Directed Expression of an Oncogene

to Sertoli Cells in Transgenic Mice

Using Mullerian Inhibiting Substance
Regulatory Sequences

Jacques J. Peschon, Richard R. Behringer*, Richard L. Cate,

Kimberly A. Harwood, Rejean L. Idzerda, Ralph L. Brinster, and
Richard D. Palmiter

Department of Biochemistry and

Howard Hughes Medical Institute (J.J.P., R.D.P.)
and Department of Pharmacology (K.A.H., R.L.I.)
University of Washington
Seattle, Washington 98195
Laboratory of Reproductive Physiology (R.R.B., R.L.B)
School of Veterinary Medicine
University of Pennsylvania
Philadelphia, Pennslylvania 19104
Department of Molecular Biology (R.L.C.)
Biogen Inc.
Cambridge, Massachusetts 02142

Mullerian inhibiting substance (MIS) is a glycoprotein large columnar cells extending approximately 70 pm
hormone expressed by Sertoli cells that induces the from the basement membrane to the lumen of the
regression of Mullerian ducts during development of seminiferous tubule (l-3). The proliferation of sperma-
the male reproductive tract. Transgenic mice carry- togonial stem cells, as well as the differentiation of
ing a fusion gene composed of human MIS transcrip- these cells into elongated spermatids, takes place
tional regulatory sequences linked to the SV40 T- within recesses created by Sertoli cell processes. Ser-
antigen gene specifically develop testicular tumors toli cells provide physical and biochemical support for
composed of a cell type histologically resembling germ cell development and facilitate the migration of
the Sertoli cell. The lack of pathology at other sites maturing germ cells from the basal toward the luminal
suggests tissue-restricted expression of the trans- compartments of the seminiferous epithelium. Addition-
gene. A cell line derived from one of the testicular ally, Sertoli cells resorb residual cytoplasmic compo-
tumors has been established that continues to ex- nents of spermatozoa (4) and aid in their release from
press markers associated with Sertoli cells, such as the seminiferous epithelium.
transferrin, sulfated glycoprotein-2, and inhibin-gB. Tight junctions between adjacent Sertoli cells com-
The cell line does not express detectable levels of prise the blood-testis barrier and result in the isolation
inhibin-cr, MIS, or FSH receptor. However, the cells of advanced stage germ cells from direct influences of
have retained forskolin responsiveness. As adult the circulatory system. Specifically, spermatogonia and
Sertoli cells cannot be propagated in vitro, the avail- early spermatocytes occupy basal positions relative to
ability of an immortal cell line displaying features the barrier, whereas maturing spermatocytes and sper-
characteristic of normal Sertoli cells should aid in matids occupy luminal positions. Thus, development of
subsequent analyses of the biology of this cell type. this latter population of cells occurs in a milieu in part
(Molecular Endocrinology 6: 1403-1411, 1992) created and regulated by the Sertoli cell. A conse-
quence of the blood-%& barrier is that various meta-
bolic requirements for meiosis and spermiogenesis
INTRODUCTION must be provided by the Sertoli cell. For example, iron
delivery to developing germ cells residing in adluminal
and luminal compartments of the seminiferous epithe-
Male germ cells differentiate in a somatic environment lium is proposed to be mediated by transferrin synthe-
composed primarily of Sertoli cells. Sertoli cells are sized within Sertoli cells (5). Both Sertoli and germ cells
express surface transferrin receptor (6), allowing for the
0888.8809/92/1403-1411$03.00/O
Molecular Endocrinology
uptake of liver-derived serum transferrin and Sertoli-
Copyright 0 1992 by The Endocrine Sowsty derived transferrin by these respective cell populations.

1403

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MOL ENDO. 1992 Vol6 No. 9
1404

