Changes in responsiveness of rat granulosa cells to prostaglandin E2 and follicle-stimulating hormone during culture

1983 ◽  
Vol 61 (6) ◽  
pp. 608-613 ◽  
Author(s):  
A. K. Goff ◽  
D. T. Armstrong
Keyword(s):  
Cyclic Amp ◽  
Granulosa Cells ◽  
Follicle Stimulating Hormone ◽  
Prostaglandin E ◽  
Dependent Manner ◽  
Isolated Cells ◽  
Progesterone Production ◽  
Dose Dependent ◽  
Stimulating Factor ◽  
Stimulation Of

The changes in responsiveness of granulosa cells to either FSH or prostaglandin E2 (PGE2), during culture of the cells, have been examined. In freshly isolated cells, FSH and PGE2 stimulated both cyclic AMP and progesterone production in a dose–dependent manner. In these cells, FSH stimulated cyclic AMP production to a greater extent than did PGE2. After the cells had been cultured for 2 days, neither FSH nor PGE2 stimulated progesterone production to any detectable extent. In these cells the ability of FSH to stimulate cyclic AMP was decreased, and that of PGE2 was increased markedly, such that PGE2 was far more effective than FSH in stimulating cyclic AMP. After culture of the cells for a further 2 days (4 days total), the FSH stimulation of cyclic AMP returned to that seen in freshly isolated cells, whereas the stimulation by PGE2 remained elevated. The acute stimulation of progesterone production could be restored by chronic exposure of the cells to either FSH or PGE2. These results demonstrate that dramatic changes in responsiveness of granulosa cells take place during culture. The results also suggest that some stimulating factor must be present to maintain the steroidogenic capabilities of the cells. Without this, although the cells are able to produce cyclic AMP in response to FSH, they cannot produce progesterone.

MATURATIONAL CHANGES IN SHEEP OVARIAN FOLLICLES: GONADOTROPHIC STIMULATION OF CYCLIC AMP PRODUCTION BY ISOLATED THECA AND GRANULOSA CELLS

1978 ◽  
Vol 89 (1) ◽  
pp. 166-172 ◽  
Author(s):  
T. J. Weiss ◽  
D. T. Armstrong ◽  
J. E. A. McIntosh ◽  
R. F. Seamark
Keyword(s):  
Cyclic Amp ◽  
Granulosa Cells ◽  
Follicle Stimulating Hormone ◽  
Adenosine Monophosphate ◽  
Ovarian Follicles ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Camp Production ◽  
Stimulation Of ◽  
Stimulating Hormone

ABSTRACT Theca and granulosa tissues isolated from sheep ovarian follicles of different sizes were incubated in the presence of human chorionic gonadotrophin (HCG; 5 IU/ml) or follicle stimulating hormone (FSH; 5 μg NIH-FSH-S11/ml) for 40 min. Changes in the total amounts of cyclic 3′,5′-adenosine monophosphate (cAMP) were used as an index of the responsiveness of these preparations to the hormones. Thecal tissue of both large (4–6 mm in diameter) and small (1–3 mm) follicles responded similarly to gonadotrophins. Granulosa cells from small follicles failed to respond to stimulation by HCG. FSH, however, consistently increased cAMP production in comparison with controls or cells treated with HCG. Granulosa cells of large follicles responded to both HCG and FSH.

scholarly journals Mastoparan promotes exocytosis and increases intracellular cyclic AMP in human platelets. Evidence for the existence of a Ge-like mechanism of secretion

1992 ◽  
Vol 281 (2) ◽  
pp. 465-472 ◽  
Author(s):  
C P D Wheeler-Jones ◽  
T Saermark ◽  
V V Kakkar ◽  
K S Authi
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
G Protein ◽  
Human Platelets ◽  
Dependent Manner ◽  
Kinase C ◽  
Reversible Electroporation ◽  
Dose Dependent ◽  
Stimulation Of

