Influence of Photoperiod, Daylength, and Feeding Schedule on Tadpole Growth and Development

The metamorphic rate of Rana pipiens tadpoles was studied under different photoperiods, daylengths, and feeding schedules. Tail resorption and hindlimb growth and development induced by immersion in 30 μg/l thyroxine (T₄) were accelerated under longer photoperiods and continuous light when 6L: 18D, 12L: 12D, 18L: 6D, and 24L regimes were compared. Constant light exposure did not produce faster development than an 18 hr photoperiod, and initially was less effective. The rate of spontaneous and T₄-induced metamorphosis was greater the shorter the day on 9L:9D, 12L: 12D, or 15L: 15D cycles, although all groups received the same overall amount of light, but in different dosages. When feeding schedule but not the LD cycle was varied, groups of tadpoles fed on 18, 24, or 30 hr regimes showed no differences in growth and development rate on 19L: 5D, and only random variations under continuous light. Differences in metamorphic rate on 18, 24, or 30 hr days are not due to the feeding schedules, but to the LD cycles. From these experiments we conclude that illumination, particularly the length and frequency of the photoperiod, affects the utilization of T₄. Development rates independent of the total amount of illumination, but related to daylength and light schedule, suggest interaction of light with an endogenous timing mechanism.     

Investigations on Myelination In Vitro: Regulation of 2',3'-Cyclic Nucleotide 3'-Phosphohydrolase by Thyroid Hormone in Cultures of Dissociated Brain Cells from Embryonic Mice

Abstract: The direct influence of l -3,3',5-triiodothyronine (T₃) on the development of 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37, CNPase) is demonstrated by using an in vitro culture system of dissociated embryonic mouse brain cells. Serum from a thyroidectomized calf, which contained low levels of T₃ (31 ng/100 ml), and thyroxine, T₄ (<1 μg/ml), was used in the culture medium in place of normal calf serum (T₃, 103 ng/100 ml; T₄, 5.7 μg/ml) to render the culture responsive to exogenously added T₃. The lower levels of enzyme activity observed in the presence of such a deficient medium could be restored to normal values by T₃ supplementation. Half-maximal effect was obtained with 2.5 ± 10⁻⁹ m -T₃. Three days of hormone treatment resulted in the maximal stimulation of CNPase. T₄ was less effective in inducing CNPase activity and the inactive analog of the hormone, reverse T₃ (3,3',5'-T₃) was ineffective. The morphological appearance of the cells was characterized by deformed (smaller size and less in number) reaggregates in the cultures, lacking hormone.     

INHIBITION OF THYROXINE-INDUCED RESORPTION OF TADPOLE TAIL BY ADENOSINE 3', 5'-CYCLIC MONOPHOSPHATE

Small segments of tail of Bufo bufo japonicus tadpoles were cultured in medium containing thyroxine (T₄) and dibutyryl cyclic AMP (dbcAMP). Like prolactin, the cyclic nucleotide blocked T₄-induced shrinkage or tail pieces. Histological study of the segments after 4-days culture revealed that dbcAMP suppressed degenerative changes induced by T₄. The inhibitory effect of prolactin on T₄-induced tail regression was promoted by caffeine, an inhibitor of adenosine 3', 5'-cyclic monophosphate (cyclic AMP)-phosphodiesterase.
The effect of prolactin on the level of cyclic AMP in the tail was also studied in vivo . Sixty min after prolactin injection, the cyclic AMP level was 2–3 times the control value. Possible involvement of cyclic AMP in the action of prolactin, which blocks tail resorption induced by T₄, was discussed.     

Effect of a light pulse during the dark on photoperiodic regulation of the rate of thyroxine-induced, spontaneous, and prolactin-inhibited metamorphosis in Rana pipiens tadpoles

