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Hypothalamo-adenohypophyseal-thyroid interrelationships in the chick embryo
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
55,
275-279 (1984)
Hypothalamo-Adenohypophyseal-Thyroid Chick Embryo
interrelationships
VI. Midgestational Adenohypophyseal Sensitivity Thyrotrophin-Releasing Hormone’ ROBERTC. THOMMES,DONJ. Department
oj’Bio/ogical
Sciences,
in the
to
WILLIAMS, AND JAMES E. WOODS
University, 1036 West Belden Accepted October 24, 1983
De Paul
Avenue,
Chicago, Illinois 60614
The functional abilities of 9.5-, 10.5-, 11.5-, and 12.5-day-old chick embryos to respond to exogenous thyrotrophin-releasing hormone (TRH) were evaluated by means of changes in plasma total thyroxine (T,). T4 concentrations were determined 3.0, 6.0, 12.0, and 24.0 hr after TRH treatment. The data of the present investigation show that chick embryo adenohypophyseal sensitivity to exogenous TRH, as evidenced by changes in plasma T, levels, increase during the 10.5 to 12.5-day incubation interval; however, the pattern and magnitude of piasma T4 response to administered TRH change markedly between Days 10.5 and 11.5 of development. This modification of the adenohypophyseal response pattern corresponds in embryonic time with the previously reported increases in immunocytochemically demonstrable TSH cells within the adenohypophysis (R. C. Thommes, J. B. Martens, W. E. Hopkins, D. A. Griesbach, D. J. Williams, M. J. Sorrentino, P. Wemke, and J. E. Woods, 1981, In ‘Ninth International Symposium of Comparative Endocrinology”; R. C. Thommes, J. B. Martens, W. E. Hopkins, M. J. Sorrentino, J. C. Caliendo, and J. E. Woods, 1983, Gen. Camp. Endocrinol. 51, 434-443).
There are a number of indications that Days 10.0-13.0 in chick embryo development are critical with regard to the emergence of adenohypophysealthyroid interactions. This hypothesis has been supported by the observations that during this period there are marked and simultaneous increases in (1) plasma iodide (Daugkras and Lachiver, 1972) and thyroidal iodine (Daug&as and Lachiver, 1972; Daug&as et al., 1976; Daug&ras-Bernard and Lachiver, 1983), (2) rates of radio-active iodide uptake (Maraud et al., 1957, 1975; HBmori et al., 1959; Mess and Straznicky, 1964; Thommes and Tonetta, 1979), (3) amounts of thyroidal monoiodotyrosine (MIT), diiodotyrosine (DIT), and thyroxine (TJ (Trunnell and Wade, 1955; Daugbras et al., 1976), (4) plasma total T4 levels (Daug&as-Ber-
nard et al., 1976; Thommes et al., 1977), as well as (5) the establishment of the feedforward (Fugo, 1940; Martindale, 1941; Daugkas-Bernard et al., 1976; Thommes et al., 1977)) and feed-backward (TixierVidal, 1958; Thommes and Tonetta, 1979) loops of the adenohypophysealthyroid component of the hypothalamo-adenohypophyseal-thyroid (H-A-T) axis. The maturation of the H-A-T axis is also reflected in changes in the histology of the adenohypophysis over developmental time. Ultrastructural analysis of chick embryo adenohypophyseal differentiation has demonstrated that on Day 6.5 two distinct cell types appear in the rostra1 pars distalis (Daikoku et al., 1974). These two cell types correspond to those previously identified by means of light microscopy as basophils (Rahn, 1939; Wingstrand, 1951; Wilson, 1952; Tixier-Vidal, 1954), one of which has been identified immunocytochemically as
1 This study was supported by a grant from the American Rivet Company, Inc., Franklin Park, Ill. 60131. 275
