Progesterone Accumulation Research Articles

Androstenedione increases cytochrome P450 aromatase messenger ribonucleic acid transcripts in nonluteinizing bovine granulosa cells

The objective of this study was to determine if androgens regulate granulosa cell steroidogenesis at physiological doses found in small bovine follicles. Bovine granulosa cells were cultured under serum-free conditions that permit the induction and maintenance of FSH-dependent estradiol secretion. Increasing androstenedione concentrations from 0.1 to 1 or 10 microM significantly increased estradiol accumulation and cytochrome P450 aromatase (P450arom) mRNA abundance. No increase in progesterone accumulation or abundance of mRNA for P450 side-chain cleavage or 3beta-hydroxysteroid dehydrogenase enzymes was observed. The addition of 0.1, 1, or 10 microM progestins or estrogens had no stimulatory effect on P450arom mRNA levels. An analysis of the 5'-untranslated region of P450arom mRNA transcripts indicated that the majority was derived from Cyp19 ovary-specific promoter 2, with some contribution from promoters 1.1 and 1.5. Transcripts from these three promoters were all significantly increased by androstenedione. Testosterone increased promoter 1.1 and 1.5-derived transcripts, but only promoter 2-derived transcripts at the highest dose tested (100 microM). Dihydrotestosterone (DHT) did not affect Cyp19 expression. Collectively, these data show that androgens may exert specific stimulatory effects on P450arom mRNA concentrations in granulosa cells. Interestingly, different androgens had different effects on Cyp19 promoter usage, suggesting differential regulation of aromatase gene expression in the developing follicle.

Developmental Sensitivity of the Bovine Corpus Luteum to Prostaglandin F2α (PGF2α) and Endothelin-1 (ET-1): Is ET-1 a Mediator of the Luteolytic Actions of PGF2α or a Tonic Inhibitor of Progesterone Secretion?1

We examined the responsiveness of large luteal cells (LLC), small luteal cells (SLC), and endothelial cells of the Day 4 and Day 10 bovine corpus luteum (CL) to prostaglandin (PG) F2alpha and endothelin (ET)-1. Using a single-cell approach, we tested the ability of each agonist to increase the cytoplasmic concentration of calcium ions ([Ca2+]i) as function of luteal development. All tested concentrations of agonists significantly (P = 0.05) increased [Ca2+]i in all cell populations isolated from Day 4 and Day 10 CL. Day 10 steroidogenic cells were more responsive than Day 4 cells to PGF2alpha and ET-1. Response amplitudes and number of responding cells were affected significantly by agonist concentration, luteal development, and cell type. Response amplitudes were greater in LLC than in SLC; responses of maximal amplitude were elicited with lower agonist concentrations in Day 10 cells than in Day 4 cells. Furthermore, on Day 10, as the concentration of PGF2alpha increased, larger percentages of SLC responded. Endothelial cells responded maximally, regardless of agonist concentration and luteal development. In experiment 2, we tested the developmental responsiveness of total dispersed and steroidogenic-enriched cells to the inhibitory actions of PGF2alpha and ET-1 on basal and LH-stimulated progesterone accumulation. The potency of PGF2alpha steroidogenic-enriched cells on Day 4 was lower than on Day 10; in contrast, the potency of ET-1 was not different. Therefore, ET-1 was a tonic inhibitor of progesterone accumulation rather than a mediator of PGF2alpha action. The lower efficacy of PGF2alpha in the early CL more likely is related to signal transduction differences associated with its receptor at these two developmental stages than to the inability of PGF2alpha to up-regulate ET-1.

Roles for prostaglandins in the steroidogenic response of human granulosa cells to high-density lipoproteins

In human granulosa-lutein cells, high-density lipoproteins (HDL) can stimulate progesterone synthesis. The objective of the present study was to establish whether prostaglandins (PGs) participate in the steroidogenic response to HDL. Both HDL and apolipoprotein AI (ApoAI) stimulated concentration-dependent increases in PGE 2, cAMP and progesterone accumulation. The minimum concentrations of HDL and ApoAI required to elevate PGE 2 production were the same as those required to stimulate cAMP accumulation and progesterone synthesis. Concentrations of PGE 2 were elevated within 10 min in cells exposed to HDL and rose progressively over 24 h, whereas cAMP and progesterone were only increased significantly after 24 h of treatment with HDL. Co-treatment with prostaglandin H synthase inhibitors (meclofenamic acid and indomethacin) abolished the cAMP and progesterone responses to both HDL and ApoAI. Hence, the ability of HDL to stimulate progesterone synthesis can be mimicked by ApoAI and appears to involve increased generation of one or more luteotrophic PGs, possibly acting via cAMP.