Since Rana pipiens tadpoles injected with thyroxine (T4) early in the dark develop more slowly than those injected in the light, we studied the effect of giving a light pulse of 1 hr early in the dark. Tadpoles injected under a 7.5-W red light bulb in a darkened room with 0.2 microgram T4 daily at 2200 hr went through metamorphosis faster on a 12L:3D:1L:8D cycle with a light pulse after injection than on a 12L:12D cycle without a light pulse, and even faster on a 12L:1.5D:1L:9.5D cycle with a light pulse before the injection. Thus a 1-hr light pulse counteracted the metamorphic delay resulting from administration of T4 in the dark, and set in motion the conditions that resulted in a more rapid response to an injection of T4. However, a 1-hr light pulse in the early dark had no effect on growth and development of older or younger untreated tadpoles or those constantly immersed in 30 micrograms/liter T4. Larvae on 21L:3D with T4 injection in the dark and on 12L:3D:1L:8D with T4 injection at 0700 hr just before the start of the main light phase progressed faster than 12L:3D:1L:8D with injection at 2200 hr in the dark before only a 1-hr light pulse. Thus the length of the light phase immediately after T4 injection was significant. There was no difference on 12L:12D and 12L:3D:1L:8D cycles in the effectiveness of daily injections of 10 micrograms prolactin (PRL) in the early dark at 2200 hr in promoting tail growth or antagonizing tail resorption induced by T4 immersion. Under these conditions, PRL utilization did not appear to be inhibited by the light pulse.     

Early larval development and metamorphosis in the summer flounder: changes in per cent whole-body water content and effects of altered thyroid status

At the end of premetamorphosis, summer flounder Paralichthys dentatus larvae had 84·1% whole-body water content (WBW), which decreased to the lowest levels (8·5%) at the start of metamorphic climax (MC). During mid- and late MC, %WBW was slightly higher (82·1%) then returned to the lowest levels at the juvenile stage. In fish treated with thyroxine (T₄-Na salt, 100 ng ml⁻¹) beginning at premetamophosis, %WBW never differed from controls of the same age throughout metamorphosis, despite an earlier start of metamorphic climax and transitional settling behaviour. This suggests that thyroid hormones do not mediate the drop in %WBW which accompanies natural metamorphosis. Thiourea (TU, 30 μg ml⁻¹) treatment of fish over the same period induced a developmental stasis in early MC which was accompanied by initially higher %WBW than controls at 33 days post-hatch, followed by a progressive decrease to abnormally low %WBW by 42 and 45 days post-hatch. Since concurrent treatment with TU+T₄ rescued the fish from both the TU-induced developmental stasis and abnormally low %WBW, these findings suggest that thyroid hormones, or thyroid hormone-mediated developmental progression, are necessary for regulating %WBW.     

Triiodothyronine Is a High-Affinity Inhibitor of Amino Acid Transport System L1 in Cultured Astrocytes

Abstract: The relationship between the transport of thyroid hormones and that of amino acids was examined by measuring the uptake of amino acids that are characteristic substrates of systems L, A, and N, and the effect of 3,3',5-triiodo-L-thyronine (T₃) on this uptake, in cultured astrocytes. Tryptophan and leucine uptakes were rapid, Na⁺-independent, and efficiently inhibited by T₃ (half-inhibition at ∼ 2 μ M ). Two Na⁺-independent L-like systems (L₁ and L₂), common to leucine and aromatic amino acids, were characterized kinetically. System L₂ had a low affinity for leucine and tryptophan ( K _m= 0.3–0.9 m M ). The high-affinity system L₁ ( K _m∼ 10 μ M for both amino acids) was competitively inhibited by T₃ with a K _i of 2–3 μ M (close to the T₃ transport K _m). Several T₃ analogues inhibited system L₁ and the T₃ transport system similarly. Glutamine uptake and α-(methylamino)isobutyric acid uptake were, respectively, two and 200 times lower than tryptophan and leucine uptakes. T₃ had little effect on the uptakes of glutamine and α-(methylamino)isobutyric acid. The results indicate that the T₃ transport system and system L₁ are related.     

Melatonin concentrations during larval and postlarval development of gilthead sea bream Sparus auratus: more than a time‐keeping molecule?

In this study, melatonin (MEL) and thyroxine (T₄) concentrations were measured during larval and postlarval development of gilthead sea bream Sparus auratus Hormones were measured in whole bodies of larvae or the head and trunk of postlarvae after 67 days of exposure to constant light, 24L:0D, constant darkness, 0L:24D or 12L:12D and in the plasma of 6 month juveniles kept under the 12L:12D, 0L:24D and 24L:0D regimes. High MEL concentrations in larvae suggested a distinct role of MEL in early organogenesis and development of S. auratus . In larvae, the gastro-intestinal tract seemed to be an important extrapineal and extraretinal source of MEL. No endogenous rhythm of MEL synthesis was demonstrated in 67 day larvae; however, in 6 month juveniles, it was evident. At early ontogenesis of S. auratus , the role of MEL is probably related mostly to the control of development and protection against free radicals, whereas its action as a time-keeping molecule develops later. The increase in T₄ concentration during the S. auratus larva–juvenile transition, i.e . between 50 and 70 days post-hatch, which was observed concurrently with the decrease of MEL concentration, may suggest an inverse relationship between T₄ and MEL.     

