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From the * Department of Biology, The University
of Dayton, Dayton, Ohio; and the Department of
Cell Biology, Georgetown University Medical Center, Washington, DC.
| Correspondence to: Marie-Claude Hofmann, PhD, Department of Biology, The University of Dayton, 300 College Park, Dayton, OH 45469-2320 (e-mail: marie-claude.hofmann{at}notes.udayton.edu). |
| Received for publication June 19, 2002; accepted for publication August 29, 2002. |
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Abstract |
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-inhibin, GATA-1, and
steroidogenic factor-1. Further, the lines express growth and differentiation
factors known to act upon germ cells in vivo and in vitro such as leukemia
inhibitory factor (LIF), transforming growth factor beta (TGF-ß), and
basic fibroblast growth factor (bFGF). Moreover, when used as feeder layers in
cocultures, at least 2 of these lines are able to maintain the viability of
type A spermatogonia for at least 7 days and to support the first steps of
spermatogonial differentiation.
Key words: Testis, immortalization, simian virus large T antigen, ecdysone, ponasterone A, growth factors
-inhibin, might also have a role in the regulation of spermatogonial
cell number and differentiation (van
Dissel-Emiliani et al, 1989;
Hakovirta et al, 1993; Mather et al, 1997).
More recently, it has been shown that spermatogonial stem cells have to fit
within a proper 3-dimensional tissue architecture in order to express specific
genes and that a physical association with Sertoli cells is necessary
(Meng et al, 2000). Thus, it
seems that a close association between spermatogonia and Sertoli cells,
coupled with the supply of specific growth factors, needs to be mimicked in
vitro to maintain germ cells in long-term cultures. Therefore, we attempted to
cultivate freshly isolated germ cells on Sertoli cell line feeder layers. We
initially worked with a Sertoli cell line immortalized with a plasmid for the
constitutive production of the simian virus 40 large tumor antigen (SV40-LTAg)
(Hofmann et al, 1992; van der Wee and Hofmann,
1999). However, upon long-term culture, the cells became
transformed and lost some of their typical characteristics, making them
unsuitable for coculture with freshly isolated germ cells. Thus, a conditional
immortalization system that allows the modulation of expression of the gene
coding for SV40-LTAg was desirable. We conditionally immortalized freshly
isolated Sertoli cells by transfecting them with 2 plasmids, p-VgRXR and
pIND-LTAg. The pVgRXR plasmid codes for the ecdysone nuclear receptor, which
forms a complex with the Drosophila steroid hormone ecdysone
(No et al, 1996). We
constructed the pIND-LTAg plasmid that contains the large T-antigen gene under
the control of an ecdysone-inducible promoter. When ponasterone A (an analog
of ecdysone) is present in the culture medium, the complex ponasterone A
receptor binds to the upstream hormone response element on the pIND-LTAg
plasmid, driving the promoter and activating the transcription of the LTAg
gene. Using this method, we established 7 Sertoli cell lines that we describe
in the present study. These lines were characterized using light and electron
microscopy, immunocytochemistry, and reverse transcriptase-polymerase chain
reaction (RT-PCR). We analyzed the expression of 16 genes known to be specific
for various cell types within the testis. All cell lines expressed genes
specific for Sertoli cells in the testis, such as SCF and sulfated
glycoprotein-2 (SGP-2). Moreover, they also expressed the genes coding for
-inhibin, GATA-1, bFGF, LIF, and TGF-ß. Thus, the pattern of gene
expression displayed by these Sertoli cell lines indicated that they could be
used as feeder layers to support the survival of type A spermatogonia in
vitro.
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Materials and Methods |
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Tissue Culture
Cells were cultivated in complete cell culture media, which consisted of
Dulbecco modified Eagle medium (DMEM) supplemented with 1 mM sodium pyruvate,
2 mM L-glutamine, 50 U/mL penicillin, 50 mg/mL streptomycin, and 100 mM
non-essential amino acids (Atlanta Biologicals, Norcross, Ga). Cells were
plated in culture dishes (Falcon, Fisher Scientific, Pittsburgh, Pa)
containing complete culture medium supplemented with 5% or 10% fetal calf
serum (FCS; Atlanta Biologicals) and incubated at 37°C and 5%
CO2 in a humidified incubator. For cultures in minimal conditions,
FCS was replaced by 5% synthetic NU serum (Fisher).
