lipoproteins to provide substrate for corticosteroid production (Faust, Goldstein ... Kovanen, Faust, Brown & Goldstein, 1979; Can et al., 1980; Turley, Andersen ...
lipoproteins and de-novo cholesterol synthesis in progesterone production by cultured bovine luteal cells
Role of
P. J.
O'Shaughnessy
and D. C. Wathes
Department of Anatomy, Medical School, University of Bristol, Bristol BS8 12TD,
U.K.
Summary. Bovine luteal cells cultured in the presence of lipoprotein-deficient serum (LPDS) produced less progesterone than did cells cultured in the presence of complete serum and, while dibutyryl cyclic AMP (dbcAMP) caused a marked stimulation of progesterone production in complete serum, it had only a small effect in LPDS. Lowdensity lipoprotein (LDL) and high-density lipoprotein (HDL) increased basal and dbcAMP-stimulated progesterone production. Cells were 7 times more sensitive to LDL than HDL although the maximum response to both lipoproteins did not differ significantly. Inhibition of de-novo cholesterol synthesis by compactin had no effect on dbcAMP-stimulated progesterone production by cells cultured with complete serum or LPDS plus lipoproteins. Basal progesterone production in complete serum was increased by compactin and this was associated with a marked inhibition of cell proliferation, both effects being reversed by mevalonic acid. Compactin caused a 50% reduction in dbcAMP-stimulated progesterone secretion by cells cultured in LPDS. These data show that bovine luteal cells are dependent upon lipoproteins to provide substrate for progesterone synthesis and that, while LDL is the preferred lipoprotein, both LDL and HDL may be of importance in vivo. Introduction
Steroid-secreting cells may derive cholesterol, the obligatory precursor of steroid hormones, from de-novo synthesis or from circulating lipoproteins. Progestagen synthesis by dispersed rat and human luteal cells in vitro depends to a large extent upon provision of cholesterol by lipoproteins (Carr et al., 1981 ; Schuler, Langenberg, Gwynn & Strauss, 1981 ; McNamara, Booth & Stansfield, 1981 ; Tureck & Strauss, 1982), de-novo cholesterol synthesis appearing to play only a minor role (Schüler, Scavo, Kirsch, Flickinger & Strauss, 1979; Schuler et al., 1981 ; Tureck & Strauss, 1982). Adrenocortical cells, from animals of several species, appear to be similarly dependent upon lipoproteins to provide substrate for corticosteroid production (Faust, Goldstein & Brown, 1977; Kovanen, Faust, Brown & Goldstein, 1979; Can et al., 1980; Turley, Andersen & Dietschy, 1981;
Simonian, White & Gill, 1982). In the rabbit, however, the corpus luteum of pregnancy derives a
significant proportion of cholesterol for progestagen production by de-novo synthesis (Kovanen, Goldstein & Brown, 1978). Similarly, Leydig cells of the rat appear to utilize de-novo synthesis rather than lipoproteins to provide cholesterol under normal conditions (Quinn, Dombrausky, Chen & Payne, 1981). In those cells that require lipoproteins for steroid synthesis, there appears to be a species dependent preference for the class of lipoprotein utilized. Rat luteal and adrenal cells use highdensity lipoprotein (HDL) in preference to low-density lipoprotein (LDL) for the provision of cholesterol substrate (Gwynne, Mahaffee, Brewer & Ney, 1976; Andersen & Dietschy, 1978; *
U.K.
