Influence of high-density lipoprotein on estradiol stimulation of luteal steroidogenesis

The aim of this investigation was to determine whether luteal cells utilize cholesterol derived from high-density lipoprotein (HDL) for steroidogenesis and whether estrogen enhances luteal utilization of exogenous sterol. Incubation of Day 15 corpora lutea (CL) with different doses of human HDL resulted in a dose-dependent increase in progesterone production. HDL in vitro enhanced the overall steroidogenic capacity. However, the percentage of increases in 17 alpha-hydroxyprogesterone, testosterone and estradiol were significantly less than that of progesterone. Day 12 hypophysectomized and hysterectomized pregnant rats were treated with either estradiol, testosterone or vehicle for 72 h. Serum pregnenolone and progesterone were markedly increased by the steroid treatment, yet in vitro production of progesterone by CL in all the groups was similar. However, in the presence of HDL in the media, only luteal tissues from steroid-treated rats increased their progesterone output. The reduced production of progesterone by luteal cells of vehicle-treated rats was not due to an accumulation of pregnenolone but to an overall reduction in exogenous sterol utilization. In summary, results of this investigation suggest 1) luteal cells of pregnant rats effectively utilize cholesterol from HDL for maximal steroidogenesis, and 2) estradiol may stimulate luteal steroidogenesis, at least in part, by affecting the incorporation or utilization of cholesterol from HDL into the cell.     

Regulation of steroidogenesis and cholesterol synthesis by prostaglandin F-2 alpha and lipoproteins in bovine luteal cells

Bovine luteal cells can utilize low density lipoprotein (LDL) or high density lipoprotein (HDL) as a source of cholesterol for steroidogenesis, and administration of PGF-2 alpha in vitro suppresses lipoprotein utilization. The objective of this study was to examine the mechanism by which PGF-2 alpha exerts this effect. Cultured bovine luteal cells received 0.25 microCi[14C]acetate/ml, to assess rates of de-novo sterol and steroid synthesis, with or without lipoproteins. Both LDL and HDL enhanced progesterone production (P less than 0.01), but caused a significant reduction in the amount of radioactivity in the cholesterol fraction. PGF-2 alpha treatment inhibited the increase in lipoprotein-induced progesterone synthesis (P less than 0.01), but did not prevent the reduction in de-novo cholesterol synthesis brought about by LDL or HDL. PGF-2 alpha alone reduced cholesterol synthesis (P less than 0.01), but it was not as effective as either LDL or HDL. Both lipoproteins and PGF-2 alpha also decreased the amount of radioactivity in the progesterone fraction (P less than 0.01), and the effect of PGF-2 alpha was similar to that of the lipoproteins. It is concluded that lipoproteins can enhance progesterone production and also suppress de-novo cholesterol synthesis in bovine luteal cells, but only the former effect of lipoproteins is inhibited by PGF-2 alpha. Therefore, it is suggested that PGF-2 alpha allows entry of lipoprotein cholesterol into the cell, but prevents utilization for steroidogenesis. In addition, PGF-2 alpha alone can suppress cholesterol synthesis, as well as decrease conversion of cholesterol to progesterone.     

Differential uptake and metabolism of free and esterified cholesterol from high-density lipoproteins in the ovary

Rat luteal cells utilize high-density lipoproteins (HDL) as a source of cholesterol for steroid synthesis. Both the free and esterified cholesterol of HDL are utilized by these cells. In this report, we have examined the relative uptake of free and esterified cholesterol of HDL by cultured rat luteal cells. Incubation of the cells with HDL labeled with [3H]cholesterol or [3H]cholesteryl linoleate resulted in 4-6-fold greater uptake of the free cholesterol compared to esterified cholesterol. The increased uptake of free cholesterol correlated with its utilization for progestin synthesis: utilization of HDL-derived free cholesterol was 3-6-fold higher than would be expected from its concentration in HDL. The differential uptake and utilization of free and esterified cholesterol was further examined using egg phosphatidylcholine liposomes containing cholesterol or cholesteryl linoleate as a probe. Liposomes containing free cholesterol were able to deliver cholesterol to luteal cells and support steroid synthesis in the absence of apolipoproteins, and the addition of apolipoprotein A-I (apo A-I) moderately increased the uptake and steroidogenesis. Similar experiments using cholesteryl linoleate/egg phosphatidylcholine liposomes showed that inclusion of apo A-I resulted in a pronounced increase in the uptake of cholesteryl linoleate and progestin synthesis. These experiments suggest that free cholesterol from HDL may be taken up by receptor-dependent and receptor-independent processes, whereas esterified cholesterol uptake requires a receptor-dependent process mediated by apolipoproteins.     