Sertoli cells mediate the hormonal control of sper- because this gene has been shown to transform a wide
matogenesis (3). The initiation of spermatogenesis in variety of cell types in transgenic mice when fused to
prepuberal animals is dependent upon pituitary-derived other promoters/enhancers (17, 18). A temperature-
FSH. This glycoprotein hormone acts on FSH receptors sensitive variant (ts1609) of T antigen was chosen
present on the Sertoli cell surface, leading to CAMP- because this might ultimately allow one to regulate T
dependent protein kinase activation and a subsequent antigen function in cell culture by growing the cells at
stimulation in the synthesis of a variety of Sertoli cell- either permissive or nonpermissive temperatures (19,
derived polypeptides. The role of FSH in the adult testis 20). The hMIS-SV40 construct was injected into fertil-
is less clear, as spermatogenesis can be maintained in ized mouse eggs, and seven transgenic mice were
hypophysectomized animals in the presence of high identified by dot hybridization of tail DNA with an SV40
levels of testosterone (7). The release of FSH from the probe.
pituitary is in turn subject to feedback regulation by two Two of the five transgene positive males, 1815-1
hormones secreted by Sertoli cells (8). Inhibin, a heter- and 1815-2, displayed grossly enlarged scrotal areas
odimeric protein composed of inhibin-cu and -p chains, and were killed at 28 and 13 weeks of age, respectively,
represses the release of FSH from pituitary gonado- for further analysis. The remaining three males were
trophs, whereas activin, a hormone composed only of maintained for several months without displaying any
inhibin-P chains, stimulates its release (9-l 1). obvious pathology. It is presumed that these males did
While the role of the Sertoli cell in the initiation and not express the transgene and thus were not subject
maintenance of spermatogenesis has been appreciated to further investigation. The Sertoli cell line described
for some time, recent experiments examining the dis- in this report was derived from 1815-2. Animal 1815-
tribution of XX and XY cells present in testes of XX<- 1 sired a litter carrying transgene-positive pups before
>XY chimeric mice suggest a critical role of the Sertoli displaying overt external abnormalities. One of his male
cell in testicular differentiation (12). It is proposed that offspring, 1815-l-1, was killed at 19 weeks of age
the testis-determining locus of the Y chromosome spe- after displaying obvious testicular pathology. Due to a
cifically induces Sertoli cell differentiation. Subsequent reduction in fertility of transgenic mice carrying active
formation of the testis and other components of the copies of the hMIS-SV40 construct, these transgenic
male reproductive tract, as well as regression of struc- lines could not be maintained.
tures associated with the female reproductive tract, are Gross pathological analysis of the three afflicted
secondary events dependent upon Sertoli cell differen- hMIS-SV40 transgenic males revealed the presence of
tiation. A polypeptide product of fetal Sertoli cells, Mul- large, bilateral testicular tumors that were heavily vas-
lerian inhibiting substance (MIS), induces the regression cularized. The testicular tumors in 1815-l were ovoid
of the Mullerian duct in vitro (13) which in females with approximate dimensions of 1.8 x 2.5 cm and had
normally gives rise to the uterus, vagina, and oviduct. a combined weight of 6.4 g. A similar phenotype was
The expression of MIS is restricted to the testis of the observed in 1815-2, whereas the testicular tumors
male and to the adult ovary of the female (14-l 6). The present within 1815-l -1 were approximately 50%
functions of MIS expression in the testis after testicular smaller. Within each animal, both testicular tumors were
differentiation and in the adult ovary are not known. symmetrical in size and external appearance and were
To develop a system in which the Sertoli cell could completely encased within the tunica albuginea. It is
be genetically manipulated in vivo, we used sequences not known at what stage during testicular development
flanking the transcription start site of the human MIS the hMIS-SV40 transgene first exerts its effects. Gross
gene to target the expression of an oncogene to Sertoli examination of other organs within each male did not
cells in transgenic mice. Male mice carrying this trans- reveal any additional pathologies.
gene specifically develop testicular tumors composed Biopsies from each of the tumors were fixed and
of a cell type histologically resembling the Sertoli cell. subject to histological analysis. Cross-sections through
From one of these tumors, we have established a cell the testicular tumor present in 1815-2 revealed a mas-
line that expresses many markers characteristic of Ser- sive presence of nongerminal cells within the seminif-
toli cells. As adult Sertoli cells comprise only a few erous tubules (Fig. 1, A and B). The proliferating popu-
percent of the cells within the testis and are postmitotic, lation of cells within the tubules was composed predom-
the availability of such a cell line should facilitate many inantly of anaplastic spindle-shaped cells. Cells
investigations into Sertoli cell function. displaying a more differentiated phenotype, with abun-
dant eosinophilic cytoplasm characteristic of normal
Sertoli cells, are observed in close proximation to the
RESULTS basal lamina in some seminiferous tubule cross-sec-
tions (Fig. 1D). Although the tubular architecture was
Human MIS-SV40 Transgenic Males Develop grossly altered, it is apparent that the afflicted cells
Testicular Tumors arose from within the seminiferous tubule and were
often contained within the boundary of the basal lamina.
We chose to fuse the 5’-flanking region of the human In several sections, masses of tumor cells were dis-
MIS (hMIS) gene to the transforming region from the lodged from the inner surface of the tubule (Fig. IB).
SV40 virus, encoding both small and large T-antigens, While this could in part represent an artifact of fixation,

