Developmental profiles of TSH, sex steroids, and their receptors in the thyroid and their relevance to thyroid growth in immature rats.

Sex steroids are reported to influence thyroid pathogenesis in human and experimental animals. However, there is no report on this phenomenon during the early developmental period. The mitotic activity of thyrocytes in rats reaches its peak by day 10 postpartum. Thyrocytes actively proliferate in immature rats during the first three postnatal weeks, during which the pre-pubertal rise in serum titers of testosterone and estradiol has been recorded. The aim of the present study was to analyze whether there is a physiological relevance between thyroid growth and sex steroids during the postnatal period. Serum and thyroid tissue hormones (TSH, testosterone, and estradiol) were assayed by liquid phase RIA, and receptors for these hormones were also quantified. The peak rate of thyrocyte proliferation was observed during the second postnatal week in rats. Since the concentrations of sex steroids and their receptors also reached a peak around this period, it is suggested that elevated sex steroids and their receptors in the thyroid might enhance thyrocyte proliferation. A positive correlation between thyroid growth indices and sex steroids and their receptors further strengthens this suggestion. This is a preliminary study, and further experimental study may strengthen this proposal. This is the first report to show the availability of sex steroids and their receptors in the thyroid glands of immature rats under normal conditions.     

Testosterone and estradiol modulate TSH-binding in the thyrocytes of Wistar rats: influence of age and sex.

Age and sex are important factors that influence thyroid pathophysiology. Though sex steroids are known to enhance thyrotropin (TSH) mRNA expression and incidence of thyroid tumours, there is no report on their effects on TSH action under normal physiological conditions. In the present study, the effects of testosterone (T) and estradiol (E2) on thyroidal TSH-receptor (TSH-R) concentration, and TSH-binding to thyrocytes (in vitro) were elucidated in immature and mature Wistar rats. Immature (10 days old) and adult (120 days old) rats of either sex were gonadectomized (GDX) and one group of GDX rats was treated with physiological doses of T and another with E2. Immature GDX rats were supplemented with the steroids for 10 days and adults were supplemented with the steroids for 30 days. While supplementation of steroids to immature rats was begun immediately after surgery, for adult rats it was started 10 days after gonadectomy. The rats were killed at the end of the experimental period. Gonadectomy significantly decreased serum TSH, and TSH-R concentration under in vivo condition and [125I]-TSH binding to thyrocytes under in vitro conditions. Supplementation of T to male and E2 to female GDX rats restored normality of the parameters. Thyrocytes of immature male rats challenged with linearly increasing doses of TSH or T (6.25-800 ng/ml) showed a dose-dependent increase in TSH-binding. However, thyrocytes of immature female rats challenged with T showed a gender-specific response. While there was a linear increase in TSH-binding in thyrocytes of males, a biphasic response was evident in thyrocytes of females. In the case of thyrocytes from adult rats, T induced a dose-dependent change in TSH-binding in males, which reached the peak in response to 12.5 ng T, and diminished thereafter. In contrast, E2 was inhibitory to TSH-binding to thyrocytes of adult male rats. On the other hand, E2 showed a clear gender-specific stimulation of TSH-binding in thyrocytes of females and an inhibition of the same in males. TSH and sex steroids upregulated TSH receptors in immature rats, whereas the effect was biphasic in adult rat thyrocytes. It is concluded from the present study that sex steroids modulate TSH-binding in rat thyrocytes, which may vary according to the age and sex of the animals.     

