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Diurnal and seasonal variations in thyroid function of neotenic tiger salamanders (Ambystoma tigrinum)
GENERAL
AND
Diurnal
COMPARATIVE
ENDOCRINOLOGY
45, 134- 137 (1981)
and Seasonal Variations in Thyroid Tiger Salamanders (Ambysfoma
Function tigrinum)
of Neotenic
No dirunal variations for plasma thyroxine (TJ in neotenic tiger salamander larvae were apparent in October, and T, levels at 1200, 1800, and 2400 hr exceed those observed in May. A significant response to ovine thyrotropin (TSH) occurred only at 0600 hr in October. Plasma T, and triiodothyronine (TJ were greatest in May at 0600 and 1800 hr, respectively, and ovine TSH significantly elevated T, and T, levels at all times except for T, at 0600 when the maximal T4 level was observed. Maximal endogenous T, (May) and maximal sensitivity to TSH (October) occurred at the end of the scotophase when plasma melatonin levels were greatest. Circulating levels of melatonin appear to be positively associated with thyroid function in this species.
The scotophasic release of pineal melatonin has been reported for many vertebrates (see Ellis, 1976) in recent years. The responsiveness of certain tissues or inductions of certain behaviors might depend upon the phasic relationships for secretion of two or more hormones (see Meier et al., 1978), and the importance of diurnal patterns in hormone secretion and target sensitivity are now recognized. We present here some preliminary evidence for seasonal differences in diurnal variations of plasma thyroid hormone levels in sexually mature larvae (neotenes) of the tiger salamander, Ambystoma tigrinum, and their relationship to the responsiveness of the thyroid gland to exogenous mammalian thyrotropin (TSH). Furthermore, we provide evidence that endogenous melatonin may not be an inhibitor of pituitary TSH release in this species. MATERIALS
AND METHODS
Sexually mature neotenes were collected in October (experiment 1) or May (experiment 2) from a small pond near Boulder, Colorado (elevation 1700 m), and were transported immediately to the laboratory. After weighing, each animal was placed in a separate, clear-plastic disposable mouse cage containing approximately 1.5 liters aged tap water. The animals were assigned to groups systematically to achieve similar mean body weights and equal sex ratios for each group and then were placed in incubators at 15”
and subjected to a 12L:12D artificial photoperiod (photophase: 0600- 1800 hr). Experiment 1. In October, eight groups of neotenes (n = 7-9) were prepared with similar mean body weights (94.0 * 2.24 to 109.3 2 2.28 g). Two groups were assigned randomly to each time (0600, 1200, 1800, or 2400 hr) for injections and sacrifice. One group at each time received daily intraperitoneal injections of 0.15 pg ovine TSH (NIH-TSH-S6, 2.47 USP units/mg) per gram of body weight in 0.05 ml vehicle (amphibian Ringer’s solution) for 3 days. The second group received vehicle for 3 days. Injections began at 0600 hr. We found earlier that there is a significant eIevation of both T3 and T, blood levels following three daily injections of TSH (Norris ef al., unpublished). This dose of ovine TSH has been shown previously to be ineffective at inducing metamorphosis in these animals when administered for longer periods (Norris et af., 1973). Animals in each group were killed and exsanguinated exactly 24 hr after the third injection, and plasma thyroxine (TJ was determined by radioimmunoassay (RIA). Experiment 2. In May male and female neotenes were separated according to body weight into four groups (mean body weights 131.4 +- 7.15 to 134.2 + 8.03 g) and were injected daily for 3 days with 0.05 ml vehicle at 0600, 1200, 1800, and 2400 hr, respectively (beginning at 1800 hr). Each group was killed 24 hr following the time of the last injection, and plasma triiodothyronine (TJ, Tq, and melatonin levels were determined by RIA. A second batch of neotenes was separated similarly into four groups (n = 7) (mean body weights: 135.4 2 4.19 to 139.9 t 6.75 g). Animals in each group received daily intraperitoneal injections of ovine TSH (0.26 pg NIH-TSHS7, 3.87 USP units/mg) per gram of body weight in 0.05 ml vehicle for 3 days at 0600, 1200, 1800, and 2400 hr, respectively. This dose of TSH will not 134
0016-6480/81/090134-04$01.00/O Copyright @ 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
135
NOTES induce metamorphosis (Norris et al., 1973). Injections began at 1800 hr. Animals were sacrificed 24 hr after the third injection, and plasma T3 and T, were determined by RIA. Radioimmunoassay. Plasma levels of T3 and T, were determined for individual samples by a modified commercial RIA system as outlined by Norris et al. (1977). The accuracy of this assay has been verified recently by a direct RIA procedure (Norman and Norris, unpublished). Plasma values for melatonin were determined by a validated RIA and were previously reported by Gern and Norris ( 1979). Statistical analyses. Thyroid hormone-level data were examined by analysis of variance (ANOVA) followed by a multiple range test (Student-Neuman Keul’s). Analysis of the melatonin data was previously reported (Gem and Norris, 1979).