Influences of photoperiod and alternate days of feeding on plasma growth hormone and thyroid hormone levels in juvenile rainbow trout

The diurnal patterns of plasma growth hormone (GH), thyroid hormone and cortisol concentrations in rainbow trout Oncorhynchus mykiss held under three photoperiod (L : D) regimes (6 : 18, 12 : 12 and 18 : 6), and fed either daily (DF) or on alternate days (ADF) with 2·0% body mass per day of a commercial trout diet were determined. The ADF groups had reduced total mass gain and specific growth rates compared with DF fish, but photoperiod had no affect on growth for either of the feeding regime groups. In the ADF groups, the mean 24 h plasma thyroxine (T₄) and triiodothyronine (T₃) concentrations were significantly lower, both on days of feeding and days of fasting, than in DF fish held under all three photoperiod regimes, but for GH, only the 18L : 6D DF group was higher than the comparable ADF groups. There were no significant differences in mean 24 h plasma cortisol concentrations of DF and ADF groups. Diurnal patterns of plasma GH, cortisol, T₄ and T₃ were found in DF fish held under all three photoperiod regimes. Increases in plasma cortisol changes were associated with the onset of the light phase; elevations in plasma GH and T₄ concentrations were more closely associated with clock time, regardless of photoperiod; increases in plasma T₃ concentrations were strongly associated with time of feeding. In ADF groups, these diurnal changes in plasma GH, T₄ and T₃ concentrations were suppressed for both the fed and fast days, and plasma cortisol concentrations were suppressed on the fasting day. The observations are discussed in terms of the proposed anabolic, catabolic and growth regulating roles of these hormones in different growth and metabolic modifying situations in teleosts.     

Evidence for Circadian Variations of Thyroid Hormone Concentrations and Type II 5'-Iodothyronine Deiodinase Activity in the Rat Central Nervous System

Abstract: The 24-h patterns of tissue thyroid hormone concentrations and type II 5'- and type III 5-iodothyronine deiodinase (5'D-II and 5D-III, respectively) activities were determined at 4-h intervals in different brain regions of male euthyroid rats entrained to a regular 12-h light/12-h dark cycle (lights on at 6:00 a.m.). Activity of 5'D-II, which catalyzes the intracellular conversion of thyroxine (T₄) to 3,3',5-triiodo- l -thyronine (T₃) in the CNS, and the tissue concentrations of both T₄ and T₃ exhibited significant daily variations in all brain regions examined. Periodic regression analysis revealed significant circadian rhythms with amplitudes ranging from 9 to 23% (for T₃) and from 15 to 40% (for T₄ and 5'D-II) of the daily mean value. 5'D-II activity showed a marked nocturnal increase (1.3–2.1-fold vs. daytime basal value), with a maximum at the end of the dark period and a minimum between noon and 4:00 p.m. 5D-III did not exhibit circadian patterns of variation in any of the brain tissues investigated. Our results disclose circadian rhythms of 5'D-II activity and thyroid hormone concentrations in discrete brain regions of rats entrained to a regular 12:12-h light-dark cycle and reveal that, in the rat CNS, T₃ biosynthesis is activated during the dark phase of the photoperiod. For all parameters under investigation, the patterns of variation observed were in part regionally specific, indicating that different regulatory mechanisms may be involved in generating the observed rhythms.     

Distribution of immunoreactive thyroxine in the endostyle and thyroid tissue of the sea lamprey Petromyzon marinus L., 1758

Anti-thyroxine (T₄) immunostaining in the endostyle and thyroid tissue during metamorphosis of sea lamprey Petromyzon marinus suggested that T₄ synthesis occurs on the luminal surface of the apical membrane of selected cells of the endostyle, and that a 'classical' endocrine thyroid function was only evident in post-transformed animals.     

The decrease in plasma melatonin at metamorphic climax in Rana catesbeiana (bullfrog) tadpoles is induced by thyroxine.