Detection of hCG Responsive Expression of the Steroidogenic Acute Regulatory Protein in Mouse Leydig Cells.

The steroidogenic acute regulatory (StAR) protein, a novel mitochondrial protein, is involved in the regulation of steroid hormone biosynthesis through its mediation of the intramitochondrial transport of the steroid substrate, cholesterol, to the cytochrome P450 cholesterol side chain cleavage (P450scc) enzyme. The expression of StAR protein is regulated by cAMP-dependent signaling in steroidogenic cells. During the course of our studies in mouse Leydig cells, we employ several methods for studying the regulation of StAR protein expression by human chorionic gonadotropin (hCG). A sensitive quantitative reverse transcription and polymerase chain reaction (RT-PCR) was utilized for determining StAR mRNA expression. Stimulation of mLTC-1 mouse Leydig tumor cells with hCG resulted in the coordinate regulation of StAR mRNA expression and progesterone accumulation in a time-response manner. The validity and accuracy of quantitative RT-PCR results in mLTC-1 cells were verified by a competitive PCR approach and were further confirmed in primary cultures of isolated mouse Leydig cells. Immunoblotting studies demonstrated an increase in the levels of the StAR protein in a concentration dependent manner following hCG stimulation in mLTC-1 cells. Northern hybridization analysis revealed three StAR transcripts, all of which were of sufficient size to encode functional StAR protein, and which were coordinately expressed in response to hCG. Collectively, the experimental approaches utilized in the present investigation allow for the demonstration and characterization of hCG mediated regulation of StAR mRNA and StAR protein expression in mouse Leydig cells.

Pitfalls in assessment of progesterone production by granulosa cells cultured in contact with silicone rubber or paraffin oil.

The influence of silicone templates, used for compartmentalization of culture dishes, on progesterone accumulation in granulosa cell cultures is studied and compared with the effect of paraffin oil, which is frequently used to cover oocyte/embryo cultures. Human granulosa-lutein cells were cultured in culture dishes compartmentalized by silicone templates, or in polystyrene plates under paraffin oil. Progesterone concentrations in the culture supernatant were compared with controls cultured in polystyrene plates. The progesterone concentration in culture supernatant was grossly reduced in silicone template cultures (2+/-0.7% of control). No inhibitory activity was identified in medium conditioned by preincubation with silicone rubber, but progesterone was absorbed from spiked medium incubated in silicone templates (recovery <2%). Progesterone concentration in culture supernatant was also reduced by a paraffin oil overlay (38+/-3% of control). From steroid spiked microdrops under oil, <2% of progesterone and 85+/-4% of estradiol was recovered. The steroidogenesis of cells cultured in silicone templates or under oil cannot be assessed correctly. It has to be considered that the concentration of lipophilic compounds may be grossly changed due to absorption by silicone rubber or paraffin oil.

Sex and season influence gonadal steroid biosynthetic pathways, end-product production and steroid conjugation in blotched blue-tongued lizards ( Tiliqua nigrolutea)

We examined differences in gonadal steroid production and biosynthetic pathway activity with changing reproductive condition and between sexes in the scincid lizard, Tiliqua nigrolutea. We observed clear seasonal and sexual variation in the production of androgens and steroid conjugates, but detected no 17β-estradiol or 5α-dihydrotestosterone produced by the gonads. An alternative steroid, more polar than estradiol, was detected: an investigation of this steroid is reported separately [Gen. Comp. Endocrinol. 129 (2002) 114]. There were seasonal and sex-related differences in steroid biosynthetic pathway activity. The Δ5 pathway metabolite, dehydroepiandrosterone, was detected only in males, and only from incubations using regressed testicular tissue. There was also a seasonal difference between the sexes in rates of progesterone accumulation, although the absence of corresponding elevated plasma concentrations suggests that the role of progesterone switches from a directly acting hormone to a precursor for others during the reproductive cycle in females. These results suggest that within the traditional view that vertebrate biosynthetic pathway activity and end-products are phylogenetically conserved, there is likely to be considerably species- and/or genus-specific variation.

