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,
From the * Surgical-Medical Research Institute and
the Departments of Surgery and
Medicine, University of Alberta, Edmonton,
Canada.
| Correspondence to: Dr Gregory S. Korbutt, Surgical-Medical Research Institute, 1074 Dentistry/Pharmacy Centre, University of Alberta, Edmonton, Alberta, Canada T6G 2N8 (e-mail: korbutt{at}ualberta.ca). |
| Received for publication January 29, 2002; accepted for publication March 20, 2002. |
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Abstract |
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Key words: Sertoli cell, peritubular myoid cell, cord formation, severe combined immunodeficient mice, testis development
It is believed that interactions between peritubular myoid and Sertoli cells are important for the formation of tubules and the development of a proper testicular environment. Using an in vitro culture system, Tung and Fritz (1980) demonstrated that when Sertoli and peritubular myoid cells isolated from pubertal rats are cocultured, they aggregate and form structures resembling germ cell-depleted seminiferous tubules. This process of tubulogenesis required the presence of both cell types since Sertoli or peritubular cells cultured alone were unable to form tubule structures (Tung and Fritz, 1980).
In this study, we have developed a novel in vivo transplantation model in which dissociated neonatal porcine Sertoli and myoid cells reaggregate and form seminiferous cords following implantation under the renal subcapsular space of immunoincompetent severe combined immunodeficient (SCID) mice. We have also extensively characterized the morphological changes that occur during the formation of these cords. Further study and experimental application of this in vivo model may provide valuable insight into intrinsic factors and mechanisms involved in testicular development, possibly by manipulating isolated testicular cells in vitro prior to implantation to observe the effect of specific factors on cord formation and development in vivo.
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Methods |
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Sertoli Cell Isolation
Neonatal porcine Sertoli cells (NPSCs) were isolated using a technique
similar to that previously described for rat Sertoli cells
(Korbutt et al, 1997).
Briefly, 1- to 3-day-old male Landrace-Yorkshire neonatal pigs were
anesthetized with Halothane, and testicles were surgically removed and placed
in 50-mL conical tubes containing cold (4°C) Hanks balanced salt solution
supplemented with 0.25% (wt/vol) fraction V bovine serum albumin (Sigma
Chemical Company, St Louis, Mo). The testes were cut into 1-mm fragments with
scissors, digested for 10 minutes at 37°C with collagenase type V (2.5
mg/mL; Sigma), and then washed 3 times with Hanks balanced salt solution. The
tissue was resuspended in calcium-free medium supplemented with 1 mM
ethyleneglycoltetraacetic acid (EGTA) and further digested with trypsin (25
µg/mL; Boehringer Mannheim, Laval, Canada) and DNase (4 µg/mL,
Boehringer) for 10 minutes at 37°C. The digest was passed through a
500-µm nylon mesh, washed with Hanks balanced salt solution, and cultured
in nontreated petri dishes (15-cm diameter) containing 60-80 x
106 cells and 35 mL of Ham F10 media supplemented with 10 mmol/L
D-glucose, 2 mmol/L L-glutamine, 50 µmol/L isobutylmethylxanthine, 0.5%
bovine serum albumin, 10 mmol/L nicotinamide, 100 U/mL penicillin, 100
µg/mL streptomycin, and 10% heat-inactivated neonatal porcine serum. Cells
were incubated for 48 hours at 37°C to allow the formation of Sertoli cell
aggregates (100- to 300- µm diameter).
Transplantation of Sertoli Cell Grafts
Prior to transplantation, the purity and number of Sertoli cells were
determined. Specifically, we assessed the number of vimentin-positive Sertoli
cells and smooth muscle alpha-actin-positive peritubular myoid cells in a
representative aliquot after dissociation of the cell aggregates using
techniques previously described for islet dissociation
(Korbutt et al, 1996). The
dispersed cell suspension was allowed to attach to Histobond adhesive
microscope slides (F.G.R. Steinmetz Inc, Surrey, BC, Canada), fixed with Bouin
solution for 30 minutes, washed with 70% ethanol, and immunostained using the
Sertoli cell marker vimentin (Korbutt et
al, 1997) or the myoid cell marker smooth muscle alpha-actin
(Tung and Fritz, 1990). In
each preparation, a minimum of 500 single cells were counted to assess the
proportion of vimentin-positive Sertoli cells and smooth muscle
alpha-actin-positive myoid cells.