0016~6480184 $I SO Copyright 0 1984 by Academic Press, Inc. All rights of repxduction in any form reserved
276
THOMMES,
WILLIAMS,
an immature thyrotroph (Thommes et al., 1981, 1983). Between days 10.5 and 11.5 there is a marked increase in the number of these TSH-immunoreactive cells, as well as the amount of immunoreactive material per cell (Thommes et aI., 1981, 1983). Also these same cells are responsive to thyrotrophin releasing hormone (TRH) as early as Day 6.5, as evidenced by heightened plasma T, levels following TRH treatment (Thommes and Hylka, 1978). Changes in the sensitivity of the adenohypophysis to exogenous TRH have been reported for the early period of chick embryo development (Day 6.5, Thommes and Hylka, 1978), as well as before, during, and immediately after hatch (Decuypere and Scanes, 1983). However, there has been no systematic study of the response of the adenohypophysis to exogenous TRH during the critical time period of Days 10.5 to 11.5 of incubation, in order to determine whether or not the striking increase in adenohypophyseal immunoreactive thyrotrophs at this embryonic time is reflected physiologically by a change in response pattern to TRH. Thus, the purpose of the present investigation was to ascertain if there is a change, during the critical time period of Days 10.5- 11.5 of embryonic development, in the sensitivity of the pituitary-thyroid component of the H-A-T axis to exogenous TRH as measured by changes in plasma total T, levels. MATERIALS
AND METHODS
All embryos were obtained from white Leghorn eggs incubated at 38 2 1.1” in a Jamesway forced-draft incubator. The age of the embryos at the time of killing is the time the eggs remained in the incubator. ‘IIvo groups of embryos were prepared. The first group received 50 pl TRH (Sigma Chemical Co., St. Louis, MO., 400 pg/lOO g body wt) and the second series receive 50 ~1 isotonic saline. Both solutions were placed on the chorioallantoic membrane (CAM) of eggs at 1.5 min intervals on Days 9.5, 10.5, 11.5, and 12.5 of development. Embryos of both series were killed at 1.5, 3.0, 6.0, 12.0, and 24 hr after treatment in the same time sequence as materials were added. Blood was drawn from extraembryonic blood vessels
AND WOODS
(Thommes and Tonetta, 1979), centrifuged at 16oOg for 10 min, and the plasma aliquoted into 100~p,l samples and then stored at - 20” until the time of T, analysis. Samples were run in duplicate when at all possible. When samples were rarely pooled, they were pooled in equialiquots. Plasma T, determinations were carried out simultaneously on control and experimental samples for each day. Thyroxine radioimmunoassay. The plasma T, radioimmunoassay method utilized in this study was that of Chopra et al. (1971), as modified by Radioassay Systems Laboratories, Inc., Carson, California. This assay is a double antibody method utilizing rabbit antithyroxine serum (anti-T,; Lot H-31, Radioassay Systems Laboratories, Inc.) and sheep anti-rabbit y-globulin (second or precipitating antibody; Lot BB-2, Radioassay Systems Laboratories, Inc). The anti-T., was diluted 1:lOOO with 1% (v/v) normal rabbit serum in 0.01 M sodium phosphosaline buffer, pH 7.6. The second antibody was used as purchased without further dilution. A standard curve was plotted with each assay, utilizing L-thyroxine (Lot 64-0134, Sigma Chemical Co.) as the standard. Statistics utilized in this study were ANOVA and Duncan’s new multiple range test (Duncan, 1955).
