The expression and biological role of the non-neuronal cholinergic system in the ovary

Functioning of the ovary depends on an interplay between hormones, locally produced growth factors and neurotransmitters. Neurotransmitters are delivered to the ovary via its sympathetic innervation and originate from intrinsic nerve cells expressing catecholaminergic and peptidergic traits. We found that the nerve fibers and nerve cells of the ovary were however not immunoreactive for the ACh-synthesizing enzyme, choline-acetyl transferase (ChAT). Immunoreactivity was instead detected in ovarian endocrine cells, namely granulosa cells (GCs), of rodents and primates. Importantly, isolated GCs produce ACh. Thus, endocrine cells are an unexpected non-neuronal source of ACh in the ovary. GCs in vivo and in vitro also contain ACh-receptors of the muscarinic subtype (MR), namely M1 and M5. MR of human GCs are functional and linked to rapid increases in intracellular calcium levels. A role of ovarian ACh/MR in the crucial process of cell proliferation is suggested by the observation that in growing follicles, ChAT-immunoreactive GCs co-express "proliferating cell nuclear antigen" (PCNA) and that cholinergic agents stimulate cell proliferation of human GCs in vitro. This proliferative effect is associated with rapid disruption of gap junction communication and phosphorylation of connexin 43. In addition, calcium-dependent channels are activated. Ongoing studies have begun to identify down-stream effects of M1/5 activation in GCs, which include, for example, expression of a transcription factor (egr-1). In summary, ovarian endocrine cells are sources and targets of ACh. We propose that an as yet unexplored intraovarian cholinergic system exists, which contributes to physiological ovarian tissue remodeling by stimulation of cell proliferation.