IO
0,
,
I
1
803 i 6.0E -E &40c
TSH
tp20-
Saline
RESULTS
Experiment I No significant variations in mean plasma T, levels occurred in October for salineinjected neotenes, but a significant response to ovine TSH was observed at 0600 hr [F(3,29) = 2.857, P < 0.051. Diurnal values for saline- and TSH-injected neotenes are depicted in Fig. 1. No differences were observed between males and females.
0’
I 1800 I
1 2400 I T
I 600 I
I 1200 I
Experiment 2 Plasma melatonin levels (Fig. 2) were significantly elevated during the scotophase
“l-r---l II
-i IO
E g 9-
51
\\
f
\\
Time
\\
FIG. 2. Diurnal variations in plasma thyroxine (T,) and triiodothyronine (TJ levels in saline- and TSHtreated neotenic Ambystomu tigrinum during May. Plasma levels of melatonin are also shown for salinetreated animals.
\\ \
I
600
I
I
1200 1800 Time of Day (hr 1
I
1
2400
FIG. 1. Diurnal variations in plasma thyroxine levels (TJ of saline- and TSH-injected neotenic Ambystomu tigrinum during October.
as evidenced by 2400- and 0600-hr samples (see Gern and Norris, 1979). Thyroxine levels in saline-treated animals were significantly elevated at 0600 hr [F(3,28) = 9.018, P < O.Ol] following the scotophasic elevation of plasma melatonin (see Fig. 2). There were no differences between 1200-, 1800-, or 2400-hr plasma samples for Tq. Max-
136
NOTES
imum T3 level (Fig. 2) was observed in 1800-hr samples [F(3,28) = 3.526, P < 0.051. Exogenous TSH had no effect on circulating T, at 0600 hr, but a significant elevation (P < 0.01) over levels determined in saline-treated neotenes was observed at 1200, 1800, 2400 hr (Fig. 2). There was no significant variation in T, levels of TSHtreated animals over time, however. Circulating T3 was elevated at all times as compared to saline-injected neotenes (P < O.Ol), and a significantly greater response occurred at 1200 hr with respect to other times [F(2,26) = 8.05, P < 0.011. No differences in the responses of males and females occurred among saline- or TSH-treated neotenes. DISCUSSION Thyroid
Hormone
Levels
Plasma T, levels in saline-treated neotenes were greater in October than in May similar to those reported by Norris et al. (1977) for freshly collected animals. Spontaneous metamorphosis (i.e., not induced by exogenous thyroid hormones or TSH) occurs with greater frequency in the fall (Platt, 1974) when endogenous levels are greater. In May there was a significant elevation of plasma T, at 0600 hr suggesting that endogenous TSH release occurred during the latter portion of the scotophase. A small but significant elevation of T3 was observed 6 hr after the T, peak (1800 hr) suggesting that much of the circulating T3 is derived peripherally from circulating T, (see Leloup and Bascaglia, 1977; Pittman, 1979). Ovine TSH produced a significant elevation of plasma T, in October as compared to saline-injected animals only at 0600 hr suggesting that the thyroid gland of these animals was most sensitive to exogenous TSH at the end of the scotophase. In May, ovine TSH caused a significant elevation of T4 except when the endogenous T4 levels were maximal (0600 hr) and the ability of the thyroid gland to release T4 was already
maximal. Furthermore, this suggests an endogenous release of TSH during the latter portion of the scotophase. In contrast, T3 levels were markedly increased by exogenous TSH at all times. The interpretation of this effect is not clear. Levels of T3 were not determined for October animals and no comparison is possible. Melatonin
