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Triiodothyronine and thyroxine levels in the thyroid and serum of the sea lamprey Petromyzon marinus L
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
31, 381-383
NOTE Triiodothyronine
and Thyroxine Levels in the Thyroid and Serum of the Sea Lamprey Petromyzon marinus L.
Triiodothyronine (T3) and thyroxine (T4) concentrations have been measured in the serum and thyroidal tissue of Pefromy~on marinus using competitive protein binding analysis and radioimmunoassay. Both hormones were found in the gland but only T3 was present in the sera of all animals sampled. T4 was present in significant quantities only in the sera of fully mature females.
Little has been reported of either the concentrations or the role of the thyroid hormones in the serum of adult lampreys (Barrington and Sage, 1971) even though it is now well established that iodine metabolism in these primitive vertebrates shows the same hormone biogenesis as that occurring in the mammalian thyroid (Clements-Merlini, 1962). Monoiodotyrosine, diiodotyrosine, triiodothyronine (T3) and thyroxine (T4) have all been detected in the endostyle of ammocoetes (Leloup, 1955) and the thyroid of adults (Larsen and Rosenkilde, 1971), and T4 has recently been assayed in the serum of the pacific hagfish and lamprey (Henderson and Lorscheider, 1975, Packard et al., 1976). Serum levels of T3 have so far not been recorded in these animals. The results presented here are for both serum and thyroidal levels of T3 and T4 in Petromyzon marinus at the spawning stage of their freshwater migration, and although the numbers analysed were small, sex differences in the hormone levels became apparent that are worthy of a more detailed examination. Sea lampreys were trapped in the River Severn at Tewkesbury, Gloucestershire, U.K. near the end of their spawning run (June-August) and in most cases because of their spent condition, they were immediately anaesthetised with MS 222 and bled from the caudal vessels. The blood
was allowed to clot at 0°C and when separated, the sera were stored at -20°C until assayed. Doubts have been raised as to the validity of assaying T4 in frozen sera (Packard et al., 1976) because of a possible loss of hormone on storage. Unless this is a particular problem with either lamprey literature serum or T4, commercial (Radiochemical Centre 1975/6) usually advocates the use of previously frozen sera. Thyroidal digests were performed by dissecting out the central region of tissue lying ventrally between the first and seventh gill slit and macerating in 2 ml 0.7% saline with 10 mg of the proteolytic enzyme, Pronase (British Drug Houses, U.K.). Digestion was effected by incubation at 37°C for several hours. Thyroxine concentrations in both serum and tissue digests were measured by competitive protein binding analysis using a commercially available kit, Thyopac 4 (Radiochemical Centre, Amersham, U.K.). Such a technique involves a preliminary separation procedure of the T4 from its binding protein and therefore measures both bound and unbound hormone levels. T4 concentrations are shown in Table 1. Triiodothyronine concentrations in serum and thyroid digests were measured by radioimmunoassay (gift of Byk-Mallinckrodt, Germany) and their values are presented in Table 1. The percentage uptake of labeled T3 by
381 Copyright Q 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
ISSN 0016-64X0
382
NOTE
TABLE CONCENTRATIONS
THE
Animal No.
I
OF THYROXINE AND TRIIODOTHYRONINE IN SERUM AND THEIR GLAND DIGESTS AND THE PERCENTAGE UPTAKE OF TRIIODOTHYRONINE
Sex
I 2 3 4 5
M M M F 1 F
6 7 8 9 10 11 12
M M M M F I F F
SERUM
OF THE
SEA
LAMPREY.