Biphasic effects of leptin in porcine granulosa cells.

The direct effects of recombinant porcine leptin on porcine granulosa cells were studied to test the hypothesis that leptin, acting through the nuclear transcription factor signal transducer and activator of transcription 3 (STAT-3), modulates sterol regulatory element-binding protein 1 (SREBP1) thereby increasing steroidogenesis. In porcine granulosa cells in culture over 48 h, leptin at 10 ng/ml increased progesterone accumulation 3-fold while it was reduced by leptin at 1000 ng/ml. Leptin had no effect on progression of granulosa cells through the cell cycle nor on the frequency of cell death. Leptin treatment at 24 or 48 h of culture resulted in dose-dependent 2- to 4-fold increases in tyrosine phosphorylation of STAT-3. Leptin had a biphasic effect on the abundance of membrane-bound and transcriptionally active forms of SREBP1. In transient transfection of primary porcine granulosa cells, the plasmid expressing the transcriptionally active form of SREPB-1 induced transcription of the key regulator of steroidogenesis, the steroidogenic acute regulatory protein (StAR). StAR transcription was also increased by the low dose of leptin and was further upregulated in the presence of the SREBP plasmid. Leptin at 1000 ng/ml inhibited SREBP1-induced StAR expression. Thus, leptin, acting through STAT-3, modulates steroidogenesis in a biphasic and dose-dependent manner, and SREBP1 induction of StAR expression may be in the cascade of regulatory events.

4-Aminopyridine Decreases Progesterone Production by Porcine Granulosa Cells

BackgroundIon channels occur as large families of related genes with cell-specific expression patterns. Granulosa cells have been shown to express voltage-gated potassium channels from more than one family. The purpose of this study was to determine the effects of 4-aminopyridine (4-AP), an antagonist of KCNA but not KCNQ channels.MethodsGranulosa cells were isolated from pig follicles and cultured with 4-AP, alone or in combination with FSH, 8-CPT-cAMP, estradiol 17β, and DIDS. Complimentary experiments determined the effects of 4-AP on the spontaneously established pig granulosa cell line PGC-2. Granulosa cell or PGC-2 function was assessed by radio-immunoassay of media progesterone accumulation. Cell viability was assessed by trypan blue exclusion. Drug-induced changes in cell membrane potential and intracellular potassium concentration were documented by spectrophotometric determination of DiBAC4(3) and PBFI fluorescence, respectively. Expression of proliferating cell nuclear antigen (PCNA) and steroidogenic acute regulatory protein (StAR) was assessed by immunoblotting. Flow cytometry was also used to examine granulosa cell viability and size.Results4-AP (2 mM) decreased progesterone accumulation in the media of serum-supplemented and serum-free granulosa cultures, but inhibited cell proliferation only under serum-free conditions. 4-AP decreased the expression of StAR, the production of cAMP and the synthesis of estradiol by PGC-2. Addition of either 8-CPT-cAMP or estradiol 17β to serum-supplemented primary cultures reduced the inhibitory effects of 4-AP. 4-AP treatment was also associated with increased cell size, increased intracellular potassium concentration, and hyperpolarization of resting membrane potential. The drug-induced hyperpolarization of resting membrane potential was prevented either by decreasing extracellular chloride or by adding DIDS to the media. DIDS also prevented 4-AP inhibition of progesterone production.Conclusion4-AP inhibits basal and FSH-stimulated progesterone production by pig granulosa cells via drug action at multiple interacting steps in the steroidogenic pathway. These inhibitory effects of 4-AP on steroidogenesis may reflect drug-induced changes in intracellular concentrations of K+and Cl- as well as granulosa cell resting membrane potential.

Insulin and IGF-I are necessary for FSH-induced cytochrome P450 aromatase but not cytochrome P450 side-chain cleavage gene expression in oestrogenic bovine granulosa cells in vitro.