In addition, to determine the number of cells transplanted in each recipient, 3 representative aliquots of the cell suspension were measured for total cellular DNA content using a Hoefer DyNa Quant 200 fluorometric assay (Amersham Pharmacia Biotech, San Francisco, Calif). Aliquots were washed with citrate buffer (150 mmol/L NaCl, 15 mmol/L citrate, and 3 mmol/L EDTA, pH 7.4), resuspended in TNE buffer (10 mM Tris, 0.2 mM NaCl, and 1 mM EDTA, pH 7.4), and sonicated. Aliquots of 10 µL were assayed in triplicate by diluting them in 2 mL of assay solution (0.1 µg/mL Hoechst 33258 in 1 x TNE) and measuring fluorescence (365 nm excitation/460 nm emission). A 6-point (0-500 ng/mL) DNA standard curve was generated using calf thymus DNA. For transplantation, aliquots consisting of 11 x 106 porcine testicular cells (6.6 pg DNA/cell) were aspirated into polyethylene tubing (PE-50), pelleted by centrifugation, and gently placed under the left renal subcapsular space of Halothane-anesthetized SCID mice (Korbutt et al, 1997).
Assessment of Tubule Formation Posttransplantation
Removal of the graft-bearing kidneys was performed for morphological
analysis at 0, 3, 7, 10, 20, 30, 40, and 60 days after transplantation (n 
3/time point, except n = 2 at 60 days). The graft-bearing kidneys were
immersed in Z-fix and embedded in paraffin. After deparaffinization and
rehydration, antigen retrieval was performed by heating slides for 15 minutes
in 0.01 M sodium citrate buffer (pH 6.0) in a microwave at full power. Tissue
sections were then immunostained as described previously
(Korbutt et al, 1997;
Akmal et al, 1998; Dufour and Kim, 1999).
Consecutive sections were incubated with 10% hydrogen peroxide to quench
endogenous peroxidases, blocked with nonspecific serum, and incubated with
primary antibody for 30 minutes. After incubation with primary antibody,
sections were incubated with the appropriate biotinylated secondary antibody
(1:200; Vector Laboratories, Burlingame, Calif) for 20 minutes, followed by
peroxidase-streptavidin, substrate-chromogen (3'3-diaminobenzidine Hcl
[DAB] or aminoethyl carbazole [AEC]), and then stained with hematoxylin (Zymed
Laboratories Inc, San Francisco, Calif). The primary antibodies used were as
follows: mouse monoclonal anti-vimentin (1:100; Dako, Carpinteria, Calif),
mouse monoclonal anti-proliferating cell nuclear antigen (anti-PCNA, 1:50;
Dako), mouse monoclonal anti-smooth muscle alpha-actin (1:50; Dako), goat
polyclonal anti-Müllerian inhibiting substance (anti-MIS) (1:100; Santa
Cruz Biotechnology, Santa Cruz, Calif), and goat polyclonal anti-DNA
transcription factor GATA-4 (1:50; Santa Cruz Biotechnology). Positive
controls included sections of 1- to 3-day-old neonatal pig testes in which
immunoreactivity for MIS, GATA-4, and vimentin was localized to Sertoli cells,
smooth muscle alpha-actin to peritubular myoid cells, and PCNA to
proliferating cells (Figure 1).
Negative controls, which showed no staining, consisted of sections incubated
without primary antibody.
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Results |
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In Vivo Cord Formation
In previous studies, we observed that testicular cells formed tubulelike
structures when cotransplanted with pancreatic islets of Langerhans underneath
the kidney capsule of mice (Suarez-Pinzon
et al, 2000). To examine the formation of these structures, NPSCs
were implanted underneath the kidney capsule of immunoincompetent SCID mice.
