Immunocytochemical demonstration of chicken hypophyseal thyrotropes and development of a radioimmunological indicator for chicken TSH using monoclonal and polyclonal homologous antibodies in a subtractive strategy.

The production and characterization of monoclonal antibodies (mAbs) toward the chicken pituitary glycoproteins are described. MAbs were first subdivided on an immunocytochemical (ICC) basis: one category of mAbs reacts with both thyrotropic and gonadotropic cells whereas another stains exclusively gonadotropic cells. The hypothesis that the former are anti-glycoprotein alpha and that the latter are anti-beta LH and/or anti-beta FSH mAbs was further elaborated in a homologous tracer binding assay. The putative anti-beta gonadotropin mAbs exclusively recognize iodinated USDA-cLH-I-1, whereas the supposed anti-alpha mAbs react with both USDA-cLH-I-1 and USDA-cFSH-I-1. The ICC localization of pituitary thyrotropes has been achieved in a double-staining protocol using an anti-LH mAb to subtract specifically the gonadotropic cells from the complete alpha-subunit-containing cell population. The result is a strictly cephalic cell population that is densely granulated in hyperthyroid and hardly detectable in hypothyroid birds. A similar subtractive approach was used to develop a thyroid-stimulating hormone (TSH)-indicator in RIA. The total titer of alpha-containing molecules is first quantified with an anti-alpha mAb. The contribution of both LH and FSH to this titer is then measured by homologous USDA-RIA and subtracted from the total alpha-immunoreactivity to give an indication of the TSH level of the sample. The direct effect of thyroid-releasing hormone on pituitary TSH release in vitro in juvenile chicks and the effect of hypo- and hyperthyroidism on circulating TSH levels in vivo in juvenile broilers have been demonstrated.

Autoradiography of pituitary cells stimulated by castration or methimazole feeding.

It has been argued that the increase in size of the pituitary of castrated cockerels is due to hypertrophy of gonadotropic cells, some of which have been converted from other pituitary basophils. A counter argument could maintain that new gonadotropes are recruited from chromophobes that are replaced by mitotic division of undifferentiated cells. Since mitosis requires thymine for DNA replication, labelled thymine (H3-thymidine) incorporated in DNA identifies new cell formation. On this basis, the chicken pituitary does not produce enough new cells to account for the size differences associated with castration or methimazole treatment.

Role of ovarian steroids in the control of moult induction in laying fowls.

1. Thirty-five Warren SSL hens were selected on the basis of variation in moulting response following a force-moult at 81 weeks of age. Fourteen hens were from a group which returned to layers mash ad libitum from day 9, while 21 came from a group with dietary restriction prolonged to day 28. Blood samples were taken on days 23 and 36, and hormone concentrations were measured. Moulting was recorded on days 0, 11, 23, 36, 52 and 68. 2. Progesterone (P4) but not oestradiol (E2) inhibited moulting during egg laying and the sharp fall in P4 concentration when laying ceased was a primary factor in the induction of moulting. 3. Thyroxine (T4) concentrations were closely correlated with all criteria of plumage renewal, but not with the onset of moulting. Although T4 was closely correlated with subsequent feather growth it is questionable whether T4 is a primer of moult-induction independent of P4. 4. Feathers which were pulled out required 12 +/- 2 d to be replaced beyond the feather-papillae stage. For remiges this timing was similar regardless of whether hens were moulting or still laying. The timing was similar for remiges and body-feathers in moulting hens, but the reappearance of body-feathers in laying hens was slightly delayed.

Effect of exogenous LH on plasma concentrations of progesterone and oestradiol in relation to the cessation of egg laying induced by different moulting methods.

