BIOLOGY OF REPRODUCTION 54, 980-986 (1996)
Interphase Chromosome Arrangement in Sertoli Cells of Adult Mice'
M. Guttenbach, 3 M.J. Martinez-Exp6sito, 3 W. Engel, 4 and M. Schmid 2 '3
InstitutfiirHumangenetik,3 Universitat Wiirzburg, Biozentrum, Am Hubland, D-97074 Warzburg, Germany
Institutfir Humangenetik,4 Universitdt Go5ttingen, 37073 Gittingen, Germany
ABSTRACT
Sertoli cells of adult male laboratory mice were examined with a number of banding techniques and by nonradioactive in situ hybridization applying different repetitive DNA probes. All banding methods revealed the typical features of mouse Sertoli cells, i.e., a central
nucleolus, usually with two chromocenters associated at diametrically opposed sides in which the centromeric regions of the chromosomes are clustered. Silver staining as well as in situ hybridization with rDNA labeled part of the chromocenters and the nucleolus,
indicating transcriptional activity of at least some of the nucleolus organizer regions. In situ hybridization with X- and Y-specific DNA
probes showed both sex chromosomes to be undercondensed in Sertoli cells. This decondensation suggests expression of sex chromosomal genes in Sertoli cells. While the X chromosome appeared to occupy a central position near one of the chromocenters, the Y
chromosome was found at the periphery of the nucleus inthe majority of cells. Hybridization with telomeric sequences resulted in strong
labeling of the chromocenters and dispersed signals at the nuclear periphery.
INTRODUCTION
tant proteins involved in spermatogenesis [19]. Sertoli cells
are mitotically inactive and exhibit a very characteristic morphology in the mouse [17] that allows an incontestable detection even in cell suspensions. Thus, Sertoli cells seem to
offer an ideal model for the analysis of interphase chromosome arrangement.
The goal of the present investigation was to examine the
organization of the postmitotic mouse Sertoli cell nucleus
in detail by application of different banding techniques as
well as by in situ hybridization with various specific DNA
probes.
Since the end of the last century when Rabl and Boveri
[1, 2] pointed out that the interphase chromosomes of certain cell types maintain their anaphase-telophase orientation, that they keep relatively fixed positions in the nucleus,
and that they remain as discrete entities throughout interphase, the interest of numerous studies has been drawn to
interphase chromosome topology. Initial approaches deduced information on interphase arrangement of certain
chromosomes by the analysis of metaphase plates (for review see [3, 4]). Later, the availability of differential banding
techniques allowed the identification of individual chromosomes/chromosome regions not only in metaphase
spreads, but also directly in interphase nuclei [5, 6]. Currently, in situ hybridization with specific DNA probes (e.g.,
[7-10]), and application of antibodies against specific nuclear or chromosomal components (e.g., [11-13]), are widely
used to study the organization of interphase nuclei from different cell types and organisms. The analyses have shown
that individual chromosomes occupy discrete domains
within the interphase nucleus [14, 15] that are at least to some
degree maintained in the course of the cell cycle [16].
Although a variety of tissues have been subjected to interphase analyses, only a few studies have been performed
on the various testis cells (e.g., [17, 18]). In addition to germ
cells, somatic cells (Sertoli cells, Leydig cells) are also found
in the mammalian testis. Sertoli cells constitute the supporting cell lineage in the mammalian testis. They have
been shown to be the site of biosynthesis of several impor-
MATERIALS AND METHODS
Preparationof Seminiferous Tubule Cells
Adult male laboratory mice (BALB/c) were used for the
present study. Immediately after the animals were killed,
the testes were dissected out. The tunica albuginea was removed, and the seminiferous tubules were squashed out in
0.075 M KCI and incubated for 40 min at 370 C. The cell
suspensions were centrifuged (1400 rpm, 8 min'), and the
pellet was fixed in ice-cold methanol:glacial acetic acid (3:1)
overnight. After three rinses with fresh fixative, the cells
were dropped onto cleaned glass slides and air dried.
