Lipid droplets in cultured luteal cells in non-pregnant sheep fed different planes of nutrition

• Novel methodology combining cell culture, fluorescent staining, confocal microscopy and Imaris software allowed for 3D reconstruction of luteal cells. Lipid droplets were quantified in rendered 3D images of luteal cells.
• The size of lipid droplets differs in large vs. small luteal cells.
• Lipid droplet dynamics in large and small luteal cells are affected by the stage of the estrous cycle and plane of nutrition in sheep.

Accumulation of lipid droplets (LD) in luteal cells likely is important for energy storage and steroidogenesis in the highly metabolically active corpus luteum (CL). The objective of this study was to determine the effect of plane of nutrition on progesterone (P4) secretion, and lipid droplet number and size in cultured ovine luteal cells. Ewes were randomly assigned to one of three nutritional groups: control (C; 100% NRC requirements, n = 9), overfed (O; 2 × C, n = 12), or underfed (U; 0.6 × C, n = 10). Superovulation was induced by follicle stimulating hormone injections. At the early and mid-luteal phases of the estrous cycle, CL were dissected from ovaries, and luteal cells isolated enzymatically. Luteal cells were incubated overnight in medium containing serum in chamber slides. Media were then changed to serum-free and after 24 h incubation, media were collected for P4 analysis, and cells were fixed in formalin and stained with BODIPY followed by DAPI staining. Z-stacks of optical sections of large and small luteal cells (LLC and SLC, respectively) were obtained using a laser-scanning microscope. Rendered 3D images of individual LLC and SLC were analyzed for cell volume, and total and individual LD volume, number and percentage of cellular volume occupied by LD by using Imaris software. Concentrations of P4 in serum and media were greater (P < 0.05) at the mid than early-luteal phase, and were not affected by nutritional plane. LD total volume and number were greater (P < 0.001) in LLC than SLC; however, mean volume of individual LD was greater (P < 0.02) in SLC than LLC. In LLC, total LD volume was greater (P < 0.02) in O than C and U ewes. In SLC, total LD volume and number was greater (P < 0.003) at the mid than early-luteal phase, and percentage of cell volume occupied by LD was greater (P < 0.002) in U than C and O ewes. These data demonstrate that both stage of luteal development and nutritional plane affect selected LD measurements and thus may affect luteal functions. Furthermore, these data confirm that LD dynamics differ among parenchymal steroidogenic luteal cell types.