ABSTRACT

Binding of piscine and mammalian gonadotrophin to plasma membranes from the ovaries of a fish, the murrel (Channa punctatus), clearly suggests that the fish ovary possesses distinct and specific binding sites for both piscine and mammalian gonadotrophins. Maximum specific binding of ¹²⁵I-labelled human chorionic gonadotrophin (¹²⁵I-hCG) and ¹²⁵I-labelled silver carp gonadotrophin (¹²⁵I-scG) was obtained at 30 °C and pH 7·5 during 2 h of incubation. In competitive binding studies, binding of radiolabelled scG was effectively inhibited by piscine gonadotrophins while LH and hCG had less effect and FSH showed no inhibition. By using plasma membrane preparations from kidney, skeletal muscle, brain and ovary it could be shown that specific binding of radiolabelled gonadotrophins was restricted to ovarian tissue. Binding characteristics of both ¹²⁵I-scG and ¹²⁵I-hCG to a preparation of murrel ovarian plasma membranes showed saturability with high affinity and low capacity. Scatchard plot analysis gave a higher dissociation constant for hCG (Kd = 235 pmol/l) than for scG (Kd= 127 pmol/l). Maximum binding capacity of scG was about twofold higher (6·27 fmol/mg protein) than that of hCG (3·76 fmol/mg protein). An increase in gonadotrophin binding resulted in a greater formation of pregnenolone from cholesterol, indicating functional relevance. At a concentration of 8 mmol/l, Ca²⁺ markedly inhibited the binding of gonadotrophin. The physiological importance of this inhibition is discussed.

J. Endocr. (1986) 111, 407–413

Abstract

Leydig cells isolated from goat testis were sonicated and pure nuclear preparations obtained for ¹²⁵I-3,5,3′-triiodothyronine (T₃)-binding assay. Under optimum assay conditions of pH 7·2 at 37 °C and 90 min of incubation, binding of ¹²⁵I-T₃ to Leydig cell nuclei reached saturation at 1·2 nmol/l concentration. A Scatchard analysis of T₃ binding exhibited a K_d of 0·535 × 10⁻⁹ mol/l and a maximum binding capacity of 1·25 pmol/mg DNA. Competitive inhibition studies showed T₃ binding to be analogue specific. The physiological relevance of T₃ binding to goat Leydig cell was examined by adding increasing concentrations of T₃ to the Leydig cell incubation (1×1⁰ cells/incubation). T₃ (10, 25 and 50 ng/ml or 4, 10 and 20 ng/incubation) resulted a dose dependent increase in androgen release and in all cases stimulation of androgen release was statistically significant (P<0·01) compared with control. Stimulation of Leydig cell androgen release by T₃ was significantly inhibited by actinomycin-D (P<0·01) and cycloheximide (P<0·01). T₃ had additive stimulatory effects on LH-augmented androgen release from Leydig cells. T₃ (50 ng/ml or 20 ng/incubation) effected a more than twofold increase in Leydig cell protein synthesis compared with control and both actinomycin-D and cycloheximide (50 μg/ml) inhibited it completely. The data indicated that the stimulatory effect of T₃ on androgen release is mediated via T₃-induced protein(s). Sub-cellular fractions obtained from T₃-treated Leydig cells showed an increase in protein synthesis in mitochondrial and soluble supernatant fractions (100 k sup) and it was only 100 k sup which stimulated androgen release from Leydig cells in separate incubations. Treatment of 100 k sup with trypsin or heat abolished its stimulatory effect. Incubation of Leydig cells with T₃ for different times showed an increase in protein synthesis prior to the stimulation of androgen release. The results therefore indicated that T₃ binding to Leydig cells induced the generation of a proteinaceous factor(s) which in turn stimulated androgen release.

Journal of Endocrinology (1994) 143, 549–556

Abstract

¹²⁵I-3,5,3′-tri-iodothyronine (T₃) binds specifically to a pure nuclear preparation from rat granulosa cells. A Scatchard analysis of T₃ binding showed a K _d of 0·65 × 10⁻⁹ mol/l and a B_max of 1·57 pmol/mg DNA. The biological relevance of T₃ binding to granulosa cells was evaluated by adding T₃ (20 ng/incubation) to granulosa cells (1 × 10₆ cells/incubation) which greatly stimulated progesterone release. T₃-stimulated progesterone release was significantly inhibited by actinomycin-D (P<0·01) and cycloheximide (P<0·01). T₃ caused about a twofold increase in granulosa cell protein synthesis as compared with the control which was inhibited by actinomycin-D and cycloheximide. The addition of T₃ to granulosa cell incubations also resulted in a more than 2·5-fold increase in mRNA. The results indicated that T₃ stimulation of progesterone release is mediated via T₃-induced protein(s) or TIP. TIP was located in the soluble supernatant fraction (100 000 g supernatant; 100 k sup) from T₃-incubated cells but could not be detected in the 100 k sup from the control cells or LH-incubated cells. TIP was purified based on its biological activity, i.e. its addition to granulosa cell incubations stimulated progesterone release into the medium. The 100 k sup from T₃-incubated granulosa cells was subjected to Sephadex G-75 gel filtration, FPLC Mono Q and FPLC Superose 6 chromatography which resulted a 273-fold purification over the starting material and a clearly homogeneous protein was obtained. SDS-PAGE of purified TIP showed it to be a 53 kDa monomer protein. Experiments conducted with radiolabelled TIP suggested internalization of TIP into the granulosa cell. The results therefore showed that T₃ induces the synthesis of mRNA and proteins in rat granulosa cells and that one of the proteins is TIP which, in turn, stimulates progesterone release from the cell, suggesting thereby that this putative protein is a novel mediator of T₃ function in the granulosa cell.