Banding Techniques
C-banding and silver staining were performed according
to the techniques of Sumner [20] and Howell and Black [21].
Hoechst 33258 staining was carried out by the method of
Hilwig and Gropp [22].
HybridizationProbes
The oligonucleotide probe (GGGTTA), and its complementary sequence (TAACCC), demonstrate the presence of
telomeric repeats in the chromosomal DNA of all verte-
Accepted November 28, 1995.
Received October 31, 1995.
'This study was supported by the Bundesministerium fr Forschung und Technologie
(grant 01 KY 9104).
2
Correspondence. FAX: 49 931 8884069.
980
CHROMOSOME ARRANGEMENT IN MOUSE SERTOLI CELLS
brates [23, 24]. These probes were end-labeled separately
with biotin-16-dUTP by means of terminal deoxynucleotidyltransferase as recommended by the supplier.
The hybrid plasmid pXlr 101A, containing one complete
rRNA gene (12 kb) from Xenopus laevis [25], binds to the
evolutionarily conserved coding sequences of the 18S + 28S
rDNA cistrons of eukaryotes.
The repetitive Y-specific probe pY353/B [26] hybridizes
to sequences distributed along the entire length of the long
arm of the mouse Y chromosome.
The repeated sequence 70-38 maps close to the centromere of the mouse X chromosome, to the locus DXWas70
[271.
A satellite DNA probe, mapping to all centromeric
regions of the mouse karyotype, was obtained by microdissection of a small marker chromosome consisting of centromeric repeats only from metaphases of a TM4 Sertoli cell
line (our unpublished data). An inverted microscope (Carl
Zeiss, Thornwood, NY) equipped with a micromanipulator
(Eppendorf, Hamburg, Germany) and phase-contrast optics
was used to isolate marker chromosomes [281. A 1-1l collecting drop (0.5 mg/ml proteinase K in 10 mM Tris-HCl
[pH 8.0], 10 mM NaCl, and 0.1% SDS) was deposited on a
siliconized slide overlaid with water-saturated liquid paraffin. Fifteen marker chromosomes were isolated and transferred to the collecting drop, which was used directly for
polymerase chain reaction (PCR). The components of the
PCR were 0.7 lpM degenerate primer (5'-CCG ACT CGA
GNN NNN NAT GTG G-3') [29], 0.2 mM dNTPs, 2.5 mM
MgC12, single-strength Taq DNA polymerase buffer, and 2.5
U Taq DNA polymerase (Promega, Madison, WI). PCR was
performed as described by Pich et al. [30].
All DNA probes were labeled with biotin-16-dUTP by
nick translation according to the method of Lichter and Cremer [31].
In Situ Hybridization
The testis preparations were denatured in 70% formamide/double-strength SSC (standard saline citrate; singlestrength SSC is 0.15 M NaCl, 0.015 M sodium citrate) at 70°C
for 1-2 min and then dehydrated by being passed through
a chilled alcohol series. Hybridization and detection of telomeric repeats were performed as described previously [32].
For the other probes, 20 tl of hybridization mixture containing 50% formamide, double-strength SSC, 5% dextran
sulfate, and 10-20 ng labeled probe was denatured for 5
min at 75C, chilled on ice, applied to each slide, and hybridized overnight at 37 0C. After three washes in 50% formamide/double-strength SSC and three washes in doublestrength SSC at room temperature, signal detection was
performed with either streptavidin-horseradish-peroxidase
and diaminobenzidine (DAB) or with fluorescein isothiocyanate (FITC)-labeled avidin. Counterstaining of the fluo-
981
rescence-detected preparations with 4,6-diamidino-2-phenylindole (DAPI) allowed the unequivocal identification of
Sertoli cells.
Scoring of Sertoli Cells
For each of the different banding techniques or hybridizations, 50 Sertoli cells were analyzed and the arrangement
of chromocenters and specific hybridization signals was
documented.
