Divergent effects of interleukin-1 beta on steroidogenesis and matrix metalloproteinase inhibitor expression and activity in cultured rat granulosa cells

Abstract

The periovulatory increase in ovarian matrix metalloproteinase inhibitor (MMPI) expression is regulated both in vitro and in vivo 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 matrix metalloproteinase inhibitor expression and activity. Using an in vitro rat granulosa cell model, these parameters were assessed in response to IL-1 beta or LH administration alone or in combination. Granulosa 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 conditions 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 granulosa cells and assessed for Northern analysis of tissue inhibitor of metalloproteinase-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 significantly (P < 0.05) increased MMPI activity in granulosa cell-conditioned medium above control values (40.9 +/- 3.0% inhibition for IL-1 beta and 67.1 +/- 5.6% inhibition for LH vs. 31.4 +/- 2.4% inhibition for 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). Methylamine hydrochloride treatment revealed that the majority of inhibitor activity in all treatment groups was derived from TIMPs. The patterns of TIMP-1, TIMP-2, and TIMP-3 messenger RNA expression among the treatment groups paralleled the TIMP-derived inhibitor activity, in that both IL-1 beta and LH alone stimulated transcript expression of all three TIMPs. In addition, an increase in progesterone production was associated 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 expression 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 MMPI expression as well as granulosa cell steroidogenesis, and that this cytokine has divergent actions in the presence and absence of LH.