Testosterone and estradiol up-regulate androgen and estrogen receptors in immature and adult rat thyroid glands in vivo

Thyroid gland is one of the non-classical target organs for sex steroids. Presence of androgen and estrogen receptors in the neoplastic and non-neoplastic thyroid glands of mammalian species is well documented. The aim of the present study is to elucidate the changes in serum and thyroidal sex steroids, and their receptors in the thyroid gland of rats from immature to adult age under gonadectomized (GDX) and sex steroids replaced conditions. Normal Wistar male and female rats from immature to adult age (day 21, 30, 45, 60 and 160 post-partum (pp)) were used in the present study. One group (I) of rats was GDX at an early age (day 10 pp) and the other group (II) at the adult age (day 120 pp). Group I rats were sacrificed at different experimental periods such as 21, 30, 45 and 60 days pp, and group II rats were sacrificed at day 160 pp. Another group of GDX rats from group I and II were replaced with physiological doses of testosterone or estradiol. Serum and thyroidal concentrations of sex steroids were estimated by RIA method and the concentrations of receptors by radioreceptor assay. Gonadectomy significantly decreased serum and thyroidal testosterone and estradiol and concentrations of androgen receptor (AR) and estrogen receptor (ER) in the thyroid. Replacement of sex steroids to GDX rats restored the normal level of sex steroids, AR and ER. Therefore, it is suggested from the present study that (i). sex steroids up-regulate their own receptors in the thyroid, (ii). sex steroids may influence thyroid growth and the proliferation of thyrocytes by modulating their receptor concentrations in the thyroid.     

Functional roles of the bone morphogenetic protein system in thyrotropin signaling in porcine thyroid cells

We uncovered a new regulation of thyrocyte function by bone morphogenetic protein (BMP) under the influence of thyrotropin (TSH) using primary culture of porcine thyrocytes. The BMP type I receptors, ALK-2 (ActRIA), -3 (BMPRIA), and -6 (BMPRIB), were expressed in porcine thyrocytes, while ALK-6 was not detected in human thyroid. Treatment with BMP-2, -4, -6, -7, and TGF-beta1 exhibited a dose-dependent suppression of DNA synthesis by porcine thyrocytes. BMP-2, -4, -6, -7, and TGF-beta1 suppressed TSH receptor mRNA expression on thyrocytes, which was consistent with their suppressive effect on TSH-induced cAMP synthesis and TSH-induced insulin-like growth factor-1 expression. Activin exhibited minimal suppression of thyrocyte DNA synthesis and did not exhibit suppressive effects on TSH receptor mRNA expression. Phosphorylated Smad1/5/8 was detected in the lysates of porcine thyrocytes treated with BMP-2, -4, -6, and -7. However, in the presence of TSH, BMP-6 and -7 failed to activate Smad1/5/8 phosphorylation and 3TP-reporter activity, whereas BMP-2 and -4 maintained clear activation of the BMP signaling regardless of the presence of TSH. This diverged regulation of thyroid BMP system by TSH is most likely due to the reduction of ALK-6 expression caused by TSH. Thus, the thyroid BMP system is functionally linked to TSH actions through modulating TSH receptor expression and TSH, in turn, selectively inhibits BMP signaling. Given that BMP system is present in human thyroid and the expression pattern of ALK-2 and BMPRII is different between follicular adenomas and normal thyroid tissues, the endogenous BMP system may be involved in regulating thyrocyte growth and TSH sensitivity of human thyroid adenomas.     

Differential response of rat skeletal muscle glycogen metabolism to testosterone and estradiol.

Although reports on sex steroids have implicated them as promoting protein synthesis and also providing extra strength to the skeletal muscle, it remains unclear whether sex steroids affect glycogen metabolism to provide energy for skeletal muscle functions, since glycogen metabolism is one of the pathways that provides energy for the skeletal muscle contraction and relaxation cycle. The purpose of the current study was to show that testosterone and estradiol act differentially on skeletal muscles from different regions, differentially with reference to glycogen metabolism. To study this hypothesis, healthy mature male Wistar rats (90-120 days of age, weighing about 180-200 g) were castrated (a bilateral orchidectomy was performed to test the significance of skeletal muscle glycogen metabolism in the absence of testosterone). One group of castrated rats was supplemented with testosterone (100 microg/100 g body weight, i.m., for 30 days from day 31 postcastration onwards). To test whether estradiol has any effect on male skeletal muscle glycogen metabolism 17beta-estradiol (5 microg/100 g body weight, i.m., for 30 days from day 31 postcastration onwards) was administered to orchidectomized rats. To test whether these sex steroids have any differential effect on skeletal muscles from different regions, skeletal muscles from the temporal region (temporalis), muscle of mastication (masseter), forearm muscle (triceps and biceps), thigh muscle (vastus lateralis and gracilis), and calf muscle (gastrocnemius and soleus) were considered. Castration enhanced blood glucose levels and decreased glycogen stores in skeletal muscle from head, jaw, forearm, thigh, and leg regions. This was accompanied by diminished activity of glycogen synthetase and enhanced activity of muscle phosphorylase. Following testosterone supplementation to castrated rats, a normal pattern of all these parameters was maintained. Estradiol administration to castrated rats did not bring about any significant alteration in any of the parameters. The data obtained suggest a stimulatory effect of testosterone on skeletal muscle glycogenesis and an inhibitory effect on glycogenolysis. Estradiol did not play any significant role in the skeletal muscle glycogen metabolism of male rats.     