and Thyroid Function
In May, Melatonin levels of salinetreated neotenes were ‘elevated during the scotophase. Although we do not have data for melatonin levels for these animals in October, we have determined in separate experiments (Gern and Norris, 1979; Gern and Norris, unpublished) that scotophasic levels of melatonin are significantly greater than photophasic levels in neotenes from this same population examined in October and November. Furthermore, it appears that all of the differences between photophasic and scotophasic melatonin levels are of pineal origin (Gern and Norris, 1979). Although there is some evidence for inhibition of thyroid activity by the pineal gland (Remy and Disclos, 1970; Gern and DeBoer, 1973; Norris and Platt, 1973), the patterns for plasma levels of thyroid hormone and sensitivity of the thyroid gland to exogenous TSH might suggest just the reverse: that melatonin stimulates TSH release or controls a rhythm that includes an activation of TSH secretion and/or thyroid gland sensitivity to TSH. The stimulatory effects of pinealectomy on thyroid function (iodide uptake) reported by Gern and DeBoer (1973) and Norris and Platt (1973) might be related to another pineal factor and not to melatonin. Prolactin reduces circulating thyroxine levels (Norris, 1978) and melatonin might influence prolactin release in these animals. The possibility that these are correlations with no causal relationship also remains. Additional studies obviously are needed to clarify this proposed causeeffect relationship, but these studies must also be concerned in more detail with the
137
NOTES
problems of diurnal and seasonal variations with respect both to secretions and to glandular sensitivities to modifying agents. ACKNOWLEDGMENTS The authors wish to thank Dr. Richard E. Jones for his critical discussions and reading of the manuscript, and the National Institutes of Health for the gift of ovine thyrotropin. We are also indebted to Dr. Mark Rollag and Dr. Gordon Niswender for the gift of melatonin antibody and to Dr. Niswender for the use of his RIA facility. This work was supported by NSF Grants PCM 7607832 and PCM 78-22335 to D.O.N. and NIH Grant NS-12257 to Charles L. Ralph.
REFERENCES Ellis, L. C., ed. (1976). Endocrine role of the pineal gland. Amer. Zool. 16, l- 101. Gem, W. A., and DeBoer, K. (1973) Pineal-thyroid relationships in neoteny. J. Colo. Wyo. Acad. Sci. 7, 32-33.
Gern, W. A., and Norris, D. 0. (1979). Plasma melatonin in the neotenic tiger salamander (Ambystoma tigrinum): Effects of photoperiod and pinealectomy. Gen. Comp. Endocrinol. 38, 393-
398.
Leloup, J., and Buscaglia, M. (1977). La triiodothyronine, hormone de la metamorphose des Amphibiens. C.R. Acad. Sci. D, 284, 2261-2263. Meier, A. H., Fivizzani, A. J., Spieler, R. E., and Horseman, N. D. (1978). Circadian hormone basis for seasonal conditions in the gulf kill&h, Fundulus grandis. In “Comparative Endocrinology” (P. J. Gaillard and H. H. Boer, eds.), pp. 141- 144. Elsevier/North-Holland, Amsterdam. Norris, D. 0. (1978). Hormonal and environmental factors involved in the determination of neoteny in urodeles. In, “Comparative Endocrinology” (P. J. Gaillard and H. H. Boer, eds.), pp. 109- 112. Elsevier/North-Holland, Amsterdam.
