Aquatic Toxicology 86 (2008) 38–48 Influence of municipal effluents on the expression of connexins in the brook trout (Salvelinus fontinalis) testis Benjamin de Montgolfier a , Michel Fournier a,b , C´ eline Audet b , David J. Marcogliese c , Daniel G. Cyr a,∗ a INRS-Institut Armand Frappier, Universit´ e du Qu´ ebec, 245 Hymus boul, Pointe-Claire, QC H9R 1G6, Canada b ISMER, Universit´ e du Qu´ ebec ` a Rimouski, 310 all´ ee des Ursulines, Rimouski, QC G5L 3A1, Canada c Fluvial Ecosystem Research Section, Aquatic Ecosystem Protection Research Division, Water Science and Technology Directorate, Science and Technology Branch, Environment Canada, St. Lawrence Centre, 105 McGill, Montreal, QC H2Y 2E7, Canada Received 11 July 2007; received in revised form 12 September 2007; accepted 21 September 2007 Abstract Gap junctions are essential for spermatogenesis. Exposure to municipal wastewater effluent can modify spermatogenesis in fish. The present aim was to determine if municipal wastewater effluent could alter the expression of testicular connexins (Cxs) in brook trout. Trout were exposed for 4 and 12 weeks to various concentrations of municipal effluent (0, 1, 10 and 20%, v/v). Hepatic vitellogenin (vtg) mRNA levels were not different between exposure groups after 4 weeks. At this time, testicular cx43 and cx31 mRNA levels increased in the 1% group, but cx30 and cx43.4 levels were not different at any concentration. Immunolocalization of each Cx did not differ between groups after 4 weeks. After 12 weeks, spermatogenesis in the 1% group was more advanced than in other groups, and hepatic vtg mRNA levels were significantly increased at the higher exposure concentrations. Testicular cx43 mRNA levels were higher than controls at all doses, while cx43.4 levels increased in a dose-dependent manner but remained lower than controls. cx31 mRNA levels were significantly lower in the 1 and 10% groups than in control and 20% group, while cx30 levels did not vary. Immunolocalization of Cxs did not differ between groups except for Cx43.4, which was expressed between spermatocytes in the 1% group. Furthermore, the Cx31 immunoreaction appeared to decrease in testicular blood vessels of fish exposed to the highest dose. Furthermore, vegf mRNA levels were unaltered by treatment at both time points. Thus, long-term exposure to environmental concentrations of wastewater effluent can alter the expression of testicular Cxs. Crown Copyright © 2007 Published by Elsevier B.V. All rights reserved. Keywords: Gap junctions; Wastewater; Spermatogenesis; Vitellogenin; Brook trout; Endocrine disruptors 1. Introduction Spermatogenesis is a complex process involving highly coor- dinated intercellular communication between Sertoli cells and developing germ cells. This type of intercellular communication is mediated by gap junctions, which allow direct communica- tion between adjacent cells (S¨ ohl and Willecke, 2004). These junctions play a critical role in the control of cell proliferation and differentiation during spermatogenesis. Gap junctions are transmembrane channels between adja- cent cells, and contain aggregates of oligomeric channels. These ∗ Corresponding author. Tel.: +1 514 630 8833; fax: +1 514 630 8850. E-mail address: Daniel.cyr@iaf.inrs.ca (D.G. Cyr). channels, or connexons, allow direct passage of regulatory molecules (<1 kDa) such as amino acids, secondary messen- gers, Ca 2+ , and cAMP, between adjacent cells (Saez et al., 1989; Vaney et al., 1998). Connexons are formed by the oligomer- ization of structurally related proteins, connexins (Cxs; Musil and Goodenough, 1991). Approximately twenty connexins have been identified and sequenced in humans, and many homologies exist with other vertebrate species (Altevogt et al., 2002; Lagree et al., 2003). It has been shown that in the mammalian testis, Cx43 is localized between adjacent Sertoli cells and between Leydig cells (Pelletier, 1995; Tan et al., 1996; Batias et al., 2000). Cx43 has been shown to be essential for spermatogenesis (Chung et al., 2001; Perez-Armendariz et al., 2001; Fiorini et al., 2004). Decreased expression of cx43 in mouse and humans is associ- 0166-445X/$ – see front matter. Crown Copyright © 2007 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2007.09.013