Abstract
Simple SummaryThe sturgeon is among the most ancient of actinopterygian fishes. Most species of sturgeon are listed as critically endangered due to habitat alteration caused by damming of rivers, pollution and overharvesting. Germ cell transplant is a useful tool to save these endangered species. To expand germ cell populations and sustain the supply for long periods for transplant, we established basal culture conditions for sturgeon germ cells. Germ cell mitotic activity has been enhanced by eliminating gonad somatic cells, supplementing with growth factor and using an alternative to fetal bovine serum. The optimal condition identified was purified germ cells cultured in serum-free medium supplemented with leukemia inhibitory factor (LIF) and glial cell line-derived neurotrophic factor (GDNF) at 21 °C. Cultured sterlet germ cells showed development after transplant into Russian sturgeon. The study provided useful information for sturgeon germ cell culture.To expand germ cell populations and provide a consistent supply for transplantation, we established basal culture conditions for sturgeon germ cells and subsequently increased their mitotic activity by eliminating gonad somatic cells, supplementing with growth factor, and replacing fetal bovine serum (FBS). The initial basal culture conditions were Leibovitz’s L-15 medium (pH 8.0) supplemented with 5% FBS (p < 0.001) at 21 °C. Proliferation of germ cells was significantly enhanced and maintained for longer periods by elimination of gonad somatic cells and culture under feeder-cell free conditions, with addition of leukemia inhibitory factor and glial-cell-derived neurotrophic factor (p < 0.001). A serum-free culture medium improved germ cell proliferation compared to the L-15 with FBS (p < 0.05). Morphology remained similar to that of fresh germ cells for at least 40 d culture. Germline-specific gene expression analysis revealed no significant changes to germ cells before and after culture. Sterlet Acipenser ruthenus germ cells cultured more than 40 days showed development after transplant into Russian sturgeon Acipenser gueldenstaedtii. Polymerase chain reaction showed 33.3% of recipient gonads to contain sterlet cells after four months. This study developed optimal culture condition for sturgeon germ cells. Germ cells after 40 d culture developed in recipient gonads. This study provided useful information for culture of sturgeon germ cells.
Highlights
Germ stem cells have the ability to self-renew as well as to differentiate into other germ cell stages.Xenotransplantation of germ cells has been conducted in fish [1]
In vitro culture of germ cells was established in Medaka Oryzias latipes [4], zebrafish Danio rerio [5], Nile tilapia Oreochromis niloticus [6] and rainbow trout Oncorhynchus mykiss [7]
To establish optimal culture conditions for sturgeon germ cells and improve their mitotic activity, we investigated the basal culture conditions for gonad cells and examined the effect of somatic cells on germ cell proliferation and assessed the influence of growth factor on germ cell mitotic activity
Summary
Germ stem cells have the ability to self-renew as well as to differentiate into other germ cell stages. Xenotransplantation of germ cells has been conducted in fish [1]. In fish, both type A spermatogonia and oogonia (probably germ stem cells) show high sex plasticity even after sexual maturation [2]. Most sturgeon species are late maturing, making culture and conservation costly and time consuming [12,13]. Germ cell culture and transplant could be an available and rapid method for surrogate production of endangered fishes with large bodies and a long life-cycle. The identity of cultured germ cells was confirmed by RT-qPCR (Quantitative real-time PCR) targeting germ cell specific genes, and the cells were transplanted into sturgeon larvae to assess their transplantability and proliferation
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