Dose response of luteinized porcine granulosa cells in vitro to prolactin: dependency on pre-exposure to human chorionic gonadotrophin

1988 ◽  
Vol 66 (10) ◽  
pp. 1337-1340 ◽  
Author(s):  
Pedro J. Chedrese ◽  
Kadaba Rajkumar ◽  
Hoa Ly ◽  
Bruce D. Murphy
Keyword(s):  
Granulosa Cells ◽  
Calf Serum ◽  
Density Lipoprotein ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Cells In Culture ◽  
The Media ◽  
Free Media ◽  
Dose Dependent

This study examined the hypothesis that human chorionic gonadotrophin (hCG) increases prolactin (PRL) stimulation of the utilization of lipoprotein-borne cholesterol by pig luteinized granulosa cells in culture. These cells, which luteinize in culture, were harvested from 6-mm or greater diameter follicles and cultured in the presence of 1% fetal calf serum and 1 μ/mL insulin for 48 h. On the third day, the media were replaced with fresh serum-free media, with the same dose of insulin, and on the following day (day 4) the media were replaced with serum- and insulin-free media. At this time (day 4) hCG was added to some cultures. On day 5, cells from the group with hCG and cells from the group without hCG were treated with graded doses of ovine PRL (0.1–3.0 μg/mL). To a second set of cells, likewise treated, 100 μg of porcine low density lipoprotein (LDL) was added. Two days later (day 7) media were sampled and replaced with media alone or media containing hormones and (or) LDL. On day 9 cultures were terminated. In the cells pre-exposed to hCG, PRL (1 μg/mL) in the presence of LDL increased progesterone production 1.7-fold (p < 0.01) on day 7 and 2.2-fold (p < 0.01) on day 9. In the granulosa cells in culture pre-exposed to hCG, the effect of PRL on LDL utilization was dose dependent and saturable at 1 μg/mL on days 7 and 9. We conclude that brief pretreatment of luteinized pig granulosa cells with hCG results in a dose-dependent PRL-induced utilization of LDL for progesterone synthesis.

scholarly journals The effect of human chorionic gonadotrophin on the expression of progesterone receptors in human luteal cells in vivo and in vitro

Reproduction ◽  
2005 ◽  
Vol 130 (1) ◽  
pp. 83-93 ◽  
Author(s):  
W Colin Duncan ◽  
Eva Gay ◽  
Jacqueline A Maybin
Keyword(s):  
Granulosa Cells ◽  
Luteal Phase ◽  
Progesterone Receptors ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Progesterone Secretion ◽  
Late Luteal Phase ◽  
Lutein Cell

The human corpus luteum expresses genomic progesterone receptors (PRs) suggesting that progesterone may have an autocrine or paracrine role in luteal function. We hypothesised that the reduction in luteal PR reported in the late-luteal phase augmented progesterone withdrawal and had a role in luteolysis. We therefore tested the hypothesis that luteal rescue with human chorionic gonadotrophin (hCG) would maintain PR expression. PR was immunolocalised to different cell types in human corpora lutea (n = 35) from different stages of the luteal phase and after luteal rescue with exogenous hCG. There was no change in the staining intensity of theca-lutein cell or stromal cell PR throughout the luteal phase or after luteal rescue. In the late-luteal phase, granulosa-lutein cell PR immunostaining was reduced (P < 0.05) but the trend to reduction was also seen after luteal rescue with hCG (P = 0.055). To further investigate the effect of hCG on granulosa-lutein cell PR expression, an in vitro model system of cultured human luteinised granulosa cells was studied. Cells were cultured for 12–13 days exposed to different patterns of hCG and aminoglutethamide to manipulate progesterone secretion (P < 0.0001). Expression of PR A/B and PR B isoforms was examined by quantitative real-time RT-PCR. PR A/B mRNA was lower (P < 0.05) after 11–13 days of culture than after 7 days of culture. This reduction could not be prevented by hCG in the presence (P < 0.05) or absence (P < 0.05) of stimulated progesterone secretion. The expression of PR B mRNA showed a similar pattern (P = 0.054). Simulated early pregnancy in vivo and hCG treatment of luteinised granulosa cells in vitro did not appear to prevent the down-regulation of PR seen during luteolysis.

Control of inhibin production by dispersed human luteal cells in vitro

1992 ◽  
Vol 4 (1) ◽  
pp. 67 ◽  
Author(s):  
HZ Wang ◽  
SH Lu ◽  
XJ Han ◽  
ZD Sun ◽  
WX Shen ◽  
...  
Keyword(s):  
Sex Steroids ◽  
Follicle Stimulating Hormone ◽  
Calf Serum ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Corpora Lutea ◽  
Luteal Cells ◽  
Oestradiol Production ◽  
Human Corpus Luteum