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Sertoli Cell Transformation 1405

Fig. 1. Testicular and Epididymal Pathology Associated with hMIS-SV40 Expression

Biopsies were taken from founder animal 1815-2 at 13 weeks of age for histological evaluation. Paraffin sections were stained
with hematoxylin and eosin. A, Cross-section through the seminiferous tubule reveals the presence of a proliferating mass of
spindle-shaped anaplastic tumor cells; bar, approximately 3.7 pm. B, Cross-section shows a population of tumor cells dislodged
from the basal lamina of the seminiferous tubule. L, Lumen of the seminiferous tubule; T, tumor cells; B, basal lamina; bar,
approximately 18.3 fim. C, Cross-section through the epididymis reveals the presence of tumor cells. T, Tumor cells: L, lumen of
the epididymis; bar, approximately 18.3 Wm. D, Cross-section through a region of the seminiferous tubule containing a population
of germ cells and epithelioid tumor cells. T, Epithelioid tumor cell; S, spermatocyte; bar, approximately 7.3 pm.

the observation that cells of similar morphology to those detect tumors originating at any other sites within these
of the testicular tumor were also present within the animals. It is unlikely that the restricted tumorigenicity
epididymides (Fig. 1C) suggests that these cells are is due to the more permissive temperature of the testes
capable of being released from the seminiferous tubule relative to core body temperature, because this same
and subsequently exit the testis. Biopsies of testicular ts1609 T antigen has been tested in transgenic mice
tumors from 1815-l and 1815-l -1 revealed essentially with its own promoter or with promoters derived from
the same phenotypes. The tumors are classified as the phenylethanolamine N-methyl transferase or P,
malignant gonadal stromal tumors composed of both genes and tumors developed in the choroid plexus,
anaplastic spindle-shaped Settoli cells as well as more adrenal medulla, and Schwann cells, respectively. The
differentiated, epithelioid Sertoli cells (Kunz, L., personal
developmental time course of choroid plexus tumor
communication).
The ability of founder animal 1815-l to sire a litter of development was indistinguishable from that observed
transgene-positive pups, and the occasional appear- with wild type T antigen (Messing, A., personal com-
ance of relatively immature germ cells in tumor cross- munication). Hence, the lack of extratesticular tumori-
sections of animal 1815-2 (Fig. 1D), indicate that func- genesis in hMISSV40 males strongly suggests that
tional germ cell maturation is possible in hMIS-SV40 functional T antigen expression is restricted to the
transgenic testes. Clearly, however, spermatogenesis testis. To confirm this, total RNA isolated from various
is not maintained in these animals. Mouse protamine 1 1815-1-1 tissues was subjected to Northern blot
(Prml) is encoded by an extremely abundant transcript analysis using a radiolabeled T antigen probe. As shown
found exclusively in round and elongated spermatids in Fig. 2, the predicted 2.5-kilobase (kb) T antigen
(21). The absence of any detectable Prml message in transcript was detected only in testicular tumor tissue.
the 1815-1-1 primary tumor (see Fig. 4D) therefore Surprisingly, a faintly hybridizing transcript migrating at
suggests a lack of germ cells that have progressed to approximately 4 kb was present in nontumor tissues.
the round spermatid stage of development. Thus, it is This transcript was considerably more abundant in tu-
proposed that hMIS-SV40 expression and the associ-
mor tissue. Several larger transcripts were also ob-
ated Sertoli cell transformation results in a cessation of
served in testicular tumor tissue. The 4-kb band does
spermatogenesis some time after the age at which
males become reproductively competent. The mainte- not represent cross-hybridization to 28s ribosomal
nance of future hMIS-SV40 transgenic lines may de- RNA as it is not present in nontransgenic tissues (see
pend upon breeding males soon after the onset of Fig. 4G, lanes 3 and 4). Additionally, it is unlikely to
sexual maturation. represent read-through transcription originating from
host sequences flanking the site of transgene integra-
Restricted Expression of the hMIS-SV40 Transgene tion, as a similar profile of hybridization was observed
in tumor cells derived from an independent founder
Although the three hMIS-SV40 transgenic males ana- male (see Fig. 4G, lane 2). We favor the possibility that
lyzed displayed marked testicular pathology, we did not the aberrant transcripts are encoded within the trans-