Between 0 and 60 days after transplantation, NPSC grafts, which were easily
identifiable underneath the kidney capsule, were examined histologically for
the presence of cord formation and development. At the time of
transplantation, the NPSCs were randomly distributed underneath the kidney
capsule (Figure 2A and F);
however, by 3 days posttransplantation, the NPSCs had organized into clusters
forming precursors to cords (Figure 2B and
G). After 7 days posttransplantation, cords similar to those found
in germ cell-depleted seminiferous tubules were evident
(Figure 2C and H). Moreover, it
was clear that the Sertoli cells were arranged with their nuclei along the
basal edge of the tubules (Figure
2H). With progression of time, the cords developed further,
becoming more defined and larger (Figure
2D, E, I, and J). No evidence of a lumen in the center of the
cords was detected at any of the time points analyzed. This is in agreement
with the lack of a lumen in the native porcine testis until after 90 days of
age (Kosco et al, 1989). A consistent progression of cord formation was
observed for all grafts at each time point.
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Epithelial Organization
Immunohistochemistry with multiple antibodies was performed to identify
Sertoli and myoid cells. MIS is only expressed in the cytoplasm of Sertoli
cells in the male (Figure 1A)
(Munsterberg and Lovell-Badge,
1991). It is known to cause regression of the müllerian ducts
during embryogenesis, thus preventing differentiation of the oviducts, uterus,
and upper vagina of the female reproductive tract
(Munsterberg and Lovell-Badge,
1991). In porcine Sertoli cells, MIS activity is low at birth,
increases to maximal levels between 10 and 19 days, and then declines to low
levels by 60 days of age (Tran et al,
1981). This decrease is suggested to be an indication of
testicular maturation in the porcine testis. To identify Sertoli cells, the
antibody against MIS was used to immunostain sections from the grafts. An
examination of MIS-immunostained sections revealed the presence of cords,
which had begun to develop 3 days after implantation
(Figure 3B and G). MIS was
clearly present in the cytoplasm of Sertoli cells for at least 60 days
(Figure 3B through E, G through
J). While not quantitative, the levels of MIS did, however, appear
to increase after 3 days posttransplantation
(Figure 3B and G) and then
decrease by day 30 after implantation
(Figure 3D and I), with further
declining levels present at 40 (data not shown) and 60 days
posttransplantation (Figure 3E and
J). This pattern is similar to the MIS expression in the
prepubertal porcine testis, suggesting the transplanted cells may be
developing along their normal pathway.
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GATA-4, a transcription factor thought to be involved in the gonadogenesis of the testis, is present in Sertoli and Leydig cell nuclei in testicular sections (Figure 1B) (McCoard et al, 2001a, b) as well as in other tissues such as the kidney and lymphocytes. Immunolocalization of GATA-4 in sections from the grafts identified Sertoli cells that were initially randomly distributed (Figure 3K) and that then began to organize into small discernible circles (Figure 3L) and finally became aligned on the basal side of the cords that resemble the seminiferous tubules present in the Sertoli cell-only or germ cell-depleted testis (Figure 3M through O) (Chakraborty, 1993).
Immunostaining was also performed using an antibody against smooth muscle alpha-actin to identify peritubular myoid cells (Figure 4A through E). Smooth muscle alpha-actin is known to be present in smooth muscle cells of blood vessels and in testicular myoid cells (Figure 1D) (Tung and Fritz, 1990). Smooth muscle alpha-actin-positive cells were randomly dispersed throughout the grafts at the time of transplantation (Figure 4A). Within 3 days after transplantation, although there were still disorganized regions, some myoid cells had begun to circle the Sertoli cells (Figure 4B). The majority of smooth muscle alpha-actin-positive cells were localized around the cords by 10 to 40 days posttransplantation (Figure 4C through E).
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Smooth muscle alpha-actin-positive cells were also detected surrounding newly formed blood vessels (Figure 4C and E). No vessels were present in the grafts at the time of transplantation; however, by day 3 posttransplantation, new vasculature was evident, with more blood vessels forming between days 3 and 10 posttransplantation (Figure 4C). These vessels were surrounded by a layer of smooth muscle alpha-actin-positive cells (Figure 4C through E). This induction of vessel formation is most likely to supply nutrients to the grafted tissue and may be induced by the Sertoli cells, which are known to produce potential angiogenic factors (Griswold, 1993; Skinner, 1993).
At higher magnification (Figure 4F through I), it was evident that the Sertoli and peritubular cells in the more mature grafts (40 days) were arranged almost exactly like the seminiferous epithelial layer in prepubertal porcine testicular cords (Tran et al, 1981). GATA-4 immunostaining clearly showed that as the Sertoli cells matured, they became polarized, with their nuclei aligning the basal edge of the tubule and their cytoplasm extending toward the center of the tubules (Figure 4G). Moreover, the peritubular cells, as identified with a smooth muscle alpha-actin antibody, were surrounding the tubules, located adjacent to the Sertoli cell nuclei (Figure 4I).
