1 Department of Development and Differentiation, Institute for Frontier Medical
Sciences, Kyoto University, Kyoto 606-8507, Japan
2 Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto
University, Kyoto 606-8501, Japan
3 The Institute of Physical and Chemical Research (RIKEN), Bioresource Center,
Ibaraki 305-0074, Japan
4 Department of Pathology and Biology of Diseases, Graduate School of Medicine,
Kyoto University, Kyoto 606-8501, Japan
* Author for correspondence (e-mail: takashi{at}mfour.med.kyoto-u.ac.jp)
Accepted 27 October 2004
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SUMMARY |
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Key words: Germ cell, Epiblast, Primordial Germ Cell (PGC), Prospermatogonia, Spermatogonia, Testis, Spermatogenesis, Transplantation, Microinsemination
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Introduction |
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Spermatogenesis is initiated shortly after birth
(Russell et al., 1990;
Meistrich and van Beek, 1993
).
Pro-spermatogonia resume mitosis as spermatogonia, at around postnatal day 5,
then enter into meiosis as spermatocytes and produce spermatids, which develop
into spermatozoa. Spermatogonial stem cells are a subpopulation of
spermatogonia and have the unique ability to self-renew as well as to
differentiate to produce spermatozoa
(Meistrich and van Beek, 1993
;
de Rooij and Russell, 2000
).
These cells continue to divide throughout the life of the animal, and can be
identified by their ability to generate and maintain colonies of
spermatogenesis following transplantation into the seminiferous tubules of
infertile recipient testes (Brinster and
Zimmermann, 1994
). Using this assay, several groups have shown
that pro-spermatogonia in developing fetal testes can differentiate into
spermatogonial stem cells when transferred into the adult testis
(Ohta et al., 2004
;
Jiang and Short, 1998
).
However, it is unknown if germline cells at earlier stages of development can
produce spermatogonial stem cells or spermatogenic colonies after
transplantation.
In this investigation, we sought to determine the potential of germline cells from earlier embryos to develop into spermatogonial stem cells, using immature recipient animals. Epiblast cells or PGCs were transplanted into infertile mouse testes and examined for their ability to re-populate the seminiferous tubules.
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Materials and methods |
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Transplantation into recipient testes
Donor cells were transplanted in histocompatible W/Wv
or Wv/Wv mice (W mice, obtained from
SLC, Shizuoka, Japan). Only 5- to 10-day-old male mice were used as
recipients. W mutants lack endogenous spermatogenesis
(Silvers, 1979), because of
mutations in the Kit gene (Nocka
et al., 1990
; Hayashi et al.,
1991
). Recipient animals were placed on ice to induce hypothermic
anesthesia, and returned to their dams after surgery
(Shinohara et al., 2001
).
Approximately 2 µl of cell suspension were introduced into each testis by
injection via the efferent duct (Ogawa et
al., 1997
).
Histological analysis
Three to four months after transplantation, the recipient testes were fixed
in 10% neutral-buffered formalin (Wako Pure Chemical Industries, Osaka, Japan)
and processed for paraffin sectioning. Sections were stained with hematoxylin
and eosin. Two histological sections were prepared from the testes of each
animal and viewed at 400x magnification to determine the extent of
spermatogenesis. The numbers of tubule cross-sections with or without
spermatogenesis (defined as the presence of multiple layers of germ cells in
the seminiferous tubule) were recorded for one histological section from each
testis. Meiosis was detected by immunofluorescence staining using
anti-synaptonemal complex protein 3 (SCP3) antibody
(Chuma and Nakatsuji, 2001) and
Alexa 488-conjugated anti-rabbit immunoglobulin G antibody (Molecular Probes,
Eugene, USA). Periodic acid Schiff (PAS) staining (Muto Pure Chemicals, Tokyo,
Japan) was carried out to examine acrosome formation in spermatids. In
experiments using Green mice, recipient testes were recovered 10 to 11 weeks
after donor cell transplantation, and analyzed by observing EGFP signals under
fluorescence microscopy. Donor cells were identified specifically because host
testis cells had no endogenous fluorescence. A cluster of germ cells was
defined as a colony when it occupied the entire circumference of the tubule
and was at least 0.1 mm long (Nagano et
al., 1999
). Cryosections of the testes fixed in 4%
paraformaldehyde in PBS were stained with Rhodamine-conjugated Peanut
agglutinin (PNA) (Vector, Burlingame, CA) for acrosomes, and with Hoechst
33258 (Sigma, St Louis, MO) for nuclei.
Microinsemination
Microinsemination was undertaken by intracytoplasmic injection into C57BL/6
x DBA/2 F1 oocytes (Kimura and
Yanagimachi, 1995). Embryos that were constructed using
spermatozoa or elongated spermatids derived from 8.5 dpc or 12.5 dpc PGCs were
transferred into the oviducts of pseudopregnant ICR females after 24 or 48
hours in culture, respectively. Live fetuses retrieved on day 19.5 were raised
by lactating ICR foster mothers.
Genotyping of offspring and bisulfite sequencing of imprinted genes
PCR fragments of the Kit gene encompassing the W point
mutation or the Wv mutation
(Nocka et al., 1990;
Hayashi et al., 1991
) were
amplified using genomic DNA from mice derived from PGC transplantation, or
from a W/Wv mouse as a control heterozygote for both
mutations. PCR primers were 5'-CATTTATCTCCTCGACAACCTTCC-3' and
5'-GCTGCTGGCTCACAATCATGGTTC-3' for W genotyping, and
5'-AGATGGCAACTCGAGACTCACCTC-3' and
5'-TGCCCCCACGCTTTGTTTTGCTAA-3' for Wv
genotyping. Amplified products were gel extracted and directly sequenced.
Bisulfite genomic sequencing of differentially methylated regions (DMRs) of
the Igf2r and H19 imprinted genes was carried out as
described (Ueda et al., 2000;
Lee et al., 2002
;
Lucifero et al., 2002
).
Briefly, genomic DNAs were isolated from the offspring derived from PGC
transplantation, and treated with sodium bisulfite, which deaminates
unmethylated cytosines to uracils, but does not affect 5-methylated cytosines.
Polymerase chain reaction amplification of each DMR from bisulfite-treated
genomic DNAs was carried out using primer sets as described
(Ueda et al., 2000
;
Lucifero et al., 2002
), and
DNA sequences were determined.
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Results |
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To confirm the donor origin of spermatogenesis, we used Green mice that ubiquitously express the EGFP transgene. Donor cells were collected from the posterior thirds of 8.5 dpc embryos that showed EGFP signals. This allowed specific identification of donor cells, because endogenous host testis cells had no detectable fluorescence. Four experiments were performed, and a total of 16 testes were microinjected with donor cells. Approximately 6 to 12 x 103 cells were transplanted into each testis. When the recipients were analyzed 10 to 11 weeks after transplantation, 4 of 16 (25%) testes had spermatogenic colonies that showed EGFP signals (Fig. 1F). The average number of colonies per testis was 0.3 ± 0.1 (mean ± s.e.m.). Histological analysis of the EGFP (+) colonies showed the presence of apparently normal spermatogenesis (Fig. 1G,H).
While these results indicate that all types of donor cells differentiated
into spermatogonial stem cells, differentiation of donor cells was not
restricted to the germline lineage. Consistent with findings of previous
studies (Illmensee and Stevens,
1979), testes receiving epiblast cells or 8.5 dpc PGCs formed
teratomas (Fig. 1I). Epiblast
cells produced larger tumors than did 8.5 dpc PGCs, and some of the
seminiferous tubules were dilated and broken. Cells from three germ layers,
including ciliated epithelium, muscle, neuron and bone, were found in these
testes. Interestingly, spermatogenesis was occasionally observed in tubules
not affected by tumorigenesis, indicating that the same population of donor
cells could cause spermatogenesis and teratogenesis. No teratomas were found
in the recipients of gonadal PGCs or pro-spermatogonia.
To examine whether germ cells generated from PGCs were fully functional, we
performed microinsemination, a technique commonly used to produce offspring
from infertile animals and humans (Kimura
and Yanagimachi, 1995; Palermo
et al., 1992
). Donor PGCs were collected from 8.5 or 12.5 dpc
embryos, and transplanted into the testes of W mice. Four months
after transplantation, spermatozoa or elongated spermatids were collected from
tubule fragments by mechanical dissociation
(Fig. 1J), and microinjected
into oocytes. The results of the microinsemination experiments are summarized
in Table 2. Spermatogenic cells
derived from 8.5 dpc PGCs appeared to be less competent for egg activation,
because a significant number of eggs receiving elongated spermatid/spermatozoa
from 8.5 dpc PGCs stayed at metaphase II or the metaphase II-anaphase
transition and did not develop into the 2-cell stage after 24 hours. However,
offspring were obtained from oocytes inseminated with spermatid/spermatozoa
derived from both stages of PGCs (Fig.
1K). Genotyping of offspring showed that they did not carry
W or Wv mutations in the Kit gene
(Nocka et al., 1990
;
Hayashi et al., 1991
),
demonstrating the offspring were derived from wild-type donor PGCs that had
been transplanted into the testis (Fig.
1L). No apparent abnormality was seen in any of the offspring, and
they were fertile. Bisulfite sequencing analysis of the offspring showed no
obvious fluctuations in the methylation status of the DMRs of paternally
methylated H19 and maternally methylated Igf2r genes
(Fig. 2).
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Discussion |
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The important factor that contributed to the results of our experiments is
the use of immature postnatal testes as recipients. Recently, Ohta et al.
(Ohta et al., 2004) showed
that pro-spermatogonia from 14.5 dpc embryos completed spermatogenesis when
transplanted in mature seminiferous tubules, while spermatogenesis did not
occur from PGCs from 12.5 dpc embryos. Another group reported that germ cells
from day 0-3 mouse pups did not show spermatogenic colonies after
transplantation into adult testes (McLean
et al., 2003
). In our study, however, epiblast cells at 6.5 dpc
and PGCs at 8.5 to 16.5 dpc produced spermatogenesis after transplantation
into immature seminiferous tubules at postnatal day 5 to 10. This difference
might simply be ascribed to structural differences; immature Sertoli cells
lack tight junctions and may allow migrating transplanted cells easier access
to the stem cell niches, which are distributed nonrandomly in the seminiferous
tubules (Chiarini-Garcia et al.,
2001
). Alternatively, immature testis may express factors that
support survival and differentiation of epiblast cells and PGCs, while mature
testis may not. Because PGCs have chemotaxic activity
(Godin et al., 1990
), we
speculate that some of the transplanted cells migrated into the niches of
immature seminiferous tubules, where they could survive. PGCs and epiblast
cells may have then switched their cell cycle fate to function as
spermatogonial stem cells. Both male and female PGCs enter into meiosis in the
absence of the male gonadal environment, but do not if they lodge there
(McLaren and Southee, 1997
;
Chuma and Nakatsuji, 2001
).
Somatic cells in the fetal testis are assumed to produce a substance that
inhibits the meiotic transition of PGCs. Given our results, it appears that
seminiferous tubules of newborn mice may also have similar meiosis-inhibiting
activity.
Erasure of parental genomic imprints commences in PGCs at around the time
of their arrival in the UGR (Szabo and
Mann, 1995; Surani,
2001
; Hajkova et al.,
2002
). However, it has not been clear whether this epigenetic
event depends on induction from the UGR or is programmed autonomously in PGCs.
As the offspring from PGC transplantations were viable and apparently healthy,
epigenetic modifications, including erasure of parental genomic imprints,
should have occurred appropriately in transplanted PGCs. This was corroborated
by the normal methylation patterns exhibited in the DMRs of the Igf2r
and H19 genes. Because the establishment of paternal methylation
proceeds after birth in the normal testis
(Ueda et al., 2000
), it
seems unlikely that the postnatal testis has the ability to erase parental
methylation. Therefore, our results suggest that PGCs may have the autonomous
program for the erasure of parental methylation before reaching the UGR.
Primordial germ cell transplantation will provide a new experimental
approach for the study of PGC development. Primordial germ cells harvested
from embryonic lethal mutants as early as gastrulation can be traced for their
differentiation capacities by transplantation into recipient testis.
Similarly, PGCs manipulated in vitro, such as those cultured with growth
factors or transfected with vectors (De
Miguel et al., 2002; Watanabe
et al., 1997
), can now be assessed for their effects on subsequent
differentiation in vivo. Such functional studies would help elucidate factors
that regulate male germline development in mammals.
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ACKNOWLEDGMENTS |
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