Norris, D. O., Duvall, D., Greendale, K., and Gem, W. A. (1977). Thyroid function in pre- and postspawning neotenic tiger salamanders (Ambystoma tigrinum). Gen. Comp. Endocrinol. 33, 512-517. Norris, D. O., Jones, R. E., and Cohen, D. (1973). Effects of mammalian gonadotropins (LH, FSH, HCG) and gonadal steroids on TSH-induced metamorphosis of Ambystoma tigrinum (Amphibia: Caudata). Gen. Comp. Endocrinol. 20, 467-473.
Norris, D. O., and Platt, J. E. (1973). Effects of pituitary hormones, melatonin, and thyroidal inhibitors on radioiodide uptake by the thyroid glands of larval and adult tiger salamanders, Ambystomu tigrinum (Amphibia: Caudata). Gen. Comp. Endocrinol. 21, 368-376. Pittman, C. S. (1979). Hormone metabolism. In “Endocrinology”, (L. J. DeGroot, G. F. Cahill, Jr., W. D. Odell, L. Martini, J. T. Potts, Jr., D. H. Nelson, E. Steinberger, and A. I. Winegrad, eds.), Vol. 1, pp. 365-372. Grune & Stratton, New York. Platt, J. E. (1974). The role of prolactin in neoteny as found in Colorado populations of the tiger salamander (Ambystoma tigrinum). Ph.D. Thesis, University of Colorado, Boulder. Remy, C., and Disclos, P. (1970). Influence de l’epiphysectomie sur le developpement de la thyroide et des gonades chez le tetards d’Alytes obstetricans. C.R. Sac. Biol. 164. 1989- 1993. DAVID 0. NORRIS WILLIAM A. GERN' KAREN GREENDALE Depurtment of Environmental, and Organismic Biology University of Colorado Boulder, Colorado 80309 Accepted February 10, 1981
Population,
l Present address: Department of Zoology and Physiology, University of Wyoming, Laramie, Wyo. 8207 1.
AND
Diurnal
COMPARATIVE
ENDOCRINOLOGY
45, 134- 137 (1981)
and Seasonal Variations in Thyroid Tiger Salamanders (Ambysfoma
Function tigrinum)
of Neotenic
No dirunal variations for plasma thyroxine (TJ in neotenic tiger salamander larvae were apparent in October, and T, levels at 1200, 1800, and 2400 hr exceed those observed in May. A significant response to ovine thyrotropin (TSH) occurred only at 0600 hr in October. Plasma T, and triiodothyronine (TJ were greatest in May at 0600 and 1800 hr, respectively, and ovine TSH significantly elevated T, and T, levels at all times except for T, at 0600 when the maximal T4 level was observed. Maximal endogenous T, (May) and maximal sensitivity to TSH (October) occurred at the end of the scotophase when plasma melatonin levels were greatest. Circulating levels of melatonin appear to be positively associated with thyroid function in this species.
