Isolation of ovine luteal cell subpopulations by flow cytometry

Differences in the characteristics of small and large luteal cells, as reported by various laboratories, may be due to species diversity and/or methodological differences in cell preparation. To evaluate whether the method of cell separation affects the properties of luteal cell subpopulations, we sorted and characterized sheep luteal cells by flow cytometry via methods previously used to investigate luteal cell subtypes from the macaque corpus luteum. Corpora lutea were obtained from superovulated ewes on Day 10 after hCG injection and enzymatically dissociated. Dispersed cells were shipped overnight on ice from the University of Arizona to the Oregon Regional Primate Research Center. Viability of cells upon arrival was ≥ 80%. When dispersed cells were analyzed by flow cytometry based on forward and 90° light scatter, three distinct subpopulations (P₁, P₂, P₃) were identified. In P₁, 35.5 ± 2.1% of cells, most (97.0 ± 0.6%; n = 3) of which were 15-22 μm in diameter, stained positive (+) for 3β-hydroxysteroid dehydrogenase (3β-HSD) activity. The remainder of P₁ cells were 3β-HSD negative and ≤ 22 μm. The size distribution of P₂ was similar to that of P₁, but P₂ contained more (53.3 ± 4.2%; n = 4) 3β-HSD (+) cells. P₃ consisted mostly (88.5 ± 4.6%; n = 3) of 3β-HSD (+) cells > 25 μm in diameter. Cell subpopulations were incubated (n = 6) at 37°C for 3 h with or without hCG (0.1-100 ng/ml), prostaglandin E₂ (PGE₂; 500 ng/ml), or dibutyryl (db)-cAMP (5 mM). Unstimulated progesterone (P) production by P₃ (large) cells (41.6 ± 10.9 ng/ml/10⁴ cells/3 h) was nearly 10-fold greater (p < 0.05) than that by P₂ (small) cells (4.6 ± 1.0 ng/ml/10⁴ cells/3 h). Secretion of P by small cells was increased (p < 0.05) ≥ 10-fold by hCG (1-100 ng/ml) and db-cAMP. Conversely, hCG (10-100 ng/ml) slightly stimulated (1.5-fold; p < 0.05) P production by large cells, whereas db-cAMP caused a greater (~2-fold; p < 0.05) increase. PGE₂ modestly elevated (~2-fold; p < 0.05) P secretion in small but not in large cells. The small P₁ cells were steroidogenically similar to P₂ cells. In summary, data on the size distribution, steroidogenic activity, and agonist sensitivity of ovine small and large luteal cells isolated by flow cytometry were, for the most part, similar to previously published data obtained from sheep cells separated by elutriation. However, the isolation, based on light scatter, of two subpopulations containing steroidogenic cells ≤ 25 μm suggests the existence of subtypes of small luteal cells. We conclude that the reported differences in the characteristics of luteal cell subpopulations from the sheep and macaque are due primarily to interspecies variation rather than to methodological differences in cell preparation. Furthermore, flow cytometry may be useful in identifying additional subtypes of ovine luteal cells.