Testicular apoptotic activity in two bio-sentinel fish species inhabiting an aquatic ecosystem in an area where continual DDT spraying occurs : utility of immunohistochemical assays

Abstract

Endocrine disrupting chemicals (EDCs) such as DDT have the ability to disrupt hormonally controlled processes, such as spermatogenesis, which is the maturation of germ cells into spermatozoa. During normal spermatogenesis, germ cell apoptosis can occur, but the degree of apoptosis within the testis could possibly be affected by exposure to EDCs. In 2004, a pilot study on the reproductive health of two freshwater fish species, Oreochromis mossambicus and Clarias gariepinus, from three impoundments in the Luvuvhu River, found concerning levels of DDT and its metabolites in both species from the Nandoni Dam, and in O. mossambicus from the Xikundu Weir. This was not surprising as a large part of the Luvuvhu River catchment is located within an area where ongoing DDT-spraying occurs for vector control purposes. Hence, in 2006, a larger WRC-funded project began to further investigate the findings from the pilot study. A subsidiary study, spanning two seasons, was initiated to investigate testicular apoptosis in fish from the polluted systems, the Nandoni Dam (ND) and the Xikundu Weir (XW), as well as a reference site, the Albasini Dam (AD), utilizing caspase-3 and TUNEL immunoexpression as apoptotic markers. In addition, three fixatives, Bouin’s Fluid (BF), Neutrally Buffered Formalin (NBF) and Paraformaldehyde (PFA), were used to determine which would be the optimal fixative for both histological and immunohistochemical assessments. Sampling occurred during season 1, the low-flow season (October 2007), during DDT spraying of the surrounding area, and season 2, the high–flow season (February 2008), two months after the DDT-spraying was completed. The testes of O. mossambicus (n = 19 season 1, n = 25 season 2) and C. gariepinus (n = 19 season 1, n = 20 season 2) were fixed in the above-mentioned fixatives, embedded in paraffin wax, prepared for immunohistochemistry, and exposed to caspase-3 antibodies and TUNEL antibodies individually. The results indicated that the residues of p,p´-DDT - DDD and - DDE were found in the fat samples of both O. mossambicus and C. gariepinus, in AD, ND and XW. Testicular apoptotic assessment using the caspase-3 assay clearly labeled spermatocytes in the process of cellular death in both seasons, in all three fixatives. When comparing the two assays, a significant difference is found between the caspase-3 and TUNEL positive cells. The results further show that, when comparing the three sampling sites, the highest amount of positive cells are found at the XW. The decrease observed in season two, in both the caspase-3 and TUNEL assay may possibly be linked to the stage of spermatogenesis, coinciding with hormonal changes associated with the different sampling seasons (i.e. breeding and non-breeding seasons). The levels of DDT found in the fat tissue, could not be correlated to an up-regulation in apoptotic cells. The results The results indicated that the choice of fixative, could affect the identification of the amount of positive cells. The utility of the caspase-3 and TUNEL assays, in conjunction with all three fixatives, proves a successful tool in assessing and quantifying modulated testicular apoptosis, creating greater research potential in the assessment of the effects of aquatic pollution. Copyright