The earliest biochemical indicators of ovarian follicle deviation in cattle include lower oestradiol and free IGF concentrations in subordinate compared with dominant follicles. We determined if decreases in FSH, IGF-I or insulin cause decreased P450 aromatase (P450arom) or P450 cholesterol side-chain cleavage (P450scc) mRNA expression in oestrogenic bovine granulosa cells in vitro. In the first experiment, cells obtained from small follicles (2-5 mm diameter) were cultured in serum-free medium supplemented with physiological concentrations of FSH, IGF-I and insulin for 4 days. A decrease in specific hormone concentration was produced by replacing 70% of spent medium with medium devoid of FSH, insulin, or insulin and IGF-I on day 4 and again on day 5 of culture. Cultures were terminated on day 7. A reduction in FSH concentrations during the last 3 days of culture decreased P450arom and P450scc mRNA levels. A reduction in insulin reduced P450arom but not P450scc mRNA levels, and a reduction of both insulin and IGF-I concentrations further decreased P450arom mRNA levels and decreased P450scc mRNA levels. In a second experiment, cells obtained from small follicles (2-5 mm diameter) were cultured with insulin (100 ng/ml) without FSH for 4 days, and then insulin was withdrawn from the culture and FSH added for a further 3 days. The withdrawal of insulin decreased (P<0.02) oestradiol accumulation and reduced P450arom mRNA to below detectable levels, but did not affect P450scc mRNA levels. The addition of FSH transiently increased oestradiol secretion and P450arom mRNA levels, but P450arom mRNA levels were undetectable at the end of the culture period. The addition of FSH significantly enhanced P450scc mRNA levels and progesterone accumulation. These data demonstrated that a reduction of insulin-like activity reduced aromatase gene expression in bovine follicles without necessarily affecting progesterone synthetic capability, and thus may initiate follicle regression in cattle at the time of follicle divergence.

Lysophosphatidic acid-induced nuclear localization of protein kinase C delta in bovine theca cells stimulated with luteinizing hormone.

The amounts of lysophospholipase D (LPLD) and the ovarian protein kinase C delta (PKCdelta) increase during the course of pregnancy. Because LPLD is involved in the production of the bioactive phospholipid lysophosphatidic acid (LPA), we examined whether stimulation with LPA would influence PKCdelta in the ovary. We used immunoblotting and immunohistochemical methods to show that stimulation of bovine theca cells with LPA leads to an unexpected redistribution of PKCdelta from the cytosol to the perinuclear area and that in the presence of LH, LPA induces a complete nuclear translocation of PKCdelta. These effects of LPA are dose dependent, can be mimicked by phorbol ester, and are inhibited by a PKCdelta inhibitor, rottlerin. Concomitantly, under the same experimental conditions both LPA and the phorbol ester PMA (4beta-phorbol-12-myristate-13-acetate) augment LH-stimulated progesterone accumulation in this cell system. This functional effect of LPA and PMA is abolished in cells pretreated with rottlerin. It is unclear whether the nuclear localization of PKCdelta indicates a specific function of the enzyme in the bovine ovary. Because PKCdelta supports a luteotropic function in rodent models, a similar role in the bovine ovary is also likely.

Potassium channel antagonists influence porcine granulosa cell proliferation, differentiation, and apoptosis.

This investigation determined the effects of K(+) channel antagonists on proliferation, differentiation, and apoptosis of porcine granulosa cells. The drugs screened for functional effects included the class III antiarrhythmic agents MK-499 and clofilium, the chromanol I(Ks) antagonist 293B, the benzodiazepine I(Ks) antagonists L-735,821 and L-768,673, and the peptidyl toxins charybdotoxin (CTX) and margatoxin (MTX). Granulosa cell proliferation and differentiation were assessed by serial measurements of cell number and progesterone accumulation in the culture media, respectively. Granulosa cell apoptosis was evaluated using flow cytometry. Additional information about drug effects was obtained by immunoblotting to detect expression of proliferating cell nuclear antigen, p27(kip1) and the caspase-3 substrate poly(ADP-ribose) polymerase. The ERG channel antagonist MK-499 had no functional effects on cultured granulosa cells. However, the broad spectrum K(+) channel antagonist clofilium decreased, in a concentration-dependent fashion, the number of viable granulosa cells cultured, and these effects were associated with induction of apoptosis. All three I(Ks) antagonists (293B, L-735,821, and L-768,673) increased basal, but not FSH-enhanced progesterone accumulation on Day 1 after treatment without affecting the number of viable cells in culture, an effect that was blocked by pimozide. In contrast, CTX and MTX increased the number of viable cells in FSH-stimulated cultures on Day 3 after treatment without affecting progesterone output per cell. These data demonstrate that selective antagonism of granulosa cell K(+) channels with distinct molecular correlates, electrophysiological properties, and expression patterns can influence differential granulosa cell proliferation, steroidogenic capability, and apoptosis. Thus, K(+) channels may represent pharmacological targets for affecting Granulosa cell function and oocyte maturation, in vivo or in vitro.