Present address:
Department of Anatomy, Royal Veterinary College, Royal College Street, London NW1 OTU,
ai, 1981; Bruot, Wiest & Collins, 1982). In contrast, human luteal cells, luteinized granulosa cells, fetal adrenal cells and placental cells preferentially utilize LDL (Winkel, Snyder, MacDonald & Simpson, 1980; Carr etai, 1980, 1981; Tureck & Strauss, 1982). Incubation of these Schuler
et
cells with HDL either has no effect or causes an actual decrease in steroid synthesis (Tureck & Strauss, 1982), perhaps due to promotion of cholesterol efflux (Wu & Baily, 1980). The relative importance of LDL, compared to HDL, in providing cholesterol substrate to bovine tissue is less certain. Simonian et al. (1982) have reported that cultured bovine adrenocortical cells require LDL as the principal source of cholesterol with HDL, at high concentrations, causing only a partial (33%) increase in steroidogenesis in the presence of adrenocorticotrophic hormone (ACTH). Bovine granulosa cells, however, appear to respond maximally to both human LDL and bovine HDL although LDL was reported to be 10 times more potent than HDL on a protein-weight basis (Savion, Laherty, Cohen, Lui & Gospodarowicz, 1982). Pate & Condon (1982) have reported that bovine luteal cells will respond to bovine LDL and HDL although the relative potency of these two lipoproteins was not examined. In this study we have examined the sensitivity of cultured bovine luteal cells to LDL and HDL and have assessed the requirement for de-novo cholesterol synthesis by use of compactin, a specific inhibitor of 3hydroxy-3-methylglutaryl coenzyme A reducíase (HMG CoA reducíase), the rate limiting enzyme in cholesterol biosynthesis (Endo, 1981). Materials and Methods Materials. Ham's F12 medium and antibiotics were purchased from Gibco Europe Ltd, Paisley, U.K. Collagenase (Worthington type CLS) was obtained from Flow Laboratories Ltd, Irvine, U.K. Compactin was a generous gift from Dr A. K. Endo (Tokyo Ono University). All other chemicals were purchased from Sigma (London), Poole, Dorset, U.K.
Preparation of lipoproteins and lipoprotein-deficient serum. Lipoproteins and lipoprotein-deficient (LPDS) were prepared from the serum of adult cows by sequential ultracentrifugation (Havel, Eder & Bragdon, 1955) using solid KBr for density adjustments. Bovine LDL was isolated
serum
density of 1-019-1-053, bovine HDL was isolated at a density of 1-075-1-215 and LPDS was isolated at a density > 1-215. Protein was measured by the method of Lowry, Rosebrough, Farr & Randall (1951) as modified by Mark well, Haas, Bieber & Tolbert (1978) and cholesterol was determined using the cholesterol oxidase method (Sigma). The protein/cholesterol ratios of LDL and HDL were 0-86 and 1-72 respectively. Lipoprotein concentrations are expressed as µg protein per ml. Both LDL and HDL migrated as homogeneous bands on polyacrylamide gel electrophoresis (Masket, Levy & Fredrickson, 1973).
at a
Culture of bovine luteal cells. Mid-cycle corpora lutea, collected fresh from cows at an abattoir, used. Luteal cells were dissociated from minced tissue of 3 or 4 corpora lutea, as previously described (O'Shaughnessy & Wathes, 1985) using Ham's Fl 2 medium containing 0-2% collagenase, 0005% DNase and 0-5% bovine serum albumin with 100 U penicillin/ml and 100 µg streptomycin/ml. Dissociated cells (2 x 105—3 x 105/dish) were cultured in 2 ml Ham's F12 medium supplemented with 10% adult bovine serum and antibiotics (O'Shaughnessy & Wathes, 1985). After 18 h the incubation medium was removed and fresh Ham's F12 medium, containing 10% complete bovine serum or LPDS, was added. The protocol followed thereafter for each experiment is described in 'Results'. Used medium was stored frozen at 20°C until assayed for progesterone by radioimmunoassay (Wathes & Porter, 1982; O'Shaughnessy & Wathes, 1985). The intra- and inter-assay coefficients of variation were 6-8% and 12-8% and the limit of sensitivity of the assay was 150 pg/ml. Cell number was measured using a haemocytometer after removal of the cells from the culture dish by trypsinization (O'Shaughnessy & Wathes, 1985). were
