Flow-cytometric enrichment of Pacific bluefin tuna type A spermatogonia based on light-scattering properties

Ichida, Kensuke; Kise, Kazuyoshi; Morita, Tetsuro; Yazawa, Ryosuke; Takeuchi, Yutaka; Yoshizaki, Goro

doi:10.1016/j.theriogenology.2017.06.022

Cited by 23 publications

(15 citation statements)

References 34 publications

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“…However, if younger fish are not available and one must use mature adults carrying large numbers of developed germ cells, germline stem cells must be enriched prior to transplantation. Several methods for isolating cell populations containing high concentrations of transplantable germline stem cells from mature testis have recently been developed by using a cell sorter (Kise et al 2012;Hayashi et al 2014;Ichida et al 2017). Further, monoclonal antibodies that specifically recognize spermatogonia containing a high concentration of germline stem cells have been recently generated (Hayashi et al, unpublished data).…”

Section: Resultsmentioning

confidence: 99%

Application of surrogate broodstock technology in aquaculture

Yoshizaki

Yazawa

2019

Fish Sci

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Surrogate broodstock technology facilitates the production of donor-derived gametes in surrogates, and comprises transplanting germ cells of a donor into recipients of a different strain or different species. The following applications of this technology are expected in the field of aquaculture: (1) the efficient and reliable production of offspring carrying superior genetic traits by transplanting donor germ cells from a single selected fish with superior traits into many recipient fish; (2) the reduction of the time required to breed fish by using a recipient species with a short generation time to produce gametes of a species with a long generation time; (3) the long-term storage of valuable species or strains as genetic resources by cryopreserving germ cells for transplantation; (4) the mass production of genetically sterile fish by transplanting germ cells of a donor fish that is sterile due to a mutation in the somatic cells into normal recipients without this mutation. It is expected that a combination of these techniques will greatly accelerate the breeding of aquaculture species. It is important to adapt surrogate broodstock technology to a wider range of fishery species and further improve the efficiency of donor-derived gamete production when using surrogate broodstock.

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Section: Resultsmentioning

confidence: 99%

Application of surrogate broodstock technology in aquaculture

Yoshizaki

Yazawa

2019

Fish Sci

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“…www.nature.com/scientificreports/ Using flow cytometry, cell specific light-scattering profiles can be detected allowing for the identification of unique populations. Using these profiles, spermatogonia have been successfully isolated in rainbow trout 31,36,40 , Japanese char 31 , masu salmon 31 and Pacific blue fin tuna 35 . Across multiple species, Vasa-positive spermatogonia have been detected in the high forward scatter region which correlated with their large size 31,35 .…”

Section: Discussionmentioning

confidence: 99%

“…Using these profiles, spermatogonia have been successfully isolated in rainbow trout 31,36,40 , Japanese char 31 , masu salmon 31 and Pacific blue fin tuna 35 . Across multiple species, Vasa-positive spermatogonia have been detected in the high forward scatter region which correlated with their large size 31,35 . We adapted the size-based flow cytometry method previously reported by Hagedorn et al 16 in a marine goby, as a means of assessing large cell viability in M.fluviatilis.…”

Section: Discussionmentioning

confidence: 99%

Cryopreservation of testicular tissue from Murray River Rainbowfish, Melanotaenia fluviatilis

Rivers

Daly

Jones³

et al. 2020

Sci Rep

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Globally, fish populations are in decline from overfishing, habitat destruction and poor water quality. Recent mass fish deaths in Australia’s Murray–Darling Basin highlight the need for improved conservation methods for endangered fish species. Cryopreservation of testicular tissue allows storage of early sperm precursor cells for use in generating new individuals via surrogacy. We describe successful isolation and cryopreservation of spermatogonia in an Australian rainbowfish. Testis histology showed rainbowfish spermatogonia are large (> 10 μm) and stain positive for Vasa, an early germ line-specific protein. Using size-based flow cytometry, testis cell suspensions were sorted through “A” (> 9 μm) and “B” gates (2–5 μm); the A gate produced significantly more Vasa-positive cells (45.0% ± 15.2%) than the “B” gate (0.0% ± 0.0%) and an unsorted control (22.9% ± 9.5%, p < 0.0001). The most successful cryoprotectant for “large cell” (> 9 μm) viability (72.6% ± 10.5%) comprised 1.3 M DMSO, 0.1 M trehalose and 1.5% BSA; cell viability was similar to fresh controls (78.8% ± 10.5%) and significantly better than other cryoprotectants (p < 0.0006). We have developed a protocol to cryopreserve rainbowfish testicular tissue and recover an enriched population of viable spermatogonia. This is the first step in developing a biobank of reproductive tissues for this family, and other Australian fish species, in the Australian Frozen Zoo.

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“…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, ).…”

Section: Discussionmentioning

confidence: 99%

“…These methods include FCM isolation based on lightscattering properties(Ichida et al, 2017;Kise et al, 2012), differential plating, Percoll gradient centrifugation(Lacerda, Batlouni, Silva, Homem, & Franca, 2006;Lacerda et al, 2010), centrifugal elutriation(Bellaiche et al, 2014), and a combination of these methods(Lacerda et al, 2010). 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, 2017;Kise et al, 2012). However, as mentioned before, FCM sorting is not useful for preparing a large number of cells for germ cell transplantation because it has to sort individual cells one by one.Differential plating can purify ASGs by >90% by the adhesion difference between ASGs and other testicular cells.…”

mentioning

confidence: 99%

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

Ichida

Hayashi

Miwa

et al. 2019

Molecular Reproduction Devel

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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.

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Flow-cytometric enrichment of Pacific bluefin tuna type A spermatogonia based on light-scattering properties

Cited by 23 publications

References 34 publications

Application of surrogate broodstock technology in aquaculture

Application of surrogate broodstock technology in aquaculture

Cryopreservation of testicular tissue from Murray River Rainbowfish, Melanotaenia fluviatilis

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

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