BIOLOGY OF REPRODUCTION 60, 515–521 (1999)
Xenogeneic Spermatogenesis Following Transplantation of Hamster Germ Cells
to Mouse Testes1
Takehiko Ogawa, Ina Dobrinski, Mary R. Avarbock, and Ralph L. Brinster2
School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6009
ABSTRACT
otic phase through which four haploid spermatids are produced from each tetraploid preleptotene spermatocyte, the
daughter cell of type B spermatogonia. Preleptotene spermatocytes give rise to leptotene spermatocytes, which are
located in the adluminal compartment of the seminiferous
tubules. During subsequent development to zygotene,
pachytene, and diplotene spermatocyte, meiotic recombination occurs. The last phase, spermiogenesis, is characterized by the dramatic morphological transformation of the
spermatogenic cell from secondary spermatocyte to spermatid and finally to spermatozoon. Spermiogenesis is subdivided into Golgi, cap, acrosome, and maturation phases,
each being subdivided into several steps on the basis of the
changes observed in the nucleus and the developing acrosome [3–5]. These final steps are complex and essential for
making functional spermatozoa that are highly specialized
to carry the haploid genome to the ovum for the creation
of a new individual.
Throughout spermatogenesis, interactions between germ
cells and somatic cells are essential. In particular, Sertoli
cells maintain continuous contact with germ cells, from
spermatogonia to spermatozoa. Spermiogenesis appears to
be particularly dependent on interaction of germ cells with
Sertoli cells. Characteristic structural features, such as ectoplasmic specializations and tubulobulbar complexes, suggest special interactions between Sertoli cells and spermatids [6]. Hormonal control of spermatogenesis also appears
to be mediated by Sertoli cells. The complexity of germ
cell differentiation, the long time frame for completion of
the process, and the close interaction between germ cells
and Sertoli cells suggest that spermatogenesis should be
highly species specific. The differences among various species in morphology of spermatozoa and time to complete
the process support that belief.
In 1994, the technique of spermatogonial transplantation
was reported, through which germ cells of a fertile mouse
can be transplanted into the seminiferous tubules of an infertile mouse to develop donor cell-derived spermatogenesis [7]. Several months after transplantation, many foci of
donor cell colonization were observed in the seminiferous
tubules of recipient mice. In the most successful transplantations, the recipient mouse could transmit the haplotype of
the donor cells to progeny [8]. A significant and surprising
extension of these studies was xenogeneic spermatogonial
transplantation [9]. Testis cells from transgenic rats were
transplanted to the testes of immunodeficient mice, and
complete rat spermatogenesis occurred in the recipient
mouse, resulting in the presence of normal-appearing rat
spermatozoa in the epididymides of recipient mice. Although there is similarity in the general appearance of rat
and mouse spermatozoa, significant differences exist between the spermatozoa of these species. The sperm heads
are distinct and the sperm tail is 40% longer in the rat.
Furthermore, the process of spermatogenesis takes 50%
longer in the rat than in the mouse. Because rat spermatogenesis could occur in the mouse testis in spite of these
It was recently demonstrated that rat spermatogenesis can
occur in the seminiferous tubules of an immunodeficient recipient mouse after transplantation of testis cells from a donor rat.
In the present study, hamster donor testis cells were transplanted to mice to determine whether xenogeneic spermatogenesis
would result. The hamster diverged at least 16 million years ago
from the mouse and produces spermatozoa that are larger than,
and have a shape distinctly different from, those of the mouse.
In four separate experiments with a total of 13 recipient mice,
hamster spermatogenesis was identified in the testes of each
mouse. Approximately 6% of the tubules examined demonstrated xenogeneic spermatogenesis. In addition, cryopreserved
hamster testis cells generated spermatogenesis in recipients.
However, abnormalities were noted in hamster spermatids and
acrosomes in seminiferous tubules of recipient mice. Hamster
spermatozoa were also found in the epididymis of recipient animals, but these spermatozoa generally lacked acrosomes, and
heads and tails were separated. Thus, defects in spermiogenesis
occur in hamster spermatogenesis in the mouse, which may reflect a limited ability of endogenous mouse Sertoli cells to support fully the larger and evolutionarily distant hamster germ
cell. The generation of spermatogenesis from frozen hamster
cells now adds this species to the mouse and rat, in which spermatogonial stem cells also can be cryopreserved. This finding
has immediate application to valuable animals of many species,
because the cells could be stored until suitable recipients are
identified or culture techniques devised to expand the stem cell
population.
INTRODUCTION
Spermatogenesis is a highly specialized and efficient
process resulting in the production of large numbers of
spermatozoa. This long and complex process depends on
precise temporal and spatial relationships between germ
cells and somatic cells and also among germ cells themselves. Through this intricate process approximately 107
spermatozoa are produced daily per gram of testis tissue in
most mammalian species from rodents to primates, including the human [1, 2].