Artificially induced cessation of egg laying caused regression of the reproductive tract in hens, as well as changes in circulating concentrations of sex steroids. Hens were bled at several stages during and after artificial moult induced by fasting or overfeeding a diet low in calcium or high in zinc. Hens received a single injection of 200 i.u. of horse LH at Day 0, 7, 21, 35 and 77 (Exp. 1) or Day 0, 8, 23, 35 and 71 (Exp. 2) after start of the treatment to induce moult. Blood samples were taken before and 20, 40 and 60 min (Exp. 1) or 15, 30 and 45 min (Exp. 2) after LH injection. Hens which were fasted or given the diet high in zinc had low plasma progesterone concentrations and the response to LH was reduced or delayed. In hens fed low calcium the reduction in plasma progesterone was less pronounced and the responsiveness to LH was more or less maintained. Conversely, there was no response of oestradiol to LH in laying hens. However, oestradiol concentrations increased in moulting hens after LH injection, due to the high oestradiol secretion from the small white follicles, since all yolky follicles were atretic.

Growth hormone stimulates the peripheral conversion of thyroxine into triiodothyronine by increasing the liver 5'-monodeiodinase activity in the fasted and normal fed chicken.

Normal fed and 2 days fasted Warren chickens were injected intravenously with 100 micrograms of ovine growth hormone (GH) and ovine prolactin and plasma concentrations of thyroid hormones were assayed prior and up to 2 h after injection. Fasting alone decreases T3, but increases T4. An injection of GH resulted in increases of plasma T3 concentrations in two fasting experiments by 40% (after 3/4 h) and 104% (after 1 h). In normal fed animals no increase is observed in the first experiment, whereas a 35% increase occurs in the second one. An injection of 100 micrograms prolactin does not influence T3 in normal fed or fasting animals. Both GH and prolactin, however, may decrease plasma concentrations of T4. In a separate experiment 50 micrograms and 200 micrograms of GH raised the decreased T3 levels after fasting by 39% and 60% respectively 1 h after injection and by 24 and 61% respectively in normal fed chicken, whereas prolactin was ineffective in this regard. Using Hisex chickens, the influence of an injection of 100 micrograms GH on plasma concentrations of thyroid hormones could be confirmed. At the same time GH increases the liver 5'-monodeiodinase activity by 330% after 1 h and by 147% after 2 h. The peroxidase activity is not influenced in normal fed chickens, but GH decreases this activity in food deprived animals after 1 h and 2 h. It is concluded that ovine GH, but not prolactin, stimulates the peripheral conversion of T4 into T3 in both normal fed and food deprived chicken and that this effect is dose dependent.

The influence of castration and methimazole on deoxyribonucleic acid content of the pituitary and thyroid glands of White Leghorn cockerels.

The pituitary gland of birds is known to increase in weight with castration and decreases with chemical thyroidectomy. Whether these changes are due to alterations in mitotic activity or to cell size changes has been examined by measurement of deoxyribonucleic acid (DNA) and protein content of these glands. The DNA content of the enlarge pituitary of castrated cockerels is the same as that in the pituitary of normal cockerels. The pituitary size increase may be due to an increase in gonadotrope size including any part of the gonadotrope population which may be obtained by recruitment of other basophils. The retardation in pituitary size and DNA content with methimazole treatment is probably due, at least in part, to growth retardation but may also be due, in part, to the conversion of larger basophils to smaller thyrotropes.

The effects of castration and/or methimazole feeding on the pituitary response to temperature extremes by cockerels.

The pituitary of goitrogen-treated White Leghorn cockerels is smaller in size than control birds and the pituitaries of castrated cockerels is nearly twice the size of control birds. The pituitary cells generated by these treatments may not be functional thyrotrophs or gonadotrophs and may not be able to respond to their usual stimuli. Low ambient temperature is a well-known stimulus to the thyroid gland acting through pituitary TSH. Cyclic-AMP-dependent protein kinase activity levels are used here as an index of cellular activity in the pituitary and thyroid glands. Castrated cockerels with or without methimazole treatment do not have an increased pituitary cAMP-dependent protein kinase activity in cold. Methimazole-treated birds have an exaggerated pituitary protein kinase response to cold stress when compared with controls. Pituitary cAMP-dependent protein kinase activity is paralleled by a similar activity increase in the thyroid gland of methimazole-treated cockerels and no increase in the thyroid of castrated birds. Castrated birds at all temperatures have an elevated thyroid cAMP-dependent protein kinase activity ratio which is interpreted as the result of removal of testosterone inhibition.