RESULTS
Differential Staining of Sertoli Cells
C-banding, Hoechst 33258, and DAPI specifically stained
the centromeric heterochromatin of all acrocentric mouse
chromosomes except the Y chromosome (Fig. 1, b, f, and
g). Because of their characteristic morphology, the Sertoli
cells could be unequivocally identified with these banding
techniques in the preparations of seminiferous tubule cells:
Sertoli cells exhibit a large central nucleolus that is flanked
for the most part by two perinucleolar satellites of highly
condensed heterochromatin. These so-called chromocenters showed dark staining after C-banding (Fig. 2, d-f) and
fluoresced brightly after staining with Hoechst 33258 (Fig.
2, g-i) and DAPI (Figs. 3, a-c, 4, a-d, and 5, a-d).
Although Giemsa stained all mouse chromosomes uniformly (Fig. la), the Giemsa-stained Sertoli cells displayed
the same characteristics as after C-banding; i.e., the chromocenters are the most darkly stained structures in the nucleus
(Fig. 2, a-c). In some of these preparations the nucleolus
appeared to be equally or even less stained than the rest of
the nuclear chromatin (Fig. 2, a, b, d-f), while in others the
nucleolus exhibited a more intense labeling (Fig. 2c). Treatment of the preparations with silver nitrate resulted in a
strong labeling of the nucleolus as well as in silver precipitations in the perinucleolar satellites (Fig. 2, k-m).
The testis preparations were scored for 50 Sertoli cells
with each banding technique. All Sertoli cells analyzed exhibited from one to three distinct round chromocenters usually associated with the nucleolus. Neither irregularly
shaped chromocenters nor nuclei with more than three
perinucleolar satellites were detected. In 13.6% of the Sertoli
cells there was only one large chromocenter; 84.2% of the
cells contained two chromocenters, usually located at diametrically opposed sides of the nucleolus; and 2.2% were
found to have three chromocenters (Fig. 6). With few exceptions (Fig. 5a), the chromocenters appeared in those nuclei to be arranged in the form of a triangle; nuclei in which
one, two (Fig. 2c), or all three chromocenters (Fig. 4b) were
directly attached to the nucleolus were found in equal numbers. In Sertoli cells with two or three chromocenters, the
size of these could vary considerably (e.g., Figs. 2i, 4c, 5a).
982
GUTTENBACH ET AL.
FIG. 1. Selected individual mouse chromosomes displaying characteristic features
after differential chromosome staining (a-c, f, g) and insitu hybridization with different probes d,e, h-k). a)Giemsa staining, b) C-banding, c)silver staining, f) DAPI
staining, g) Hoechst 33258 staining. Insitu hybridization with d) rDNA, e) X-specific
DNA probe, h) telomeric sequences, i) mouse satellite DNA, and k)Y-specific DNA
probe.
FIG. 3. Insitu hybridization of mouse Sertoli cells with telomeric probe (a'-c') and
counterstaining of the same cells with DAPI (a-c). The clustering of telomeric signals
in the chromocenters is indicated by arrows (a'-c'). Bar represents 10 gm.
In Situ Hybridization with Different DNA Probes
Specificity of hybridization was controlled by analysis of
metaphase chromosomes present in the preparations of
seminiferous tubule cells (Fig. 1, d, e, h-k). Counterstaining
of the fluorescence-labeled preparations with DAPI allowed
the reliable identification of Sertoli cells.
Telomeric Sequences
With the oligonucleotide probe used, the telomeric
regions of all mouse chromosomes were distinctly labeled
(Fig. Ih). In the Sertoli cells the telomeric sequences of the
short arms appeared to be clustered in the chromocenters.