Regulation of the corpus luteum by protein kinase C. II. Inhibition of lipoprotein-stimulated steroidogenesis by prostaglandin F2 alpha

This study was designed to examine the antisteroidogenic action of prostaglandin (PG) F2 alpha on ovine luteal cells in vitro. Purified populations of large and small steroidogenic luteal cells were treated with lipoproteins, luteinizing hormone (LH), and/or PGF2 alpha. To investigate the involvement of the protein kinase C (PKC) pathway in hormone action, luteal cells were made PKC-deficient by treatment for 12 h with 1 microM phorbol-12-myristate-13-acetate. Progesterone production by nonstimulated large and LH-stimulated small luteal cells was significantly increased by treatment with high- and low-density lipoprotein (HDL, 5-fold increase; LDL, 2-fold increase). PGF2 alpha inhibited (p less than 0.0001) progesterone production by HDL-stimulated large luteal cells in a dose-dependent manner, with 60 nM causing maximal inhibition. No effect of PGF2 alpha (20nM-20 microM) was found on production of progesterone by HDL-stimulated, PKC-deficient large cells or by LH- and HDL-stimulated small luteal cells. These results suggest that PGF2 alpha has a direct antisteroidogenic effect on the large luteal cell that is mediated through the PKC second messenger pathway.     

Lipoprotein-stimulated and estradiol-maintained progesterone secretion by dissociated rabbit luteal cells in vitro

Elevated activity of 3-hydroxy-3-methyglutaryl coenzyme A reductase (HMG-CoA reductase) was observed in the rabbit ovary and corpus luteum during pregnancy. Based on this study, it was proposed that de novo cholesterol synthesis rather than the uptake of exogenous plasma cholesterol (lipoproteins) was of primary importance in providing steroid substrate for progesterone synthesis by the rabbit luteal cell. Using a perifusion system, the present study challenges this hypothesis by demonstrating that both low- and high-density lipoproteins (at protein concentrations of 100 micrograms/ml and 50 micrograms/ml, respectively) were able to acutely stimulate progesterone production by dissociated rabbit luteal cells. The increase in progesterone synthesis was due to increased cholesterol substrate and not to protein-enhanced progesterone release. The ability of luteal cells to respond to lipoproteins was dependent on both dose- and sequence of treatment, with high-density lipoprotein (HDL) being unable to stimulate progesterone production if preceded by perifusion with low-density lipoprotein (LDL) or HDL. In addition, 17 beta-estradiol appeared to regulate lipoprotein utilization by attenuating the LDL response after 1 h of perifusion. We conclude that lipoproteins may provide cholesterol substrate for progesterone biosynthesis in vitro and that 17 beta-estradiol, in addition to maintaining progesterone production by luteal cells, may also regulate lipoprotein utilization. Thus, maintenance of steady progesterone secretion in response to estradiol supercedes that of LDL-stimulated progesterone secretion by rabbit luteal cells in vitro. This study suggests an interaction between estrogen and lipoproteins that may prove physiologically important in regulating progesterone production by rabbit luteal cells in vivo.     

