg LHRH a threefold increase in plasma concentrations of testosterone was observed in sham-ectomized frogs (up to 10.3 2 1.2 rig/ml). This high value was main-
tained during 2 hr, and was still significantly elevated from controls 7.5 hr after injection (P < 0.005). No influence on testosterone levels was found in hypox animals following injection of LHRH. 3. T3 and T4 content of the thyroid gland A survey of the thyroid
hormone
con-
278
JACOBS
1
0
2
ET AL.
I
4
8
I,
II
I
I
I
h204u/fs etter21n,ectlo:
FIG . 2. Plasma concentrations of T4 (means + SEM) in Rana temporaria following injection of frog Ringer solution (0; n = 10 unless indicated otherwise on the figure) or 0.5 and 5 pg LHRH (0; n = 13) on 2 consecutive days. * P < 0.05; ** P < 0.01 (ANOVA).
tents, measured 7 hr (first experiment), 4 hr (second experiment), and 7.5 hr (third experiment) after injection, is presented in Table 1, together with the respective body weights . The amount of glandular T, was not influenced by any of the experimental treatments used. T, content assayed in the glands of the control R. ridibundu frogs was significantly higher (P < 0.05) compared to the LHRH-(but not TRH-) injected animals. However this effect was no longer seen in R. esculenta nor R. temporaria. DISCUSSION
The present study indicates that intravenously injected mammalian LHRH is able to raise circulating levels of T, (but not of T3) in three frog species. As expected, plasma testosterone concentrations were elevated as well (in R. esculenta) (McCreery et al., 1982; Moore et al., 1982; Licht et al., 1984). Synthetic TRH also caused an increase in plasma T4 when injected in R. ridibunda, confirming a previous study (Dar-r-as and Kuhn, 1982) and in R. temporaria (Darras and Kuhn, unpublished observation). Since only two different doses of LHRH (1 and 10 pg) and one dose of TRH (50 pg) were tested (first experiment), no definite
conclusions regarding the relative potencies of LHRH and TRH can be drawn. The fact that 8.5 nmol LHRH (10 pg) was as effective as 138 nmol TRH (50 pg) in R. ridibunda in November may be an important observation. However, one has to consider the possibility that 50 pg TRH may also have acted in an inhibitory way regarding lower effective doses. Seasonal variations in the response caused by LHRH and TRH may occur as well, but our study does not permit any conclusions in this regard. On the other hand, our results indicate that in R. ridibunda 1 pg LHRH was also sufficient for stimulating the release of T4 (fourfold elevation of the preinjection value after 2 hr), and since in a previous study (Darras and Kuhn, 1982) it was found that the minimal effective dose of TRH in the same species was also around 1 kg (3.3-fold elevation of the preinjection value), one may conclude that LHRH is at least as potent as TRH to increase plasma T, concentrations in this species. Immunological and chromatographic evidence suggests that the mammalian form of GnRH is present in the anuran brain (Alpert et al., 1976; Eiden and Eskay, 1980; King and Millar, 1980; Sherwood, 1986; Sherwood et al., 1986). It is known that 1 pg of LHRH will cause ovulation in Xenopus (Thornton and Geschwind, 1974). In R. es-
LHRH
STIMULATES
FROG
279
THYROID
7.5
4
hours
Testosterone nglml
after
InJectlo~
T
10 -
8-
6-
4-
0.2
-i
-sc=pm-=$--==== lo t b ’ 0
2
4
0 hours
after
lnjectk.%
2
4
hours
after
InjectI::
FIG. 3. Plasma concentrations of T,, T4, and testosterone (means rt SEM) in pars distalis (- - -) and sham-ectomized (-) frogs (Rana esculenta) following injection of 10 kg LHRH (0; n = resp. 13, 13) or frog Ringer solution (0; II = resp. 11, 9, unless indicated otherwise on the figure). * P < 0.05; *’ P < 0.025; *** P < 0.005 (ANOVA).
