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Laboratory for Experimental Medicine and Endocrinology, Onderwijs en Navorsing, Gasthuisberg, Catholic University of Leuven, B-3000 Leuven, Belgium
Address all correspondence and requests for reprints to: Prof. Dr. G. Verhoeven, Laboratory for Experimental Medicine and Endocrinology, Onderwijs en Navorsing, Gasthuisberg, B-3000 Leuven, Belgium. E-mail: guido.verhoeven{at}med.kuleuven.ac.be
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(Her-) and Her-ß stimulate transferrin and
androgen-binding protein production by cultured rat Sertoli
cells up to 2.5-fold. These effects are more pronounced than those of
previously identified growth factors active in this assay, such as
insulin-like growth factor I and basic fibroblast growth factor.
Combination with these factors results in additive effects and in
marked morphological changes in the Sertoli cell cultures, including
formation of tubule-like structures. Stimulation of androgen-binding
protein secretion is paralleled by increased levels of the
corresponding messenger RNA. This parallelism was less consistent for
transferrin. Semiquantitative RT-PCR indicates that the expression of
NDF- and NDF-ß is more pronounced in peritubular cells than in
Leydig or Sertoli cells. Conversely, the main receptors for
heregulins/NDFs, HER-3 and HER-4, are predominantly expressed in
Sertoli cells. A displacement assay confirms the presence of
high-affinity binding sites for [125I]Her-ß on intact
Sertoli cells and reveals parallel displacement curves for Her-ß,
Her-, and concentrated peritubular cell-conditioned medium (PTCM;
estimated ED50 values: 1 ng/ml, 50 ng/ml, and 130 µg
protein/ml, respectively), indicating that peritubular cells secrete
one or more factors able to compete for heregulin receptors. It is
concluded that heregulins/NDFs may play a role in
mesenchymal-epithelial interactions in the testis. Estimates of the
concentrations of heregulins in PTCM, however, make it unlikely that
they contribute significantly to the effects observed with low
concentrations of PTCM and ascribed to the putative mediator PModS
(peritubular factor that modulates Sertoli cell function). Further
investigations will be required to define the exact role of heregulins
in the testis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One of the most intriguing paracrine agonists affecting Sertoli cell function is PModS (peritubular factor that modulates Sertoli cell function) (8, 9, 10, 11). This factor is supposed to be responsible for the marked effects of peritubular cell-conditioned medium (PTCM) on Sertoli cells. Moreover, peritubular myoid cells are target cells for androgens, and some in vitro data suggest that the production of PModS is increased by androgens (8, 11). This has led to the hypothesis that at least some of the effects of androgens on Sertoli cells are mediated indirectly by changes in PModS production.
Two factors with PModS activity have been purified from PTCM (9), but their identity and their exact physiological role remain elusive. Moreover, recent data suggest that factors with PModS-like activity and physicochemical characteristics may be produced by a variety of myoid and nonmyoid mesenchymal cells and cell lines (12, 13, 14, 15). Furthermore, effects observed with concentrated conditioned media often exceed those observed with PModS-enriched preparations, and it cannot be excluded that the effects ascribed to PModS may actually be the result of a combination of factors.
Searching for known growth factors that might contribute to PModS effects, our attention was drawn to the heregulins [also named neu differentiation factors (NDFs) and neuregulins]. In fact, these factors share a number of characteristics with PModS, including their high molecular size (44 kDa), their affinity for heparin, and their involvement in mesenchymal-epithelial interactions (16, 17, 18, 19, 20). In the present paper, we demonstrate that heregulins/NDFs are produced by peritubular cells and that they are able to affect Sertoli cell morphology and function.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and -ß [epidermal growth factor (EGF) domain]
were obtained from R&D Systems (Minneapolis, MN).
