Reproduction Abstracts

Introduction: Lipopolysaccharide (LPS) is a pathogen-associated molecular pattern (PAMP), expressed by gram-negative bacteria. The specific receptor for LPS on host cells is TLR4, one of 11 members of the TLR family involved in innate immunity. Here we investigated i) changes in GC/TC expression of TLR4 during follicle development, ii) The effect of LPS on oestradiol secretion from non-luteinised granulosa cells (GC) and androstendione from non-luteinised theca cells (TC), iii) whether LPS exerts its effect via TLR4 and iv) the effect of LPS on theca and stromal cell (SC) migration.

Methods: GC and TC were isolated from bovine antral follicles (1–18 mm) and RNA extracts used for RT-qPCR analysis of relative gene expression (normalized to β-actin). GC and TC isolated from 4 to 6 mm follicles were cultured (serum-free) for four days with/without LPS and TLR4 inhibitor in the presence/absence of FSH (GC) or LH (TC). Media were assayed for steroids by ELISA and cell-lysates used for RT-qPCR. For wound-healing assays, TC and SC were cultured in 10% serum, a ‘scratch’ made in the near-confluent monolayer and fresh media added with/without LPS. Cell migration (% wound closure) was assessed over 24 h by time-lapse microscopy.

Results and discussion: Both cell-type and follicle category affected (P<0.001) levels of TLR4 mRNA during follicle development. Expression increased with follicle size in both cell types. LPS suppressed FSH-induced estradiol secretion by GC (P<0.01) and LH-induced androstendione secretion by TC (P<0.01). In GC, LPS down-regulated (P<0.001) CYP19A1 and up-regulated TNF-α and GPR77 expression. In TC, LPS down-regulated CYP17, INSL3 and StAR (P<0.001) while upregulating TNFR1 and NFkB (P<0.001). The inhibitory effect of LPS on GC/TC steroid secretion was blocked by TLR4 inhibitor. However, LPS had no significant effect on either TC or SC migration. Results confirm a profound inhibitory action of LPS on follicular steroidogenesis but it did not affect cell migration.