Relationship between steroid excretion patterns and breast cancer incidence in Israeli women of various origins.

A nationwide study of the steroid excretion patterns in postmenopausal Israeli migrant women demonstrated differences between high- and low-risk groups for breast cancer in the following variables: age at first parturition, number of pregnancies, number of live births, height, and weight. The direction of the differnces was in line with those observed for breast cancer patients. The groups also differed in the exretion of estriol, 17-ketosteroids, and allotetrahydrocortisol. Multiple regression analysis revealed that the exretion of estriol was significantly lower in population groups in whom breast cancer incidence was high. Possibly this trend--which has also been observed in adolescent and premenopausal women--reflected environmental influences on peripheral estrogen metabolism.

Triiodothyronine binding in rat anterior pituitary, posterior pituitary, median eminence and brain.

In vivo studies of the exchange of tracer [125-I]-L-triiodothyronine (T3) between plasma (P), and the anterior pituitary (AP), posterior pituitary (PP), median eminence (ME) and the frontal lobes of the brain (B), in the rat show that from 2.5 h onwards the concentration of new [125I]T3 in AP, PP and B were parellel to that of the plasma, with a t1/2 of 7.4 h; the t1/2 for ME was 10.3 h. The extrapolation of these curves to zero time was assumed to indicate the relative concentration of T3 per unit weight in terms of total body T3. T3 content of these tissues was determined by radioimmunoassay. The values obtained validated the steady state parameters derived from the radio-isotopic measurements. As an indicator of the concentration gradient between tissue and plasma the organ/plasma (O/P) ratio was calculated; these data indicate that under steady state conditions, the order of T3 concentration is AP greater than PP greater than ME greater than B. Binding studies have shown that AP and PP contain "specific," saturable binders while ME and B do not. Evaluation of the binding parameters of the high affinity binders in both AP and PP gave similar association constants. These associations constants, when corrected for the binding strength of T3 to plasma proteins, resulted in values similar to those of neuclear T3 binders.

Suppression of thyrotropin by 3,5-dimethyl-3'-isopropyl-L-thyronine in euthyroid and hypothyroid rats.

The effect of 3,5-dimethyl-3'-isopropyl-L-thyronine (DIMIT) a nonhalogenated thyroid hormone analog, on plasma and pituitary TSH levels was studied in euthyroid and 5-day-thyroidectomized (T-5d) rats. The minimal dose of DIMIT which suppressed basal plasma TSH for a 24-h period was 5.3 micrograms/100 g BW in the euthyroid rats and 5.2 micrograms/100 g BW in the T-5d group. The equipotent doses of T3 were 1.0 and 1.5 micrograms/100 g BW, respectively. Thus, the apparent DIMIT to T3 molar ratio ranged from 6.6 in the hypothyroid to 10 in the euthyroid group. The suppression of TRH-induced TSH release in the euthyroid rats required a high dose of DIMIT (9.2 micrograms/100 g BW), resulting in a DIMIT to T3 molar ratio of 17. Time-course studies comparing the effects of equipotent doses of DIMIT and T3 on TSH release rates in euthyroid and T-5d rats have shown a maximal suppression by 3--24 h after the administration of both T3 and DIMIT. However, the recovery from the effect was slower after T3. TSH synthesis in the T-5d group was similarly suppressed by DIMIT and T3. DIMIT has significant thyromimetic activity in euthyroid and hypothyroid rats, as evidenced by the suppresion of TSH synthesis and release.

Striated muscle fibers differentiate in primary cultures of adult anterior pituitary cells.

Anterior pituitary cells from adult male rats were cultured on a natural extracellular matrix (ECM). From the 5th day, spindle-shaped cells, fusing to form elongated fibers, were observed among the epithelial cells. These fibers later increased in size and number and were identified as striated muscle fibers, based on their multi-nucleation, cross-striation and rhythmic contraction. Striated muscle development was further validated by the appearance of cholinergic-nicotinic receptors, as demonstrated by the binding of 125I-alpha bungarotoxin (a-Btx). The effect of various supplements added to the cultures on a-Btx binding was used as a measure of muscle cell differentiation. ECM and serum were essential for myotube formation. Insulin (or IGF-I) and cortisol synergistically caused a 20-fold increase in myogenesis. FGF and EGF were effective only in the presence of insulin and cortisol. The data suggest that: (a) cells, derived from adult pituitary, differentiate, in primary culture, into striated muscle fibers; (b) the induction of this differentiation is multi-factorial; ECM, serum, insulin and glucocorticoids facilitate this myogenesis.

The synthesis and release of TSH in the thyroidectomized rat: c) the effect of T3 on hypothyroidism of varying duration.

The effect of thyroid hormones on TSH synthesis and release from the anterior pituitary of the rat, at the various stages of hypothyroidism was studied by injecting L-Triiodothyronine (T3) iv (1.5 microgram/100 g b.w.) to rats 5 days and 3 months post thyroidectomy (Tx-5d and Tx-3m rats). The changes in plasma and pituitary TSH levels, as well as in the metabolic clearance rate (MCR) of the TSH were followed for 3 days after the T3 injection. From these data TSH synthesis and release rates were determined. TSH release was inhibited in both experimental groups between 3 and 24 h after the injection. TSH synthesis was suppressed in the Tx-5d group to 35% of the initial value. However in the Tx-3m group it was stimulated to approximately 270%, 24 h after the T3. The data suggest that thyroid hormones may affect the synthesis of TSH in two opposite pathways, the first through the stimulation of protein synthesis and the second, inhibitory, as part of the feedback mechanism. Under short-term hypothyroidism, when protein synthesis is not yet diminished, the administration of thyroid hormones results in the inhibition of both TSH synthesis and release through the feedback regulation. In prolonged hypothyroidism, however, when protein synthesis is depressed, the effect of thyroid hormones on the stimulation of TSH synthesis predominates.

