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From the * Center for Reproduction and
Development, Monash Institute of Medical Research, Monash University, Clayton,
Australia; CSIRO Livestock Industries,
Armidale, Australia; Australian Research
Council Centre of Excellence in Biotechnology and Development, Australia; and
Department, of Environment and Natural Sciences,
Graduate School of Environment and Information Sciences, Yokohama National
University, Yokohama, Japan.
| Correspondence to: Dr Zhen Zhang, Center for Reproduction and Development, Monash Institute of Medical Research, Monash University, 27–31 Wright St Clayton, VIC 3168, Australia (e-mail: zhen.zhang{at}med.monash.edu.au). |
| Received for publication November 2, 2007; accepted for publication February 5, 2008. |
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Abstract |
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Key words: Bovine spermatogonia, cell aggregates, xenotransplantation, antibodies
Clinical trials for human germ cell transplantation after cancer therapy have been reported as underway (Radford et al, 1999), but no outcome has been described yet. Germ cell transplantation has been extended into several other species including some livestock (ie, goats, pigs, and bulls; Hill and Dobrinski, 2006). In one encouraging success, a donor germ cellsired offspring was produced following a goat-to-goat germ cell transplantation (Honaramooz et al, 2003). Transplantation between species (xenotransplantation) has yielded variable success. Reciprocal transplantation between mice and rats appears to work, but the mouse testis seems unable to support domestic animal spermatogenesis (Dobrinski et al, 2000; Izadyar et al, 2002b; Oatley et al, 2002). Nonrodent donor cells rarely develop beyond spermatogonia in recipient mouse testes, indicating either that there are factors that support foreign germ cell development that are lacking in the mouse testis, or alternatively, the mouse testis contains factors that prevent the differentiation of nonrodent germ cells. Resolution of these issues is required to develop germ cell xenotransplantation into a useful experimental and ultimately commercial option. The bovine model is a large animal model that may provide key information on both these issues.
A critical requirement for assessing the success of male germ cell transplantation in nonrodent species is the ability to distinguish donor cells from host cells after transplantation. In rodents, this has largely been overcome by the use of rodent cells expressing either green fluorescent protein or β-galactosidase (Tan, 1991; Nakanishi et al, 1999). When cells without a marker are transplanted into a mutant mouse lacking endogenous spermatogenesis, such as the W strain, any subsequent spermatogenesis can be deduced as arising from donor cells (Ogawa et al, 2000; Ohta et al, 2000). Transplantation between different inbred rat strains with distinct fur colors also makes it relatively easy to assess transplant success using DNA microsatellite analysis and inspection of offspring (Zhang et al, 2003).
The colonization of bull donor cells after transplantation into busulfan-treated or irradiated testes of nude mice has been assessed using a species-specific antibody (Dobrinski et al, 2000), anti-PGP9.5 antibody (Oatley et al, 2004b), the lectin DBA as a species-specific gonocyte marker (Izadyar et al, 2002b; Izadyar et al, 2003; Goel et al, 2007), or a vital cell dye, PKH26 (Honaramooz et al, 2002). However, since all donor cells used for the bull-to-mouse transplantation were collected from immature animals, the donor cell population would have contained immature somatic cells, such as peritubular myoid cells and Sertoli cells, which could proliferate in the host testes. Whether bull germ cells formed colonies in mouse hosts that contained bovine somatic cells was not clarified. This highlights the need to identify suitable reagents for the precise identification of both donor and recipient germ and somatic cell types following transplantation of bull testicular cells into recipient mice. Our previous studies have demonstrated the value of species-specific antibody reactivity in distinguishing donor cells and host cells (Zhang et al, 2006, 2007).
In the present study, we first screened antibodies previously shown in rodents to react specifically with Sertoli cells, germ cells, or peritubular myoid cells for their capacity to serve as cell-specific markers in developing and adult bull testes. Then we performed culture, grafting, and transplantation studies using isolated bovine testicular cells or tissue fragments into mouse recipients. Evaluation using those antibodies allowed accurate identification of specific bovine testicular cell types following these experiments, providing clear evidence that immature bovine Sertoli cells colonize and form tubular structures in the mouse testes after transplantation and after grafting. These outcomes provide a solid platform for future studies involving xenotransplantation.
