Lipopolysaccharide (LPS) inhibits steroid production in theca cells of bovine follicles in vitro: distinct effect of LPS on theca cell function in pre- and post-selection follicles

Abstract

In postpartum dairy cows, lipopolysaccharide (LPS) derived from gram-negative bacteria such as Escherichia coli causes uterine inflammation and leads to ovarian dysfunction. The aim of this study was to determine the effect of LPS on steroid production in bovine theca cells at different stages of follicular development. Theca cells isolated from pre- and post-selection follicles (PRFs, <8.5 mm in diameter, and POFs, >8.5 mm in diameter, respectively) of bovine ovaries were exposed to LPS under luteinizing hormone (LH) conditions, estradiol (E2) conditions or both conditions in vitro. Bovine theca cells expressed the LPS receptor gene complex: Toll-like receptor 4 (TLR4), CD14 and MD2. LPS suppressed progesterone (P4) and androstenedione (A4) production with downregulation of steroidogenic enzyme transcripts when theca cells were stimulated with LH. By contrast, LPS did not affect P4 or A4 production when theca cells were stimulated with E2. P4 and A4 production in theca cells from PRFs was suppressed by LPS as early as at 48 h of culture, whereas the effect of LPS on theca cells from POFs was observed at 96 h of culture. The results demonstrate that LPS inhibits steroid production in theca cells under LH conditions. Moreover, theca cells from POFs showed a slower response to LPS compared with that of theca cells from PRFs, which might imply a distinct effect of LPS on follicles at different developmental stages. These findings suggest a possible mechanism of ovarian dysfunction and subsequent infertility in cows with endometritis.

Fig. 1.

mRNA expression of (a) TLR4, (b) CD14 and (c) MD2 in the theca cells of pre-selection follicles (PRFs; <8.5 mm, n = 5) and post-selection follicles (POFs; >8.5 mm , n = 5). All values are means ± standard error of the mean. Values with different letters (a, b) are significantly different between groups (P < 0.05).

Fig. 2.

Effect of lipopolysaccharide (LPS) on number of viable theca cells isolated from post-selection follicles (>8.5 mm; a–c) and pre-selection follicles (<8.5 mm; d–f) during term 1 (white circles, 0–48 h) and term 2 (black triangles, 48–96 h). Theca cells were stimulated with 2.5 ng/ml luteinizing hormone (LH; a, d), 100 ng/ml estradiol (E2; b, e) or LH and E2 (c, f). All values are means ± standard error of the mean of three independent experiments.

Fig. 3.

Effect of lipopolysaccharide (LPS) on the production of progesterone (P4; a–c) and androstenedione (A4; d–f) in bovine theca cells from post-selection follicles (>8.5 mm) during term 1 (white circles, 0–48 h) and term 2 (black triangles, 48–96 h). Theca cells were stimulated with 2.5 ng/ml luteinizing hormone (LH; a, d), 100 ng/ml estradiol (E2; b, e) or LH and E2 (c, f). Data are expressed as the percentage of control (100%) steroid accumulation in the culture medium. All values are means ± standard error of the mean of three independent experiments. Values with different letters (a, b) are significantly different between groups (P < 0.05).

Fig. 4.

Effect of lipopolysaccharide (LPS) on the mRNA expression of StAR (a–c) CYP17 (d–f) and LHr (d–f) in bovine theca cells from post-selection follicles (>8.5 mm) at 96 h of culture. Theca cells were stimulated with 2.5 ng/ml luteinizing hormone (LH; a, d, g), 100 ng/ml estradiol (E2; b, e, h) or LH and E2 (c, f, i). All values are means ± standard error of the mean of three independent experiments. Values with different letters (a, b) are significantly different between groups (P < 0.05).

Fig. 5.

Effect of lipopolysaccharide (LPS) on the production of progesterone (P4; a-c) and androstenedione (A4; d-f) in bovine theca cells from pre-selection follicles (<8.5 mm) during term 1 (white circles, 0–48 h) and term 2 (black triangles, 48–96 h). Theca cells were stimulated with 2.5 ng/ml luteinizing hormone (LH; a, d), 100 ng/ml estradiol (E2; b, e) or LH and E2 (c, f). Data are expressed as the percentage of control (100%) steroid accumulation in the culture medium. All values are means ± standard error of the mean of three independent experiments. Values with different letters (a, b) are different between groups (P < 0.05)

Fig. 6.

Effect of lipopolysaccharide (LPS) on the mRNA expression of StAR (a–c), CYP17 (d–f) and LHr (d–f) in bovine theca cells from pre-selection follicles (<8.5 mm) at 48 h of culture. Theca cells were stimulated with 2.5 ng/ml luteinizing hormone (LH; a, d, g), 100 ng/ml estradiol (E2; b, e, h) or LH and E2 (c, f, i). All values are means ± standard error of the mean of three independent experiments. Values with different letters (a, b) are significantly different between groups (P < 0.05).