Effects of thyrotropin and N6,O2′-dibutyryl cyclic 3′,5′-adenosine monophosphate on prostaglandin levels in thyroid

We have shown that TSH increases PG levels in isolated bovine thyroid cells. We now report that TSH also increases PG levels in rat and mouse thyroid, and that these effects may be mediated via cyclic AMP. PG and cyclic AMP levels in intact rat and mouse thyroid lobes were measured by radioimmunoassay. During 60-min incubations at 37°C, 25 mU/ml TSH effected a 75–83% increase in PGE₁ and PGF_2α ”equivalents“ in rat thyroid; parallel measurements of endogenous cyclic AMP in these intact thyroid lobes revealed that maximal TSH-induced increase in cyclic AMP also required 60-min incubations. In mouse thyroid, 5 mU/ml TSH increased PGE₁ and PGF_2α levels 38–82% above basal; this TSH effect was evident within 15 min of incubation, thus mimicking the time-course of TSH-induced increase in mouse thyroid cyclic AMP. Exogenous DBcAMP, 0.5 to 3 mM, effected a dose-related increase in mouse thyroid PG levels. The stimulatory effects of both TSH and DBcAMP on mouse thyroid PG levels were abolished by aspirin and indomethacin. These studies suggest that TSH-induced increase in endogenous PG levels in thyroid may be mediated by cyclic AMP.     

Regulation of the phosphatidylinositol 3-kinase,Akt/protein kinase B,FRAP/mammalian target of rapamycin,and ribosomal S6 kinase 1 signaling pathways by thyroid-stimulating hormone (TSH) and stimulating type TSH receptor antibodies in the thyroid gland

Thyroid-stimulating hormone (TSH) regulates the growth and differentiation of thyrocytes by activating the TSH receptor (TSHR). This study investigated the roles of the phosphatidylinositol 3-kinase (PI3K), PDK1, FRAP/mammalian target of rapamycin, and ribosomal S6 kinase 1 (S6K1) signaling mechanism by which TSH and the stimulating type TSHR antibodies regulate thyrocyte proliferation and the follicle activities in vitro and in vivo. The TSHR immunoprecipitates exhibited PI3K activity, which was higher in the cells treated with either TSH or 8-bromo-cAMP. TSH and cAMP increased the tyrosine phosphorylation of TSHR and the association between TSHR and the p85alpha regulatory subunit of PI3K. TSH induced a redistribution of PDK1 from the cytoplasm to the plasma membrane in the cells in a PI3K- and protein kinase A-dependent manner. TSH induced the PDK1-dependent phosphorylation of S6K1 but did not induce Akt/protein kinase B phosphorylation. The TSH-induced S6K1 phosphorylation was inhibited by a dominant negative p85alpha regulatory subunit or by the PI3K inhibitors wortmannin and LY294002. Rapamycin inhibited the phosphorylation of S6K1 in the cells treated with either TSH or 8-bromo-cAMP. The stimulating type TSHR antibodies from patients with Graves disease also induced S6K1 activation, whereas the blocking type TSHR antibodies from patients with primary myxedema inhibited TSH- but not the insulin-induced phosphorylation of S6K1. In addition, rapamycin treatment in vivo inhibited the TSH-stimulated thyroid follicle hyperplasia and follicle activity. These findings suggest an interaction between TSHR and PI3K, which is stimulated by TSH and cAMP and might involve the downstream S6K1 but not Akt/protein kinase B. This pathway may play a role in the TSH/stimulating type TSH receptor antibody-mediated thyrocyte proliferation in vitro and in the response to TSH in vivo.     

