DIVERGENT EFFECTS OF INTERLEUKIN-1-BETA ON STEROIDOGENESIS AND MATRIXMETALLOPROTEINASE INHIBITOR EXPRESSION AND ACTIVITY IN CULTURED RAT GRANULOSA-CELLS

The periovulatory increase in ovarian matrix metalloproteinase inhibit or (MMPI) expression is regulated both in vitro and in uivo by LH, but the intermediary steps in this process are uncertain. The purpose of this experiment was to determine whether interleukin-1 beta (IL-1 beta ), a known modulator of MMPI expression in other systems and one that is induced by LH in the ovary, is capable of regulating granulosa matr ix metalloproteinase inhibitor expression and activity. Using an in vi tro rat granulosa cell model, these parameters were assessed in respon se to IL-1 beta or LH administration alone or in combination. Granulos a cells were obtained from 24-day-old immature female rats primed with 20 IU PMSG at 22 days of age. Cells were cultured under serum-free co nditions for 24 h at 37 C in the presence of medium (control), LH (100 ng/ml), IL-1 beta (10 ng/ml), or LH plus IL-1 beta. MMPI activity in the conditioned medium was assessed using a colorimetric assay (n = 6) , whereas progesterone and estrogen concentrations in the conditioned medium were determined by RIA (n = 6). RNA was isolated from the granu losa cells and assessed for Northern analysis of tissue inhibitor of m etalloproteinase-1 (TIMP-1; n = 4), TIMP-2 (n = 3), and TIMP-3 (n = 3) expression. When added to granulosa cells, IL-1 beta and LH each sign ificantly (P < 0.05) increased MMPI activity in granulosa cell-conditi oned medium above control values (40.9 +/- 3.0% inhibition for IL-1 be ta and 67.1 +/- 5.6% inhibition for LH vs. 31.4 +/- 2.4% inhibition fo r controls). When added in combination, IL-1 beta had no effect on LH- stimulated inhibitor activity (67.1 +/- 5.6% inhibition vs. 69.9 +/- 5 .1% inhibition for LH and LH plus IL-1 beta, respectively). Methylamin e hydrochloride treatment revealed that the majority of inhibitor acti vity in all treatment groups was derived from TIMPs. The patterns of T IMP-1, TIMP-2, and TIMP-3 messenger RNA expression among the treatment groups paralleled the TIMP-derived inhibitor activity, in that both I L-1 beta and LH alone stimulated transcript expression of all three TI MPs. In addition, an increase in progesterone production was associate d with IL-1 beta-stimulated (1.22-fold over control values; P = 0.0006 ) and LH-stimulated (9.6-fold over control values; P = 0.007) MMPI exp ression and activity. Lastly, IL-1 beta and LH significantly (P < 0.05 ) decreased estrogen production by approximately 33% compared to that in cultures with LH only. It is concluded from the current study that IL-1 beta is a mediator of MMPT expression as well as granulosa cell s teroidogenesis, and that this cytokine has divergent actions in the pr esence and absence of LH.