Journal of Endocrinology (1996) 150, 309–318

Blood samples collected from 29 women (aged between 19 and 35 years) during the luteal phase of the menstrual cycle (between days 18 and 23 of the cycle) showed that deficiency in thyroid hormone level is related to a decrease in progesterone (P4) secretion. To observe the effect of thyroid hormone on human ovarian luteal cells, 3,5,3'-triiodothyronine (T3; 125 ng/ml) was added to luteal cells in vitro. T3 significantly stimulated progesterone release (P < 0.01) from luteal cells and this could be blocked by cycloheximide, indicating a protein mediator for the T3 effect. The T3 stimulatory effect was inhibited by anti-T3 antibody suggesting specificity of T3 action. Addition of T3 caused a more than threefold increase in cellular protein synthesis which was inhibited by cycloheximide. Preparation of partially purified thyroid hormone-induced factor (TIF) (from peak II of Sephadex G 100 chromatography of T3-incubated cells), and its addition to luteal cell incubations caused a significant increase in P4 release (P < 0.05). Incubation with trypsin or treatment with heat destroyed the stimulatory effect of TIF on P4 release, indicating the proteinaceous nature of TIF. Purified thyroid hormone-induced protein. (TIP) from rat granulosa cells and fish ovarian follicles greatly stimulated P4 release from human luteal cells. These results suggest that T3 stimulation of P4 release from human luteal cells is not direct, but is mediated through a putative protein factor, which appears to be a protein conserved through evolution as far as its biological activity is concerned.

ABSTRACT

Quantitative evaluation of the different varieties of germ cells at stage VII of the seminiferous epithelium cycle, namely type-A spermatogonia (ASg), preleptotene spermatocytes (pLSc), mid-pachytene spermatocytes (mPSc) and step 7 spermatids (7Sd), along with radioimmunoassay of plasma gonadotrophins (FSH and LH), testosterone and testicular testosterone were performed in Wistar rats following treatment with aldrin (polycyclic chlorinated hydrocarbon insecticide) for approximately one (13 days) or two cycles (26 days) of the seminiferous epithelium.

Extensive degeneration of all varieties of germ cells at stage VII, reduction in the sperm count and significant reductions in plasma concentrations of LH and testosterone were observed following aldrin treatment. The reduction in plasma concentrations of FSH was statistically significant only after treatment for two cycles. The inhibitory effect of aldrin on plasma gonadotrophins, testosterone levels, testicular testosterone content and numbers of 7Sd and ASg was maximum after treatment for two cycles. Administration of human chorionic gonadotrophin along with aldrin treatment for two cycles partially prevented the degeneration of germ cells and enhanced testosterone production.

The results indicate that aldrin may have a direct inhibitory influence on gonadotrophin release, but the possibility of a direct action of the insecticide at the level of the testes is also discussed.

J. Endocr. (1988) 119, 75–81

Experiments were carried out to identify the altered genes in hyperthyroid rat heart and their influence on the functions of cardiac myocytes. Chronic treatment of rats with 3,5,3' triiodo-L-thyronine (T3) resulted in a prominent increase in the size of the left ventricle with increased wall thickness and reduced chamber volume leading to concentric cardiac hypertrophy. The heart weight to body weight ratio (HW/BW) in hyperthyroid rats was increased by about 58% over that of normal rats. Using cDNA microarray comprising 588 genes, we compared the differences in mRNA expression of hyperthyroid and normal rat heart. Based on a threshold of greater than 10% change, about 37 genes were found to be regulated by T3. Further analyses by Western blotting, Northern blotting and real-time quantitative RT-PCR of some of the genes confirmed the microarray results. The T3-altered genes encode various types of proteins related to metabolism, matrix and cytoskeletal structures, growth factors, transcription factors, Ca(2+)-channels etc. The physiological significance of one of these altered proteins in hyperthyroid heart, insulin-responsive glucose transporter (GLUT) type 4 (GLUT4), was studied in detail. The expression of GLUT4 was drastically reduced in the ventricular tissues of hyperthyroid heart. Insulin-induced glucose uptake in hyperthyroid cardiomyocytes was reduced significantly, indicating the impaired glucose transport in cardiac cells. Interestingly, a few genes such as GLUT4, cytochrome P450 isoforms, superoxide dismutase (SOD), collagens, matrix metalloproteinases (MMP), tissue inhibitors of matrix metalloproteinases etc. which had not been reported earlier were found to be altered in hyperthyroid heart. Our results show some new aspects of hyperthyroid heart which will be important in assessing the pathophysiology of hypertrophied cardiomyocytes.