The changes in adenohypophysial and hypothalamic content and in hypothalamic release of dopamine and thyrotrophin-releasing hormone (TRH) into the hypophysial portal system during the suckling-induced release of prolactin were investigated. An increase in peripheral plasma levels of prolactin was induced by mammary nerve stimulation in urethane-anaesthetized and by suckling in unanaesthetized lactating rats. In the unanaesthetized rat, suckling caused a decrease of dopamine levels in hypothalamus and adenohypophysis and a short-lasting small increase in hypothalamic TRH. Mammary nerve stimulation induced a transient decrease in dopamine levels and an increase in TRH levels in hypophysial stalk blood. To assess the significance of the observed changes in dopamine and TRH levels for prolactin release, these changes in dopamine and TRH were mimicked in lactating rats anaesthetized with urethane and pretreated with α-methyl-p-tyrosine (AMpT, a competitive inhibitor of catecholamine synthesis). Reducing hypothalamic dopamine secretion by treatment with AMpT increased peripheral plasma levels of prolactin from 15 to 477 ng/ml; an infusion with dopamine, resulting in plasma levels similar to those measured in hypophysial stalk plasma, reduced plasma levels of prolactin to 127 ng/ml. Neither a 50% reduction in dopamine infusion rate for 15 min nor administration of 100 ng TRH caused an appreciable change in plasma prolactin levels. However, when dopamine infusion was reduced by 50% for 15 min just before TRH was injected, then an increase in plasma levels of prolactin from 172 to 492 ng/ml was observed. Thus, the effectiveness of TRH in releasing prolactin in the lactating rat was enhanced when a transient decrease of dopamine levels occurred before treatment with TRH. It is concluded that the changes observed in dopamine and TRH levels in hypophysial stalk blood are involved in the suckling-induced prolactin release in an important manner.

Department of Internal Medicine III and Clinical Endocrinology, Medical Faculty, Erasmus University, Rotterdam, The Netherlands

(Received 19 November 1976)

Recently data have suggested that in man the contribution to the overall turnover of thyroxine (T₄) of pathways along which this prohormone is converted either into metabolically active 3,3′,5-tri-iodothyronine (T₃) or into inactive 3,3′,5′-tri-iodothyronine (reverse T₃, rT₃) is under physiological control. We describe here the synthesis of [¹²⁵I]rT₃ and the production and characterization of antisera raised against an rT ₃-bovine serum albumin (rT₃-BSA) conjugate.

3,3′,5′-Tri-iodo-l-thyronine-BSA, prepared essentially according to the method of Olivier, Parker, Brasfield & Parker (1968), l-rT₃ and 3,3′-di-iodo-l-thyronine (3,3′-T₂) were obtained by courtesy of Dr E. Scheiffele (Henning GmbH., Berlin); l-T₄ and l-T₃ were purchased from Sigma Chemical Co. (St Louis, Missouri, U.S.A.); 3-iodo-l-tyrosine (MIT) and 3,5-di-iodo-l-tyrosine (DIT) were obtained from Calbiochem AG (Lucerne, Switzerland); Na¹²⁵I (sp.act. approximately 14 mCi/μg) from The Radiochemical Centre (Amersham) and goat anti-rabbit

SUMMARY

Radioimmunoassays for the measurement of the 1–34 human parathyroid hormone fragment (1–34 hPTH) were developed using antisera raised in rabbits against synthetic 1–34 hPTH-N (amino acid sequence proposed by Niall). Binding of ¹²⁵I-labelled 1–34 hPTH-N to these antisera was optimal at pH 5·5. Limits of detection varied between 25 and 200 pg/ml. Cross-reactivity of 1–34 bovine PTH was substantial in all assays; 1–34 hPTH-B (structure proposed by Brewer), 1–84 hPTH and 1–29 hPTH cross-reacted only with antisera from one animal. 1–29 Human PTH was obtained from partial hydrolysis of both 1–84 hPTH and 1–34 hPTH-N. Production of 1–29 hPTH from 1–84 hPTH was demonstrated by comparison of the elution profiles of the reaction product and 1–29 bovine PTH on Sephadex G-50. Thus, evidence was obtained that position 30 in native hPTH is occupied by an aspartic acid residue.

