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.

Effects of growth hormone and triiodothyronine on insulin-induced progesterone production by granulosa cells from prepubertal gilts

1992 ◽  
Vol 72 (3) ◽  
pp. 589-593 ◽  
Author(s):  
R. N. Kirkwood ◽  
P. A. Thacker ◽  
K. Rajkumar
Keyword(s):  
Growth Hormone ◽  
Granulosa Cells ◽  
Culture Medium ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Bovine Insulin ◽  
Dependent Manner ◽  
Progesterone Production ◽  
Porcine Granulosa Cells

Two experiments were performed using granulosa cells from medium-sized follicles (2–4 mm) derived from prepubertal gilts. Cells were cultured in a serum-free medium at a density of either 1 or 2 × 106 viable cells per well (experiments 1 and 2, respectively). For exp. 1, porcine growth hormone (pGH) (0 or 100 ng mL−1) was included in the culture medium from the time of plating, and low-density lipoprotein (LDL) (100 μg mL−1) was added at 72 h. For exp. 2, granulosa cells were plated in a culture medium containing either pGH (0 or 100 ng mL−1) or triiodothyronine (T3) (0 or 5 ng mL−1) or both pGH T3; LDL was not included. For both experiments, after 24 h of culture, bovine insulin at 0, 10, 100 or 1000 ng mL−1 was included in the medium. Hormones were replaced at 48 and 72 h, and the cultures were terminated at 96 h. Results from exp. 1 indicated that insulin increased (P < 0.01) progesterone production in a dose-dependent manner, both in the presence and absence of LDL. This response was augmented (P < 0.01) by co-culture with pGH. Results from exp. 2 confirmed the augmenting effect of pGH (P < 0.01). It was further observed that T3 increased (P < 0.01) progesterone production when cultured with insulin at 1000 ng mL−1, but at lower insulin-inclusion levels, results were equivocal. The progesterone production response was greatest (P < 0.01) when cells were cultured with both pGH and T3 at insulin levels of 100 or 1000 ng mL−1. There appeared to be little relationship between the media concentrations of insulin-like growth factor 1 and progesterone. The present results suggest that relatively high levels of pGH and T3 will enhance the in vitro steroidogenic capabilities of porcine granulosa cells. Key words: Granulosa cells, GH, T3, insulin

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.

scholarly journals Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1-Mediated Autophagy in Human Granulosa Cells as an Alternative of Programmed Cell Death

Endocrinology ◽  
2006 ◽  
Vol 147 (8) ◽  
pp. 3851-3860 ◽  
Author(s):  
Nicole Duerrschmidt ◽  
Olga Zabirnyk ◽  
Marcin Nowicki ◽  
Albert Ricken ◽  
Fayez A. Hmeidan ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density ◽  
Dependent Manner ◽  
Oxidized Low Density Lipoprotein ◽  
Time Point

The LOX-1 receptor, identified on endothelial cells, mediates the uptake of oxidized low-density lipoprotein (oxLDL). The oxLDL-dependent LOX-1 activation causes endothelial cell apoptosis. We here investigated the presence of LOX-1 in granulosa cells from patients under in vitro fertilization therapy. We were interested in the oxLDL-dependent LOX-1 receptor biology, in particular in the induction of apoptosis. In the human ovary, LOX-1 was localized in regressing antral follicles. In granulosa cell cultures, oxLDL-induced mRNA expression of LOX-1 in a time- and dose-dependent manner. The LOX-1 inhibitors (anti-LOX-1 antibody and κ-carrageenan) abrogated the up-regulation of LOX-1. The oxLDL (100 μg/ml) treatment caused the autophagy form of programmed cell death: 1) reorganization of the actin cytoskeleton at the 6-h time point; 2) uptake of YO-PRO, a marker for the early step of programmed cell death, before propidium iodide staining to signify necrosis; 3) absence of apoptotic bodies and cleaved caspase-3; 4) abundant vacuole formation at the ultrastructural level; and 5) decrease of the autophagosome marker protein MAP LC3-I at the 6-h time point indicative of autophagosome formation. We conclude that follicular atresia is not under the exclusive control of apoptosis. The LOX-1-dependent autophagy represents an alternate form of programmed cell death. Obese women with high blood levels of oxLDL may display an increased rate of autophagic granulosa cell death.

Evidence for differences in low density lipoprotein processing by porcine granulosa and luteal cells in vitro: effect of addition of serum for plating of granulosa cells on lipoprotein metabolism

1989 ◽  
Vol 122 (2) ◽  
pp. 557-564 ◽  
Author(s):  
K. Rajkumar ◽  
H. Ly ◽  
P. W. Schott ◽  
B. Njaa ◽  
B. D. Murphy
Keyword(s):  
Granulosa Cells ◽  
Time Course ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density ◽  
Luteal Cells ◽  
Serum Withdrawal ◽  
Progesterone Secretion ◽  
Vitro Effect