RESULTS On Day 9.5 of incubation there was a statistically significant increase in plasma T, levels 1.5 hr after TRH treatment (Table 1, Fig. 1). Plasma T4 levels were not statistically different when comparisons were made between control and experimental groups after 3.0, 6.0, 12.0, and 24.0 hr. On Day 10.5 significant increases in plasma T, concentrations were seen at 1.5, 3.0, and 12.0 hr after TRH treatment. On Days 11.5 and 12.5, mean plasma T, levels of embryos treated with TRH were significantly different from the corresponding saline-administered embryos at every time interval studied (Table 1, Fig. 1). On Day 11.5 the mean percentage increase of T, levels in TRH-treated embryos was 180%, with a range of 169 to 187%. Day 12.5 embryos treated with TRH for 1.5 hr showed the greatest response, as evidenced by an increase of 524% in plasma T, levels (12.06 ng T,/ml) when compared to vehicle controls of the same time period (2.30 ng T4/ ml). The smallest percentage increase (137.3%) on this day was seen at 24.0 hr
THYROTROPHIN-RELEASING
PLASMA
HORMONE-CHICK
277
EMBRYO
TABLE 1 T4 LEVELS IN SALINE- AND TRH-TREATED CHICK EMBRYOS Iteatment
Age at time of sacrifice (hr)
Saline” (Mean f SE)
TRH” (Mean + SE)
9.5 +
1.5 3.0 6.0 12.0 24.0
(7)b (8) (8) (5) (10)
0.51 0.98 1.14 0.90 0.52
f f k f T
0.17 0.32 0.14 0.31 0.22
(ll)b (10) (7) (6) (12)
1.60 0.32 0.66 0.53 0.24
f 2 i -t +
0.17’ O.lod 0.22d 0.29d O.Ogd
10.5 +
1.5 3.0 6.0 12.0 24.0
(II) (10) (11) (14) (10)
0.21 0.20 1.43 0.71 2.33
_c f f i r
0.05 0.07 0.27 0.26 0.36
(13) (II) (11) (14) (7)
1.09 0.43 1.03 1.82 2.10
i f f -c -t
0.25’ 0.07’ 0.19d 0.46’ 0.30d
11.5 +
1.5 3.0 6.0 12.0 24.0
(9) (9) (11) (12) (9)
3.96 3.89 3.42 2.60 1.64
-t 2 f i r
0.68 0.69 0.57 0.39 0.24
(12) (12) (12) (13) (11)
6.95 6.58 6.24 4.87 3.06
+ 2 f 2 ?
0.55’ 0.67’ 0.86’ 0.45’ 0.41’
12.5 +
1.5 3.0 6.0 12.0 24.0
(7) (9) (12) (13) (8)
2.30 5.22 2.19 5.23 7.72
zk -c + 2 +
0.48 0.77 0.41 0.58 0.83
(9) 12.06 f 2.10’ (10) 12.32 IT 1.69 (13) 6.17 5 1.48’ (11) 8.65 ? 0.81’ (9) 10.60 5 0.97’
n TRH-treated chicks received 400 CLgilOD g body wt; controls received 50 ~1 isotomc satme. b Number of samples. ’ Statistically different from the corresponding salinetreated individual at the 5% level as determined by ANOVA and the Duncan new multiple range test. d Not statistically different from the corresponding salinetreated individual at the 5% level as determined by ANOVA and the Duncan new multiple range test.
posttreatment, thus exhibiting greater variation than that observed on Day 11.5. With the exception of Day 11.5 the greatest relative response to TRH treatment on each day studied was seen 1.5 hr after administration. Therefore, the data of the present study (Table 1, Fig. 1) demonstrate that between Days 10.5 and 11.5 in the embryonic development of the domestic fowl there is a change in the response pattern of the adenohypophysis to exogenous TRH. The saline-treated groups exhibit a marked statistically significant increase in plasma T, when one compares the composite means of Days 10.5 and 11.5 (0.97 vs 3.27 rig/ml, respectively) (P < 0.05). DISCUSSION The data of the present
investigation
FIG.
1. Plasma
T., in TRH-treated
chick
embryos.