Cell Isolation, Transfection, and Subcloning
Animal investigations were conducted according to the National Research
Council Guide for the Care and Use of Laboratory Animals. After the
testes were removed from 6-day-old BALB/c mice, the seminiferous tubules were
reduced to a single-cell suspension using a series of enzymatic digestions
(Bellve et al, 1977; Hadley et
al, 1985,
1990). Cotransfections with
the pIND-LTAg plasmid and the pVgRXR plasmids were performed with the
Lipofectin technique (GIBCO/Life Technologies, Grand Island, NY), and the
transfected cells were selected with the antibiotics zeocin (Invitrogen) and
G418 (GIBCO/Life Technologies). We obtained 2 cell lines, G5 and J5, that
showed notably different morphologies. The G5 cell line was subcloned by 2
rounds of limiting dilutions in 96-well plates. Seven
subclones—SG5-1.13, SG5-2, G5F2, G5G2, G5B1, G5B2, and G5B3—were
selected on the basis of morphological resemblance to Sertoli cells in tissue
culture (cytoplasmic projections, lipid inclusions, and indented nuclei) using
phase-contrast microscopy. These cells were maintained in culture with
complete culture medium with the addition of 5% NU serum (Fisher), ponasterone
A (5 µM) (Invitrogen, La Jolla, Calif), zeocin (100 µg/mL) (Invitrogen),
and G418 (100 µg/mL) (GIBCO/Life Technologies).
Inactivation/Reactivation of the Inducible Oncogene LTAg
The G5 and J5 cell lines were cultured in LabTek chambers (Fisher) with
complete culture media supplemented with 10% FCS, ponasterone A (5 µM),
zeocin (100 µg/mL), and G418 (100 µg/mL). Immunocytochemistry was
initially performed on these cells to confirm the expression of the LTAg, the
oncoprotein produced by the SV40-LTAg gene. MDA-231, a breast carcinoma cell
line, was used as a negative control for LTAg expression, and MDA-231 clone B,
a clone of the MDA-231 cell line transfected with pIND-LTAg and pVgRXR, served
as the positive control. The cell lines were cultivated in complete culture
media with 10% FCS. The cells were fixed with ice-cold methanol for 20 minutes
and then incubated with a purified mouse anti-LTAg primary antibody
(Calbiochem, San Diego, Calif) at a dilution of 1:500, 1:1000, or 1:2000 or no
primary antibody (negative control). The secondary antibody was a biotinylated
anti-mouse immunoglobulin G made in the horse (Vector Laboratories,
Burlingame, Calif). The streptavidin-alkaline phosphatase technique (Vector)
was used to visualize the presence of LTAg, and observations were made using
brightfield microscopy (IMT microscope, Olympus Corp, Melville, NY).
Once LTAg expression was confirmed in the G5 and J5 cell lines, the cells were cultured without ponasterone A for 10 days, and immunocytochemistry was performed to determine if the LTAg gene had been inactivated. Then, ponasterone A was again added to the cultures for either 1 day or 7 days, the cells were fixed, and immunocytochemistry for LTAg was performed to determine how long it took for the oncogene to be reactivated.
Growth Curves
The SF7 (Hofmann et al,
1992) and SG5-2 cell lines were counted and seeded at 20 000 cells
per dish in 18 different 60-mm tissue culture dishes with complete cell
culture media and 5% FCS. For the next 6 days, 3 of the dishes for each cell
line were trypsinized, and the cells were counted using trypan blue exclusion.
The cell numbers are presented as mean plus or minus standard deviation.
