Lectin‐binding pattern as tool to identify and enrich specific primary testis cells of the tilapia (Oreochromis niloticus) and medaka (Oryzias latipes)

Abstract

Cell type-specific lectin binding is a useful tool for the analysis of developing systems. We describe the binding pattern of 21 different fluorescein isothiocyanate (FITC)-labelled lectins to the testis of two model teleost species, the medaka (Oryzias latipes) and the tilapia (Oreochromis niloticus). The analysis of the binding pattern was carried out on tissue sections (medaka and tilapia) and using primary culture cells (only tilapia). Lectin binding was studied by confocal microscopy and for histological analysis some sections were, in addition, stained with bodipy to gain additional information concerning the cytological organization of the cystic mode of spermatogenesis in fish. The observed differences in lectin staining of different cell types in primary cultures were quantified by flow cytometry. Only few lectins bound specifically to haploid cells while the reaction to diploid or tetraploid cells was generally stronger. However, the extracellular material around the haploid spermatids and spermatozoa in spermatocysts showed a strong staining reaction with several lectins (e.g., Phaseolus vulgaris Erythro agglutinin). The apparent differences in the cellular lectin-binding pattern can be used to identify particular cell types, to monitor their differentiation in vitro or to enrich particular cell types from heterogeneous cultures using magnetic beads coated with anti-FITC antibodies. Using the latter approach, we show that it is possible to enrich for gonial cells and at the same time deplete the preparation for haploid cells and Sertoli cells.

Molecular characterization and expression profile of the estrogen receptor α gene during different reproductive phases in Monopterus albus

“…On one hand, genes encoding immune-related proteins also encode hormone receptor-related proteins. A large number of immune-related proteins, such as lectins37, lysozyme, hepcidin and complement components3839, including ERα, have been identified in fish gonads. Massartr found that ERα was expressed in the immune organs of O. mykis s40.…”

The effects of estrogenic and androgenic endocrine disruptors on the immune system of fish: a review

“…A number of the genes that encode for immune proteins are co-expressed with those that encode for sex-steroid receptors. For example, lectins (Tokalov and Gutzeit 2007;Swain and Kayak 2009), lysozyme, hepcidin and complement component 3 (Saurabh and Sahoo 2008;Chaves-Pozo et al 2008), Toll-like receptors and interleukins (Kumari et al 2009;Yu et al 2009) are abundantly found in the gonadal tissues. Conversely, ER and AR are widely distributed in immune tissues, such as the spleen, liver, and anterior kidney leukocytes (Slater et al 1995;Todo et al 1999;Lynn et al 2008;Liu et al 2009;Shved et al 2009), suggesting these organs may be targets for sex-steroids.…”

oct4‐EGFP reporter gene expression marks the stem cells in embryonic development and in adult gonads of transgenic medaka

“…4). In non‐transgenic cultures, different cell populations can be recognized by forward scatter (FSC) analysis (Tokalov and Gutzeit, 2007) in which the smallest cells with low FSC can be recognized as spermatids (St) and spermatozoa (Sz). The large spermatogonia, characterized by high FSC, exhibit the strongest fluorescence in oct4 ‐EGFP fish and comprise 1–3% of all primary testis cells (Fig.…”

“…Primary testis cells of oct4 ‐EGFP transgenic medaka and non‐transgenic medaka were isolated as described (Tokalov and Gutzeit, 2007) without Percoll gradient centrifugation. To determine their DNA content, cells were stained with Hoechst 33342 (5 µg/ml; Life Technologies) for 30 min, washed once, and kept in DMEM (Biochrom, Germany) supplemented with 2% fetal calf serum (PAA, Austria).…”