A novel approach to quantifying ovarian cell lipid content and lipid accumulation in vitro by confocal microscopy in lean women undergoing ovarian stimulation for in vitro fertilization (IVF)

Abstract

Purpose: To quantify intracellular lipid levels in cumulus cells (CCs) and mural granulosa cells (MGCs) of lean women undergoing gonadotropin therapy for in vitro fertilization (IVF), based upon different cell preparation methods.

Methods: CCs and MGCs from 16 lean women undergoing ovarian stimulation for IVF were studied. Cells were pooled by cell type, with each type of cell separated into two groups for determination of initial lipid content (Method 1) and subsequent lipid accumulation in vitro (Method 2). Cells for initial lipid content were immediately fixed at the time of the oocyte retrieval with 4% paraformaldehyde in suspension, while those for subsequent lipid accumulation in vitro were cultured for 4 h with 5% fetal calf serum and then fixed. Cells were treated with lipid fluorescent dye BODIPY® FL C16 and nuclear marker DAPI. Intracellular lipid was quantified by confocal microscopy, using ImageJ software analysis.

Results: There was no significant effect of cell type (P = 0.2) or cell type-cell preparation method interaction (P = 0.8) on cell area (Method 1: CC 99.7 ± 5.1, MGC 132.8 ± 5.8; Method 2: CC 221.9 ± 30.4, MGC 265.1 ± 48.5 μm(2)). The mean area of all cells combined was significantly less for cells prepared by Method 1 (116.2 ± 4.9 μm(2)) vs. Method 2 (243.5 ± 22.5 μm(2), P < 0.00005). Intracellular lipid level, however, was significantly altered by cell preparation method (P < 0.05; cell preparation method-cell type interaction, P < 0.00001). Initial lipid content was significantly lower in CC (74.5 ± 9.3) than MGC (136.3 ± 16.7 fluorescence/cell area, P < 0.00005), while subsequent lipid accumulation in vitro was significantly higher in CC (154.0 ± 9.1) than MGC (104.6 ± 9.9 fluorescence/cell area, P < 0.00001). The relatively diminished initial CC lipid content compared to subsequent CC lipid accumulation in vitro (P < 0.00001), and the opposite pattern for MGC (P < 0.05), significantly lowered the CC/MGC lipid ratio in Method 1 (0.55 ± 0.04) vs. Method 2 (1.58 ± 0.10, P < 0.00001).

Conclusions: Differential uptake or utilization of lipid by CC and MGC occurs during oocyte maturation and steroidogenesis, respectively, with the amount of lipid present in ovarian cells a function of both the follicular microenvironment at the time of the oocyte retrieval and the capacity of these cells to accumulate lipid in vitro over time.

Fig. 1

Overlap images of BODIPY® FL C₁₆ and DAPI. Method 1: Initial lipid content of a cumulus cells and b mural granulosa cells; Method 2: Lipid accumulation in vitro of c cumulus cells and d mural granulosa cells. Overlap and single channel images were taken with a confocal x63 oil objective, and quantification was performed on single channel BODIPY® FL C₁₆ images

Fig. 2

a Mean ovarian cell fluorescence and b ovarian cell area based upon cell preparation method. The amount of ovarian cell lipid was significantly altered by cell type and cell preparation method. Initial lipid content by Method 1 was significantly lower in cumulus than mural granulosa cells; lipid accumulation in vitro by Method 2 was significantly higher in cumulus than mural granulosa cells (cell type effect). For cumulus cells, initial lipid content by Method 1was lower than lipid accumulation in vitro by Method 2, while the opposite pattern was true for mural granulosa cells (cell preparation method effect). Mean cell area of cumulus and mural granulosa cells combined was significantly less for all cells prepared by Method 1 (initial lipid content) compared to Method 2 (lipid accumulation in vitro). *, P < 0.00005; **, P < 0.00001 cumulus vs. mural granulosa cell; †, P < 0.05; ††, P < 0.00005; †††, P < 0.00001 Method 1 vs. Method 2