Preparation of Sertoli cells, peritubular cells, and Leydig
cells
Sertoli cells and peritubular cells were prepared essentially as
described previously (11, 13). Routinely, 40 testes, derived from
19-day-old Wistar rats, were decapsulated and digested for 1 h at
32 C with trypsin (2.5 mg/ml) and deoxyribonuclease (DNase; 10 µg/ml)
in a shaking water bath (140 oscillations/min). Tryptic action was
stopped by the addition of 0.5% (wt/vol) soybean trypsin inhibitor.
The tubular fragments were allowed to sediment, the supernatant was
eliminated, and the sedimented fragments were washed three times with
PBS-A [Ca2+- and Mg2+-free PBS (Dulbecco’s
formula), 1.5 mg/ml D-glucose, 100 µg/ml streptomycin
sulfate, and 100 U/ml penicillin G]. A peritubular cell-enriched
fraction was obtained by digesting the tubular fragments with PBS-A
supplemented with collagenase (1 mg/ml), hyaluronidase (1 mg/ml), and
DNase (10 µg/ml) for 20 min at 32 C. The supernatant of this
incubation (peritubular fraction) was passed through a nylon screen
(Nytal, 75 µm; Swiss Silk Bolting Co., Zurich,
Switzerland).
To obtain highly purified Sertoli cells, remaining tubular fragments
were digested once more with hyaluronidase (1 mg/ml) and DNase (10
µg/ml) for approximately 60 min (120 oscillations/min). The
efficiency of this procedure in removing remaining peritubular cells
was monitored continuously by phase-contrast microscopy, and incubation
times were adapted to obtain optimal results. The final Sertoli cell
aggregates were dispersed, and the cells were plated and maintained in
24-well culture plates (5 x 105 cells/well) in
serum-free medium A (RPMI-1640 supplemented with 22.5 mM
HEPES, 4.3 mM L-glutamine, 100 µg/ml
streptomycin sulfate, and 100 U/ml penicillin G), as described
previously (13). Immunocytochemical staining for -smooth muscle
isoactin (21) revealed less than 0.5% contamination with peritubular
cells.
The peritubular cell fraction, obtained as described above, was
resuspended in medium B (medium A supplemented with 10% FCS), and
cells were divided over two 175-cm2 culture flasks
(Nunc). After 5 h, the cells were washed twice with 10 ml
medium B to remove contaminating Sertoli cells. Fresh medium B was
added, and incubation was continued for 3 days at 32 C in a humidified
incubator gassed with 5% CO2. After this period, the
monolayer was briefly trypsinized (0.05% trypsin and 0.02% EDTA), and
the cells were replated at half-density. This procedure was repeated
twice, and 9-day-old peritubular cells were used in the experiments to
be described. All these cells stained positive for -smooth muscle
isoactin.
The Leydig cell fraction was obtained from interstitial cell preparations after collagenase dispersion of decapsulated testes from 19-day-old Wistar rats and fractionation on discontinuous Percoll density gradients, as described previously (22). The cells were maintained in serum-free medium A.
Collection of PTCM and Sertoli cell-conditioned medium
(SCCM)
Precultured peritubular cells, prepared as described above, were
washed three times with serum-free medium A. Thereafter, incubation was
continued in the same medium. Twenty-four hours later, the cultures
were washed once more, and fresh serum-free medium was added. From then
on, conditioned media were collected every 2 or 3 days for 10–14 days.
Conditioned media were centrifuged at 3000 x g for 10
min, benzamidine (0.1 mM) and phenylmethylsulfonylfluoride
(25 µM) were added, and media were stored at -20 C.
Sertoli cells were prepared as described above. On day 4, the cells were washed three times with serum-free medium A. The cells were incubated for another 72 h in the same medium; and on day 7, conditioned media were collected and stored at -20 C.
Measurement of PModS-like activity in conditioned media
The ability of conditioned media to stimulate transferrin
secretion was evaluated by exposing freshly prepared Sertoli cells, for
7 days, to conditioned media. Culture media were replaced on day 4, and
incubation was ended on day 7. Routinely, transferrin was measured in
media collected on day 7. In some experiments, ABP was measured in the
same media.