Cellular Proliferation
Gross morphological analysis at the time of graft removal indicated that
there was marked growth of the transplanted tissue as the cords developed.
Porcine Sertoli cells in the native testis have been shown to proliferate
during postnatal testicular development until puberty, which occurs between
100 and 120 days of age in the pig (Tran
et al, 1981; Franca et al,
2000). To determine whether the grafted Sertoli cells are
proliferating, serial sections were immunostained for vimentin
(Figure 5A through E) and PCNA
(Figure 5F through J). Vimentin
is an intermediate filament protein that was demonstrated to be present in the
cytoplasm of Sertoli cells (Figure
1C) (Korbutt et al,
1997). PCNA is involved in DNA replication and has been shown to
be localized to proliferating cells (Bravo
et al., 1987; Prelich et al,
1987; Hall et al,
1990). Immunostaining of the transplanted tissue with an
anti-vimentin antibody showed vimentin localized to the cytoplasm of Sertoli
cells within the tubules and also in the interstitial cells
(Figure 5A through E).
Immunostaining with an anti-PCNA antibody showed that Sertoli cells were
positive for PCNA, indicating that they were proliferating throughout 0 to 60
days posttransplantation (Figure 5F through
J, data not shown). In addition, some interstitial cells were also
proliferating (Figure 5H through
J).
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Discussion |
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While the process by which cords form under the kidney capsule is not identical to the formation of cords in the embryonic testis, it is strikingly similar. During cord formation, cells from the mesonephros migrate into the male gonad and interact with the aggregating Sertoli and germ cells (Magre and Jost, 1980, 1991; Jost et al, 1981; Buehr et al, 1993; Tilmann and Capel, 1999). It is presumed that these migrating cells are peritubular myoid cells. Consistent with these results, the myoid cells in the graft also migrated and surrounded the aggregating Sertoli cells. As the graft matured, the myoid cells were juxtaposed to Sertoli cell nuclei that were present on the basal region of the cords. The Sertoli cells in normal seminiferous tubules have this relationship with peritubular cells and also are polarized, with their cytoplasm extending into the apical region of the tubular structures (Russell et al, 1990). The cellular organization of the cords in the grafts is similar to the morphological arrangement of cells found in Sertoli cell-only or germ cell-depleted seminiferous tubules of the testis (Chakraborty, 1993).
Other studies have demonstrated the formation of tubulelike structures in vitro (Tung and Fritz, 1980; Hadley et al, 1985); however, to our knowledge, our transplantation model is the first description of an in vivo model. Using an in vitro culture system, Tung and Fritz (1980) demonstrated that pubertal Sertoli/peritubular cocultures are able to aggregate and form structures resembling germ cell-depleted seminiferous tubules. These in vitro studies have demonstrated that mesenchymal—epithelial (myoid—Sertoli) cell interactions are important for the deposition of the extracellular matrix components that are required for tubule formation (Tung and Fritz, 1987). This process of tubulogenesis required the production of laminin by Sertoli cells, since anti-laminin antibodies prevented the formation of tubules (Hadley et al, 1990; Tung and Fritz, 1994). These factors required for tubule formation in the in vitro system have since been shown to be required for cord formation during embryonic testis development (Buehr et al, 1993; Marinos et al, 1995; Tilmann and Capel, 1999). Our in vivo model provides an excellent alternative model to study the cellular factors, secretory products, and mechanisms responsible for interaction of cells during cord formation in the testis. In contrast to in vitro models, the present in vivo model allows for a more physiological environment, thereby permitting the testicular cells to interact with other endogenous systems.
Similar to the in vitro system (Tung and Fritz, 1980), the Sertoli and myoid cells within the graft appear to direct the formation of cords, indicating that the developmental signals required for cord formation are intrinsic characteristics of the Sertoli and myoid cells. In addition, we have observed similar structures in other transplant sites such as the subcutaneous space, the epididymal fat pad, and the omental pouch (data not shown). These results suggest that it is not the renal environment but rather the cells within the graft that direct the formation of cord structures.
