ABSTRACT

Hydrogen peroxide (H₂O₂) is an essential substrate for the peroxidase reaction in thyroid hormone biosynthesis. We demonstrated the production of H₂O₂ from porcine thyroid cells stimulated with extracellular ATP, using a scopoletin–horseradish peroxidase (HRP) system. Incubation of isolated cells for 1 day in the presence of 10% (v/v) newborn calf serum was necessary for the detection of induction by ATP of H₂O₂ production. The rate of H₂O₂ production induced by the addition of ATP increased in a dose-dependent manner, and the concentration of ATP required for half-maximum stimulation was about 10 μmol/l. ADP and GTP were also effective, but only at higher concentrations than ATP. In the absence of extracellular Ca²⁺, the production rate was very low.

Production of H₂O₂ from thyroid cells was also measured by a method which discriminated between H₂O₂ and superoxide anion (O₂ ⁻); in this, diacetyl-deuteroheme-substituted HRP was employed as the trapping agent for both O₂ metabolites. The thyroid cells produced H₂O₂, but not O₂ ⁻, when the cells were stimulated by extracellular ATP.

Journal of Endocrinology (1990) 126, 283–287

ABSTRACT

Isolated porcine thyroid follicular cells were cultured on a collagen-coated Millipore filter to form a monolayer. The monolayer could translocate ¹²⁵I added in the medium beneath the filter (basal medium) into the medium above the monolayer (apical medium) and form an iodide concentration gradient of several-fold. Transcellular iodide pump activity was observed when the cells were cultured with TSH in the basal medium. In the absence of TSH, the translocation of iodide was very slow. The concentration of TSH required to activate the iodide pump was 0·1–0·3 mU/ml. Addition of ClO₄ ⁻ to the basal medium inhibited transcellular transport, whilst addition of ClO₄ ⁻ to the apical medium was much less effective.

Constituents labelled with ¹²⁵I in the apical medium were analysed. The amount of protein-bound ¹²⁵I measured by acid precipitation was 3–8% of the total radioactivity. The residual radioactivity was found to be iodide ion by paper chromatography. Further analysis by sodium dodecylsulphate–polyacrylamide gel electrophoresis revealed that most of the ¹²⁵I-labelled protein was at the position of bovine serum albumin which had been added to the culture medium.

The monolayer culture of cells on collagen-coated filter would be a useful experimental system for analysing thyroid cell functions for which the cell polarity is essential.

Journal of Endocrinology (1990) 126, 275–281

Abstract

The functional capacity of the recombinant human FSH (hFSH) receptor was tested on the basis of gonadotrophin stimulation of cyclic AMP (cAMP) production by transient transfections of 293 cells and stable transfections of Chinese hamster ovary (CHO) cells. A CHO cell line expressed with the hFSH receptor cDNA covering the entire amino acid coding region revealed the presence of FSH binding site (K _d 6·2 × 10⁻¹⁰ m) on the plasma membrane. Treatment of transfected cells with hFSH induced dose-dependent increases in intracellular cAMP production. These results indicate that the hFSH receptor functionally couples with endogenous adenylyl cyclase. Although rat FSH also induced dose-dependent increases in cAMP production, bovine FSH was effective only at high doses and human chorionic gonadotropin did not alter cAMP levels compared with control values.

Northern blot analysis with a cRNA probe derived from hFSH receptor cDNA indicated the presence of two common FSH receptor mRNA transcripts (2·4 and 4·1 kb) in RNA prepared from a human ovary and transfected cell lines.

Preincubation of CHO cells expressing a functional hFSH receptor (CHO-FSHR) with FSH for 16 h decreased the subsequent cAMP production resulting from a 30-min pulse of FSH stimulation. These results indicate that desensitization of the adenylyl cyclase response to FSH stimulation occurs in CHO-FSHR cells. This cell line therefore provides a tool with which to pursue detailed studies on the molecular basis of FSH-induced desensitization.

