Flow-Cytometric Isolation and Enrichment of Teleost Type A Spermatogonia Based on Light-Scattering Properties1

Abstract

The transplantation of germ cells is a powerful tool both for studying their development and for reproductive biotechnology. An intraperitoneal germ cell transplantation system was recently developed for use in several teleost species. Donor germ cells transplanted into the peritoneal cavity of hatchlings migrated toward and were incorporated into the recipient's genital ridges, where they underwent gametogenesis. Among male germ cells, only type A spermatogonia were capable of colonizing the recipient gonads, unlike those at more advanced stages. The enrichment of type A spermatogonia is therefore important to achieve efficient donor-cell incorporation and subsequent donor-derived gametogenesis. Here we established a simple and rapid system of isolation and enrichment for fish type A spermatogonia, using flow cytometry. Type A spermatogonia were found to have distinctive forward and side light scatter properties compared to that with other types of testicular cell. Based on these characteristics, we were able to isolate and enrich type A spermatogonia by using flow cytometry. After intraperitoneal transplantation, the enriched type A spermatogonia could be successfully incorporated into the recipient genital ridges. This flow cytometry approach using forward and side light scatter was also found to be applicable to other salmonid and sciaenid species, suggesting that it could be a powerful tool for isolating and enriching transplantable type A spermatogonia in a wide range of teleosts. We expect this method to contribute significantly to germ cell biology and biotechnology.

TALEN‐mediated gene targeting in porcine spermatogonia

“…Isolating spermatogonia by flow cytometry using light scatter properties has been reported previously in teleost fish where a cell population highly enriched with undifferentiated type A spermatogonia (∼93% VASA positive) could be obtained from immature rainbow trout testis (Kise et al, ). In the fish study, researchers used a transgenic rainbow trout line that carries a Vasa‐GFP reporter to set a germ cell gate on the light scatter plot and sorted type A spermatogonia from immature non‐transgenic males based on light scatter properties.…”

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

“…Therefore, methods of isolating and enriching ASGs without the use of transgenic fish are required to apply the germ cell transplantation system to aquaculture and conservation. These methods include FCM isolation based on light‐scattering properties (Ichida et al, ; Kise et al, ), differential plating (Shikina et al, ), Percoll gradient centrifugation (Lacerda, Batlouni, Silva, Homem, & Franca, ; Lacerda et al, ), centrifugal elutriation (Bellaiche et al, ), and a combination of these methods (Lacerda et al, ). We previously reported that FCM dependent on light‐scattering properties makes it possible to isolate and enrich ASGs to 73–93% in several teleosts (Ichida et al, ; Kise et al, ).…”

“…RNA probes were synthesized from vasa cDNA fragments of rainbow trout. Probe synthesis and hybridization were performed according to the method described by Kise et al (2012) and Sawatari, Shikina, Takeuchi, and Yoshizaki (2007). A minimum of 100 cells for each specimen was counted to calculate the proportion of vasapositive and vasa-negative cells, and the proportions were expressed as a percentage of total testicular cells.…”

Biological Characteristics of Fish Germ Cells and their Application to Developmental Biotechnology

“…2010), which could enable the enrichment of ASG from fish testes using specific antibody‐mediated flow cytometry or magnetic cell sorting. Finally, we recently developed a simple flow cytometry protocol to enrich ASG using light scattering properties (Kise et al. 2012).…”