Control of progesterone production in small and large bovine luteal cells separated by flow cytometry

Corpora lutea were collected from Holstein heifers on Days 10 and 12 of the oestrous cycle and the cells were dispersed with collagenase. The dispersed cells were separated into preparations of highly purified (90-99%) small (less than 20 microns) and large (greater than 25 microns) luteal cells by unit gravity sedimentation and fluorescence-activated cell sorting. Net progesterone accumulation by 1 x 10(5) small cells and 1 x 10(3) large cells during 2 and 4 h incubations, respectively, were measured after additions of LH, PGF-2 alpha, and phorbol esters, alone and in combination. Progesterone synthesis was increased (P less than 0.05) by phorbol dibutyrate (PBt2) or PGF-2 alpha (P less than 0.05) in small, but not in large, luteal cells (10.1 +/- 3.0 and 18.1 +/- 5.0 ng/10(5) cells for 0 and 50 nM-PBt2, and 19.9 +/- 3.2 and 44.2 +/- 9.3 ng/10(5) cells for 0 and 1 microgram PGF-2 alpha/ml). The previously reported stimulatory effects of PKC activation and PGF-2 alpha addition to total dispersed cell preparations are therefore entirely attributable to the small, theca-derived cells. Small cells responded to low levels of LH (9.1 +/- 1.1, 69.0 +/- 5.4 and 154.7 +/- 41.4 ng/10(5) cells for 0, 1 and 5 ng LH/ml, respectively, P less than 0.05), while large cells responded only to high levels of LH (1635 +/- 318, 2662 +/- 459 and 3386 +/- 335 pg/10(3) cells for 0, 100 and 1000 ng LH/ml, respectively, P less than 0.05). PGF-2 alpha inhibited LH-, 8-Br-cAMP- and forskolin-stimulated progesterone synthesis in the large cells (3052 +/- 380, 3498 +/- 418, 3202 +/- 391 pg/10(3) cells for 1 microgram LH/ml, and 0.5 mM-8-Br-cAMP, and 1 microM-forskolin respectively and 1750 +/- 487, 2255 +/- 468, 2165 +/- 442 pg/10(3) cells for PGF-2 alpha + LH, PGF-2 alpha + 8-Br-cAMP and PGF-2 alpha + forskolin, respectively), indicating that the inhibitory effect of PGF-2 alpha on progesterone synthesis in large cells occurs at a site distal to cAMP generation. These results suggest that the large cells are the targets of the luteolytic effects of PGF-2 alpha, while the small cells are responsible for the previously reported luteotrophic effect of PGF-2 alpha in vitro.