Spermatogenesis can be divided into three phases. In the
first phase, the spermatogonial stem cells divide to provide
daughter spermatogonia that undergo differentiation
through a series of subsequent divisions. Spermatogonia are
located on the basement membrane of the seminiferous tubules, separated from the adluminal compartment of the
tubule by tight junctions between Sertoli cells (blood-testis
barrier). The second phase of spermatogenesis is the meiAccepted September 25, 1998.
Received August 18, 1998.
1
Supported by the National Institute of Health (NICHD 36504), U.S.
Department of Agriculture/NRI Competitive Grants Program (95–37205–
2353), Commonwealth and General Assembly of Pennsylvania, and the
Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation.
2
Correspondence: R.L. Brinster, School of Veterinary Medicine, University of Pennsylvania, 3850 Baltimore Avenue, Philadelphia, PA 19104–
6009. FAX: 215 898 0667.
515
516
OGAWA ET AL.
significant differences in sperm morphology and developmental timing, and in spite of the fact that the two species
phylogenetically diverged 10–11 million years ago, it
seemed possible that xenogeneic transplantation might be
achieved for other species.
In order to explore and develop this approach, we chose
the Syrian golden hamster as a donor animal because of
the widespread interest in hamsters as experimental animals
in both reproductive and nonreproductive research. In addition, hamster spermatozoa are distinct in size and shape
from those of the mouse or rat. The hamster spermatozoon
is larger than the mouse spermatozoon and is characterized
by a large, hook-shaped head with a prominent acrosomal
cap as well as a long, thick tail [10]. Thus, using the hamster as a donor species represents a logical and important
extension of xenogeneic spermatogonial transplantation to
a species that is significantly more divergent from the
mouse, in physiology and sperm structure, than is the rat.
The studies described here were done to investigate whether hamster spermatogenesis from donor cells can occur in
the seminiferous tubules of recipient mice.
MATERIALS AND METHODS
Donor Cell Preparation
Syrian golden hamsters (Harlan Sprague Dawley, Inc.,
Indianapolis, IN), between 1 and 2 mo of age, were used
as the source of donor testis cells. Spermatogenesis was
fully developed in all donor testes. The animals were maintained in an air-conditioned environment at 228C on a 14L:
10D photoperiod before they were killed. All experimental
procedures were approved by the Animal Care and Use
Committee at the University of Pennsylvania. Testis cells
were collected by a two-step enzymatic digestion previously described [11], with minor modifications. Briefly, the
tunica albuginea was manually removed from the testes.
The exposed seminiferous tubules were then dissociated
with collagenase (1 mg/ml; type IV, Sigma Chemical Co.,
St. Louis, MO) in 10 ml of Hanks’ Balanced Salt Solution
without calcium and magnesium (HBSS) at 378C for 5–10
min. DNase I (7 mg/ml) in HBSS was added as needed
(approximately 100 ml) to further dissociate tubules. After
rinsing 2–4 times in HBSS, the tissue was digested with
0.25% trypsin and 1 mM EDTA in 10 ml of HBSS at 378C
for 5–10 min. Fetal bovine serum (1–2 ml) was added to
stop enzymatic digestion. The resulting cell suspension was
filtered through a nylon mesh with 60-mm pore size (Tetko
Inc., Kansas City, MO), and the cells were collected by
centrifugation, 600 3 g for 5 min at 168C. The pellet was
resuspended in Dulbecco’s Modified Eagle’s medium
(DMEM) containing 10% fetal bovine serum (FBS), 2 mM
glutamine, 6 mM lactate, 0.5 mM pyruvate, 30 mg/L penicillin, and 50 mg/L streptomycin (designated DMEM-C)
to a final concentration of 100 to 250 3 106 cells/ml. Viability of cells was greater than 95% as determined by trypan blue exclusion.
Donor Cell Freezing
Several aliquots of hamster testis cells collected as described above were frozen and kept in liquid nitrogen for
transplantation at a later date. The freezing procedure was
essentially the same as for cultured somatic cells [12, 13].
First, collected hamster testis cells were suspended in
DMEM-C at a concentration of 56 3 106 cells/ml. Freezing
medium (FBS, DMEM-C, DMSO in a ratio of 1:3:1) was
added slowly at a volume equal to that of the original cell
suspension and mixed. Cells were aliquoted at 1.0 ml per
freezing vial, placed in an insulated container at 2708C for
at least 12 h, and stored in liquid nitrogen (21968C). The
cells were thawed by swirling in a 378C water bath, and
DMEM-C was added slowly to three times the volume in
the vial. This cell suspension was centrifuged at 600 3 g
for 5 min at 168C, and the pellet was resuspended in
DMEM-C to a cell concentration of 129 3 106 cells/ml for
transplantation. Viability of cells after freezing and thawing
was 43%.