Steroidogenesis in cumulus cells of bovine cumulus-oocyte-complexes matured in vitro with BSA and different concentrations of steroids.

The present in vitro experiments were designed to evaluate the ability of bovine cumulus-oocyte-complexes (COCs) to produce steroids and also to evaluate the modulatory effects of added estradiol, progesterone and testosterone on the steroidogenic activity of COCs. Considerable estradiol accumulation was observed in the control maturation medium for in vitro maturation of bovine COCs during the 24h of maturation (P<0.05). When testosterone was added to the medium at various concentrations, a slight estradiol accumulation occurred, which, however, was lower (P<0.05) than that observed in the control medium. Slight estradiol accumulation was observed in maturation medium containing progesterone at concentrations of 2.5, 5.0 and 10.0 microg/ml, but these increases were less (P<0.05) than those observed in the control medium. However, in the presence of 1.0 microg/ml progesterone, estradiol accumulation was equal to that of the control medium (P>0.05). Progesterone accumulation (P<0.05) was observed in the control medium for in vitro maturation of bovine COCs. When estradiol was added to the maturation medium, progesterone accumulation was observed, but was significant (P<0.05) only when the medium was supplemented with the lesser concentrations of estradiol utilized in the experiment (1.0 microg/ml). The results demonstrated that (1) cumulus cells of bovine COCs are able to secrete estradiol and progesterone in culture systems for in vitro maturation, and this steroidogenesis is modulated by the steroids progesterone, testosterone and estradiol, and (2) the addition of estradiol to the in vitro maturation medium of bovine oocytes should be reviewed, since cumulus cells of COCs have been demonstrated to secrete estradiol in the maturation medium.     

Interaction of thyroid hormone and sex steroids at the rabbit reticulocyte membrane in vitro: control by 17 beta-estradiol and testosterone of thyroid hormone-responsive Ca2+-ATPase activity

Physiological concentrations (10(-10) M) of L-thyroxine and triiodo-L-thyronine were found in vitro to enhance Ca2+-ATPase activity in reticulocyte-enriched red cell membranes from female rabbits and to inhibit this enzyme in the male reticulocyte. Cross-incubation experiments with reticulocyte-enriched red cells and plasma from the opposite sex demonstrated that this sex-specific membrane response to thyroid hormone was transferable by plasma. Similar experiments with intact reticulocytes exposed to physiological concentrations (10(-11) M) of testosterone and 17 beta-estradiol indicated that the plasma factors were the sex steroids. That is, incubation in vitro with testosterone converted female-source reticulocytes to male-type responsiveness to thyroid hormone (inhibition of Ca2+-ATPase activity); incubation with estradiol converted male-source reticulocyte-enriched red cells to female-type responsiveness (stimulation by iodothyronines of membrane Ca2+-ATPase activity). Similar results were obtained when reticulocyte ghosts were incubated with testosterone and 17 beta-estradiol prior to determination of membrane enzyme activity. Etiocholanolone (5 beta-androstan-3 alpha-ol-17-one) and testosterone were equipotent, but 5 alpha-dihydrotestosterone had little activity in this system. Estrone and estradiol were equipotent, but estriol had no permissive effect on the stimulation by iodothyronine of reticulocyte membrane Ca2+-ATPase activity. Expression of thyroid hormone action in vitro on Ca2+-ATPase activity in the rabbit reticulocyte is determined at the membrane level by testosterone and estrogen. The structure-activity relationships of the sex steroids for this membrane action are different than those reported for nuclear actions of the steroids.     