—
Statistical analysis. Results were assessed by analysis of variance and the Newman-Keul test after logarithmic transformation of the data to avoid heterogeneity of variance. Data from single experiments are illustrated here although the effects described have been observed in two or more experiments. Each experiment used cells from 3 or 4 cows and replicate independent incubations were performed within each treatment to assess error. Results
Bovine luteal cells cultured for 72 h in LPDS produced markedly lower amounts of progesterone than did cells cultured in complete serum (Table 1). In the presence of complete serum, dbcAMP caused a marked stimulation of progesterone production whereas, in LPDS, dbcAMP caused only a slight stimulation of production. When cells were incubated for longer periods in LPDS, dbcAMP had no effect on progesterone production (data not shown). Addition of LDL (30 µg protein/ml) or HDL (60 µg protein/ml) to cells cultured in LPDS caused a marked increase in dbcAMP-stimulated progesterone production (Table 1). At the concentrations tested, LDL appeared to be more potent than HDL. To assess the relative potency of LDL and HDL in providing substrate for luteal progesterone synthesis the effects of increasing concentrations of lipoproteins were tested (Text-fig. 1). Both LDL and HDL caused a concentration-dependent increase in basal and dbcAMPstimulated progesterone secretion. The ED50 of LDL for dbcAMP-stimulated progesterone production was 11-25 ± 5-7 µg/ml (mean ± s.e.m. of 2 experiments) while the ED50 of HDL was 79 ± 16 (4 experiments). In a separate experiment, the effect of 200 µg LDL/ml did not differ significantly from the effect of 300 µg HDL/ml (1522 ± 74 and 1603 ± 103 ng progesterone/105 cells, respectively ; 2). High concentrations of HDL ( 1000 µg/ml) caused a decline in dbcAMPstimulated progesterone production and this effect was associated with a marked retraction and rounding-up of luteal cells in the culture dish. =
Table 1.
Progesterone secretion by bovine luteal cells cultured in 10% complete serum, LPDS or LPDS plus lipoproteins Progesterone (ng/105 cells) Basal
Complete
serum
LPDS LPDS + HDL (60 µg/ml) LPDS + LDL (30 µß/ 1)
208 110 138 159
± ± ± ±
dbcAMP (1 mM) 21 13 8 20
2406 140 523 1278
± ± ± ±
206 16 97 251
Bovine luteal cells were cultured in 10% complete serum for 18 h then 10% complete serum or LPDS with or without dbcAMP for 24 h. The medium was changed, HDL or LDL added, and progesterone secretion measured after a 48h incubation under the conditions indicated above. Results are mean ± of duplicate independent incubations.
s.e.m.
The effects of compactin on luteal cell proliferation in 10% complete serum are shown in Textfig. 2(a). Addition of 5 or 10 µ -compactin caused cells to retract and round-up in the culture dish within 48 h. After a further 48 h, compactin at these concentrations caused cells to detach from the culture dish. Lower concentrations of compactin (2-5 and 1-25 µ ) had no effect on morphology during the first 48 h of incubation although they did have an inhibitory effect on cell proliferation. During a second 48-h incubation, however, these concentrations of compactin also caused cell detachment. Morphology and proliferation of cells cultured in the presence of mevalonic acid (10
2000
S
1600
1200
S
800
o
400
30
10
100
30
LDL^g/ml)
HDLWml)
1. Sensitivity of cultured bovine luteal cells to lipoproteins. Bovine luteal cells were cultured in Ham's F12 medium plus 10% complete serum for 18 h. The culture medium was replaced by 10% LPDS with or without dbcAMP. After incubation for another 24 h the medium was changed and various amount of HDL (a) or LDL (b) were added. Progesterone secretion was measured after a further 48-h incubation with ( - ) or without ( - ) 1 mM-dbcAMP. Results are mean ± s.e.m. of duplicate independent incubations.