culenta a total of three subcutaneous injections, each containing 300 ng of a longacting LHRH analog, raised the testicular testosterone content, an effect which was abolished after hypophysectomy (Pierantoni et al., 1984). Our hypophysectomy experiments on R. esculenta revealed that the elevation of plasma testosterone was mediated through release of a gonadotropin,
probably LH (Licht, 1979), and not via direct action of LHRH on the steroidogenesis in the testis as was demonstrated in vitro in R. pipiens (Segal and Adjuwon, 1979). In the bullfrog (5450 g) a dose of 2.5 and 5 pg LHRH was able to increase plasma gonadotropins (FSH and LH), but its duration of action was short (less than 1 hr) (Daniels and Licht, 1980). In view of these results, a
280
JACOBS
lo-pg dose of LHRH as used in R. escuZenta in order to increase T4 and testosterone must be considered high, whereas 1 pg as used in R. ridibunda would be more physiological. The results clearly indicate that LHRH stimulates the release of T, via the pituitary. There are however several possibilities for the peptide to exert its action. (a) A direct effect of testosterone upon the thyroid gland (or pituitary) should not be excluded since a stimulatory influence of sex steroids on thyroid activity has been reported in teleostean fishes (Sage, 1973; Chakraborti and Bhattacharya, 1984), the chick embryo (Hoshino et al., 1987), and in mammals (see Galton, 1971). However, the fact that in our study using R. esculenta testosterone and T4 were released simultaneously after LHRH injection and that T4 was already decreasing 4 hr after injection to reach control values when the testosterone level was still significantly elevated is not favorable for this hypothesis. (b) LHRH may act as a neurotransmitter to effect other hypothalamic hormones, e.g., TRH, leading to TSH secretion. A small amount of salmonid-like GnRH has been detected in the brain of some anuran and urodelan species (Sherwood et al., 1986). This GnRH is similar to that found in the bullfrog retina (Eiden et al., 1982) and sympathetic ganglia (Eiden and Eskay, 1980; Jan and Jan, 1982; Jones et al., 1984) and has been proposed to have a neurotransmitter function (Sherwood et al., 1986). (c) Paracrine interactions in the pituitary may lead to an LHRH-induced activation of the thyrotropes. (d) LHRH may stimulate the thyrotropes directly, apart from the gonadotropes. Since in this assumption TSH, LH, and FSH will be increased, the animal should be capable of distinguishing between endogenous thyrotropins and gonadotropins. In the bullfrog it was shown that homologous LH and FSH had relatively lit-
ET AL.
tle effect on the thyroid gland, while homologous TSH stimulated thyroxine release and accelerated metamorphosis (MacKenzie et al., 1978). In addition MacKenzie and Licht (1984) found no clear indication that the thyroid gland of the leopard frog (Rana pipiens) or the tree frog (Hyla regilla) would respond to bullfrog LH. So it could be possible that the receptors on the thyrotropes of a frog do not discriminate between TRH and LHRH (e.g., by reacting with the identical pyroglutamyl-histidyl residue), that two different kinds of receptors are present on the same cell, or that two types of TSH cells occur. The last two possibilities would give the animal the opportunity to use its thyroidal and gonadal axis simultaneously as well as independently if necessary (e.g., during metamorphosis or short periods of greater T, need). Injection of LHRH into R. temporaria did not cause the same pronounced T, increase as in the other frogs, although about the same doses, relative to the body weight, were used. Only the higher concentration (5 pg/animal) was effective in four of the nine animals tested. This may be due to species or seasonal differences in receptor sensitivity or number. While plasma T, concentrations were very significantly decreased 4 days after removal of the pars distalis in R. esculenta, the influence on plasma T, levels was much slower, probably because they had been sustained by peripheral T4 conversion. The rise of circulating T, which was noticed (paired t test) 4 hr after injection of Ringer and LHRH in R. ridibunda, and 2 hr after administration of LHRH in shamectomized R. esculenta may be caused by the existence of a circadian rhythm in the T,-S-monodeiodinating activity. Kidneys dissected in the evening from winterfrogs (adapted to room temperature) exhibited a higher T4 converting activity in vitro than when removed in the morning or at noon (P < 0.001) (Jacobs and Kuhn, unpublished observation). In addition, in the hypophy-
LHRH
STIMULATES
FROG
TABLE T4 AND T, CONTENT
Expt. 1: R. ridibunda Ringer LHRH TRH Expt. 2: R. temporaria Ringer LHRH Expt. 3: R. esculenta Sham: Ringer LHRH Hypophysectomized:
OF THE THYROID
Ringer LHRH
281
THYROID
1
GLANDS REMOVED (SEE RESULTS)
AT THE END OF EACH EXPERIMENT
Body weight (g)
T, (rig/thyroid)
T3 (&thyroid)
60.7 + 3.5” (10)’ 63.7 + 3.0 (10) 61.0 ? 3.7 (8)
1858 + 323 (9) 1357 + 160 (9) 1502 k 295 (8)
13.1 + 1.7 (9) 9.0 + 0.8 (9) 8.8 + 1.3 (8)
28.1 + 2.7 (10) 31.7 k 2.4 (13)
837 + 93 (8) 947 k 151 (9)
9.7 2 1.4 (8) 8.2 2 1.2 (9)
50.3 49.5 49.4 51.5
2 2 k +
2.5 (9) 1.6 (13) 2.0 (11) 2.3 (13)
1441 1339 1785 1438
+ 224 (9) + 130 (13) +- 195 (9) + 172 (13)
3.6 4.0 6.5 4.3
2 2 k 2
0.8 0.6 1.1 0.9
(9) (13) (9) (13)
a Means + SEM. ’ Number of animals.