Recent studies have shown that mastoparan, an amphiphilic peptide derived from wasp venom, accelerates guanine nucleotide exchange and GTPase activity of purified GTP-binding proteins. In the present study we have examined the functional consequences of exposure of intact human platelets to mastoparan. Mastoparan promoted rapid (less than or equal to 1 min) dose-dependent increases in 5-hydroxy[14C]tryptamine and beta-thromboglobulin release from dense-granule and alpha-granule populations respectively. The exocytotic response did not result from a lytic effect of mastoparan and occurred in the complete absence of platelet shape change and aggregation. Liberation of [3H]arachidonate and increases in cytosolic [Ca2+] (detected with fura 2) were not observed in platelets stimulated with mastoparan. Similarly, in platelets preloaded with [3H]inositol during reversible electroporation, mastoparan did not cause the accumulation of [3H]inositol phosphates. Mastoparan-induced secretion was unaffected by preincubation with either the protein kinase C inhibitor staurosporine (10 nM-10 microM) or prostacyclin (PGI2; 100 ng/ml) and was not accompanied by phosphorylation of the 45 kDa protein kinase C substrate or the 20 kDa protein normally associated with platelet activation. The G-protein inhibitor guanosine 5′-[beta-thio]diphosphate (GDP[S]; 1 mM) attenuated the secretion induced by mastoparan in both intact and saponin-permeabilized platelets. Encapsulation of GDP[S] during reversible permeabilization inhibited mastoparan-induced secretion, providing evidence for an intracellular action of GDP[S]. In all these studies thrombin (0.05-0.2 unit/ml) elicited characteristic responses, and thrombin-induced secretion was inhibited by staurosporine, PGI2 and GDP[S]. Mastoparan also increased intra-platelet cyclic AMP in a dose-dependent manner. Mastoparan and PGI2 increased 32P incorporation into a protein of approx. 24 kDa, whereas phosphorylation of a 50 kDa substrate was only seen in PGI2-stimulated platelets. These results indicate that mastoparan promotes secretion by a mechanism which does not involve stimulation of phospholipase C and suggest that the secretory event may result either from a direct fusogenic action of mastoparan and/or from stimulation of the putative exocytosis-linked G-protein, Ge.

Bovine granulosa cell culture for assessment of potency and specificity of antibodies to pregnant mares' serum gonadotrophin

1996 ◽  
Vol 134 (4) ◽  
pp. 497-500 ◽  
Author(s):  
Mehmet Kuran ◽  
Peter J Broadbent ◽  
JS Morley Hutchinson
Keyword(s):  
Cell Culture ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Culture System ◽  
Dependent Manner ◽  
Progesterone Production ◽  
Neutralizing Capacity ◽  
Dose Dependent

Kuran M, Broadbent PJ, Hutchinson JSM. Bovine granulosa cell culture for assessment of potency and specificity of antibodies to pregnant mares' serum gonadotrophin. Eur J Endocrinol 1996;134:497–500. ISSN 0804–4643 Antibodies to pregnant mares' serum gonadotrophin (PMSG) neutralize the effect of PMSG in vivo and increase the number of transferable embryos when administered at the optimum time relative to the preovulatory luteinizing hormone (LH) surge in PMSG-stimulated cows. The objective of the present study was to investigate the possible use of bovine granulosa cells in a serum-free culture system as a bioassay for antibodies to PMSG. Granulosa cells (2–3 × 105 viable cells) were cultured with varying doses of PMSG and/or an anti-PMSG for 4 days. Whilst progesterone production (ng/μg DNA) of granulosa cells was stimulated by PMSG (p < 0.01) in a dose-dependent manner, increasing amounts of anti-PMSG neutralized (p < 0.01) this stimulatory effect of either follicle-stimulating hormone or LH on progesterone production of bovine granulosa cells in vitro. The bovine granulosa cell culture system is a potential in vitro bioassay method for testing the specificity and the neutralizing capacity of different anti-PMSG preparations. Mehmet Kuran, Ondokuz Mayis Universitesi, Ziraat Fakultesi, Zootekni Bolumu, 55149 Samsun, Turkey

scholarly journals Stimulation of production of prostaglandin E in gingival cells exposed to products of human blood mononuclear cells

