Reproduction Abstracts

Introduction: Cyclic ovarian function involves continual tissue remodelling. During ovulation macrophages invade the ovary and secret pro-inflammatory cytokines such us TNF- and IL6 that have local actions on ovarian cells. Here we investigated i) the effect of macrophages on secretion of oestradiol by granulosa cells (TC) and androstenedione by theca cells (TC) and ii) the effect of macrophages on TC and stroma cell (SC) migration was also assessed using an in vitro ‘wound healing’ assay.

Methods: Bovine monocyte-derived macrophages were prepared from citrated blood. GC and TC were isolated from 4 to 6 mm follicles and cultured (serum-free) for 4 days with/without macrophages in the presence/absence of LH (TC) or FSH (GC). Media were assayed for steroids and RNA extracted for gene expression analysis using RT-qPCR (normalized to β-actin). For wound-healing (‘scratch’) assays TC and cortical SC were cultured (10% serum) with/without macrophages for 2 days. A ‘scratch’ was made in the near confluent monolayer and cell migration (% wound closure) assessed over 24 h.

Results and discussion: Macrophages suppressed FSH-induced estradiol secretion by GC (P<0.0001) and LH-induced androstendione secretion by TC (P<0.001). The inhibitory action of macrophages on GC was accompanied by down-regulation (P<0.001) of CYP19A1 (tenfold).and up-regulation of TNC and SLPI expression. In TC, macrophages reduced expression of several transcripts including INSL3 (~15-fold), CYP17A1 (approximately sixfold), LHR (fivefold), and HSD3B (fivefold). Co-culture of both TC and SC with macrophages accelerated wound healing (P<0.001) while dexamethasone (10⁻⁸–10⁻⁶ M) retarded wound healing. Interestingly, exogenous TNC also accelerated wound healing in both SC and TC monolayers (P<0.0001) suggesting an involvement in the response to macrophages.

Supported by HCED-Iraq.