Porcine granulosa cells in suspension culture. II. Luteinization and hCG responsiveness

1981 ◽  
Vol 240 (6) ◽  
pp. E622-E629 ◽  
Author(s):  
A. R. LaBarbera ◽  
R. J. Ryan
Keyword(s):  
Adenylate Cyclase ◽  
Granulosa Cells ◽  
Culture Medium ◽  
Cultured Cells ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Short Term ◽  
Progesterone Secretion ◽  
Porcine Granulosa Cells ◽  
Ovine Prolactin

Granulosa cells from small follicles were cultured as suspensions in spinner flasks for 10 days in the absence or presence of follicle-stimulating hormone (FSH). With or without FSH, the cultured cells ultrastructurally resembled luteinized cells to different degrees. FSH increased progesterone accumulation in the culture medium. Ovine prolactin potentiated the effect of FSH in terms of the quantity of progesterone produced and the duration of accumulation. FSH increased acute human chorionic gonadotropin (hCG)-responsive progesterone secretion in short-term incubations of cultured granulosa cells. Responsiveness of FSH-cultured cells was maximal at day 4; that of control cultured cells was maximal at day 6. Adenylate cyclase activity of homogenates of cells cultured for 4, 6, or 8 days was measured. FSH induced in cultured cells an hCG sensitivity of the adenylate cyclase enzyme. These results indicate that FSH induced hCG-responsive progesterone secretion and hCG-responsive adenylate cyclase activity that correlate with ultrastructural signs of luteinization and with the previously reported FSH induction of hCG receptors.

scholarly journals Forskolin refractoriness. Exposure to the diterpene alters guanine nucleotide-dependent adenylate cyclase and calcium-uptake activity of cells cultured from the rat aorta

1987 ◽  
Vol 241 (2) ◽  
pp. 463-467 ◽  
Author(s):  
J F Krall ◽  
N Jamgotchian
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Cultured Cells ◽  
Rat Aorta ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Morphological Properties ◽  
Guanine Nucleotide ◽  
Intact Cells ◽  
Uptake Activity

Cells with the morphological properties of endothelial cells were cultured from the rat aorta. The cultured cells accumulated 45Ca2+ from the medium in a manner which was stimulated by forskolin and by 8-bromo-cyclic AMP. Pretreating the cultures for 20 h with forskolin diminished forskolin-dependent Ca2+-uptake activity. Adenylate cyclase activity of cultured cell homogenates was stimulated by guanosine 5′-[beta, gamma-imido]triphosphate (p[NH]ppG) and forskolin, and by isoprenaline in the presence, but not in the absence, of guanine nucleotide. p[NH]ppG increased forskolin sensitivity and caused a leftward shift in the forskolin dose-response curve. Pretreating the cultured cells with forskolin for 20 h, conditions that decreased forskolin-dependent Ca2+ uptake, increased basal and guanine nucleotide-dependent adenylate cyclase activity, but not forskolin-dependent activity determined in the absence of p[NH]ppG. Forskolin pretreatment diminished p[NH]ppG's capacity to increase forskolin sensitivity, but did not have a significant effect on either the sensitivity of adenylate cyclase to p[NH]ppG or its responsiveness to isoprenaline. These results suggest that the Ca2+-uptake mechanism is cyclic AMP-dependent and that guanine nucleotides mediated forskolin-dependent cyclic AMP production by the intact cells. In addition, there may be different guanine nucleotide requirements for hormone-receptor coupling and forskolin activation.

Porcine granulosa cell conditioned media as autocrine regulator of progesterone secretion

1993 ◽  
Vol 5 (1) ◽  
pp. 95
Author(s):  
RT Denkova ◽  
IG Ivanov ◽  
LN Kanchev
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Cultured Cells ◽  
Primary Cultures ◽  
Inhibitory Effect ◽  
Conditioned Media ◽  
Progesterone Secretion ◽  
Porcine Granulosa Cells ◽  
Porcine Granulosa Cell

