Isolation and transplantation of sturgeon early-stage germ cells

Abstract

We report, for the first time, a series of baseline techniques comprising isolation and transplantation of female and male early-stage germ cells in sturgeon to generate a germline chimera as a potential tool for surrogate reproduction and gene banking. Cells were dissociated from testis, characterized by mostly spermatogonia, and from ovary, exclusively comprising oogonia and previtellogenic oocytes, of Acipenser baerii, using 0.3% trypsin (2 hours, 23 °C) dissolved in PBS, isotonic with blood plasma. The dissociated germ cells were sorted by Percoll gradient centrifugation followed by immunolabeling with germ cell-specific vasa antibody DDX4, while 10% to 30% Percoll solution contained 79.4% and 70.8% labeled testicular and ovarian cells. Sorted germ cells were transplanted into a cavity close to a presumptive genital ridge of newly hatched heterospecific Acipenser ruthenus larvae with fluorescein isothiocyanate-labeled endogenous primordial germ cells. The transplanted germ cells were randomly distributed in the body cavity through 30-day posttransplantation (dpt). Subsequently, the cells were organized into genital ridges 50 dpt and proliferated 90 dpt. The number of both transplanted and endogenous germ cells significantly increased from 18.1, 22.2, and 29.1 (30 dpt) to 108.5, 90.8, and 118.5 (90 dpt) in ovarian, testicular, and endogenous germ cells, respectively (P < 0.05). The efficiency of transplantation was 60% (counted 90 dpt).

Identification and sexually dimorphic expression of vasa isoforms in Dabry′s sturgeon (Acipenser dabryanus), and functional analysis of vasa 3′-untranslated region

“…Moreover, a PGC transplantation assay between sturgeon and goldfish proved that PGC migration machinery is conserved between these two distantly related species (Saito et al 2014). The ability of chimeric mRNA to label and trace PGCs in Dabry's sturgeon is extremely useful for PGC cryopreservation and the conservation of endangered species by germ cell transplantation technologies (Okutsu et al 2006;Psenicka et al 2015;Saito and Psenicka 2015;Yoshizaki et al 2005).…”

Sterilization of sterlet Acipenser ruthenus by using knockdown agent, antisense morpholino oligonucleotide, against dead end gene

“…Another possibility is the transplantation of subsequent stages of PGCs, the SG/OG. The advantage of SG/OG in comparison with PGCs is in their easy isolation from testes or ovary to obtain a large number of cells which can be directly microinjected into the body cavity of recipient fry to counteract the potential migration inability of transplanted germ cells [23,25]. To induce germ line chimera producing donor's gametes only, the host should be sterile.…”

Novel Technique for Visualizing Primordial Germ Cells in Sturgeons (Acipenser ruthenus, A. gueldenstaedtii, A. baerii, and Huso huso)1

“…We have already shown that sturgeon PGCs can be isolated using an enzymatic solution and the isolated cells can be transplanted into different species; furthermore, transplanted PGCs can migrate to the genital ridge even in a phylogenetically distant xenogeneic host [18]. Additionally, we established a technique for isolating spermatogonia and oogonia using Percoll gradient layers; transplantation of these cells into the body cavity of hatched larvae showed that they can proliferate, migrate and settle at the host gonadal ridge [36]. In this transplantation experiment, the PGCs of the host were labeled with FITC, while the transplanted donor germ cells were labeled with the common cell-labeling reagent PKH-26; this allowed us to check that the donor PGCs localized to the developing host gonad.…”