Steroidogenic effects of lipoproteins and 25-hydroxycholesterol on luteal and ovarian cells: a comparison of two pseudopregnant rat models

Steroidogenesis was compared between luteal cells from immature pseudopregnant (PSP) rats induced by either 5 IU pregnant mare serum gonadotropin (PMSG) alone or 50 IU PMSG combined with 25 IU human chorionic gonadotropin (hCG). It was also determined whether differences in steroidogenesis existed when the entire ovary (ovarian cells) or just luteal cells from Day 4 PSP rats were exposed in vitro to lipoproteins or 25-hydroxycholesterol (25-OH chol). In the absence of luteinizing hormone (LH), basal steroid accumulation, especially progesterone (P4) was around fourfold greater in luteal cells from rats treated with PMSG alone than from rats receiving PMSG-hCG. However, serum P4 and LH were about fivefold greater in the latter group. It is therefore likely that net cellular cholesterol uptake per luteal cell is lower in the PMSG-hCG treated rats, but this is offset by a much greater mass and number of corpora lutea. Lipoproteins (HDL and LDL) and 25-OH chol stimulated in vitro luteal steroidogenesis from rats treated with PMSG alone or PMSG-hCG, and their responses were virtually identical. Therefore, luteal steroidogenesis in the rat always depends on exogenous cholesterol even though treatment in the preovulatory period with PMS or PMSG-hCG and serum LH and follicle-stimulating hormone (FSH) levels on Day 4 PSP are very different. When ovarian cells from PMSG-hCG treated rats were incubated with LH plus HDL or 25-OHP, the production of 20 alpha-DHP was considerably greater than luteal cell production which may be due to a contribution from nonluteal cells. Indeed, about 30% of the cells in the PMSG-hCG group represent nonluteal components as estimated by weight and deoxyribonucleic acid content.     

Luteal progesterone synthesis and high-density lipoprotein. Evidence implicating a pronase-sensitive HDL-binding site in the mechanism by which HDL supports luteal progesterone synthesis

We have shown previously that corpus luteum cells isolated from the superovulated ovaries of rats treated with 4-amino-pyrazolo[3,4-d]pyrimidine constitute a suitable experimental system by which to investigate the mechanism in which plasma high-density lipoprotein (HDL) plays a role in luteal cellular progesterone synthesis. In the present study, the rate of luteal cellular progesterone synthesis was shown to be stimulated by 125I-labelled HDL up to about 70% of the rate achieved in the presence of native HDL. The concentration of HDL needed for half-maximal stimulation of progesterone synthesis in the presence of lutropin was not significantly different irrespective of whether radioiodinated HDL or unlabelled HDL was used. Experimental conditions for studying the binding of 125I-labelled HDL to isolated luteal cells have been defined and cellular binding affinity and binding capacity have been measured. Exposure of the luteal cells to pronase virtually abolished their capacity to bind 125I-HDL and made them unable to respond to added HDL by increasing their rate of progesterone synthesis in the presence of lutropin. Control experiments showed this effect of pronase on cellular progesterone synthesis not to be due to destruction of cellular lutropin receptors, nor to general cellular damage. This evidence supports the view that luteal cellular binding of HDL is part of the mechanism by which HDL acts in luteal progesterone synthesis. Cellular binding capacity and affinity for 125I-labelled HDL were the same irrespective of whether or not lutropin was present during incubation. Furthermore, the binding capacity and affinity of cells from the ovaries of rats not treated with 4-amino-pyrazolo[3,4-d]pyrimidine were the same as in luteal cells isolated from rats that had been treated.     

Effects of high density lipoprotein containing high or low beta-carotene concentrations on progesterone production and beta-carotene uptake and depletion by bovine luteal cells

