University of Miyazaki, RIKEN, Kyoto University,
Shiga University of Medical Science, Hokkaido
UniversityOocyte and Sperm from iPSCsof an Endangered Species-Sexual flexibility of endangered germ cells-Key Points● Derivation of iPSCs from the endangered species Tokudaia osimensis.
● Generation of mouse/T. osimensis interspecific chimeras.

● Production of oocyte and sperm from T. osimensis.

● Discovery of sexual flexibility of XO female T. osimensis germ cells.

To preserve biodiversity, we must spare no effort to prevent theextinction of endangered species. The derivation of inducedpluripotent stem cells (iPSCs) from such species is seen as apotentially effective insurance against extinction. Here, we
generate interspecific chimeras that have endangered species
cells in their whole body including germ cells.
We have successfully established iPSCs from an endangered
species, Tokudaia osimensis. Because of a unique sex
chromosome composition XO/XO, female T. osimensis iPSCs
could contribute and survive as germ cells in adult interspecificovary and testis, which suggests the acquisition of sexual
flexibility of germ cells according to their unique evolution.
These results provide an attractive research tool to explore thesex determination after disappearance of the Y chromosome
according to mammalian evolution. This is the first report toderive live animals and germ cells including endangered
species cells, and indicates the potential reduction of extinction
risk of an endangered species through the use of iPSCs.

Graphical Summary
Fibroblast cells from a tail tip

T. osimensis (♀: XO)

Derivation of iPSCs

Preparation ofrecipient embryos
Mouse

Inject iPSCs tomouse embryos

Transplantationof interspecificembryos

Pseudopregnant
mouse

Interspecific chimeras with endangered cells in their whole body were born
FemalechimeraMalechimera

T. osimensis

oocyte（ ）Ovary

Mouse oocytes

T. osimensis
spermatids（ ）Testis

Mouse spermatids

Successful generation of germ cells from an endangered species, T. osimensis

Objectivs
One in every four animal species is threatened by extinction.
Although it is crucial to save animal species by preserving theirnatural environments, a number of conservation strategies need tobe implemented. If an animal species becomes extinct, it is very
difficult to rewind such extinction. One of potentially most effective
candidates for reducing the risk of extinction of endangered
species is the derivation of induced pluripotent stem cells (iPSCs)
from a small piece of somatic cell tissue. If iPSCs from threatened
endangered species could be derived, it may provide a valuable
way to save their genome or cell resource and to produce germ
cells. Moreover, their iPSCs provide information about theirinteresting characteristics without any reduction of the individual.

Tokudaia osimensis● A small rodent from the Amami-OshimaIsland in Japan● An endangered species (EN)
● Chromosome: 2n=25, XO/XO
Photo：Miyazaki City Phoenix ZooResultsResults

To establish iPSCs from a wild individual T. osimensis, a small tip of
the tail was obtained from a live female and was used for fibroblast
cell culture. We released the captured T. osimensis into the fieldafter recording the individual characteristics and giving first aid to
the injured tail. We established iPSCs of T. osimensis andsuccessfully detected their specific culture condition (Fig. 1). The
derivation of allogeneic preimplantation embryos from an
endangered species as a recipient is impractical. We attempted toprepare preimplantation embryos and to generate interspecificchimeras using mice as the host and recipient for embryotransplantation. T. osimensis iPSCs effectively contributed to thebody of interspecific chimeras (Fig. 2) with germline transmission(Fig. 3). Finally, XO female iPSCs could differentiate into male germ
cells in the interspecific mouse testis and survive for more than 7weeks after birth (Fig. 4).
Fig. 1Fig. 2

Fig. 1: T. osimensis iPSCs
Fig. 2: Interspecific chimerawith brown hair fromT. osimensis cells

200µm

Fig. 3ovary

Fig. 420µm

testisFig. 3: Detection of T. osimensis
oocyte in adult interspecificovary (arrow)
Fig. 4: Detection of T. osimensis
spermatids in adult interspecifictestis (arrowheads)

SignificanceSexual flexibility of endangered T. osimensis germ cells
In most mammals, female (XX) cells cannot differentiate and remain

as spermatids in the male reproductive environment. Because Yrelated genes have the critical involvements to raise and maintainas male germ cells. However, T. osimensis have lost their Y
chromosome and evolved to generate males without Y-related
genes. In this research, we found that female (XO) T. osimensis cells
can differentiate and maintain as spermatids according to thetesticular contribution, which suggests the acquisition of sexual
flexibility of T. osimensis germ cells by their evolution. These results
raise some interesting questions about mammalian evolution,
epigenetic regulation, sex determination, and gamete genesis ofthe unique animal species.
iPSC technology as insurance against possible extinction
Because many species are now threatened with extinction, it is
crucial to preserve biodiversity. We have successfully generated
oocyte and spermatids from a small piece of tissue of a wild
endangered individual. We demonstrate that iPSC technology is apotentially effective strategy for conserving endangered species
from likely extinction.
Note
If endangered species are successfully brought back by iPSC
technology in the future, they must be maintained only in strictly
managed environments as a precious bioresource.
They should not be released into the wild.

Next aims
Effective generation of sperm and oocytes of T. osimensis in vitro .
We aim to generate sperm and oocytes efficiently from thisendangered species in vitro. If we can produce germ cells in vitro,
we will try to generate embryos and individuals of this endangeredspecies.
Developmental fate of XX, XO, and OO embryos.
If we can generate T. osimensis embryos,T. osimensis iPSCs (♀| XO)

their sex chromosome constitution is
supposed to consist of XX, XO, and OO.
We have been much interested in theirGermcells

X

developmental fate and their epigenetic？

regulations.

EmbryosXXOXOXO XO？

OO

Sex chromosome constitution of the embryos

Gonadal development (Sex determination)
In this research, we have elucidated the sexual flexibility of germ
cells of T. osimensis. We would like to clarify the mechanism for thesex determination which T. osimensis obtained during its evolution.
Generation of iPSCs and germ cells from the other endangeredspecies.
We aim to generate true naïve iPSCs from the other endangeredspecies,

suchmuenninke.

asTokudaia tokunoshimensis and Tokudaia

Publication
Flexible adaptation of male germ cells from female iPSCsof endangered Tokudaia osimensis.
Arata Honda, Narantsog Choijookhuu, Haruna Izu, Yoshihiro
Kawano, Mizuho Inokuchi, Kimiko Honsho, Ah-Reum Lee,
Hiroki Nabekura, Hiroshi Ohta, Tomoyuki Tsukiyama, Yasuhide
Ohinata, Asato Kuroiwa, Yoshitaka Hishikawa, Mitinori Saitou,
Takamichi Jogahara, Chihiro Koshimoto.
Science Advances (2017) 3: e1602179
Research groups
University of Miyazaki
Arata Honda, Narantsog Choijookhuu, Haruna Izu,
Yoshihiro Kawano, Mizuho Inokuchi, Kimiko Honsho,
Ah-Reum Lee, Hiroki Nabekura, Yoshitaka
Hishikawa, Takamichi Jogahara, Chihiro Koshimoto.
RIKEN
Arata Honda, Yasuhide Ohinata
Kyoto University
Hiroshi Ohta, Mitinori Saitou
Shiga University of Medical Science
Tomoyuki Tsukiyama
Hokkaido University
Asato Kuroiwa
Contact
Arata Honda, Ph.D.
Tel/Fax: (+81)985-85-9866

E-mail: a-honda(at)med.miyazaki-u.ac.jp*Change (at) in e-mail address to @