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Material and Methods |
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Animals and Tissue Collection
Bovine testes were obtained from 8 prepubertal (2–4 mo old) Bos
taurus bull calves (Holstein and Angus breeds) for use in culture,
explant, and transplant experiments (CSIRO Cattle Breeding Laboratory,
Armidale, Australia). Testes from animals of this age are expected to weigh
less than 25 grams, with the most advanced germ cell-type spermatogonia or
gonocytes (Herrid et al,
2006). Testes were collected by castration under general
anesthetic (0.1 mg/kg xylazine, followed by 3 mg/kg ketamine), washed in
sterile Dulbecco phospate-buffered saline (DPBS; GIBCO, Carlsbad, California),
then maintained on ice overnight in PBS with antibiotics. Samples from these
animals and from 1 adult Bos taurus bull (Angus breed) were also used
for immunohistochemistry. The adult bull testis was collected from an abattoir
and delivered to the laboratory within 2 hours.
CBA or BALB/c nude mice were purchased from Monash Central Animal Services and maintained in the low barrier animal house of Monash Medical Centre (Clayton, Australia). All investigations conformed to the NHMRC/CSIRO/AAC Code of Practice for the Care and Use of Animals for Experimental Purposes and were approved by the Monash University Standing Committee on Ethics in Animal Experimentation or by the CSIRO Animal Ethics Committee, Armidale, Australia.
Upon delivery to the laboratory, the testis was rinsed 3 times with sterile DPBS and weighed. The tunica albuginea was removed, connective tissues were excised, and the testis was cut into small (0.5 cm3) fragments. These were used for fresh cell and tissue fragment collections (only for immature testes) and for histologic studies by fixing fragments in Bouin fixative or 4% paraformaldehyde solutions at 4°C overnight.
Cell Collection and Culture
Approximately 1–10 g of bovine testis tissue was dissociated into
cell suspensions using methods described previously
(Zhang et al, 2003) with minor
modifications. Briefly, tissues were incubated in 0.1% collagenase, 0.05%
hyaluronidase, and 0.05% DNase I in Dulbecco Modified Eagle Medium/F12 medium
(DMEM/F12 medium; GIBCO) for 20 minutes twice at 35°C in a shaking water
bath. Between steps, tubules were allowed to settle, then the medium
containing the enzymes was replaced following 2 further rinses with
calcium-free DPBS. Single tubules were digested in 0.075% trypsin in DPBS
containing 1 mM EDTA and DNase I for 10 minutes at 35°C in a shaking water
bath. The cells were collected by centrifugation and resuspended in DMEM/F12
medium. After cell counting, these fresh bovine testicular cells were used for
cell aggregation culture or left on ice prior to transplantation.
Individual drops of 30 µL of the final cell suspension containing an average of 42 x 104 primary bovine cells (between 11.4 x 104 and 64.4 x 104; n = 3) were placed onto the cover of a 100-mm Petri dish, with 25–30 drops loaded per dish. The cover was inverted and placed over the bottom of the Petri dish containing sterile water. These hanging drops were cultured at 37°C in 5% CO2 in air for a maximum of 7 days without medium change. The cultured aggregates were used for grafts into the testis and under the back skin of nude mice. Aggregates were examined by immunocytochemistry after fixation in 2% paraformaldehyde in PBS at 4°C overnight.
Tissue Culture
Tunica-free fragments of around 2–3 mm3 were cultured on a
0.4 µm-pore cell culture insert membrane (Falcon; BD Biosciences, North
Ryde, Australia) floating on medium at 37°C in 5% CO2 in air.
The medium was changed every 3 days. The cultured tissue fragments were used
for xenografts and for immunohistochemistry after fixation in 2%
paraformaldehyde in PBS at 4°C overnight.
Preparation of Recipient Mice, Donor Cells, and Cell Transfer
Nude mice (6–8 wk old) were injected intraperitoneally (IP) with a
single dose of busulfan at 30–35 mg/kg. Preparation of busulfan solution
was as described previously (Zhang et al,
2007). Mice were used as recipients for germ cell transplantation
4–6 weeks after busulfan injection.
Freshly dispersed bovine testicular cells were combined with trypan blue (0.02%–0.04%). The final cell concentration for each experiment is listed in Table 1.
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Injection of cells was performed as described previously (Zhang et al, 2006, 2007). Recipient mice were anesthetized by peritoneal injection of Avertin (20 mg/mL, 0.3 mL/per adult mouse). The testis was exposed via lower central abdominal incision. Each testis was injected with 10–15 µL of donor cells. Recipient mice were killed 2–3 months after transplantation. The recipient testes were removed and fixed in 4% paraformaldehyde in PBS at 4°C overnight.
