ABSTRACT
The cholinergic system consists of acetylcholine (ACh), its synthesising enzyme, choline acetyltransferase (CHAT), transporters such as the high-affinity choline transporter (SLC5A7; also known as ChT1), vesicular ACh transporter (SLC18A3; also known as VAChT), organic cation transporters (SLC22s; also known as OCTs), the nicotinic ACh receptors (CHRN; also known as nAChR) and muscarinic ACh receptors. The cholinergic system is not restricted to neurons but plays an important role in the structure and function of non-neuronal tissues such as epithelia and the immune system. Using molecular and immunohistochemical techniques, we show in this study that non-neuronal cells in the parenchyma of rat testis express mRNAs for Chat, Slc18a3, Slc5a7 and Slc22a2 as well as for the CHRN subunits in locations completely lacking any form of innervation, as demonstrated by the absence of protein gene product 9.5 labelling. We found differentially expressed mRNAs for eight α and three ß subunits of CHRN in testis. Expression of the α7-subunit of CHRN was widespread in spermatogonia, spermatocytes within seminiferous tubules as well as within Sertoli cells. Spermatogonia and spermatocytes also expressed the α4-subunit of CHRN. The presence of ACh in testicular parenchyma (TP), capsule and isolated germ cells could be demonstrated by HPLC. Taken together, our results reveal the presence of a non-neuronal cholinergic system in rat TP suggesting a potentially important role for non-neuronal ACh and its receptors in germ cell differentiation.
Subject(s)
Acetylcholine/metabolism , Choline O-Acetyltransferase/metabolism , Testis/metabolism , Animals , Cells, Cultured , Cholinergic Neurons/cytology , Cholinergic Neurons/metabolism , Immunohistochemistry , In Situ Hybridization , Male , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Organic Cation Transport Proteins/genetics , Organic Cation Transport Proteins/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Subunits/genetics , Protein Subunits/metabolism , RNA, Messenger/metabolism , Rats , Rats, Inbred WF , Receptors, Cholinergic/genetics , Receptors, Cholinergic/metabolism , Sertoli Cells/cytology , Sertoli Cells/metabolism , Spermatogenesis , Spermatozoa/cytology , Spermatozoa/metabolism , Testis/cytology , Testis/innervation , Vesicular Acetylcholine Transport Proteins/genetics , Vesicular Acetylcholine Transport Proteins/metabolismABSTRACT
While the comet assay is used to detect DNA damage in isolated cells following exposure to chemicals in vitro, few publications report the use of the procedure in liver cells isolated from mice. Our initial efforts to use the assay to assess DNA damage in mouse hepatocytes maintained on collagen-coated dishes were hampered by high levels of baseline damage in controls, which appeared to result from mechanical damage sustained during the dislodgement of adherent cells in the early stages of the assay protocol. Here we describe an efficient version of the comet assay in cultured mouse hepatocytes that involves careful recovery of cells using a "scraping" buffer supplemented with 10% high purity grade DMSO. Use of this buffer strongly diminished the frequency of false positives. Using the industrial reagent styrene as a positive control in the optimised procedure, non-cytotoxic concentrations of this substance (2.5-10 mM) significantly increased mean comet tail length, area, and moment. Co-incubation with the CYP inhibitor SKF-525A strongly attenuated these effects of styrene. Collectively, these findings confirm this method is highly suitable for the detection of DNA damage by bioactivation-dependent compounds in freshly isolated mouse hepatocytes.