Recipient Mice and Donor Cell Transplantation
NCr Swiss nude (nu/nu) mice (Taconic, Germantown,
NY) 10–20 wk of age were used as recipient animals to
avoid immunological rejection of donor cells. The mice
were treated with busulfan (40 mg/kg) at least 4 wk before
donor cell transplantation to deplete endogenous germ cells
in the testes [7]. Because this dose of busulfan is toxic to
the hematopoietic system, the mice received bone marrow
transplantation from nontreated nude mice. Donor bone
marrow cells were collected from a nude mouse by flushing
the marrow of femurs and tibiae with DMEM-C. A volume
of 0.25 ml, containing 3–6 3 106 cells/ml, was injected
into the jugular vein of recipient mice 3 days after busulfan
treatment [9]. For transplantation of testis cells, the seminiferous tubules of recipient mice were filled with the donor
hamster cell suspension by injection through the efferent
ducts as described previously [14].
Analysis of the Recipient Testes and Epididymides
Between 89 and 457 days after donor cell transplantation, the recipient mice were killed by CO2 inhalation, and
both testes and epididymides were removed. In most cases,
one testis was fixed in 10% neutral buffered formalin
(NBF) for 24 h and processed for paraffin embedding and
sectioning. The other testis was snap-frozen in liquid nitrogen and stored at 2708C for use at a later date in a different
experiment. Four histological sections with a 25-mm interval between sections were made from the testis of each
mouse, and the sections were stained with periodic acidSchiff (PAS) and hematoxylin. Each slide was examined at
3400 magnification for hamster spermatogenesis. To determine the extent of hamster spermatogenesis in recipient
mouse testes, the number of tubule cross sections containing hamster, mouse, or no spermatogenesis was recorded
for one section from each testis. Spermatozoa were recovered from the epididymis and vas deferens of recipient mice
and fixed in NBF. The spermatozoa suspension was stained
by addition of an equal volume of 100 mg/ml Hoechst
33258 [15] and examined at 3630 magnification by fluorescence and phase-contrast microscopy. Hamster and
mouse spermatozoa were identified based on sperm head
morphology and tail size, and up to 2000 spermatozoa were
analyzed per sample.
RESULTS
Hamster Spermatogenesis in Mouse Testes
In the first experiments, testis cells were collected and
within 5 h injected into the seminiferous tubules of the
recipient nude mice. Approximately 80–90% of the surface
tubules of the recipient testes were filled with the donor
cell suspension as judged with the aid of trypan blue added
to the solution as an indicator [14]. Surface filling of tu-
517
HAMSTER SPERMATOGENESIS IN MOUSE TESTES
TABLE 1. Spermatogenesis from hamster testis cells transplanted into recipient nude mouse testes.
Donor cell
concentration
Experiment (cells 3 106/ml)
1
218
2
198
3
226
4
137
Recipient
mouse
Time to
analysis
(days)*
1515
1517
116
120
1518
1559
1560
1561
163
415
146
457
1574
291
1575
99
1576
1578
1852
1853
1854
144
114
125
134
128
Type of spermatogenesis in seminiferous tubules of recipient (%)† Hamster sperm in
epididymis and
Hamster
Mouse
None
vas deferens‡
11
3
18
11
2
7
9
12
3
4
2
5
3
10
1
4
2
(8)
(3)
(18)
(11)
(2)
(11)
(12)
(11)
(2)
(2)
(2)
(4)
(4)
(15)
(1)
(4)
(3)
50
67
73
55
26
64
74
47
55
77
34
57
76
56
19
19
47
(38)
(61)
(73)
(53)
(20)
(105)
(104)
(43)
(40)
(41)
(32)
(51)
(102)
(83)
(23)
(20)
(76)
39
30
9
34
72
29
17
41
42
19
63
38
21
34
80
77
51
(29)
(27)
(9)
(33)
(56)
(47)
(24)
(37)
(31)
(10)
(59)
(34)
(29)
(51)
(100)
(84)
(82)
1/70
10/500
1/5
0/931
6/1678
3/1174
0/545
1/700
2/1831
0/2000
0/1724
0/32
0/1853
* Number of days from injection of donor cells to analysis of the recipient testis for presence of spermatogenesis.
† In a histological section of each recipient mouse testis, the number of seminiferous tubules containing hamster, mouse, and no spermatogenesis was
counted; only one testis of each mouse was available for histological analysis except #1517, #1561, #1574, and #1575, in which both testes were
available. The numbers in parentheses represent the number of tubules that contained the designated type of spermatogenesis.
‡ Numerator, number of hamster spermatozoa; denominator, number of total spermatozoa including both mouse and hamster. In all cases, spermatozoa
recovered from epididymis and vas deferens of both sides were pooled.
bules with dye was regarded as reflecting the degree of
donor cell suspension distribution throughout the tubules.
