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

Abstract

In the fish germ cell transplantation system, only type A spermatogonia (ASGs) and oogonia are known to be incorporated into the recipient genital ridges, where they undergo gametogenesis. Therefore, high colonization efficiency can be achieved by enriching undifferentiated germ cells out of whole testicular cells. In this study, we used magnetic‐activated cell sorting (MACS) for enriching undifferentiated germ cells of rainbow trout using a monoclonal antibody that recognizes a specific antigen located on the germ cell membrane. We screened the antibodies to be used for MACS by performing immunohistochemistry on rainbow trout gonads. Two antibodies, nos. 172 and 189, showed strong signals for ASGs and oogonia. Next, we performed MACS with antibody no. 172 using gonadal cells isolated from vasa‐gfp rainbow trout showing GFP in undifferentiated germ cells. We found that GFP‐positive cells are highly enriched in antibody no. 172‐positive fractions. Finally, to examine the transplantability of MACS‐enriched cells, we intraperitoneally transplanted sorted or unsorted cells into recipient larvae. We observed that transplantability of sorted cells, particularly ovarian cells, were significantly higher than that of unsorted cells. Therefore, MACS with antibody no. 172 could enrich ASGs and oogonia and become a powerful tool to improve transplantation efficiency in salmonids.

Cryopreservation of testicular tissue from Murray River Rainbowfish, Melanotaenia fluviatilis

“…Whole testis cell suspensions have been successfully transplanted into surrogates via germ cell surrogacy 17 , 19 , 29 , 30 , but an enriched sample of spermatogonial cells, particularly type A spermatogonia, is preferred as this can improve the success rate of surrogacy experiments 31 . There are several methods for isolating spermatogonial cells from a gonadal cell suspension including gradient centrifugation 32 , 33 and elutriation 34 , fluorescent activated sorting (FACS) 14 , 35 , 36 and magnetic activated sorting (MACS) 37 – 39 . Gradient based sorting, while the most cost effective, may be difficult to use in species with smaller gonads and a low spermatogonia cell number if bands are not easily identified in the centrifuged column.…”

“…In addition, in the absence of a broadly applicable cell surface marker to detect live spermatogonia, this is often restricted to transgenic species 14 , 36 . Similarly, MACS faces the same issues regarding antibody development to detect spermatogonia, although success has been reported in some species 37 – 39 . Ultimately, an isolation method that is specific but does not require the use of transgenic fish or antibodies is what will be most applicable in endangered species.…”

Spermatogonial Stem Cells in Fish: Characterization, Isolation, Enrichment, and Recent Advances of In Vitro Culture Systems

“…The method consisted of generating monoclonal antibodies (mAb) to cell-surface molecules of rainbow trout type A und spermatogonia by inoculating enriched live A und spermatogonia into mice and screening with a combination of cell enzyme-linked immunosorbent assay, live-cell staining, and flow cytometry (FCM) [75]. Among the obtained antibodies, two (numbers 80 and 95) were capable of specifically labelling A und spermatogonia of rainbow trout and zebrafish [75], while other antibodies (numbers 172 and 189) showed strong signals for type A spermatogonia and the oogonia of several species of salmonid [76]. By using these antibodies with fluorescence-activated cell sorting (FACS) [75] or magnetic-activated cell sorting (MACS) [77], it was possible to enrich A und spermatogonia and increase transplant success rate in selected teleosts.…”

Enhanced Enrichment of Medaka Ovarian Germline Stem Cells by a Combination of Density Gradient Centrifugation and Differential Plating

“…The first two methods are optimal for the specific isolation of OGSCs. However, specific markers for fish OGSCs have not yet been identified [ 9 ], and commercially available transgenic fish expressing reporter proteins governed by an OGSC-specific promoter have yet to be developed. Thus, the only currently available and easily accessible method for fish OGSC isolation is based on a cellular property for which there are two main cell separation methods.…”

“…During these procedures, the effects of each experimental treatment on OGSC enrichment were evaluated on the basis of cellular morphology and gene expression levels of OGSC-specific Nanos2 and germ cell-specific Vasa . Although several studies have previously used Vasa gene expression as a parameter to evaluate GSC enrichment [ 4 , 9 , 20 ], Vasa is a germ cell marker expressed in all types of germ cells from GSCs to gametes [ 22 ] and thus cannot provide accurate data for GSC enrichment. By contrast, Nanos2 is expressed only in OGSCs [ 2 ] and we, therefore, speculated that the use of Nanos2 as well as Vasa to evaluate OGSC enrichment could provide more reliable data than the use of Vasa alone.…”