Estradiol is responsible for reduced renal prostaglandin dehydrogenase activity in female rats

The contribution of sex steroids to sex-related differences in renal prostaglandin dehydrogenase activity and urinary prostaglandin excretion was examined in 7-8-week-old male and female rats subjected to sham-operation or gonadectomy at 3 weeks of age. Rats were injected subcutaneously twice over a 6-day interval with vehicle (peanut oil, 0.5 mg/kg) or with depot forms of testosterone (10 mg/kg), estradiol (0.1 mg/kg), progesterone (5 mg/kg), or with estradiol and progesterone combined (0.1 and 5 mg/kg). After the second injection, 24-h urine samples were collected for prostaglandin measurement by radioimmunoassay; the rats were killed, and renal and pulmonary prostaglandin dehydrogenase activities were determined by radiochemical assay. Renal prostaglandin dehydrogenase activity was 10-times higher in intact male rats than in intact females. Gonadectomy increased renal prostaglandin dehydrogenase activity 4-fold in females, but had no effect in males; estradiol, alone or combined with progesterone, markedly suppressed renal prostaglandin dehydrogenase activity in both sexes, while testosterone or progesterone alone had no effect. Pulmonary prostaglandin dehydrogenase did not differ between the sexes and was unaffected by gonadectomy or sex-steroid treatment. Intact female sham-operated rats excreted 70-100% more prostaglandin E2, prostaglandin F2 alpha, and 6-keto-prostaglandin F1 alpha in urine than did males; gonadectomy abolished the difference in urinary prostaglandin E2 excretion. Estradiol decreased urinary prostaglandin E2 in females but not in males; treatment with other sex steroids did not alter urinary prostaglandin excretion.     

Estrogen effects on thyroid iodide uptake and thyroperoxidase activity in normal and ovariectomized rats

Sex steroids interfere with the pituitary-thyroid axis function, although the reports have been controversial and no conclusive data is available. Some previous reports indicate that estradiol might also regulate thyroid function through a direct action on the thyrocytes. In this report, we examined the effects of low and high doses of estradiol administered to control and ovariectomized adult female rats and to pre-pubertal females. We demonstrate that estradiol administration to both intact adult and pre-pubertal females causes a significant increase in the relative thyroid weight. Serum T3 is significantly decreased in ovariectomized rats, and is normalized by estrogen replacement. Neither doses of estrogen produced a significant change in serum TSH and total T4 in ovariectomized, adult intact and pre-pubertal rats. The highest, supraphysiological, estradiol dose produced a significant increase in thyroid iodide uptake in ovariectomized and in pre-pubertal rats, but not in control adult females. Thyroperoxidase activity was significantly higher in intact adult rats treated with both estradiol doses and in ovariectomized rats treated with the highest estradiol dose. Since serum TSH levels were not significantly changed, we suggest a direct action of estradiol on the thyroid gland, which depends on the age and on the previous gonad status of the animal.     

Adaptive reactions of the endocrine system of children living under conditions of contrasting photoperiods

Age-related changes in the levels of pituitary, thyroid, and sex hormones and their dependence on the day length were studied in 242 healthy 10-to 16-year-old boys born and permanently living in the southern part of Arkhangel’sk oblast. It was found that the serum levels of thyroid hormones, gonadotropins, and sex steroids underwent circannual changes. The minimum concentrations of thyroxine, testosterone, and estradiol and the maximum concentrations of triiodothyronine, lutcinizing and follicle-stimulating hormones, and progesterone were associated with the shortest days. In contrast, the longest days were associated with the maximum concentrations of thyroxine, testosterone, and estradiol and the minimum concentrations of triiodothyronine, luteinizing and follicle-stimulating hormones, and progesterone. A factor dependence of the thyroxine, triiodothyronine, testosterone, estradiol, and prolactin concentrations on the day length was found in the group examined. This dependence was more pronounced during the second half of adolescence.     