demonstrate that in the chick embryo adenohypophyseal sensitivity to exogenous TRH, as evidenced by changes in plasma T, levels, increases during the 10.5-12.5 day incubation period. More specificially, the pattern and magnitude of plasma T4 response to administered TRH change markedly between Days 10.5 and 11.5, corresponding in time with the previously reported increased in immunocytochemically demonstrable TSH cells within the adenohypophysis (Thommes et al., 1981, 1983). The marked increase in circulating total T, levels which characterizes the salinetreated controls during the period of 10.5 to 11.5 days of incubation reconfirms the earlier observations of Daugeras-Bernard (1976) and Thommes et al. (1977) where similar increases were observed in normal, intack chick embryos. Several explanations have been advanced to explain the marked increase in thyroidal activity which normally occurs during the Day 10.5- 12.5 embryonic period
278
THOMMES,
WILLIAMS,
AND WOODS
(Thommes and Hylka, 1978). One hypothesis which previously seemed attractive was that this Day 10.5-11.5 period is the time of the beginnings of the initial sensitivity of the pituitary and/or thyroid to TRH and TSH, respectively. This is apparently not the case, since these glands are already sensitive to their respective trophic hormones at least as early as Day 6.5 (Thommes and Hylka, 1978). Similarly, it had also been suggested that the increase in thyroid activity during Days 10.0-13.0 might be due to the initial synthesis and/or release of TRH by the hypothalamus and/ or TSH by the adenohypophysis. The results of Thommes et al. (1981), however, have indicated that immunocytochemically demonstrable TRH-containing perikarya are present in the hypothalamic rudiment as early as Day 4.5 while immunoreactive TSH cells are present in the pars distalis by Day 6.5. The amounts of immunochemitally demonstrable TRH in the hypothalamus and in the median eminence increase gradually (Thommes et al., 1981) with no distinct period of marked increase. The number of immunocytochemically demonstrable thyrotrophs in the adenohypophysis, on the other hand, increase markedly during the Days 10.5-11.5 incubation interval (Thommes et af., 1981, 1983). Thus, the data of the present investigation strongly support the hypothesis that the developmental increase in adenohypophyseal TSH-producing cells during the Days 10.511.5 embryonic period is of functional significance. However, whether or not TRH is involved in the in vivo regulation of these TSH-producing cells remains problematic.
Daugtras, N., and Lachiver, F. (1972). Evolution de I’iode, 1271, total thyroidien chez l’embryon de poulet au tours de l’incubation. J. Embryol. Exp.
REFERENCES
Rahn, H. (1939). The development of the chick pituitary with special reference to the cellular differentiation of the pars buccalis. 1. Morpho/. 64,
Chopra, I. J., Nelson, J. C., Solomon, D. H., and Beall, G. N. (1971). Production of antibodies specifically bonding triiodothyronine and thyroxine. J. C/in.
Endocrinol.
Metab.
32, 299-308.
Daikoku, S., Ikeuchi, C., and Nakagawa, H. (1974). Development of the hypothalamohypophysial unit in the chick. Gen. Camp. Endocrinol. 23, 256915 &,_1.
Morphol.
27, 615-622.
Daugeras, N., Brisson, A., Lapointe, F., and Lachiver, F. (1976). Thyroidal iodine metabolism during the development of the chick embryo. Endocrinology
98, 1321-1331.
Daugeras-Bernard, N., and Lachiver, E (1983). Effect of hypophysectomy by partial decapitation and treatment with thiourea on iodine metabolism in the developing chick embryo. J. Endocrinol. 98, 113-119. Daugeras-Bernard, N., Leloup, J., and Lachiver, F. (1976). Evolution de la thyroxinemie au cours du developpment de l’embryon de poulet. Influence de l’hypophysectomie. C. R. Acad. Sci. (Paris) 283,
1325-1327.
Decuypere, E., and Scanes, C. G. (1983). Variation in the release of thyroxine, triiodothyronine and growth hormone in response to thyrotrophin releasing hormone during development of the domestic fowl. Acta Endocrinol (Copenhagen) 102, 220-223.
Duncan, D. B. (1955). Multiple range and multiple F tests. Biometrics 11, l-42. Fugo, N. W., (1940). Effects of hypophysectomy in the chick embryo. J. Exp. Zoo!. 85, 271-291. Hamori, J.. Mess, B., and Sz&kely, G. Y. (1959). Onset of thyroidal 1131 accumulation in normal and decapitated chick embryos. Acta Biol. Acad. Sci. Hung.
10, 207-213.
Maraud, R., Audine. M., and Stoll, R. (1975). Sur l’existence de relations hypothalamo-hypophysaires chez l’embryon de poulet. C. R. Seances Sot. Biol.
Ses Fil.
169, 923-927.
Maraud, R., Stoll, R., and Blanquet, P. (1957). Sur le role de I’hypophyse dans la concentration du radioiode par la thyroide de l’embryon de poulet. C. R. Seances
Sot.
Biol.
Ses Fil.
151, 572-574.
Mess, B.. and Straznicky, K. (1964). Functional differentiation of the thyroid gland investigated by 1131 autoradiography in decapitated chick embryos bearing hypophyseal homografts. Acta Biol.