Grafts of Tissue Fragments or Hanging Drop Cell Aggregates
Two to 3 linear incisions of about 0.5 cm were made on each side of the
midline in the dorsal skin of the anesthetized recipient mouse. A small space
was made within the fascia by blunt dissection under the incision. The fresh
or cultured tissues or cell aggregates were implanted into the spaces under
the skin. One to 3 tissue fragments or 10–16 hanging drops were
implanted in each site, and the incision site was closed by suturing. Grafts
with tissues from 5 different donors were performed on 3 different days into 4
recipients (Table 2). Two mice
were castrated at the same time they were used as recipients. Hanging drop
aggregates were also grafted into the testes of a nude mouse. For this mouse,
the incision was made in the low-middle abdomen, and the testes were exposed.
A very small hole was made on the testis tunica using a surgical blade, and
6–10 aggregates were inserted within the interstitial space under a
dissecting microscope. The tunica incision was not sutured, but the abdominal
incision was sutured. Recipient mice were killed 2–3 months later, and
the grafts on the back and the testes containing cell aggregate grafts were
removed and fixed in 4% paraformaldehyde in PBS at 4°C overnight.
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Immunohistologic Assessment
For histologic analysis, the fixed tissues were rinsed 3 times in 70%
(vol/vol) ethanol followed by routine processing and paraffin embedding.
Serial sections of 4–5 µm thick were prepared using routine dewaxing
and rehydration procedures. Antigen retrieval was performed in 0.01M citrate
buffer (pH 6.0) by heating in a microwave using full power for 3 minutes, then
70% power for 6 minutes. Sections were allowed to cool for 1 hour. All
sections were treated for 10 minutes with 0.1% Triton X-100 in Tris-buffered
saline (TBS). To reduce nonspecific background staining, sections were treated
with 10% (wt/vol) bovine serum albumin in TBS for 4 hours or with a blocking
reagent solution (Roche, Indianapolis, Indiana) for 30 minutes at room
temperature (RT) following the manufacturer's instructions. Sections were then
incubated with 1 or 2 different primary antibodies (see
Table 3) at 4°C overnight,
followed by application of secondary antibodies at RT for 30–60 minutes.
For immunohistochemistry, antibody binding was detected with an avidinbiotin
complex (ABC Elite kits; Vector Laboratories, Burlingame, California) and a
color reaction product developed following addition of
3,3'-diaminobenzidine tetrahydrochloride (DAB; DAKO, Kingsgrove,
Australia). These sections were counterstained with hematoxylin prior to
mounting with DPX Mountant. For immunofluorescence double staining, Alexa
Fluor antibodies (Alexa 488 or 546 Goat anti-rabbit, Alexa 488 or 546 rabbit
anti-mouse, Alexa 546 goat anti-rat; Molecular Probes Inc, Eugene, Oregon)
were used at a 1:300 dilution in TBS. The lectin of Dolichos biflorus
agglutinin (DBA) was supplied conjugated with either fluorescein
isothiocyanate (FITC) or with biotin (Table
3). Sections were counterstained with the DNA fluorescent stain,
DAPI, in mounting medium (Vector Laboratories). After photography of the
fluorescent staining, sections in some slides were washed in DPBS, then the
biotin-ABC staining procedure was repeated, starting from the secondary
antibody incubation.
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For mouse recipient testes exhibiting donor bovine cell colonization, the proportion of tubules with bovine Sertoli cells or spermatogonia was determined by counting 4–10 testis sections situated 600–900 µm apart within each recipient mouse testis.
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Results |
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Binding of the bovine germ cell marker, DBA lectin, was detected in the cytoplasm of gonocytes and spermatogonia of bovine testes at all ages, but it was absent from all cells of mouse testes (Figure 1A through C). The protein gene product 9.5 (PGP9.5) was readily observed in both the cytoplasm and nucleus of gonocytes and spermatogonia in bovine testes. In contrast, the PGP9.5 signal was relatively faint in cells along the inside of the basement membrane of the mouse seminiferous tubule epithelium (Figure 1D through F). Sertoli cells and spermatogonia exhibited weak positive staining for PGP9.5 (Figure 1F arrow and arrowhead). The presence of vasa-homolog protein (vasa) was evident in the cytoplasm of spermatogonia, spermatocytes, and spermatids in both bull and mouse testes (Figure 1G through I). Germ cell nuclear antigen 1 (GCNA1) protein was detected in the nucleus of mouse, but not bovine, germ cells from spermatogonia to spermatids (data not shown; Zhang et al, 2006).
