Zebrafish Germline Chimeras Produced by Transplantation of Ovarian Germ Cells into Sterile Host Larvae1

Abstract

High frequency production of zebrafish germline chimeras was achieved by transplanting ovarian germ cells into sterile Danio hybrid recipients. Ovarian germ cells were obtained from 3-mo-old adult Tg(vasa:DsRed2-vasa);Tg(bactin:EGFP) double transgenic zebrafish by discontinuous Percoll gradient centrifugation. An average of 755 ± 108 DsRed-positive germ cells was recovered from each female. For transplantations, a total of approximately 620 ± 242 EGFP-positive cells of which 12 ± 4.7 were DsRed-positive germ cells were introduced into the abdominal cavity under the swim bladder of 2-wk-old sterile hybrid larvae. Six weeks after transplantation, a total of 10 recipients, obtained from 2 different transplantations, were examined, and 2 individuals (20%) were identified that possessed a large number of DsRed- and EGFP-positive cells in the gonadal region. The transplanted ovarian germ cells successfully colonized the gonads and differentiated into sperm in the male hybrid recipients. Of 67 adult recipients, 12 (18%) male chimeric fish reproduced and generated normal offspring when paired with wild-type zebrafish females. The fertilization efficiency ranged from 23% to 56%. Although the fertile male chimeras were generated by transplantation of ovarian germ cells, the F1 generation produced by the male chimeras contained both male and female progeny, indicating that male sex determination in zebrafish is not controlled by sex chromosome heterogamy. Our findings indicate that a population of ovarian germ cells that are present in the ovary of adult zebrafish can function as germline stem cells, able to proliferate and differentiate into testicular germ cells and functional sperm in male recipients. The high frequency of germline chimera formation achieved with the ovarian germ cells and the convenience of identifying the chimeras in the sterile host background should make this transplantation system useful for performing genetic manipulations in zebrafish.

Identification of promoter elements responsible for gonad-specific expression of zebrafish Deadend and its application to ovarian germ cell derivation

“…This culture system provides an initial step for searching and studying the proliferation and maintenance of zebrafish ovarian germ cells in culture which is valuable in understanding ovarian germline stem cell biology and identifying crucial growth factors for their self-renewal, stemness and proliferation. Together with germ cell transplantation technology that we have developed (Wong et al, 2011), our system is useful for future success in establishing germline-competent ovarian germline stem cell cultures.…”

“…The resulting ovarian cell suspension was filtered through a 60-mm mesh followed by a 37-mm mesh and partially purified using a discontinuous Percoll (GE Healthcare) gradient (20% and 40%) according to a published protocol (Wong et al, 2011). The cell fraction was re-suspended in LDF medium (Collodi et al, 1992) and transferred into a plate that contained a confluent monolayer of growth-arrested feeder cells.…”

“…Cultured ovarian germ cells were trypsinized, washed and re-suspended in 50 ml of L-15 medium, and immediately transplanted into two-week-old infertile zebrafish larvae treated with dnd antisense morpholino (Slanchev et al, 2005) using a published method (Wong et al, 2011). Four weeks after transplantation, all the recipients were examined and 3 DsRed-positive recipients were euthanized, and the status of the transplanted ovarian germ cells and the developmental stage of the gonads were evaluated.…”

Analysis of a novel gene, Sdgc, reveals sex chromosome-dependent differences of medaka germ cells prior to gonad formation

“…In rainbow trout and zebrafish, isolated oogonia and spermatogonia from mature gonads can recolonize the undifferentiated gonads of embryos and produce either sperm or eggs, depending on the sex of the surrounding somatic cells (Okutsu et al, 2006;Wong et al, 2011;Yoshizaki et al, 2010).…”

Isolation and cryopreservation of early stages of germ cells of tench (Tinca tinca)

“…A disadvantage of these early stages of germ cells (SG/OG and their subsequent stages) lies in limited migratory activity to the required position in the gonads when these cells are transplanted during embryonic stage. Therefore SG/ OG must be transplanted into the peritoneal cavity near the genital ridge of hatched fry or directly into the gonads (Yoshikazi et al 2010;Wong et al 2011;Nobrega et al 2012). The transplantation of PGCs/SG/OG into a closely related species, creating a germ-line chimera, could enhance fish production for research as well as aquaculture.…”