Some effects of photoperiod on the pituitary/thyroid axis of castrated cockerels.

Pituitary cyclic adenosine monophosphate-dependent protein kinase (cAMP-PK) activity ratios of castrated cockerels on a short photoperiod are highest 2 weeks after testosterone removal in castrated birds and remain high through a 3rd week. Castrated cockerels on long photoperiods have a decline in cAMP-PK activity ratios after an elevation at 1 week following testosterone removal. This is interpreted to mean that the pituitaries of cockerels on short photoperiods are sensitive to testosterone inhibition but that this sensitivity disappears in long photoperiod-stimulated birds. Long daylengths also appear to enhance thyroid activity in castrated cockerels, although thyroid cAMP-PK activity ratios increase in the thyroid of all cockerels following the removal of testosterone (castration). These results suggest that testosterone has an inhibitory effect on both the pituitary and thyroid glands.

Muscle fiber types in the dystrophic puboischiofemoralis of commercial broilers.

Muscle fiber types, their diameters, lipid content, and nuclei distributions were studied in normal and necrotic puboischiofemoral muscles from commercial broiler chickens of the Hubbard strain. Three categories of puboischiofemoral fiber morphology (alpha R, beta R, and alpha W) occur in the cranial and caudal portions of the pars lateralis and in the cranial, medial, and caudal portions of the pars medialis. The greatest amount of necrosis occurred in the caudal portion of the pars medialis of the puboischiofemoralis. A fiber type characterized by a greatly enlarged and rounded or oval cross-section, decreased fat content, and internalized and clustered nuclei was present in necrotic muscle but not in normal muscle.

Age-related changes in pituitary/thyroid function in chickens.

Pituitary cyclic AMP-dependent protein kinase (cAMP-PK) activity ratios are not increased in response to thyrotropin-releasing hormone (TRH) in old chickens (retired White Leghorn breeders). This reduced responsiveness may be due to reduced hypothalamic function, reduced thyrotrope function, or to a reduction in TRH membrane receptors. The thyroid cAMP-PK activity ratio of old males does not respond to TRH treatment but the thyroid of old females does have an increased activity ratio ater TRH injection. Both males and females have a much higher basal cAMP-PK activity ratio than young birds. This higher basal level is thought to be due to an increased thyroid-stimulating hormone (TSH) stimulation, and the failure of old males to increase activity after TRH injections may be due to a loss of the thyroid's ability to respond to direct TRH stimulation.

Comparative effects of three goitrogenic treatments on White Leghorn chickens.

White Leghorn chickens respond to dietary goitrogenic compounds by forming either colloidal or hyperplastic goiters. Thiourea does not cause increases in DNA above that seen in controls, although thyroid weight increases. This may be due to a secondary toxic effect of thiourea on the chick thyroid, which inhibits cellular replication. Both methimazole- and propylthiouracil- (PTU) fed birds exhibit cellular hyperplasia and increases in DNA amounts beyond control levels. The rapid thyroid growth in methimazole-treated birds from 30 to 70 days of age is partially due to follicular colloid accumulation. Growth of the thyroid in PTU-fed birds is thought to be due to cellular hyperplasia and hypertrophy, since DNA values are greatest in these glands. Histological studies of the thyroid glands confirm the results of DNA and protein assays for all three goitrogen-treated groups. The ultrastructural changes associated with prolonged stimulation of the chick thyroid are an increase in the amount of primary and secondary lysosomes, microvilli, and mitochondria, and a dilation of the endoplasmic reticulum and Golgi apparatus. It is thought that micropinocytosis is a more important endocytotic process during goiter development in the chick than is phagocytosis.

The effect of rapeseed meal and methimazole on levels of plasma hormones in growing broiler cockerels.