sectomized frogs fluctuating T, concentrations were also observed, whereby the original slight difference between hypox and sham animals was abolished. The results of the present experiments may again raise the question of whether GnRH was the original controlling hormone of a gonadotropin which influenced both thyroid gland and gonads, while TRH evolved later to control a gonadotropindeduced thyrotropin. Until now only few data supported this hypothesis. In the female sea lamprey plasma T4 was raised after injection of a LHRH analog (Sower et al., 1985). However, other studies demonstrated that in fish, a rise in blood gonadotropin after injection of LHRH was not accompanied by a change in thyroid activity (Brown et al., 1985; MacKenzie et al., 1987). Perifused pituitaries of turtles did not secrete TSH during LHRH stimulation (Licht and Porter, 1985). It is also generally accepted that the mammalian thyrotrope is not directly stimulated by LHRH. Only after chronic LHRH treatment a slight increase in plasma T4 was observed, perhaps due to changes in ovarian estrogen secretion (Fraser, 1983). ACKNOWLEDGMENTS The skillful technical assistance of Mrs. F. Voets,
Ms. L. Noterdaeme, and Mr. W. Van Ham is gratefully acknowledged. G. Jacobs was supported by the National Fund for Scientific Research (Belgium). G. Vandorpe was supported by the Belgian IWONL (Instituut tot aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw).
REFERENCES Alpert, L. C., Brawer, J. R., Jackson, I. M. D., and Reichlin, S. (1976). Localization of LHRH in neurons in frog brain (Rana pipiens and Rana catesbeiana). Endocrinology 98, 910-921. Brown, C. L., Grau, E. G., and Stetson, M. H. (1985). Functional specificity of gonadotropin and thyrotropin in Fundulus heteroclitus. Gen. Comp. Endocrinol.
58, 252-258.
Chakraborti, P., and Bhattacharya, S. (1984). Plasma thyroxine levels in freshwater perch: Influence of season, gonadotropins, and gonadal hormones. Gen. Comp. Endocrinol. 53, 179-186. Daniels, E., and Licht, P. (1980). Effects of gonadotropin-releasing hormone on the levels of plasma gonadotropins (FSH and LH) in the bullfrog, Rana
catesbeiana.
Gen.
Comp.
Endocrinol.
42,
455-463. Darras, V. M., and Kuhn, E. R. (1982). Increased plasma levels of thyroid hormones in a frog Rana ridibunda following intravenous administration of TRH. Gen. Comp. Endocrinol. 48, 469-475. Dickhoff, W. W., and Darling, D. S. (1983). Evolution of thyroid function and its control in lower vertebrates. Amer. Zool. 23, 697-707. Eiden, L. E., and Eskay, R. L. (1980). Characterization of LRF-like immunoreactivity in the frog sympathetic ganglia: Non-identity with LRF decapeptide. Neuropeptides 1, 29-37.
282
JACOBS
Eiden, L. E., Loumaye, E., Sherwood, N., and Eskay, R. L. (1982). Two chemically and immunologically distinct forms of luteinizing hormonereleasing hormone are differentially expressed in frog neural tissues. Peptides 3, 323-327. Etkin, W., and Gona, A. G. (1968). Failure of mammalian thyrotropin-releasing factor preparation to elicit metamorphic responses in tadpoles. Endocrinology 82, 1067-1068. Etkin, W., and Kim, Y. S. (1971). Effects of TRH on tadpole tissue. Amer. Zool. 11, 654. (Abstract 148) Fraser, H. M. (1983). Effect of treatment for 1 year with a luteinizing hormone-releasing hormone agonist on ovarian, thyroidal, and adrenal function and menstruation in the stumptailed monkey (Mucca
arctoides).
