In order to elucidate the mechanism of thyroid hormone action on the ovary, direct effects of L-thyroxine (T₄) or L-triiodothyronine (T₃) on steroidogenic enzyme induction were investigated in vitro using a monolayer culture system of porcine granulosa cells obtained from small follicles.
The cells were cultured in the absence or presence of porcine FSH (20ng/ml) for 6 days, with or without T₄ or T₃, under sparsely (4%) serum supplemented condition. The mechanism of thyroid hormone action on the granulosa cells was studied by testing the capability of thyroid hormone to enhance the steroidogenesis in response to exogenously provided substrates. Concomitant treatment with FSH (20ng/ml) and T₄ (10^-7 M) caused a further increased production of progesterone in response to the addition of pregnenolone compared to that in the absence of pregnenolone. The same treatment with FSH and T₄ also caused a further increased production of estrone in response to the addition of androstenedione. Concomitant treatment with 10^-9 M T₃ demonstrated similar stimulatory effects on the steroidogenesis in cultured granulosa cells. T₄ or T₃ alone without FSH was incapable of exhibiting these stimulatory effects.
Furthermore, aromatase activity in cultured granulosa cells assessed by the release of tritiated water from [1β-³H, 4-¹⁴ C] androstenedione was significantly higher in the cells treated concomitantly with FSH (20ng/ml) and T₄ (10^-7M) than that in the cells treated with FSH alone.
These results suggest that thyroid hormone synergizes with FSH and increases FSH-mediated induction of 3β-hydroxysteroid dehydrogenase (3β-HSD) and aromatase activity in immature granulosa cells. Since the effective dose of T₄ and T₃ observed in our studies is in the physiological range of circulating total levels of T₄ and T₃, it can be concluded that the synergism between FSH and thyroid hormone is of physiological importance to the full expression of FSH actions in the functional differentiation of immature granulosa cells.

View full abstract

In order to clarify the onset of insulin secretion and the regulatory mechanism of its receptor induction in fetus, blood glucose, serum insulin and its hepatic receptor in situ in fetal rats (D₁₈-D₂₁) were measured and the changes of the levels of insulin and its receptor after the direct injection of glucose (1g/kg) to fetal rats in utero were investigated.
In fetal rats (D₁₈-D₂₁), both serum insulin and glucose levels increased as pregnancy progresses and specific binding of insulin in fetal liver microsomal membranes increased on D21 of gestation, mainly due to the increase in binding affinity rather than binding capacity.
After the direct injection of glucose to the fetus in utero, the rapid increase in serum insulin and the rapid decrease in insulin specific binding to liver microsomal membranes were observed in a part of D₂₀ and all of D₂₁ fetal rats, which was mainly due to the decrease of binding capacity. This suggests that the acute elevation of endogenous insulin level followed by the decrease of insulin specific binding in fetal rat liver is based on the down regulation mechanism of insulin receptor, because the amounts of insulin separated from liver microsomal membranes were less than one twentieth of the insulin concentration which are enough to decrease the binding capacity of hepatic receptor theoretically. In addition, the dissociation of ¹²⁵I-insulin from liver microsomal membranes of glucose-treated rats were indistinguishable from that of control.
From these results, it can be concluded that the onset of glucose-stimulated insulin secretion appears in fetal rats on D₂₀ of gestation and the elevation of endogeneous insulin rapidly down-regulates the number of hepatic insulin receptor in fetal rats.