The ability of porcine granulosa cells to release a progesterone inhibiting substance(s) was examined in vitro. Granulosa cells (SGCs, MGCs and LGCs) were harvested from small, medium or large antral follicles respectively. The effect of granulosa cell conditioned media obtained from small follicles (SGCCM) was studied in the culture of LGCs by estimation of progesterone secretion; the conditioned media evoked the inhibition of progesterone secretion by the LGCs. SGCCM produced by various numbers of cultured granulosa cells showed a dose-related inhibition of progesterone production. A maximum inhibitory effect was noted when a 5-fold concentration of SGCCM was used. The addition of SGCCM had no effect on the growth of the cultured cells. The factor(s) inhibiting progesterone secretion appeared to be a nonsteroidal substance of molecular mass greater than 10 kDa and was heat-stable and trypsin-sensitive. The data presented support the suggestion that the conditioned media generated by primary cultures of SGCs contain nonsteroidal regulators capable of inhibiting progesterone secretion by cultured LGCs; this inhibitory activity can play an important autocrine regulatory role in the process of follicular differentiation.

scholarly journals Calmodulin activation of adenylate cyclase in the mouse B16 melanoma

1984 ◽  
Vol 224 (2) ◽  
pp. 453-460 ◽  
Author(s):  
S Mac Neil ◽  
S W Walker ◽  
H J Senior ◽  
A Pollock ◽  
B L Brown ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Adenylate Cyclase ◽  
Metal Ion ◽  
Cultured Cells ◽  
Marked Change ◽  
Earth Metal ◽  
B16 Melanoma ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Maximal Stimulation

Calmodulin antagonists inhibited hormone-stimulated cyclic AMP accumulation in both cultured cells and cell lysates of mouse B16 melanoma. Particulate preparations of B16 melanoma contained 34-45% of total cell calmodulin, which could not be dissociated by extensive washing irrespective of the presence of EGTA in the buffer. The adenylate cyclase activity in such preparations was unaffected by the addition of exogenous calmodulin. However, the rare-earth-metal ion La3+, which can mimic or replace Ca2+ in many systems, produced an immediate inhibition of agonist-stimulated adenylate cyclase activity and preincubation of particulate preparations was La3+ followed by washing with La3+-free buffer dissociated calmodulin (96% loss) from particulate preparations. The loss of calmodulin from particulate preparations was associated with a decrease in agonist responsiveness (74%) and a marked change in the Ca2+-sensitivity of the enzyme, low concentrations of calcium (approx. 10 nM) now failing to stimulate enzyme activity, high concentrations of calcium (greater than or equal to 100 nM) producing greater-than-normal inhibition of enzyme activity. Direct activation of adenylate cyclase by the addition of pure calmodulin was now demonstrable in such calmodulin-depleted particulate preparations. Half-maximal stimulation of agonist-responsive adenylate cyclase occurred at 80 nM-calmodulin in the presence of 10 microM free Ca2+. Maximal stimulation by calmodulin (at 300-600 nM) restored enzyme activity to 89 +/- 5% (mean +/- S.E.M., n = 7) of the activity in untreated, calmodulin-intact, preparations.

Effects of FSH and LH on adenylate cyclase activity in rat granulosa cell membranes during follicular maturation

1985 ◽  
Vol 109 (2) ◽  
pp. 258-265
Author(s):  
K. Nordenström ◽  
S. Rosberg ◽  
P. Roos
Keyword(s):  
Adenylate Cyclase ◽  
Granulosa Cells ◽  
Dose Response ◽  
Response Curve ◽  
Dose Response Curve ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Follicular Maturation ◽  
Maximal Stimulation ◽  
Clear Increase