The effect of rapid and depot testosterone and estradiol on spatial performance in water maze

Men and women differ in some cognitive functions including spatial abilities. These differences seem to be affected by sex steroids, but the results are controversial. The aim of this work is to describe the effects of rapid or depot testosterone and estradiol on spatial memory in rats. Thirty-two adult male Wistar rats were divided into 6 groups. Five groups were gonadectomized, and one group was left as control. Castrated groups received sterile oil, testosterone isobutyras, testosterone propionate, estradiol dipropionate or estradiol benzoate. We evaluated spatial performance (escape latency, overall improvement, and time in the quadrant after platform removal) of the rats in a spatial water maze. Animals receiving exogenous sex steroids showed higher plasma concentrations of the particular hormones. Experimental groups improved during the acquisition spatial trials in the water maze. No significant differences between the groups during probe trial were found. In overall improvement, the testosterone depot and estradiol depot groups showed less improvement in comparison to the control groups (P<0.05). No differences in respect to administered hormones were found in corresponding receptor gene expression in hippocampus. In conclusion, exogenous testosterone affects spatial memory of adult castrated males.     

In vitro maturation, fertilization and early cleavage rates of bovine fetal oocytes

The in vitro developmental competence of oocytes harvested from 3 to 6 mm follicles from ovaries of 7.5 months to term fetuses and adult cows was compared. Cumulus oocyte complexes (COCs) were washed and placed in 200 microl droplets of maturation medium 199, supplemented with 10 microg/ml FSH, 10 microg/ml LH, 1.5 microg/ml estradiol, 75 microg/ml streptomycin, 100 IU/ml penicillin, 10 mM Hepes, and 10% fetal bovine serum (FBS) under oil and incubated for 24 h at 39 degrees C and 5% CO2. Matured oocytes were exposed to frozen-thawed TALP swim-up, heparin-capacitated sperm (20 h, 39 degrees C, 5% CO2). Presumptive zygotes were cultured in medium 199 containing 8 mg/ml BSA-V, 100 IU/ml penicillin G, 75 microg/ml streptomycin, and 10 mM Hepes (48 h, 39 degrees C, 5% CO2). Oocytes/embryos were fixed, stained with DAPI, and evaluated under fluorescent microscopy to assess maturation, fertilization, and subsequent embryonic development. There was a difference (P<0.05) between fetal and adult cow oocytes for in vitro maturation (IVM; 80.1% versus 92.0%), fertilization (69.3% versus 79.9%), and cleavage rates (36.7% versus 49.9%), respectively. Poor IVM, fertilization and embryonic development of fetal oocytes may be due to a higher incidence of blockage at germinal vesicle (GV) and metaphase-I (M-I) stage after IVM (12.0% versus 2.3% for fetal versus adult oocytes, respectively, P<0.05). Although the IVF results with fetal oocytes are poorer than with adult cow oocytes, they were still high enough to be considered for use in research and when death of the dam and/or fetus is pre-mature or sudden.     

Thyroid stimulating effect of exogenous vasopressin and oxytocin in hypophysectomized rats during immobilization stress

Male Wistar rats were hypophysectomized 1 week before restraint stress. The hypophysectomy caused a decrease of blood vasopressin (30%, P less than 0.05) and a diminution of the thyroid activity (the thyrocyte height lowered to 43%, P less than 0.01). The TSH concentration was about normal and remained constant during the experiment. After 20 min of the restraint stress, the vasopressin concentration reached 178% (P less than 0.01), but the thyroid did not response in rats with the intact hypophysis. In the hypophysectomized rats, the restraint stress caused neither essential changes of the blood vasopressin nor the thyroid function as compared with the hypophysectomized control. An injection of vasopressin (5.0 ng/100 g) or oxytocin (15.0 ng/100 g) resulted in a slight activation of the thyroid in the hypophysectomized rats but significantly stimulated in when combined with the restraint stress; vasopressin injection led to an increase of the thyrocyte height to 152% (P less than 0.01), oxytocin--to 126% (P less than 0.05). Thus, in hypophysectomized rats, vasopressin and oxytocin can influence the thyroid directly. Stressful conditions facilitate the thyroid stimulating effect of these nonapeptide neurohormones.     