P-glycoprotein-170 inhibition significantly reduces cortisol and ciclosporin efflux from human intestinal epithelial cells and T lymphocytes.

To assess the role of P-glycoprotein-170 (P-gp) in transporting cortisol and ciclosporin from human intestinal epithelium and T lymphocytes. The effect of P-gp inhibitors (verapamil, 0-100 microM; PSC 833, 0-20 microM) on the intracellular accumulation of 3H-cortisol and 3H-ciclosporin was studied in confluent layers of human Caco-2 cells (n=6), a P-gp-dependent absorptive intestinal epithelial cell phenotype, and moderately resistant MDRhigh CEM/VBL 100 T cells (n=6). The transport of 3H-vinblastine, a strong multidrug resistance (MDR) substrate, and 3H-progesterone, a poor MDR substrate, was also studied. Caco-2 cells had a 2.4-, 6.6-, 6.7- and 1.03-fold higher net basal to apical transport (efflux) of 3H-cortisol, 3H-ciclosporin, 3H-vinblastine and 3H-progesterone, respectively. PSC 833 (20 microM) reduced cortisol efflux by 69% (0.23 +/- 0.04 to 0.07 +/- 0.01 pmol/cm2/h, P < 0.05) and ciclosporin efflux by 76% (11.1 +/- 1.4 to 2.7 +/- 0.6 pmol/cm2/h, P < 0.001). MDRlow CEM T cells had a 1.4-, 1.9-, 3.2- and 1.02-fold higher intracellular accumulation of cortisol, ciclosporin, vinblastine and progesterone than MDRhigh CEM/VBL 100 T cells. Increasing concentrations of PSC 833 (> 0.1 microM) and verapamil (> 1 microM) restored the intracellular level of 3H-cortisol and 3H-ciclosporin in MDRhigh CEM/VBL 100 T cells to that of MDRlow CEM cells with little change in accumulation in the MDRlow parental cell line. P-gp inhibitors significantly increase intracellular cortisol and ciclosporin levels in human intestinal epithelium and T lymphocytes in a dose-dependent manner, demonstrating a potential mechanism for overcoming poor response to immunosuppressant therapy in refractory inflammatory bowel disease.

Effects of progestins on progesterone synthesis in a stable porcine granulosa cell line: control of transcriptional activity of the cytochrome p450 side-chain cleavage gene.

The purpose of the present study was to examine the effects of progestins on progesterone synthesis and expression of the cytochrome P450 cholesterol side-chain cleavage gene (P450(scc)) in a stable porcine granulosa cell line, the JC-410. Cells were incubated for 48 h with the synthetic progestogen-levornorgestrel with or without RU486 (progesterone and glucocorticoid receptor antagonist) or RWJ26819 (progesterone agonist without affinity to glucocorticoid receptors). Both levonorgestrel and RU486 enhanced progesterone accumulation in a dose-dependent manner. RU486 did not antagonize the effects of levonorgestrel, and RWJ26819 had no effect on progesterone production in cultured JC-410 cells. Progesterone and levonorgestrel increased steady state P450(scc) mRNA levels after 3-6 h of treatment. Progesterone and RU486 at 0.1, 1, and 10 microM increased the transcription rate of P450(scc) transiently expressed in JC-410 cells after 18 h of incubation; 30 microM had no effect, and 100 microM suppressed transcription. Levonorgestrel did not affect transcription of the P450(scc) gene, and RWJ26819 reduced its transcription. Progesterone and RU486 significantly decreased the number of cells and total protein content after 72 and 24 h of incubation, respectively. Levonorgestrel had no effect, whereas RWJ26819 increased (24 h) but subsequently reduced (72 h) cell number and protein content. The present results indicate that progestins are capable of directly modulating progesterone biosynthesis in porcine JC-410 granulosa cells. These effects may be exerted in part through the regulation of P450(scc) gene expression. Ostensible differences exist between progesterone and its synthetic analogues in the control of progesterone secretion in the stable porcine granulosa cell line in vitro.