RNA extraction and Northern blot analysis
Sertoli cells were seeded in five 55-cm2 dishes
(15 x 106 cells/dish) and pretreated as specified in
the text. Total RNA was extracted using the method of Chirgwin et
al. (23). Northern blot analysis was performed on RNA samples (20
µg/lane), separated on a 1% (wt/vol) agarose gel containing
formaldehyde, as described by Sambrook et al. (24). The RNA
was transferred to a Biotrans (+) nylon membrane and hybridized
successively with a probe for transferrin, ABP, and 18S RNA, as
described previously (25).
Reverse transcriptase-PCR amplification
Total RNA was extracted from 20-day-old rat testes and purified
testicular cells (Leydig cells, peritubular myoid cells, and
Sertoli cells) using the method of Chirgwin et al. (23). One
microgram of target RNA was reverse transcribed with Superscript II
reverse transcriptase (Life Technologies,
Merelbeke, Belgium) at 42 C for 50 min in the presence of random
primers. Semiquantitative PCR was performed for NDF-, NDF-ß,
HER-2, HER-3, HER-4, ABP, and ß-actin. The primers used for the PCR
reactions and the cycling parameters are summarized in Table 1
. The PCR products were separated by
agarose gel electrophoresis and visualized by ethidium bromide
staining. The authenticity of all amplified bands was confirmed by
ligation in pGEM-T (Promega Corp., Madison, WI) and
by sequencing using Autoread sequencing kits (Pharmacia,
Uppsala, Sweden) and an A.L.F. automated sequencer
(Pharmacia).
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Heregulin binding assay
Freshly prepared Sertoli cells were seeded in 24-well dishes
(5 x 105 cells/well) in medium A. The medium was
changed on day 4, and Her-ß receptor binding was studied in the
Sertoli cells on day 7. For the binding assay, cells were washed twice
with PBS and preincubated in assay-buffer (medium A with 0.1% BSA) for
2 h at 32 C. The plates were cooled on ice, and the cultures were
incubated in assay buffer with both [125I]Her-ß
(105 cpm) and different concentrations of unlabeled
Her-ß. Similar displacement studies were performed with Her-,
PTCM, SCCM, and BSA. For the displacement experiments with BSA, the BSA
(0.1%) was omitted from the assay-buffer. With Her-ß, maximal
displacement was observed at about 20 ng/ml. Binding equilibrium was
reached already after 4 h, and displaceable binding remained
essentially constant during the first 24 h. Thereafter, binding
diminished mainly as a consequence of Sertoli cell detachment.
Routinely, measurements were performed after 20 h of incubation at
0 C. The cultures were washed four times with ice-cold PBS containing
0.1% (wt/vol) BSA to remove unbound [125I]Her-ß.
Finally, cells were lysed in 1 ml 0.1 M NaOH, and
radioactivity was determined in a LKB Wallac (Turku,
Finland) 1277 gamma counter.
Analytical methods
Transferrin was measured by an RIA using
125I-labeled rat transferrin and a rabbit antiserum against
rat transferrin developed in our laboratory. Antibody-bound and free
transferrin were separated using donkey antirabbit Ig-coated cellulose
(Sac-Cel, IDS, Boldon, UK). Details of the method have been described
previously (13). The assay displays no cross-reaction with bovine
transferrin.
ABP was measured using a filter disc assay developed in our laboratory
(26). Briefly, extensively dialyzed conditioned media were equilibrated
with [3H]5-dihydrotestosterone in the absence or
presence of a 100-fold excess unlabeled steroid. Binding was measured
by adsorption of the 5-dihydrotestosterone-ABP complex to
diethylaminoethyl-cellulose filter paper, at pH 8.5, using a 1225
manifold from Millipore Corp. (Bedford, MA).
Radioactivity retained on the filters was measured in a liquid
scintillation counter.
Cell DNA was measured, at the end of the experiment, using the method of Labarca and Paigen (27).
