Sex determination without the Y Chromosome in two Japanese rodents
Tokudaia osimensis osimensis and Tokudaia osimensis spp.
National Institute of Bioscience and Human-Technology, 1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
Experimental Animal Center, School of Medicine, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889-1601, Japan
Received: 2 June 2000 / Accepted: 24 August 2000
Abstract. Both males and females of the species of spinous coun-
try-rats (Tokudaia osimensis osimensis, T. o. o., Rodentia: Muri-
dae), which live on Amami Oshima Island, a southern Japanese
island, have 25 chromosomes. Another species of spinous country-
rats (Tokudaia osimensis spp., T. o. spp., which live on Toku-
noshima Island 40 km south of Amami Oshima Island, also have
an odd number of chromosomes, 45. Karyotypes of males and
females by the G-band method were indistinguishable in both
populations. The lesser number of chromosomes (25) of T. o. o. is
likely to be a result of Robertsonian fusions of 45 chromosomes of
T. o. spp. that seem to be the offspring of another spinous country-
rat Tokudaia osimensis muenninki (T. o. m.), which live on Oki-
nawa Island and have 44 chromosomes including the X and Y
Chrs. The lengths of the non-paired, putative X-Chr of T. o. o. and
T. o. spp. occupied roughly 3.2% and 1.7% of the total lengths,
respectively, hinting at translocation or exchange of a part of the X
Chr and thus in violation of Ohno’s Law. Southern blot analysis
with murine Sry as a probe indicated that these two animals do not
have Sry. When Zfx from T. o. spp. was used as a probe, both males
and females of T. o. o. and T. o. spp. showed two bands, suggesting
possible translocation of Zfy from the Y Chr. Comparison of physi-
cal characteristics, constituents of chromosomes, and sex-
determination methods of these three Tokudaia country-rat popu-
lations suggests that each is endemic to each island and constitutes
an independent species. These specialized species would provide
us with clues to elucidate the mechanisms of primary sex deter-
mination and karyotype evolution in mammals.
In mammals, males are heterozygotes having the X and Y Chr, and
the masculinization process is genetically regulated by the pres-
ence or absence of a single gene on the Y Chr, Sry/SRY (sex-
determining region Y; Sinclair et al., 1990). There are rare excep-
tions of sex-determination, not involving the Y Chr. Two vole
species, Ellobius lutescens and E. tancrei (Rodentia: Arvicolinae)
were the first such exceptions reported (Just et al. 1995). Males
and females of E. lutescens have an identical 2n ס 17, XO karyo-
type. Males and females of E. tancrei, E. talpinus, and E. alaicus
have unusual sex chromosome constitutions from 2n ס 32, XX to
54, XX (see Vogel et al. 1998).
Spinous country-rats (Tokudaia osimensis muenninki (T. o. m.)
on Okinawa Island (Okinawa prefecture, Japan) have 44 chromo-
somes with the standard XX/XY sex-determining system
(Tsuchiya et al. 1989). However, the chromosome number is 2n ס
25 for those on Amami Oshima Island, Kagoshima prefecture,
Japan [Tokudaia osimensis osimensis (T. o. o.); Honda et al., 1977]
and 2n ס 45 for those on Tokunosima Island, Kagoshima prefec-
ture [Tokudaia osimensis spp. (T. o. spp.); Honda et al., 1978].
Karyotypes of males (XO) and females (XO) of these rodents are
These spinous country-rats live on tiny islands; urban devel-
opment, environmental disruption, homeless dogs and cats, and in
particular on Amami Oshima Island, introduction of mongooses to
eradicate venomous pit vipers habu (Trimeresurus flavoviridis)
have adversely impacted their habitats, and they live under harsh
conditions. The three spinous country-rats of this study have been
registered in the Red List presented by the Environment Agency of
Japan as endangered species that are on the verge of extinction.
Recently, Soullier et al. (1998) reported that Sry was not am-
plified from T. o. o. by PCR, while clear Sry bands were detected
from phylogenetically closely related Apodemus species. Here we
show that neither T. o. o. nor T. o. spp. has Sry and suggest that a
part of the Y Chr is translocated to the X Chr or an autosome in
both country-rat species.
Materials and Methods
A male and a female of T. o. o. and T. o. spp. had been captured
with traps for investigation before these animals were listed in the Red List
as endangered species.
DNA and cell preparation.
Animals were killed by bleeding under
anesthesia, and the liver and lung were removed. DNA was isolated from
the liver by the phenol method (Sambrook et al. 1989). DNA from mouse
and bovine livers was prepared by the same method.
The lung was used to obtain cultured cells. Small pieces of
lung tissue were cut with scissors and placed on the substrata of tissue-
culture dishes. After approximately 30 min, Eagle’s minimum essential
medium supplemented with 10% fetal bovine serum and antibiotics (peni-
cillin, 100 U/ml and streptomycin, 100 g/ml) was added to the dishes.
Dishes were incubated in a CO
incubator at 37°C. After several days,
fibroblast-like cells had proliferated in the cultures and were harvested by
trypsinization. Aliquots of cells were stored in liquid nitrogen until use
(later, cells were lost by accidental evaporation of liquid nitrogen).
Colcemid (0.1 g/ml) was added to cultures and, after 2 h,
cells were harvested by trypsinization. Chromosome specimens were pre-
pared by the air-dry method; G-banding patterns were obtained by the
Cloning of a part of Zfx from T. o. spp.
Cloning of a part of Zfx from
T. o. spp. was described previously (Xiao et al. 1998). The sequence of this
fragment is available; the GSDB, DDBJ, EMBL, and NCBI accession
number is D83489 (since the Y Chr is lacking in T. o. spp., this sequence
was registered as Zfx; there remains, however, the possibility that this is Zfy
translocated from the Y Chr).
Correspondence to: S. Sutou; E-mail: email@example.com
Mammalian Genome 12, 17–21 (2001).
© Springer-Verlag New York Inc. 2001