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.

Prolactin, LH, and estradiol-17β in utilization of lipoprotein substrate by porcine granulosa cells in vitro

1988 ◽  
Vol 66 (5) ◽  
pp. 561-566 ◽  
Author(s):  
K. Rajkumar ◽  
P. Klingshorn ◽  
P. J. Chedrese ◽  
B. D. Murphy
Keyword(s):  
Granulosa Cell ◽  
Cell Cultures ◽  
Granulosa Cells ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density ◽  
Porcine Granulosa Cells ◽  
Progesterone Synthesis ◽  
Serum Free Conditions

Porcine granulosa cells cultured under serum free conditions responded by increased progesterone secretion to the addition of the leuteotropic hormones, LH, prolactin, and estradiol. Provision of extracellular substrate for steroidogenesis in the form of porcine high density lipoprotein or low density lipoprotein enhanced progesterone accumulation by granulosa cell cultures. Estradiol, LH, and prolactin all greatly increased progesterone accumulation in the presence of either high or low density lipoproteins. Increases in progesterone accumulation following addition of prolactin or LH in combination with estradiol suggested the presence of a synergistic interaction among leuteotropins. Pre-exposure of granulosa cell cultures to estradiol increased the subsequent stimulatory effect of prolactin on lipoprotein utilization. It is concluded that all three leuteotropins function to enhance and may interact in the utilization of extracellular lipoprotein substrate for progesterone synthesis.

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.

The effects of phospholipase C on oestradiol and progesterone secretion in porcine granulosa cells cultured in vitro

2019 ◽  
Vol 54 (9) ◽  
pp. 1236-1243 ◽  
Author(s):  
Huali Chen ◽  
Youfu Yang ◽  
Youlin Wang ◽  
Yamei He ◽  
Jiaxin Duan ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Granulosa Cells ◽  
Progesterone Secretion ◽  
Porcine Granulosa Cells ◽  
Cells Cultured

Inhibitory Effect of Transferrin on Progesterone Production in the Granulosa Cell of Humans in vivo and Porcine Granulosa Cell in vitro

1995 ◽  
Vol 40 (1) ◽  
pp. 1-4 ◽  
Author(s):  
Yasushi Kawano ◽  
Hisashi Narahara ◽  
Kenji Miyamura ◽  
Kumato Mifune ◽  
Isao Miyakawa
Keyword(s):  
Granulosa Cell ◽  
Inhibitory Effect ◽  
Progesterone Production ◽  
Porcine Granulosa Cell

Comparison of the facilitative roles of insulin and insulin-like growth factor I in the functional differentiation of granulosa cells: in vitro studies with the porcine model

1988 ◽  
Vol 117 (2) ◽  
pp. 230-240 ◽  
Author(s):  
Takeshi Maruo ◽  
Masato Hayashi ◽  
Hiroya Matsuo ◽  
Yasuo Ueda ◽  
Hajime Morikawa ◽  
...  
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Binding Sites ◽  
Physiological Concentration ◽  
Physiological Level ◽  
Estradiol Secretion ◽  
Porcine Granulosa Cells ◽  
Igf I ◽  
Hcg Receptor

Abstract. The facilitative effects of insulin and IGF-I were compared in vitro with regard to induction of differentiated functions of porcine granulosa cells. The monolayers were maintained under serum-free conditions in the absence or presence of porcine FSH (20 μg/l), with or without graded doses of insulin or IGF-I. Concurrent treatment with IGF-I and FSH produced morphological differentiation and augmented LH/hCG receptor binding together with an enhancement in progesterone and estradiol secretion relative to treatment with FSH alone. IGF-I alone was incapable of exhibiting these effects. Insulin synergized with FSH to facilitate the granulosa cell functions except estradiol secretion. Maximal effective dose of IGF-I was 100 μg/l which is within the physiological concentration in vivo, whereas that of insulin was 1.0 mg/l, which is 1000-fold higher than the physiological level. Although the maximal effective doses of IGF-I and insulin produced a comparable increment in progesterone secretion and LH/hCG receptor induction, combined treatment with IGF-I and insulin did not prove additive. [125I]IGF-I binding revealed that specific IGF-I receptors with two classes of binding sites are present on porcine granulosa cells. No distinct differences were detected between IGF-I receptors of granulosa cells from small, medium and large follicles. Insulin was approximately 100-fold less active than IGF-I in competing for [125I]IGF-I binding. These findings suggest that porcine granulosa cells possess specific IGF-I binding sites which may mediate the cytodifferentiative actions of insulin-like peptides. Since IGF-I is more potent than insulin in amplifying the actions of FSH and maximally exerts the cytodifferentiative effects at the physiological concentration, it is likely that IGF-I plays the more important role in granulosa cell differentiation in synergy with FSH.

