ABSTRACT

White Leghorn male chicks of 40 days of age were fasted for 5 days and then refed. Blood samples were collected from these chicks before, during and after fasting and serum levels of GH and insulin-like growth factor-I (IGF-I) and serum IGF-I-binding activity were determined. The fasting-induced reduction in body weight was accompanied by a significant rise in circulating GH and fall in IGF-I, coupled with increased serum IGF-I-binding activity. When pooled serum was chromatographed under neutral conditions, IGF-I binding activity and IGF-I immunoreactivity were mainly associated with a large (M _r= 150 000) and a small protein (M _r=30 000). Fasting induced a marked increase in the IGF-I-binding activity of the 30 kDa IGF-I-binding protein (IGFBP) and refeeding restored activity to the normal levels seen before fasting. Ligand blotting of serumbinding proteins with ¹²⁵I-labelled IGF-I, after first subjecting the samples to polyacrylamide gel electrophoresis and transfer to nitrocellulose, revealed that four IGFBPs (M _r=20 000, 30 000, 35 900 and 41 000) were present in chicken serum, and that the ¹²⁵I-labelled IGF-I binding of the 30 kDa monomer was increased by fasting and restored to normal by refeeding in agreement with gel filtration profiles of IGF-Ibinding activity. Western blot analysis suggested that the 30 kDa IGFBP is homologous to IGFBP-2 found in mammalian blood plasma. The results show that IGFBPs in chicken serum and their responses to fasting are similar to those in mammals.

Journal of Endocrinology (1993) 139, 363–370

Hormonal responsiveness in peripheral tissues is variable in patients with resistance to thyroid hormone (RTH). One cause of this may be differential interaction of RTH mutants of thyroid hormone receptor beta (TR beta) with TR auxiliary proteins (TRAPs). We used gel shift mobility assays to examine the interaction of wild-type and mutant TR beta s with retinoid X receptors (RXRs) and endogenous TRAPs. Some mutants showed reduced homodimerization but retained heterodimerization with recombinant RXRs. Wild-type TR beta formed heterodimeric complexes with multiple TRAPs in nuclear extracts of rat tissues, but RTH mutants showed variably altered heterodimerization with each TRAP. With liver nuclear extract, all mutants with impaired homodimerization also showed impaired TR beta-TRAP heterodimerization. Thus heterodimerizations with RXRs and TRAPs are differently affected by RTH mutations. Our results suggest that multiple TRAPs are expressed in tissue-specific patterns. The variability of TR beta heterodimerization with TRAPs may account, in part, for the variable tissue responsiveness in RTH.

ABSTRACT

Using a polyclonal antibody against a synthetic fusion protein corresponding to 167 amino acids of the N-terminal region of the human renal mineralocorticoid receptor (MR), an immunohistochemical study was performed to investigate the intraglandular and intracellular localization of the receptor in human kidney, salivary gland, pancreas, mammary gland and sweat gland both at the light and electron microscopic levels. In the kidney, immunoreactivity was observed in distal convoluted tubules, branches of Henle's loop, and collecting tubules in the renal cortex, and papillary and Henle's loops' ducts in the renal medulla. No significant differences in the distribution of immunoreactivity were observed using different fixatives (10% neutral formalin, 100% methanol, 4% paraformaldehyde, PLP (periodate–lysine–2% paraformaldehyde) solution and Zamboni solution) and processing methods (paraffin embedding and frozen sectioning). Immunoreactivity in the kidney was observed in both the cytoplasm and nucleus, with cytoplasmic staining predominant, regardless of the methods of tissue preparation. Immunoelectron microscopy, employing a pre-embedding method, demonstrated the presence of immunoreaction precipitates in nuclei, endoplasmic reticulum, perinuclear cisternae, free cytoplasm and cell membranes. In nuclei, immunoreactivity was observed in euchromatin but not in heterochromatin, which is consistent with an association of MR with specific DNA regulatory elements located in transcriptionally active euchromatin. In other organs, MR was expressed in cells of the excretory ductal system where mineralocorticoids are known to play a role in electrolyte homeostasis.