Each of the perinucleolar bodies usually contained one
brightly fluorescing spot. In some cases, however, two or
three discrete telomeric signals could be discerned in one
chromocenter (Fig. 3, a'-c'). Besides the strong signals in
the heterochromatin bodies, small fluorescing dots were
scattered throughout the entire surface of the nuclei, probably reflecting the telomeric regions of the long arms of the
mouse chromosomes. In preparations detected with streptavidin-horseradish-peroxidase and DAB, these signals
were even more prominent.
Satellite DNA
FIG. 2. Differentially stained mouse Sertoli cells. a-c) Giemsa staining, d-f) C-banding, g-i) Hoechst fluorescence, k-m) silver staining. Chromocenters are indicated by
arrows (k-m). Bar represents 10 pm.
Hybridization with mouse satellite DNA resulted in a
strong labeling of the centromeric heterochromatin of all
mouse chromosomes (Fig. i) except the Y. In this way Sertoli cells showed a hybridization pattern identical to the pattern obtained with DAPI staining (Fig. 4a, a', b, b'): the
perinucleolar heterochromatin bodies were brightly labeled
CHROMOSOME ARRANGEMENT IN MOUSE SERTOLI CELLS
983
FIG. 4. Mouse Sertoli cells hybridized with mouse satellite DNA (a', b') and rDNA Ic', d') and counterstaining of the same cells with DAPI (a-d). Hybridization signals in
chromocenters (a', b') and nucleoli (c', d') are indicated by arrows. Bar represents 10 pm.
FIG. 5. In situ hybridization of mouse Sertoli cells with Y-specific (a', b') and X-specific (c', d') DNA probes. The cells were
counterstained with DAPI (a-d). The highly decondensed signals of the Y- (a', b') and the X-chromosome (c', d') are indicated by
arrows. Bar represents 10 lm.
with FITC in their entirety. Distinguishing individual centromeres was not possible.
18S+28S rDNA
In metaphase spreads present in the testis preparations,
six chromosomes showed hybridization to probe pXlrlOlA.
The signals were found in the pericentromeric regions of
these chromosomes (Fig. Id) and colocalized with the sites
detected by silver staining. In some Sertoli nuclei the chromocenters exhibited strong FITC fluorescence (Fig. 4c'),
while in others no labeling of the perinucleolar bodies was
obvious (Fig. 4d'). In all Sertoli cells, however, the nucleolar
region was covered by dispersed and elongated hybridization signals (Fig. 4, c', d'), indicating decondensation of the
984
GUTTENBACH ET AL.
FIG. 6. Incidence of the different centromere patterns inmouse Sertoli cells.
nucleolus organizer regions (NOR) of at least some NORbearing chromosomes.
Y Chromosome
The binding sites of the probe pY353/B were distributed
along the entire long arm of the mouse Y chromosome (Fig.
lk). As in our previous study [33], the Y chromosome was
found to be decondensed in all Sertoli cells examined, as
indicated by a very diffuse, granular signal (Fig. 5a'). In
some cells the FITC labeling pattern had a necklace-like
appearance (Fig. 5b'). The hybridization signals, representing the Y chromosome domains, were found to be localized
at the periphery of the nucleus in the majority of cells.
X Chromosome
Probe 70 -38 specifically detected locus DXWas70 in the
mice used in the present study. Autosomal sequences did
not hybridize with the probe. DXWas70 maps close to the
centromere of the mouse X chromosome (Fig. le). In all
Sertoli cells the hybridization signals of the X chromosome
were in close proximity to one of the chromocenters (Fig.
5, c' and d'). A diffuse, often necklace-like signal was observed, pointing to an undercondensation of at least the X
chromosomal locus DXWas70.
DISCUSSION
A number of differential banding techniques, as well as
in situ hybridization with DNA probes specific for several
chromosome regions, have been applied in the present
study to reveal in detail the chromosome arrangement in
Sertoli cells of adult laboratory mice. In cell suspensions of
seminiferous tubule cells, the nuclear and chromosomal
characteristics can be used as clues to distinguish the various cell types from one another. Sertoli cells generally show
one large central nucleolus with a clustering of the centromeric heterochromatin of the chromosomes, most frequently
in two chromocenters at its periphery [17, 34].
