Lipopolysaccharide Inhibits in Vitro Luteinizing Hormone-Stimulated Rat Ovarian Granulosa Cell Estradiol but Not Progesterone Secretion1

Abstract

Endotoxin, known as lipopolysaccharide (LPS), is a component of gram-negative bacterial cell walls and is a potent immunostimulator, inducing the release of several cytokines, such as tumor necrosis factor alpha (TNF alpha) and interleukins (IL) 1, 6, and 8. A previous study with immature rats revealed that exogenous administration of LPS inhibits ovarian estradiol secretion in response to eCG. The present study was undertaken in order to determine whether LPS could directly inhibit rat granulosa cell (GC) steroid secretion. GC were collected and purified from 26-day-old hypophysectomized female rats (hypophysectomy on Day 23). Purified GC were highly responsive to FSH (1-100 ng/ml), leading to increased estradiol, progesterone, and cAMP accumulation in culture media. GC were also capable of binding 125I-labeled hCG and were responsive to LH stimulation. Treatment of GC with LPS (1-100 ng/ml) led to a significant (p < 0.01) dose-dependent decrease in LH-stimulated estradiol accumulation in culture media (maximum 75% inhibition). However, treatment of GC with LPS had no significant effect on FSH-stimulated progesterone or estradiol, or LH-stimulated progesterone accumulation in culture media. GC stimulated with 8-bromo cAMP were also insensitive to the effects of LPS. LPS had no significant effect on 125I-labeled hCG binding to GC homogenates, nor did it have any significant effect on FSH or LH-stimulated cAMP accumulation. Treatment of both FSH and LH-stimulated GC with LPS was associated with an increase in IL-6 bioactivity in culture media. This effect could be blocked with the nonreceptor tyrosine kinase inhibitor herbimycin A. TNF alpha bioactivity was undetectable with or without LPS challenge. Direct challenge of GC with recombinant murine IL-6 had no effect on either FSH or LH-stimulated estradiol whereas TNF alpha inhibited FSH-stimulated estradiol secretion. Collectively, these results suggest that the inhibitory effects of LPS were not mediated by either IL-6 or TNF alpha. Treatment of GC with the epidermal growth factor receptor tyrosine kinase inhibitor, tyrphostin A46, blocked the inhibitory effects of LPS on steroid secretion and was associated with an increased cAMP accumulation in culture media. The results indicate that LPS inhibits in vitro GC estradiol secretion. This effect appears to be restricted to the LH-stimulated aromatization of androgens to estrogen and may involve a tyrosine kinase signaling pathway.