View full abstract

In thirty-one patients with various pituitary disorders, their serum TSH levels were reevaluated using two kinds of highly sensitive immunoradiometric assays (IRMAs). TSH levels were measured by radioimmunoassay (RIA) supplied by Daiichi Radioisotope Laboratory and the IRMAs, which were RIA-gnost TSH Ultrasensitive and SUCROSEP TSH IRMA using two types of monoclonal antibodies. The sensitivities of them were 0.08 and 0.1μU/ml, respectively. Free thyroxine and free triiodothyronine levels were measured by RIAs (Amerlex kits). There was a significant correlation between basal TSH levels and the maximum increase in serum TSH above baseline using the RIA and the IRMAs when responses to TRH (500μg i.v.) was determined, but no correlation between basal TSH and thyroid free hormone levels. In their TRH tests, three patients showed absent responses to TRH which were measured by the RIA, although by the IRMAs slight responses were observed. In two patients, exaggerated responses to TRH were observed and the increase of TSH levels measured by the IRMAs showed 143-210% higher than that of the RIA. The IRMAs were able to handle their peak levels time in all the patients with responses, however, in some patients the RIA was not sensitive enough to distinguish it. These results indicated that measurement of TSH levels by the IRMAs might be useful in the evaluation of the TSH secretion capacity in patients with various pituitary disorders.

View full abstract

The purpose of this study was to estimate the sympatho-adrenomedullary activity in young patients with borderline hypertension (BHT, n=23), compared with age-matched normotensive subjects (NT, n=9), so that two studies were performed as follows : they were subjected to isometric stress, by maintaining handgrip at the 30% level of maximal voluntary contraction for three minutes. With the exercise blood pressure and pulse rate increased to the same degree in BHT as in NT. In contrast, the response of plasma total catecholamine (plasma epinephrine plus norepinephrine) at the end of this isometric exercise was greater in BHT than in NT (93.0 ± 12.6 in BHT vs. 47.1 ± 15.4pg/ml in NT). Moreover, the effects of intravenous glucagon injection (1 USP unit) were studied in twelve subjects of BHT (n=12) and all of NT (n=9). The injection of glucagon induced a transient increase in pulse rate, but there was no significant difference in the elevation of pulse rate with glucagon between BHT and NT. Plasma epinephrine also increased temporarily, and returned to the baseline within ten minutes after injection. The increments of plasma epinephrine at two and three minutes after injection were significantly greater in BHT than those in NT : 44.1 ± 12.3 vs. 5.1 ± 4.4pg/ml, and 68.9 ± 13.2 vs. 32.1 ± 8.9pg/ml, respectively. Thus, patients with borderline hypertension had the augmented response of plasma catecholamine to both isometric exercise and glucagon stimulation. Evidence presented suggests that the responses of sympathetic nervous system and adrenal medulla to stress are increased in young patients with borderline hypertension. Moreover, the augmented response of sympatho-adrenomedullary system to stress may be involved in the development of essential hypertension.

View full abstract

Anti-thyroglobulin antibody (aTg) synthesis by the lymphocytes in the peripheral blood and the thyroid gland were studied in patients with Hashimoto's disease (HD) or Graves' disease (GD), all in euthyroid states, to clarify the mechanism of autoantibody synthesis.
The ability of the lymphocytes to synthesize aTg was analyzed in the culture system of lymphocytes incubated in a concentration of 1 × 10⁶ cells/ml for 7 days at 37°C in 5% CO₂-95% air. The B cells alone were also cultured in the absence of T cells or PWM to estimate their abilities on spontaneous aTg synthesis. The regulatory functions of T cells on aTg synthesis by B cells were investigated in cross-combination cultures of B cells and an equal number of T cells. The concentration of aTg and total IgG in cultured medium were measured by sensitive enzyme immunoassay developed by us, and the capacity on aTg synthesis was expressed as aTg/IgG ratio (aTg%). The surface markers of lymphocytes in the peripheral blood and the thyroid gland were determined by flowcytometry using mouse monoclonal antibodies (CD3, CD4, CD8, OKIal, CD20 and Leu7).
These results were obtained as follows : 1) All the lymphocytes from the peripheral blood, thyroid gland and bone marrow of HD patients synthesized much more aTg (3.1 ± 1.6, 2.2 ± 0.9, 1.5 ± 0.5%, respectively) than those from normal peripheral blood lymphocytes (1.0 ± 0.9%). This hyper-function of aTg synthesis by the lymphocytes in HD patients was caused by the presence of activated B cells and the predominance of helper T cells.2) Both the lymphocytes from the peripheral blood and the thyroid gland in GD patients synthesized the same level of aTg (0.7 ± 0.7%) as in normal subjects. However, the lympho cytes from bone marrow and lymph nodes, which were indicated by Benner et al. to play a main role in antibody synthesis after immunization with antigen, were involved in aTg synthesis (1.8 ± 1.2, 5.4 ± 1.1%, respectively). 3) There was no correlation between aTg synthesis and CD4⁺/CD8⁺ ratio of the peripheral blood lymphocytes in AITD patients.
These results suggest that aTg synthesis in HD patients is an expression of abnormal immune reaction due to the presence of aTg specific activated B cells and dysfunction of regulatory T cells, and that aTg production by the lymphocytes from the bone marrow and lymph nodes in GD patients is resulted from a normal immune response to the high level of thyroglobulin in the blood. The CD4⁺/CD8⁺ ratio of the lymphocytes from the patients does not reflect their ability to synthesize a specific antibody.