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The Sertoli cell markers, WT1 and Mullerian-Inhibiting Substance (MIS), were detected in the Sertoli cell nucleus and cytoplasm, respectively, in both bull and mouse testes (Figure 1M through O for WT1 in bull and mouse; Figure 2B, D for MIS in bull only). Vimentin was also detected in the cytoplasm of Sertoli cells in bull but not mouse testes (Figure 1J through L). The newly reported Sertoli cell marker, NONO (Kuwahara et al, 2006), was only detected in the nucleus of mouse Sertoli cells, with no reactivity to this antibody observed in bull Sertoli cells (Figure 1P through R).
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The 
-actin smooth muscle (SMA) protein was detected in smooth
muscle cells of blood vessels and in peritubular myoid cells in the testes of
both bull (Figure 3B and J) and
mouse (not shown).
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SMA were readily detected
(Figure 3B and D) in all
grafted tissues. Only 2 of 5 bovine tissue grafts contained tubules with a few
single or paired germ cells stained positively by the anti-vasa antibody, with
germ cells present in 15.6% and 23% of tubules in these grafts
(Figure 3C). We noted that
those bovine grafts with germ cells present were only those grafted onto mice
that had been castrated; bovine tissues grafted onto intact mice contained no
detectable germ cells (Table
2). However, there was no indication that these surviving bovine
germ cells differentiated during the 2–3 months after the graft was
implanted, as judged by their location, the number of cells residing together
(single or paired), and the ratio of cytoplasm to nucleus in these cells
(Figure 3C;
Russell et al, 1990). The
culture period of 5 days prior to grafting did not prevent the survival of
germ cells within the graft for the duration of the subsequent experiment.
We also cultured testicular cells derived from 3 of these 5 testes in
hanging drops for 3–7 days (Table
2). When observed 3 hours after initial drop formation, the bovine
cells remained as single cells (Figure
3E); however, when assessed 3 days later, these cells had formed
aggregates (Figure 3F).
Following 5 days of culture, both germ and Sertoli cells were present in the
aggregates, as demonstrated by anti-vasa and anti-vimentin staining
(Figure 3G and H). These
aggregates at culture day 3 were grafted into 2 testes of a single mouse and
under the dorsal skin of 3 nude mice. When they were removed 2–3 months
later, the aggregated cell grafts implanted under the skin had disappeared in
1 mouse. The surviving cell aggregates placed under the skin at each site
appeared to form 1 tissue mass per site
(Figure 3I). Viewed using
immunohistology, these tissue masses contained tubular structures exhibiting
the normal arrangement of both Sertoli cells and peritubular myoid cells, as
revealed by staining with anti-SMA and anti-vimentin antibodies in all
grafts (Figure 3J and L).
However, no germ cells or cells stained by anti-vasa antibody were found in
any of the grafts (Figure 3K).
The aggregates implanted inside the testis survived and formed tubular
structures, but they did not grow as large as the grafts placed subcutaneously
in the back. Using mouse or bovine cell-specific antibodies, we identified
Sertoli cells in 1 or 2 tubules very close to the tunica albuginea that
stained positive with the anti-vimentin antibody
(Figure 3M) but negative using
the anti-NONO antibody (Figure
3N), indicating that these tubules had formed from bovine cells.
In contrast, Sertoli cells in the adjacent tubules did not stain with the
anti-vimentin antibody but were recognized by the anti-NONO antibody,
indicating they were of mouse origin
(Figure 3M and N). No germ
cells were found within the bovine tubules, while many mouse germ cells at
different stages of maturation were present in the mouse tubules, as
demonstrated by anti-vasa and anti-GCNA1 antibody staining respectively
(Figure 3O and P).
Bovine Testicular Cells Transplanted Into Mouse Testes
Freshly collected bovine testicular cells from 2 prepubertal animals were
injected into 5 testes of nude mice that had been treated with busulfan
4–5 weeks previously. The majority of the injected bovine testicular
cells were Sertoli cells (80% and 90% vimentin-positive cells from each
preparation) and about 10% were germ cells (8% and 12% vasa-positive;
Table 1). The recipient mice
were killed and testes removed 2 months after transplantation. Using serial
sections and double-staining with different antibody combinations, some
Sertoli cells stained positive for vimentin in a few tubules
(Figure 4A, C, E, and H), and
these vimentin-positive cells were not stained by the anti-NONO antibody
(Figure 4B, F, G, and H).