Melatonin decreases in the plasma of Rana catesbeiana (bullfrog) tadpoles at the climax of metamorphosis when the thyroxine (T(4)) level peaks. Since melatonin inhibited thyroid function in vitro, it would be of interest to determine if the decline in plasma melatonin permits greater thyroid hormone secretion, or if the increasing levels of T(4) cause the climactic decrease in plasma melatonin. The reciprocal effects of administering T(4) or melatonin just prior to metamorphic climax were examined in tadpoles kept at 22 degrees C on an 18L:6D cycle. If melatonin functions as a thyroid antagonist at later metamorphic stages, administration of melatonin should decrease plasma T(4), whereas if T(4) causes the decline in plasma melatonin, T(4) treatment of tadpoles prior to climax should induce the climactic melatonin decrease prematurely. Once daily injection of 40 microg melatonin for 5 days at 19.30 h had no effect on metamorphic progress, or on plasma T(4) or melatonin levels, except for a transient rise in melatonin just after the injection. Immersion in 2.2x10(-4) M melatonin for 6 days accelerated metamorphosis and decreased plasma melatonin, but had no effect on plasma T(4). Administration of T(4) by injection of 0.2 microg, or immersion in a 6.3x10(-8) M solution accelerated metamorphosis more than melatonin immersion, raised plasma T(4) to climax levels, and induced a decrease in plasma melatonin. We conclude that rapid clearance of exogenous melatonin from the circulation in these experiments did not allow it to affect plasma T(4), and that there is clear evidence that the rise in T(4) induces the climax decrease in plasma melatonin. The finding that immersion in a high level of melatonin can lower plasma melatonin and accelerate metamorphosis, whereas a single daily injection does not, provides an explanation for some of the contradictory reports in the literature concerning melatonin's effect on tadpole metamorphic progress.     

Effects of supplemental UV-B radiation on growth and leaf photosynthetic reactions of soybean (Glycine max)

Experiments were conducted under greenhouse conditions to investigate the effects of enhanced UV-B radiation (280 to 320 nm) on height, fresh and dry weights, leaf chlorophyll and carotenoids, CO₂ uptake rates, and Hill activity in soybean ( Glycine max L. cv. Bragg). Plants were exposed for 6 h continuously from midmorning to midafternoon each day to UV-B radiation which was provided by Westinghouse FS-40 sun lamps filtered with 0.127-mm cellulose acetate film (UV-B enhanced) or 0.127-mm Mylar S film (UV-B Mylar control). Three different UV-B enhanced radiation levels were tested: 1.09 (treatment T₁), 1.36 (treatment T₂), and 1.83 (treatment T₃) UV-B sun equivalent units (UV-B_sec) where 1 UV-B_sec= 15.98 mW·m⁻² of solar UV-B obtained by applying EXP -[(α-265)/21]², a weighting function that simulates the DNA absorption spectrum, to the UV-B lamp systems. These UV-B levels correspond to a calculated decrease in stratospheric ozone content of 6%, 21%, and 36% for treatment T₁, T₂, and T₃, respectively.
Daily exposure of soybean plants to UV-B radiation significantly decreased height, fresh and dry weights, leaf chlorophyll and carotenoid contents, and CO₂ uptake rates. Leaf pigment extracted in 80% acetone from UV-B-treated soybean plants showed considerable increase in absorption in the wavelength region of 330 to 400 nm with increased UV-B radiation levels. Chloroplast preparations from leaves of T₂ and T₃ plants showed significant reductions in Hill reaction measurements.     

Influence of Growth Hormone and Thyroxine On Cell Kinetics In the Proximal Tibial Growth Plate of Snell Dwarf Mice

Abstract. In Snell dwarf mice, the influence of short-term treatment with human growth hormone (hGH) or thyroxine on the proliferative and sulphation activity of the proximal tibial growth plate was studied. By autoradiographic methods, the [³H]methylthymidine incorporation after a single injection was measured, after 2 hr incorporation time. the labelling index was calculated and the number of labelled mitoses was counted. In addition, the distribution of the labelled nuclei over the proliferating and degenerating zones was determined by continuous labelling for 25 and 73 hr.
In untreated dwarf mice after [³H]-methylthymidine administration, the number of labelled nuclei in the growth plate is low. Labelling occurs, as expected, mainly in the cells of the proliferative zones. the number of labelled nuclei in control dwarf mice was similar after 25 and 73 hr continuous labelling. This suggests that many cells are in a resting G_o or prolonged G₁ phase. Both hGH and T₄ treatment induce a significant increase of the number of labelled nuclei per growth plate and of the number of mitoses. Since hormonal treatment induces a small number of mitoses after 2 hr incorporation of the label, the minimal G₂ phase of the cell cycle is less than 2 hr. In addition, treatment with hGH and T₄ stimulates chondrocytes in the zone of proliferative and hypertrophic cells to actively incorporate [³⁵S]-sulphate.     