Prtrom~orz
Thyroxine concn. units: nmolesiliter for serum: &gland for extract
Condition Not mature
morinus
Triiodothyronine concn. units: nmoles/liter for serum; &gland for extract
% T3 uptake by serum (relative to human euthyroid serum)
Not detectable
-
23.2 23.2 Not detectable Not detectable 139 104.2 69.5
1.65 I .77 1.72 1.50 0.50 1.15 0.55
90.4 96 57.6 71.2 40 35.9 40.9
Human standard Thyroid extract
IN THYROID
0.54 0.525 0.37 0.46 0.32
1
Fully mature
LEVEIS B\
92-117 F
Not mature
M
serum was measured using the commercially available kit, Thyopac 3 (Radiochemical Centre). Such an assay measures the capacity of the serum proteins to bind the labeled T3 and in mammals is a reflection of the degree of prior saturation of proteins with endogenous thyroid hormones. In the results presented in Table 1, the standard is the normal range for a euthyroid human serum. Values below 92% binding generally indicate a hyperthyroid condition and above 117%, a hypothyroid condition. Although obviously not comparable with regards to species, this test will be used to draw some conclusions about the thyroidal status of the sea lamprey. Values for the T4 concentrations in the sera of 4 fully mature (spawning or spent) males range from undetectable to 23.2 nmoles/liter. Assays on 5 early freshwater run lampreys (not mature), in all cases,
0.69
4
0.58
3
-
showed undetectable T4 levels whatever the sex. Three spent or spawning females, on the other hand, showed elevated T4 concentrations ranging from 69.5 to 139 nmoles/liter. With the exception of these high values in the females, there is general agreement with the results of Fontaine and Leloup (1950) and Packard er al. (1976) in that T4 levels are very low in the sea lamprey. T3 was detected in all animals assayed, even in those with no recorded T4. In the fully mature animals. concentrations appeared to be higher in the males [ 1.66 -c 0.1 nmole/liter (? lSD)] than in the females [0.73 ‘-+ 0.3 nmole/liter (+ 1 SD)]. The immature animals had values of 0.45 + 0.09 nmolefliter (+ 1 SD). The values quoted for T3 and T4 in the thyroid tissue serve only to confirm the presence of the hormones in the tissue. It is
383
NOTE
possible that the T3 values quoted for the tissue are high because of (1) possible cross-reactivity of T4 with anti-T3 (although this is less than 0.5%) and (2) deiodination of T4 to T3 in the digest. The T3 uptake values, when compared to the human standard, indicates either a high free thyroid hormone concentration or low levels of a thyroid binding protein in the serum. In the case of the mature females, the high thyroid hormone levels would explain the results, whereas in the males a reduced level of serum binding proteins is a possible explanation. The provisional conclusions of this study are that both T3 and T4 are present in easily detectable amounts in the thyroid gland of P. marinus and that during anadromous migration, very low levels of T4 are released into the circulation. At spawning, particularly in the females, there is a marked increase in T4 concentration in the serum. T3 levels, in common with all vertebrates, are in low concentration but are nevertheless detectable. Whether there is an inverse relationship between T3 and T4 in the serum as implied by the results in Table 1 will need to be verified using larger numbers of animals. It is possible, however, that T3 may be the more important physiological hormone in these primitive animals. The physiological significance of the high T4 levels in the serum of the mature females is unknown.
REFERENCES Barrington. E. .I. W., and Sage, M. (1971). The endostyle and thyroid gland. In “The Biology of Lampreys” (M. W. Hardisty and I. C. Potter, eds.). Academic Press, New York. Clements-Merlini, M. (1962). Altered metabolism of 1’“’ by the endostyle and notochord of ammocoete larvae. II. Effects of treatment with thiourea or potassium thiocyanate. Gen. Camp. Endocrinol. 2, 361-368. Fontaine. M.. and Leloup, J. (1950). L’iodemie d’un Cyclostome marin (Pefromvzon marinus L.) au moment de sa migration reproductice. C.R. Acnd. Sci. 230, 1538-1539. Henderson, N. E., and Lorscheider, F. L. (1975). Thyroxine and protein bound iodine concentrations in plasma of the Pacific Hagfish Eptcrtrefus .stouti (Cyclostomata). Camp. Biochrrn. Physiol. SlA, 723-726. Larsen, L. 0.. and Rosenkilde, P. (1971). Iodine metabolism in normal. hypophysectomised and thyrotropin-treated river lampreys, Lampeira Jluviatilis (Gray) L. (Cyclostomata). Gerf. Camp. Endocrinol. 17, 94-104. Leloup. J. (1955). Metabolisme de I’iode et fonctionnement endostylaire chez I’ammocoete de Lampetrcr planeri. .I. Physiol. 47, 671-677. Packard, G. C.. Packard, M. J., and Gorbman. A. (1976). Serum thyroxine concentrations in the Pacific Hagfish and Lamprey and the Leopard Frog. Grn. Camp. Endocrinol. 28, 365-367. Radiochemical Centre Leaflet I.M. 74 (1975/6) T3 R.I.A. Kit for the immunoassay of serum T3 (liothyronine). DAVID School of’ Biological Sciences University of Buth Bath. BA2 7AY, England Accepted September 24, 1976
J. HORNSEY
AND
COMPARATIVE
ENDOCRINOLOGY
31, 381-383
NOTE Triiodothyronine
and Thyroxine Levels in the Thyroid and Serum of the Sea Lamprey Petromyzon marinus L.
