Use of Low-Density and High-Density Lipoproteins in Undifferentiated Porcine Granulosa Cells

BIOLOGY OF REPRODUCI1ON Use 41, 855-861 (1989) of Low-Density and High-Density Lipoproteins in Undifferentiated Porcine Granulosa Cells1 K. RAJKUMAR,2 H. LY, P. W. SCHOTF, Reproductive Department Biology and B. D. MURPHY Research Unit of Obstetrics and Gynecology University of Saskatchewan Saskatchewan, Canada ABSTRACT Granulosa cells aspirated from medium-sized porcine ovarian follicles (3 5 mm) in short-term incubation responded to the addition of both low-density lipoprotein (LDL) and high-density lipoprotein (HDL) with increased accumulation of progesterone. HDL wa,s more potent than L.DL in enhancing progesterone secretion. When granulasa cells were cultured under serum-free conditions for 72 h, HDL but not LDL exhibited a dosedependent enhancement ofproge.sterone secretion. Addition of insulin to the cells greatly potensiated the silmulatoty effect of LOL on progesterone accwnulation, while the response to HDL was only slightly increased. Granulosa cells in culture degraded LDL but not HDL. Addition of insulin enhancedLDL degradation. Exposure of cells in culture to chioroquine, an inhibitor of lysosomal function, completely blocked LOL degradation and LDLinduced stimulation of steroidogenesis. The stinudatory effect of HDL was not affected by chioroquine. We interpret these findings to indicate that granukisa cells derive cholesterol from LDL by means of lysosomal degradation, which is not required for use of cholesterol from HDL. Monensin, a carboxylic ionophore that interrupts recycling of LOL receptors, prevented LDL-enhanced progesterone accumulation but not HDL-induced stimulation. This provides evidence that HDL-i nduced stimulation of steroidogenesis does not involve LDL receptors. We conclude that HDL present infollicularfluid is capable of providing cholesterol to granulosa cells for steroidogenesis. The stimulatory effect of HDL is not due to the presence of apoprotein E, an HDL component that binds to the LDL receptor. A unique HDL pathway that does not involve LDL receptors and lysosomal degradation may operate in porcine granulosa cells. - INTRODUCTION been postulated that this restricted access to the avascular compartment of the follicle may limit the supply of lipoprotein-carned cholesterol for in vivo use by granulosa cells (Simpson et a!., 1980). Tureck and Strauss (1982) reported that addition of HDL reduces steroid production by human granulosa cells in culture. In immature poreine granulosa cells, swine HDL but not human HDL has a stimulatory effect on steroidogenesis (Veldhuis et a!., 1984). It is not known whether swine HDL contains traces of approprotein E (apo-E) and stimulates steroidogenesis by binding to LDL receptors, or if HDL pathway is operative in swine granulosa cells. The mechanisms by which LDL and HDL deliver cholesterol to steroid-secreting cells are different (Paavola et a!., 1985). The LDL receptor recognizes both apoprotein B (apo-B) and apo-E, (Innerarity and Mahley, 1978), while HDL receptors specifically recognize both apoprotein A-I and apoprotein A-I! (Hwang and Menon, 1985). After initial binding of lipoproteins to specific receptors, LDL and HDL follow distinctly different pathways (for review, see Murphy and Silavin, All steroidogenic tissues are capable of synthesizing cholesterol de novo. Uptake of lipoprotein-canied cholesterol is the principal means by which ovaries acquire substrate for steroidogenesis (Rosenbium et a!., 1981; Gwynne and Strauss, 1982; Murphy et al., 1985; Murphy and Silavin, 1989). The lipoprotein class that delivers cholesterol to steroidogenic cells depends upon the species and the experimental conditions. Under various circumstances, low-density lipoprotein (LDL), highdensity lipoprotein (HDL), or both, can supply cholesterol (Strauss et a!., 1981). In Graafian follicles prior to ovulation, HDL but not LDL gains access to the avascular antral fluid compartment in which granulosa cells reside (Chang et a!., 1976; Veldhuis et al., 1984). It has Accepted Received Council lime 19, 1989. Febru&y 27. 1989. study was funded by grant of Canada requ to B.D. MA-9245 from Medical Research Murphy. 855 Saskatoon, RAJKUMAR 856 1989). In a recent MATERIALS Lipoprotein Isolation AND and METhODS Characterization LDL was isolated from serum collected from prepubertal sows, and HDL was separated from follicular fluid collected from pre-ovulatory follicles by potassium bromide density-gradient ultracentrifugation (‘Ferpstra et a!., 1981). The LDL band appeared in the density range between 1.03 and 1.06 g/ml, and the HDL band was found in the range of 1.08-1.20 g/ml. Puiifled lipoproteins from serum and fofficular fluid were dia!yzed against phosphate-buffered saline (PBS) with 0.01% ethylenediaminetetraacetate (EDTA) using an An#{252}conDiaflo ultrafiltration cell (Amicon Corp., Lexington, MA) with a PM-30 membrane and a positive nitrogen pressure. LDL and HDL were characterized by sodiwn dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, L.aemmli, 1970), which was then stained with Coomassie Brilliant Blue. To determine whether follicular fluid HDL contained apo-E, HDL was subjected to heparin-manganese precipitation (protein: hepann: manganese chloride ratio of 2.3:1:4.8) as described by Mahley and Innerarity (1977). After incubation of Hl)L with heparin-manganese, the precipitated lipoproteins were removed by centrifugation and the supeniatant fraction was dialyzed against 5% barium chloride to remove heparin. Lipoprotein in the supematant was then washed with PBS with 0.01% EDTA and concentrated with Amicon ultrafiltration. The protein pattern of HDL after the heparinmanganese precipitation was characterized by SDS- PAGE. When same treatment, tatet Cell Incubation ‘I-labeled LDL was subjected 92% of the radioactivity was to the precipi- Studies Granulosa cells were aspirated aseptically from medium-sized porcine ovarian follicles (3-5 mm) Cells were separated from follicular fluid by centrifugation (200 x g) and were washed three times in Eagle’s minimum essential medium (MEM, GIBCO, Grand Island, NY). The cells were then preincubated at 37#{176}C for 1 h under an atmosphere of 95% 02:5% CO2. At the end of incubation, the cells were centrifuged at 200 x g and the resuspended pellet was filtered through a silk screen to remove the cell clumps. Approximately 1 x 106 cells were added to plastic tubes either with or without graded doses of LDL or HDL and incubated for 4 h at 37#{176}C. At the end of incubation, the cells were centrifuged and media were separated for estimation of progesterone by a radioinimunoassay technique (Rajkumar et a!., 1985). The cell pellet was then washed with PBS and dissolved in 0.5 N NaOH for estimation of protein (Lowry et a!., 1951). Effect of Insulin on Utilization of LDLand borne Cholesterol for Progesterone Secretion by Granulosa Cells in Culture HDL- Granulosa cells from medium-sized fofficles of porcine ovaries (...truncated)