Ovarian follicular cells have innate immune capabilities that modulate their endocrine function

Abstract

Oestrogens are pivotal in ovarian follicular growth, development and function, with fundamental roles in steroidogenesis, nurturing the oocyte and ovulation. Infections with bacteria such as Escherichia coli cause infertility in mammals at least in part by perturbing ovarian follicle function, characterised by suppression of oestradiol production. Ovarian follicle granulosa cells produce oestradiol by aromatisation of androstenedione from the theca cells, under the regulation of gonadotrophins such as FSH. Many of the effects of E. coli are mediated by its surface molecule lipopolysaccharide (LPS) binding to the Toll-like receptor-4 (TLR4), CD14, MD-2 receptor complex on immune cells, but immune cells are not present inside ovarian follicles. The present study tested the hypothesis that granulosa cells express the TLR4 complex and LPS directly perturbs their secretion of oestradiol. Granulosa cells from recruited or dominant follicles are exposed to LPS in vivo and when they were cultured in the absence of immune cell contamination in vitro they produced less oestradiol when challenged with LPS, although theca cell androstenedione production was unchanged. The suppression of oestradiol production by LPS was associated with down-regulation of transcripts for aromatase in granulosa cells, and did not affect cell survival. Furthermore, these cells expressed TLR4, CD14 and MD-2 transcripts throughout the key stages of follicle growth and development. It appears that granulosa cells have an immune capability to detect bacterial infection, which perturbs follicle steroidogenesis, and this is a likely mechanism by which ovarian follicle growth and function is perturbed during bacterial infection.

Figure 1

Androstenedione production of theca cells isolated from (A) small (<4 mm diameter), (B) medium (4–8 mm diameter) or (C) large (>8 mm diameter) bovine follicles. Theca cells were treated with LPS at the concentrations indicated. After 48 h treatment, supernatants were harvested and androstenedione production was measured by RIA. The number of theca cells (×10⁶/ml) after 96 h culture are indicated (D to F). Numerical values are presented as the mean+s.e.m. of three experiments.

Figure 2

Oestradiol and progesterone production of granulosa cells isolated from (A and D) small (<4 mm diameter), (B and E) medium (4–8 mm diameter) or (C and F) large (>8 mm diameter) bovine follicles. Granulosa cells were treated with LPS at the concentrations indicated. After 48 h treatment, supernatants were harvested and steroid production was measured by RIA. *P<0.05 compared with control, within follicle size. Numerical values are presented as the mean+s.e.m. of three experiments.

Figure 3

Oestradiol and progesterone production of granulosa cells isolated from (A and D) small (<4 mm diameter), (B and E) medium (4–8 mm diameter) or (C and F) large (>8 mm diameter) bovine follicles. Granulosa cells were treated with LPS at the concentrations indicated. After 96 h treatment, supernatants were harvested and steroid production was measured by RIA. The number of granulosa cells (×10⁶/ml) after 96 h culture are indicated (G to I). *P<0.05 compared with control, within follicle size. Numerical values are presented as the mean+s.e.m. of three experiments.

Figure 4

Analysis of gene expression by granulosa cells associated with reproductive function. Granulosa cells isolated from medium (4–8 mm diameter) or large (>8 mm diameter) follicles were stimulated with 10 μg/ml LPS for 48 h and harvested, RNA was isolated and reverse transcribed as described in Materials and Methods. cDNA was analysed for the presence of (A) aromatase, FSHR, LHR, ERα, and (B) NOS2, IL-1α and TNFα transcripts using the indicated primer pairs (Table 1). A representative result is shown (n=3). (C) PCR bands were analysed and are represented as LPS treatment (open bar) relative to nil control (closed bar) for granulosa cells isolated from large (>8 mm diameter) follicles. Results are presented as the mean+s.e.m. of three experiments. *P<0.05 compared with control.

Figure 5

Analysis of LPS receptor complex and immune mediator gene expression by granulosa cells. Granulosa cells isolated from medium (4–8 mm diameter) or large (>8 mm diameter) follicles were stimulated with 10 μg/ml LPS for 48 h and harvested, RNA was isolated and reverse transcribed as described in Materials and Methods. cDNA was analysed for the presence of (A) CD45, (B) TLR4, CD14, MD-2, GAPDH, using the indicated primer pairs (Table 1). A representative result is shown (n=3).