TSH synthesis and release in the thyroidectomized rat: b) effect of T3.

The time and dose dependent effects of T3 on the anterior pituitary (AP) TSH release and synthesis were studied in rats 5 days after thyroidectomy (T-5d). T3, in various doses, was injected iv to animals which were kileed at various times later. Plasma and AP TSH were measured. T3 lowered plasma TSH to a minimum within 3 h. An inverse linear relationship was found between T3, up to 634 +/- 134 ng/100 g b.w. (mean +/- SD) and plasma TSH. Higher doses of T3 did not lower plasma TSH any further. At T3 of 634 +/- 134 ng/100 g b.w. the specific AP binder was calculated to be 68% to 90% saturated; thus suggesting a relationship between occupancy by T3 of the AP binder and the regulation of TSH release. The TSH release and synthesis rates were compared after 0.5 micrograms, 1 microgram and 25 micrograms T3. All three doses of T3 suppressed the release as well as the synthesis. The effect of T3 on the synthesis rate, however, lagged behind the effect on the release both in time and magnitude.

Thyrotrophin and growth hormone secretion and cell morphology in hypothyroid pituitary cells cultured on a natural extracellular matrix.

Hypothyroid rat pituitary cells were cultured on an extracellular matrix (ECM) produced by corneal endothelial cells. ECM markedly affected the following parameters. 1) Cell attachment, spreading and proliferation were improved, resulting in a more rapid formation of confluent cell monolayers. 2) In the presence of ECM the production of both thyrotrophin (TSH) and growth hormone (GH) was larger than in its absence. Immunofluorescence studies revealed that 20% of the cells in these monolayers were thyrotrophs; consequently, TSH content was higher while GH level was lower than in control cultures of euthyroid pituitaries. These data suggest that the in vivo physiological differences are maintained by the conditions of the culture. 3) ECM enabled the response of both the somatotrophs and the thyrotrophs to thyrotrophin releasing hormone (TRH) and the cells remained responsive for at least 10 days. Maximal stimulation (1.3- to 6-fold) was obtained with 14 nM of TRH. In the absence of ECM the cells failed to respond to TRH. 4) Cell morphology was examined by scanning electron microscopy (SEM). Cells grown on ECM were characteristically epithelial, whereas those cultured on plastic plates had a fibroblast-like appearance. Four types of cells were identified in the epithelial cell monolayers by the appearance of their surface. TRH induced a 2-fold increase in the number of cells covered with microvilli and a corresponding decrease in the number of smooth surfaced cells. This suggests that hormone secretion is associated with the formation of microvilli.

Serum reduces the TSH content in rat pituitary cells in culture.

Euthyroid rat pituitary cells were plated on a natural extracellular matrix (ECM) and cultured in either a medium supplemented with hormones and growth factors (SM) or in an unsupplemented medium (USM). Hormone supplementation resulted in a marked increase in the number of cells and in a less pronounced increase in the TSH content. Addition of 10% dehormonized serum (DHS) to the medium caused a further increase in the cell number as well as a significant elevation in the GH content but, surprisingly, it caused also a dramatic decrease in the TSH content. Time course experiments revealed that the serum doubled the rate of suppression of the TSH. DHS prepared from rat serum was as effective as that prepared from foetal calf serum and heat-inactivation of the serum did not change its suppressive effect on the production of TSH. This effect was confined to the serum fraction which was precipitable with 45% ammonium sulphate but did not bind to Con-A. It is suggested that a high molecular weight protein fraction of serum reduces the number of thyrotrophs in the culture and/or decreases the rate of TSH production by these cells.

TSH binding proteins in rat and human serum.

When serum of hypothyroid rats was fractionated on a Sephadex G-100 column, most of the immunoreactive TSH was found as a front running peak, together with the high molecular weight serum proteins. Similarly, a rTSH preparation (10 mU), chromatographed in the presence of 1 ml of normal rat serum also migrated at the front, however, when a high load of TSH (4.4 U) was added to 1 ml of serum, two immunoreactive peaks were found, suggesting the saturation of the front running fraction. Immunoelectrophoresis and autoradiography of rat or human sera containing the respective 125I-labelled TSHs showed binding of the labelled TSH to IgG, alpha-2-macroglobulin, and to a third unidentified protein, migrating near the albumin line. In order to determine if the bound TSH is biologically active, the high molecular weight protein fraction was separated from hypothyroid rat serum by 45% ammonium sulphate precipitation. Immunoreactivity was determined by RIA and the biological activity was determined, in vitro, by the stimulation of 99Tc uptake by FRTL-5 cells. The 45% ammonium sulphate precipitate contained almost all of the immunoreactivity and the bioactivity of the TSH of whole serum. These results indicate that: a) The endogenous circulating TSH in the hypothyroid rat exists mainly in a protein-bound form and this protein-bound TSH contains most of the hormonal bioactivity of the serum. b) Exogenous TSH binds to serum proteins in euthyroid and hypothyroid rats and in humans. There are three protein fractions in these sera that bind TSH, one of which is an immunoglobulin. The occurrence of TSH binding immunoglobulins may involve autoimmune mechanisms.