1981 ◽  
Vol 198 (2) ◽  
pp. 391-396 ◽  
Author(s):  
S M D'Souza ◽  
D J Englis ◽  
A Clark ◽  
R G Russell
Keyword(s):  
Mononuclear Cell ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Gingival Tissue ◽  
Prostaglandin E ◽  
Dependent Manner ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Gingival Cells ◽  
Stimulation Of

1. Supernatant media from cultures of unstimulated human peripheral blood mononuclear cells contained one or more factors that increased by several hundred-fold the production of prostaglandin E by fibroblast-like cells derived from both inflamed and normal human gingival tissue. 2. This stimulation occurred in a dose-dependent manner and was completely inhibited by 14 microM-indomethacin. 3. Responsiveness to the factor declined as the age of the cell culture increased. 4. An increase in prostaglandin E production was first observed after a 2h exposure to the mononuclear cell factor(s) and could be prevented by cycloheximide. 5. Brief exposure (0.5 and 1.0 h) to mononuclear cell factor did not increase prostaglandin E production by the cells in a subsequent 72 h incubation in the absence of mononuclear cell factor. 6. Addition of arachidonate (10 microM and 15 microM) further enhanced stimulation of prostaglandin E production in response to mononuclear cell factor. 7. The stimulatory activity was resistant to digestion by trypsin, but was heat-labile, so that only 17% remained after treatment at 56 degrees C for 30 min.

scholarly journals Role of leukemia inhibitor factor (LIF) in decidualisation of murine uterine stromal cells in vitro

2004 ◽  
Vol 181 (3) ◽  
pp. 477-492 ◽  
Author(s):  
AA Fouladi Nashta ◽  
CV Andreu ◽  
N Nijjar ◽  
JK Heath ◽  
SJ Kimber
Keyword(s):  
In Vitro Culture ◽  
Stromal Cells ◽  
Control Level ◽  
Calf Serum ◽  
Prostaglandin E ◽  
Dependent Manner ◽  
Alp Activity ◽  
Dose Dependent ◽  
In Cells

Decidualisation of uterine stromal cells is a prerequisite for implantation of the embryo in mice. Here we have used an in vitro culture system in which stromal cells decidualise as indicated by a number of markers, including an increase in alkaline phosphatase (ALP) activity. The latter was used as a quantitative marker of decidualisation in the presence of low (2%) fetal calf serum. Prostaglandin E(2) (PGE(2)), which is known to induce decidualisation, increased ALP activity, and this effect was blocked in a dose-dependent manner by indomethacin. Leukemia inhibitory factor (LIF) was then examined, but it had no effect on PGE(2) secretion. However, LIF suppressed ALP activity in a dose-dependent manner in the presence of 2% serum, while an inhibitor of LIF that competes for binding to its receptor reversed the effect of LIF and increased ALP activity above the control level. In serum-free cultures, stromal cells differentiated rapidly, and no differences were observed between LIF-treated and untreated cultures. Stromal cells produce LIF during in vitro culture, and this peaked at 48 h. Freshly collected stromal cells from both day-2 and -4 pregnant mice expressed mRNA for the LIF receptor, and the transcript level was higher in cells isolated on day 4. However, no differences were observed in the relative levels of transcripts in cells from day 2 and day 4 after culture, nor were there differences between the LIF-treated cultures and controls. Therefore, in this study, we have shown that LIF suppresses decidualisation of murine uterine stromal cells in the presence of serum, this is not due to the regulation of PGE(2) secretion by stromal cells.

