EVALUATION OF IN-VITRO CULTURE CONDITIONS TO DEMONSTRATE PREGNANCY-DEPENDENT CHANGES IN LUTEAL FUNCTION IN THE PIG

This study was designed to evaluate the most effective in vitro cultur e conditions for study of pregnancy-dependent changes in LH-stimulated steroidogenesis in porcine luteal tissue. Mixed luteal cells recovere d from the CL of cyclic (Day 12) and pregnant (Day 12 and 16) pigs wer e incubated either in Eagle's minimum essential medium (MEM) or in MEM supplemented with different concentrations of porcine or calf serum o r BSA, or were incubated in a defined medium (DM) containing 2 mu g/ml insulin, 40 ng/ml hydrocortisone, 5 mu g/ml transferrin, and 10(-7) M thyroxine. Cultures were treated with various doses of porcine LH (0. 1, 1, 10, 100 ng/ml). Additionally, large and small luteal cells of Da y 12 cyclic and pregnant pigs were incubated for 4 h in medium 199 sup plemented with 2% porcine serum in the absence or presence of various concentrations of LH. Overall, progesterone production by the luteal c ells of Day 12 pregnant pigs was significantly (p < 0.05) higher than that by cells of the cyclic pigs. However, the luteal cells of pregnan t Day 16 pigs released significantly (p < 0.05) less progesterone than the other groups. LH had a stimulatory effect (p < 0.001) on progeste rone release by mixed luteal cells that was independent of the type of medium supplementation or reproductive status. Porcine serum, calf se rum, and BSA (p < 0.001, p < 0.001, and p < 0.05, respectively) also s timulated progesterone production. However, the doses of porcine and c alf serum or BSA that stimulated progesterone release were dependent o n the reproductive status of the pig. Progesterone production by large luteal cells always exceeded (p < 0.001) that of small cells and was stimulated by LH (p < 0.007). Small luteal cells of both cyclic and pr egnant (Day 12) gilts did not respond to LH treatment, and small lutea l cells of cyclic pigs produced significantly (p < 0.04) more progeste rone than those of pregnant pigs. Our results demonstrate differences in the steroidogenic potential of the CL of pregnant and cyclic pigs o n Day 12. LH influenced steroidogenesis in vitro in luteal cells obtai ned from early-pregnant pigs and from pigs in the midluteal phase of t he estrous cycle. Our data support the suggestion that an interaction between small and large luteal cells enhances response to LH stimulati on in vitro, but the exact physiological role of porcine small and lar ge luteal cells still needs further study.