Immunocytochemistry for c-kit, -Actin, GATA-1, and
3ß-ol-Hydroxysteroid-Dehydrogenase
Cells were grown on FCS-coated coverslips until 80% confluency and fixed
with ice-cold methanol for 5 minutes. After washing with phosphate-buffered
saline (PBS), the cells were stained with a mouse monoclonal -actin
antibody at a dilution of 1:100 (Roche Molecular Biochemicals, Indianapolis,
Ind), a rabbit polyclonal 3ß-ol-hydroxysteroid-dehydrogenase antibody at
a dilution of 1:200 to 1:500 (a kind gift from J. I. Masson, University of
Edinburgh, United Kingdom), a rabbit polyclonal c-kit antibody at a dilution
of 1:50 to 1:200 (Santa Cruz Biotechnology, Santa Cruz, Calif), or a rabbit
GATA-1 antibody at a dilution of 1:100 (Santa Cruz Biotechnology). The sites
of reaction of the primary antibodies were shown by biotinylated secondary
antibodies and the streptavidin-peroxidase technique (Zymed, San Francisco,
Calif) or by the immunofluorescence technique.
Cytochemistry for Alkaline Phosphatase
In order to visualize the expression of alkaline phosphatase (AP), the
cells were cultured in LabTek chambers (Fisher), fixed in situ with 10%
formaldehyde (Sigma Chemical Co, St Louis, Mo) in PBS for 10 minutes at
4°C, and then washed twice with 0.2 M Tris buffer (pH 8.9). The substrate
reagent, prepared fresh, consisted of 0.01% naphthol-AS-MX phosphate and 0.06%
fast violet B salt (Sigma) in 0.1 M Tris buffer (pH 8.9). The staining
solution was immediately filtered and then incubated with the fixed cells for
45 minutes at 37°C. After incubation, the cells were washed with distilled
water and observed under brightfield microscopy (IMT-2 microscope, Olympus).
In this assay, a red color at the plasma membrane indicates the expression of
AP. NIH 3T3 fibroblasts were used as a negative control since they do not
express this enzyme.
RT-PCR
In order to determine the phenotypes of the 7 sublines of G5, RT-PCR was
performed using primers for genes specifically expressed by various cell types
within the testis. Total RNA was collected from the subclones using TRI
REAGENT according to the manufacturer's protocol (Molecular Research Center
Inc, Cincinnati, Ohio). Total RNA samples were treated with 1 U/1 µg RNA of
RQ1 RNase-free DNase (Promega, Madison, Wis) to degrade any genomic DNA
present. Complementary DNA (cDNA) was synthesized from 5 µg of the total
RNA using SuperScript II RT and oligo(dT) primers to selectively reverse
transcribe messenger RNA (mRNA; GIBCO/Life Technologies).
Two microliters of the cDNA obtained from each subclone was amplified for 40 cycles (denaturation at 94°C for 30 seconds, annealing at specific temperatures for 45 seconds, and elongation at 72°C for 45 seconds) using primers specific for various genes expressed by different testicular cell types. As a positive control and to check for the presence of contaminating genomic DNA, PCR primers for cyclophilin, a housekeeping gene, were also used. The primers were designed using published sequences from mouse or other mammalian species (Stratagene, La Jolla, Calif) (Walther et al, 1996; Schrans-Stassen et al, 1999). The primers and PCR conditions used were as follows.
-Inhibin, produced by Sertoli cells (5'-GCA ATG GAT GGG GAA
GGT GG-3' [sense strand] and 5'-GGT GGC TGC GTA TGT GTT
GG-3' [antisense strand], with an annealing temperature of 54°C),
for a PCR product fragment of 237 bp
(Walther et al, 1996);
As negative controls, PCR was performed on RT reaction products obtained
without the use of RT. All PCR reaction products were electrophoresed on 2%
agarose gels made in 1x Tris-boric acid-EDTA buffer (TBE, pH 8.0) with
0.01% ethidium bromide in a running buffer consisting of TBE with 0.01%
ethidium bromide. Ten microliters of PCR product and 2 
L of 6x
loading dye were loaded into the gel and electrophoresed for 32 minutes at 100
V, 200 mA, and 600 W. Results were viewed on an ultraviolet light lamp
table.
Light and Electron Microscopy
Cells were cultivated in 60-mm petri dishes until 80% confluency, washed
with PBS, and treated with 0.05% trypsin-EDTA in PBS (Atlanta Biologicals).