However, all Sertoli cells, whether vimentin- or NONO-positive, were stained
by the anti-WT1 antibody (Figure 4C, G, and
H). These results demonstrate that bovine Sertoli cells survive in
mouse testes following transplantation and form epithelial structures within
the seminiferous tubules. We also noted that those tubules having some Sertoli
cells recognized by the anti-vimentin antibody always also have a small
proportion of vimentin-negative and NONO-positive Sertoli cells
(Figure 4C, G, and H),
indicating that both mouse and bovine Sertoli cells can reside adjacent to
each other within the tubular epithelium. However, only rarely were bovine
germ cells that reacted positively to the lectin DBA present within the mouse
tubules that also contained bovine Sertoli cells
(Figure 4A, B, F, and I). When
double-staining of DBA and vasa was performed in the serial sections, a
DBA-positive cell was also vasa-positive
(Figure 4D), but most germ
cells in adjacent tubules, presumably endogenous mouse germ cells, were not
stained by DBA. However, all germ cells were stained by anti-mvh antibody
(Figure 4E, F, and N). None of
those bovine donor germ cells appeared to have differentiated based on the
morphology of their chromatin. We stained both new serial sections and also
those sections previously examined by immunofluorescence using the biotin-ABC
immunohistochemistry procedure. The results were identical to those obtained
when fluorescent staining alone was used: Some Sertoli cells stained positive
with the anti-vimentin antibody (Figure 4K
and O) and negative with the anti-NONO antibody
(Figure 4J), yet all Sertoli
cells were recognized by the WT1 antibody
(Figure 4L and P). While only
some germ cells were recognized by the lectin DBA
(Figure 4I and M), all germ
cells were stained by the anti-vasa antibody
(Figure 4N). A pair of adjacent
donor germ cells stained by DBA was occasionally found in these sections
(Figure 4M).
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Discussion |
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The antibody that recognizes PGP9.5 (PGP9.5 is also named ubiquitin C-terminal hydrolase L through 1 [UCH-L1]; Wrobel et al, 1996; Kon et al, 1999), has previously been used as a gonocyte and spermatogonial marker for bull-to-mouse testicular cell transplantation (Oatley et al, 2004b). In our studies, this protein was detected with a strong signal in spermatogonia in bovine testes but yielded only a faint signal with mouse cells along the basement membrane of seminiferous tubules. Both Sertoli cells and spermatogonia in mouse testes exhibited this weak signal. Our results are consistent with previous studies in bull and mouse testes (Wrobel et al, 1996; Kon et al, 1999). Despite these differences, we did not assess donor cell colonization based on anti-PGP9.5 antibody staining because DBA provided a more reliable way to distinguish between germ cells of these species.
DBA has been used as a marker for prespermatogonia, the precursors of bovine spermatogonia present until the onset of spermatogenesis at week 30 of age (Ertl and Wrobel, 1992). Due to the lack of its binding to mouse germ cells, it has been used as a marker of bull donor germ cells in mouse testes after transplantation (Izadyar et al, 2002a). In our study, all DBA-stained cells showed overlap with some, but not all, of the vasa-stained cells in bovine testis. It can be explained because the vasa protein was detected in both undifferentiated and differentiated germ cells, whereas the DBA-positive cells are only the undifferentiated spermatogonia. Our bovine testicular cell transplantation results revealed that very few bovine germ cells colonized and survived in mouse testes after transplantation, with only 1% of tubules having single or paired bull spermatogonia, similar to previous reports (Dobrinski et al, 2000; Izadyar et al, 2002b; Oatley et al, 2002). The bull donor germ cells reactive to DBA were always found as single or occasionally paired cells within mouse testes, and they do not appear to be differentiated based on morphological criteria including chromatin configuration, position within the epithelium, and number of syncitial cells (de Rooij and Russell, 2000). Although they were stained positive by anti-vasa antibody, no progeny cells were evident.
Interspecific male testicular cell transplantation into mouse testes with donor cells from nonrodent species performed previously (Dobrinski et al, 2000; Nagano et al, 2001; Izadyar et al, 2002b; Oatley et al, 2002) has consistently shown that spermatogonial stem cells from nonrodent species colonize and survive but do not differentiate in the mouse seminiferous epithelium. However, these studies did not examine whether donor Sertoli cells were present in mouse testes and whether the donor Sertoli cells would support donor germ cell differentiation. In mouse-to-mouse spermatogonial transplantation, injection of donor cells with or without additional mitotic Sertoli cells did not enhance the ability of the donor spermatogenic cells to colonize the recipient mouse testis (Brinster and Avarbock, 1994). A key finding from the present study is that almost all bovine spermatogonia were embedded within bovine Sertoli cells (Figure 3A, I, K, M, and O), suggesting this may be favorable or required for foreign germ cell survival. However, these germ cells did not differentiate during the 2-month period. This may indicate that either bovine Sertoli cells in mouse testis are not fully functional, or that possibly some factors produced by the mouse testis cannot support bovine Sertoli or germ cell function or both. Another possibility is that the mouse testis actively produces substances that inhibit the development of foreign Sertoli cells and/or the differentiation of foreign germ cells.
