Hormonal regulation of messenger ribonucleic acid expression for steroidogenic factor-1, steroidogenic acute regulatory protein, and cytochrome P450 side-chain cleavage in bovine luteal cells. (1/519)

To examine hormonal regulation of genes pertinent to luteal steroidogenesis, bovine theca and granulosa cells derived from preovulatory follicles were cultured with various combinations of forskolin and insulin. On Day 8 of culture, progesterone production was measured, and mRNA levels of steroidogenic factor-1 (SF-1), cytochrome P450 side-chain cleavage enzyme (P450scc), and steroidogenic acute regulatory protein (StAR) were determined by means of semiquantitative reverse transcription-polymerase chain reaction. Notably, the combination of forskolin plus insulin stimulated progesterone production in luteinized theca cells. This was probably a result of a synergistic interaction between forskolin and insulin, observed on both StAR and P450scc mRNA levels. However, in luteinized granulosa cells (LGC), forskolin and insulin each independently were able to up-regulate the levels of P450scc and StAR mRNA levels, respectively. Moreover, insulin alone was sufficient to maintain the high steady-state levels of StAR mRNA in LGC. Both insulin and insulin-like growth factor I enhanced StAR gene expression in LGC. SF-1 was constitutively expressed in bovine luteal cells; its amounts did not vary between the two luteal cell types or with hormonal treatments. In summary, this study demonstrates a distinct, cell-type specific regulation of StAR and P450scc mRNA in the two bovine luteal cell types.  (+info)

Luteinizing hormone inhibits conversion of pregnenolone to progesterone in luteal cells from rats on day 19 of pregnancy. (2/519)

We have previously reported that intrabursal ovarian administration of LH at the end of pregnancy in rats induces a decrease in luteal progesterone (P4) synthesis and an increase in P4 metabolism. However, whether this local luteolytic effect of LH is exerted directly on luteal cells or on other structures, such as follicular or stromal cells, to modify luteal function is unknown. The aim of the present study was to determine the effect of LH on isolated luteal cells obtained on Day 19 of pregnancy. Incubation of luteal cells with 1, 10, 100, or 1000 ng/ml of ovine LH (oLH) for 6 h did not modify basal P4 production. The addition to the culture medium of 22(R)-hydroxycholesterol (22R-HC, 10 microgram/ml), a membrane-permeable P4 precursor, or pregnenolone (10(-2) microM) induced a significant increase in P4 accumulation in the medium in relation to the control value. When luteal cells were preincubated for 2 h with oLH, a significant (p < 0.01) reduction in the 22R-HC- or pregnenolone-stimulated P4 accumulation was observed. Incubation of luteal cells with dibutyryl cAMP (1 mM, a cAMP analogue) plus isobutylmethylxanthine (1 mM, a phosphodiesterase inhibitor) also inhibited pregnenolone-stimulated P4 accumulation. Incubation with an inositol triphosphate synthesis inhibitor, neomycin (1 mM), or an inhibitor of intracellular Ca2+ mobilization, (8,9-N, N-diethylamino)octyl-3,4,5-trimethoxybenzoate (1 mM), did not prevent the decrease in pregnenolone-stimulated P4 secretion induced by oLH. It was concluded that the luteolytic action of LH in late pregnancy is due, at least in part, to a direct action on the luteal cells and that an increase in intracellular cAMP level might mediate this effect.  (+info)

Identification of 17-methyl-18-norandrosta-5,13(17-dien-3beta-ol, the C19 fragment formed by adrenal side chain cleavage of a 20-aryl analog of (20S)-20-hydroxycholesterol. (3/519)