Effect of non aromatizable androgens on LHRH and TRH responses in primary testicular failure

We have assessed the gonadotropin, TSH and PRL responses to the non aromatizable androgens, mesterolone and fluoxymestrone, in 27 patients with primary testicular failure. All patients were given a bolus of LHRH (100 micrograms) and TRH (200 micrograms) at zero time. Nine subjects received a further bolus of TRH at 30 mins. The latter were then given mesterolone 150 mg daily for 6 weeks. The remaining subjects received fluoxymesterone 5 mg daily for 4 weeks and 10 mg daily for 2 weeks. On the last day of the androgen administration, the subjects were re-challenged with LHRH and TRH according to the identical protocol. When compared to controls, the patients had normal circulating levels of testosterone, estradiol, PRL and thyroid hormones. However, basal LH, FSH and TSH levels, as well as gonadotropin responses to LHRH and TSH and PRL responses to TRH, were increased. Mesterolone administration produced no changes in steroids, thyroid hormones, gonadotropins nor PRL. There was, however, a reduction in the integrated and incremental TSH secretion after TRH. Fluoxymesterone administration was accompanied by a reduction in thyroid binding globulin (with associated decreases in T3 and increases in T3 resin uptake). The free T4 index was unaltered, which implies that thyroid function was unchanged. In addition, during fluoxymesterone administration, there was a reduction in testosterone, gonadotropins and LH response to LHRH. Basal TSH did not vary, but there was a reduction in the peak and integrated TSH response to TRH. PRL levels were unaltered during fluoxymesterone treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct influence of melatonin on the thyroid and comparison with prolactin

Melatonin administered in vivo had previously been shown to inhibit thyroid cell proliferation and subsequent in vitro thyroxine (T(4)) secretion in anuran tadpoles. Melatonin in vitro also directly reduced the sensitivity of the thyroid to thyrotropin (TSH). The present work sought to determine whether melatonin directly affected baseline, unstimulated T(4) secretion, and to compare its effect with that of prolactin (PRL). Thyroids from larval Rana catesbeiana or adult Rana pipiens were incubated in control or melatonin (0.01 to 100 microg/ml) media. Melatonin directly inhibited T(4) secretion by thyroids from both tadpoles and frogs at all concentrations of melatonin used and at both prometamorphic and climax tadpole stages. PRL, used in vitro at 10 microg/ml, did not influence the response of the thyroid to TSH (0.2 microg/ml) in young tadpoles, or the baseline secretion of T(4) by thyroids at any stage of larval life except climax, when T(4) secretion was significantly decreased by the third day of culture. Thus although both melatonin and PRL have been shown to antagonize the action of T(4) in vitro, and to decrease metamorphic rate, melatonin is a much more effective thyroid gland inhibitor than PRL.     

Optimization of culture conditions for determining hepatobiliary disposition of taurocholate in sandwich-cultured rat hepatocytes

Summary This study was undertaken to examine the influence of time and volume of collagen overlay, type of media, and media additives on taurocholate (TC) accumulation and biliary excretion in hepatocytes cultured in a collagen-sandwich configuration. Hepatocytes were isolated from male Wistar rats by in situ perfusion with collagenase, seeded onto collagencoated 60-mm dishes, overlaid with gelled collagen, and cultured for 4 d. Experiments to examine the influence of time and volume of collagen overlay were conducted in Dulbecco's modified Eagle's medium (DMEM)+1.0μM dexamethasone (DEX)+5% fetal bovine serum (FBS). Hepatocytes were overlaid at 0 h with 0.1 or 0.2 ml collagen, or at 24 h with 0.1 or 0.2 ml collagen. The influence of media type and additives was examined in hepatocytes overlaid at 0 h with 0.2 ml collagen and incubated in DMEM+0.1μM DEX, DMEM+0.1μM DEX+5% FBS, Williams' medium E+0.1μM DEX+1% ITS^Θ+, DMEM +1.0μM DEX, DMEM+1.0 μM DEX+5% FBS, or modified Chee's medium (MCM)+0.1 μM DEX+1% ITS^Г+. [³H] TC accumulation by hepatocytes in Hank's balanced salt solution (HBSS) and Ca²⁺-free HBSS was measured, and the biliary-excretion index (BEI: percentage of accumulated TC localized in the canalicular compartment) was calculated. Light microscopy and carboxydichlorofluorescein fluorescence were employed to examine the cellular and canalicular morphologies. The volume of collagen used for both the substratum and the overlay did not affect TC accumulation or biliary excretion. The BEI tended to be higher in cells overlaid at 24 h (BEI=0.649 [0.1 ml collagen]; BEI=0.659 [0.2 ml collagen]) compared with those overlaid at 0 h after seeding (BEI=0.538 [0.1 ml collagen]; BEI=0.517 [0.2 ml collagen]), although the differences were not statistically significant. Hepatocytes cultured in MCM produced consistently the lowest BEI of TC (BEI=0.396). Differing DEX concentration (0.1 μM versus 1.0 μM) with or without 5% FBS did not appear to have a significant effect on the BEI of TC.     

