Establishment of novel monoclonal antibodies for identification of type A spermatogonia in teleosts†

Abstract

We recently established a germ cell transplantation system in salmonids. Donor germ cells transplanted into the body cavity of recipient embryos migrate toward and are incorporated into the recipient gonad, where they undergo gametogenesis. Among the various types of testicular germ cells, only type A spermatogonia (A-SG) can be incorporated into the recipient gonads. Enriching for A-SG is therefore important for improving the efficiency of germ cell transplantation. To enrich for A-SG, an antibody against a cell surface marker is a convenient and powerful approach used in mammals; however, little is known about cell surface markers for A-SG in fish. To that end, we have produced novel monoclonal antibodies (mAbs) against cell-surface molecules of rainbow trout (Oncorhynchus mykiss) A-SG. We inoculated mice with living A-SG isolated from pvasa-GFP transgenic rainbow trout using GFP-dependent flow cytometry. By fusing lymph node cells of the inoculated mice with myeloma cells, we generated 576 hybridomas. To identify hybridomas that produce mAbs capable of labeling A-SG preferentially and effectively, we screened them using cell ELISA, fluorescence microscopy, and flow cytometry. We thereby identified two mAbs that can label A-SG. By using flow cytometry with these two antibodies, we could enrich for A-SG with transplantability to recipient gonads from amongst total testicular cells. Furthermore, one of these mAbs could also label zebrafish (Danio rerio) spermatogonia. Thus, we expect these monoclonal antibodies to be powerful tools for germ cell biology and biotechnology.

Enrichment of transplantable germ cells in salmonids using a novel monoclonal antibody by magnetic‐activated cell sorting

“…To identify suitable antibodies for MACS, immunohistochemistry (IHC) was performed on paraffin sections of gonads isolated from 12‐month‐old immature rainbow trout using 11 monoclonal antibodies (nos. 23, 80, 87, 95, 97, 115, 137, 162, 172, 179, and 189) that can recognize the cell surface antigen of rainbow trout germ cells (Hayashi et al, ). A particularly clear and germ‐cell‐specific signal was observed when using antibody nos.…”

“…In this study, from the antibody library made by directly inoculating ASGs of rainbow trout into mice (Hayashi et al, ), we identified two novel antibodies that can clearly recognize the cell surface antigen of germ cell lineages. Further, by applying antibody no.…”

“…We previously established monoclonal antibodies (nos. 80 and 95) that can recognize ASGs specifically (Hayashi et al, ). However, when the MACS with antibody nos.…”

“…In this study, from the antibody library made by directly inoculating ASGs of rainbow trout into mice (Hayashi et al, 2019) It is possible that antibodies bound to the cell surface inhibit some cell functions, since cell surface proteins or sugar chains could be masked by the antibodies. Although in this study, MACS-enriched germ cells using antibody no.…”

“…When conducting the immunoreactions, the following primary antibodies were used: nos. 23, 80, 87, 95, 97, 115, 137, 162, 172, 179, and 189 (Hayashi et al, ). All antibodies were diluted with Can Get Signal immunostain solution B (Toyobo Co., Osaka, Japan) in 1:10, 1:100, and 1:1,000 ratios.…”