Giemsa staining and C-banding resulted in identical
banding patterns: the chromocenters were always heavily
stained, while the nucleolus appeared as a faint, almost invisible structure in some preparations and as a distinctly
stained organelle in others. The fluorescent dyes DAPI and
Hoechst 33258 caused brightly fluorescing heterochromatin
bodies, the nucleolus exhibiting again only faint staining.
Neither of the banding methods nor in situ hybridization
with satellite DNA permitted distinction between individual
centromeres within the perinucleolar bodies. The heterochromatic regions of the chromosomes involved appeared
to be completely fused.
The evaluation of 50 Sertoli nuclei with each banding
technique revealed a clustering of the centromeric regions
into one, two, or three perinucleolar chromocenters of variable size per nucleus. While investigating the arrangement
of centromeres in mouse Sertoli cells by C-banding, Hsu et
al. [171 described the existence of up to seven heterochromatin blocks (large and tiny ones) in Sertoli cells. The mean
number of heterochromatin bodies detected per nucleus
was four. Since in the present study not more than three
blocks were observed, the question arises whether individual differences between animals or strains exist or whether
the higher numbers detected in this earlier investigation are
simply due to misidentification of tubule sheath cells.
The Sertoli cells showed intense silver labeling of the
nucleolus as well as silver deposits in the perinucleolar bodies. Silver-positive NOR proteins are specific markers for
transcriptionally active nucleolar sites during interphase;
they thus indicate expression of the rRNA genes in mouse
Sertoli cells. The typical mammalian nucleolus consists of
three main structures: the fibrillar centers, the dense fibrillar
component (DFC), and the granular component. In human
Sertoli cells the DFC has been shown to be the predominant
site of the transcribed rRNA genes [35, 361. It is likely that
rRNA transcription in the mouse also takes part in the I)FC.
Moreover, in cultured human cells the argyrophilic proteins
were found to be organized as a twisted necklace structure
within interphase nuclei [37]. But with the methods applied
in the present study, no fine structures of the silver deposits
could be discerned.
A labeling pattern similar to that obtained with silver
staining was produced by hybridization of the cells with
18S + 28S rDNA. Here the dispersed signals in the nucleolar
region point to an undercondensation of the NORs associated with transcriptional activity. Since the metabolic state
of cells is closely linked with nucleolar transcriptional activity (protein synthesis requires cytoplasmic ribosomes consisting of rRNA), the active NORs underscore the role of
Sertoli cells in the testis as sources of important proteins.
In situ hybridization with the telomeric probes usually
produced one distinct spot in each of the chromocenters
and small, dispersed signals at the periphery of the nuclei.
This pattern, which is seen in all Sertoli cells, recalls again
a chromosome orientation suggested by Rahl [1]. Since the
centromeric regions of the mouse chromosomes are clustered in the chromocenters, the telomeric sequences of the
short arms are probably located in the perinucleolar heterochromatin bodies. It seems likely that the long arms of the
chromosomes extend to the outside of the nucleus with
CHROMOSOME ARRANGEMENT IN MOUSE SERTOLI CELLS
their telomeres attached to the nuclear membrane, thus producing the dispersed labeling over the whole nucleus.
However, the possibility that some chromosomes form
loops, locating their long arm telomeres in the center of the
nucleus as well, can not be excluded.
In a few cells, not only one but up to three individual
telomeric signals were detected in the perinucleolar bodies.
A similar pattern was observed by Haaf et al. [38] employing
antikinetochore antibodies. They found two types of Sertoli
cells: those with type I nuclei in which discrete centromeres/kinetochores were discernible within the chromocenters, and those with type II nuclei in which centromeres
were fused and no individual centromeres were distinguished. Parallel incubation of the cells with anti-RNA polymerase I antibodies revealed a strong nucleolar immunofluorescence (transcription of rRNA genes) only in type II cells.