After trypsin inhibition with 1% (vol/vol) FCS in PBS, the cell samples were
centrifuged at 200 x g. The pellets were carefully resuspended
in 1 mL of 5% glutaraldehyde in 0.2 M s-collidine buffer, pH 7.4, and then
transferred into microcentrifuge tubes. Glutaraldehyde and s-collidine buffer
were purchased from Electron Microscopy Sciences (Fort Washington, Pa). After
a 30-minute fixation with occasional inversion of the tube, the cells were
centrifuged at 200 x g and washed 3 times with 0.2 M collidine
buffer. The samples were then post-fixed in a final concentration of 1%
OsO4 in 1.5% potassium ferrocyanide for 1 hour at room temperature
and washed for 15 minutes with 0.2 M s-collidine buffer. After dehydration in
increasing concentrations of ethanol, the samples were cleared in propylene
oxide and then embedded in Epon (Electron Microscopy Sciences). One-micrometer
sections were cut with an LKB microtome, stained with toluidine blue, and
examined by light microscopy. Thin sections were stained with uranyl acetate
and lead citrate and examined by electron microscopy.
Cocultures of Germ Cells and Sertoli Cells
Sertoli cells of the lines SG5-1.13 and SG5-2 were seeded in 24-well plates
at a concentration of 2 x 104 cells per well in 1 mL of
culture medium. Type A spermatogonia were isolated using the STAPUT method
(Dym et al, 1995), and a
single-cell suspension was immediately seeded with the Sertoli cells at a
concentration of 4 x 104 cells/well. The culture medium used
was DMEM completed as described above. However, in this case, 5% synthetic NU
serum (Becton-Dickinson, Sparks, Md) was used instead of FCS to provide
defined culture conditions. Three days after seeding, the cocultures were
fixed and stained with a rabbit polyclonal anti—c-kit antibody (Santa
Cruz Biotechnology) at a dilution of 1:100 to visualize the spermatogonia. The
sites of reaction of the primary antibody were shown by a biotinylated
secondary antibody and the streptavidin-peroxidase technique (Zymed). For
comparison, a single-cell suspension of type A spermatogonia was seeded on
adherent 6-day-old primary Sertoli cells or NIH 3T3 cells using the same
culture conditions.
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Results |
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After the 2 testicular cell lines, G5 and J5, were cultured for 10 days without ponasterone A, many of the cells entered a crisis state and died. However, 10% of the original cell populations survived, and these cells continued to express LTAg without the addition of ponasterone A in the culture medium. After 6 months of continuous culture, the ability to turn off the expression of LTAg in these cells was lost. Since both lines exhibited a mixed phenotype, the G5 line was further subcloned by limiting dilution, resulting in 7 cell lines exhibiting Sertoli celllike morphology in tissue culture. While the expression of the LTAg remained constitutive in all of the lines, their rate of proliferation was markedly lower than the rate of proliferation of the SF7 Sertoli cell line previously established (Hofmann et al, 1992). SF7 cells were immortalized using the LTAg under the permanent control of the SV40 promoter. Figure 2 compares the rate of proliferation of the cell line SF7, which expresses a high level of LTAg, to the rate of proliferation of the SG5-2 cell line, a subclone of G5.
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Characterization of the Cell Lines by Immunocytochemistry and
Phase-Contrast Microscopy
The J5 and G5 cell lines were stained by immunocytochemistry for the
presence of the following markers: c-kit, -actin, and
3ß-ol-hydroxysteroid-dehydrogenase. None of the lines expressed the c-kit
receptor or 3ß-ol-hydroxysteroid-dehydrogenase, excluding the presence of
differentiating spermatogonia and Leydig cells using this method (data not
shown). However, about 50% of the J5 cells expressed -actin, indicating
the presence of peritubular myoid cells. This cell line was not further
characterized.