Acad.
Sci. Hung.
15, 77-85.
Martindale, E (1941). Initiation and early development of thyrotropic function in the incubating chick. Anat.
Rec.
79, 373-393.
483-517.
Thommes, R. C., and Hylka. V. W. (1978). Hypothalamo-adenohypophyseal-thyroid interrelationships in the chick embryo. I. TRH and TSH sensitivity. Gen. Comp. Endocrinol. 34, 193-200. Thommes, R. C., Martens, J. B., Hopkins, W. E.,
THYROTROPHIN-RELEASING Griesbach, D. A., Williams, D. J., Sorrentino, M. J., Wemke, P, and Woods, J. E. (1981). Thyroid function and regulation in the chick embryo. 9th International Symposium of Comparative Endocrinology. Thommes, R. C., Martens, J. B., Hopkins, W. E., Sorrentino, M. J., Caliendo, J. C., and Woods, J. E. (1983). Hypothalamo-adenohypophysealthyroid interrelationships in the chick embryo. IV. Immunocytochemical demonstration of TSH in the hypophyseal pars distalis. Gen. Camp. Endocrinol. 51, 434-443. Thommes, R. C., and Tonetta, S. A. (1979). Hypothalamo-adenohypophyseal-thyroid interrelationships in the chick embryo. II. Effects of thiourea treatment on plasma total thyroxine levels and thyroidali25 I uptake. Gen. Comp. Endocrinol. 37, 167-176. Thommes, R. C., Vieth, R. L., and Levasseur, S. (1977). The effects of hypophysectomy by means of surgical decapitation on thyroid function in the
HORMONE-CHICK
EMBRYO
279
developing chick embryo. I. Plasma thyroxine. Gen. Comp. Endorinol. 31, 29-36. Tixier-Vidal, A. (1954). Etude histophysiologique de l’hypophyse anterieur de l’embryon de poulet. Arch. Anot. Microsc. Morphol. Exp. 43, 163- 186. Tixier-Vidal, A. (1958). Etude histophysiologique des relations hypophyse et thyroide chez l’embryon de poulet. Arch. Anat. Microsc. Morphol. Exp. 47, 17-340. Trunnell, J. B., and Wade, P. (1955). Factors governing the development of the chick embryo thyroid. II. Chronology of the synthesis of iodinated compounds studied by chromatographic analysis. .I. Clin. Endocrinol. Metab. 15, 107-117. Wilson, M. E. (1952). The embryological and cytological basis of regional patterns in the definitive epithelial hypophysis of the chick. Amer. J. Anat. 91, I-50. Wingstrand, K. G. (1951). “The Structure and Development of the Avian Pituitary,” Hakan Ohlssons Boktryckeri. Lund, Sweden.
AND
COMPARATIVE
ENDOCRINOLOGY
55,
275-279 (1984)
Hypothalamo-Adenohypophyseal-Thyroid Chick Embryo
interrelationships
VI. Midgestational Adenohypophyseal Sensitivity Thyrotrophin-Releasing Hormone’ ROBERTC. THOMMES,DONJ. Department
oj’Bio/ogical
Sciences,
in the
to
WILLIAMS, AND JAMES E. WOODS
University, 1036 West Belden Accepted October 24, 1983
De Paul
Avenue,
Chicago, Illinois 60614
The functional abilities of 9.5-, 10.5-, 11.5-, and 12.5-day-old chick embryos to respond to exogenous thyrotrophin-releasing hormone (TRH) were evaluated by means of changes in plasma total thyroxine (T,). T4 concentrations were determined 3.0, 6.0, 12.0, and 24.0 hr after TRH treatment. The data of the present investigation show that chick embryo adenohypophyseal sensitivity to exogenous TRH, as evidenced by changes in plasma T, levels, increase during the 10.5 to 12.5-day incubation interval; however, the pattern and magnitude of piasma T4 response to administered TRH change markedly between Days 10.5 and 11.5 of development. This modification of the adenohypophyseal response pattern corresponds in embryonic time with the previously reported increases in immunocytochemically demonstrable TSH cells within the adenohypophysis (R. C. Thommes, J. B. Martens, W. E. Hopkins, D. A. Griesbach, D. J. Williams, M. J. Sorrentino, P. Wemke, and J. E. Woods, 1981, In ‘Ninth International Symposium of Comparative Endocrinology”; R. C. Thommes, J. B. Martens, W. E. Hopkins, M. J. Sorrentino, J. C. Caliendo, and J. E. Woods, 1983, Gen. Camp. Endocrinol. 51, 434-443).