Statistical analysis
All the data shown were confirmed in at least two independent
experiments. Statistical analysis was performed using one-way ANOVA
supplemented by Tukey’s studentized range test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Her-ß using exactly
the same experimental conditions as those selected for the PModS
bioassay (12, 13, 14, 15). Transferrin secretion during the last 3 days of the
7-day incubation period was used as a parameter of activity. IGF-I and
bFGF, two growth factors previously shown to be active in this bioassay
(14), were included as a positive control (Fig. 1).
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and Her-ß stimulated transferrin secretion more
than 2-fold, an effect which exceeded that of IGF-I and bFGF. The
heregulins were active from a concentration of 1 ng/ml and above; and
at low concentrations, Her-ß tended to be more active than Her-.
Repetition of the same dose-response studies in PTCM (at a
concentration resulting in less than 3-fold stimulation of transferrin
secretion) demonstrated additive effects between the active factors in
the conditioned medium and the studied growth factors but no
cooperativity (data not shown). This was confirmed in an independent
experiment (Fig. 2) in which a maximally
effective concentration of Her-ß was added to increasing
concentrations of PTCM. A very potent pool of PTCM was used, resulting
in a 10-fold stimulation of transferrin secretion at a protein
concentration of 200 µg/ml. Additive effects of Her-ß were observed
at all concentrations of PTCM studied; although at high concentrations
(from 100 µg/ml on), differences were no longer statistically
significant.
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(upper panel)
shows that Her- stimulated not only transferrin but also ABP
secretion. Moreover, the effect of Her- (a 2.5-fold stimulation of
transferrin and ABP secretion) was markedly enhanced by combination
with IGF-I and bFGF (a 5-fold stimulation of transferrin and a 3.5-fold
stimulation of ABP production). Northern blotting experiments (Fig. 3, middle and lower panel) demonstrated an increase
in ABP messenger RNA (mRNA) expression comparable with that observed
for the corresponding protein. Such a parallelism was not observed for
transferrin. In fact, a clear increase in transferrin mRNA was evident
only in cells stimulated with the combination of growth factors,
suggesting that at least part of the effect on transferrin secretion
may be at the translational level. Comparable results were obtained
when the same experiments were performed with Her-ß (results not
shown).
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and Her-ß also provoked
morphological changes in Sertoli cell cultures (Fig. 4). Heregulins alone induced focal
clustering of cells and appearance of cell-free regions in the
monolayers. Again, Her-ß tended to be more active than Her-. IGF-I
and bFGF induced no morphological changes (not shown); but when
combined with one or both of the heregulins, ball- or tubule-like
structures were formed, comparable with those observed with
concentrated preparations of PTCM (15).
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and NDF-ß
(the rat analogs of the heregulins) in the testis and in testicular
somatic cells. The number of cycles was adapted to permit
semiquantitative comparison of the degree of expression in different
RNA extracts. ABP, a well-known marker of Sertoli cells, was included
to test the validity of this approach. The quality of the complementary
DNA (cDNA) derived from the different RNA preparations was evaluated by
PCR for ß-actin (Fig. 5).
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and NDF-ß
mRNA expression was most pronounced in peritubular cells, but lower
levels were also observed in Leydig cells and in complete testicular
tissue. No differences in the expression of the NDFs could be
demonstrated in peritubular cells treated for 9 days with testosterone
(1 µM) and in control cells (data not shown).
Expression of heregulin/NDF receptors by rat somatic testicular
cells
Using the same experimental setup as outlined in the previous
paragraph, we also compared the expression of the receptor molecules
HER-2, HER-3, and HER-4 in complete testicular tissue and in the
mentioned somatic cell types (Fig. 6).
HER-2 displayed comparable levels of expression in all RNA preparations
investigated. HER-3 was found mainly in unfractionated testis tissue
and in Sertoli cells with minor expression in peritubular and Leydig
cells. HER-4 was predominantly expressed in Sertoli cells.
