The task of the mammalian testis is the production and controlled release of androgens and the development of germ cells. Impairment of these processes e.g. by trauma, inflammation or idiopathic reasons can result in sub- or infertility. Some of these insults can cause intense pain. Surprisingly, the innervation of the testis is not well characterised in spite of the presence of archetypical neurotransmitter such as acetylcholine (ACh). Of note, ACh has been shown to play an important role in many cellular processes including the male reproductive tract.Therefore, the aim of this project was to investigate if the presence of ACh is based on neuronal or non-neuronal origin and in case of the latter which cells would have the molecular components to synthesise and respond to ACh.Adjacent mesenteric tissue (meso) connecting testis, epididymis and vas deferens were isolated from Wistar Furth rats. Nerve fibres were detected using multiple labelling immunohistochemistry (IHC). Nf200-positive-labelled sensory nerve fibres within the meso-structures were identified positive for the known nociceptive markers CGRP, TRPV1, IB4 and VGluT1. Primary afferent innervation of the mesodeferens was examined using retrograde transport of Cholera toxin-B. Positive dorsal root ganglia neurons (DRG) were further analysed using IHC. Our data confirm the DRG lumbar 1 as major origin of nociceptive nerve fibres, which reach the testis via testicular artery or vas deferens.Due to the absence of nerve fibres within the rat testicular parenchyma (TP), a neuronal origin of ACh can be excluded. Thus, the presence of molecules related to the ACh-system were investigated within the TP and testicular capsule (TC) using qRT-PCR and subsequently for isolated testicular macrophages (TM), peritubular cells (PTC) and Sertoli cells (SC) using standard RT-PCR.The mRNA expression of ACh-receptors and molecules necessary for ACh synthesis (choline acetyltransferase, ChAT; high-affinity choline transporter-1, ChT1) and release (vesicular ACh transporter, VAChT; organic cation transporter, OCT2) were investigated in rat TP, TC and subsequently in TM, SC and PTC. Except for the absence of alpha6 and beta4 in TP all analysed molecules of the ACh-system are present in TC and TP. The presence of ChAT-, ChT1- and VAChT- mRNAs was supported by IHC. ChAT and alpha7 mRNAs were determined by in situ hybridisation.Most components of the cholinergic system were present in isolated testicular cells (TM, SC, PTC), but showed cell specific differences mainly in nAChR subunits. TM and SC were stimulated with nicotine and muscarine, which both did not reveal any change of the intracellular calcium concentration ([Ca2+]i). The MR-stimulation of PTC induced a rise in [Ca2+]i, whereas there was no response to nicotine. This metabotropic response in PTC as seen in neurons was blocked by the muscarinic-antagonist atropine. However, nicotine and muscarine given 2 min prior to ATP significantly changed the agonist-induced rise of [Ca2+]i in SC and TM. These data demonstrate that TM and SC have nAChR and MR with functions distinct from the known neuronal-like ones.Based on the established role of the cholinergic anti-inflammatory pathway, the influence of the ACh-system was analysed in a chronic inflammatory model i.e. autoimmune orchitis. Samples from TP of 3 groups stimulated with saline buffer, Freund&
#8223;s adjuvant or testicular homogenate were analysed using qRT-PCR. The orchitis group showed a reduced mRNA expression profile for: alpha3- alpha5, alpha7, alpha10, beta2, beta3, M4R, M5R, ChAT, ChT1 and OCT2. No changes in the mRNA expression level were observed for alpha6, beta1, beta4, M1R-M3R and VAChT.Taken together, molecules necessary for synthesis, release, binding and uptake of ACh are present in rat testicular parenchyma and isolated testicular cells (TM, SC and PTC). Peritubular cells show neuronal-like activation via MR, whereas indirect influences on purinergic-receptor-signalling via signalling pathways distinct from the nervous system are demonstrated in TM and SC.
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