Seven cell lines resulted from the subcloning of G5 and were designated
SG5-1.13, SG5-2, G5B1, G5B2, G5B3, G5F2, and G5G2. These new lines were
cultivated for more than 2 years. In phase-contrast microscopy, the SG5-1.13
and SG5-2 cell lines appeared mostly spindle shaped. Cells from both lines
exhibited some lipid inclusions within the cytoplasm. All cell lines showed
cytoplasmic projections resembling those of primary Sertoli cells
(Figure 3A and B). The SG5-1.13
and SG5-2 lines were again stained by immunocytochemistry for c-kit,
-actin, desmin, and 3-ß-ol-hydroxysteroid-dehydrogenase. The cells
were negative for these markers, which are specific for germ cells,
peritubular cells, and Leydig cells (data not shown).
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Expression of AP
Cytochemistry for AP was performed to confirm the presence or absence of
peritubular myoid cells. In this assay, AP activity is shown by the presence
of a red precipitate at the cell membrane of positive cells. AP activity is a
characteristic feature of peritubular myoid cells
(Anthony and Skinner, 1989) and
germ cells in early stages of development
(Narisawa et al, 1992). All
cell lines were negative for
AP.
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Cellular Morphology in Light and Electron Microscopy
Toluidine blue—stained 1-µm-thick sections confirmed that the
SG5-1.13 and SG5-2 cell lines had many of the morphological characteristics of
in vivo Sertoli cells. In particular, the SG5-2 cell line consists of a
homogenous population of cells that are 8-9 µm in diameter with an
irregular outline (Figure 4A).
Electron microscopy analysis revealed that the nucleus is indented and
contains dense nucleoli, some in association with the nuclear envelope
(Figure 4B). The cytoplasm
contains many spherical and elongated mitochondria, some lipid inclusions, and
some phagosomes. The SG5-1.13 cells have a very similar morphology.
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Characterization of the Cell Lines Using RT-PCR
The Table summarizes the results of the RT-PCR experiments. Total RNA was
extracted from the cell lines and from freshly isolated Sertoli cells as a
control (6-day-old testes) (Figure
5). There was a PCR product for the housekeeping gene cyclophilin
in all of the cell lines and freshly isolated Sertoli cells, indicating the
presence of cDNA in every sample after RT. All of the Sertoli
cell—specific cDNAs tested were present in the freshly isolated Sertoli
cells (positive control). The cDNAs for the androgen and FSH receptors were
absent in all cell lines. The cDNA for SCF and SGP-2, genes specifically
expressed by Sertoli cells in the testis, was present in all of the cell
lines. TGF-ß, -inhibin, and LIF cDNAs were obtained from all of
the cell lines with the exception of the G5B1 cell line. SF-1 and GATA-1 cDNAs
were present in all of the cell lines. bFGF cDNA was present in all cell lines
with the exception of SG5-1.13 and G5G2. There were no specific PCR bands for
the LH receptor and 3ß-ol-hydroxysteroid-dehydrogenase, genes
specifically expressed by Leydig cells, in any of the cell lines. Also, there
was no PCR band for the c-kit receptor, indicating that none of the lines were
of germ cell origin.
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Culture of Freshly Isolated Type A Spermatogonia on Sertoli Cell
Monolayers
All cell lines expressed growth factors that are known to induce germ cell
survival and growth in cocultures, except for SG5-1.13 and G5G2, which do not
produce bFGF (Resnick et al,
1992). We thus assessed whether SG5-1.13 and another cell line,
SG5-2, were able to maintain the survival of isolated type A spermatogonia in
vitro. In vivo, undifferentiated spermatogonia proliferate and differentiate
synchronously as Apaired (Apr) spermatogonia in clusters
of 2 germ cells and as Aaligned (Aal) spermatogonia in
rows of 4, 8, or 16 germ cells. At this stage, these cells remain connected by
intercellular bridges (de Rooij and
Russell, 2000). We observed that, in minimal culture conditions,
freshly isolated type A spermatogonia could survive for at least 7 days, and
they were able to differentiate into both Apr and Aal
cells in cocultures with both Sertoli cell lines
(Figure 6A and B). This result
is similar to what is observed when type A spermatogonia are cultivated on a
feeder layer of primary Sertoli cells
(Figure 6C and D). In our
hands, the germ cells never attached to the NIH 3T3 cells.