Luteal cells were isolated from mid-luteal heifer ovaries by collagenase digestion. Cells were cultured with DMEM/Ham's F12 medium in serum pre-treated plastic culture dishes for periods of up to 11 days. As beta-carotene is almost completely insoluble in all polar solvents, it was added to cultures in either dimethyl sulphoxide (DMSO), tetrahydrofuran (THF) or as high-density lipoprotein (HDL) containing high or low beta-carotene concentrations. Medium was replaced after 24 h, thereafter medium was changed every 48 h. Treatment of cells with DMSO alone or with beta-carotene (5 micromol/l) in DMSO both resulted in significant (P<0.01) stimulation of progesterone production. beta-Carotene (5 micromol/l) in THF did not alter progesterone production but 50 micromol/l beta-carotene in THF resulted in significant inhibition (P<0.02) of progesterone production on days 3 and 7. Cultures were also supplemented with bovine HDL preparations containing equal concentrations of cholesterol (25 microg/ml) but high or low beta-carotene (12.4 or 0.44 microg/mg of cholesterol). Both HDL preparations significantly stimulated progesterone production (P<0. 001) but the high beta-carotene HDL was significantly (P<0.02) more effective than the low beta-carotene HDL. However, when given together with bovine luteinizing hormone (bLH) or dibutyryl cAMP (dbcAMP), the high beta-carotene HDL stimulated progesterone production less than did the low HDL (P<0.01). Uptake and depletion of beta-carotene by luteal cells were also examined in culture. beta-Carotene supplementation increased luteal cell beta-carotene from an initial level of 373 ng per 10(6) cells to 2030 ng per 10(6) cells by day 6. In contrast, the levels in control cells decreased to 14% of starting values during the same period. Cells treated with HDL containing high beta-carotene on day 1 or days 1 and 3 were then incubated with or without bLH or dbcAMP for a further 2 days to investigate the effect of bLH and dbcAMP on depletion of beta-carotene by luteal cells. beta-Carotene depletion in the luteal cells was significantly higher (P<0.05) in LH- and dbcAMP-treated cells than in the control cells in both groups. These results indicate that the use of solvents such as DMSO or THF may have undesirable effects due to alteration of cell membrane permeability. Supplementation with bLH or dbcAMP may increase the metabolism of beta-carotene in luteal cells. bLH or dbcAMP together with high beta-carotene HDL may, when combined with the effect of increased beta-carotene metabolism, give less stimulation than with low beta-carotene HDL.     

In vitro differentiation of bovine theca and granulosa cells into small and large luteal-like cells: morphological and functional characteristics

This study was undertaken to investigate whether bovine granulosa and theca interna cells could be luteinized in vitro into luteal-like cells. Granulosa and theca cells were cultured for 9 days in the presence of forskolin (10 microM), insulin (2 micrograms/ml), insulin-like growth factor I (100 ng/ml), or a combination of these agents. During the first day of culture, granulosa and theca cells secreted estradiol and androstenedione, respectively; progesterone rose only after 3-5 days in culture and reached a maximum on the ninth day of culture. Cells incubated in the presence of forskolin plus insulin exhibited morphological and functional characteristics of luteal cells isolated from the corpus luteum. It was found that cell diameter, basal and stimulated progesterone secretion, and pattern of cell replication for both cell types were comparable to those of luteal cells. Numerous lipid droplets and intensified mitochondrial adrenodoxin staining also indicated active steroidogenesis in luteinized cells. After 9 days in culture, stimulants were withdrawn, and the culture proceeded in basal medium for an additional 5 days; elevated progesterone levels were maintained by luteinized granulosa cells (LGC), whereas in contrast a dramatic drop in progesterone production was observed in luteinized theca cells (LTC). On Day 9, cells were challenged for 3 h with LH (10 ng/ml), forskolin (10 microM), or cholera toxin (100 ng/ml), resulting in a 4-fold increase in progesterone secretion by LTC; the same treatments failed to stimulate progesterone in LGC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipoprotein augmentation of human chorionic gonadotropin and prolactin stimulated progesterone synthesis by rat luteal cells