Putative activation of the peroxisome proliferator-activated receptor gamma impairs androgen and enhances progesterone biosynthesis in primary cultures of porcine theca cells.

Ovarian theca cells are the predominant source of gonadotropin-stimulated androgen biosynthesis in vivo. Troglitazone (TG), a synthetic agonist of the peroxisome proliferator-activated receptor gamma (PPARgamma) and a thiazolidinedione used to treat insulin resistance, decreases serum androgen concentrations in women with hyperthecosis and/or polycystic ovary syndrome. Using reverse transcription-polymerase chain reaction (RT-PCR), we demonstrated the presence of PPARgamma mRNA in the porcine ovary. Since activation of ovarian PPARgamma may alter hormone-stimulated steroidogenesis in vitro, we cultured porcine theca cells for 48 h in the presence of two different PPARgamma ligands, TG and 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2). Putative TG-mediated activation of PPARgamma resulted in a 53%-69% decrease in LH- and/or insulin-stimulated androstenedione and testosterone accumulation. Although TG reduced 3-isobutylmethylxanthine-enhanced LH-stimulated cAMP accumulation by 74%-78%, it did not alter basal cAMP concentrations. Exposure to 8Br-cAMP did not overcome the TG-induced inhibition of androgen accumulation. In contrast, TG administration amplified basal and hormone-stimulated progesterone accumulation, particularly in the presence of insulin, without altering levels of 17alpha-hydroxyprogesterone. The putative natural PPARgamma ligand, 15d-PGJ2, inhibited androgen biosynthesis and stimulated progesterone production. RT-PCR-based amplification of cytochrome P450 cholesterol side-chain cleavage (CYP11A) and cytochrome P450 17alpha-hydroxylase/C-17,20-lyase (CYP17) transcripts indicated that TG moderately enhanced expression of these genes. However, TG did not affect CYP17 protein expression. We conclude that putative ligand-mediated activation of PPARgamma decreases LH- and/or insulin-driven theca cell androgen production by impairing the ability of CYP17 to synthesize androstenedione from available progestins. The corresponding augmentation of progesterone production could suggest that PPARgamma activation induces theca cell differentiation toward a progestin-synthesizing phenotype.

Steroidogenesis in cumulus cells of bovine cumulus–oocyte-complexes matured in vitro with BSA and different concentrations of steroids

The present in vitro experiments were designed to evaluate the ability of bovine cumulus–oocyte-complexes (COCs) to produce steroids and also to evaluate the modulatory effects of added estradiol, progesterone and testosterone on the steroidogenic activity of COCs. Considerable estradiol accumulation was observed in the control maturation medium for in vitro maturation of bovine COCs during the 24 h of maturation ( P<0.05). When testosterone was added to the medium at various concentrations, a slight estradiol accumulation occurred, which, however, was lower ( P<0.05) than that observed in the control medium. Slight estradiol accumulation was observed in maturation medium containing progesterone at concentrations of 2.5, 5.0 and 10.0 μg/ml, but these increases were less ( P<0.05) than those observed in the control medium. However, in the presence of 1.0 μg/ml progesterone, estradiol accumulation was equal to that of the control medium ( P>0.05). Progesterone accumulation ( P<0.05) was observed in the control medium for in vitro maturation of bovine COCs. When estradiol was added to the maturation medium, progesterone accumulation was observed, but was significant ( P<0.05) only when the medium was supplemented with the lesser concentrations of estradiol utilized in the experiment (1.0 μg/ml). The results demonstrated that (1) cumulus cells of bovine COCs are able to secrete estradiol and progesterone in culture systems for in vitro maturation, and this steroidogenesis is modulated by the steroids progesterone, testosterone and estradiol, and (2) the addition of estradiol to the in vitro maturation medium of bovine oocytes should be reviewed, since cumulus cells of COCs have been demonstrated to secrete estradiol in the maturation medium.

Secretory activity of bovine ovarian granulosa cells transfected with sense and antisense insulin-like growth factor (IGF) binding protein-3 and the response to IGF-I, GH, LH, oxytocin and oestradiol.