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Discussion |
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In situ, Sertoli cells exhibit a typical epithelial morphology and are highly polarized, with a tall, columnar cytoplasm extending from the basal part of the seminiferous epithelium to the lumen. Their nuclei are usually localized close to the basement membrane. It is, however, well known that Sertoli cells lose their characteristic morphology after enzymatic digestion of the tissue. The cells become rounded once their connections to the basal membrane and the other cells have been severed. Bellve et al (1977) have described the morphology of a pure preparation of Sertoli cells from the testes of prepubertal mice by light and electron microcopy. After enzymatic digestion, the cells became rounded, with an irregular cytoplasmic contour, an indented nucleus, and heterochromatin in association with the nuclear membrane. The Sertoli cell lines that we established exhibit the same characteristics in light and electron microscopy analysis.
Freshly isolated Sertoli cells are also known to lose their typical morphology in culture when conventionally cultivated on a plastic substratum. Hadley et al (1985) reported that the Sertoli cell phenotype was best maintained when the cells were cultivated on a reconstituted basement membrane such as Matrigel. Moreover, when cultivated within the gel, Sertoli cells were also able to form cord-like structures. The cell lines that we describe in the present study behave in a similar way; they spread when cultivated directly on plastic but form cords when cultivated on Matrigel. This behavior has already been described in a previous report (van der Wee et al, 2001).
Several paracrine growth factors produced by Sertoli cells in vivo have
been recently identified, and their role in germ cell survival and
proliferation has been demonstrated. In particular, the transmembrane form of
SCF, the ligand for c-kit, supports the survival of primordial germ cells
(Dolci et al, 1991), while the
soluble form stimulates the proliferation of primordial germ cells and DNA
synthesis in type A spermatogonia (Rossi
et al, 1993; Dym et al,
1995; Dirami et al,
1999; Feng et al,
2000). LIF is a survival and proliferation factor for primordial
germ cells and gonocytes (Matsui et al,
1991; De Miguel et al,
1996; Nikolova et al,
1997). LIF has also been shown to maintain long-term cultures of
embryonic stem cells in an undifferentiated stage
(Smith et al, 1988; Williams et al, 1988) and
might have a similar function in maintaining the stem cell potential of
certain spermatogonia (Piquet-Pellorce et
al, 2000). Another factor important for the survival and
proliferation of primordial germ cells in culture is bFGF
(Matsui et al, 1992;
Resnick et al, 1992), which is
produced by Sertoli cells. Sertoli cells are also the primary source of
testicular inhibin, a paracrine regulator of various cell types
(Mather et al, 1997).
-Inhibin might also have a role in the regulation of spermatogonial
cell number and differentiation (van
Dissel-Emiliani et al, 1989;
Hakovirta et al, 1993;
Mather et al, 1997). TGF-ß produced by Sertoli cells has also been linked to germ cell
differentiation (Mullaney and Skinner,
1993; Fritz, 1994;
Kierszenbaum, 1994). Thus,
although their function in spermatogenesis has not been clearly elucidated,
any of these molecules, alone or in combination, could play a crucial role in
spermatogonia proliferation and/or differentiation. Therefore, the Sertoli
cell lines we established were screened specifically for the expression of
these growth factors. We show here that, in addition to the morphological
characteristics of Sertoli cells in light and electron microscopy analysis,
these cell lines exhibit a molecular phenotype similar to that of Sertoli
cells in vivo. The genes expressed include SGP-2, -inhibin, and GATA-1,
as well as genes coding for major growth and differentiation factors such as
SCF, LIF, bFGF, and TGF-ß. Moreover, the expression of the orphan nuclear
receptor SF-1 by all of the cell lines indicates that transactivation of
male-specific genes could occur in these cells, which might influence the
development of germ cells in coculture. However, the lack of expression of
androgen and FSH receptors suggests that these cells may not be suitable for
modeling the hormonal control of spermatogenesis.