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, Her-ß, or proteins derived from concentrated PTCM. As a
control, competition was also performed with proteins from SCCM and
with BSA (Fig. 7).
[125I]Her-ß bound efficiently to Sertoli cells, and
displacement was observed with Her-, Her-ß, and
PTCM-derived proteins. The displacement curves displayed
parallelism, and the apparent ED50 values were 1 ng/ml for
Her-ß, 50 ng/ml for Her-, and 130 µg/ml for PTCM. Only minor
displacement was observed with proteins derived from SCCM, whereas BSA
tended to enhance rather than to decrease binding.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The finding that heregulins/NDFs are produced in the rat testis
corroborates earlier findings on the expression of these growth factors
in the genital ridge and the gonadal primordium of the mouse (19) and
in the human testis (17). The observation that heregulins/NDFs are
mainly expressed in peritubular myoid cells and, to a lesser extent, in
Leydig cells is also in accordance with in situ
hybridization studies showing interstitial expression in the testes of
neonatal mice (19). It is interesting to note that, at least at the
mRNA level, peritubular cells express both NDF- (generally related
to mesenchymal-epithelial interactions) and NDF-ß (usually related to
neuronal development) (18).
Under conditions routinely employed in bioassays for PModS (7-day
exposure), both Her- and Her-ß (recombinant human EGF-domains)
stimulate transferrin secretion 2- to 3-fold. Such a degree of
stimulation is comparable with that observed with androgens, FSH, or
IL-1ß and is higher than that observed with IGF-I, bFGF, and IL-6
tested under the same conditions (28, 11, 29, 30). EGF (as well as a
whole series of other growth factors and cytokines) is inactive in this
bioassay (14). Moreover, the heregulins stimulate not only transferrin
but also ABP secretion; and their effects are additive with those of
IGF-I and bFGF, resulting in an up-to-5-fold stimulation of the
secretion of these proteins. For ABP, the effects at the protein level
are paralleled by changes in the level of the corresponding mRNA. For
transferrrin, this parallelism is not so obvious, suggesting that at
least part of the effects may be located at the translational level.
This would be in accordance with recent studies showing that the
increase in transferrin biosynthesis by rat Sertoli cells during
testicular development is also mainly caused by an increase in the
translation rate of transferrin mRNA (31). Interestingly, the effects
of heregulins/NDFs on Sertoli cell secretory function are accompanied
by morphological changes in the Sertoli cell monolayers. These changes
culminate in cord formation when the heregulins are combined with IGF-I
and bFGF. Similar morphological effects have been observed with other
agonists acting on Sertoli cells, including PTCM (15), cytokines (28),
activin (32), and laminin (7). These morphological effects are striking
and consistent, but their significance may differ for the various
agonists mentioned. Laminin and PTCM may act by changing the ability of
the cells to deposit a basal lamina; and this, in turn, may favor
tubule formation (7). For the other agonists mentioned, the effects
described in vitro are mainly observed at high
concentrations and/or when various factors are combined. The underlying
mechanisms and the potential physiological significance require further
investigation.
Although the HER-2 receptor was originally used to identify heregulin family members, it is now known that only the HER-3 and HER-4 receptors truly bind heregulins (33). HER-3 lacks an intrinsic tyrosine kinase domain; but heterodimerization, with other receptors of the HER family (such as HER-2), results in cooperative effects on ligand binding (34) and on receptor phosphorylation (33, 35). HER-4 has a classic intrinsic tyrosine kinase domain; but, as is the case with HER-3, it is also proposed to form heterodimers with other HER members, and HER-4 phosphorylation may induce phosphorylation of HER-2 (33, 36).