ABSTRACT The present studies were carried out to compare the low density lipoprotein (LDL) metabolism by freshly isolated immature porcine granulosa cells with that by luteal cells. Furthermore, we have examined the effect of serum used for plating of granulosa cells on lipoprotein degradation and utilization. In incubation studies, addition of LDL as an exogenous substrate had a mild stimulatory effect on progesterone accumulation by granulosa cells, while it exhibited a dose-dependent stimulatory effect on luteal cells. When granulosa and luteal cells were incubated with 125I-labelled LDL, membrane binding of LDL occurred in both cell types, but only luteal cells were capable of internalizing the bound LDL. Granulosa cells in incubation degraded LDL much less in comparison with luteal cells, and the amount varied with the maturity of the cells. When granulosa cells were plated with graded amounts of serum which was withdrawn for 48 h following plating, they exhibited enhanced LDL degradation in a serum concentration-dependent fashion. Addition of serum for plating selectively enhanced utilization of LDL, but not high density lipoprotein (HDL) for progesterone accumulation by the cells in culture. Time-course studies on LDL degradation by granulosa cells following serum withdrawal indicate that the ability of cells to degrade LDL decreased in a time-dependent fashion. Serum withdrawal selectively decreased utilization of LDL but not HDL for progesterone secretion. It is concluded that immature granulosa cells have a limited capability to utilize cholesterol carried by LDL. However, when cultured in the presence of serum, cells acquire the ability to utilize more efficiently LDL-carried cholesterol for progesterone secretion which is then lost following long-term withdrawal of serum from culture. Journal of Endocrinology (1989) 122, 557–564

scholarly journals Cholesterol requirement of P3-X63-Ag8 and X63-Ag8.653 mouse myeloma cells for growth in vitro.

1987 ◽  
Vol 165 (6) ◽  
pp. 1761-1766 ◽  
Author(s):  
J D Sato ◽  
T Kawamoto ◽  
T Okamoto
Keyword(s):  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density ◽  
Myeloma Cells ◽  
Serum Free ◽  
Low Concentrations ◽  
Absolute Requirement ◽  
Serum Free Conditions ◽  
Mouse Myeloma

P3-X63-Ag8 and X63-Ag8.653 mouse myeloma cells have an absolute requirement for cholesterol for growth under serum-free conditions. This requirement can be satisfied by low density lipoprotein at 2-6 micrograms/ml or by BSA-bound cholesterol at 5-10 micrograms/ml. Cholesterol-independent variants can be selected after prolonged growth in low concentrations of serum.

scholarly journals Expression of Scavenger Receptor-BI and Low-Density Lipoprotein Receptor and Differential Use of Lipoproteins to Support Early Steroidogenesis in Luteinizing Macaque Granulosa Cells

Endocrinology ◽  
2009 ◽  
Vol 150 (2) ◽  
pp. 957-965 ◽  
Author(s):  
Mary Cherian-Shaw ◽  
Muraly Puttabyatappa ◽  
Erin Greason ◽  
Annabelle Rodriguez ◽  
Catherine A. VandeVoort ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Low Density Lipoprotein ◽  
Scavenger Receptor ◽  
Density Lipoprotein ◽  
Low Density ◽  
Lipoprotein Receptor ◽  
Low Density Lipoprotein Receptor ◽  
Scavenger Receptor Bi ◽  
Progesterone Synthesis ◽  
Density Lipoprotein Receptor

An ovulatory hCG stimulus to rhesus macaques undergoing controlled ovarian stimulation protocols results in a rapid and sustained increase in progesterone synthesis. The use of lipoproteins as a substrate for progesterone synthesis remains unclear, and the expression of lipoprotein receptors [very-low-density lipoprotein receptor (VLDLR), low-density lipoprotein receptor (LDLR), and scavenger receptor-BI (SR-BI)] soon after human chorionic gonadotropin (hCG) (&lt;12 h) has not been characterized. This study investigated lipoprotein receptor expression and lipoprotein (VLDL, LDL, and HDL) support of steroidogenesis during luteinization of macaque granulosa cells. Granulosa cells were aspirated from rhesus monkeys undergoing controlled ovarian stimulation before or up to 24 h after an ovulatory hCG stimulus. The expression of VLDLR decreased within 3 h of hCG, whereas LDLR and SR-BI increased at 3 and 12 h, respectively. Granulosa cells isolated before hCG were cultured for 24 h in the presence of FSH or FSH plus hCG with or without VLDL, LDL, or HDL. Progesterone levels increased in the presence of hCG regardless of lipoprotein addition, although LDL, but not HDL, further augmented hCG-induced progesterone. Other cells were cultured with FSH or FSH plus hCG without an exogenous source of lipoprotein for 24 h, followed by an additional 24 h culture with or without lipoproteins. Cells treated with hCG in the absence of any lipoprotein were unable to maintain progesterone levels through 48 h, whereas LDL (but not HDL) sustained progesterone synthesis. These data suggest that an ovulatory stimulus rapidly mobilizes stored cholesterol esters for use as a progesterone substrate and that as these are depleted, new cholesterol esters are obtained through an LDLR- and/or SR-BI-mediated mechanism. An ovulatory stimulus to primate granulosa cells increases the expression of low-density lipoprotein (LDL) receptor and scavenger receptor class B, type I, while LDL serves as the primary substrate for progesterone synthesis.

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