Frontiers in Marine Science (Oct 2015)
Analysis of testicular germ cell apoptosis in Cobitis loaches (Cobitidae, Teleostei) from diploid-tetraploid populations
Abstract
Taxa of the genus Cobitis are distributed in Europe mainly in populations composed of individuals at different ploidy level. The species occur in Poland include the spined loach C. taenia and the Danubian loach C. elongatoides, triploid Cobitis females and tetraploid males and females. Previous morphological study has demonstrated abnormalities in spermatogenesis of the Cobitis tetraploid individuals associated with lack of sperm in their gonads. So far, however, the mechanism responsible for the gonadal sterility has not been explained. Over the past years, there is a growing evidence on incidence of apoptosis during fish spermatogenesis, however there is no data on the process of apoptosis in the male gonad of the natural polyploid. The aim of the study was to analyze apoptosis in the gonads of diploid and tetraploid Cobitis males. The expression of caspase-3 was analyzed by fluorescent immunohistochemistry using first rabbit, polyclonal anty-caspase-3 antibody (1:400) and second anti-rabbit antibody conjugated with Alexa Fluor 488 (1:2000). Number of TUNEL-positive cells was determined using fluorescence method based on the ability of terminal deoxynucleotidyl transferase (TdT) to incorporate fluorescein-labelled deoxyuridine triphosphate (dUTP) to the 3'-OH termini of the nuclear DNA strand breaks (TUNEL assay). Analysis of microscopic images showed no sperm in the testes of tetraploid Cobitis males. The highest expression of caspase-3 as well as the highest number of TUNEL-positive cells were demonstrated in the testes of the Danubian spined loach C. elongatoides compared to spined loach C. taenia and tetraploid males from diploid-polyploid population. The relatively low immunoexpression of caspase-3 and low number of TUNEL-positive cells do not unequivocally confirm that apoptosis disturb spermatogenesis in tetraploid males of Cobitis. Further studies are needed to explore the mechanisms behind such gonadal abnormality.
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