Abstract. Adenylate cyclase activity was measured in membranes prepared of granulosa cells isolated from PMSG-treated immature rats and the effects of ovine, highly purified human and rat gonadotrophins were compared. Furthermore, a comparison of the effects of the human preparations (hLH, hFSH) on the adenylate cyclase activity in membranes prepared from granulosa cells isolated at different stages of follicular maturation, was performed. The adenylate cyclase in membranes of immature granulosa cells was stimulable with FSH but not with LH, while in pre-ovulatory granulosa cell membranes, both gonadotrophins were stimulatory with FSH generally being more effective than LH. Surprisingly, the dose-response curve for ovine LH (oLH) was biphasic with a plateau at a level of adenylate cyclase activity corresponding to the maximal stimulatory effect of hCG. With increasing oLH concentrations the response resumed and the maximal stimulation corresponded to that of oFSH. With highly purified rat gonadotrophins the FSH response was significantly higher than the response to LH at all concentrations tested. Using highly purified human gonadotrophins the maximal FSH response was 50% higher than the maximal LH response and by adding increasing concentrations of hFSH to a maximally stimulatory concentration of hLH it was possible to mimic the biphasic dose-response curve for oLH. When the membranes were prepared from ganulosa cells isolated after the pro-oestrus LH/FSH surge there was clear increase in the sensitivity of the adenylate cyclase to stimulation with LH although the maximal response was unaffected. The sensitivity to FSH was not altered. These data indicate that FSH gives a higher maximal stimulation of the adenylate cyclase in rat granulosa cells than does LH, and the biphasic dose-response curve seen with oLH most probably is due to a FSH contamination in the LH preparation used. After in vivo exposure to the pro-oestrus LH/FSH surge no desensitization was observed but instead a clear increase in LH sensitivity. It thus seems as if the primary FSH sensitive cell under the influence of the LH/FSH surge turns to a primary LH sensitive one.

Effect of long-term and short-term diabetes on the parathyroid hormone sensitive rat renal adenylate cyclase: correlation with vitamin D metabolism

1988 ◽  
Vol 66 (10) ◽  
pp. 1313-1318 ◽  
Author(s):  
L. G. Rao ◽  
M. S. Kung
Keyword(s):  
Vitamin D ◽  
Parathyroid Hormone ◽  
Adenylate Cyclase ◽  
Diabetic Rats ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Short Term ◽  
Vitamin D Metabolism ◽  
Male Wistar Rats

In short-term experiments, male Wistar rats were made diabetic for 10 days with a single injection of streptozotocin (65 mg/kg body weight). One group of diabetic rats was treated with insulin for 3 days prior to sacrifice. In long-term experiments, vitamin D replete or vitamin D depleted rats were made diabetic for 6 weeks. Criteria for diabetes were loss of weight, glycosuria (Tes-Tape), and hyperglycemia. In long-term diabetic rats the activity of renal mitochondrial 25-hydroxyvitamin D3 (25-(OH)D3) 1α-hydroxylase was significantly decreased and that of 25-(OH)D3 24-hydroxylase increased. However, the parathyroid hormone (PTH) sensitive renal adenylate cyclase activity of diabetic rats was not different from that of the nondiabetic rats in either the vitamin D replete group or the vitamin D depleted group. On the other hand, the PTH-sensitive renal adenylate cyclase activity was significantly higher in short-term diabetic rats than in control and insulin-treated rats. These differences were observed at doses of 10−8 to 10−5 M of PTH. This study has demonstrated for the first time that there are differences in the PTH-sensitive adenylate cyclase response between long-term and short-term diabetic rats. The hypersensitivity to PTH of the renal adenylate cyclase observed in short-term diabetic rats probably represents a response to insulin deficiency during the early development of diabetes mellitus in the rats.

Cytochemical Evidence for Presynatic β-Adrenergic Regulation of Secretion in the Developing Rat Submandibular Gland

1977 ◽  
Vol 35 ◽  
pp. 430-431
Author(s):  
L.S. Cutler
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Submandibular Gland ◽  
Neonatal Rat ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Parotid Glands ◽  
Adrenergic Agonist ◽  
Rat Submandibular Gland

Many studies previously have shown that the B-adrenergic agonist isoproterenol and the a-adrenergic agonist norepinephrine will stimulate secretion by the adult rat submandibular (SMG) and parotid glands. Recent data from several laboratories indicates that adrenergic agonists bind to specific receptors on the secretory cell surface and stimulate membrane associated adenylate cyclase activity which generates cyclic AMP. The production of cyclic AMP apparently initiates a cascade of events which culminates in exocytosis. During recent studies in our laboratory it was observed that the adenylate cyclase activity in plasma membrane fractions derived from the prenatal and early neonatal rat submandibular gland was retractile to stimulation by isoproterenol but was stimulated by norepinephrine. In addition, in vitro secretion studies indicated that these prenatal and neonatal glands would not secrete peroxidase in response to isoproterenol but would secrete in response to norepinephrine. In contrast to these in vitro observations, it has been shown that the injection of isoproterenol into the living newborn rat results in secretion of peroxidase by the SMG (1).

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