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 β 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.
S U Schirmer, I Eckhardt, H Lau, J Klein, Y C DeGraaf, K S Lips, C Pineau, I L Gibbins, W Kummer, A Meinhardt and R V Haberberger
D Rath, S Barcikowski, S de Graaf, W Garrels, R Grossfeld, S Klein, W Knabe, C Knorr, W Kues, H Meyer, J Michl, G Moench-Tegeder, C Rehbock, U Taylor and S Washausen
Pre-selection of spermatozoa based on the relative DNA difference between X- and Y-chromosome bearing populations by flow cytometry is an established method that has been introduced into commercial cattle production. Although several important improvements have increased the sort efficiency, the fertilising ability of sexed spermatozoa based on offspring per insemination is still behind farmers' expectations. The main stress factors, especially on mitochondria, that reduce the lifespan of spermatozoa are described, and new technical as well as biological solutions to maintain the natural sperm integrity and to increase the sorting efficiency are discussed. Among these methods are the identification of Y-chromosome bearing spermatozoa by bi-functionalised gold nanoparticles and triplex hybridisation in vivo as well as new laser-controlled deflection system that replaces the deflection of spermatozoa in the electrostatic field. Additionally, as well as a new nonsurgical transfer system of spermatozoa into the oviduct of cows has been developed and allows a significant reduction of spermatozoa per transfer. Altogether, the improvements made in the recent years will allow a broader use of sex-sorted spermatozoa even in those species that require more cells than cows and sheep.