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Formation and cultivation of medaka primordial germ cells

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Abstract

Primordial germ cell (PGC) formation is pivotal for fertility. Mammalian PGCs are epigenetically induced without the need for maternal factors and can also be derived in culture from pluripotent stem cells. In egg-laying animals such as Drosophila and zebrafish, PGCs are specified by maternal germ plasm factors without the need for inducing factors. In these organisms, PGC formation and cultivation in vitro from indeterminate embryonic cells have not been possible. Here, we report PGC formation and cultivation in vitro from blastomeres dissociated from midblastula embryos (MBEs) of the fish medaka (Oryzias latipes). PGCs were identified by using germ-cell-specific green fluorescent protein (GFP) expression from a transgene under the control of the vasa promoter. Embryo perturbation was exploited to study PGC formation in vivo, and dissociated MBE cells were cultivated under various conditions to study PGC formation in vitro. Perturbation of somatic development did not prevent PGC formation in live embryos. Dissociated MBE blastomeres formed PGCs in the absence of normal somatic structures and of known inducing factors. Most importantly, under culture conditions conducive to stem cell derivation, some dissociated MBE blastomeres produced GFP-positive PGC-like cells. These GFP-positive cells contained genuine PGCs, as they expressed PGC markers and migrated into the embryonic gonad to generate germline chimeras. Our data thus provide evidence for PGC preformation in medaka and demonstrate, for the first time, that PGC formation and derivation can be obtained in culture from early embryos of medaka as a lower vertebrate model.

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Abbreviations

bFGF:

Basic fibroblast growth factor

BMP4:

Bone morphogenetic protein 4

dpc or hpc:

Day(s) or hour(s) post culture

dpf or hpf:

Day(s) or hour(s) post fertilization

ES cell:

Embryonic stem cell

FBS:

Fetal bovine serum

FS:

Fish serum from rainbow trout

GFP:

Green fluorescent protein

MBE:

Midblastula embryo

MEE:

Medaka embryo extract

PR:

Fusion between puromycin acetyltransferase and red fluorescent protein

PBS:

Phosphate-buffered saline

PBST:

Tween-20 (0.1%) in PBS

pPGC:

Presumptive primordial germ cell

RT-PCR:

Reverse transcription plus polymerase chain reaction

3′-UTR:

3′ Untranslated region

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Acknowledgments

We thank J. Deng for fish breeding.

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Authors and Affiliations

  1. Department of Biological Sciences, National University of Singapore, Science Drive 4, Singapore, 117543, Singapore

    Zhendong Li, Mingyou Li, Ni Hong & Yunhan Hong

  2. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, 201306, China

    Mingyou Li & Yunhan Hong

  3. School of Marine Sciences, Sun Yat-Sen University, Guangzhou, 501275, China

    Meisheng Yi

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  1. Zhendong Li
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  2. Mingyou Li
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  3. Ni Hong
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  4. Meisheng Yi
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  5. Yunhan Hong
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Correspondence to Meisheng Yi or Yunhan Hong.

Additional information

This work was supported by grants to Y.H. from the National Research Foundation Singapore (grant NRF-CRP7-2010-03) and to M.Y. from the National Natural Science Foundation of China (grant 31271576).

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Fig. S1

PGC formation in vivo is independent of somatic development. Vg embryos at the 1-cell stage were non-injected or injected with MOpw (an antisense morpholino oligo) and monitored for somatic and PGC development at 4 dpf. a, b Control embryo. c-g MOpw-injected embryos, showing PGC formation in the absence of somatic structures (red fluorescence attributable to the co-injected dye, ey eye, od oil droplet, pc pigment cell, gr gonad region). Bars 100 μm. (TIFF 6795 kb)

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Li, Z., Li, M., Hong, N. et al. Formation and cultivation of medaka primordial germ cells. Cell Tissue Res 357, 71–81 (2014). https://doi.org/10.1007/s00441-014-1867-z

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