The scotophasic release of pineal melatonin has been reported for many vertebrates (see Ellis, 1976) in recent years. The responsiveness of certain tissues or inductions of certain behaviors might depend upon the phasic relationships for secretion of two or more hormones (see Meier et al., 1978), and the importance of diurnal patterns in hormone secretion and target sensitivity are now recognized. We present here some preliminary evidence for seasonal differences in diurnal variations of plasma thyroid hormone levels in sexually mature larvae (neotenes) of the tiger salamander, Ambystoma tigrinum, and their relationship to the responsiveness of the thyroid gland to exogenous mammalian thyrotropin (TSH). Furthermore, we provide evidence that endogenous melatonin may not be an inhibitor of pituitary TSH release in this species. MATERIALS
AND METHODS
Sexually mature neotenes were collected in October (experiment 1) or May (experiment 2) from a small pond near Boulder, Colorado (elevation 1700 m), and were transported immediately to the laboratory. After weighing, each animal was placed in a separate, clear-plastic disposable mouse cage containing approximately 1.5 liters aged tap water. The animals were assigned to groups systematically to achieve similar mean body weights and equal sex ratios for each group and then were placed in incubators at 15”
and subjected to a 12L:12D artificial photoperiod (photophase: 0600- 1800 hr). Experiment 1. In October, eight groups of neotenes (n = 7-9) were prepared with similar mean body weights (94.0 * 2.24 to 109.3 2 2.28 g). Two groups were assigned randomly to each time (0600, 1200, 1800, or 2400 hr) for injections and sacrifice. One group at each time received daily intraperitoneal injections of 0.15 pg ovine TSH (NIH-TSH-S6, 2.47 USP units/mg) per gram of body weight in 0.05 ml vehicle (amphibian Ringer’s solution) for 3 days. The second group received vehicle for 3 days. Injections began at 0600 hr. We found earlier that there is a significant eIevation of both T3 and T, blood levels following three daily injections of TSH (Norris ef al., unpublished). This dose of ovine TSH has been shown previously to be ineffective at inducing metamorphosis in these animals when administered for longer periods (Norris et af., 1973). Animals in each group were killed and exsanguinated exactly 24 hr after the third injection, and plasma thyroxine (TJ was determined by radioimmunoassay (RIA). Experiment 2. In May male and female neotenes were separated according to body weight into four groups (mean body weights 131.4 +- 7.15 to 134.2 + 8.03 g) and were injected daily for 3 days with 0.05 ml vehicle at 0600, 1200, 1800, and 2400 hr, respectively (beginning at 1800 hr). Each group was killed 24 hr following the time of the last injection, and plasma triiodothyronine (TJ, Tq, and melatonin levels were determined by RIA. A second batch of neotenes was separated similarly into four groups (n = 7) (mean body weights: 135.4 2 4.19 to 139.9 t 6.75 g). Animals in each group received daily intraperitoneal injections of ovine TSH (0.26 pg NIH-TSHS7, 3.87 USP units/mg) per gram of body weight in 0.05 ml vehicle for 3 days at 0600, 1200, 1800, and 2400 hr, respectively. This dose of TSH will not 134
0016-6480/81/090134-04$01.00/O Copyright @ 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
135
NOTES induce metamorphosis (Norris et al., 1973). Injections began at 1800 hr. Animals were sacrificed 24 hr after the third injection, and plasma T3 and T, were determined by RIA. Radioimmunoassay. Plasma levels of T3 and T, were determined for individual samples by a modified commercial RIA system as outlined by Norris et al. (1977). The accuracy of this assay has been verified recently by a direct RIA procedure (Norman and Norris, unpublished). Plasma values for melatonin were determined by a validated RIA and were previously reported by Gern and Norris ( 1979). Statistical analyses. Thyroid hormone-level data were examined by analysis of variance (ANOVA) followed by a multiple range test (Student-Neuman Keul’s). Analysis of the melatonin data was previously reported (Gem and Norris, 1979).
IO
0,
,
I
1
803 i 6.0E -E &40c
TSH
tp20-
Saline
RESULTS
Experiment I No significant variations in mean plasma T, levels occurred in October for salineinjected neotenes, but a significant response to ovine TSH was observed at 0600 hr [F(3,29) = 2.857, P < 0.051. Diurnal values for saline- and TSH-injected neotenes are depicted in Fig. 1. No differences were observed between males and females.
0’
I 1800 I
1 2400 I T
I 600 I
I 1200 I
Experiment 2 Plasma melatonin levels (Fig. 2) were significantly elevated during the scotophase
“l-r---l II
-i IO
E g 9-
51
\\
f
\\
Time
\\
FIG. 2. Diurnal variations in plasma thyroxine (T,) and triiodothyronine (TJ levels in saline- and TSHtreated neotenic Ambystomu tigrinum during May. Plasma levels of melatonin are also shown for salinetreated animals.