The production of inhibin in vitro by dispersed cells from early to mid (Days 16-19) and late stage (Day 23) human corpus luteum (CL) was examined, and the effects of human chorionic gonadotrophin (hCG), follicle stimulating hormone (FSH), oestradiol and testosterone on inhibin production were determined. Corpora lutea from five subjects in the early to mid luteal stage and three subjects in late luteal stage were dispersed with enzymes and the luteal cells cultured in medium supplemented with 5% calf serum and either FSH (1, 10 or 100 ng mL-1), oestradiol-17 beta (2.5, 5 or 10 micrograms mL-1) or testosterone (0.25, 1 or 5 micrograms mL-1) with or without hCG (1 I.U. mL-1). Cells were cultured for 1 to 3 days without changes of medium, and the concentrations of progesterone, oestradiol and immunoreactive inhibin in the medium were measured by radioimmunoassay. Cells from both types of CL produced inhibin in vitro under basal conditions, but only cells from early to mid CLs responded to hCG with a significant increase in inhibin production. Both progesterone and oestradiol production were stimulated by hCG in both groups of CL. Inhibin concentrations in the cell cultures declined with time in culture, particularly in the late CL group, whereas the concentration of steroids increased. Neither FSH, oestradiol nor testosterone significantly changed inhibin production in either CL group. It was concluded that inhibin production by human luteal cells in vitro is influenced by the age of the CL, and is dependent on LH (hCG) but not on FSH or sex steroids.

Effect of prolactin and cyclic AMP on 125I-labelled low density lipoprotein uptake and metabolism by luteinized porcine granulosa cells in culture

1988 ◽  
Vol 66 (11) ◽  
pp. 1450-1454 ◽  
Author(s):  
Kadaba Rajkumar ◽  
Hoa Ly ◽  
Pedro J. Chedrese ◽  
Bruce D. Murphy
Keyword(s):  
Granulosa Cells ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density ◽  
Surface Binding ◽  
Cells In Culture ◽  
Porcine Granulosa Cells ◽  
Lipoprotein Uptake ◽  
Free Media ◽  
Uptake And Metabolism

The present study examines the effect of prolactin (PRL) and N6-21-O-dibutyryladenosine 3′5′-cyclic monophosphate (cAMP) on low density lipoprotein (LDL) uptake and metabolism by luteinized porcine granulosa cells in culture. Granulosa cells from preovulatory follicles were plated with 1% serum and 1 μg/mL of insulin for the first 48 h. Following plating (day 3) the cells were cultured in serum-free media with the same dose of insulin. The next day the medium was replaced with serum- and insulin-free medium, and to some cultures 1.23 IU/mL of human chorionic gonadotropin (hCG) was added. On day 5 the medium was again replaced and graded amounts of PRL (0, 0.03, 0.3, and 3 μg/mL) were added. Following 48 h of incubation with PRL, 20 μg/mL of 125I-labelled LDL was added to cultures. Surface-bound, internalized, and degraded LDLs were quantitated at 12 h following addition of LDL. To examine the effect of cAMP on LDL metabolism, the cells were exposed for 24 h to cAMP (3 mM) on day 6 of culture. PRL had a stimulatory effect on LDL degradation by luteinized granulosa cells. Pre-exposure of cells to hCG augmented the stimulatory effect of PRL. Addition of cAMP also enhanced LDL degradation by luteinized granulosa cells. Both PRL and cAMP increased surface binding of LDL in cells pre-exposed to hCG, but there was no effect on internalization. The increase in cell surface binding of LDL with PRL and cAMP was less than their effect on LDL degradation. It is concluded that PRL and cAMP enhance LDL metabolism in luteinized porcine granulosa cells by increasing surface binding of LDL and by a post-receptor mechanism. PRL may play a role in the supply of LDL-carried cholesterol for steroidogenesis by luteinized granulosa cells.

Effects of growth hormone on FSH, insulin and triiodothyronine-mediated estradiol production by granulosa cells from prepubertal gilts in vitro

1993 ◽  
Vol 73 (2) ◽  
pp. 443-447 ◽  
Author(s):  
K. Rajkumar ◽  
R. N. Kirkwood ◽  
P. A. Thacker
Keyword(s):  
Growth Hormone ◽  
Granulosa Cells ◽  
Serum Free Medium ◽  
Free Medium ◽  
Viable Cells ◽  
Porcine Granulosa Cells ◽  
The Media ◽  
Dose Dependent ◽  
Production Response

Three experiments were performed using porcine granulosa cells from medium-sized follicles (2–4 mm) cultured in serum-free medium at a density of 2 × 106 viable cells per well. Cells were cultured in the presence of combinations of FSH, insulin, growth hormone (GH) and triiodothyronine (T3), and subsequent estradiol (E2) production was monitored. In the presence of insulin, a dose-dependent E2 production response to FSH was observed (exp. 1). However, in all experiments, E2 production was reduced (P < 0.05) by the inclusion of GH in the media. The inclusion of T3 augmented the E2 response to insulin, an effect attenuated by GH. We conclude that GH, at the dose employed, is inhibitory to E2 production in granulosa cells of medium-sized ovarian follicles. Key words: Granulosa cells, growth hormone, estradiol

Thyroid hormone as a regulator of basal and human chorionic gonadotrophin-stimulated steroidogenesis by cultured porcine theca and granulosa cells isolated at different stages of the follicular phase