Vimentin is an intermediate filament protein in Sertoli cells, and its synthesis is regulated by follicle-stimulating hormone (FSH; DePhilip and Kierszenbaum, 1982; Sasaki et al, 1998) and androgen (Wang et al, 2006). The Non-POU-domain-containing, octamer binding protein (NONO) forms a complex with the androgen receptor and functions as a coactivator for the receptor (Kuwahara et al, 2006). Testosterone regulates spermatogenesis via the androgen receptor present in Sertoli cells and by Sertoli cell products that influence developing germ cells (De Gendt et al, 2004; Hill et al, 2004). Although bull and mouse Sertoli cells can coexist within recipient mouse seminiferous tubules, whether the bull Sertoli cells function as they would in their native testis remains unknown; however, clearly they continue to produce a range of proteins that can be recognized by appropriate antisera. This raises the possibility that successful xenotransplantation may also require investigation of peritubular or Leydig cell-derived factors for driving spermatogenic differentiation. Yoshida et al (2007) demonstrated recently that reconstitution of stem cell niches for transplanted spermatogonial stem cells would involve the vascular network accompanying Leydig and other interstitial cells. This possibly explains that even under the presence of bovine Sertoli cells in mouse host testis, the bovine spermatogonia stem cells would not differentiate after transplantation because the newly formed stem cell niches may not be integrated or may be composed of mouse interstitial cells that are functionally species-specific.
In this study, testis fragments from 5 different animals were cultured for 0–5 days before grafting. Germ cells are typically maintained during such short-term culture (Figure 3G; Oatley et al, 2004b). However, only a few tubules in the fragment grafts contained single or paired spermatogonia (average 7.7%; Table 2) 2 months after grafting with no germ cell differentiation evident. The 2-month period following grafting might be too short to observe spermatogonial differentiation in the grafted fragment (Rathi et al, 2005), so a longer time course study is underway. Even though greater success was achieved after castration of the host, it is unlikely that testosterone is a key factor affecting germ cell survival and differentiation, given its nonspecific nature. It is also possible that other factors produced by the testis may have an effect. However, because only 2 host mice had been castrated, these data are viewed as preliminary. Previous results (Oatley et al, 2004a; Oatley et al, 2005) revealed that germ cells were able to differentiate in bull testis tissues xenografted in castrated mice whose circulating testosterone levels had been maintained by the grafted bull testis tissue for 4 weeks. This indicates that the prevailing endocrine environment could either directly or indirectly affect donor-derived spermatogenesis (Rathi et al, 2005). The ages of our bovine donors were estimated at around 2–4 months based on our previous publication (Herrid et al, 2007) and are similar to those used in another study (Oatley et al, 2005).
Immature Sertoli cells are known to form cords or tubular structures in extratesticular sites following culture (Gassei et al, 2006) or following ectopic grafting to the kidney (Dufour et al, 2002), brain (Sanberg et al, 1996), or under the skin (Honaramooz et al, 2007). Because of the immunosuppressive features of Sertoli cells, they can survive long-term in allografts and xenografts in wild type and immunodefective recipients (Hemendinger et al, 2002; Dufour et al, 2003). In our study, cultured testicular cells aggregated within hanging drops in 3 days. Sertoli cells and germ cells were present in the aggregates at least for 5 days. However, no germ cells were found 2 months after grafting. Previous reports showed that porcine spermatogonia survive as long as 25 weeks in the cell pellets grafted under the back skin of castrated nude or SCID mice (Honaramooz et al, 2007). These suggest that the mouse systemic environment does not support bull germ cell survival and development.
In summary, we have characterized a range of reagents that identify various types of testicular cells, providing tools to distinguish bull spermatogonia and Sertoli cells from mouse cells and to document bovine germ cell and somatic cell colonization and survival in mouse testes after transplantation. This study provides a firm basis for studies designed to define the critical barriers to, and drivers of, successful xenotransplantation of spermatogonia across all species.
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Acknowledgments |
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Footnotes |
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