Journal of Endocrinology (1994) 141, 369–375

Abstract

The acquisition of FSH receptor during preantral folliculogenesis is believed to be a key step in the subsequent development of follicles. We examined the interaction between activin and cAMP in FSH receptor induction in rat granulosa cells by measuring ¹²⁵I-FSH binding and FSH receptor mRNA. In the ¹²⁵I-FSH binding study, 0·2 mm 8-Br-cAMP and 1 μm forskolin were maximally effective in FSH receptor induction (169 and 220% respectively of control), while higher concentrations gave attenuated responses. It appears that cAMP has ambivalent effects on FSH receptor induction depending on the concentration and length of exposure. Activin alone dramatically increased the number of FSH receptors (314% of control). Moreover, synergistic effects of activin and 8-Br-cAMP or forskolin were observed on FSH receptor induction: a combination of activin (80 ng/ml) and low doses of 8-Br-cAMP (0·2 mm) or forskolin (1 μm) was most effective (160 or 140% of that induced by activin alone) and receptor levels reached a maximum at 24 h. These levels then markedly decreased after 72 h of incubation. Northern blot analysis revealed that the combination of activin (80 ng/ml) and 8-Br-cAMP (0·2 mm) or forskolin (1 μm) increased FSH receptor mRNA to about 140% of that induced by activin alone. These results indicate that activin and cAMP induced FSH receptor synergistically. However, activin did not enhance the production of cAMP induced by forskolin. In addition, a protein kinase A inhibitor (H89) (2 μm), which inhibited the effects of forskolin, had no effect on the action of activin. Taken together, the present findings suggest that the action of activin is not via a cAMP pathway, and that activin works co-operatively with cAMP on folliculogenesis.

Journal of Endocrinology (1995) 147, 103–110

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

The present study investigated possible sites through which ACTH or corticosterone inhibit progesterone secretion in pregnant rats, and the role of placental factors in blocking the inhibitory effect. The number of conceptuses was adjusted to one (1C group) or more than ten (FC group) on day 7 of pregnancy by aspirating the desired number. Serum concentrations of progesterone, testosterone and oestradiol were significantly (P<0·01) lower on day 15 in the 1C group than in the FC group. Corpora lutea (CL) obtained on day 15 were incubated for 6 h with corticosterone or ACTH. Corticosterone (1 μmol/l) significantly (P<0·05) inhibited progesterone secretion in the IC group but not in the FC group. The inhibitory effect of corticosterone in the IC group was completely blocked by co-addition of 1 μmol testosterone/l or 1 μmol oestradiol/l but not by 1 μmol dihydrotestosterone/l. ACTH (1 μg/l–1 mg/l) had no direct effect on progesterone secretion in either the IC or the FC groups, although ACTH apparently decreases progesterone secretion in vivo. Placentae obtained from rats of the FC group on day 15 were incubated for 24 h with or without ACTH (1 mg/l). The supernatant after placental incubation without ACTH significantly (P<0·01) increased progesterone secretion by the CL in both the IC and FC groups, and also eliminated the inhibitory effect of corticosterone in the IC group. The supernatant after placental incubation with ACTH also increased progesterone secretion in the FC group as effectively as the supernatant from the control incubation, but it had no effect in the IC group. It is concluded that corticosterone directly inhibits progesterone secretion by the CL, whereas the inhibitory effect of ACTH is mediated through the placenta. The results indicate that these inhibitory effects of corticosterone or ACTH are eliminated if the CL has been exposed to enough placental hormones before day 15 of pregnancy.

Journal of Endocrinology (1991) 129, 405–410

ABSTRACT

Preovulatory follicles from adult hamsters on the morning of pro-oestrus were used in this study. Serotonin stimulated oestradiol production by preovulatory follicles during a 5-h incubation in 1 ml Krebs–Ringer bicarbonate glucose medium containing isobutylmethylxanthine (0.1 mmol/l; IBMX) and androstenedione (1 μmol/l). The enhanced oestradiol production by serotonin was dependent on the dose of IBMX and androstenedione. Mianserin, a serotonin type-1 and serotonin type-2 receptor antagonists, prevented the serotonin-enhanced oestradiol production in a dose-dependent manner. Ketanserin, a specific serotonin type-2 receptor antagonist, was ineffective in blocking the action of serotonin, indicating that the effect of serotonin was mediated by the serotonin type-1 receptor. In the presence of androstenedione (1 μmol/l), serotonin was unable to enhance oestradiol production in isolated granulosa cells. It was also unable to enhance oestradiol production in early atretic follicles; atresia was induced experimentally by an injection of phenobarbital in order to prevent ovulation.

The data indicate that serotonin stimulates oestradiol production by hamster preovulatory follicles in vitro. The mechanism of action of serotonin involves an intact healthy follicle, a serotonin type-1 receptor and possibly cyclic AMP. The increased oestradiol secretion might be related to increased androgen production by the follicle and increased permeability (leakiness) of the follicle to androstenedione which serves as substrate for aromatization to oestradiol by the granulosa cell.