Incubation of (20R)-20-phenyl-5-pregnene-3beta,20-diol, an aromatic analog of (23S)-20-hydroxycholesterol, with an adrenal mitochondrial preparation leads to the formation of four compounds: pregnenolone, phenol, a C8 ketone, acetophenone, and a nonpolar C19 compound. This latter compound has now been identified by reverse isotope dilution analysis and by gas chromatography/mass spectrometry as 17-methyl-18-norandrosta-5,13(17)-dien-3beta-ol. From these results it is evident that enzymatic fission of the C-17,20 bond of this synthetic derivative occurs. On the other hand, when (20S)-20-hydroxy[21-14C]cholesterol was used as substrate, the analogous cleavage did not take place. Thus, substitution of an aromatic group on C-20 facilitates side chain cleavage between that carbon atom and the nucleus whereas neither of the naturally occuring precursors, cholesterol or its 20-hydroxylated counterpart, are metabolized to a C8 fragment.  (+info)

Enzymatic properties of vesicle-reconstituted human cytochrome P450SCC (CYP11A1) differences in functioning of the mitochondrial electron-transfer chain using human and bovine adrenodoxin and activation by cardiolipin. (4/519)

The recently reported heterologous expression and purification of both human cytochrome P450SCC and adrenodoxin [Woods, S.T., Sadleir, J., Downs, T., Triantopoulos, T., Haedlam, M.J. & Tuckey, R.C. (1998) Arch. Biochem. Biophys. 353, 109-115] has enabled us to perform studies with the membrane-reconstituted human enzymes to better understand the side-chain cleavage reaction in humans. Human P450SCC was successfully reconstituted into dioleoylphosphatidylcholine vesicles with and without cardiolipin and its enzymatic properties characterized in the membrane-bound state. Enhancement of the P450SCC activity and significant activation by cardiolipin were observed when human adrenodoxin instead of bovine adrenodoxin was used as electron donor. In the absence of cardiolipin, Km for cholesterol was decreased twice in the case of human adrenodoxin indicating enhanced cholesterol binding. On the other hand, in the presence of cardiolipin in the membrane both Km and V for cholesterol were decreased with human adrenodoxin as electron donor. Kinetic analysis of the interaction between human P450SCC and its redox partners provided evidence for enhanced binding of the human electron donor to human P450SCC indicated by both an increased V and decreased Kd for human adrenodoxin compared with the values with bovine adrenodoxin. Because no similar effects were observed in Tween 20 micelles, these results suggest that the phospholipid membrane may play an important role in the interaction of human adrenodoxin with human P450SCC.  (+info)

Dynamics of periovulatory steroidogenesis in the rhesus monkey follicle after ovarian stimulation. (5/519)

The temporal relationships and regulation of events in the primate follicle during the periovulatory interval are poorly understood. This study was designed to elucidate the dynamics of steroid synthesis in the macaque follicle during ovarian stimulation cycles in which serum/follicular fluid aspirates were collected at precise intervals before (0 h) and after (up to 36 h) administration of the ovulatory human chorionic gonadotrophin (HCG) bolus. Serum concentrations of progesterone increased (P < 0.05) within 30 min, and follicular fluid progesterone concentrations were elevated 180-fold within 12 h, of HCG injection, and remained elevated until the time of ovulation. In contrast, 17beta-oestradiol concentrations increased initially, but then declined (P < 0.05) by 36 h post-HCG. Acute incubation of granulosa cells with and without steroidogenic substrates demonstrated that: (i) 3beta-hydroxysteroid dehydrogenase and aromatase activities were present in equivalent amounts before and after HCG; whereas (ii) P450 side-chain cleavage activity increased (P < 0.05) within 12 h of HCG; and (iii) exogenous low-density lipoprotein and cholesterol were not utilized for steroidogenesis. This model should be useful for further studies on ovulation and luteinization in primates, and enable elucidation of the local actions of progesterone and other steroids at specific time points during the periovulatory interval.  (+info)

Paracrine glucocorticoid activity produced by mouse thymic epithelial cells. (6/519)