Porcine granulosa cell desensitization: prolonged FSH-responsive cAMP production in vitro

1983 ◽  
Vol 244 (5) ◽  
pp. E435-E441
Author(s):  
A. R. LaBarbera ◽  
A. E. Fisher
Keyword(s):  
Cholera Toxin ◽  
Granulosa Cells ◽  
Prolonged Exposure ◽  
Phosphodiesterase Inhibitor ◽  
Toxin Production ◽  
Camp Production ◽  
Porcine Granulosa Cells ◽  
Incubation Experiments ◽  
Porcine Granulosa Cell

Granulosa cells from small (1-2 mm) follicles of porcine ovaries produced cAMP in response to follicle-stimulating hormone (FSH); they produced little cAMP in response to human chorionic gonadotropin (hCG) or to cholera toxin. Production, estimated by quantification of total cAMP (medium + cells) by radioimmunoassay, appeared to be episodic in the absence or presence of the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (MIX) and continued for at least 20 h. Results of dual incubation experiments with thorough washing of cells between incubations indicated that cells remained responsive to FSH, to hCG, and to cholera toxin after prior exposure to homologous stimulator. Preincubation of cells for 3 h in the absence of stimulator enhanced FSH responsiveness. In experiments with heterologous stimulators in two incubations, first, preincubation with FSH enhanced subsequent hCG and cholera toxin responsiveness; and second, preincubation with hCG or cholera toxin did not affect FSH responsiveness. Desensitization of FSH-activated adenylate cyclase was never greater than 35%; the degree of attenuation was dependent on FSH concentration and duration of exposure to FSH. Cells perifused with medium containing MIX and a maximally effective concentration of FSH for 5 h released cAMP continuously. In summary, the results indicated that porcine granulosa cells do not become unresponsive to FSH after prolonged exposure to FSH in vitro.

Uncoupling between proliferation and differentiation of ovine granulosa cells in vitro

1994 ◽  
Vol 142 (3) ◽  
pp. 497-510 ◽  
Author(s):  
D Monniaux ◽  
C Pisselet ◽  
J Fontaine
Keyword(s):  
Cell Proliferation ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Follicular Growth ◽  
Proliferating Cells ◽  
Progesterone Secretion ◽  
Proliferation And Differentiation ◽  
Igf I