Text-fig.
(b)
114
18
66
114
Hours in culture
Effects of compactin on proliferation of bovine luteal cells in culture. Bovine luteal cells were cultured in Ham's F12 medium plus 10% complete serum for 18 h. The medium was then renewed and contained 0 < - >. 1-25 (D-D), 2-5 ( - ), 5 (V-V) or 10 (O-O) µ -compactin alone (a) or with 10 mM-mevalonic acid (b). Cell numbers were measured after a further 48- or 96-h incubation. Results in both figures are the mean of duplicate independent incubations from the same experiment.
Text-fig. 2.
m\t) and various concentrations of compactin did not differ markedly from cells cultured with mevalonic acid alone (Text-fig. 2b). Compacin (3 µ ) had no effect on dbcAMP-stimulated progesterone production and caused an actual increase in basal progesterone production in the presence of 10% complete serum (Table 2).
Table 2. Effect of
compactin
on
progesterone secretion by cultured bovine luteal cells
Progesterone Compactin (3 µ )
Control Basal
Complete Complete
serum serum
plus
mevalonic acid (10 mM) LPDS LPDS plus mevalonic acid LPDS plus HDL
(200 ag/ml) LPDS plus LDL (50 µ / 1)
dbcAMP
(1 mM)
Basal
dbcAMP
(1 mM)
320 ± 15a 417 ± 36a
3029 ± 141" 3264 ± 192"
665 ± 26c 400 ± 41a
2894 ± 103b 2778 ± 217"
116 ± 2a 168 ± 7a
211 ± 6» 259 ± 36b
84 ± 5C 126 ± 18c
111 ± 5a 292 ± 29b
378 ± 4a
1436 ± 78b
656 ± 38c
1736 ± 251»
310 ± 7a
916 ± 40b
513 ± 81c
1038 ± 96b
The experimental procedure was as described in Table 1 except that mevalonic acid and/or compactin were added the final 48-h incubation as indicated. Results are mean ± s.e.m. of duplicate independent incubations. Within each row a different superscript indicates a significant difference in progesterone secretion (P < 005).
during
In the presence of LPDS, compactin inhibited dbcAMP-stimulated progesterone production although the effect on basal production was less marked. These inhibitory effects of compactin on dbcAMP-stimulated production were overcome by the addition of mevalonic acid, HDL or LDL. In the presence of HDL or LDL compactin again caused an increase in basal progesterone secretion compared to control.
Discussion Data presented in this report indicate that bovine luteal cells depend upon lipoproteins to provide substrate for progesterone synthesis and that de-novo cholesterol synthesis is unimportant in the presence of an exogenous cholesterol source. As previously reported by Pate & Condon (1982), cultured bovine luteal cells respond to both LDL and HDL although the present study shows that LDL is 7 times more potent on a protein-weight basis and 3-5 times more potent on a basis of cholesterol content. While cells are more sensitive to LDL than HDL, it is not clear which of these two lipoproteins plays the more important role in vivo since HDL is the major lipoprotein class present in bovine serum (Jonas, 1972; Raphael, Dimick & Puppione, 1973). The ratio of HDL to LDL varies with the lactational state of the animal although the data of Raphael et al. (1973) indicate that this ratio (on a basis of cholesterol ester content) does not fall below 10-9 and may rise to 26-7. This suggests that HDL may play an important role in providing cholesterol to the bovine corpus luteum. It is unlikely that the effects of HDL observed in this study are due to HDL interaction with the LDL receptor since bovine HDL does not contain apoprotein E (Savion et al., 1982) which is known to play a role in the interaction of HDL with the LDL receptor and metabolic pathway (Innerarity, Pitas & Mahley, 1980). Comparison between this report and the study of Savion et al. (1982) suggests that the relative potency of LDL, compared to HDL, is similar in the luteal and granulosa cells of cows although luteal cells appear to be more sensitive to both lipoproteins. Like those of some other mammals, the corpus luteum of the cow contains large and small luteal cells (Donaldson & Hansel, 1955) which differ in their basal progesterone secretion and responsiveness to LH (Ursely & Leymarie, 1979). The large cells of the early corpus luteum are derived from the granulosa cells while the small cells are of thecal origin (Alila, 1983) and it is possible that responsiveness to lipoproteins may differ between the cell types. The apparent difference in sensitivity between granulosa and luteal cells to lipoproteins may, therefore, be due to the presence of luteinized thecal cells in the corpus luteum. It
