title: Native and modified (acetylated) low density lipoprotein-supported steroidogenesis by macaque granulosa cells collected before and after the ovulatory stimulus: correlation with fluorescent lipoprotein uptake.
abstract: We recently reported that uptake of fluorescent-tagged low density lipoprotein (DiI-LDL) by macaque granulosa cells (GC) was greatly enhanced within 27 h of an ovulatory stimulus (hCG injection). The present study was designed to determine whether increased DiI-LDL uptake correlated with an increased capacity for LDL-supported steroidogenesis. We also tested whether modified [acetylated (ac)] LDL or high density lipoprotein (HDL), which are not ligands for the LDL receptor, supported progesterone (P) production. Beginning at menses, adult female rhesus macaques were treated with human (h) FSH and hLH for 9 days to promote development of multiple follicles. On day 10, monkeys were injected with hCG (1000 IU) or received no ovulatory stimulus. Large follicles were aspirated on day 10 (nonluteinized GC) or 27-34 h after hCG administration (luteinizing GC). GC (2 x 10(4)/0.2 ml) were cultured in Dulbecco's Modified Eagle's Medium-Ham's F-12 plus insulin, transferrin, H2SeO3, and aprotinin, with 0-100 micrograms hLDL, ac-hLDL, or hHDL. P concentrations in medium were determined by RIA. LDL (1-25 micrograms/ml) dose-dependently increased (up to 15-fold; P < 0.05) basal and hCG-stimulated P production by luteinized GC on days 1-8 of culture. However, LDL (25 micrograms/ml) did not alter basal P production by nonluteinized GC and increased (2-fold; P < 0.05) hCG-stimulated P secretion only on days 4-8. Basal and hCG-stimulated P production by luteinized GC were initially (days 1-2) increased (up to 2-fold; P < 0.05), but later (days 6-8) suppressed (P < 0.05) in a dose-dependent manner by 1-100 micrograms ac-LDL/ml. Ac-LDL did not alter basal or hCG-stimulated P production by nonluteinized GC. HDL (1-100 micrograms/ml) did not alter P production by either luteinized or nonluteinized GC. The number of viable luteinized GC on day 8 was reduced (30-50%; P < 0.05) after exposure to 10 micrograms ac-LDL/ml or more, whereas only the highest dose (100 micrograms/ml) of LDL or HDL reduced cell survival. Ac-LDL did not alter the survival of nonluteinized GC in culture. Flow cytometric analyses using fluorescent-tagged lipoproteins (DiI-LDL/DiI-ac-LDL) demonstrated the uptake of both native and ac-LDL by luteinized GC. Uptake of DiI-LDL was competitively suppressed in a dose-dependent manner by unlabeled LDL, but not by ac-LDL. In contrast, DiI-ac-LDL uptake was competitively inhibited by both ac-LDL and LDL.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Division of Reproductive Sciences, Oregon Regional Primate Research Center, Beaverton 97006.
authors.forename: J D
authors.initials: JD
authors.lastname: Brannian
authors.forename: R L
authors.initials: RL
authors.lastname: Stouffer
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D001692:Biological Transport; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000494:pharmacology*; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology*; D006107:Granulosa Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008075:Lipoproteins, HDL / Q000378:metabolism; D008077:Lipoproteins, LDL / Q000378:metabolism / Q000494:pharmacology*; D007986:Luteinizing Hormone / Q000494:pharmacology*; D008253:Macaca mulatta; D010053:Ovary / Q000187:drug effects / Q000502:physiology*; D011374:Progesterone / Q000096:biosynthesis*; D013997:Time Factors
chemical_list: D006063:Chorionic Gonadotropin; D008075:Lipoproteins, HDL; D008077:Lipoproteins, LDL; C032629:acetyl-LDL; D011374:Progesterone; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone