Human Reproduction, Vol.25, No.5 pp. 1113– 1122, 2010
Advanced Access publication on February 19, 2010 doi:10.1093/humrep/deq001
ORIGINAL ARTICLE Andrology
Xenografting of testicular tissue from
an infant human donor results in
accelerated testicular maturation
Y. Sato 1,3,6, S. Nozawa1, M. Yoshiike1, M. Arai 1,4, C. Sasaki 2,
and T. Iwamoto 1,5
1
Department of Urology, St. Marianna University School of Medicine, Kawasaki, Japan 2Institute for Ultrastructural Morphology, St. Marianna
University School of Medicine, Kawasaki, Japan 3Present address: Department of Histology and Cell Biology, Graduate School of Biomedical
Science, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan 4Present address: Unit for Molecular Neurobiology of Learning
and Memory, Okinawa Institute of Science and Technology Promotion Corporation, 12-24 Suzaki, Uruma, Okinawa 904-2234, Japan
5
Present address: Center for Infertility and IVF, International University of Health and Welfare Hospital, Iguchi, Nasushiobara, Tochigi
329-2763, Japan
6
Correspondence address. Department of Histology and Cell Biology, Graduate School of Biomedical Science, Nagasaki University, 1-12-4
Sakamoto, Nagasaki, 852-8523, Japan. E-mail: [email protected]
Submitted on August 5 2008; resubmitted on December 17 2009; accepted on January 4 2010
background: Grafting of testicular tissue into immunodeficient mice has been used to differentiate the neonatal testes from different
animal species up to the level of complete spermatogenesis; however, this approach has not been successful for human testicular tissue. The
aim of this study was to evaluate the capacity for differentiation of infant human testicular tissue grafts.
methods and results: Testicular tissue from a 3-month-old patient with testicular cancer was grafted into immunodeficient nude
mice. At the time of grafting, A spermatogonia were the only germ cells present in the testicular tissue. B spermatogonia and first spermatocytes were observed at 7 months and 1 year after grafting, respectively. Positive immunostaining with antibodies against BOULE and
CDC25A suggested that spermatocytes in the graft were not arrested but in meiosis. Furthermore, ultrastructural and immunohistochemical
analyses showed that the onset of both Sertoli cell maturation and partial differentiation of Leydig cells preceded the appearance of spermatocytes. Differentiation of testicular cells was accelerated compared with in vivo development.
conclusions: Spermatogenesis in the xenograft of infant human testicular tissues proceeded successfully from the stage of spermatogonial stem cells until pachytene spermatocyte formation. The differentiation of Sertoli cells and Leydig cells was reproduced in a
manner similar to that in normal testicular development. Grafting of infant human testicular tissue may be a powerful tool to examine
the early period of human spermatogenesis and may pave the way for fertility preservation among infant patients.
Key words: fertility / human / spermatogenesis / testis / xenograft
Introduction
Male infertility is principally dependent on testicular maturation and
sperm production. There are many common features in spermatogenesis between humans and other animals, although some substantial
differences between them have also been reported (Clermont,
1972; Yanaihara and Troen, 1972; Amann, 2008). Therefore, the
results from animal experiments cannot always be extrapolated to
humans. Thus, there is a need for more favorable models to study
human spermatogenesis.
In recent years, ectopic grafting of immature testicular tissues from
various mammalian species under the back skin of immunodeficient
mice has been developed as a new strategy for preserving testicular
function and generating mature spermatozoa (Honaramooz et al.,
2002, 2004; Oatley et al., 2005; Schlatt et al., 2002; Snedaker
et al., 2004). Despite success in these approaches, this technique is
associated with a high risk of cancer relapse or retroviral infection if
applied to patients. However, xenografting is useful for simultaneous
detection of malignant cell contamination and spermatogonial potential in testicular xenografts collected for fertility preservation (Hou
et al., 2007). It has been reported that the recovery of spermatogenesis in a graft depends on the age of the donor (Schlatt et al., 2002).
Spermatogenesis in a xenograft might provide a model for the study of
human spermatogenesis and could represent a viable approach for fertility preservation in cancer patients, if it is possible to select appropriate donor tissue under optimum host conditions.
& The Author 2010. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
For Permissions, please email: [email protected]
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Unlike successful studies on various animal species, spermatogenesis in a testicular graft from common marmoset with structural similarities in the arrangement of the germinal epithelium to humans was
found not to proceed beyond the spermatogonial stage (Wistuba
et al., 2003, 2004, 2006; Luetjens et al., 2005). Autologous grafting
of immature marmoset testicular tissue showed complete spermatogenesis only in scrotal transplants (Wistuba et al., 2006; Luetjens
et al., 2008). However, in the case of rhesus monkeys, xenografting
of testicular tissue from juvenile animals produced functional sperm
(Honaramooz et al., 2004). Although grafting with adult human testicular tissue derived from clinical material could maintain only a few
spermatogonia (Geens et al., 2006; Schlatt et al., 2006), fetal human
testicular grafts have shown differentiation of gonocytes into spermatogonia and Sertoli cells (Yu et al., 2006). As for the grafting of prepubertal human testicular tissue, spermatogonia and Sertoli cells were
preserved (Goossens et al., 2008) and differentiation of spermatogenic
cells up to pahcytene spermatocytes was observed (Wyns et al.,
2008). To the best of our knowledge, there are no reports of
studies using neonatal or infant human testicular tissues for this xenografting system.
The testis tissue from juvenile donors contains immature seminiferous tubules or tubules with focal spermatogenesis (Wyns et al., 2008).
Compared with juvenile donor tissue, all the spermatogonia, Sertoli
cells and Leydig cells show a more undifferentiated status in the testicular tissue from 3-month-old infants. Considering the efficacy of grafting immature testicular tissues from various animal species, infant
human testicular tissue could be expected to induce spermatogenesis.
Therefore, this infant tissue was grafted into nude mice and the spermatogenesis in the graft was analyzed by immunohistochemistry, morphometry and electron microscopy.
Materials and Methods
Tissue donor
Infant human testicular tissue was obtained from a 3-month-old male
patient undergoing orchiectomy for a testicular tumor (hemangioma)
after written informed consent was obtained from his parents to use
some of his tissue for this research. As well as use for grafting, a small
part of the excised tissue was used for morphological and histological
evaluations. The tissue for grafting was kept in ice-cold PBS for periods
not exceeding 4 h. It was confirmed afterwards by histopathological evaluation that the donor tissue for grafting contained no tumorous regions.
This study was approved by the Ethical Committee/Institutional Review
Board of St. Marianna University School of Medicine.
Recipient animals and surgical procedures
Seven-week-old immunodeficient male nude mice (BALB/cAJCL-nu/nu;
CLEA Japan, Kawasaki) were used as the recipients (n ¼ 5). Mice were
anesthetized using Nembutal, sodium pentobarbital (Dainippon Sumitomo
Pharma Co., Ltd, Osaka, Japan) and castrated by bilateral orchiectomy
through abdominal incisions. Immediately after castration, six to eight testicular fragments (sizes ranged from 0.5 to 1 mm3) were placed in each
incision made under the dorsal skin on either side of the dorsal midline
using forceps and the incisions were sutured using 4-0 braided silk
(Matsuda Co., Ltd, Tokyo). Animal experiments in this study were
approved by the Animal Research Committee of St. Marianna University
of Medicine.
Sato et al.
Histological and ultrastructural analyses
Specimens for morphological evaluation of the testicular tissue obtained
before and after grafting were prepared by basically the same procedure.
At four time points (1, 2, 4 and 7 months after grafting), one or two randomly selected grafts were biopsied from the recipient mice under
anesthesia. One year after grafting, all the recipient mice were sacrificed
and all remaining grafts were recovered. A large proportion of the testicular specimens were cut and fixed using Bouin’s fluid and embedded in paraffin. Five-micrometer-thick sections were stained with hematoxylin and
eosin in a standard fashion for light microscopic observations or used
for immunohistochemical evaluation. For the ultrastructural study, small
fragments of the specimens were fixed with 1% glutaraldehyde in 0.2 M
cacodylate buffer overnight, post-fixed in 1% osmium tetroxide in the
buffer for 1 h after washing, dehydrated through a graded alcohol series
and embedded with Epon812 (EPON812 kit, TAAB, USA). Ultrathin sections counterstained with lead acetate were observed under transmission
electron microscope TEM-1200EX (JOEL DATUM Ltd., Tokyo, Japan) at
an 80-kV accelerating voltage.
Immunohistochemistry
Deparaffinized and rehydrated sections were incubated in 3% H2O2/
MeOH for 10 min to suppress endogenous peroxidase activity after treatment for antigen retrieval with 10 mM sodium citrate/distilled water (pH
6.0) in an autoclave at 1208C for 10 min, as appropriate. Non-specific
binding was blocked by incubation with 10% goat serum in PBS for 1 h.
Primary rabbit polyclonal antibodies against BOULE (H-89, 1:100),
CDC25A (144, 1:200), androgen receptor (AR) (N-20, 1:200) or
3b-hydroxysteroid dehydrogenase (3b-HSD; H-143, 1:25; all from Santa
Cruz Biotech, CA, USA) diluted with 1% BSA in PBS, were applied to
the slides and incubated for 2 h (CDC25A) or overnight (BOULE, AR,
3b-HSD). Rabbit IgG (Dako, Japan) adjusted to a working concentration
for each primary antibody was used for a negative control. After several
washes in PBS, the sections were incubated with anti-rabbit IgG (Fab0 )
labeled with amino acid polymer-peroxidase complex [Histofine simple
stain MAX PO (R); Nichirei Co., Tokyo, Japan] for 1 h. All incubations
of the sections with blocking serum, IgG or antibodies were performed
in a humidified chamber at room temperature. The sections were then
washed and the peroxidase complex detected by incubation with
aminoethyl-carbazol (AEC; Histofine simple stain AEC solution; Nichirei
Co.) in accordance with the manufacturer’s instructions. Sections were
counterstained with hematoxylin, mounted in aqueous permanent mounting solution (Nichirei Co.) and observed under the microscope. Control
sections showing complete spermatogenesis (mean Johnsen score 7.4 –
9.4) were made from three patient biopsies diagnosed with obstructive
azoospermia in two of the cases and oligozoospermia with varicocele in
the third (age range, 27 – 38 years old), which were obtained after
informed consent was provided for this research.
Results
Spermatogenic differentiation of
transplanted infant human tissue
There was a marked increase in the size of the recovered grafts, typically 2–4 mm in diameter, reflecting about 10 –50-fold growth. Surviving grafts contained different features of seminiferous tubules in which
differentiated spermatogenic cells up to spermatogonia (33.3%) and
spermatocytes (35.4%) were observed as well as Sertoli cell only patterns (18.8%) and atrophic changes (12.5%; Table I).
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Differentiation of immature human testis in xenograft
Table I Seminiferous tubules in the grafts showing variable degrees of spermatogenic activity.
Animal
Graft survival
Tubule atrophy
Sertoli cell only
Spermatogonia
Spermatocyte
...........................................................
Leptotene
Zygotene
Pachytene
.............................................................................................................................................................................................
1
4/6
66.7%
1/4
25%
1/4
25%
2/4
50%
0/4
0/4
0%
0/4
2
6/6
100%
0/6
0%
3/6
50%
2/6
33.3%
1/6
0/6
16.7%
0/6
3
4/6
66.7%
0/4
0%
0/4
0%
0/4
0%
0/4
0/4
100%
4/4
4
4/6
66.7%
1/4
25%
0/4
0%
2/4
50%
1/4
0/4
25%
0/4
Mean
75.0%
12.5%
18.8%
33.3%
35.4%
The number of grafts showing respective stages as the most advanced stage of spermatogenesis are indicated.
After grafting, the diameter of seminiferous tubules increased, the
interstitial compartments of the tissue became enlarged and clusters
of Leydig cells were observed (Fig.1A– C). Apale spermatogonia (latedifferentiated A spermatogonia) and B spermatogonia were first
found at 2 months and 7 months (Fig. 1B0 ) after grafting, respectively,
although only Adark spermatogonia (early undifferentiated A spermatogonia) were contained in the testicular tissue before grafting (Fig. 1A0 ).
One year after grafting, pachytene spermatocytes were observed as the
most advanced type of germ cell (Fig. 1C0 ). Four of five mice survived
and one died at 7 months after grafting because of an accident. The survival rate of the testicular grafts after 1 year was 75.0% (n ¼ 24, Table I).
Sertoli cell differentiation of transplanted
infant human tissue
Two months after grafting, Sertoli cells showed an immature phenotype:
ovoid nuclei with a small nucleolus (Nistal et al., 1982). Four months after
grafting, the shape of nuclei had changed (Fig. 2A). On the basis of the
appearance of the nucleus and cytoplasm, Sertoli cells were divided
into two types: a dark type characterized by protruded electron-dense
cytoplasm and irregular-shaped dark nucleus, and a clear type of
Sertoli cells showing clear cytoplasm with an elongated nucleus
(Fig. 2A), which demonstrated typical differentiation of Sertoli cells
during the immature period (Vilar, 1970). Seven months after grafting,
most of the Sertoli cells showed a clear cytoplasm with irregular-shaped
nuclei (Fig. 2B). Each nucleus included a prominent nucleolus surrounded
by nucleolonema, as found in mature Sertoli cells, and the cytoplasm
showed well-developed rough endoplasmic reticula and abundant
mitochondria (Fig. 2C and D), as shown in the later period of immature
Sertoli cells (Nistal et al., 1982). Desmosomes were observed, but tight
junctions were not found at 7 months after grafting (Fig. 2D). Tight
junctions between Sertoli cells were encountered at 1 year after grafting
(Fig. 2E), as normally observed with mature Sertoli cells in late prepubertal testes (Nistal et al., 1982; Kerr, 1991).
Leydig cell differentiation of transplanted
infant human tissue
There were clusters of immature Leydig cells in the testicular interstitium of the grafted tissue at 7 months after transplantation (Fig. 3A).
Immature Leydig cells were classified into three categories as they
differentiated from fetal-type through infantile-type into partially
differentiated-type cells (Nistal et al., 1986). The nuclei showed a multilobulated morphology (Fig. 3A) that is usually observed in infantiletype Leydig cells (Nistal et al., 1986). The cytoplasm possessed mitochondria with well-developed tubular cristae and peroxisome-like
bodies (Fig. 3B), as shown in partially differentiated Leydig cells
(Nistal et al., 1986). Smooth endoplasmic reticulum was observed,
but it was not abundant (Fig. 3B). Leydig cells showed the secretion
of lipids in the graft (Fig. 3B). One year after grafting, the numbers
of partially differentiated Leydig cells, usually observed during the
late pubertal period in vivo (Prince, 1990; Habert et al., 2001), had
increased, and in addition characteristics of a more advanced stage
in partially differentiated Leydig cells such as lipid droplets, lysosomes
and multivesicular bodies were increased in frequency in the cytoplasm (Fig. 3C and C0 ).
Immunohistochemistry
BOULE, a highly conserved key regulator of meiosis in male germ cells
(Luetjens et al., 2004), was localized in the cytoplasm of spematocytes
(leptotene to pachytene) in the testicular tissue 1 year after grafting,
and was similar to that in adult testis (Fig. 4A and C). However, no
BOULE staining was observed in the infant donor testis (Fig. 4B). Furthermore, strong immunoreactivity of CDC25A, which has a crucial
function in the completion of the meiotic M-Phase (Wolgemuth
et al., 2002), was detected in the cytoplasm of spermatocytes 1
year after grafting; a similar staining pattern was observed in adult
spermatocytes (leptotene to pachytene; Fig. 4D and F). Weak staining
of CDC25A in spermatogonia was commonly observed in the donor,
graft and adult tissues (Fig. 4D–F). Both molecules were detected in
spermatocytes of the graft simultaneously.
The Sertoli cells in the graft after 1 year exhibited strong staining for
AR, indicating that these cells possessed the ability to functionally
respond to androgens (Fig. 5A) and showed a staining pattern similar
to those in the adult control tissues (Fig. 5C), whereas the infant
donor control tissues showed no staining (Fig. 5B). Myoid cells and
Leydig cells had the same localization of AR in all cases (Fig. 5A– C).
Thus, the Sertoli cells of infant testicular tissue were considered to
have differentiated into more mature cells in the graft within 1 year.
The Leydig cells in the tissue 1 year after grafting exhibited a specific
cytoplasmic staining for 3b-HSD exclusively in the cytoplasm (Fig. 5D).
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Sato et al.
Figure 1 Histological appearance of human testicular tissue from the infant patient (A, before grafting, 3 months of age; A0 , magnification of A; B, 7
months after grafting; B0 , magnification of B; C, 1 year after grafting; C0 , magnification of C).
After grafting, the diameter of seminiferous tubules increased, the interstitial compartments became wide and the clusters of Leydig cells were observed. Adark spermatogonia (Asp) were observed before grafting (A0 ), B spermatogonia (Bsp) were observed at 7 months after grafting, and pachytene spermatocytes (Pach) at 1 year after
grafting were observed, as the most advanced spermatogenic stages. mc, myoid cell; sc, Sertoli cell; lc, Leydig cell; Zyg, zygotene spermatocyte. Bars: 100 mm (A, B, C),
15 mm (A0 , B0 , C0 ).
The adult control exhibited 3b-HSD staining in the Leydig cells
(Fig. 5F) although the donor infant control showed only faint staining
in the cytoplasm (Fig. 5E). Leydig cells in the testicular graft were
mature enough to start steroidogenesis within 1 year.
Negative control specimens using rabbit IgG in place of anti-serum
showed no staining for every antibody examined in all cases (data not
shown).
Discussion
The present study showed that human testicular tissue derived from
a 3-month-old boy survived as xenografts in recipient mice for at
least 1 year, and germ cell differentiation and somatic cell maturation
occurred.
In the microenvironment of the grafts, A spermatogonia, which
were initially contained in the infant donor testis, differentiated into
B spermatogonia in 7 months and into the first spermatocytes in 1
year. On the other hand, human germ cell differentiation into B spermatogonia and first spermatocytes is normally observed at 4–5 years
of age and at 8–10 years of age (late prepubertal period), respectively.
Compared with normal testicular development, differentiation of
the spermatogenic cells in immature human testicular grafts was
accelerated (Fig. 6) similarly to that for immature testes from pigs
and rhesus monkeys (Honaramooz et al., 2002, 2004). Accelerated
Differentiation of immature human testis in xenograft
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Figure 2 Ultrastructure of Sertoli cells from the infant patient after grafting (A, 4 months after grafting; B– D, 7 months after grafting; E, 1 year after
grafting).
Two types of Sertoli cell were observed at 4 months after grafting: Sertoli cells with clear cytoplasm and elongated nuclei (cSC) and Sertoli cells with dark elongated
cytoplasm and irregular nuclei (dSC) (A). At 7 months after grafting, all Sertoli cells showed clear cytoplasm and irregular-shaped nuclei (B). The nucleus contained a
nucleolus (nl) and nucleolonema (nu), and well-developed rough endoplasmic reticula (rer) and many mitochondria were observed in the cytoplasm (C, D). Between
the junction of Sertoli cells, desmosomes were present (arrows) (D). Tight junctions between Sertoli cells were observed at 1 year after grafting (arrows) (E). Bars:
4 mm (A), 5 mm (B), 2 mm (C, D), 1 mm (E).
differentiation of spermatogenic cells in the graft might be a consequence of a change in the timing of maturation of Sertoli cells and/
or Leydig cells in the testicular tissue after grafting.
Sertoli cells have crucial roles in the development and differentiation
of germ cells (de Kretser et al., 1998; Silva et al., 2002). Human Sertoli
cells have been reported to start proliferation between the late prepubertal period (8– 10 years of age) to puberty (10–13 years of age)
with some characteristic morphological and other changes (Nistal
et al., 1982; Vilar, 1970; Kerr, 1991; Schulze, 1988; Sharpe et al.,
2003). AR expression in Sertoli cells is suppressed during the neonatal
and infantile periods, and is then expressed in the Sertoli cells after late
puberty and during adulthood (Sharpe et al., 2003). The present
study explored whether Sertoli cells in infant human testicular grafts
have the capacity to support germ cell differentiation. Morphological
investigations demonstrated that Sertoli cells in the graft showed
immature features when observed at 2 months after grafting, and
exhibited more mature characteristics 1 year after grafting. From the
expression of AR, the differentiation state of Sertoli cells in the testicular tissue 1 year after grafting was thought to be equivalent to that in
the late pubertal stage in vivo, suggesting that the differentiation of
Sertoli cells in the infant human testicular graft was also accelerated,
in addition to that of spermatogenic cells in the graft (Fig. 6).
Leydig cells are important for the secretion of steroid hormones
necessary for spermatogenesis (Habert et al., 2001). Mature functional
Leydig cells exhibiting a steroid-producing morphology are predominantly present in the fetal and adult periods (Prince, 1984; Habert
et al., 2001). Infantile inactive type, small Leydig cells present from
the neonatal to prepubertal period (from birth to 10 years of age)
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Sato et al.
Figure 3 Ultrastructure of Leydig cells from the infant patient after grafting (A, B, 7 months after grafting; C, C0 , 1 year after grafting).
Multilobulated infantile-type Leydig cells (lc) with lipid droplets (arrows) were clustered at the interstitium (A). Mitochondria with tubular cristae (mt) were characterized
as partially differentiated Leydig cells (B). Several signs of partially differentiated Leydig cells were increased in prominence 1 year after grafting: many lipid droplets (L),
lysosomes, multivesicular bodies (mv) and mitochondria with tubular cristae (C, C0 ). rer, rough endoplasmic reticulumn. Bars: 10 mm (A), 1 mm (B), 2 mm (C), 1 mm (C0 ).
N, nucleus; ser, smooth endoplasmic reticulum; pl, peroxisome-like body.
and partially differentiated Leydig cell appear and can be observed in
the later prepubertal period (from 6 to 8 years of age; Nistal et al.,
1986; Prince, 1990). Ultrastructural observations of the human testicular graft revealed that infantile-type Leydig cells were predominantly
present until 7 months after grafting; however, a large number of partially differentiated Leydig cells, as found in the late prepubertal period
in vivo, were observed in the specimen 1 year after grafting. In addition,
the expression of 3b-HSD, a marker of steroidogenic activity,
indicated that Leydig cells in the graft after 1 year started typical
differentiation of mature functional Leydig cells, as normally shown
in the early pubertal period in vivo. These results showed that the
differentiation of Leydig cells in this infant human graft was also accelerated, similar to those of spermatogenic cells and Sertoli cells.
However, the production of testosterone in the recipients was
expected to be lower because seminal vesicles of the castrated recipient mice were 10 times smaller than those of the intact controls (data
not shown). Testosterone production is influenced by the size of the
functional Leydig cell population. The number of mature, active Leydig
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Differentiation of immature human testis in xenograft
Figure 4 Expression of BOULE and CDC25A in testicular tissue from an infant patient after grafting.
(A– C) Expression of BOULE. (D – F) Expression of CDC25A. (A, D) Infant testicular tissue 1 year after grafting. (B, E) Infant testicular tissue before grafting. (C, F) Adult
testicular tissue. Arrows: strong expression of BOULE (A, C) and CDC25A (D, F) in pachytene spermatocytes. Arrowheads: weak expression of CDC25A (D, E, F) in
spermatogonia. Bar: 50 mm.
cells contained in the graft may have been insufficient for testosterone
production to recover the size of the seminal vesicles in the castrated
recipients.
The time required for differentiation of spermatogenic cells in immature testicular xenografts has been found to be different among animal
species used as donor sources. The rate of spermatogenesis is accelerated in testicular xenografts from pigs, sheep and rhesus monkeys but
not in those from cats or cattle, compared with that in the donor
species (Honaramooz et al., 2002, 2004; Oatley et al., 2004, 2005;
Snedaker et al., 2004). The length of the spermatogenic cycle is conserved in porcine and ovine testis xenografts and the cycle length is
thought to be inherent to the germ cell genotype. Therefore, these
results suggest that accelerated spermatogenesis in xenografts is not
attributable to a shortened length of spermatogenic cycle but probably
to changes in the endocrine environment (Zeng et al., 2006).
Gonadotrophins are known as possible factors affecting the maturational drive of testicular cells in grafts. In human post-natal development, serum gonadotrophins, FSH and LH, are at low levels
throughout the prepubertal period, after gonadotrophins in infants
increase to a peak at around 4–10 weeks of age (Grumbach,
2005). It has been shown that boys with central precocious puberty
may reach full testis maturation owing to excess gonadotrophins at
a very early age (Kakarla and Bradshaw, 2003). In contrast, total gonadotrophin deficiency has been found to delay puberty and cause the
testes to remain in an immature state (Grumbach, 2005), demonstrating the importance of the induction due to gonadotrophins. Castration
of the host leads to increased gonadotrophins levels (Schlatt et al.,
2003). This situation may resemble the hormonal environment of pubertal onset for the infant graft, affecting testicular maturation and
leading to acceleration of the differentiation processes.
Depending on the maturation state of the donor testis, the responsiveness to gonadotrophins in grafts differs. In the rhesus monkey as a
donor species, exogenous supplementation of gonadotrophins is
needed for infant testicular grafts but not for juvenile ones
(Honaramooz et al., 2004; Rathi et al., 2008). It was recently shown
that human testicular tissues that are orthotopically transplanted
from prepubertal boys into mouse hosts developed up to pachytene
spermatocytes without supplementation of gonadotrophins
(Wyns et al., 2008). The present results indicate that grafts obtained
from a 3-month-old boy showed the same differentiation patterns
as mentioned above and proceeded to the stage of pachytene spermatocytes, which suggests that the developmental status at the time of
grafting does not affect the potential for graft development in prepubertal human testis. However, because species-specific differences in
gonadotrophins could also be involved in insufficient germ cell maturation, it may be appropriate to use human gonadotrophin supplementation in a human model to further investigate the potential of infant
testicular tissue.
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Sato et al.
Figure 5 Expression of AR and 3b-HSD in the testicular tissue from an infant patient after grafting.
(A– C) Expression of AR. (D – F) Expression of 3b-HSD. (A, D) Infant testicular tissue 1 year after grafting. (B, E) Infant testicular tissue before grafting. (C, F) Adult
testicular tissue. Arrows: strong expression of AR in the Sertoli cells (A, C) and 3b-HSD in Leydig cells (D, F). Expression of AR was also observed in Leydig cells
(LC) and myoid cells (MC) (A, B, C). Almost no expression of 3b-HSD was observed in the infant tissue (E). Bar: 50 mm.
Figure 6 Schematic drawing of accelerated differentiation of testicular cells in the graft.
Solid lines with arrowheads show the appearance of each cell type for developing spermatogonia/spermatocytes and Sertoli cells in the graft. Dotted lines
show the appearance of only a small number of these cell types. Black lines
represent differentiation in vivo and the gray lines represent that in the grafts.
SG, spermatogonia; Ad SG, Adark spermatogonia; Ap SG, Apale spermatogonia;
B SG, B spermatogonia; SPC, spermatocyte.
Unlike xenografting of immature testicular tissue from many other
species (Honaramooz et al., 2002, 2004; Schlatt et al., 2002; Oatley
et al., 2005; Snedaker et al., 2004), human spermatogonial stem
cells from infant testicular tissue could not differentiate into complete
spermatozoa and the most advanced spermatogenic stage was pachytene spermatocyte. It has been shown that in the case of human testis
with meiotic arrest, in which BOULE is absent, CDC25A is concomitantly lacking and the lack of both BOULE and CDC25A results in
meiotic arrest (Luetjens et al., 2004). According to the expression
of BOULE and CDC25A in spermatocytes in the present study, spermatogenesis in testicular tissue 1 year after grafting was thought not to
be arrested but in meiosis. In other words, the grafted infant tissue still
might have the ability to promote further differentiation beyond the
spermatocyte stage.
The duration of the prepubertal period differs among mammalian
species; humans need 8–10 years before entering the pubertal
period, which is much longer than that in other mammalian species.
Even if differentiation is accelerated in the graft, a period of 1 year
may be insufficient for further differentiation of all the cell types.
The life span of a mouse may be too short to sustain human spermatogenesis in a xenograft. For further study, it might be necessary to
undertake transexplantation of grown grafts or xenografting of
Differentiation of immature human testis in xenograft
various stages of donor testicular tissue in order to examine the efficacy of this xenografting system in human testicular development. The
present results have a limitation in that a single testis donor may not
be representative; therefore, more studies using human tissue are
needed in the near future.
In conclusion, the present study was able to differentiate infant
human spermatogenic cells up to pachytene spermatocytes from the
spermatogonial stem cells using a xenografting system. The differentiation of Sertoli cells and Leydig cells could be reproduced in the
graft in a similar manner to that in infant testicular development in
vivo. Infant human testicular tissue showed a higher potential for development; therefore, xenografting using this tissue may provide a promising strategy for germline preservation in prepubertal cancer patients.
Especially with regard to hormonal manipulation of germ cell developments, the present system using infant human testicular tissue is useful
to make up for the differences in fertility preservation strategies
between humans and other animals (Mitchell et al., 2009). Furthermore, this technique may be a powerful tool accessible for the
study human spermatogenesis at least up to meiosis.
Acknowledgements
We are grateful to Dr T. Takahashi at the St. Marianna University
School of Medicine for providing the surgical specimens, and to Ms
T. Katayama and the members of the animal facility of St. Marianna
University of Medicine for animal care. We also thank Dr
I. Doblinski, Dr A. Honaramooz at the University of Pennsylvania
School of Veterinary Medicine and Dr M. Itoh at the Central Institute
for Experimental Animals for technical advice with regard to grafting.
Funding
Support for this research came from Core Research for Evolutional
Science and Technology, Japan Science and Technology Corporation
to T.I., a Grant-in-aid for Science and Research (C) from the Japan
Society for the Promotion of Science to T.I (15591719) and a
Grant-in-aid for Young Scientists (B) from the Ministry of Education,
Culture Sports, Science and Technology to Y.S. (17791095).
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