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MOL ENDO. Vol6 No. 9
1406

9.6
6.6

4.3

2.3

Fig. 3. Morphological Phenotype of MSC-1 Cells in Culture

A, MSC-1 cells that had been continuously maintained in
culture for 2 months were fixed and stained with hematoxylin.
Fig. 2. Restricted Expression of the hMIS-SV40 Transgene All cells within the culture immunostained positively for SV40
Total RNA was extracted from various tissues of 1815-l - T-antigen (data not shown). E, Large epithelioid-shaped tumor
cells; S, small spindle-shaped tumor cells; bar, approximately
1 at 19 weeks of age and subject to Northern blot analysis
24 fim. B, MSC-1 cells maintained in culture for 10 days after
using an SV40 large T antigen probe. Each lane contains 10
pg total RNA. For comparison, 10 rg total RNA extracted from placement of the primary tumor into culture were labeled for
24 h with [3H]thymidine, fixed, and stained for SV40 T-antigen.
the 18152-derived MSC-1 cell line is included. The sizes, in
The same culture was subsequently coated with emulsion,
kilobases, of various HindlllX mol wt markers are shown on
the left. exposed for 1 week, and photographed. The color reagent
used to detect T-antigen immune complexes remains visible
after emulsion treatment. A, T antigen-positive nucleus; D, T
antigen-positive nucleus displaying intensely dark grains indic-
gene but do not encode functional T antigen protein. In ative of [3H]thymidine incorporation; bar, approximately 7.3
accord with this hypothesis, the 4-kb transcript present pm.
in all tissues, as well as the larger transcripts present
in tumor tissue, hybridized to hMlS 5’-flanking se-
quences lying upstream of the hMlS TATA homology within the primary tumor is not known. T antigen mes-
(data not shown). sage was readily detected in total RNA isolated from
the cultured cells (Fig. 4G, lane 2) and all cells displayed
Derivation of a Cell Line from Testicular Tumor positive nuclear immunostaining for T antigen (Fig. 38).
Tissue Additionally, both populations of cells incorporated [3H]
thymidine (Fig. 3B and data not shown). We have not
Immortal cell lines displaying differentiated phenotypes attempted to clone the large and small cells, although
can be derived from T antigen-induced tumors in trans- their relative abundance has remained unchanged
genie mice (20, 22, 23). Normal adult Sertoli cells do throughout in vitro propagation. We have not taken
not divide in vivo or when placed into cell culture. It was advantage of the temperature sensitivity of the hMIS-
therefore of interest to determine if hMIS-SV40 Sertoli SV40 transgene to determine the extent to which im-
cells could be propagated in vitro and continue to mortalization of these cells is dependent upon functional
express markers characteristic of normal Sertoli cells. T antigen expression, as has been demonstrated in a
Testicular tumor tissue excised from 1815-2 at 13 similar system (20). However, we have propagated the
weeks of age was mechanically and enzymatically dis- cells at 37 C for 1-2 weeks without any obvious
aggregated to generate a cell suspension that was changes in morphology or growth rate. We refer to the
plated out on standard tissue culture plates in Dulbec- 1815-2 cultured testicular tumor cell line as MSC-1.
co’s modified Eagle’s medium (DMEM) supplemented
with 10% heat-inactivated fetal calf serum and main- Expression of Settoli Cell Markers by MSC-1 Cells
tained at 32 C to approximate normal testicular tem-
perature and to assure full activity of the temperature- To determine if the cultured cells continue to express
sensitive T antigen gene. After 9 days, the cultures markers characteristic of normal Sertoli cells, we ex-
contained a morphologically heterogeneous population amined total RNA by Northern blot analysis using a
of cells consisting of large, epithelioid-shaped cells dis- variety of cDNA probes encoding products known to
playing extensive cytoskeletal processes as well as be expressed by Sertoli cells. The MSC-1 cells used
smaller spindle-shaped cells (Fig. 3A). The relationship for these experiments had been continuously main-
between the morphologically distinct populations of tained in culture for several months.
cells within the MSC-1 cultures, and those present Transferrin and sulfated glycoprotein-2 (SGP-2) are