This experiment was repeated on four separate days using
similar procedures to assure repeatability. The animals were
killed 99–457 days after the cell transplantation, and testes
were examined for the presence of hamster spermatogenesis. In the golden hamster and in the mouse, 35 days are
necessary for a type A1 spermatogonium to form a mature
spermatozoon. Thus, the interval from transplantation to
analysis represented 2.5–13 times the duration of hamster
spermatogenesis. This assured that there was adequate time
for maturation of hamster spermatids and spermatozoa,
which are necessary to distinguish hamster from mouse
spermatogenesis. In addition, the longer intervals would reflect the ability of foreign spermatogenesis to persist. The
results of these four experiments are shown in Table 1.
Hamster spermatogenesis was generated from donor
stem cells in all recipient mouse testes. In order to estimate
the degree of colonization, a cross section of the seminiferous tubules of each testis was examined, and the contents
were classified as hamster, mouse, or no spermatogenesis.
The morphology of hamster germ cells during spermiogenesis is characterized by round spermatids displaying large
acrosomal caps that stain brightly with PAS reagent (Fig.
1E). In addition, elongated spermatids and spermatozoa
have a long and hook-shaped head (Fig. 1B). On the basis
of these criteria, hamster spermatogenesis was identified on
average in 6.3% of tubules examined with a range of 1–
18% in individual testes. Clearly, this technique underestimates the extent of hamster spermatogenesis because stages
of hamster spermatogenesis that do not contain round or
elongated spermatids or spermatozoa would not be identified as hamster and were therefore classified as mouse spermatogenesis. The areas of mouse spermatogenesis resulted
from endogenous recipient stem cells that were not destroyed by busulfan treatment. This regeneration of endogenous recipient spermatogenesis is a general phenomenon
resulting from the survival ability of the spermatogonial
stem cell. In recipient nude mice, regeneration of endogenous spermatogenesis is common, because the dose of bu-
sulfan tolerated by the animal is limited due to the compromised immune system.
Hamster spermatogenesis in recipient mouse testes
showed several morphological abnormalities. Spermatogonia, spermatocytes, and round spermatids appeared morphologically normal. However, abnormalities in head shape
of elongated spermatids were frequently observed, and in
some cases the acrosomal cap of these cells had marked
deformities, such as a ballooned shape (Fig. 1F). Furthermore, the positional arrangement of hamster elongated
spermatids in mouse seminiferous tubules appeared poorly
organized (Fig. 1D) compared to the pattern in hamster
testes (Fig. 1B), and the number of elongated spermatids
appeared reduced compared to the number of round spermatids present.
Cryopreserved Hamster Testis Cells Generated
Xenogeneic Spermatogenesis
To determine whether hamster spermatogenic cells can
be stored for long periods, testis cells were collected and
cryopreserved in liquid nitrogen for 18 days and then
thawed for transplantations. Viability of thawed hamster
cells was only 43% compared to more than 95% for freshly
collected testis cells. Nonetheless, the cryopreserved hamster testis cells colonized 5 of 6 recipient testes (Table 2).
However, only 1% or 2% of the cross sections of recipient
seminiferous tubules observed contained evidence of spermatogenesis. This appears to be fewer than the 6.3% observed with freshly collected cells. The morphological appearance of hamster spermatogenesis in the recipient mouse
seminiferous tubules appeared identical to that seen after
transplantation of fresh cells. Abnormalities in the early differentiation stages of hamster spermatogenesis, spermatogonial differentiation and meiotic stages, were not observed. However, acrosomal and head shape defects in
elongated spermatids and spermatozoa were similar to those
present after transplantation of freshly collected testis cells
described above.
518
OGAWA ET AL.
FIG. 1. Histological appearance of hamster spermatogenesis in mouse seminiferous tubules. A) Normal spermatogenesis in
a control mouse. B) Normal spermatogenesis in a control hamster showing prominent acrosome and hook-shaped head of
elongated spermatids. C) Recipient mouse
testis with both hamster spermatogenesis,
in the lower right, and endogenous mouse
spermatogenesis, in the upper left. D)
Hamster spermatogenesis in a recipient
mouse testis. Characteristic hamster spermatozoa are present, but the cellular arrangement is less organized compared to
that in the control (B). E) Hamster spermatogenesis in recipient mouse testis
showing prominent acrosomes characteristic of hamster round spermatids. F) Hamster spermatogenesis in the mouse testis
showing abnormal shape of acrosome
with ballooned appearance (arrows). Stain,
PAS and hematoxylin; A–D bar 5 30 mm;
E, F bar 5 20 mm.
Hamster Spermatozoa in Mouse Epididymides
To determine whether normal hamster spermatozoa were
produced, liberated into the tubules, and transported to the
epididymis, the contents of the vas deferens and epididymis
were collected and carefully examined. In many cases, the
number of spermatozoa examined was limited because
spermatogenesis was inefficient. Although tubules in the
testis may show histological evidence of spermatogenesis,
production of spermatozoa may be low because the process
is inefficient or not widespread throughout the testis. Nonetheless, hamster spermatozoa were found in approximately
one half of the samples examined: 7 of 13 from fresh testis
cell transplantations and 2 of 4 from injection of cryopreserved hamster cells (Tables 1 and 2).
The morphological characteristics of the hamster spermatozoa observed (31 in total) were not completely normal.
In Figure 2, the first four panels show mouse (Fig. 2, A
and B) and hamster (Fig. 2, C and D) spermatozoa to illustrate normal morphology. Figure 2, E–G, shows spermatozoa collected from recipient mouse epididymides. Basically, the general head and tail morphology of the hamster
spermatozoa was characteristic of the species. The head
size and shape were clearly identifiable, and the tail was
large and distinct from that of mouse spermatozoa. Two
TABLE 2. Spermatogenesis from cryopreserved hamster testis cells transplanted into mouse testes.
Donor cell
concentration*
(cells 3 106/ml)
Time to analysis
(days)†
1872
1873
129
129
89
163
1874
129
174
1876
129
174
Recipient
mouse
Type of spermatogenesis in seminiferous tubules of recipient (%)‡
Hamster
1
1
2
2
0
2
(1)
(1)
(3)
(2)
(0)
(1)
Mouse
21
35
67
16
0
96
(29)
(34)
(100)
(14)
(0)
(53)
None
78
64
31
82
100
2
(109)
(61)
(46)
(73)
(78)
(1)
Hamster sperm in
epididymis and
vas deferens§
6/80
0/525
0/0
1/2000
* Donor hamster testis cells were frozen and kept in liquid nitrogen as described in the text, for 18 days before they were thawed and injected into
the testes of recipients. Cell viability was 43%. Donor cell concentration is for live cells.
† Number of days from injection of donor cells to analysis of the recipient testis for presence of spermatogenesis.
‡ In a histological section of each recipient mouse testis, the number of seminiferous tubules containing hamster, mouse, and no spermatogenesis was
counted; in mouse #1872 and #1876, only one testis was available for analysis. The numbers in parentheses represent the number of tubules that
contained the designated type of spermatogenesis.
§ Numerator, number of hamster spermatozoa; denominator, number of total spermatozoa including both mouse and hamster. In all cases, spermatozoa
recovered from epididymis and vas deferens of both sides were pooled.
HAMSTER SPERMATOGENESIS IN MOUSE TESTES
519
FIG. 2. Epididymal spermatozoa collected from recipient mice. A, C, and E are
phase-contrast, and B, D, F, and G are
fluorescence photomicrographs. A and B)
Normal mouse spermatozoon, with short,
rounded, sickle-shaped head from recipient mouse. C and D) Normal hamster
spermatozoon, with long, straight body
and hooked head with large acrosomal
cap from control hamster for comparison.
Note large thick tail. E and F) Spermatozoon from epididymis of recipient mouse
#1517, with distinct hamster morphology.
G) Spermatozoa from epididymis of recipient mouse #1576 showing characteristic
features of both hamster (arrow) and
mouse (arrowhead) spermatozoa. Bar 5
20 mm.
main abnormalities were noted. First, most hamster spermatozoa observed had head and tail separated. This was
not seen in the mouse spermatozoa from the same samples
and therefore was not related to a general disturbance of
the testicular or epididymal environment. Second, the acrosome of the hamster spermatozoa was absent or was
poorly formed and abnormal in appearance. While the number of hamster spermatozoa examined was small, the consistency of observations among the samples strongly suggests that they represent specific cellular defects. No differences in the abnormalities were observed between hamster spermatozoa arising from frozen and fresh testis cells.
DISCUSSION
The present study demonstrates unequivocally that hamster spermatogenesis occurs in the seminiferous tubules of
an immunodeficient mouse after transplantation of donor
testis cells. In earlier experiments it was found that rat testis
cells transplanted to mouse seminiferous tubules would establish rat spermatogenesis [9]. At least two factors raised
the question whether similar xenogeneic transplantations
would be successful in hamster cells. First, the morphology
of the hamster spermatozoon is characterized by greater
differences from the mouse than is the structure of the rat
spermatozoon. The hamster spermatozoon has a large hookshaped head with a very prominent acrosome; the total area
of the head and acrosome is at least 60% larger than in the
mouse, whereas in the rat, the sperm head is only about
15% larger than in the mouse. In addition, the hamster
sperm tail is approximately 60% longer than and twice the
diameter of the mouse sperm tail. The sperm tail in the rat
is about 40% longer and 40% larger in diameter than in the
mouse. Thus, the difference in the structure to be supported
by Sertoli cells in the mouse tubule is greater between
mouse and hamster than between mouse and rat. Second,
the rat diverged in evolution 10–11 million years (Myr)
ago, whereas the mouse-hamster separation was approximately 16 Myr ago [16]. A recent report has suggested that
this evolutionary divergence may be even greater, 41 Myr
for mouse-rat and 66 Myr for mouse-hamster [17]. While
these later figures have been questioned, the molecular evidence has convinced some. However, whichever figure is
correct, the hamster has been separated at least 16 Myr
from the mouse. This is similar to the separation between
the bovine and caprine species [18] and represents three
times the 5 Myr that separate man and chimpanzee [19].
Thus, success of the hamster-to-mouse transplantation, despite large structural dissimilarities of the spermatozoa and
the long evolutionary separation, is important in assessing
the possibility of success for similar transplantation among
domestic animals, or from wild and endangered species to
recipient domestic animals that might serve as hosts following immunological suppression.
Successful spermatogenesis is highly dependent on
structural associations and functional interactions between
Sertoli cells and all stages of germ cells. It has been reported that up to 50 different germ cells, which may have
developed from several separate stem cells, can be sup-
520
OGAWA ET AL.
ported by a single Sertoli cell that rests on the basement
membrane and extends to the lumen of the seminiferous
tubule [20]. Furthermore, most germ cells interact with
more than one Sertoli cell. Specialized junctional complexes between germ cells and Sertoli cells are common, and
spermatids occupy a cavity or crypt in the Sertoli cell [21].
Thus, the interaction among the cells is intimate and is
believed to be essential for the differentiation process. This
relationship assumes an interesting and important significance in xenogeneic transplantation. It has been clearly
documented that rat germ cells transplanted to mouse seminiferous tubules are supported by mouse Sertoli cells [9,
22]. In ultrastructural observations of spermatogenesis in
mouse tubules, rat germ cells were always found with
mouse Sertoli cells, and no evidence of rat Sertoli cells was
ever identified in recipient mouse testes [22]. Furthermore,
the time necessary for rat spermatogenesis completion in
mouse recipients is 52 days, identical to that found for donor rat testes [23]. Therefore, the germ cell dominates and
controls the germ cell differentiation process; the Sertoli
cell appears to function as a relatively passive somatic supporting cell. Because the hamster donor cell population
contains Sertoli cells, they have the opportunity to colonize
the recipient mouse seminiferous tubule. However, the very
strong evidence from rat-to-mouse transplantations suggests
that this does not occur. Furthermore, the donor cell population contains only Sertoli cells that have stopped dividing, because of the donor animal’s age [24], and they would
therefore have little opportunity to colonize the mouse
basement membrane already occupied by endogenous Sertoli cells. Thus, it is likely that most if not all xenogeneic
hamster spermatogenesis represents differentiation of hamster germ cells supported by mouse Sertoli cells. A similar
situation in rat-to-mouse transplantations results in relatively normal rat spermatogenesis and the production of normal-appearing rat spermatozoa [22].
The situation in hamster cell transplantations appears to
be different. While normal spermatogonial division and differentiation were observed, and while spermatocyte meiosis
proceeded without obvious defects, spermiogenesis of hamster germ cells showed clear abnormalities of head and acrosome development. Furthermore, no normal hamster
spermatozoa were found in the epididymal content analysis.
Prominent defects were the absence of normal acrosomes
and the separation of head and tail. These findings indicate
abnormalities in the xenogeneic hamster spermiogenesis
process and suggest that the Sertoli cell plays a significant
supportive and perhaps species-specific role in its interaction with germ cells that may influence final spermatozoa
differentiation stages. Ultrastructural analyses are planned
to elucidate in more detail the abnormalities that occur during spermiogenesis in recipient mice at several time intervals following transplantation of hamster testis cells. It
seems that the greater evolutionary divergence time and/or
structural difference between hamster and mouse as compared to rat and mouse allows the role of the Sertoli cell
to become more obvious. Perhaps it is not surprising that
the first abnormalities observed with xenogeneic transplantation should be in spermiogenesis, since the process is extremely complicated and species specific [4, 5]. As the phylogenetic separation of donor and recipient is increased and
the spermatozoal characteristics reflect a greater difference,
abnormalities may be seen in earlier stages of spermatogenesis. Inadequate or incorrect growth factors, cytokines,
or surface molecule configurations [25] in a recipient testis
are likely to play a critical role in determining whether
donor stem cells can survive and undergo differentiation.
It is impossible to predict which donor-recipient combinations may succeed, because inadequate information exists
about the complex processes involved.