Regulation of oxytocin secretion by the ovine corpus luteum: effect of activators of protein kinase C

1990 ◽  
Vol 124 (2) ◽  
pp. 225-232 ◽  
Author(s):  
J. J. Hirst ◽  
G. E. Rice ◽  
G. Jenkin ◽  
G. D. Thorburn
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Corpus Luteum ◽  
Phospholipase C ◽  
Tissue Slices ◽  
Kinase C ◽  
Luteal Tissue ◽  
Dose Dependent ◽  
Stimulation Of

ABSTRACT The effect of protein kinase C activation and dibutyryl cyclic AMP on oxytocin secretion by ovine luteal tissue slices was investigated. Several putative regulators of luteal oxytocin secretion were also examined. Oxytocin was secreted by luteal tissue slices at a basal rate of 234·4 ± 32·8 pmol/g per h (n = 24) during 60-min incubations.Activators of protein kinase C: phorbol 12,13-dibutyrate (n = 8), phorbol 12-myristate,13-acetate (n = 4) and 1,2-didecanoylglycerol (n = 5), caused a dose-dependent stimulation of oxytocin secretion in the presence of a calcium ionophore (A23187; 0·2 μmol/l). Phospholipase C (PLC; 50–250 units/l) also caused a dose-dependent stimulation of oxytocin secretion by luteal slices. Phospholipase C-stimulated oxytocin secretion was potentiated by the addition of an inhibitor of diacylglycerol kinase (R59 022; n = 4). These data suggest that the activation of protein kinase C has a role in the stimulation of luteal oxytocin secretion. The results are also consistent with the involvement of protein kinase C in PLC-stimulated oxytocin secretion. The cyclic AMP second messenger system does not appear to be involved in the control of oxytocin secretion by the corpus luteum. Journal of Endocrinology (1990) 124, 225–232

Effects of recombinant bovine somatotrophin, insulin-like growth factor-I and insulin on bovine granulosa cell steroidogenesis in vitro

1994 ◽  
Vol 143 (1) ◽  
pp. 157-164 ◽  
Author(s):  
J G Gong ◽  
D McBride ◽  
T A Bramley ◽  
R Webb
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Dependent Manner ◽  
Serum Free ◽  
Factor I ◽  
Igf I ◽  
Serum Free Conditions ◽  
Dose Dependent ◽  
Bovine Somatotrophin ◽  
Cells Cultured

Abstract Our previous studies have demonstrated that physiological concentrations of metabolic hormones, including recombinant bovine somatotrophin (BST), insulin-like growth factor-I (IGF-I) and insulin, can significantly stimulate the proliferation of bovine granulosa cells cultured under serum-free conditions. In this study we investigated the effects of these factors on bovine granulosa cell steroidogenesis using the same culture system. Bovine granulosa cells were obtained from antral follicles classified into three size classes: small, <5 mm; medium-sized, 5–10 mm and large, >10 mm in diameter. Whilst not affecting steroidogenesis by granulosa cells from small and medium-sized follicles, BST (10–1000 ng/ml) stimulated the secretion of both oestradiol and progesterone by granulosa cells from large follicles in a dose-dependent manner. Insulin (1–1000 ng/ml) and IGF-I (10–1000 ng/ml) stimulated the secretion of oestradiol and progesterone by granulosa cells from all three size categories of follicles in a dose-dependent manner. FSH (200 ng/ml) alone increased progesterone secretion by granulosa cells from all three size classes of follicles, but had no effect on oestradiol secretion by granulosa cells. Both IGF-I (200 ng/ml) and insulin (30 ng/ml) acted in synergy with FSH (200 ng/ml) to stimulate steroidogenesis by granulosa cells from all three size categories of follicles, but no such interaction was observed between BST (50 ng/ml) and FSH (200 ng/ml). In conclusion, BST, IGF-I and insulin significantly influence the steroidogenic activity of bovine granulosa cells cultured under serum-free conditions. However, unlike their effects on cell proliferation, the minimal effective concentrations of these factors required to stimulate granulosa cell steroidogenesis were higher than those observed in our previous studies in vivo. Journal of Endocrinology (1994) 143, 157–164

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