\\ \
I
600
I
I
1200 1800 Time of Day (hr 1
I
1
2400
FIG. 1. Diurnal variations in plasma thyroxine levels (TJ of saline- and TSH-injected neotenic Ambystomu tigrinum during October.
as evidenced by 2400- and 0600-hr samples (see Gern and Norris, 1979). Thyroxine levels in saline-treated animals were significantly elevated at 0600 hr [F(3,28) = 9.018, P < O.Ol] following the scotophasic elevation of plasma melatonin (see Fig. 2). There were no differences between 1200-, 1800-, or 2400-hr plasma samples for Tq. Max-
136
NOTES
imum T3 level (Fig. 2) was observed in 1800-hr samples [F(3,28) = 3.526, P < 0.051. Exogenous TSH had no effect on circulating T, at 0600 hr, but a significant elevation (P < 0.01) over levels determined in saline-treated neotenes was observed at 1200, 1800, 2400 hr (Fig. 2). There was no significant variation in T, levels of TSHtreated animals over time, however. Circulating T3 was elevated at all times as compared to saline-injected neotenes (P < O.Ol), and a significantly greater response occurred at 1200 hr with respect to other times [F(2,26) = 8.05, P < 0.011. No differences in the responses of males and females occurred among saline- or TSH-treated neotenes. DISCUSSION Thyroid
Hormone
Levels
Plasma T, levels in saline-treated neotenes were greater in October than in May similar to those reported by Norris et al. (1977) for freshly collected animals. Spontaneous metamorphosis (i.e., not induced by exogenous thyroid hormones or TSH) occurs with greater frequency in the fall (Platt, 1974) when endogenous levels are greater. In May there was a significant elevation of plasma T, at 0600 hr suggesting that endogenous TSH release occurred during the latter portion of the scotophase. A small but significant elevation of T3 was observed 6 hr after the T, peak (1800 hr) suggesting that much of the circulating T3 is derived peripherally from circulating T, (see Leloup and Bascaglia, 1977; Pittman, 1979). Ovine TSH produced a significant elevation of plasma T, in October as compared to saline-injected animals only at 0600 hr suggesting that the thyroid gland of these animals was most sensitive to exogenous TSH at the end of the scotophase. In May, ovine TSH caused a significant elevation of T4 except when the endogenous T4 levels were maximal (0600 hr) and the ability of the thyroid gland to release T4 was already
maximal. Furthermore, this suggests an endogenous release of TSH during the latter portion of the scotophase. In contrast, T3 levels were markedly increased by exogenous TSH at all times. The interpretation of this effect is not clear. Levels of T3 were not determined for October animals and no comparison is possible. Melatonin
and Thyroid Function
In May, Melatonin levels of salinetreated neotenes were ‘elevated during the scotophase. Although we do not have data for melatonin levels for these animals in October, we have determined in separate experiments (Gern and Norris, 1979; Gern and Norris, unpublished) that scotophasic levels of melatonin are significantly greater than photophasic levels in neotenes from this same population examined in October and November. Furthermore, it appears that all of the differences between photophasic and scotophasic melatonin levels are of pineal origin (Gern and Norris, 1979). Although there is some evidence for inhibition of thyroid activity by the pineal gland (Remy and Disclos, 1970; Gern and DeBoer, 1973; Norris and Platt, 1973), the patterns for plasma levels of thyroid hormone and sensitivity of the thyroid gland to exogenous TSH might suggest just the reverse: that melatonin stimulates TSH release or controls a rhythm that includes an activation of TSH secretion and/or thyroid gland sensitivity to TSH. The stimulatory effects of pinealectomy on thyroid function (iodide uptake) reported by Gern and DeBoer (1973) and Norris and Platt (1973) might be related to another pineal factor and not to melatonin. Prolactin reduces circulating thyroxine levels (Norris, 1978) and melatonin might influence prolactin release in these animals. The possibility that these are correlations with no causal relationship also remains. Additional studies obviously are needed to clarify this proposed causeeffect relationship, but these studies must also be concerned in more detail with the
137
NOTES
problems of diurnal and seasonal variations with respect both to secretions and to glandular sensitivities to modifying agents. ACKNOWLEDGMENTS The authors wish to thank Dr. Richard E. Jones for his critical discussions and reading of the manuscript, and the National Institutes of Health for the gift of ovine thyrotropin. We are also indebted to Dr. Mark Rollag and Dr. Gordon Niswender for the gift of melatonin antibody and to Dr. Niswender for the use of his RIA facility. This work was supported by NSF Grants PCM 7607832 and PCM 78-22335 to D.O.N. and NIH Grant NS-12257 to Charles L. Ralph.