The successful colonization of recipient seminiferous tubules by cryopreserved hamster donor cells is an encouraging extension of studies performed with mouse and rat
transplantations. In each of these three species, donor spermatogonial stem cells can be preserved at 21968C for long
periods, and spermatogenesis resulted after transplantation
([13]; unpublished results). However, the extent of colonization from cryopreserved donor cells appears to be lower
than with freshly collected cells. The results presented in
Tables 1 and 2 suggest that this is true for hamster cells,
and similar observations have been made for the mouse and
rat (unpublished results). The important aspect of the cryopreservation studies is that the evidence is now extremely
strong that spermatogonial stem cells of most, if not all,
mammalian species can be preserved for long periods. The
cryopreservation of mature spermatozoa from a number of
species is routinely performed for subsequent use in artificial insemination or in vitro fertilization [26–28]. However,
the procedure varies for each species and must be determined empirically in each case. Furthermore, frozen spermatozoa cannot replicate, and each cell is only one haploid
combination of the male gamete complement. In contrast,
cryopreservation of spermatogonial stem cells follows a
simple procedure established for cultured somatic cells, and
the same protocol appears to be similarly applicable to all
the species examined [12, 13]. More importantly, cryopreserved spermatogonial stem cells can proliferate and undergo meiotic recombination during their development in
the recipient testis, thereby reestablishing the entire genetic
potential of the donor male. Thus, the stem cells of valuable
or unique males could now be frozen for later use after
techniques of culture and gene modification are developed.
The present study suggests that the cell-cell interactions
between spermatogenic cells and Sertoli cells are rather
flexible and can be functionally competent between cells
from different species, but with limitations as species divergence increases. This is surprising considering the many
structural and functional relationships that have been described [6, 29]. One would expect these intricate interactions to be rigidly controlled and very specific, thereby not
allowing minor mismatch of cell surface recognition molecules. The extent of difference that will be tolerated between germ cells and Sertoli cells in cell-cell interactions
and allow successful spermatogenesis is not known, nor is
it known which specific molecules are responsible for these
interactions. However, on the basis of the success of the
studies reported here, it seems worthwhile to explore the
technique of xenogeneic spermatogonial transplantation in
a wide range of animals. With the development of efficient
culture systems for spermatogonial stem cells, it should
eventually be possible to expand the stem cells in vitro and
modify their genetic composition before transplantation to
host testes. Using a smaller, more readily available animal
as xenogeneic recipient would facilitate the generation of
spermatozoa from larger farm animals or endangered species. The resulting spermatozoa could be used for in vitro
fertilization or intracytoplasmic sperm injection. This approach to the generation of transgenic animals through manipulation of the male germ line would be useful in animal
species in which embryonic stem cell and nuclear transfer
technology have not been perfected. Furthermore, xenogeneic spermatogonial transplantation will provide a new av-
HAMSTER SPERMATOGENESIS IN MOUSE TESTES
enue for the study of unique aspects of spermatogenesis.
As techniques improve, the combination of spermatogonial
stem cell culture, genetic manipulation, cryopreservation,
and transplantation will represent a powerful approach in
biology, medicine, and agriculture.
14.
15.
ACKNOWLEDGMENTS
16.
We thank our colleagues for discussions and suggestions. In addition,
we are grateful to C. Freeman and R. Naroznowski for assistance with animal maintenance and experimentation, and to J. Hayden for photography.
17.
REFERENCES
18.
1. Amann RP. Sperm production rates. In: Johnson AD, Gomes WR,
Vandemark NL (eds.), The Testis, vol.1. New York: Academic Press;
1970: 433–482.
2. Wing TY, Christensen AK. Morphometric studies on rat seminiferous
tubules. Am J Anat 1982; 165:13–25.
3. Leblond CP, Clermont Y. Spermiogenesis of rat, hamster and guinea
pig as revealed by the ‘‘periodic acid-fuchsin sulfurous acid’’ technique. Am J Anat 1952; 90:167–215.
4. Clermont Y, Oko R, Hermo L. Cell biology of mammalian spermiogenesis. In: Desjardins C, Ewing LL (eds.), Cell and Molecular Biology of the Testis. Oxford: Oxford University Press; 1993: 332–376.
5. Meistrich ML. Nuclear morphogenesis during spermiogenesis. In: de
Kretser D (ed.), Molecular Biology of the Male Reproductive System.
San Diego, CA: Academic Press; 1993: 67–97.
6. Russell LD. Morphological and functional evidence for Sertoli-germ
cell relationships. In: Russell LD, Griswold MD (eds.), The Sertoli
Cell. Clearwater, FL: Cache River Press; 1993: 365–390.
7. Brinster RL, Zimmermann JW. Spermatogenesis following male
germ-cell transplantation. Proc Natl Acad Sci USA 1994; 91:11298–
11302.