Journal of Endocrinology (1992) 132, 305–310

The syndrome of resistance to thyroid hormone (RTH) is an inherited disorder involving a mutation of the thyroid hormone receptor (TR) gene. Mutant (m) TR inhibits wild-type (wt) TR functions in a dominant negative manner, and this dominant negative effect (DNE) is a crucial factor in RTH pathogenesis. The molecular mechanism of the DNE is still unclear, although several possibilities (including competition between wt- and mTRs at the T(3) response element (TRE), sequestration of TR-associated protein(s) and titration out of functional TR) have been considered. Here we report that the DNE of mTRs is strongly correlated with their binding avidity for the retinoid X receptor (RXR), and especially for corepressor SMRT (silencing mediator for retinoid and thyroid hormone receptor), but not for the nuclear receptor corepressor, NCoR. The DNE of six natural TRs and four artificially constructed mTRs was assayed using a TR reporter gene containing TRE-DR4 (DR=direct repeat), TRE-pal (pal=palindrome) or TRE-lap (lap=inverted palindrome) in CV1 cells treated with 10 nM T(3). Of the mTRs examined, F451X (with a carboxy-terminal 11-amino-acid truncation) identified in a patient with RTH exhibited the strongest DNE on all TREs. The binding affinities between mTRs and corepressors SMRT or NCoR were quantified using a two-hybrid interference assay system consisting of VP16-TR(LBD) (LBD=ligand binding domain) and Gal4(DBD)-SMRT (DBD=DNA binding domain), or Gal4(DBD)-NCoR respectively, together with the Gal4 reporter gene. In this assay, VP16-TR(LBD) and Gal4(DBD)-SMRT (or Gal4 (DBD)-NCoR) interact with each other and trans-activate the Gal4 reporter gene. When an equal amount of mTR is coexpressed, it reduces the transcriptional activity of the reporter gene, depending on its binding avidity for a corepressor. A very strong correlation was observed between the SMRT-binding activity and the potency of the DNE among six natural mTRs and also among all mTRs, including four artificially constructed ones. The relationship between NCoR and DNE, however, was not significant. When we assayed the binding avidity of mTRs for RXR by using a two-hybrid assay system consisting of Gal4(DBD)-RXR(LBD) and VP16-TR(LBD), a significant correlation between DNE and binding avidity for the RXR was also observed. These results suggest that a corepressor plays an important role in DNE pathogenesis.

Human thyroid hormone receptor (TR) is encoded by two distinct genes, TR alpha and TR beta. TR heterodimerizes with retinoid X receptor (RXR) and binds efficiently to the thyroid hormone (T(3)) response element (TRE) of target genes. In the absence of T(3), unliganded TR suppresses the basal promoter activity of positively regulated genes (silencing). Silencing mediator for retinoid and thyroid hormone receptors (SMRT) and nuclear receptor co-repressor (N-CoR) interact with unliganded TR and function as corepressor proteins. Previously, we found beta F451X with carboxyl (C)-terminal 11-amino acid deletion had stronger silencing potency than wild-type TR beta 1 and beta E449X with C-terminal 13-amino acid deletion on a subset of TREs. In the present study, to assess the isoform-specific effects of the C-terminal truncations on TR silencing, we constructed two mutant TR alpha 1s (alpha F397X and alpha E395X) with the same respective C-terminal truncations as beta F451X and beta E449X and analysed their silencing activities. Unlike beta F451X and beta E449X, alpha F397X and alpha E395X showed similarly stronger silencing potency than wild-type TR alpha 1. We further studied the abilities of wild-type and the mutant TR beta 1s and alpha 1s on RXR and co-repressor binding by a two-hybrid interference assay. beta F451X had significantly stronger abilities to bind to RXR and SMRT than did wild-type TR beta 1 and beta E449X. In contrast, wild-type TR alpha 1, alpha F397X and alpha E395X showed similar abilities to bind to RXR and SMRT. beta E449X and alpha E395X, which have identical C-terminal truncation, showed less ability to bind to N-CoR than did wild-type TR beta 1 and beta F451X and wild-type TR alpha 1 and alpha F397X respectively. These results indicate that an identical C-terminal truncation gives rise to different effects on TR beta 1 and alpha1 with respect to silencing potency, RXR binding and SMRT binding. The difference in the silencing potency among wild-type TR beta 1, beta F451X and beta E449X correlated well with the difference in the ability to bind co-repressor SMRT.