Abstract Granulosa cells of ovarian follicles both proliferate and undergo differentiation. In vivo, an inverse relationship between proliferation and steroidogenesis is observed. However, both processes can be enhanced by insulin-like growth factor-I (IGF-I) in vitro. Studies were undertaken in the ewe to understand the mechanisms controlling the balance between proliferation and differentiation in cultured granulosa cells from antral follicles better. For this purpose, granulosa cells from ovine small follicles (1–3 mm in diameter) and large follicles (5–7 mm in diameter) were compared for progesterone secretion, cytochrome P450 side-chain cleavage (P450scc) expression and their proportions of non-proliferating (G0) cells, in response to IGF-I and FSH stimulation in vitro. IGF-I mainly enhanced the proliferation of granulosa cells from small follicles but it strongly increased progesterone secretion and P450scc expression in granulosa cells from large follicles, in synergy with FSH. Blocking granulosa cell proliferation by the administration of colcemid or aphidicolin had no effect or a weak stimulating effect on progesterone secretion. At the beginning of the culture period, the proportion of non-proliferating cells, estimated by continuous [3H]thymidine labelling experiments, was clearly higher in large than in small follicles (91% vs 30%, P<0·001). For both cell types, treatment with IGF-I in vitro reduced the proportion of non-proliferating cells at 72 h of culture (40% vs 70% respectively in IGF-I-stimulated and unstimulated cells from large follicles, P<0·001, and 17% vs 30% respectively in IGF-I-stimulated and unstimulated cells from small follicles, P<0·001). Treatment with FSH had no effect on the proportion of non-proliferating cells. As revealed by immunohistochemistry experiments, IGF-I, in synergy with FSH, clearly increased the percentage of cells expressing P450scc enzyme and the intensity of staining in granulosa cells from large follicles. Unexpectedly, heavily stained cells in mitosis were observed in IGF-I-stimulated cells from large follicles after 96 h of culture, suggesting that dividing cells might also produce progesterone. Overall, these results support the hypothesis that the growth-promoting and the cytodifferentiative effects of IGF-I are clearly distinct. Moreover, they suggest that uncoupling between proliferation and steroidogenesis may occur in cultured ovine granulosa cells. The loss of proliferative activity accompanying terminal follicular growth in vivo could be reversed in vitro. During terminal follicular growth in vivo, the existence of an active mechanism inhibiting granulosa cell proliferation, and unrelated to terminal differentiation, is therefore strongly suspected. Journal of Endocrinology (1994) 142, 497–510

Modulation of differentiation of rat granulosa cells in vitro by interferon-γ

1992 ◽  
Vol 133 (1) ◽  
pp. 131-139 ◽  
Author(s):  
S. Xiao ◽  
J. K. Findlay
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Cell Function ◽  
Inhibitory Effect ◽  
Interferon Γ ◽  
Detectable Effect ◽  
Aromatase Activity ◽  
Dependent Manner ◽  
Ifn Γ

ABSTRACT The effects of recombinant rat interferon-γ (rRaIFN-γ) and rat IFN (RaIFN, a mixture of IFN-γ and -α) on basal and FSH-induced ovarian granulosa cell function were studied. Granulosa cells were harvested from diethylstilboestrol-treated immature rats and cultured (2 × 105 viable cells/well per 0·5 ml) in serumfree medium with or without treatment for 48 h. In the presence of FSH (20 ng/ml), rRaIFN-γ (10–1000 U/ml) significantly inhibited FSH-stimulated aromatase activity (76·4 ± 2·3% maximum inhibition compared with FSH treatment alone), inhibin (40·4 ± 3·7%), progesterone (47·7 ± 8·6%) and 20α-hydroxypregn-4-en-3-one (20α-OHP) (51·8±1·7%) production in a dose-dependent manner. Furthermore, rRaIFN-γ inhibited FSH- and forskolin (FSK; 30 μmol/l)-induced extracellular cAMP accumulation (46·0 ± 6·6% and 29·1 ± 7·3% respectively). The inhibitory effect of rRaIFN-γ on FSK-induced cAMP was accompanied by decreased FSK-induced aromatase activity, inhibin, progesterone and 20α-OHP production. rRaIFN-γ had no detectable effect on aromatase activity, progesterone production and 20α-OHP production in the absence of FSH, but significantly stimulated basal inhibin production by 1·5-fold. rRaIFN-γ alone also caused a small but significant increase in basal levels of cAMP. The timecourse studies showed that FSH-induced aromatase activity and inhibin production were consistently suppressed by rRaIFN-γ, FSH-induced progesterone and 20α-OHP were inhibited at 1 and 2 days and then stimulated on days 3, 4 and 5 relative to FSH alone. There was no difference in DNA content between treatment and non-treatment wells during 5 days of culture. RaIFN had similar effects to rRaIFN-γ. We conclude that IFN-γ can inhibit FSH-induced granulosa cell differentiation and that, in the absence of FSH, IFN-γ stimulated undifferentiated granulosa cells to produce more inhibin. The mechanism of its action is likely to involve changes in cAMP production. Journal of Endocrinology (1992) 133, 131–139

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