Assessment of fish spermatogenesis through high-quality immunofluorescence against fish testicular samples with an antibody set

A complete evaluation of the spermatogenetic status of a fish by accurately identifying different types of spermatogenic cells is useful not only for reproductive studies but also for genetic breeding. For this task, it is required to establish a simple and practical experimental procedure, to obtain repeatable, high-quality imaging data. Here, we have developed antibodies against the zebrafish ( Danio rerio ) spermatogenesis-related proteins, including Ddx4, Piwil1, Sycp3, and Pcna, and an integrated method for high-quality and high-through output immunofluorescence on testis sections of different fish species. We accurately identified different spermatogenic cells at different stages. Type-A spermatogonia can be identified by the highest expression of Ddx4 and Piwil1 among different spermatogenic cells. Type-B spermatogonia is identified by the second highest expression of Ddx4 and the highest expression of Pcna among different spermatogenic cells. Spermatids can be distinguished from spermatozoa by the expression of Piwil1. The different subtypes of primary spermatocytes (SPC-I) can be identified by co-staining of Sycp3 and Pcna. Leptotene SPC-I show polar expression of both Sycp3 and Pcna at the same side of the nucleus. All the antibodies were tested for practicality in four fish species, Chinese rare minnow ( Gobiocypris rarus ), common carp ( Cyprinus carpio ), blunt snout bream ( Megalobrama amblycephala ), and rice field eel ( Monopterus albus ). Using this method and the antibody sets, we were able to precisely and accurately evaluate the spermatogenetic status in different fish species. Highlights A practical method for high-quality immunofluorescence against fish testicular samples has been developed. An antibody set of Ddx4, Piwil1, Sycp3 and Pcna have been developed for identification of a variety of spermatogenic cells in different fish species. Antibodies against zebrafish proteins have been tested in the four fish species, Chinese rare minnow, common carp, blunt snout bream and rice field eel.