There are a number of indications that Days 10.0-13.0 in chick embryo development are critical with regard to the emergence of adenohypophysealthyroid interactions. This hypothesis has been supported by the observations that during this period there are marked and simultaneous increases in (1) plasma iodide (Daugkras and Lachiver, 1972) and thyroidal iodine (Daug&as and Lachiver, 1972; Daug&as et al., 1976; Daug&ras-Bernard and Lachiver, 1983), (2) rates of radio-active iodide uptake (Maraud et al., 1957, 1975; HBmori et al., 1959; Mess and Straznicky, 1964; Thommes and Tonetta, 1979), (3) amounts of thyroidal monoiodotyrosine (MIT), diiodotyrosine (DIT), and thyroxine (TJ (Trunnell and Wade, 1955; Daugbras et al., 1976), (4) plasma total T4 levels (Daug&as-Ber-
nard et al., 1976; Thommes et al., 1977), as well as (5) the establishment of the feedforward (Fugo, 1940; Martindale, 1941; Daugkas-Bernard et al., 1976; Thommes et al., 1977)) and feed-backward (TixierVidal, 1958; Thommes and Tonetta, 1979) loops of the adenohypophysealthyroid component of the hypothalamo-adenohypophyseal-thyroid (H-A-T) axis. The maturation of the H-A-T axis is also reflected in changes in the histology of the adenohypophysis over developmental time. Ultrastructural analysis of chick embryo adenohypophyseal differentiation has demonstrated that on Day 6.5 two distinct cell types appear in the rostra1 pars distalis (Daikoku et al., 1974). These two cell types correspond to those previously identified by means of light microscopy as basophils (Rahn, 1939; Wingstrand, 1951; Wilson, 1952; Tixier-Vidal, 1954), one of which has been identified immunocytochemically as
1 This study was supported by a grant from the American Rivet Company, Inc., Franklin Park, Ill. 60131. 275
0016~6480184 $I SO Copyright 0 1984 by Academic Press, Inc. All rights of repxduction in any form reserved
276
THOMMES,
WILLIAMS,
an immature thyrotroph (Thommes et al., 1981, 1983). Between days 10.5 and 11.5 there is a marked increase in the number of these TSH-immunoreactive cells, as well as the amount of immunoreactive material per cell (Thommes et aI., 1981, 1983). Also these same cells are responsive to thyrotrophin releasing hormone (TRH) as early as Day 6.5, as evidenced by heightened plasma T, levels following TRH treatment (Thommes and Hylka, 1978). Changes in the sensitivity of the adenohypophysis to exogenous TRH have been reported for the early period of chick embryo development (Day 6.5, Thommes and Hylka, 1978), as well as before, during, and immediately after hatch (Decuypere and Scanes, 1983). However, there has been no systematic study of the response of the adenohypophysis to exogenous TRH during the critical time period of Days 10.5 to 11.5 of incubation, in order to determine whether or not the striking increase in adenohypophyseal immunoreactive thyrotrophs at this embryonic time is reflected physiologically by a change in response pattern to TRH. Thus, the purpose of the present investigation was to ascertain if there is a change, during the critical time period of Days 10.5- 11.5 of embryonic development, in the sensitivity of the pituitary-thyroid component of the H-A-T axis to exogenous TRH as measured by changes in plasma total T, levels. MATERIALS
AND METHODS