This study was conducted to evaluate the effect of active immunization against inhibin on hormonal levels and the ovulation rate in goats. Ten adult Shiba goats (Capra hircus) in two groups were used in this study. The first group was injected with inhibin vaccine (immunized, n=5) and the second group was injected with Freund's adjuvant (control, n=5) followed by three booster injections at 4-week intervals. After the third booster injection, three consecutive periods of oestrus were induced using prostaglandin F(2alpha) at intervals of 11 days. Blood samples were collected at 2-6 h intervals and the ovaries were monitored using B-mode ultrasonography. All inhibin-immunized goats generated antibodies that bound (125)I-labelled bovine inhibin and their FSH concentrations were significantly higher than corresponding values in the control group. Also, inhibin-immunized goats had significantly higher preovulatory oestradiol-17beta (P<0.01) and higher concentrations of progesterone in the luteal phase (P<0.05). Immunization of goats against inhibin resulted in a significant (P<0.01) increase in ovulation rate (control: 1.7+/-0.3 vs immunized: 7.6+/-1.1). These results demonstrate that active immunization against inhibin enhances ovarian follicular development and ovulation rate by promoting an increase in pituitary FSH secretion. Therefore, immunization against inhibin may be a useful alternative to the conventional approach of superovulation in goats.

Abstract

Clinical resistance to thyroid hormone (RTH) has been classified into generalized resistance to thyroid hormone (GRTH) and pituitary resistance to thyroid hormone (PRTH) types. Since similar mutations have been identified in tri-iodothyronine (T₃) receptor (TR) β gene in GRTH and PRTH, and since considerable overlap has been seen in the clinical manifestations in patients with GRTH and PRTH, two subtypes of RTH are now considered to be a continuous spectrum with the same genetic defect. A point mutation at amino acid Arg 338 to Trp (R338W) which we identified in a patient with PRTH is very interesting, since R338W has been found in several other patients with PRTH, raising the possibility that this mutation may tend to associate with a phenotype of PRTH.

In our previous study, we found that R338W had relatively less impaired transcriptional potency, weaker dominant negative activity on various T₃ response elements and poor homodimer formation, as compared with another GRTH mutant. In this study, to investigate the functional properties of R338W further, especially in terms of the relation between transcriptional activity and dimer formations, we introduced the R338W mutation into the mutant receptors, K443E and F451X, constructing the double mutants, R338W/K443E and R338W/F451X. Both R338W/K443E and R338W/F451X showed negligible T₃ binding and transcriptional activities. The dominant negative activities of K443E and F451X were, however, significantly weakened by introducing the R338W mutation. As a control, a double mutant G345R/K443E was constructed by introducing a point mutation, G345R, located in the same exon 9 as R338W, into the K443E mutant. Dominant negative activity did not differ between G345R/K443E and K443E. Homodimer formation was significantly reduced in the double mutants containing R338W, but not G345R.

In summary, introducing the R338W mutation, but not G345R, into the mutant TR significantly weakened the dominant negative activity, despite further impairment of the T₃ binding and transcriptional activities.

Journal of Endocrinology (1996) 151, 293–300

Abstract

This paper describes a novel mutant mouse that has been spontaneously derived from the Snell's dwarf (DW/J) mouse. It was named the 'growth-retarded mouse' because of a characteristic growth pause followed by the delayed onset of pubertal growth. The onset of the increase in pituitary GH content that normally occurs concomitant with pubertal growth was also delayed in the growth-retarded mice. The serum concentration of thyroxine was very low in these mice from the neonatal period through adulthood, and a supplement of tri-iodothyronine was effective in shortening the growth pause and commencing the suppressed pubertal growth. Histological and immunohistochemical studies revealed that the anterior pituitary gland of the growth-retarded mouse contains clustered unusual chromophobic cells which are not reactive to various antisera against anterior pituitary hormones and the gland becomes enlarged with age. Breeding data indicated that these characteristics of the mice show an autosomal recessive inheritance and the gene responsible was designated as 'grm'. Partial linkage analysis utilizing microsatellite polymorphism demonstrated that the grm gene does not identify with the lit or hyt genes. Based on comparison of the hormonal status and growth pattern between growth-retarded, dwarf and normal mice, we have suggested the existence of a mutual interaction, possibly positive feedback regulation, between the pituitary and thyroid glands, that develops or matures the hormonal network which is responsible for rapid somatic growth and metabolic changes at puberty in mice.

Journal of Endocrinology (1994) 142, 435–446