View full abstract

The aim of this study is to elucidate the role of “β-endorphin (β-End) & β-lipotropin (β-LPH)” on the regulation of gonadotropin (Gn) secretion. We investigated the relationships between immunoreactive β-End plus β-LPH and Gn in peripheral plasma of normal menstruating women, 1) during periovulatory period, especially at the time of Gn surge, 2) at the estrogen induced Gn surge (positive feedback) and 3) at the time of hypoxic stress.
Plasma concentrations of β-End plus β-LPH were measured as immunoreactive β-End (i-β-End) by RIA after extraction with Sep-Pak C₁₈.
First, we assessed pulsatile Gn secretions during periovulatory period in 19 normal women every 10 minutes for 4 hours, some women were at the time of Gn surge in its ascending limb, plateau of the peak, and descending limb of the surge. Meanwhile, circadian variations of plasma i-β-End levels of these subjects were assessed at 4 hours' interval on the same day. In two subjects, on the day before the onset of LH surge, significantly low (p<0.05) basal and peak levels of i-β-End were observed, although the basic patterns of circadian rhythm were preserved.
Secondly, changes of plasma i-β-End levels during estrogen (estradiol benzoate 1mg i.m.) induced positive feedback tests were evaluated in 16 normal women and 6 hypothalamic amenorrheic women by daily blood sampling. In normal subjects, small but significant increases (p<0.05) of plasma i-β-End were observed when Gn showed initial decreases at 48 hours after injection. Subsequently at 72 hours, however, plasma i-β-End decreased precipitously at the time of Gn surge. On the other hand, in hypothalamic amenorrheic women who were devoid of Gn surge, no significant changes of plasma i-β-End levels were observed.
The transient decreases of plasma i-β-End just prior to the Gn surge support the idea that i-β-End exerts tonic inhibition on the onset of Gn surge and the disappearance of its inhibition might trigger the positive Gn surge. And it was also suggested that release mechanisms of both i-β-End and Gn are impaired in hypothalamic amenorrhea.
Thirdly, in acute hypoxic stress experiment, 5 normal female volunteers were placed in hypobaric (500 mbar) condition in which oxygen supply is a half of atmosphere, simultaneous blood samplings of Gn, prolactin and i-β-End were performed every 15 minutes for 3 hours. The concomitant increases of plasma prolactin and i-β-End levels were observed in 3 women, while pulsatile Gn secretions were not disturbed. This evidence suggests the acute increase of i-β-End secretion does not necessarily suppress the basal pattern of pulsatile Gn release.
Altogether, it was suggested that i-β3-End (β-End & β-LPH) is not substantially related to the tonic release mechanism of pulsatile Gn secretion, but is rather related to the cyclic release mechanism of Gn surge.

View full abstract