Johnson et al (1996a,b) have developed a transplantation model in which they transplant the entire testis into the pinna of the ear. In this model, they demonstrate that the Sertoli cells are able to develop normally, although this development is slightly delayed (Johnson et al, 1996b). They even report instances of complete spermatogenesis in some of the tubules; however, most of the tubules were devoid of germ cells (Johnson et al, 1996b). They further demonstrate the effects of hypophysectomy, sex of the host, and number of transplanted testes on Sertoli cell proliferation and circulating hormone levels (Johnson et al, 1996a). These studies indicate that transplantation of the testis is a valid model to study testicular development and validate our in vivo model for the study of testicular morphogenesis. Furthermore, our model will be useful because it allows the isolated cells to be manipulated in vitro prior to transplantation to examine the outcome during testis development.
GATA-4, a member of the GATA transcription factor family, has been shown to be important in cell differentiation and organ development (Simon, 1995; Kuo et al, 1997; Molkentin et al, 1997) as well as in the up-regulation of MIS in the testis (Viger et al, 1998; Tremblay and Viger, 1999; Wantanabe et al, 2000). In our model, GATA-4 was detected in the transplanted Sertoli cells at all time points analyzed, which is consistent with its role in testicular development. In the normal testis, it is expressed in Sertoli cells prior to cord formation, throughout puberty, and into adulthood, suggesting it may be involved in testis development (McCoard et al, 2001a,b).
The presence of MIS in the grafts also may be important since MIS is thought not only to regulate regression of the Müllerian ducts but also to be a marker of Sertoli cell function during testis differentiation (Vigier et al, 1987; Behringer et al, 1990). MIS is expressed in the gonads in a sexually dimorphic manner (Munsterberg and Lovell-Badge, 1991); it is present in Sertoli cells after cord formation, continues to be expressed throughout embryonic development, and then declines to low levels after birth (Tran et al, 1977). In pigs, MIS is again up-regulated to high levels between postnatal days 10 and 19 and steadily declines to low levels after 60 days (Tran et al, 1981). MIS expression in the transplanted Sertoli cells was similar to that in the native porcine testis, suggesting the Sertoli cells in this in vivo model are developing as in the prepubertal testis.
Sertoli cell proliferation in the grafts also paralleled that in the native
testis (Orth, 1982;
Franca et al, 2000). During
testicular development, Sertoli cells proliferate maximally in the embryo,
continue to proliferate at birth, and cease to divide after puberty
(Orth, 1982; Franca et al, 2000), which is
around 100-120 days of age in the pig
(Tran et al, 1981). This
growth is essential for male fertility since the number of Sertoli cells
defines the sperm capacity of the adult testis. Consistent with these results,
transplanted neonatal Sertoli cells in the grafts were found to proliferate
normally at all time points after transplantation up to 60 days. Many factors
can regulate testis growth, including follicle-stimulating hormone, epidermal
growth factor, calf serum, nerve growth factor, neurotropin-3, and
transforming growth factor-
(Cupp et al,
1999a,
b,
2000;
Levine et al, 2000). It
remains to be seen if the factors that regulate growth in the native testis
are the same factors that regulate growth in our in vivo model system. In
order to study testicular growth with this model, Sertoli cells engineered to
produce specific inhibitors to growth factors believed to be required for
Sertoli cell proliferation can be transplanted and the grafts examined for the
effect. This model would allow for similar studies in cord formation and other
developmental processes.
In summary, it is remarkable that transplanted porcine Sertoli and myoid cells were able to reorganize into cords similar to the cellular organization in the seminiferous tubules from Sertoli cell-only or germ cell-depleted testes after implantation underneath the kidney capsule of SCID mice. Equally interesting is the expression of GATA-4 and MIS as well as the proliferation of the Sertoli cells, which mimicked the expression patterns and growth of Sertoli cells in the native porcine testis. These results suggest the transplanted testicular cells were able to develop normally in this ectopic site, independent of the local environment and even across species barriers (ie, pig to mouse). Therefore, this in vivo model of cord formation and prepubertal development represents a novel system to study morphogenesis and growth during prepubertal testicular development. In addition, prepubertal testicular development in the pig is delayed when compared to the rodent models normally studied, allowing a more detailed analysis of the changes that occur during prepubertal development.
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Acknowledgments |
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Footnotes |
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