Endocrinology
112, 245-253.
Galton, V. A. (1971). Thyroxine metabolism and thyroid function in rats following administration of estrogen. Endocrinology 88, 976-982. Gona, A. G., and Gona, 0. (1974). Failure of synthetic TRF to elicit metamorphosis in frog tadpoles or red-spotted newts. Gen. Comp. Endocrinol. 24, 223-225. Hoshino, S., Huybrechts, L. M., Darras, V. M., Decuypere, E., and Kuhn, E. R. (1987). Thyrotrophic and peripheral activity of estradiol in the chick embryo. Med. Sci. Res. 15, 1409-1410. Hurlburt, M. E. (1977). Role of the thyroid gland in ovarian maturation of the goldfish, Carussius uurutus L. Cunud. J. Zoo/. 55, 1906-1913. Jackson, I. M. D., and Bolaffi, J. L. (1983). Phylogenetic distribution of TRH: Significance and function. In “Thyrotropin-releasing hormone” (E. C. Griffiths and G. W. Bennett, Eds.), p. 191-202. Raven Press, New York. Jackson, I. M. D., and Reichlin, S. (1974). Thyrotropin-releasing hormone (TRH): Distribution in hypothalamic and extrahypothalamic brain tissue of mammalian and submammalian Chordates. Endocrinology
95, 854862.
Jacobs, G. F. M., Michielsen, R. P. A., and Kuhn, E. R. (1988). Thyroxine and triiodothyronine in plasma and thyroids of the neotenic and metamorphosed axolotl Ambystomu mexicunum: Influence of TRH injections. Gen. Comp. Endocrinol 70, 145-151. Jan, L. Y., and Jan, Y. N. (1982). Peptidergic transmission in sympathetic ganglia of the frog. J. Physiol.
327, 219-246.
Jones, S. W., Adams, P. R., Brownstein, M. J., and Rivier, J. E. (1984). Teleost luteinizing hormonereleasing hormone: Action on bullfrog sympathetic ganglia is consistent with role as neurotransmitter. J. Neurosci. 4, 420-429. King, J. A., and Millar, R. P. (1980). Comparative aspects of luteinizing hormone-releasing hormone
ET AL.
structure and function in vertebrate phylogeny. Endocrinology
106, 707-7 17.
Kuhn, E. R., Gevaerts, H., Jacobs, G., and Vandorpe, G. (1987). Reproductive cycle, thyroxine and corticosterone in females of the giant swamp frog Dicroglossus occipitalis at the equator. Gen. Comp. Endocrinol. 66, 137-144. Licht, P. (1979). Reproductive endocrinology of reptiles and amphibians: Gonadotropins. Annu. Rev. Physiol.
41, 337-351.
Licht, P., Millar, R., King, J. A. McCreery, B. R., Mendonca, M. T., Bona-Gallo, A., and Lofts, B. (1984). Effects of chicken and mammalian gonadotropin-releasing hormones (GnRH) on in vivo pituitary gonadotropin release in amphibians and reptiles. Gen. Comp. Endocrinol. 54, 89-96. Licht, P., and Porter, D. A. (1985). In vivo and in vitro responses to gonadotropin releasing hormone in the turtle, Chrysemys pictu, in relation to sex and reproductive stage. Gen. Comp. Endocrinol. 60, 75-85. MacKenzie, D. S., and Licht, P. (1984). Studies on the specificity of thyroid response to pituitary glycoprotein hormones. Gen. Comp. Endocrinol. 56, 156-166. MacKenzie, D. S., Licht, P., and Papkoff, H. (1978). Thyrotropin from amphibian (Ranu catesbeiana) pituitaries and evidence for heterothyrotropic activity of bullfrog luteinizing hormone in reptiles.Gen.
Comp.
MacKenzie, D. Breton, B. in goldfish ing thyroid
Endocrinol.
36, 566-574.