Previous data have suggested that glucocorticoids (GCs) are involved in the differentiation of thymocytes into mature T cells. In this report we demonstrate that the mouse thymic epithelial cells (TEC) express the cytochrome P450 hydroxylases Cyp11A1, Cyp21, and Cyp11B1. These enzymes, in combination with 3beta-hydroxysteroid dehydrogenase (3betaHSD), convert cholesterol into corticosterone, the major GC in rodents. In addition, when TEC were cocultured with 'reporter cells' containing the glucocorticoid receptor (GR) and a GR-dependent reporter gene, a specific induction of reporter gene activity was observed. Induction of reporter gene activity was blocked when the TEC and reporter cells were incubated in the presence of the Cyp11B1 inhibitor metyrapone or the 3betaHSD inhibitor trilostane, as well as by the GR antagonist RU486. Coculturing of TEC with thymocytes induced apoptosis in the latter, which was partially blocked by the enzyme inhibitors and RU486. We conclude that TEC secrete a GC hormone activity and suggest a paracrine role for this in thymocyte development.  (+info)

Steroidogenic enzyme expression in human corpora lutea in the presence and absence of exogenous human chorionic gonadotrophin (HCG). (7/519)

In a human conception cycle, the expected decline in progesterone production by the corpus luteum during the late luteal phase is prevented by human chorionic gonadotrophin (HCG) secreted by the implanting blastocyst. This study investigated the expression of components of the synthetic pathway for progesterone in human corpora lutea in the presence and absence of HCG in vivo. Corpora lutea were obtained from: (i) normally cycling women at the time of hysterectomy and classified on the basis of the urinary luteinizing hormone (LH) surge as early (n = 3), mid- (n = 3), or late luteal (n = 3); or (ii) women who had received daily doubling doses of HCG (n = 3) to 'rescue' the corpus luteum. Expression patterns of steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) were investigated by Northern blotting, in-situ hybridization and immunohistochemistry. Luteal 'rescue' with HCG was associated with the continued expression of these components. In the late luteal phase, in the absence of HCG, expression remained but was more variable. The expression of 3beta-HSD mRNA was significantly reduced during the luteal phase (P<0.01). In conclusion, during luteal 'rescue', HCG acts to maintain the steroidogenic pathway. In the absence of HCG, the decline in progesterone production begins in the presence of the main components of the steroidogenic pathway. While unlikely to initiate this decline, the altered expression levels of these components, particularly that of 3beta-HSD, may contribute to the continued reduction in progesterone production.  (+info)

Growth factor modulation of steroidogenic acute regulatory protein and luteinization in the pig ovary. (8/519)

In vivo and in vitro luteinization were investigated in the porcine ovary, with emphasis on expression of steroidogenic acute regulatory protein (StAR). StAR mRNA and protein as well as cytochrome P450 side-chain cleavage mRNA (P450scc) increased during the luteal phase in the corpus luteum (CL) and were absent in regressed CL. Cytochrome P450 aromatase mRNA (P450arom) was not detectable at any time in CL. In vitro luteinization of granulosa cells occurred over 96 h in culture, during which P450arom mRNA was present at 1 h after cell isolation but not detectable at 6 h; and P450scc and StAR mRNAs were first detectable at 6 h and 48 h, respectively. Incubation of cultures with insulin-like growth factor I (IGF-I, 10 ng/ml), dibutyryl cAMP (cAMP, 300 microM), or their combination, induced measurable StAR mRNA at 24 h (p < 0.05), increased progesterone accumulation at 48 h, and elevated both StAR and P450scc expression through 96 h. Incubation of luteinized granulosa cells with epidermal growth factor (EGF, 10 nM) changed their phenotype from epithelioid to fibroblastic, eliminated steady-state StAR expression, and interfered with cAMP induction of StAR mRNA and progesterone accumulation. EGF had little apparent effect on P450scc mRNA abundance. It is concluded that StAR expression characterizes luteinization, and early luteinization is induced by cAMP and IGF-I in vitro. Further, EGF induces a morphological and functional phenotype that appears similar to an earlier stage of granulosa cell function.  (+info)

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