Differential uptake of fluorescent-tagged low density lipoprotein by cells from the primate corpus luteum: isolation and characterization of subtypes of small and large luteal cells.
Three enriched populations of cells [C alpha, non-steroidogenic cells less than or equal to 15 microns in diameter; R1, small (less than or equal to 15 microns) steroidogenic cells; and R3, large (greater than 20 microns) steroidogenic cells] have been isolated from the macque corpus luteum using flow cytometry based on light scatter properties. To determine whether the cell populations differ in their ability to bind and internalize low-density lipoprotein (LDL), collagenase-dispersed cells were prepared from the corpus luteum of rhesus monkeys at midluteal phase of the menstrual cycle. Cells were incubated in Hams F-10 medium containing fluorescent-tagged LDL (DiI-LDL). Optimal labeling occurred at 10 micrograms DiI-LDL/10(6) cells.ml incubated for 20 min at 37 degrees C. Labeled cells were analyzed and sorted by flow cytometry based on light scatter and fluorescence. Only 8.2 +/- 1.0% (n = 10) of C alpha cells exhibited fluorescence intensity greater than the autofluorescence of unlabeled cells. In contrast, 52.3 +/- 3.4% of R1 cells and 83.9 +/- 2.3% of R3 cells were fluorescent. Uptake of DiI-LDL was competitively inhibited when cells were conincubated with either unlabeled monkey or human LDL, but human high-density lipoprotein and very low-density lipoprotein were less effective. Progesterone (P) production by fluorescent [DiI-LDL(+)] R1 luteal cells was increased (P less than 0.001) in the presence of pregnenolone, but not human (h) CG, consistent with earlier results for the R1 population. Surprisingly, basal P production by the nonfluorescent [DiI-LDL(-)] R1 cells was similar to that by fluorescent cells and was stimulated by both hCG (P less than 0.01) and pregnenolone (P less than 0.001). Basal P production by DiI-LDL(+) R3 cells was nearly 10-fold greater than that by DiI-LDL(-) R3 cells. P secretion by both DiI-LDL(+) and (-) R3 cells was stimulated by hCG (P less than 0.01) and pregnenolone (P less than 0.001). DiI-LDL(-) C alpha cells produced barely detectable levels of P, but DiI-LDL(+) C alpha cells secreted P at levels similar to R1 cells. We conclude that: 1) multiparameter (light scatter and fluorescence) cell sorting is a useful method for separating enriched populations of cells from the corpus luteum; and 2) small and large luteal cell populations from the macaque corpus luteum consist of subtypes of steroidogenic cells that differ in lipoprotein uptake and/or gonadotropin sensitivity.
Brannian
J D
JD
Division of Reproductive Biology, Oregon Regional Primate Research Center, Beaverton 97006.
Shiigi
S M
SM
Stouffer
R L
RL
D000818:Animals; D001667:Binding, Competitive; D002232:Carbocyanines; D002469:Cell Separation; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D005260:Female; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008184:Luteal Cells / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D008253:Macaca mulatta; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D011284:Pregnenolone / Q000494:pharmacology; D011374:Progesterone / Q000096:biosynthesis
D002232:Carbocyanines; D006063:Chorionic Gonadotropin; D005456:Fluorescent Dyes; D008077:Lipoproteins, LDL; C024286:3,3'-dioctadecylindocarbocyanine; D011374:Progesterone; D011284:Pregnenolone