Specific visualization of live type A spermatogonia of Pacific bluefin tuna using fluorescent dye-conjugated antibodies †.

Specific visualization of live type A spermatogonia of Pacific bluefin tuna using fluorescent dye-conjugated antibodies†. Biol Reprod. 2019 Apr 01;: Authors: Ichida K, Kawamura W, Miwa M, Iwasaki Y, Kubokawa T, Hayashi M, Yazawa R, Yoshizaki G Abstract During our previous work toward establishing surrogate broodstock that can produce donor-derived gametes by germ cell transplantation, we found that only type A spermatogonia (ASGs) have the potency to colonize recipient gonads. Therefore, the ability to visualize ASGs specifically would allow the sequential analysis of donor cell behavior in the recipient gonads. Here we produced monoclonal antibodies that could recognize the cell surface antigens of ASGs in Pacific bluefin tuna (Thunnus orientalis), with the aim of visualizing live ASGs. We generated monoclonal antibodies by inoculating Pacific bluefin tuna testicular cells containing ASGs into mice and then screened them using cell-based enzyme-linked immunosorbent assay (ELISA), immunocytochemistry, flow cytometry (FCM), and immunohistochemistry, which resulted in the selection of two antibodies (Nos. 152 and 180) from a pool of 1152 antibodies. We directly labeled these antibodies with fluorescent dye, which allowed ASG-like cells to be visualized in a one-step procedure using immunocytochemistry. Molecular marker analyses against the FCM-sorted fluorescent cells confirmed that ASGs were highly enriched in the antibody-positive ...
Source: Biology of Reproduction - Category: Reproduction Medicine Authors: Tags: Biol Reprod Source Type: research