A collagenase dispersed cell suspension from PMSG-hCG primed immature rats responded to exogenously added hCG, cholera enteroxin, prolactin, and 8-Bromocyclic-AMP with increase in progesterone production in a dose dependent manner, and this stimulation was augmented by the plasma lipoprotein fractions hHDL and hLDL. The responsiveness to low doses of prolactin was not apparent when lipoprotein fractions were not included in the assay mixture. When the incubation mixture contained either LDL or HDL, the stimulatory effect of prolactin on progesterone production was evident at 5 and 10 micrograms prolactin/ml of the incubation mixture. Progesterone production, both basal and hormone stimulated, was maximum on day 7 of pseudopregnancy. Although the extent of hCG and prolactin stimulation of progesterone production and its potentiation by lipoprotein fractions was observed to be higher on days 3 and 5 than that seen on day 7, the net amount of progesterone produced was highest on day 7. The basal as well as hormone and lipoprotein stimulated progesterone production started to decline after day 7, reaching a nadir on day 14. These experiments show that prolactin is effective in stimulating progesterone production by rat luteal cells in vitro and that lipoprotein fractions, LDL and HDL further potentiate this response. This study further suggests that it is important to include LDL or HDL as a source of cholesterol for in vitro experiments in which the steroidogenic response of luteal cells to exogenous stimuli is tested.     

Effects of in vivo and in vitro administration of prostaglandin F2 alpha on lipoprotein utilization in cultured bovine luteal cells

Two experiments were conducted to determine if a loss in the ability to utilize lipoprotein-cholesterol is one mechanism whereby prostaglandin F2 alpha (PGF2 alpha) decreases steroidogenesis in bovine luteal cells. In the first experiment, serum-free cultures of bovine luteal cells were treated with PGF2 alpha (100 ng/ml) for 5 days prior to addition of lipoproteins. Exposure to PGF2 alpha completely suppressed low-density lipoprotein (LDL)- and high-density lipoprotein (HDL)-stimulated progesterone production (p less than 0.01) compared to control (no PGF2 alpha) cultures. Luteal cells cultured in the presence of LDL + luteinizing hormone (LH, 10 ng/ml) + PGF2 alpha produced significantly less progesterone than luteal cells cultured with LDL + LH (p less than 0.05). Treatment with PGF2 alpha had no significant effect on HDL + LH-stimulated progesterone synthesis. In the second experiment, cows were injected with a luteolytic dose of PGF2 alpha (25 mg), and the corpora lutea were removed at 0 (no PG), 1, 4, or 12 h post-injection. Dissociated luteal cells were placed in culture for 7 days, either with or without LH (10 ng/ml), and lipoproteins were added on Days 5-7. LH stimulation of progesterone production was apparent in cultures obtained at 0 and 12 (p less than 0.05) but not 1 and 4 h post-PGF2 alpha. Addition of either LDL or HDL increased progesterone synthesis in all cultures, regardless of time following in vivo administration of PGF2 alpha. It is concluded that PGF2 alpha can inhibit bovine luteal cell utilization of either LDL or HDL in vitro. However, luteal cell utilization of lipoproteins in vitro is not adversely affected by in vivo exposure to PGF2 alpha, if collected within 12 h post-PGF2 alpha.     

Receptor-mediated gonadotropin action in the ovary. Demonstration of acute dependence of rat luteal cells on exogenously supplied steroid precursor (sterols) for gonadotropin-induced steroidogenesis

Incubation of luteal cells with human, horse and rat sera, but not bovine sera resulted in enhanced basal and hCG-stimulated progesterone accumulation. The stimulatory effect of human or rat sera on basal, hCG- or 8 Br-cyclic AMP-induced progesterone synthesis in luteal cells was evident within 15-30 min after incubation, reaching a maximum after 3-4 h. The stimulatory effects of hCG and/or sera were blocked by inhibitors of RNA and protein synthesis. Similarly, lysosomotropic agents, chloroquine (100 microM) and ammonium chloride (10 mM), partly blocked the steroidogenic response of luteal cells to hCG and/or human or rat sera. Incubation of cells in the presence of 2-deoxyglucose, sodium azide and phenylmethylsulfonyl fluoride resulted in partial inhibition of progesterone secretion in response to hCG or sera. Fractionation of human or rat sera into various lipoprotein fractions demonstrated that LDL and HDL most effectively supported and potentiated the steroidogenic response to hCG. Lipoprotein-deficient serum, however, did not alter gonadotropin-induced steroid production. Incubation of luteal cells with increasing concentrations of h-LDL and h-HDL enhanced both basal and hCG-mediated steroidogenesis in a dose-related manner, although very high concentrations of these lipoproteins were inhibitory. Further, [3H]cholesterol from [3H]cholesteryl linoleate-LDL was incorporated into luteal cell progesterone and the extent of this incorporation was enhanced by hCG. Addition of excess unlabeled h-LDL, h-HDL, as well as r-HDL, drastically reduced the incorporation of radioactive label into progesterone. These studies suggest that (a) serum potentiation of steroidogenesis was due to presence of lipoproteins, mainly LDL and HDL, and (b) the lipoprotein-bound cholesterol is delivered into the luteal cells and utilized for steroidogenesis.     