Journal of Endocrinology (1990) 125, 433–438

Abstract

The effect of FSH on the induction of FSH receptors in granulosa cells is believed to be mediated, at least in part, by the cAMP second messenger system. We examined the effect of activin and cAMP on FSH receptor expression in this culture system. Steady-state levels of FSH receptor mRNA, analyzed by Northern blot hybridization, increased 3·5-fold in response to 24-h incubation with activin and 1·7-fold with 12-h incubation with 8-bromoadenosine 3,5-cyclic monophosphate (8-Br-cAMP; 0·2 mm). We have investigated whether 8-Br-cAMP- and/or activin-induced increases in FSH receptor mRNA levels are the result of increased transcription and/or altered mRNA stability. The rates of FSH receptor mRNA gene transcription, assessed by nuclear run-on transcription assay, increased 3-fold in cells treated with activin and 1·5-fold in cells treated with 8-Br-cAMP for 2 h. To examine the degradation rates of FSH receptor mRNA transcripts, granulosa cells were preincubated with 8-Br-cAMP, activin, or medium alone for 6 h. After the preincubation period, 5 μm actinomycin-D or 200 μm 5,6-dichloro-1-β-ribofuranosyl benzimidazole were added to arrest new RNA synthesis. The decay curves for the 2·4 kb FSH receptor mRNA transcript in granulosa cells were not significantly different in the absence or presence of 8-Br-cAMP. Activin, on the other hand, significantly altered the slope of the FSH receptor mRNA decay curve and increased the half-life of the 2·4 kb FSH receptor mRNA transcript. These data provide evidence that cAMP induces FSH receptor mRNA levels by stimulating the transcription rate and that activin increases FSH receptor mRNA levels both by stimulating transcription rates and by stabilizing the FSH receptor mRNA transcripts.

Journal of Endocrinology (1997) 153, 465–473

Abstract

The purpose of this study was to examine the possible mechanism through which RU486 induces luteolysis during the late-luteal phase in pseudopregnant (PSP) rats. PSP rats received a subcutaneous injection of RU486 in sesame oil (5 mg/kg body weight) or sesame oil alone once a day between day 9 and day 11 of pseudopregnancy. Serial blood samples were collected on days 5, 9, 10, 11 and 12 and assayed for progesterone content. To examine the possible action of RU486 through a uterine and/or a pituitary (prolactin-dependent) mechanism, PSP rats and chronic hysterectomized PSP rats which had been hysterectomized before PSP induction received a subcutaneous injection of RU486 in sesame oil (5 mg/kg body weight), sesame oil alone, prolactin in 50% polyvinylpyrrolidone (15 IU/day), or RU486 and prolactin once a day between day 9 and day 11 of pseudopregnancy. Serial blood samples were collected on days 5, 9, 10 and 11 and assayed for progesterone content. Blood samples were also collected at 0400 h on day 12 and used for prolactin and progesterone determinations. To examine the direct effect of RU486 on corpus luteum and/or pituitary, hysterectomized rats underwent hypophysectomy and pituitary autotransplantation on dioestrus 1 and received a subcutaneous injection of RU486 in sesame oil or sesame oil alone for 3 days between day 21 and day 23 after surgery. Serial blood samples were collected on days 10, 21, 22, 23 and 24 and assayed for progesterone and prolactin contents.

In ordinary PSP rats, serum progesterone levels were significantly (P<0·01) lower in the RU486-treated group than in the control group (9 ± 1 vs 53 ± 7 ng/ml; mean ± s.e.m.) on day 11. Serum prolactin levels at 0400 h on day 12 of pseudopregnancy were significantly (P<0·05) lower in the RU486-treated group than in the control group (16 ±4 vs 154 ±44 ng/ml; mean ± s.e.m.). The concomitant prolactin treatment reversed the luteolytic effects of RU486 on day 11 of pseudopregnancy. In hysterectomized PSP rats, RU486 also suppressed serum prolactin levels, and the concomitant prolactin treatment again reversed the luteolytic effects of RU486. In hysterectomized rats which were hypophysectomized and pituitary autotransplanted, RU486 treatment did not induce any significant changes in serum progesterone and prolactin levels.

These results indicated that RU486 induced luteolysis during the late-luteal phase in PSP rats by suppressing prolactin secretion via a hypothalamic mechanism.

Journal of Endocrinology (1996) 150, 93–98