Triiodothyronine (T3) and thyroxine (T4) concentrations have been measured in the serum and thyroidal tissue of Pefromy~on marinus using competitive protein binding analysis and radioimmunoassay. Both hormones were found in the gland but only T3 was present in the sera of all animals sampled. T4 was present in significant quantities only in the sera of fully mature females.
Little has been reported of either the concentrations or the role of the thyroid hormones in the serum of adult lampreys (Barrington and Sage, 1971) even though it is now well established that iodine metabolism in these primitive vertebrates shows the same hormone biogenesis as that occurring in the mammalian thyroid (Clements-Merlini, 1962). Monoiodotyrosine, diiodotyrosine, triiodothyronine (T3) and thyroxine (T4) have all been detected in the endostyle of ammocoetes (Leloup, 1955) and the thyroid of adults (Larsen and Rosenkilde, 1971), and T4 has recently been assayed in the serum of the pacific hagfish and lamprey (Henderson and Lorscheider, 1975, Packard et al., 1976). Serum levels of T3 have so far not been recorded in these animals. The results presented here are for both serum and thyroidal levels of T3 and T4 in Petromyzon marinus at the spawning stage of their freshwater migration, and although the numbers analysed were small, sex differences in the hormone levels became apparent that are worthy of a more detailed examination. Sea lampreys were trapped in the River Severn at Tewkesbury, Gloucestershire, U.K. near the end of their spawning run (June-August) and in most cases because of their spent condition, they were immediately anaesthetised with MS 222 and bled from the caudal vessels. The blood
was allowed to clot at 0°C and when separated, the sera were stored at -20°C until assayed. Doubts have been raised as to the validity of assaying T4 in frozen sera (Packard et al., 1976) because of a possible loss of hormone on storage. Unless this is a particular problem with either lamprey literature serum or T4, commercial (Radiochemical Centre 1975/6) usually advocates the use of previously frozen sera. Thyroidal digests were performed by dissecting out the central region of tissue lying ventrally between the first and seventh gill slit and macerating in 2 ml 0.7% saline with 10 mg of the proteolytic enzyme, Pronase (British Drug Houses, U.K.). Digestion was effected by incubation at 37°C for several hours. Thyroxine concentrations in both serum and tissue digests were measured by competitive protein binding analysis using a commercially available kit, Thyopac 4 (Radiochemical Centre, Amersham, U.K.). Such a technique involves a preliminary separation procedure of the T4 from its binding protein and therefore measures both bound and unbound hormone levels. T4 concentrations are shown in Table 1. Triiodothyronine concentrations in serum and thyroid digests were measured by radioimmunoassay (gift of Byk-Mallinckrodt, Germany) and their values are presented in Table 1. The percentage uptake of labeled T3 by
381 Copyright Q 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
ISSN 0016-64X0
382
NOTE
TABLE CONCENTRATIONS
THE
Animal No.
I
OF THYROXINE AND TRIIODOTHYRONINE IN SERUM AND THEIR GLAND DIGESTS AND THE PERCENTAGE UPTAKE OF TRIIODOTHYRONINE
Sex
I 2 3 4 5
M M M F 1 F
6 7 8 9 10 11 12
M M M M F I F F
SERUM
OF THE
SEA
LAMPREY.