ABSTRACT

The changes in hypothalamic release of dopamine and thyrotrophin-releasing hormone (TRH) into the hypophysial portal vascular system during an oestrogen-stimulated surge of prolactin in ovariectomized rats were investigated.

A single injection of 5 μg oestradiol benzoate resulted in a reliable increase in the plasma levels of prolactin during the afternoon 3 days later. Anaesthesia did not block this afternoon surge of prolactin, although its magnitude was only half of that of unanaesthetized rats. Before and during this surge, hypophysial stalk blood was collected into methanol to analyse the hypothalamic release of dopamine and TRH. Immunoreactive TRH in these methanolic extracts eluted as a single peak with the same retention time as authentic TRH on reverse-phase high performance liquid chromatography. In comparison to the morning values, levels of dopamine decreased and those of TRH increased in hypophysial stalk blood by 50 and 240% respectively. These data indicate that hypothalamic dopamine and TRH may be involved in the afternoon surge of prolactin.

Daily treatment with parachlorophenylalanine, an inhibitor of serotonin synthesis, reduced the hypothalamic release of TRH by 50%, but did not prevent the afternoon surge of prolactin and TRH induced by oestradiol benzoate.

J. Endocr. (1985) 105, 107–112

The development of a highly sensitive and specific radioimmunoassay for 3,3′-di-iodothyronine (3,3′-T₂) is described. The assay was applied to the measurement of 3,3′-T₂ in unextracted human serum and used 8-anilino-l-naphthalene-sulphonic acid to inhibit the binding of 3,3′-T₂ to serum transport proteins. The lower limit of detection of the assay was 2 fmol 3,3′-T₂ per tube, which corresponded to 10 pmol 3,3′-T₂/l serum. The mean concentration of 3,3′-T₂ in normal serum was found to be 23 pmol/l, which is considerably lower than most values reported previously. Evidence is presented which suggests that the cross-reactivity of tri-iodothyronine with the antiserum to 3,3′-T₂ is an important factor in the measurement of serum concentrations of 3,3′-T₂ by radioimmunoassay.

Department of Internal Medicine III and Clinical Endocrinology, Medical Faculty, Erasmus University, Rotterdam, and * Laboratory ofEndocrinological Chemistry, Municipal Hospital 'Bergweg', Rotterdam, The Netherlands

(Received 2 June 1975)

The availability of synthetic human parathyroid hormone (hPTH) fragments (Andreatta, Hartmann, Jöhl, Kamber, Maier, Riniker, Rittel & Sieber, 1973; Tregear, Van Rietschoten, Greene, Niall, Keutmann, Parsons, O'Riordan & Potts, 1974) has enabled the production of region specific antibodies and thus the development of region specific radioimmunoassays (RIA). This is particularly meaningful with respect to the heterogeneity of immunoreactive forms of PTH in the circulation (Arnaud, Goldsmith, Bordier, Sizemore, Larsen & Gilkinson, 1974). The peptides were synthesized according to the sequences proposed for the first 34 amino acids by Brewer, Fairwell, Ronan, Sizemore & Arnaud (1972) (referred to as 1–34 hPTH-B) and Niall, Sauer, Jacobs, Keutmann, Segre, O'Riordan, Aurbach & Potts (1974) (1–34 hPTH-N), which differ in positions 22, 28 and 30.