1996 ◽  
Vol 8 (6) ◽  
pp. 961 ◽  
Author(s):  
EL Gregoraszczuk ◽  
M Skalka
Keyword(s):  
Thyroid Hormone ◽  
Granulosa Cells ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Follicular Cells ◽  
Steroid Secretion ◽  
Follicle Size ◽  
Androgen Secretion

To characterize thyroid hormone action in the ovary, the direct effect of triiodothyronine (T3) was investigated in vitro using a culture system of porcine theca cells (Tcs) and granulosa cells (Gcs) in mono- and co-culture (GT), the latter resembling follicles in vivo. The cells were cultured in the absence or presence of human chorionic gonadotrophin (hCG) with or without T3 (10(-7), 10(-9) or 10(-11) M). Follicular cells were obtained from follicles of different size (small, medium and large), and steroid secretion into the culture medium was detected by radioimmunoassay. T3 alone did not influence steroid secretion by Tcs and Gcs isolated from follicles that were small and medium in size. In preovulatory follicles, an increase in basal androgen secretion and a simultaneous decrease in oestradiol secretion were observed with Tcs and Gcs in both mono- and co-culture. T3 together with hCG decreased hCG-stimulated androgen secretion in Tcs isolated from medium-sized follicles and had a simultaneous stimulatory effect on hCG-stimulated oestradiol secretion by Gcs. In cultures of follicular cells obtained from large follicles, T3 decreased hCG-stimulated secretion of both androgen and oestrogen by Tcs and simultaneously stimulated oestradiol secretion in GT co-cultures. Thus, the interaction of T3 with gonadotrophin hormone modulated follicular steroidogenesis, depending on follicle size and cell type used in culture. The observed T3-induced increase in basal androgen secretion by Tcs could account for the atresia of follicles, since it is accompanied by a decrease in oestradiol secretion in GT co-culture. In its co-activity with hCG, an adequate level of T3 prevents excessive androgen production by Tcs, probably influencing aromatization processes in the follicle. An increase in hCG-stimulated oestradiol secretion in GT co-cultures is then observed. Further investigations are required to clarify whether this is linked with an effect on the aromatization processes occurring in the follicle.

scholarly journals Regulation of adiponectin and its receptors in rat ovary by human chorionic gonadotrophin treatment and potential involvement of adiponectin in granulosa cell steroidogenesis

Reproduction ◽  
2007 ◽  
Vol 133 (4) ◽  
pp. 719-731 ◽  
Author(s):  
Christine Chabrolle ◽  
Lucie Tosca ◽  
Joélle Dupont
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Protein Levels ◽  
Cellular Expression ◽  
Adiponectin Mrna ◽  
Igf I ◽  
Rat Ovary

In mammals, adiponectin and its receptors (AdipoR1 and AdipoR2) mRNAs are expressed in various tissues. However, the cellular expression and the role of adiponectin system have never been investigated in rat ovary. Here, we report the presence of adiponectin, AdipoR1 and AdipoR2 in rat ovaries, and we have investigated its role in granulosa cells. Using RT-PCR and western blot, we show that the mRNAs and proteins for adiponectin, AdipoR1 and AdipoR2 are found in the ovaries. Immunohistochemistry localized adiponectin, AdipoR1 and AdipoR2 in theca-interstitial T-I cells, corpus luteum, oocyte and less abundantly in granulosa cells. In the KGN human granulosa cell line, adiponectin mRNA and protein were undetectable; AdipoR2 was weakly expressed, whereas AdipoR1 was clearly present. Human chorionic gonadotrophin (hCG) injection (48 h) after pregnant mare serum gonadotrophin (PMSG) injection (24 h) in immature rats increased the level of adiponectin (protein) by about threefold (P< 0.05) and those of AdipoR1 by threefold (mRNA,P< 0.05) and 1.5-fold (protein,P< 0.05) in ovary, whereas the mRNA and protein levels of AdipoR2 were unchanged. Interestingly, hCG injection (48 h) after the PMSG treatment (24 h) decreased plasma adiponectin levels and increased insulin plasma levels.In vitroin primary rat granulosa cells, human adiponectin recombinant (5 μg/ml) in the presence or absence of follicle-stimulating hormone (10−8M, 48 h) had no effect on the steroidogenesis. However, it increased progesterone secretion (P< 0.05) by about twofold and oestradiol production (P< 0.05) by about 1.6-fold in response to insulin-like growth factor-I (IGF-I) (10−8M). Furthermore, it improved IGF-I-induced IGF-I receptor-β subunit tyrosine phosphorylation and ERK1/2 phosphorylation. In basal state, human adiponectin recombinant also increased rapidly but transiently the ERK1/2, p38 and Akt phosphorylations, whereas it increased more lately the adenosine 5′-monophosphate-activated protein kinase (AMPK) phosphorylation. Thus, AdipoR1 and AdipoR2 are regulated by hCG treatment in rat ovary and adiponectin enhances IGF-I-induced steroidogenesis in granulosa cells.

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