Thus, the authors concluded that the nucleolar transcriptional activity in mouse Sertoli cells is dependent on centromere arrangement and that centromere fusion is a prerequisite for nucleolus function. Since in the present study
hybridization with the telomeric sequences resulted in small
discrete signals (located close to the centromere, and comparable in size to those obtained by kinetochore labeling),
those nuclei in which the telomeric signals were not totally
fused could possibly be related to the type I nuclei described by Haaf et al. [38].
None of the techniques applied in the present study to
analyze the chromosome arrangement in postmitotic mouse
Sertoli cells permit the identification of specific individual
centromeres in the chromocenters. It would be of interest
to know whether more or less the same chromosomes/centromeres always participate in one chromocenter or whether
the centromere distribution varies from cell to cell. Thus, for
example, differing arrangement of chromosomes could affect
cellular activity. To resolve this problem, however, specific
centromeric probes would be necessary in order to specifically detect single chromosomes.
Organization of the Sex Chromosomes
The probe 70 -38 was used by Disteche and Adler [39]
to localize the X chromosome in interphase nuclei of mouse
bone marrow cells and fibroblasts. In the majority of cells
the signal was found at the outer region of the nuclei. Conversely, in the present investigation the X chromosomal locus DXWas70 was always located in close association with
one of the heterochromatin bodies in mouse Sertoli cells,
indicating involvement of the X chromosome centromere in
the organization of this chromocenter.
In human interphase nuclei a frequent colocalization of
the Y chromosome and the nucleolus has been observed
[401. The same is true for mouse spermatids [33]. However,
in the majority of mouse Sertoli cells the Y chromosomal
signal was detected in the outer region of the nucleus. It
985
appears likely that the lack of satellite DNA in the centromeric region of the Y chromosome prevents fusion of the Y
centromere with the other chromosomes into heterochromatin bodies. In this way the Y chromosome, in contrast to
the other chromosomes, is free to occupy a position rather
distant from any chromocenter.
The hybridization signals of the X and the Y chromosomes were found to be clearly undercondensed. Decondensation of the Y chromosomes seemed to be more pronounced than in the X, but this can be explained by the
different binding modus of the two probes used: while
pY353/B delineates the whole Y chromosome by hybridizing to its entire long arm, the X chromosomal probe binds
to a relatively small region close to the centromere. Thus,
the additional use of a painting probe could provide further
information concerning the condensation status of the X.
Regarding the data obtained by Kofman-Alfaro et al. [41], it
appears likely that the mouse X chromosome is also decondensed in its whole length. Using a painting probe specific
for the human X chromosome, these authors found the X
to be extremely decondensed in Sertoli cells of healthy
males, whereas it was condensed in males with spermatogenic impairment. The same was found to be true for the
human [42] and mouse Y chromosome ([33, 43] and our
unpublished data). If chromosome decondensation is tantamount to transcriptional activity, expression of X and Y
chromosomal genes is to be expected in Sertoli cells. Probe
pY353/B was shown to identify testis-specific transcripts in
the mouse [44]. But since these transcripts are confined to
the round spermatid stage of spermiogenesis [451, expression of other Y chromosomal genes is likely to occur in
mouse Sertoli cells.
To summarize, the present data show that chromosomes
are arranged in a very specific way that does not vary between different Sertoli cells of the mouse. The strong decondensation of X and Y hybridization signals suggests transcription of sex chromosomal genes in Sertoli cells of adult
mice, and silver staining as well as rDNA hybridization indicates transcription of the ribosomal genes. The probably
active transcription stage of the sex chromosomes and the
NOR adds to the importance of Sertoli cells as sites of the
biosynthesis of several important proteins in the testis.
ACKNOWLEDGMENT
We would like to thank Dr. Christine Disteche for kindly providing the X-specific probe
70-38.
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