Abstract
Natriuretic peptides (NPs) have been reported to have critical roles in follicular development and oocyte maturation in rodents. This study aimed to extend our current understanding of NP-mediated signalling pathways and mechanisms of action in the follicles of a monovulatory species. Ovine granulosa cells (GCs) and theca cells (TCs) were cultured under conditions designed to allow gonadotrophin-stimulated cell differentiation. Gene expression analysis was performed by qualitative (q)PCR for NPs and NPRs (between 16 and 96 h of culture) and VEGF120 and VEGF164 (between 16 and 144 h of culture). A qualitative analysis of the production of NP/NPR family members and NP ligand/receptor associations was carried out utilising a highly sensitive immunological approach known as 'proximity ligation assay' (PLA). All NPRs were observed in GCs, while NPRA was absent in TCs. In GCs, gene expression of NPRA, NPRB and NPRC was apparent but only active BNP and CNP and not ANP, were detected. Also in GCs, ANP but not CNP was able to significantly (P < 0.05) reduce oestradiol and increase (P < 0.05) progesterone. Inhibition of VEGF164 by ANP and CNP (P < 0.01) after 48 h of culture preceded up-regulation of VEGF120 by ANP (P < 0.01) after 144 h, but not CNP. Taken together, these findings appear to demonstrate that NP responsiveness in the GC compartment of sheep follicles is multi-facilitated, utilising both autocrine and paracrine stimulation pathways.
Highlights
It is well established that members of the natriuretic peptide (NP) superfamily of ligands and their receptors (NPRs) are expressed in several locations within the female reproductive system of a number of poly-ovular mammals, such as rat (Gutkowska et al 1993), mice (Tsai et al 2005) and pigs (Zhang et al 2005)
Utilising a ‘single target’ proximity ligation assay (PLA, Methods), NP receptors A, B and C appeared abundant in cultured ovine granulosa cells (GCs) (Fig. 2)
In cultured ovine theca cells (TCs), NPRB and NPRC appeared abundant, but in these cells cluster formation or cell-cell proximity was not noted to be a prerequisite, and the A-type receptors were found to be absent or very poorly represented. These outcomes broadly matched the findings of the immuno-histochemical (IHC) study oNPPA/ANP oNPPB/BNP oNPPC/CNP oNPR1/oNPRA oNPR2/oNPRB Eukaryotic 18S rRNA
Summary
It is well established that members of the natriuretic peptide (NP) superfamily of ligands and their receptors (NPRs) are expressed in several locations within the female reproductive system of a number of poly-ovular mammals, such as rat (Gutkowska et al 1993), mice (Tsai et al 2005) and pigs (Zhang et al 2005). They have been reported in various compartments of the ovary, including follicular fluid, theca cells (TCs), granulosa cells (GCs), the oocyte and the corpus luteum, and this suggests they may have important roles in ovarian function.
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