Receptor-mediated gonadotropin action in the ovary. Action of cytoskeletal element-disrupting agents on gonadotropin-induced steroidogenesis in rat luteal cells.

The role of the cellular cytoskeletal system of microtubules and microfilaments on gonadotropin-stimulated progesterone production by isolated rat luteal cells has been investigated. Exposure of luteal cells to human choriogonadotropin resulted in a stimulation of cyclic AMP (4-7-fold) and progesterone (3-4-fold) responses.l Incubation of cells with the microfilament modifier cytochalasin B inhibited the gonadotropin-induced steroidogenesis in a dose- and time-dependent manner. The effect of cytochalasin B on basal production of steroid was less pronounced. Cytochalasin B also inhibited the accumulation of progesterone in response to lutropin, cholera enterotoxin, dibutyryl cyclic AMP and 8-bromo cyclic AMP. The inhibition of steroidogenesis by cytochalasin B was not due to (a) inhibition of 125I-labelled human choriogonadotropin binding to luteal cells, (b) inhibition of gonadotropin-stimulated cyclic AMP formation or (c) a general cytotoxic effect and/or inhibition of protein biosynthesis. Cytochalasin D, like cytochalasin B, inhibited gonadotropin- and 8-bromo cyclic AMP-stimulated steroidogenesis. Although cytochalasin B also blocked the transport of 3-O-methyl-glucose into luteal cells, cytochalasin D was without such an effect. Increasing glucose concentration in the medium, or using pyruvate as an alternative energy source, failed to reverse the inhibitory effect of cytochalasin B. The anti-microtubular agent colchicine failed to modulate synthesis and release of progesterone by luteal cells in response to human choriogonadotropin. These studies suggest that the cellular microfilaments may be involved in the regulation of gonadotropin-induced steroidogenesis. In contrast, microtubules appear to be not directly involved in this process.     

Role of prostaglandin E2 in basal and noradrenaline-induced progesterone secretion by the bovine corpus luteum

The role of prostaglandin E2 (PGE2) in basal and noradrenaline (NA)-stimulated utilization of high density lipoprotein (HDL) as a source of cholesterol for progesterone synthesis was examined. In Experiment 1, a cannula was inserted into the aorta abdominalis through the coccygeal artery (cranial to the origin of the ovarian artery) in mature heifers, to facilitate infusion of NA (4 mg/30 min; n = 3) on day 10 of the estrous cycle. Three other heifers were similarly cannulated to serve as control. Before, during, and after NA or saline infusion, blood samples from the vena cava were collected every 5-15 min for analysis of PGE2, progesterone, and cholesterol. Each NA infusion stimulated (P < 0.01) secretion of both hormones in heifers. Short-duration increases (P < 0.05) in progesterone were observed due to the infusion of NA while cholesterol was not altered significantly. In addition, increases in PGE2 concentrations (P < 0.05) compared to controls were seen after NA infusion. Therefore, we used an in vitro model to verify the effect of PGE2 on HDL utilization by luteal cells from day 5 to 10 of the estrous cycle. In the preliminary experiment, 10(-6) M of PGE2 out of four different doses examined was selected for further studies, since it evoked the highest release of progesterone. In the next experiment, it was found that HDL increases progesterone secretion by luteal cells and both PGE2 and LH increased (P < 0.05) the response to HDL while NA did not. In the last in vitro experiment, progesterone stimulated PGE2 secretion by luteal cells. In conclusion, PGE2 may be directly involved in the utilization of cholesterol from HDL for progesterone synthesis. Furthermore, PGE2 may influence NA-stimulated progesterone secretion by the corpus luteum (CL). It is concluded that there is a positive feedback loop between progesterone and luteal PGE2 during days 5-10 of the estrous cycle.     

