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Search for other papers by A Bandyopadhyay in
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Search for other papers by P Roy in
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Search for other papers by S Bhattacharya in
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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 Bmax 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
Search for other papers by T Forster in
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Search for other papers by D Roy in
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Search for other papers by P Ghazal in
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Microarrays are a powerful method for the global analysis of gene or protein content and expression, opening up new horizons in molecular and physiological systems. This review focuses on the critical aspects of acquiring meaningful data for analysis following fluorescence-based target hybridisation to arrays. Although microarray technology is adaptable to the analysis of a range of biomolecules (DNA, RNA, protein, carbohydrates and lipids), the scheme presented here is applicable primarily to customised DNA arrays fabricated using long oligomer or cDNA probes. Rather than provide a comprehensive review of microarray technology and analysis techniques, both of which are large and complex areas, the aim of this paper is to provide a restricted overview, highlighting salient features to provide initial guidance in terms of pitfalls in planning and executing array projects. We outline standard operating procedures, which help streamline the analysis of microarray data resulting from a diversity of array formats and biological systems. We hope that this overview will provide practical initial guidance for those embarking on microarray studies.
Search for other papers by M Datta in
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Search for other papers by S Bhattacharya in
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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.
Search for other papers by A. R. Sheth in
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Search for other papers by P. R. Sheth in
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Search for other papers by R. Roy in
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Inhibin administered to adult male rats delayed the in-vivo pituitary responsiveness to thyrotrophin releasing hormone (TRH) as observed in terms of prolactin release in the serum. It also decreased the sensitivity of the pituitary gland to TRH, in terms of TSH release. However, inhibin alone did not alter the serum levels of prolactin and TSH, although it significantly suppressed serum FSH levels. In addition, the inhibin effect on FSH release was blocked by TRH.
Search for other papers by P L Sensky in
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Search for other papers by C H Roy in
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Search for other papers by R J Barnes in
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Search for other papers by M F Heath in
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Abstract
Continuous infusion of cortisol into adrenalectomized fetal sheep during the last 72 h of gestation (term=145 ± 2 days) produced a significant (P<0·05) rise in fetal serum tri-iodothyronine (T3) mean ± s.e.m. concentrations from 398 ± 65 to 1340 ± 238 ng/l. A concurrent decrease in plasma thyroxine (T4) levels was observed in three out of four animals. No significant changes in the concentrations of either hormone were noted prior to the start of cortisol infusion. The plasma concentrations of cortisol, T3 and T4 at term were similar to those in untreated full-term lambs. Adrenalectomized fetuses not given cortisol infusions still had low levels of T3 at term, with no increase being observed. The results suggest that cortisol plays an important role in the increase of fetal plasma T3 observed towards the end of gestation. This is probably achieved by the stimulation of the monodeiodination of T4 to T3 in the peripheral tissues.
Journal of Endocrinology (1994) 140, 79–83
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Search for other papers by P R Provost in
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Search for other papers by R Roy in
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Search for other papers by M-C Gauthier in
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Search for other papers by A Bélanger in
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Abstract
In addition to dehydroepiandrosterone (DHEA) sulfate (S), the human plasma also contains a second form of DHEA ester: DHEA-fatty acid esters (DHEA-FA). In the human adult, the plasma concentrations of DHEA-FA, DHEA and DHEAS are in the range of 6, 12 and 2000 nm respectively. Although the adrenal is responsible for almost all production of DHEAS in the circulation, DHEA-FA is formed from DHEA by an enzyme present in the circulation. Our work has clearly demonstrated that lecithincholesterol acyltransferase, localized on high density lipoprotein, is responsible for DHEA-FA production. Once DHEA-FA is formed, it is subsequently transferred to very low density lipoprotein (VLDL) and low density lipoprotein (LDL), like cholesteryl esters. Plasma lipoproteins contain at least 90% of circulating DHEA-FA of which 40% are found in the LDL fraction. Analysis of the fatty acid composition of tritiated DHEA-FA-labelled LDL ([3H]DHEA-FA-LDL) indicated the prevalence of DHEA-linoleate/palmitoleate and DHEA-oleate. Treatment of [3 H]steroid-FA-LDL with charcoal does not remove radioactivity, thus suggesting that the non-polar steroid is incorporated into the central non-polar core of the lipoproteins. Incubation of [3H]DHEA-FA-LDL with ZR-75–1 breast cancer cells produced a time-dependent increase in labeled non-conjugated steroids in the cell culture medium, whereas the levels of tritiated DHEA-FA decreased. Lipoidal radioactivity in cells increased with time, but non-conjugated radioactivity associated with the cells showed no such increase. HPLC analysis of the culture medium indicated the presence of tritiated DHEA and androst-5-ene-3β, 17β-diol. Our study indicates that circulating DHEA-FA incorporated into lipoproteins may indeed act as a substrate for potent steroid formation following their entry into steroid target cells.
Journal of Endocrinology (1996) 150, S119–S124