Prtrom~orz
Thyroxine concn. units: nmolesiliter for serum: &gland for extract
Condition Not mature
morinus
Triiodothyronine concn. units: nmoles/liter for serum; &gland for extract
% T3 uptake by serum (relative to human euthyroid serum)
Not detectable
-
23.2 23.2 Not detectable Not detectable 139 104.2 69.5
1.65 I .77 1.72 1.50 0.50 1.15 0.55
90.4 96 57.6 71.2 40 35.9 40.9
Human standard Thyroid extract
IN THYROID
0.54 0.525 0.37 0.46 0.32
1
Fully mature
LEVEIS B\
92-117 F
Not mature
M
serum was measured using the commercially available kit, Thyopac 3 (Radiochemical Centre). Such an assay measures the capacity of the serum proteins to bind the labeled T3 and in mammals is a reflection of the degree of prior saturation of proteins with endogenous thyroid hormones. In the results presented in Table 1, the standard is the normal range for a euthyroid human serum. Values below 92% binding generally indicate a hyperthyroid condition and above 117%, a hypothyroid condition. Although obviously not comparable with regards to species, this test will be used to draw some conclusions about the thyroidal status of the sea lamprey. Values for the T4 concentrations in the sera of 4 fully mature (spawning or spent) males range from undetectable to 23.2 nmoles/liter. Assays on 5 early freshwater run lampreys (not mature), in all cases,
0.69
4
0.58
3
-
showed undetectable T4 levels whatever the sex. Three spent or spawning females, on the other hand, showed elevated T4 concentrations ranging from 69.5 to 139 nmoles/liter. With the exception of these high values in the females, there is general agreement with the results of Fontaine and Leloup (1950) and Packard er al. (1976) in that T4 levels are very low in the sea lamprey. T3 was detected in all animals assayed, even in those with no recorded T4. In the fully mature animals. concentrations appeared to be higher in the males [ 1.66 -c 0.1 nmole/liter (? lSD)] than in the females [0.73 ‘-+ 0.3 nmole/liter (+ 1 SD)]. The immature animals had values of 0.45 + 0.09 nmolefliter (+ 1 SD). The values quoted for T3 and T4 in the thyroid tissue serve only to confirm the presence of the hormones in the tissue. It is
383
NOTE
possible that the T3 values quoted for the tissue are high because of (1) possible cross-reactivity of T4 with anti-T3 (although this is less than 0.5%) and (2) deiodination of T4 to T3 in the digest. The T3 uptake values, when compared to the human standard, indicates either a high free thyroid hormone concentration or low levels of a thyroid binding protein in the serum. In the case of the mature females, the high thyroid hormone levels would explain the results, whereas in the males a reduced level of serum binding proteins is a possible explanation. The provisional conclusions of this study are that both T3 and T4 are present in easily detectable amounts in the thyroid gland of P. marinus and that during anadromous migration, very low levels of T4 are released into the circulation. At spawning, particularly in the females, there is a marked increase in T4 concentration in the serum. T3 levels, in common with all vertebrates, are in low concentration but are nevertheless detectable. Whether there is an inverse relationship between T3 and T4 in the serum as implied by the results in Table 1 will need to be verified using larger numbers of animals. It is possible, however, that T3 may be the more important physiological hormone in these primitive animals. The physiological significance of the high T4 levels in the serum of the mature females is unknown.
REFERENCES Barrington. E. .I. W., and Sage, M. (1971). The endostyle and thyroid gland. In “The Biology of Lampreys” (M. W. Hardisty and I. C. Potter, eds.). Academic Press, New York. Clements-Merlini, M. (1962). Altered metabolism of 1’“’ by the endostyle and notochord of ammocoete larvae. II. Effects of treatment with thiourea or potassium thiocyanate. Gen. Camp. Endocrinol. 2, 361-368. Fontaine. M.. and Leloup, J. (1950). L’iodemie d’un Cyclostome marin (Pefromvzon marinus L.) au moment de sa migration reproductice. C.R. Acnd. Sci. 230, 1538-1539. Henderson, N. E., and Lorscheider, F. L. (1975). Thyroxine and protein bound iodine concentrations in plasma of the Pacific Hagfish Eptcrtrefus .stouti (Cyclostomata). Camp. Biochrrn. Physiol. SlA, 723-726. Larsen, L. 0.. and Rosenkilde, P. (1971). Iodine metabolism in normal. hypophysectomised and thyrotropin-treated river lampreys, Lampeira Jluviatilis (Gray) L. (Cyclostomata). Gerf. Camp. Endocrinol. 17, 94-104. Leloup. J. (1955). Metabolisme de I’iode et fonctionnement endostylaire chez I’ammocoete de Lampetrcr planeri. .I. Physiol. 47, 671-677. Packard, G. C.. Packard, M. J., and Gorbman. A. (1976). Serum thyroxine concentrations in the Pacific Hagfish and Lamprey and the Leopard Frog. Grn. Camp. Endocrinol. 28, 365-367. Radiochemical Centre Leaflet I.M. 74 (1975/6) T3 R.I.A. Kit for the immunoassay of serum T3 (liothyronine). DAVID School of’ Biological Sciences University of Buth Bath. BA2 7AY, England Accepted September 24, 1976
J. HORNSEY