REFERENCES Ellis, L. C., ed. (1976). Endocrine role of the pineal gland. Amer. Zool. 16, l- 101. Gem, W. A., and DeBoer, K. (1973) Pineal-thyroid relationships in neoteny. J. Colo. Wyo. Acad. Sci. 7, 32-33.
Gern, W. A., and Norris, D. 0. (1979). Plasma melatonin in the neotenic tiger salamander (Ambystoma tigrinum): Effects of photoperiod and pinealectomy. Gen. Comp. Endocrinol. 38, 393-
398.
Leloup, J., and Buscaglia, M. (1977). La triiodothyronine, hormone de la metamorphose des Amphibiens. C.R. Acad. Sci. D, 284, 2261-2263. Meier, A. H., Fivizzani, A. J., Spieler, R. E., and Horseman, N. D. (1978). Circadian hormone basis for seasonal conditions in the gulf kill&h, Fundulus grandis. In “Comparative Endocrinology” (P. J. Gaillard and H. H. Boer, eds.), pp. 141- 144. Elsevier/North-Holland, Amsterdam. Norris, D. 0. (1978). Hormonal and environmental factors involved in the determination of neoteny in urodeles. In, “Comparative Endocrinology” (P. J. Gaillard and H. H. Boer, eds.), pp. 109- 112. Elsevier/North-Holland, Amsterdam.
Norris, D. O., Duvall, D., Greendale, K., and Gem, W. A. (1977). Thyroid function in pre- and postspawning neotenic tiger salamanders (Ambystoma tigrinum). Gen. Comp. Endocrinol. 33, 512-517. Norris, D. O., Jones, R. E., and Cohen, D. (1973). Effects of mammalian gonadotropins (LH, FSH, HCG) and gonadal steroids on TSH-induced metamorphosis of Ambystoma tigrinum (Amphibia: Caudata). Gen. Comp. Endocrinol. 20, 467-473.
Norris, D. O., and Platt, J. E. (1973). Effects of pituitary hormones, melatonin, and thyroidal inhibitors on radioiodide uptake by the thyroid glands of larval and adult tiger salamanders, Ambystomu tigrinum (Amphibia: Caudata). Gen. Comp. Endocrinol. 21, 368-376. Pittman, C. S. (1979). Hormone metabolism. In “Endocrinology”, (L. J. DeGroot, G. F. Cahill, Jr., W. D. Odell, L. Martini, J. T. Potts, Jr., D. H. Nelson, E. Steinberger, and A. I. Winegrad, eds.), Vol. 1, pp. 365-372. Grune & Stratton, New York. Platt, J. E. (1974). The role of prolactin in neoteny as found in Colorado populations of the tiger salamander (Ambystoma tigrinum). Ph.D. Thesis, University of Colorado, Boulder. Remy, C., and Disclos, P. (1970). Influence de l’epiphysectomie sur le developpement de la thyroide et des gonades chez le tetards d’Alytes obstetricans. C.R. Sac. Biol. 164. 1989- 1993. DAVID 0. NORRIS WILLIAM A. GERN' KAREN GREENDALE Depurtment of Environmental, and Organismic Biology University of Colorado Boulder, Colorado 80309 Accepted February 10, 1981
Population,
l Present address: Department of Zoology and Physiology, University of Wyoming, Laramie, Wyo. 8207 1.