Four-day-old bovine corpus luteum: progesterone production and identification of matrix metalloproteinase activity in vitro

Little is known about matrix metalloproteinases (MMPs), enzymes that are required for the structural remodeling and angiogenesis that occur in the corpus luteum (CL) during the first several days postovulation. In fact, little attention has focused on early CL function including the regulation of progesterone (P4) production. Thus, the objective of the present study was 1) to investigate the effects of insulin, LH, and dibutyryl cAMP on P4 production and cell numbers and 2) to identify MMPs in the 4-day-old CL, with use of a defined culture system. Cultures were seeded with either 1 X 10(6) or 0.5 X 10(6) cells. All cultures containing insulin had higher P4 levels and cell numbers (p < 0.05) than those without. In cultures containing insulin, basal P4 levels were high throughout the culture period. Furthermore, neither LH nor dibutyryl cAMP stimulated P4 production (p > 0.05) at a seeding density of 1 X 10(6), whereas they stimulated P4 production (p < 0.05) at a seeding density of 0.5 X 10(6) on Days 6 and 8 of culture. In conditioned medium of control cultures seeded with 0.5 X 10(6) cells, substrate gel electrophoresis (zymography) showed two intense bands that migrated at Mr approximately 97 000 and approximately 65 000-64 000, while two weaker ones migrated at Mr approximately 88 000 and approximately 64 000-63 000. The molecular weights of the Mr approximately 97 000 and approximately 88 000 species were consistent with MMP-9 family members, while the molecular weights of the Mr approximately 65 000-64 000 and approximately 64 000-63 000 species were consistent with MMP2 family members. Incubation with 1,10 phenanthroline verified that these enzymes were bona fide MMPs. Furthermore, treatment of conditioned medium with p-aminophenylmercuric acetate (APMA) diminished the intensity of the Mr approximately 97 000 and approximately 65 000-64 000 bands and enhanced the Mr approximately 63 000-62 000 band, indicating that the Mr approximately 97 000 and approximately 65 000-64 000 species were latent forms of MMP-9 and MMP-2, respectively. In addition, no differences in enzyme activity were observed between control and LH samples, while only the Mr approximately 97 000 band decreased significantly in intensity over the 8-day culture period, as corroborated by densitometry. In summary, cells from 4-day-old CL secreted high levels of basal P4, and these levels and cell numbers required insulin. Furthermore, at the lower seeding density and in the presence of insulin, LH and dibutyryl cAMP significantly stimulated P4 over controls on Days 6 and 8 of culture. In addition, MMP-9-like and MMP-2-like enzymes were identified in conditioned medium, and their activity was not regulated by LH. Thus, the present findings suggested that luteal cells from 4-day-old CL produce P4 and are a potential source of MMP-like enzymes.