The data summarized in Fig. 6
suggest that HER-2 is expressed to a
comparable degree in Leydig cells, peritubular cells, and Sertoli
cells. Both HER-4 and HER-3 are preferentially expressed in Sertoli
cells, although some expression of HER-3 is also noted in peritubular
and Leydig cells. The presence of functional receptors on Sertoli cells
is confirmed not only by the above described biochemical and
morphological effects but also by the demonstration of high-affinity
binding on the membranes of intact Sertoli cells. Estimated
ED50 values for displacement of bound
[125I]Her-ß were 1 ng/ml (0.1 nM) for
Her-ß and 50 ng/ml (7 nM) for Her-. These values are
in accordance with ED50 values reported for the recombinant
molecules by the providing company using a breast tumor cell line
bioassay (0.5–2 ng/ml and 20–40 ng/ml). A slightly lower (10-fold)
difference for the apparent affinities of NDF-ß and NDF- for
receptors on T47D mammary carcinoma cells has also been reported
previously (37). Concentrated PTCM competes for
[125I]Her-ß binding, suggesting that it contains
heregulins/NDFs. Much less competition is observed with SCCM. Taking
into account the molecular mass of native heregulin (44 kDa) and the
ED50 value observed for displacement of
[125I]Her-ß by PTCM (130 µg protein/ml), one may
estimate that, at this protein concentration, the concentrated PTCM
contains the equivalent of 308 ng/ml Her- or 4.5 ng/ml Her-ß. It
should be stressed that this can be only a crude estimate because the
actual ligand(s) in PTCM are unknown and because the affinity of native
ligands may differ considerably from that of the recombinant
EGF-domains used in the displacement assay (37, 38).
The above described data strongly support the contention that heregulins/NDFs play a role in mesenchymal-epithelial interactions in the testis. Further experiments will be required, however, to delineate their exact physiological function. In fact, nearly all the present experiments have been performed on cultured cells derived from 19-day-old testes. The heregulins and their receptors, however, represent an extremely versatile and dynamic signaling system with changing patterns of expression and function during cell fate determination and morphogenic processes (39). Accordingly, it is not unlikely that the activity and the role of the involved ligands and receptors may be affected by cell culture conditions and gonadal stage of development.
One of the important questions to be addressed concerns the potential
implication of heregulins/NDFs in the effects ascribed to PModS. From
the data available, it is evident that heregulins mimic some of the
most striking biochemical effects of PModS, notably the stimulation of
ABP and transferrin secretion. Moreover, they also induce the
morphological changes observed with concentrated preparations of PTCM
and with partially purified fractions of PModS. Furthermore, heregulins
share with PModS their affinity for heparin and their high molecular
size (16, 17). It should be noted, however, that even at maximally
effective concentrations, heregulins provoke only a 2- to 3-fold
stimulation of transferrin secretion, whereas a 5- to 10-fold
stimulation is observed with concentrated PTCM and, according to
Skinner et al., also with purified PModS (9). Moreover, the
displacement data shown in Fig. 7 suggest that the concentration of
heregulins/NDFs in PTCM is rather low, a finding that is corroborated
by the observation that heregulins display additive effects, even at
high concentrations of PTCM (Fig. 2). Finally, whereas production of
PModS is assumed to be regulated by androgens, we have failed to
demonstrate effects of androgens on heregulin expression. The latter
finding should be interpreted with caution, however. In fact, RT-PCR is
a poor technique to demonstrate effects that may be quite small.
Moreover, effects of androgens on PModS production in cultured
peritubular cells may strongly depend on culture conditions (8); and
although we consistently observed androgen stimulation in earlier
experiments (11, 12, 13), we have been unable to confirm such effects in
more recent studies for reasons that, despite intensive research,
remain elusive. It may be concluded that heregulins may mimick several
of the morphological and biochemical effects of PModS. The low
concentrations of heregulins in PTCM and the mentioned studies on the
additivity of the effects of PTCM and heregulins, however, make it
unlikely that heregulins contribute significantly to the effects
usually ascribed to PModS.
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Acknowledgments |
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Footnotes |
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2 A Senior Research Assistant of the Fund for Scientific
Research-Flanders (Belgium).
Received September 17, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-smooth muscle
isoactin is a specific differentiation marker. Biol Reprod 42:351–365[Abstract]
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