S., Sokolowska, M., Peter, R. E., and (1987). Increased gonadotropin levels do not result in alterations in circulathormone levels. Gen. Comp. Endocrinol. 67, 202-2 13. McCreery, B. R., Licht, P., Barnes, R., Rivier, J. E., and Vale, W. W. (1982). Actions of agonistic and antagonistic analogs of gonadotropin releasing hormone (Gn-RH) in the bullfrog Runu cutesbeiunu. Gen. Comp. Endocrinol. 46, 511-520. Moore, F. L., Miller, L. J., Spielvogel, S. P., Kubiak, T., and Folkers, K. (1982). Luteinizing hormonereleasing hormone in the reproductive behavior of a male amphibian. Neuroendocrinology 35, 212216. Peyrot, A., Pons, G., and Vellano, C. (1974). Etude du metaboiisme thyroi’dien du triton Crete par l’association in vitro de thyroide et d’hypophyse. Effet de la TRH. Gen. Comp. Endocrinol. 22, 374. (Abstract 106) Pierantoni, R., Iela, L., d’Istria, M., Fasano, S., Rastogi, R. K., and Delrio, G. (1984). Seasonal testosterone profile and testicular responsiveness to pituitary factors and gonadotrophin releasing hormone during two different phases of the sexual cycle of the frog (Ranu eculentu). J. Endocrinol. 102, 387-392.
LHRH
STIMULATES
Porter, D. A., and Licht, P. (1986). Effects of temperature and mode of delivery on responses to gonadotropin-releasing hormone by superfused frog pituitaries. Gen. Comp. Endocrinol. 63, 236-244. Rosenkilde, P. (1979). The thyroid hormones in Amphibia. In “Hormones and Evolution” (E. J. W. Barrington, Ed.), Vol. I, p. 437-491. Academic Press, New York. Sage, M. (1973). The evolution of thyroidal function in fishes. Amer. Zool. 13, 899-905. Schultheiss, H. (1978). The influence of purified amphibian pituitary hormones, pituitary grafts and TRH on thyroid activity and metamorphosis. In “Proceedings, Eighth Int. Symp. Comp. Endocrinol., Amsterdam,” Abstr. p. 75. Segal, S. J., and Adejuwon, C. A. (1979). Direct effect of LH-RH on testicular steroidogenesis in Rana pipiens. Biolog. Bull. 157, 393-394. Sherwood, N. M. (1986). Evolution of a neuropeptide family: Gonadotropin-releasing hormone. Amer. Zoo/. 26, 1041-1054. Sherwood, N. M., Zoeller, R. T., and Moore, F. L. (1986). Multiple forms of gonadotropin-releasing hormone in amphibian brains. Gen. Comp. Endocrinol. 61, 313-322. Shiomi, K., Hachiya, T., Yoshimura, M. (1974). Effect of synthetic TRH (thyrotropin releasing hormone) on tadpole metamorphosis. Excerpta Med. Sect. 3 30, 234. (Abstract 1231)
FROG
283
THYROID
Sower, S. A. Plisetskaya, E., and Gorbman A. (1985). Steroid and thyroid hormone profiles following a single injection of partly purified salmon gonadotropin or GnRH analogues in male and female sea lamprey. J. Exp. Zool. 235, 403-408. Suzuki, S., and Suzuki, M. (1981). Plasma thyroxine and triiodothyronine levels during and after metamorphosis of amphibians. In “Proceedings, Ninth Int. Symp. Comp. Endocrinol., Hong Kong,” Abstr. PlO. Taurog, A., Oliver, C., Eskay, R. L., Porter, J. C., and McKenzie, J. M. (1974). The role of TRH in the neoteny of the mexican axolotl (Ambystoma mexicunum).
Gen.
Comp.
Endocrinol.
24, 267-
279. Thornton, V. F., and Geschwind, I. I. (1974). Hypothalamic control of gonadotropin release in Amphibia: Evidence from studies of gonadotropin release in vitro and in vivo. Gen. Comp. Endocrinol. 23, 294-301. Vandesande, F., and Aspeslagh, M. R. (1974). Failure of thyrotropin-releasing hormone to increase ‘25I uptake by the thyroid in Runu temporuriu. Gen Comp.
Endocrinol.
23, 355-356.
Vandorpe, G., Jacobs, G. F. M., and Kuhn, E. R. (1987). Seasonal changes of the 5’-monodeiodination activity in kidney and skin homogenates of male Runu ridibundu: Relation to plasma thyroxine (T4) and testosterone. Gen. Comp. Endocrinol. 68, 163-169.