A number of Sertoli cell lines have been already established. However, none
of them expresses the FSH receptor stably, except for a line reported by
Walther et al (1996), where it
was transcribed at very low levels. The Sertoli cell line MSC-1 has been
specifically transfected with an expression vector for the FSH receptor,
allowing the cells to recover this original characteristic
(Peschon et al, 1992; Eskola et al, 1998). The
testicular cell line TM4, derived after multiple passages in culture, has been
used in many studies but does not show a very differentiated phenotype
(Mather and Phillips, 1984). Sertoli cell lines have been established using the SV40 or the polyoma T
antigen (Hofmann et al, 1992;
Peschon et al, 1992; Paquis-Flucklinger et al,
1993; McGuinness et al,
1994; Walther et al,
1996). Some of these lines express growth factors, in particular
SCF, but their ability to promote the survival and differentiation of the type
A spermatogonia in vitro has not been reported. Several murine Sertoli cell
lines have been established using a temperature-sensitive mutant of LTAg
(Boekelheide et al, 1993; Roberts et al, 1995;
Walther et al, 1996;
Jiang et al, 1997). These
Sertoli cells show signs of differentiation when LTAg is turned off at
40°C (nonpermissive temperature). However, this temperature is unsuitable
for the culture of germ cells, which proliferate and differentiate at the
optimal temperature of 34°C. The Sertoli cell lines that we established
can be used in a defined medium at the critical temperature of 34°C, and
they express a combination of growth factors that have not been described in
other Sertoli cell lines. Although some of these factors are produced by STO
fibroblasts, the latter cannot always be used to study somatic cell—germ
cell interactions since they do not express Sertoli cell—specific genes
such as -inhibin, SF-1, SGP-2, and GATA-1.
In order to study cell fate decisions in the early phases of spermatogenesis at the molecular level, an in vitro system must be devised whereby type A spermatogonia, including the testis stem cells, can be cultured for a prolonged period of time. The availability of type A spermatogonia that can be maintained in vitro while retaining their differentiation potential is highly desirable. In vitro culture of these cells is essential for the introduction of foreign genes in order to study spermatogenesis at the molecular level as well as for preliminary studies leading to germ line gene therapy. In this paper, we present the characterization of several Sertoli cell lines that could be used as feeder layers for the survival and early differentiation steps of primary type A spermatogonia in vitro. In particular, the lines SG5-2 and SG5-1.13 seem to be able to maintain the viability of spermatogonia, since these germ cells still express the c-kit receptor after several days of coculture in serum-free medium. In a previous study, we showed that the cell line SG5-1.13 was able to maintain the replication potential of freshly isolated type A spermatogonia for at least 1 week using bromodeoxyuridine incorporation (van der Wee et al, 2001). In the present study, we show that type A spermatogonia isolated in a single-cell suspension are able to form paired and aligned structures when cocultivated with SG5-1.13 and SG5-2. This indicates that these cell lines, like primary Sertoli cells, can sustain the early stages of spermatogonial differentiation in vitro. In the absence of primary Sertoli cells, or in the absence of these cell lines, the formation of Aal spermatogonia never occurs. Also, when we used NIH 3T3 fibroblasts as feeder layers, the germ cells never attached.
In summary, we believe that the Sertoli cell lines described in the present report represent a valuable experimental tool because they can grow in minimal serum conditions at the critical temperature of 34°C. In addition, their growth rate is slow, and they produce a combination of growth and differentiation factors that have not been reported in previously established Sertoli cell lines. Such features make these lines suitable for cocultures with undifferentiated spermatogonia and also suitable for the study of germ cell—Sertoli cell interactions.
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Footnotes |
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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De Felici M, Pesce M. Growth factors in mouse primordial germ cell migration and proliferation. Prog Growth Factor Res.1994; 5:135 -143.[Medline]
De Miguel MP, De Boer-Brouwer M, Paniagua R, van den Hurk R, de Rooij DG, van Dissel-Emiliani FM. Leukemia inhibitory factor and ciliary neurotropic factor promote the survival of Sertoli cells and gonocytes in coculture system. Endocrinology.1996; 137:1885 -1893.[Abstract]
De Rooij DG, Russell LD. All you wanted to know about spermatogonia but were afraid to ask. J Androl.2000; 21:776 -798.[Medline]
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