Testosterone modulates cardiomyocyte Ca(2+) handling and contractile function

The extent to which sex differences in cardiac function may be attributed to the direct myocardial influence of testosterone is unclear. In this study the effects of gonadal testosterone withdrawal (GDX) and replacement (GDX+T) in rats, on cardiomyocyte shortening and intracellular Ca(2+) handling was investigated (0.5 Hz, 25 oC). At all extracellular [Ca(2+)] tested (0.5-2.0 mM), the Ca(2+) transient amplitude was significantly reduced (by approximately 50 %) in myocytes of GDX rats two weeks post-gonadectomy. The time course of Ca(2+) transient decay was significantly prolonged in GDX myocytes (tau, 455+/-80 ms) compared with intact (279+/-23 ms) and GDX+T (277+/-19 ms). Maximum shortening of GDX myocytes was markedly reduced (by more than 60 %) and relaxation significantly delayed (by more than 35 %) compared with intact and GDX+T groups. Thus testosterone replacement completely reversed the cardiomyocyte hypocontractility induced by gonadectomy. These results provide direct evidence for a role of testosterone in regulating functional Ca(2+) handling and contractility in the heart.     

Effect of sex and prior exposure to a cafeteria diet on the distribution of sex hormones between plasma and blood cells

It is generally assumed that steroid hormones are carried in the blood free and/or bound to plasma proteins. We investigated whether blood cells were also able to bind/carry sex-related hormones: estrone, estradiol, DHEA and testosterone. Wistar male and female rats were fed a cafeteria diet for 30 days, which induced overweight. The rats were fed the standard rat diet for 15 additional days to minimize the immediate effects of excess ingested energy. Controls were always kept on standard diet. After the rats were killed, their blood was used for 1) measuring plasma hormone levels, 2) determining the binding of labeled hormones to washed red blood cells (RBC), 3) incubating whole blood with labeled hormones and determining the distribution of label between plasma and packed cells, discounting the trapped plasma volume, 4) determining free plasma hormone using labeled hormones, both through membrane ultrafiltration and dextran-charcoal removal. The results were computed individually for each rat. Cells retained up to 32% estrone, and down to 10% of testosterone, with marked differences due to sex and diet (the latter only for estrogens, not for DHEA and testosterone). Sex and diet also affected the concentrations of all hormones, with no significant diet effects for estradiol and DHEA, but with considerable interaction between both factors. Binding to RBC was non-specific for all hormones. Estrogen distribution in plasma compartments was affected by sex and diet. In conclusion: a) there is a large non-specific RBC-carried compartment for estrone, estradiol, DHEA and testosterone deeply affected by sex; b) Prior exposure to a cafeteria (hyperlipidic) diet induced hormone distribution changes, affected by sex, which hint at sex-related structural differences in RBC membranes; c) We postulate that the RBC compartment may contribute to maintain free (i.e., fully active) sex hormone levels in a way similar to plasma proteins non-specific binding.