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 > or = 80%. When dispersed cells were analyzed by flow cytometry based on forward and 90 degrees 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 microns in diameter, stained positive (+) for 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity. The remainder of P1 cells were 3 beta-HSD negative and < or = 22 microns. The size distribution of P2 was similar to that of P1, but P2 contained more (53.3 +/- 4.2%; n = 4) 3 beta-HSD (+) cells. P3 consisted mostly (88.5 +/- 4.6%; n = 3) of 3 beta-HSD (+) cells > 25 microns in diameter. Cell subpopulations were incubated (n = 6) at 37 degrees 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).(ABSTRACT TRUNCATED AT 250 WORDS)