is also possible that luteinization leads to an increased sensitivity of granulosa cells to lipoproteins. Previous studies have shown that LH or hCG acts to increase the number of receptors for LDL in the human corpus luteum (Carr et ai, 1981) and that LDL receptor number is highest in the midluteal phase (Ohashi, Carr & Simpson, 1982). After ovulation a primary effect of LH may therefore be to increase LDL receptors, thus increasing uptake of cholesterol for progesterone synthesis. Whether a similar mechanism exists to increase sensitivity to HDL in the bovine ovary remains to be determined. In contrast to granulosa and luteal cells, bovine adrenocortical cells show only a limited responsiveness to HDL, compared to LDL (Simonian et al., 1982). The sensitivity of these cells to LDL however, is markedly greater than the sensitivity of luteal cells (ED50 1-1 µg/ml compared to 11-25 µg/ml). There appears, therefore, to be a difference in the utilization of lipoproteins by the adrenal cortex and the ovary in the cow. It has been shown that inhibition of HMG CoA reductase by compactin leads to decreased proliferation and survival of several cell lines in culture (Kaneko, Hazama-Shimada & Endo, 1978 ; Quesney-Huneeus, Wiley & Siperstein, 1979; Habenicht, Glomset & Ross, 1980). The present results show that compactin has a similar inhibitory effect on bovine luteal cells in primary culture. The antagonism of this effect by mevalonic acid suggests that compactin is acting specifically to inhibit HMG CoA reductase. Since the effect of compactin was observed in the presence of complete serum it indicates that a product of mevalonic acid metabolism, other than cholesterol, is required for cell survival and proliferation. The nature of this essential factor is unknown at
present. The lack of an effect of compactin on dbcAMP-stimulated progesterone secretion by luteal cells cultured in complete serum, or LPDS plus lipoproteins, indicates that de-novo cholesterol synthesis is unimportant in maintenance of steroidogenesis by cells cultured in the presence of lipoproteins. The apparent increase in basal progesterone secretion caused by compactin may be related to inhibition of cell proliferation since proliferation and expression of differentiated function tend to be exclusive (Orly, Sato & Erickson, 1980). Such an effect would not be observed in the presence of dbcAMP because luteal cell proliferation is already markedly inhibited by this nucleotide (Gospodarowicz & Gospodarowicz, 1975; O'Shaughnessy & Wathes, 1985). Compactin caused a 50% reduction in progesterone synthesis by cells cultured in LPDS alone, indicating that luteal cells have a limited ability, in the absence of lipoproteins, to provide cholesterol for progestagen synthesis both by utilization of stored cholesterol and by de-novo cholesterol synthesis. In conclusion, this study indicates that bovine luteal cells depend upon lipoproteins to maintain progesterone synthesis and that, while LDL appears to be the preferred substrate, both lipoproteins may be utilized in vivo. Unlike the rat and the human, sensitivity of bovine steroidogenic tissue to LDL and HDL does not correspond to the circulating levels of these lipoproteins and the importance of HDL in vivo may depend upon the circulating levels of LDL. This work was supported by a grant from F. Hoffman-La Roche & Co., Basel, Switzerland. We thank Dr A. K. Endo for the generous donation of compactin and Mrs S. D. Birkett for advice on
lipoprotein electrophoresis.
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