All embryos were obtained from white Leghorn eggs incubated at 38 2 1.1” in a Jamesway forced-draft incubator. The age of the embryos at the time of killing is the time the eggs remained in the incubator. ‘IIvo groups of embryos were prepared. The first group received 50 pl TRH (Sigma Chemical Co., St. Louis, MO., 400 pg/lOO g body wt) and the second series receive 50 ~1 isotonic saline. Both solutions were placed on the chorioallantoic membrane (CAM) of eggs at 1.5 min intervals on Days 9.5, 10.5, 11.5, and 12.5 of development. Embryos of both series were killed at 1.5, 3.0, 6.0, 12.0, and 24 hr after treatment in the same time sequence as materials were added. Blood was drawn from extraembryonic blood vessels
AND WOODS
(Thommes and Tonetta, 1979), centrifuged at 16oOg for 10 min, and the plasma aliquoted into 100~p,l samples and then stored at - 20” until the time of T, analysis. Samples were run in duplicate when at all possible. When samples were rarely pooled, they were pooled in equialiquots. Plasma T, determinations were carried out simultaneously on control and experimental samples for each day. Thyroxine radioimmunoassay. The plasma T, radioimmunoassay method utilized in this study was that of Chopra et al. (1971), as modified by Radioassay Systems Laboratories, Inc., Carson, California. This assay is a double antibody method utilizing rabbit antithyroxine serum (anti-T,; Lot H-31, Radioassay Systems Laboratories, Inc.) and sheep anti-rabbit y-globulin (second or precipitating antibody; Lot BB-2, Radioassay Systems Laboratories, Inc). The anti-T., was diluted 1:lOOO with 1% (v/v) normal rabbit serum in 0.01 M sodium phosphosaline buffer, pH 7.6. The second antibody was used as purchased without further dilution. A standard curve was plotted with each assay, utilizing L-thyroxine (Lot 64-0134, Sigma Chemical Co.) as the standard. Statistics utilized in this study were ANOVA and Duncan’s new multiple range test (Duncan, 1955).
RESULTS On Day 9.5 of incubation there was a statistically significant increase in plasma T, levels 1.5 hr after TRH treatment (Table 1, Fig. 1). Plasma T4 levels were not statistically different when comparisons were made between control and experimental groups after 3.0, 6.0, 12.0, and 24.0 hr. On Day 10.5 significant increases in plasma T, concentrations were seen at 1.5, 3.0, and 12.0 hr after TRH treatment. On Days 11.5 and 12.5, mean plasma T, levels of embryos treated with TRH were significantly different from the corresponding saline-administered embryos at every time interval studied (Table 1, Fig. 1). On Day 11.5 the mean percentage increase of T, levels in TRH-treated embryos was 180%, with a range of 169 to 187%. Day 12.5 embryos treated with TRH for 1.5 hr showed the greatest response, as evidenced by an increase of 524% in plasma T, levels (12.06 ng T,/ml) when compared to vehicle controls of the same time period (2.30 ng T4/ ml). The smallest percentage increase (137.3%) on this day was seen at 24.0 hr
THYROTROPHIN-RELEASING
PLASMA
HORMONE-CHICK
277
EMBRYO
TABLE 1 T4 LEVELS IN SALINE- AND TRH-TREATED CHICK EMBRYOS Iteatment
Age at time of sacrifice (hr)
Saline” (Mean f SE)
TRH” (Mean + SE)
9.5 +
1.5 3.0 6.0 12.0 24.0
(7)b (8) (8) (5) (10)
0.51 0.98 1.14 0.90 0.52
f f k f T
0.17 0.32 0.14 0.31 0.22
(ll)b (10) (7) (6) (12)
1.60 0.32 0.66 0.53 0.24
f 2 i -t +
0.17’ O.lod 0.22d 0.29d O.Ogd
10.5 +
1.5 3.0 6.0 12.0 24.0
(II) (10) (11) (14) (10)
0.21 0.20 1.43 0.71 2.33
_c f f i r
0.05 0.07 0.27 0.26 0.36
(13) (II) (11) (14) (7)
1.09 0.43 1.03 1.82 2.10
i f f -c -t
0.25’ 0.07’ 0.19d 0.46’ 0.30d
11.5 +
1.5 3.0 6.0 12.0 24.0
(9) (9) (11) (12) (9)
3.96 3.89 3.42 2.60 1.64
-t 2 f i r
0.68 0.69 0.57 0.39 0.24
(12) (12) (12) (13) (11)
6.95 6.58 6.24 4.87 3.06
+ 2 f 2 ?