The aim of our in vitro experiments was to examine if IGF binding protein (IGFBP)-3 is involved in control of bovine ovarian secretory activity. For this purpose we performed the transfection of bovine granulosa cells with cDNA sense and antisense constructs increasing or inhibiting IGFBP-3 synthesis. The release of IGFBP-3, progesterone, oxytocin, IGF-I and prostaglandins F (PGF) and E (PGE) by control and transfected cells was compared. The transfected ovarian cells were cultured with and without bLH (100 ng/ml), bGH (100 ng/ml), IGF-I (10 ng/ml), oxytocin (10 ng/ml) and oestradiol-17beta (100 ng/ml). The concentration of IGFBP-3 produced was assessed using ligand and western blotting and secretion of progesterone, oxytocin, IGF-I, PGF and PGE was evaluated using RIA/IRMA techniques. Transfection of cells with the sense IGFBP-3 cDNA construct resulted in the expected increase in IGFBP-3 release, whereas the antisense IGFBP-3 construct induced the expected reduction in IGFBP-3 output. The granulosa cells transfected to overexpress IGFBP-3 had an increase in IGF-I, PGF and PGE release, and a decrease in basal and hormone- or growth factor-induced accumulation of progesterone and oxytocin. The granulosa cells transfected to have reduced IGFBP-3 expression gave primarily significant opposite findings. The present results suggest the involvement of IGFBP-3 in control of bovine ovarian steroid, peptide hormone, growth factor and prostaglandin release. IGFBP-3 is a physiological stimulator of IGF-I and prostaglandin release and an inhibitor of steroid and peptide hormone output.

Gonadotropin-induced accumulation of 4,4-dimethylsterols in mouse ovaries and its temporal relation to meiosis.

The resumption of oocyte meiosis is triggered by a number of 4,4-dimethylsterols termed meiosis-activating sterols (MAS). The levels of meiosis active (follicular fluid [FF]-MAS and bull testes [T]-MAS) and inactive (lanosterol) 4,4-dimethylsterols, free cholesterol, and progesterone were determined in gonadotropin-primed prepubertal mouse ovaries in vivo by high-performance liquid chromatography. Ovaries responded to an ovulatory stimulation by increasing their content of 4,4-dimethylsterols but not of free cholesterol. The ovarian 4,4-dimethylsterol response was followed with regard to time and dose-response to the gonadotropins and the resumption of meiosis was evaluated using histologic sections. All 4,4-dimethylsterols accumulated in a time-dependent manner in gonadotropin-primed mice after a subsequent stimulation with hCG. The peak of 4,4-dimethylsterol accumulation appeared postmeiotically but coincided roughly with ovulation, and the resumption of meiosis was triggered when the intraovarian level of MAS was <20% of its maximum. The ovarian accumulation of progesterone preceded the 4,4-dimethylsterol accumulation. The FF-MAS accumulation displayed a dose-response maximum with respect to hCG, and a variation of the follicular priming regime revealed that, in contrast to progesterone production, 4,4-dimethylsterol accumulation is dependent on previous follicular growth beyond the gonadotropin-dependent stage. The FF-MAS was not liberated from esterified stores during the accumulatory response and appeared to be synthesized de novo from a precursor (or precursors) metabolically upstream to lanosterol. The data remain inconsistent with a model in which MAS is regarded as the physiological trigger of meiosis. The 4,4-dimethylsterol accumulation is suggested to influence maturation processes by affecting membrane sterol composition.

Oxytocin inhibits LH-stimulated production of androstenedione by bovine theca cells

Oxytocin secretion by bovine granulosa cells increases dramatically after the LH/FSH surge. We have shown that oxytocin stimulates progesterone secretion and inhibits FSH-stimulated estradiol secretion in vitro by granulosa cells from bovine preovulatory follicles obtained before the LH/FSH surge. To determine if oxytocin regulates LH-stimulated steroid production by bovine theca interna cells, theca cells were isolated from preovulatory follicles obtained before the LH surge and were cultured for 4 days in the presence or absence of LH (2 or 4 ng/ml), without or with graded doses of oxytocin (125–1000 ng/ml). LH increased accumulation of androstenedione and progesterone. Oxytocin inhibited LH-stimulated androstenedione production, but had no effect on LH-stimulated progesterone production by cultured theca interna. The next objective was to determine if oxytocin regulates LH-stimulated steroidogenesis by modulating the levels of mRNA for steroidogenic enzymes and/or Steroidogenic Acute Regulatory protein (StAR). Low doses of LH alone increased the levels of mRNA for P450 17α-hydroxylase (17α-OH), 3β-hydroxysteroid dehydrogenase (3β-HSD) and cytochrome P450 side-chain cleavage, but not for StAR. In contrast, the effects of oxytocin on LH-stimulated androstenedione production were not associated with changes in the levels of mRNA for steroidogenic enzymes or StAR. These results suggest that oxytocin may play a paracrine role in regulating the follicular/luteal phase shift in steroidogenesis by decreasing androstenedione secretion by theca cells of the ovulatory follicle and that this effect is not mediated by changes in the levels of mRNA for steroidogenic enzymes and StAR.