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Sertoli Cell Transformation 1407

secretory products synthesized by a variety of cell types testis, whereas this transcript is relatively rare in the
but whose expression within the rat testis is restricted adult testis (14-16). As the MSC-1 line continues to
to Sertoli ceils (1, 24, 25). As shown in Fig. 4A, the express T antigen, presumably under the control of
MSC-1 cells expressed very high levels of transferrin hMlS transcriptional regulatory sequences, we antici-
mRNA relative to those found in the adult testis of a pated that the cell line would also express endogenous
nontransgenic control mouse. The level of transferrin MIS. Although MIS message was observed in the pri-
mRNA present in the primary tumor is lower than that mary tumor and in control adult testis, it was undetect-
observed in the MSC-1 line. However, this tumor is able in the MSC-1 line (Fig. 4C).
derived from animal 1815-l -1, whereas the cell line is Inhibin-cy, -@A, and -pB subunits are expressed by
derived from 1815-2. Additionally, the tumor represents Sertoli cells and play a role in the regulation of FSH
a more heterogeneous population of cells relative to secretion by pituitary gonadotrophs (8, 26). Extrago-
the MSC-1 line; many of the cell types present within nadal expression of these subunits is also observed in
the tumor may not express transferrin. Both the tumor the mouse (27). As shown in Fig. 4E, both the primary
and the MSC-1 line expressed SGPP mRNA (Fig. 4B). tumor and the MSC-1 cells express inhibin-olB message
However, in this case, expression of SGP-2 in the MSC- at levels comparable to those observed in control adult
1 line was diminished relative to that observed in the testis. In contrast, inhibin-cumessage is detected in the
primary tumor and control testis. primary tumor but not in the MSC-1 line (Fig. 4F). These
High levels of MIS message are detected in the fetal data suggest that the primary tumor is capable of
producing both inhibin (composed of LY-and P-subunits)
and activin (composed only of P-subunits), whereas the
MSC-1 line might only be capable of producing activin.
Expression of the Rlla regulatory subunit of CAMP-
dependent protein kinase is induced in Sertoli cells after
exposure to FSH (28). Although the FSH receptor
(FSHR) is expressed by fetal and adult Sertoli cells in
vitro and in vivo (29) the MSC-1 cell line does not
express any detectable FSHR message (Griswold, M.,
personal communication). Thus, FSHR engagement
was simulated by forskolin-mediated elevation of intra-
cellular CAMP levels. As shown in Fig. 5A, the level of
-
RII, message is induced by forskolin in MSC-1 cells.
2.0 -il Densitometric scans consistently showed at least a 7-
fold induction at 16 h.

C / ' I" DISCUSSION

2.3 - 4.3

Previous research on the molecular biology of Sertoli

2.3 cell function has relied upon descriptions of specific
gene activity in Sertoli cells (l), chimeric mice in which
testicular cell types are composed of different geno-
types (12, 30), and the in vitro manipulation of either
primary Sertoli cells or established, putative Sertoli cell
lines (1, 31). In this report, we demonstrate the feasi-
bility of targeting heterologous gene expression to Ser-
toli cells in vivo using the hMlS promoter. Male mice
expressing an oncogene under the transcriptional con-
trol of hMlS transcriptional regulatory sequences spe-
Fig. 4. Expression of Various Markers by the Cultured MSC- cifically develop testicular tumors composed of Sertoli
1 Cell Line cells. These cells can be propagated in vitro and con-
Total RNA was extracted from MS&l cells that had been tinue to express markers associated with Sertoli cells.
continuously maintained in culture for several months and The MIS gene is expressed in both fetal and adult
subject to Northern blot analysis using the following probes: Sertoli cells and in postnatal granulosa cells of females.
A, transferrin; B, sulfated glycoprotein-2; C, MIS; D, protamine-
Sertoli and granulosa cells share a variety of biochemi-
1; E, inhibin-@B; F, inhibin-a; G, SV40 T antigen; and H, actin.
Hybridization to the actin probe verifies the integrity of RNA
cal properties and have been proposed to arise from a
samples in each lane. Each lane contains 10 fig total RNA. In common precursor (32). Although tissue-specific
each panel, the RNA samples were loaded as follows: lane 1, expression of MIS mRNA in the adult mouse has not
1815-l -1 tumor; lane 2, MSC-1 cell line; lane 3, control adult been documented, the bovine gene is expressed exclu-
mouse testis; and lane 4, control adult mouse liver. sively in the testis and in the ovary (14,15). Additionally,

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MOL ENDO. 1992
1408
FORSKOLIN TREATMENT (HOURS)
0 4 6 IO
A that purified Sertoli cells derived from newborn calf
B -28s
Fig. 5. Induction of Rlla Expression by
Cells
Total RNA was extracted from MSC-1
cultured in the presence of 10 FM forskolin
times and subject to Northern blot analysis
probe. Each lane contains 10 pg total RNA
staining of ribosomal RNA indicates that
intact RNA were loaded in each lane (B).
in situ hybridization analysis of MIS expression during
embryonic development in male mice reveals a Sertoli
cell-specific pattern of expression (16). Male mice car-
rying an hMIS-SV40 transgene specifically develop Ser-
toli cell tumors. In addition, several transgenic females
developed ovarian tumors composed of granulosa cells
(Behringer, R. R., J. J. Peschon, R. L. Brinster, and R.
D. Palmiter, unpublished data). The observation that
Sertoli and granulosa cell lineages are specifically af-
fected in hMIS-SV40 transgenic mice suggests that the
transcriptional elements dictating this specificity are
contained within 2 kb 5’ of the start of hMlS message
start site. However, attempts at targeting the expres-
sion of a /acZ reporter gene to Sertoli cells using these
hMlS sequences, or 7.5 kb 5’-sequence flanking the
transcription initiation site of the mouse MIS gene, have
thus far been unsuccessful (Behringer, R. R., R. D.
Palmiter, and R. L. Brinster, unpublished data). The
phenotypic consequences of T antigen expression may
represent a more sensitive method of detecting MIS
transgene activity than /acZ expression. It is therefore
possible that while MIS Y-flanking sequences function
properly in a qualitative sense, additional information is
required for efficient expression. Elevated MIS trans-
gene expression may require additional sequence infor-
mation and might also be dependent upon a specific
locus-control region, analogous to that described for
the P-globin locus (33).
Endogenous MIS expression is relatively high in the
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Vol6 No. 9
fetal testis and declines significantly after birth (14-l 6).
16 24 Expression of the MIS gene in either primary fetal Sertoli
cultures or established adult Sertoli cell lines has not
been evaluated, although it has been demonstrated
testes rapidly lose the capacity to produce MIS in
culture (34,35). As the MSC-1 cell line expresses SV40
-28s T-antigen message, presumably under the control of
the MIS promoter, it is surprising that endogenous MIS
message is not detected in these cells. Although it is
-18s possible that the endogenous MIS locus is subject to
gene inactivation during Sertoli cell maturation, it is
difficult to reconcile this with the observation that the
adult testis as well as the 1815-I-l primary tumor
continue to express MIS message. Perhaps the expres-
sion of MIS observed in the normal adult testis or in the
primary tumor is contributed by a cell type other than
the Sertoli cell. It is also plausible that sustained expres-
sion of MIS by adult Sertoli cells requires the presence
of germ cells. Alternatively, there may exist heteroge-
-18s
neity within populations of Sertoli cells with respect to
Forskolin in MSC-1
MIS expression that is not mimicked by the hMlS
transgene. Interestingly, a similar phenomenon was
cells that had been observed in a retinal cell line isolated from a SV40 T-
for the indicated antigen-induced tumor in transgenic mice (23).
using a murine RllB For an in vitro Sertoli cell line to be of use in the
(A). Methylene blue analysis of Sertoli cell function, it must display features
equal amounts of characteristic of normal Sertoli cells in vivo. The dem-
onstration that the MSC-1 line expresses both trans-
ferrin and SGP-2, products known to be specifically
expressed within the testis by Sertoli cells (24, 25)
indicates that this line maintains part of the program of
gene expression associated with Sertoli cells. Addition-
ally, the induction of Rlla expression by forskolin in
MSC-1 cells indicates that although the cells do not
express FSH receptor, they are capable of appropri-
ately responding to elevated CAMP levels. Thus, the
signaling components normally associated with FSHR
are present and function within MSC-1 cells.
The profile of inhibin subunit expression by MSC-1
cells suggests that the cells are only capable of produc-
ing activin. FSH induces inhibin expression by Sertoli
cells (26) whereas activin promotes FSH release by
pituitary gonadotrophs. Hence, the inability to produce
inhibin may reflect an inability of these cells to respond
to FSH. However, MSC-1 cells treated with forskolin
for up to 24 h still fail to express inhibin-cu message
(Hat-wood, K. A., R. L. Idzerda, and J. J. Peschon,
unpublished data). As it is possible that forskolin treat-
ment does not fully mimic FSHR activation, it will be
interesting to determine if additional characteristics of
FSH responsiveness can be imparted upon MSC-1 cells
by transfection of a functional FSHR gene, and to what
extent the transfectants will respond to FSH by altera-
tions in inhibin subunit expression.
Many aspects of Sertoli cell function are difficult to
address due to the inability of Sertoli cells to replicate
and survive in long-term culture systems. The ability to
immortalize Sertoli cells in vivo using oncogenes under
the transcriptional control of genes specifically ex-
Sertoli Cell Transformation 1409

pressed in Sertoli cells represents a potentially powerful Cell Culture and lmmunostaining
approach to generating established Sertoli cell lines. It
may also be possible to establish such lines from trans- The cell line described in this report was derived from testicular
tumor tissue excised from hMISSV40 founder animal 1815
genie mice expressing oncogenes linked to pan-acting 2 at 13 weeks of age. The tumor was minced with iridectomy
transcriptional control elements, provided that these scissors, rinsed in DMEM, and incubated for 20 min at 37 C
elements function in Sertoli cells (20). An immortal FSH- with agitation in the presence of 300 U/ml collagenase (stock
responsive Sertoli cell line (TM-4) has been previously 4176; Cooper Biomedical, Malvern, PA) and 50 ig/ml DNasel
(D-5025: Siama. St. Louis. MO). The susoension was further
isolated from prepuberal murine testicular cultures (31). dispersed u’sing’ a Pasteur pipette. Large’ cellular aggregates
However, only limited data are available concerning the were subjected to an additional 20 min incubation as described
ability of this cell line to express other markers associ- above. The resulting cell suspension was plated onto standard
ated with Sertoli cell function. Additionally, Sertoli cell tissue culture plates in DMEM supplemented with 10% heat-
lines isolated from prepuberal testicular sources may inactivated fetal calf serum and incubated at 32-33 C in a
humidified atmosphere of 95% sir/5% CO*. Media was re-
differ profoundly from those isolated from sexually ma- placed every 3 days. Cells were passaged at dilutions not
ture animals. greater than l/5 by incubating for 1 O-l 5 min at 32 C in dispase
A homogeneous population of Sertoli cells cultured (40235: Collaborative Research. Bedford. MA). Cultures to be
as a monolayer on nontreated plastic clearly does not photographed were fixed on the plates and stained with he-
matoxylin.
accurately reflect normal Sertoli cell architecture in vivo. Cell replication was assessed by [3H]thymidine incorpora-
Sertoli cells are normally columnar and are maintained tion and autoradiography as described (39). Cultures were
in close contact with neighboring Sertoli cells as well immunostained for SV40 large T antigen using a rabbit anti-
as maturing germ cells. It has been previously demon- large T antigen antibody as described (39). Antigen-antibody
complexes were visualized using reagents and suggested
strated that the expression of Sertoli cell markers by
protocols provided with Vectastain ABC (Vector Laboratories,
primary cultures can be influenced by Sertoli cell mor- Burlingame, CA) and Zymed (San Francisco, CA) SABC kits.
phology (36) as well as by the presence of germ cells
(37). Although these parameters may be irrelevant for RNA Extraction and Analysis
certain aspects of Sertoli cell function and analysis, they
may result in an increased phenotypic resemblance RNA was extracted from tissues and cultured cells by homog-
between Sertoli cells in viva and immortal Sertoli cells enization in guanidinium isothiocyanate, followed by precipi-
tation in lithium chloride as described (40). RNA samples were
in vitro. Under the appropriate culture conditions, im-
denatured and electrophoresed in 1% agarose gels containing
mature rat Sertoli cells can adopt a columnar morphol- 2.2 M formaldehyde and transferred to nitrocellulose (41). Blots
ogy, organize into testicular cords, and support a limited were hybridized at 42 C in 50% formamide, 10% dextran
amount of germ cell development (36). The ability to sulfate. -, salmon soerm DNA. 50 mM NaPi. 3x SSC
100 us/ml
duplicate these findings using immortal cells would (1XSSC=0.15~NaCI,0.~15~Nacitrate,pH7.0)and0.1%
sodium dodecyl sulfate (SDS) to radioactive DNA probes pre-
greatly facilitate the identification of Sertoli cell-derived pared by random-primed labeling (42). Blots were washed at
structural and diffusible factors responsible for germ 68 C in 2x SSC, 0.1% NaPi, 0.1% SDS, and at 50 C in 0.5%
cell proliferation and maturation. SDS, 0.1% NaPi, 0.5 mM EDTA, 5 mM Tris-Cl, pH 7.5. For the
experiment presented in Fig. 5, total RNA was isolated by hot
phenol extraction, electrophoresed as described above, and
transferred to Hybond-N (Amersham, Arlington Heights, IL).
MATERIALS AND METHODS The blot was stained in 0.4% methylene blue/0.5% sodium
acetate, pH 5.0, and subsequently hybridized at 63 C in 50%
Plasmid Construction and Generation of Transgenic Mice formamide, 5x SSC, 50 mM NaPi, 0.5% SDS, 5X Denhardt’s,
and 100 pg/ml salmon sperm DNA to a radioactive riboprobe
hMlS sequences 5’ of the hMlS transcription start site were made from an RII, cDNA fragment. The blot was washed in
fused to a Stul-BarnHI restriction fragment encoding the 0.1 x SSC, 0.2% SDS at 68 C.
tsl609 temperature-sensitive derivative of SV40 large T anti-
gen (19). The fusion was made at the Aflll site at position -27
Acknowledgments
nucleotide (nt) with respect to the hMlS site of transcription
initiation (14) and extended an additional 2 kb 5’ of this site.
The Aflll site encompasses the putative hMlS TATA homology; We thank our colleagues for critical review of the manuscript:
digestion with Aflll, treatment with the Klenow fragment of Glenda Froelick for tissue sectioning and staining; Drs. Carol
Escherichia co/i DNA polymerase, and subsequent ligation to Quaife and Larry Kunz for histological analysis of testicular
the Stul site of SV40 results in a single base alteration in this tumors; Cyndy Gartside for performing the [3H]thymidine in-
homology. corporation assay; Diane Allen, Mary Avarbock, Lynda Munar.
The resulting construct, hMIS-SV40, was excised from vec- and Felicity Oram for technical assistance; Drs. M. Griswold,
tor sequences and microinjected into the male pronucleus of S. McKnight, S. Bhasin, and C. Rubin for kindly providing us
fertilized hybrid eggs as described (38). Transgenic mice were with the SGP-2, transferrin, inhibin subunit, and RII, probes,
identified by tail DNA dot analysis using an SV40 probe. All respectively; and Drs. M. Griswold and P. Donahoe for many
animal experimentation was conducted in accord with stand- helpful suggestions during the course of this work.
ards outlined in the NIH Guide for the Care and Use of
Laboratory Animals.
Received March 17,1992. Revision received May 19,1992.
Histopathology Accepted June 30, 1992.
Address requests for reprints to: Dr. Jacques Peschon,
Testicular tumor segments and epididymides were collected, lmmunex Corporation, 51 University Street, Seattle, Washing-
fixed in Carnoy’s reagent, and embedded in paraffin for routine ton 98101.
staining with hematoxylin and eosin. This work was supported in part by NIH Grants HD-09172

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MOL ENDO. 1992 Vol6 No. 9
1410

(to R.D.P.), HD-23657 (to R.L.B.), HD-12629 (to R.L.I.) and 19. Tevethia MJ, Ripper LW 1977 Biology of Simian Virus 40
Training Grant GM-07750 (to K.A.H.). (SV40) transplantation antigen (TrAg). Virology 81 :192-
* Current address: University of Texas, M.D. Anderson Can- 211
cer Center, Department of Molecular Genetics, 1515 Hol- 20. Jat PS, Noble MD, Ataliotis P, Tanaka Y, Yannoutsos N,
combe Boulevard, Houston, Texas 77030. Larsen L, Kioussis D 1991 Direct derivation of condition-
ally immortal cell lines from an H-2Kb-tsA58 transgenic
mouse. Proc Natl Acad Sci USA 88:5096-5100
21. Kleene KC, Distel RJ, Hecht NB 1985 Nucleotide se-
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Sertoli Cell Transformation 1411

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