Evidence for the preferential utilization of esterified cholesterol in progesterone production by rabbit corpora lutea

Our previous studies show that lipoproteins stimulate progesterone secretion by rabbit luteal cells in vitro and that estradiol modifies this effect. This study examines the relationship between estradiol and serum lipoproteins for progesterone production by rabbit corpora lutea in vivo. Using morphometric analysis, we determined that estrogen treatment of hysterectomized pseudopregnant (E-hyst) rabbits increased luteal lipid volume by mid-pseudopregnancy without altering serum progesterone levels. Treatment of E-hyst rabbits with 4-amino-3,4,pyrazolo pyrimidine (APP) during early to mid-pseudopregnancy reduced serum cholesterol levels without decreasing serum progesterone concentrations. However, 3-hydroxy-3 methyl glutaryl-CoA reductase activity was increased. Thus, in the presence of exogenous estrogen, serum cholesterol is esterified and stored rather than converted directly into progesterone. APP-treatment of E-hyst rabbits during late-pseudopregnancy, when estrogen receptor levels are low, increased serum progesterone levels and reduced intracellular lipid content. Thus, stored lipid is the primary source of cholesterol for progesterone synthesis. In addition, estrogen, via estrogen receptor, is important in maintaining steady progesterone output despite fluctuations in serum lipoprotein levels. A working model for cholesterol utilization by rabbit luteal cells is presented, which suggests that stored cholesterol esters, derived from both endogenous and exogenous sources, is the key source or cholesterol for progesterone production. Furthermore, we propose that estradiol regulates the uptake and storage of cholesterol and its rate of metabolism into progesterone.     

Localization of prostaglandin F2 alpha inhibition of lipoprotein use by bovine luteal cells.

Prostaglandin F2 alpha (PGF2 alpha) inhibits lipoprotein-stimulated progesterone production by bovine luteal cells in vitro and the objective of this study was to localize the site of action of PGF2 alpha. Cultured bovine luteal cells were treated with PGF2 alpha for seven days, and then with either lipoproteins or 25-hydroxycholesterol in the presence of aminoglutethimide (which inhibits cholesterol side-chain cleavage) for the final 48 h. The effects of PGF2 alpha on progesterone production, cellular cholesterol content, mitochondrial cholesterol content and cholesterol side-chain cleavage activity were determined. As expected, PGF2 alpha inhibited (P less than 0.05) lipoprotein-stimulated progesterone production. However, PGF2 alpha did not inhibit low-density lipoprotein-stimulated, or high density lipoprotein-stimulated, increases in cellular cholesterol (P less than 0.05) or inhibit lipoprotein-induced increases in mitochondrial cholesterol content (P less than 0.05). Additionally, cholesterol content of mitochondria increased (P less than 0.05) in the presence of PGF2 alpha alone. To determine if the PGF2 alpha-induced inhibition of steroidogenesis occurred at, or after, the side-chain cleavage reaction, we treated cells with the readily diffusable sterol, 25-hydroxycholesterol. Prostaglandin F2 alpha did not inhibit 25-hydroxycholesterol-stimulated progesterone production (P less than 0.05). Prostaglandin F2 alpha may therefore exert its luteolytic effect at a site after cholesterol transport to the mitochondria but before cholesterol side-chain cleavage.     