Effects of androstenedione, insulin and luteinizing hormone on steroidogenesis in human granulosa luteal cells.

The present study was conducted to investigate the effect of androstenedione, insulin and LH on human granulosa cell oestrogen and progesterone production. We postulated that elevated concentrations of androstenedione, insulin and LH may be important modulators of granulosa cell steroidogenesis. Granulosa cells obtained in connection with IVF procedures were cultured for a total of 4 days in serum-free medium containing androstenedione (10(-6) mol/l). We tested the effect of androstenedione (10(-5) mol/l) on insulin (0-800 microIU/ml), LH (1-10 ng/ml) as well as on insulin + LH-stimulated oestrogen and progesterone production. Insulin increased the basal secretion of steroid hormones, and furthermore augmented LH-stimulated oestrogen and progesterone accumulation in granulosa cell cultures. Androstenedione (10(-5) mol/l) stimulated basal oestrogen production, but significantly reduced (32-58%) insulin + LH-stimulated oestrogen and progesterone secretion (P < 0.05). These results suggest that high androstenedione concentrations may act directly to impair insulin augmentation of LH-stimulated oestradiol and progesterone production in cultured human granulosa luteal cells. This is compatible with the hypothesis that high androgen levels may inhibit oestrogen production in polycystic ovary follicles, and as such may contribute to anovulation and infertility in women with polycystic ovary syndrome.

Coeliac ganglion adrenergic activity modifies ovarian progesterone during pregnancy: its inter-relationship with LH.

Most of the fibres that constitute the superior ovarian nerve (SON) originate at the neuronal bodies of the coeliac ganglion and innervate rat ovarian stroma cells. The purpose of this work was to study the part played by innervation on ovarian release of progesterone on day 15 and at the end of pregnancy in an integrated in vitro system known as the coeliac ganglion-SON-ovary system. We also investigated, in the same system, whether there is some kind of inter-relationship between the effect of adrenergic agents and LH on progesterone release on day 15 of pregnancy. The coeliac ganglion and the ovary were incubated in separate compartments, linked by the SON. The ovary was immersed in 2 ml buffer solution (ovarian compartment) and the coeliac ganglion was immersed in 2 ml of a different buffer solution (ganglion compartment). Under these conditions, the accumulation of progesterone in the ovarian compartment medium was used as an endpoint. Conditions were standardised on day 15 of pregnancy, when the decrease in the release of ovarian progesterone caused by non-specific stimulation on the ganglion with KCl (56 mM) demonstrated the functional integrity of the system. Neural influence was evaluated by the addition of adrenergic agents at a concentration of 10(-6)M to the coeliac ganglion. On day 15 of pregnancy, noradrenaline and propranolol increased progesterone release while phentolamine diminished it. The existence of ganglionic tone was assessed by analysing progesterone basal levels at different stages of pregnancy. The highest secretion of progesterone was found to take place on day 15, diminishing as pregnancy advanced. In addition, adrenergic neural participation was studied during the physiological luteolysis occurring at the end of pregnancy. Major findings were that noradrenaline increased ovarian accumulation of progesterone on day 19 and decreased it on day 20, while propranolol and phentolamine diminished progesterone release on both days. In additional studies, some neuroendocrine aspects were investigated at a peripheral level. The addition of LH only to the ovarian compartment did not affect progesterone secretion. However, when LH in the ovarian compartment was accompanied by noradrenaline, propranolol or phentolamine in the ganglion compartment, the release of progesterone decreased. It can be concluded that modifications of the neural state of the coeliac ganglion affect ovarian progesterone secretion and the physiology of pregnancy via the SON. The results may confirm that the coeliac ganglion-SON-ovary system provides a direct link between the autonomic nervous system and physiological events during pregnancy.