Two

Thyroid hormones (THs) are essential for normal fetal development, with even mild perturbation in maternal thyroid status in early pregnancy being associated with neurodevelopmental delay in children. Transplacental transfer of maternal THs is critical, with increasing evidence suggesting a role for 3,3′,5-tri-iodothyronine (T3) in development and function of the placenta itself, as well as in development of the central nervous and other organ systems. Intrauterine growth restriction (IUGR) is associated with fetal hypothyroxinaemia, a factor that may contribute to neurodevelopmental delay. The recent description of monocarboxylate transporter 8 (MCT8) as a powerful and specific TH membrane transporter, and the association of MCT8 mutations with profound neurodevelopmental delay, led us to explore MCT8 expression in placenta. We describe the expression of MCT8 in normal human placenta throughout gestation, and in normal third-trimester placenta compared with that associated with IUGR using quantitative reverse transcriptase PCR. MCT8 mRNA was detected in placenta from early first trimester, with a significant increase with advancing gestation (P=0.007). In the early third trimester, MCT8 mRNA was increased in IUGR placenta compared with normal samples matched for gestational age (P<0.05), but there was no difference between IUGR and normal placenta in the late third trimester. Western immunoblotting findings in IUGR and normal placentae were in accord with mRNA data. MCT8 immunostaining was demonstrated in villous cytotrophoblast and syncytiotrophoblast as well as extravillous trophoblast cells from the first trimester onwards with increasingly widespread immunoreactivity seen with advancing gestation. In conclusion, expression of MCT8 in placenta from early gestation is compatible with an important role in TH transport during fetal development and a specific role in placental development. Altered expression in placenta associated with IUGR may reflect a compensatory mechanism attempting to increase T3 uptake by trophoblast cells.

Abstract

TRH-like peptides share the N- and C-terminal amino acids with TRH (pGlu-His-Pro-NH₂) but differ in the middle amino acid residue. One of them, pGlu-Glu-Pro-NH₂ (<EEP-NH₂; EEP) is present in the rat pituitary gland, but its biological significance is unknown. We investigated the localization and regulation of this tripeptide in the rat pituitary gland. To distinguish between TRH and EEP two antisera were used for RIA: specificity of antiserum 4319 for the TRH-like peptides pGlu-Phe-Pro-NH₂ and EEP was equal to or greater than that for TRH, whereas antiserum 8880 is TRH-specific. Our RIA data showed the presence of a TRH-like peptide in the anterior pituitary gland (AP) and of TRH in the posterior pituitary gland (PP). The TRH-like peptide in the AP was identified on anion-exchange chromatography and subsequent HPLC as EEP. Pathophysiological conditions such as altered thyroid and adrenal status and suckling did not affect pituitary gland levels of EEP. In general, however, there is a clear sex difference: levels of EEP are higher in male than in female rats. In both sexes gonadectomy leads to a substantial two- to threefold rise in EEP levels, abolishing the sex difference. Testosterone administration to gonadectomized male rats normalizes levels of EEP again. Disulfiram, an inhibitor of the enzyme peptidylglycine α-amidating monooxygenase, reduced levels of EEP in the AP by approximately 50%. In conclusion: 1) the TRH-like peptide EEP is present in the AP, whereas TRH is confined to the PP, 2) levels of EEP in the AP are regulated by sex steroids, 3) EEP is actively amidated in the AP and thus seems to be produced from a glycine-extended progenitor sequence.

Journal of Endocrinology (1995) 145, 43–49

Abstract

We investigated the effects of diabetes mellitus on the hypothalamo-hypophysial-thyroid axis in male (R×U) F₁ and R-Amsterdam rats, which were found to respond to streptozotocin (STZ)-induced diabetes mellitus with no or marked increases, respectively, in plasma corticosterone. Males received STZ (65 mg/kg i.v.) or vehicle, and were killed 1, 2 or 3 weeks later. At all times studied, STZ-induced diabetes mellitus resulted in reduced plasma TSH, thyroxine (T₄) and 3,5,3′-tri-iodothyronine (T₃). Since the dialyzable T₄ fraction increased after STZ, probably as a result of decreased T₄-binding prealbumin, plasma free T₄ was not altered during diabetes. In contrast, both free T₃ and its dialyzable fraction decreased during diabetes, which was associated with an increase in T₄-binding globulin. Hepatic activity of type I deiodinase decreased and T₄ UDP-glucuronyltransferase increased after STZ treatment. Thus, the lowered plasma T₃ during diabetes may be due to decreased hepatic T₄ to T₃ conversion.