Metamorphic rate as a function of the light/dark cycle in Rana pipiens larvae

1. The rate of development of Rana pipiens tadpoles in spontaneous and thyroxine (T4)-induced metamorphosis was studied on light/dark (LD) cycles in which the photophase was held constant while the scotophase was progressively extended or vice versa. 2. Metamorphic rate fluctuated in both types of experiments as the LD cycle lengthened. However, the pattern of resonance differed with the length of the photophase. For example, with an 8 hr light phase, development rate slowed and then increased as the cycle was extended from 24 to to 36 hr, whereas with a 12 hr photophase the reverse took place. 3. The findings are compatible with the occurrence of a rhythm of light sensitivity in photoperiodic time measurement in this amphibian. 4. From the viewpoint of hormonal mechanisms, it is suggested that photoperiodic effects on metamorphic rate result from different patterns of melatonin secretion under the various LD cycles, since melatonin can modify the action of T4 in metamorphosis.     

Stage-specific polypeptide and villin expression during thyroid-hormone-induced substitution of the amphibian intestinal epithelium

Abstract. Treatment of anuran tadpoles with 5 n M 3,3',5-triiodo- l -thyronine (T₃) results in the complete substitution of the intestinal epithelium. We have examined the developmental pattern of protein synthesis in Alytes obstetricans intestinal epithelium using two-dimensional gel electrophoresis. Four different types of changes have been observed. The group I polypeptides (M_r:41 500; 44 500; 51 500; 55000 and 101000) are only synthesized during the first week of hormonal treatment. They are specific of the primary (larval) epithelium. On the other hand, polypeptides referred to as Group II (M_r: 47000; 48000; 58000; 66500, pl 5.2; 99500 and 102000) are not detected until day 8. They are characteristic of the secondary tissue. Polypeptides of Group III (M_r: 42000, pl 5.15 and 5.25; 42500, 47500, pl 5.25 and 5.55) expressed between the 6th and 8th day of T₃ treatment, are specific of growing stem cells. During this critical period, Group IV polypeptides (M_r: 63500; 66500, pl 6.35; 105000, pl 5.5 and 5.55) are not synthesized. The protein of M_r 105000 (pI 5.5 and 5.55) is immunologically related to villin, a core protein of intestinal microvilli. Expression of this protein has been analyzed by immunoreplica and immunocytochemical procedures during differentiation of basal stem cells into secondary absorptive epithelial cells. The results have been compared to that obtained during spontaneous metamorphosis [19].     

Characterization of the Thyroid Hormone Transport System of Cerebrocortical Rat Neurons in Primary Culture

Abstract: The uptake of 3',3,5-triiodo- l -thyronine (T₃) and l -thyroxine (T₄) by primary cultures derived from rat brain hemispheres was studied under initial velocity conditions, at 25°C. Uptake of both hormones was carrier mediated and obeyed simple Michaelis-Menten kinetics. The K _m of T₃ uptake was very similar to that of T₄, and did not vary significantly from day 1 to 4 in culture (310–400 n M ). The maximal velocity ( V _max) of T₃ uptake nearly doubled between day 1 and 4 of culture (41 ± 3 vs. 70 ± 5 pmol/min/mg of DNA, respectively). The V _max of T₄ uptake did not change (28 ± 8 and 31 ± 4 pmol/min/mg of DNA on days 1 and 4, respectively). The rank order of unlabeled thyroid hormone analogues to compete with labeled T₃ or T₄ uptakes were the same (T₃ > T₄ > 3',5',3-triiodo- l -thyronine > 3',3,5-triiodo- d -thyronine > triiodothyroacetic acid), indicating that the transport system is stereospecific. Unlabeled T₄ was a stronger competitor of labeled T₄ uptake than of labeled T₃ uptake, whereas unlabeled T₃ had the same potency for both processes. These results suggest that T₃ and T₄ are transported either by two distinct carriers or by the same carrier bearing separate binding sites for each hormone. They also indicate that the efficiency of T₃ uptake increases during neuronal maturation.     

