Zebrafish germline chimeras produced by transplantation of ovarian germ cells into sterile host larvae

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.

FIG. 1.

Production of sterile Danio hybrid fish. A) Hybrid fish were produced by in vitro fertilization of zebrafish eggs with pearl danio sperm. Gonad development in the adult hybrid male (B), zebrafish male (C), hybrid female (D), and zebrafish female (E). Transverse sections of gonads from adult hybrid male (F), Bouin-paraffin section, zebrafish male (G), hybrid female (H), and Bouin-paraffin section, zebrafish female (I). Fluorescence photomicrographs showing the expression of Vasa protein (red) in hybrid testis (J) and hybrid ovary (K); normal rabbit serum as controls in hybrid testis (L) and hybrid ovary (M). Sg = spermatogonium; St = spermatid; S = spermatozoa; Oo = oogonia; PO = primary oocytes; SgOII = secondary-growth oocyte phase II; SgOIII = secondary-growth oocyte phase III. Bars = 20 μm (F–H, J–L, M) and 100 μm (I).

FIG. 2.

A) Photomicrograph showing the expression of DsRed (white arrows) in ovarian germ cells in the ovary of a Tg(vasa:DsRed2-vasa);Tg(bactin:EGFP) double transgenic zebrafish. B) Same ovary shown at higher magnification. Bar = 100 μm.

FIG. 3.

The isolation and transplantation of ovarian germ cells. DsRed-expressing ovarian cells from Tg(vasa:DsRed2-vasa);Tg(bactin:EGFP) double transgenic zebrafish were partially purified by discontinuous Percoll gradient centrifugation. A) The number of DsRed-positive cells obtained from each gradient fraction (mean ± SD). B) Isolated ovarian germ cells were transplanted into the abdominal cavity between the swim bladder (SB) and gastrointestinal (GI) tract of 2-wk-old sterile hybrid larvae. Location of transplanted cells (dotted lines). Length of larva = 5 mm.

FIG. 4.

A) Photomicrograph showing the incorporation of transplanted ovarian germ cells into the gonad of a recipient larva 5 days after transplantation. The arrows point to individual DsRed-positive cells. Bar = 100 μm. B) The number of recipient larvae with up to 5 DsRed-positive cells present in the gonadal region 5 days after transplantation. Data from three transplantation experiments (T1–T3) are shown.

FIG. 5.

Colonization of the recipient gonad by transplanted ovarian germ cells. A) Fluorescence photomicrograph of a recipient gonad 6 wk after transplantation showing that the DsRed-expressing ovarian germ cells have proliferated and completely colonized the tissue (A1–A3). A1: red fluorescence, A2: green fluorescence, A3: bright field. Fluorescence photomicrograph of a nonchimeric recipient (A4–A6). A4: red fluorescence, A5: green fluorescence, A6: bright field. Area enclosed inside the dotted lines represents the region of gonadal tissues. B) After reaching sexual maturity at 3 mo of age, the germline chimeric fish possessed one fully developed testis that contained a large number of DsRed-expressing cells derived from the transplanted germ cells (B1 inset, gonads were dissected for fluorescence detection). The other testis in the chimeric fish (black arrow) was underdeveloped. C) Transverse section of the fully developed testis from a fertile male chimera that contains spermatid (St) and spermatozoa (S). Bars = 100 μm (A3, A6, B1) and 20 μm (C).

FIG. 6.

A) Fluorescence photomicrograph of an F1 embryo produced by a germline chimeric father showing EGFP expression throughout its body indicating that the embryo was produced from sperm derived from the transplanted Tg(vasa:DsRed2-vasa);Tg(bactin:EGFP) double transgenic ovarian germ cells. Because vasa-DsRed expression in the embryo is from maternal RNA, DsRed expression was not observed in the F1 embryos. B, C) The F1 adult fish possessed paired gonads that were of normal size (B, ovary; C, testis) and expressed DsRed (B1 inset and C1 inset, gonads were dissected for fluorescence detection) throughout the tissue. D) The F2 embryos produced by mating heterozygous F1 siblings did express maternal vasa-DsRed in the gonadal region (white arrows) and EGFP expression (black arrows) throughout their bodies in approximately 75% of the F2 embryos. Right bottom corner: schematic drawing of the area where the photo was taken. Bars = 200 μm.