The effects of feeding a heat treated rapeseed meal, which has goitrogenic properties, on the concentrations of plasma pituitary and thyroid gland hormones was investigated in broiler cockerels of between 3 and 10 weeks of age. For purposes of comparison, two other groups were included in the study; one was fed the goitrogen, methimazole, and the other a normal control diet. The hormones measured were thyroxine (T4), triiodothyronine (T3), growth hormone (GH), prolactin, and luteinizing hormone (LH). In birds fed methimazole the thyroid glands were greatly enlarged, the concentrations of plasma T4 and T3 were depressed and the concentrations of growth hormone, prolactin, and LH were elevated. The high level of plasma LH in the birds fed methimazole was not due to the absence of sufficient concentrations of plasma testosterone to exert a negative feedback effect. Although the inclusion of rapeseed meal in the diet caused the thyroid glands to enlarge, the concentrations of all the hormones studied, with the exception of T3, were similar to those in the control birds. However, there was a tendency, which was more pronounced in birds of between 3 and 5 weeks of age, for rapeseed meal to depress the concentrations of plasma T4, GH, and LH and to increase the concentration of plasma prolactin. The most significant observation was that between 3 and 5 weeks of age the inclusion of rapeseed meal in the diet significantly (P less than .001) depressed the concentration of plasma T3.

Localization of cells producting thyroid stimulating hormone in the pituitary gland of the domestic drake.

Cells binding anti-bovine TASH beta serum were found exclusively in the rostral lobe of the adenohypophysis of the drake using the peroxidase-antiperoxidase complex unlabelled antibody method. The specificity of the binding of the anti-serum to TSH cells was established by relating the morphology and relative abundance of immunochemically stained cells to the TSH content of the adenohypophysis after experimentally altering the activity of the pituitary-thyroid axis. The TSH activity of the adenohypophysis was assessed indirectly, by the weight of the thyroid glands, and directly, by bioassay. As determined by bioassay, the TSH content of the rostral lobe of the adenohypophysis was much greater than that of the caudal lobe. Compared with control drakes, immunochemically stained cells in birds fed a goitrogen, methimazole, seemed to be enlarged and were closer together, while the stained cells in drakes injected with thyroxine were shrunken and less intensely stained. The TSH content of the adenohypophysis was increased in drakes fed methimazole. Castration did not alter the TSH content of the adenohypophysis or change the morphology of immunochemically stained cells. These observations suggest that in the drake: 1) anti-bovine TSH beta serum binds specifically to TSH cells; 2) the TSH cells occur in the rostral and not in the caudal lobe of the adenohypophysis; and 3) the activity of TSH cells is not inhibited by the feedback effects of gonadal steroids.

Reproductive tissue activity in hypothyroid or heat stressed hens.

The ability of the reproductive tract of the White Leghorn hen under heat stress or methimazole thyroid blockage to assimilate 32P was examined. Both treatments increase the uptake of 32P by the shell gland and egg shell but only heal stress increases 32P uptake by the ovaries and reduces shell thickness. The greater shell thinness under the heat stress is related to an increased deposition of phosphate in the shell. Gilbert (1967) has reported an increased serum calcium, due to calcification inhibitions by phosphate, leading to an increased gonadotropin secretion. Thyroid blockage appears to stimulate TSH and gonadotropin LH secretion but in the absence of phosphate inhibition of calcification shell thickness is not affected. It is also speculated that hypothyroidism does not stimulate FSH secretion and therefore ovarian tissues is not activated by this condition.

The effect of hypothalamic lesions on thyroid-stimulating hormone concentration in the chicken pituitary.

Studies of thyroid activity following hypothalamic lesions have provided indirect evidence of TSH influence on the pars distalis of birds. The present study examined the influence of hypothalamic lesions on TSH assays of the rostral and caudal lobes of the pars distalis. Lesions of the septomesencephalic tract increased TSH levels in the rostral lobe but had no influence on the caudal lobe. Lesions of the supraoptic or ventrolateral nuclei neither stimulated nor depressed TSH in either lobe of the pars distalis.