Median eminence content of TRH increased after STZ, suggesting that hypothalamic TRH release is reduced during diabetes and that this is not caused by impaired synthesis or axonal transport of TRH to the median eminence. Hypothalamic proTRH mRNA did not change in diabetic (R×U) F₁ rats during the period of observation, but was lower in R-Amsterdam rats 3 weeks after STZ. Similarly, pituitary TSH and TSHβ mRNA had decreased in R-Amsterdam rats by 1 week after STZ treatment, but did not change in (R×U) F₁ rats. The difference between the responses in diabetic R-Amsterdam and (R×U) F₁ rats may be explained on the basis of plasma corticosterone levels which increased in R-Amsterdam rats only. Hypothalamic TRH content was not affected by diabetes mellitus, but the hypothalami of diabetic rats released less TRH in vitro than those of control rats. Moreover, insulin had a positive effect on TRH release in vitro.

In conclusion, the reduced hypothalamic TRH release during diabetes is probably not caused by decreases in TRH synthesis or transport to the median eminence, but seems to be due to impaired TRH release from the median eminence which may be related to the lack of insulin. Inhibition of proTRH and TSHβ gene expression in diabetic R-Amsterdam rats is not a primary event but appears to be secondary to enhanced adrenal activity in these animals during diabetes.

Journal of Endocrinology (1997) 153, 259–267

Abstract

This study describes the effects of litter size and acute suckling on the synthesis and release of hypothalamic TRH, as indirectly estimated by determination of hypothalamic prothyrotrophin-releasing hormone (proTRH) mRNA and median eminence TRH content. The effects of litter size (five or ten pups) were studied throughout lactation, while suckling-induced acute changes were analyzed on day 13 of lactation in dams with ten pups. In view of the enhanced adrenal activity during lactation and recent evidence that corticosteroids have negative effects on hypothalamic TRH, we also studied adrenalectomized (ADX) dams treated with corticosterone to maintain basal plasma corticosterone levels.

In addition to an increased plasma level of prolactin (PRL), adrenal weight and plasma corticosterone increased, while plasma TSH, tri-iodothyronine (T₃), thyroxine (T₄) and free T₄ (FT₄) levels decreased during lactation. Litter size correlated positively with plasma PRL, adrenal weight and plasma corticosterone. No effect of litter size was observed on plasma T₃, but rats with ten pups had lower plasma TSH, T₄ and FT₄ than rats with a five-pup litter. Compared with dioestrous rats, lactating rats showed an increased hypothalamic proTRH mRNA content on day 2, but not on days 8 and 15 of lactation. Median eminence TRH in lactating rats gradually increased until day 15 and decreased thereafter. Acute suckling, after a 6-h separation of mother and pups, rapidly increased plasma PRL and corticosterone in the mothers, but had no effects on plasma TSH and thyroid hormone levels. Hypothalamic proTRH mRNA increased twofold after 0·5 h of suckling, and then gradually returned to presuckling values after 6 h. Compared with sham-operated rats, corticosterone-substituted ADX rats with ten pups had increased plasma PRL and TSH, hypothalamic proTRH mRNA and pituitary TSH β mRNA on day 15 of lactation. Moreover, while acute suckling did not enhance TSH release in sham-operated rats, it provoked not only PRL but also TSH release in corticosterone-substituted ADX dams.

It is concluded that suckling exerts a rapid, positive effect on hypothalamic proTRH mRNA content. However, the concurrent enhanced adrenal activity has negative effects on hypothalamic proTRH gene expression resulting in a suppressed hypophysial-thyroid axis during lactation. While TRH appears to play a role in PRL release during the first days of lactation and during acute suckling, TRH seems not important in maintaining PRL secretion during continued suckling.

Journal of Endocrinology (1996) 148, 325–336