0.55’ 0.67’ 0.86’ 0.45’ 0.41’
12.5 +
1.5 3.0 6.0 12.0 24.0
(7) (9) (12) (13) (8)
2.30 5.22 2.19 5.23 7.72
zk -c + 2 +
0.48 0.77 0.41 0.58 0.83
(9) 12.06 f 2.10’ (10) 12.32 IT 1.69 (13) 6.17 5 1.48’ (11) 8.65 ? 0.81’ (9) 10.60 5 0.97’
n TRH-treated chicks received 400 CLgilOD g body wt; controls received 50 ~1 isotomc satme. b Number of samples. ’ Statistically different from the corresponding salinetreated individual at the 5% level as determined by ANOVA and the Duncan new multiple range test. d Not statistically different from the corresponding salinetreated individual at the 5% level as determined by ANOVA and the Duncan new multiple range test.
posttreatment, thus exhibiting greater variation than that observed on Day 11.5. With the exception of Day 11.5 the greatest relative response to TRH treatment on each day studied was seen 1.5 hr after administration. Therefore, the data of the present study (Table 1, Fig. 1) demonstrate that between Days 10.5 and 11.5 in the embryonic development of the domestic fowl there is a change in the response pattern of the adenohypophysis to exogenous TRH. The saline-treated groups exhibit a marked statistically significant increase in plasma T, when one compares the composite means of Days 10.5 and 11.5 (0.97 vs 3.27 rig/ml, respectively) (P < 0.05). DISCUSSION The data of the present
investigation
FIG.
1. Plasma
T., in TRH-treated
chick
embryos.
demonstrate that in the chick embryo adenohypophyseal sensitivity to exogenous TRH, as evidenced by changes in plasma T, levels, increases during the 10.5-12.5 day incubation period. More specificially, the pattern and magnitude of plasma T4 response to administered TRH change markedly between Days 10.5 and 11.5, corresponding in time with the previously reported increased in immunocytochemically demonstrable TSH cells within the adenohypophysis (Thommes et al., 1981, 1983). The marked increase in circulating total T, levels which characterizes the salinetreated controls during the period of 10.5 to 11.5 days of incubation reconfirms the earlier observations of Daugeras-Bernard (1976) and Thommes et al. (1977) where similar increases were observed in normal, intack chick embryos. Several explanations have been advanced to explain the marked increase in thyroidal activity which normally occurs during the Day 10.5- 12.5 embryonic period
278
THOMMES,
WILLIAMS,
AND WOODS
(Thommes and Hylka, 1978). One hypothesis which previously seemed attractive was that this Day 10.5-11.5 period is the time of the beginnings of the initial sensitivity of the pituitary and/or thyroid to TRH and TSH, respectively. This is apparently not the case, since these glands are already sensitive to their respective trophic hormones at least as early as Day 6.5 (Thommes and Hylka, 1978). Similarly, it had also been suggested that the increase in thyroid activity during Days 10.0-13.0 might be due to the initial synthesis and/or release of TRH by the hypothalamus and/ or TSH by the adenohypophysis. The results of Thommes et al. (1981), however, have indicated that immunocytochemically demonstrable TRH-containing perikarya are present in the hypothalamic rudiment as early as Day 4.5 while immunoreactive TSH cells are present in the pars distalis by Day 6.5. The amounts of immunochemitally demonstrable TRH in the hypothalamus and in the median eminence increase gradually (Thommes et al., 1981) with no distinct period of marked increase. The number of immunocytochemically demonstrable thyrotrophs in the adenohypophysis, on the other hand, increase markedly during the Days 10.5-11.5 incubation interval (Thommes et af., 1981, 1983). Thus, the data of the present investigation strongly support the hypothesis that the developmental increase in adenohypophyseal TSH-producing cells during the Days 10.511.5 embryonic period is of functional significance. However, whether or not TRH is involved in the in vivo regulation of these TSH-producing cells remains problematic.
Daugtras, N., and Lachiver, F. (1972). Evolution de I’iode, 1271, total thyroidien chez l’embryon de poulet au tours de l’incubation. J. Embryol. Exp.
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