The role of high density lipoproteins in rat adrenal cholesterol metabolism and steroidogenesis

Addition of rat or human high density lipoproteins (HDL) or human low density lipoproteins (LDL) to rat adrenocortical cells in vitro was found to enhance steroid production and increase cell cholesterol content. These effects of HDL were not observed in cultured mouse Y-1 adrenal cells, suggesting that rat adrenal cells possess a specific mechanism for uptake of HDL cholesterol not found in Y-1 cells. The effects of HDL were most marked on cells previously stimulated with adrenocorticotropin (ACTH) and depleted of their endogenous cholesterol stores. Such cells were prepared either by treatment in vivo with 4-aminopyrazolopyrimidine or in vitro with ACTH (10(-7) M) in lipoprotein-poor media. Steroid production by treated cells exhibited a saturable dependence on media HDL concentration. In addition to enhancing ACTH stimulated steroid production, addition of HDL also resulted in a saturable concentration-dependent increase in cell cholesterol content. Both aminoglutethimide and cycloheximide were found to inhibit HDL-enhanced steroid production. Finally, addition of HDL to short term incubations (5 1/2 h) of ACTH-treated cells caused no change in the rate of incorporation of 14C-acetate into cholesterol or corticosterone. These results indicate that rat adrenocortical cells possess a specific, saturable, ACTH-dependent mechanism for uptake of HDL cholesterol. Moreover, cellular uptake of HDL cholesterol exceeded by at least 4-fold the amount of cholesterol associated with HDL apoprotein degraded by the cells, suggesting that utilization of HDL cholesterol does not require endocytosis and lysosomal degradation of the entire HDL particle.     

Function of abnormal corpora lutea in vivo after GnRH-induced ovulation in the anoestrous ewe

Anoestrous Romney Marsh ewes with and without progesterone treatment (+P, -P) were treated with small-dose (250 ng) multiple injections of GnRH at 2-h intervals for 48 h. Animals were slaughtered on Days 4, 5, 7 and 11 after the end of GnRH treatment and luteal function was assessed by the measurement of daily plasma progesterone concentrations. In all animals which ovulated (29/32, 91%) peripheral progesterone concentrations rose to 0.5-1.0 ng/ml within 3 days of the end of GnRH treatment. In 7/7 (100%) +P animals and 5/22 (23%) -P animals, progesterone concentrations continued to rise and were maintained at levels greater than 1.5 ng/ml until slaughter. In the remaining -P animals, plasma progesterone concentrations declined to reach basal levels by Day 5. Corpora lutea recovered from these animals showed signs of premature regression on Day 5 and were fully regressed by Day 7. Progesterone priming delayed the occurrence of the LH surge which occurred 39.1 +/- 3.6 h after the end of GnRH treatment in the +P animals compared to 20.2 +/- 1.74 h (P less than 0.001) in the -P animals in which luteal function was abnormal and 22.4 +/- 4.35 h in the -P animals in which luteal function was normal. These results show that abnormal luteal function occurs in the majority of GnRH-treated ewes in the absence of progesterone pretreatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of lipoproteins and luteotrophins on progesterone accumulation by luteal cells from the pregnant pig

The roles of prolactin (Prl) and LH in the maintenance of luteal function in pregnant pigs were investigated. Luteal cells from pigs between days 70 to 95 of pregnancy were dissociated and incubated for 4 h. In the absence of exogenous cholesterol, LH exhibited a dose-dependent stimulatory effect on progesterone secretion. Prl had a mild stimulatory effect on progesterone accumulation and at lower doses Prl potentiated the response to LH. Low density lipoprotein (LDL) but not high density lipoprotein (HDL) had a mild stimulatory effect on progesterone secretion. When exogenous cholesterol was provided as the substrate in the form of LDL or HDL, Prl had a striking stimulatory effect on progesterone secretion. When 25-hydroxycholesterol which bypasses the lipoprotein receptor was provided as the substrate, Prl failed to stimulate progesterone accumulation. The stimulatory effect of LH was potentiated when LDL, HDL, or 25-hydroxycholesterol were present. The results of this study suggest that LH increases the uptake of exogenous cholesterol in the form of lipoproteins and enhances the utilization of internalized cholesterol for progesterone synthesis. Prl appears to stimulate progesterone synthesis by enhancing the uptake of lipoproteins.