Heterotrophic tobacco cell cultures during greening

Structural changes in cells and plastids are described that occur during the greening of an initially dark-grown cell suspension of tobacco ( Nicotiana tabacum L. cv. Xanthi). The pattern of cell growth during greening, expressed in dry weight or cell number, showed a classical sigmoid curve with a lag phase (T₀–T₂), an exponential phase (T₃–T₉) and a stationary phase (T₁₀–T₂₁). Achlorophyllous vacuolated cells (T₀), obtained after 3 culture cycles in the dark, contained amyloplasts devoid of lamellae. Exposure to light brought about an enrichment in cytoplasm and an amyloplast to proplastid transformation (starch loss) accompanied by chlorophyll synthesis. By T₃, many cells appeared meristematic and contained dividing proplastids with rudimentary single lamellae typical of those in intact meristematic leaf cells. As cell division occurred (T₃ to T₉), plastids replicated and their internal membrane system developed progressively into defined grana-intergrana thylakoids. By the stationary phase of cell growth (T₁₄), the lamellar system had reached a highly structured grana-intergrana network typical of higher plant chloroplasts. We have emphasized the analogies between the sequence of events (proplastid to chloroplast transition) during the greening of tobacco cells and that in developing intact leaves; in this respect the cell cultures provide a useful material for studies dealing with the biogenesis of structural or physiological events.     

Dose-related effects of 17βoestradiol (E2) on liver weight, plasma E2, protein, calcium and thyroid hormone levels, and measurement of the binding of thyroid hormones to vitellogenin in rainbow trout, Salmo gairdneri Richardson

Administration of 17β-oestradiol (E₂) to rainbow trout, in the form of hydrogenated coconut oil implants produced a stable, long-term elevation in plasma E₂ levels. The elevation was doserelated (over the range 1–10mg kg-¹ body weight) both 4 and 8 weeks after implantation. Dose-related increases were also observed with respect to liver weight-body weight ratios and plasma protein levels. Plasma T₃ and total calcium levels were depressed and elevated, respectively, by E₂ treatment but the responses were not linearly related to the dose of E₂ administered; there was no significant effect of E₂ on plasma T₄ levels.
E₂ induced a shift in the binding of T₃ to plasma proteins, with T₃ binding to smaller molecular weight proteins; neither T₄ nor T₃ bound to vitellogenin which was present at high levels in the plasma of E₂-treated fish.     

Effect of rearing density on thyroid and interrenal gland activity and plasma and hepatic metabolite levels in rainbow trout, Salmo gairdneri Richardson

There were significant inverse correlations between rearing density of rainbow trout, Salmo gairdneri Richardson, and final body weight, plasma L-thyroxine (T₄), trüodo-L-tryronine (T₃), cortisol and protein concentrations, plasma T₄/T₃ ratios and thyroid epithelial cell height. In addition, hepatosomatic indices and plasma free fatty acid concentrations were higher in fish reared at low (134 g 1⁻¹) density compared with groups reared at medium (210g1⁻¹) or high density (299g 1⁻¹), and the post-feeding (3.5-4h) elevation in plasma glucose and triglyceride levels evident in trout maintained at low rearing density was not found in those fish reared at higher densities. There were no significant effects of rearing density on hematocrit, carcass composition, hepatic glycogen and lipid levels and interregnal nucleus size.     

Thyroid Hormone Replacement Restores Circulating Enkephalin Concentrations in Hypophysectomized Fetal Sheep

Abstract: We have investigated the effects of fetal hypophysectomy (HX) with or without thyroxine (T₄) replacement on the plasma concentrations of free methionine-enkephalin (free Met-Enk), noradrenaline, and adrenaline in late gestation sheep fetus. Plasma adrenaline concentrations were significantly higher in intact fetal sheep (1.05 ± 0.12 pmol/L) between 125 and 140 days of gestation when compared with the HX + saline (0.64 ± 0.10 pmol/L) and HX + T₄ (0.61 ± 0.08 pmol/L) groups. During the first 15 days of the T₄ or saline infusion, the plasma concentrations of free Met-Enk were significantly higher in the HX + T₄ group (392 ± 40 pmol/L) than in the HX + saline group (299 ± 43 pmol/L). At this stage of gestation, however, circulating concentrations of free Met-Enk were significantly higher in intact fetal sheep (556 ± 51 pmol/L) than in either of the HX groups. Between 125 and 140 days of gestation, plasma free Met-Enk concentrations were similar and significantly higher in the intact and HX + T₄ groups than those measured in the HX + saline fetal sheep. We conclude that the decrease in circulating free Met-Enk concentrations after removal of the fetal pituitary is primarily a consequence of functional athyroidism.