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

John D. Brannian, Stanley M. Shiigi, Richard Stouffer

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    14 Citations (Scopus)

    Abstract

    Three enriched populations of cells [Cα, non-steroidogenic cells ≤15 μm in diameter; R1, small (≤15 μm) steroidogenic cells; and R3, large (>20 μm) steroidogenic cells] have been isolated from the macaque 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 μg DiI-LDL/106 cells·ml incubated for 20 min at 37 C. Labeled cells were analyzed and sorted by flow cytometry based on light scatter and fluorescence. Only 8.2 ± 1.0% (n = 10) of Cα 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 coincubated 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 <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 <0.01) and pregnenolone (P <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 <0.01) and pregnenolone (P <0.001). DiI-LDL(-) Ca cells produced barely detectable levels of P, but DiI-LDL(H-) Ca 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.

    Original languageEnglish (US)
    Pages (from-to)3247-3253
    Number of pages7
    JournalEndocrinology
    Volume129
    Issue number6
    StatePublished - Dec 1991

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    Luteal Cells
    Corpus Luteum
    LDL Lipoproteins
    Primates
    Pregnenolone
    Population
    Fluorescence
    Macaca
    Light
    Flow Cytometry

    ASJC Scopus subject areas

    • Endocrinology
    • Endocrinology, Diabetes and Metabolism

    Cite this

    @article{312dee4b2efb47adbc3a9e69677d6421,
    title = "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",
    abstract = "Three enriched populations of cells [Cα, non-steroidogenic cells ≤15 μm in diameter; R1, small (≤15 μm) steroidogenic cells; and R3, large (>20 μm) steroidogenic cells] have been isolated from the macaque 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 μg DiI-LDL/106 cells·ml incubated for 20 min at 37 C. Labeled cells were analyzed and sorted by flow cytometry based on light scatter and fluorescence. Only 8.2 ± 1.0{\%} (n = 10) of Cα 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 coincubated 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 <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 <0.01) and pregnenolone (P <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 <0.01) and pregnenolone (P <0.001). DiI-LDL(-) Ca cells produced barely detectable levels of P, but DiI-LDL(H-) Ca 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.",
    author = "Brannian, {John D.} and Shiigi, {Stanley M.} and Richard Stouffer",
    year = "1991",
    month = "12",
    language = "English (US)",
    volume = "129",
    pages = "3247--3253",
    journal = "Endocrinology",
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    publisher = "The Endocrine Society",
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    }

    TY - JOUR

    T1 - Differential uptake of fluorescent-tagged low density lipoprotein by cells from the primate corpus luteum

    T2 - Isolation and characterization of subtypes of small and large luteal cells

    AU - Brannian, John D.

    AU - Shiigi, Stanley M.

    AU - Stouffer, Richard

    PY - 1991/12

    Y1 - 1991/12

    N2 - Three enriched populations of cells [Cα, non-steroidogenic cells ≤15 μm in diameter; R1, small (≤15 μm) steroidogenic cells; and R3, large (>20 μm) steroidogenic cells] have been isolated from the macaque 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 μg DiI-LDL/106 cells·ml incubated for 20 min at 37 C. Labeled cells were analyzed and sorted by flow cytometry based on light scatter and fluorescence. Only 8.2 ± 1.0% (n = 10) of Cα 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 coincubated 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 <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 <0.01) and pregnenolone (P <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 <0.01) and pregnenolone (P <0.001). DiI-LDL(-) Ca cells produced barely detectable levels of P, but DiI-LDL(H-) Ca 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.

    AB - Three enriched populations of cells [Cα, non-steroidogenic cells ≤15 μm in diameter; R1, small (≤15 μm) steroidogenic cells; and R3, large (>20 μm) steroidogenic cells] have been isolated from the macaque 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 μg DiI-LDL/106 cells·ml incubated for 20 min at 37 C. Labeled cells were analyzed and sorted by flow cytometry based on light scatter and fluorescence. Only 8.2 ± 1.0% (n = 10) of Cα 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 coincubated 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 <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 <0.01) and pregnenolone (P <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 <0.01) and pregnenolone (P <0.001). DiI-LDL(-) Ca cells produced barely detectable levels of P, but DiI-LDL(H-) Ca 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.

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