8. Brinster RL, Avarbock MR. Germline transmission of donor haplotype following spermatogonial transplantation. Proc Natl Acad Sci
USA 1994; 91:11303–11307.
9. Clouthier DE, Avarbock MR, Shanna DM, Hammer RE, Brinster RL.
Rat spermatogenesis in mouse testis. Nature 1996; 381:418–421.
10. Hammer CE. The semen. In: Hafez ESE (ed.), Reproduction and
Breeding Techniques for Laboratory Animals. Philadelphia: Lea &
Febiger; 1970: 56–73.
11. Bellvé AR, Cavicchia JC, Millette CF, O’Brien DA, Bhatnagar YM,
Dym M. Spermatogenic cells of the prepuberal mouse. Isolation and
morphological characterization. J Cell Biol 1977; 74:68–85.
12. Robertson EJ. Embryo-derived stem cell lines. In: Robertson EJ (ed.),
Teratocarcinomas and Embryonic Stem Cells: A Practical Approach.
Oxford: IRL Press; 1987: 71–112.
13. Avarbock MR, Brinster CJ, Brinster RL. Reconstitution of spermato-
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
521
genesis from frozen spermatogonial stem cells. Nat Med 1996; 2:693–
696.
Ogawa T, Arechaga JM, Avarbock MR, Brinster RL. Transplantation
of testis germinal cells into mouse seminiferous tubules. Int J Dev
Biol 1997; 41:111–122.
Hogan B, Beddington R, Constantini F, Lacy E. Manipulating the
Mouse Embryo: A Laboratory Manual, 2nd ed. Plainview: Cold
Spring Harbor Laboratory Press; 1994.
Catzeflis FM, Dickerman AW, Michaux J, Kirsch JAW. DNA hybridization and rodent phylogeny. In: Szalay FS, Novacek MJ, McKenna
MC (eds.), Mammal Phylogeny (Placentals), vol 2. New York: Springer Verlag; 1993: 159–172.
Kumar S, Hedges SB. A molecular timescale for vertebrate evolution.
Nature 1998; 392:917–920.
Miyamoto MM, Kraus F, Laipis PJ, Tannhauser SM, Webb SD. Mitochondrial DNA phylogenies within artiodactyla. In: Szalay FS, Novacek MJ, McKenna MC (eds.), Mammal Phylogeny (Placentals), vol
2. New York: Springer Verlag; 1993: 268–281.
Takahata N, Satta Y. Evolution of the primate lineage leading to modern humans: phylogenetic and demographic inferences from DNA sequences. Proc Natl Acad Sci USA 1997; 94:4811–4815.
Weber JE, Russell LD, Wong V, Peterson RN. Three-dimensional reconstruction of a rat stage V Sertoli cell: II. Morphometry of SertoliSertoli and Sertoli-germ cell relationships. Am J Anat 1983; 167:163–
179.
Russell LD, Ettlin RA, Hikim AP, Clegg ED. Mammalian spermatogenesis. In: Histological and Histopathological Evaluation of the Testis. Clearwater, FL: Cache River Press; 1990: 1–40.
Russell LD, Brinster RL. Ultrastructural observation of spermatogenesis following transplantation of rat testis cells into mouse seminiferous tubules. J Androl 1996; 17:615–627.
Franca LR, Ogawa T, Avarbock MR, Brinster RL, Russell LD. Germ
cell genotype controls cell cycle during spermatogenesis. Biol Reprod
1998; 59:1371–1377.
Gondos B, Berndston WE. Postnatal and pubertal development. In:
Russell LD, Griswold MD (eds.), The Sertoli Cell. Clearwater. FL:
Cache River Press; 1993: 116–154.
Skinner MK. Secretion of growth factors and other regulatory factors.
In: Russell LD, Griswold MD (eds.), The Sertoli Cell. Clearwater, FL:
Cache River Press; 1993: 237–247.
Tada N, Sato M, Yamanoi J, Mizogori T, Kasai K, Ogawa S. Cryopreservation of mouse spermatozoa in the presence of raffinose and
glycerol. J Reprod Fertil 1990; 89:511–516.
Watson PF. Artificial insemination and the preservation of semen. In:
Lamming GE (ed.), Marshall’s Physiology of Reproduction, vol. 2,
4th ed. Edinburgh: Churchill Livingstone; 1990: 747–869.
Hafez ESE. Preservation and cryopreservation of gametes and embryos. In: Hafez ESE (ed.), Reproduction in Farm Animals, 6th ed.
Philadelphia, PA: Lea & Febiger; 1993: 503–525.
Sharpe RM. Regulation of spermatogenesis. In: Knobil E, Neill JD
(eds.), The Physiology of Reproduction, 2nd Edition. New York: Raven Press, Ltd.; 1994: 1363–1434.