Regulation of glypican-1, syndecan-1 and syndecan-4 mRNAs
expression by follicle-stimulating hormone, cAMP increase
and calcium influx during rat Sertoli cell development
Sylvie Brucato, Jean Bocquet and Corinne Villers
Laboratoire de Biochimie IRBA, UPRES, Universite
´
de Caen, France
In seminiferous tubules, Sertoli cells provide structural and
nutritional support for the developing germinal cells. Cell-
to-cell signaling and cell adhesion require proteoglycans
expressed at the cell membrane. A preliminary biochemical
and structural approach indicated that cell surface proteo-
glycans are mostly heparan sulfate proteoglycans (HSPG).
Glypican-1, syndecans-1 and -4 were identified using a
molecular approach. Their differential regulation was dem-
onstrated in immature rat Sertoli cells. Follicle-stimulating
hormone (FSH) is the main regulator of Sertoli cell function.
Signal transduction triggered by FSH involves both an
increased intracellular cAMP synthesis and a calcium influx.
This study demonstrates that FSH, through its second
messengers (increase in intracellular cAMP and intracellular
calcium), downregulated the glypican-1 mRNA expression
in Sertoli cells from 20-day-old rats. On the other hand,
syndecan-1 mRNA expression is not modulated by FSH as
it would result from the antagonistic effects of increased
intracellular cAMP and intracellular calcium levels. Finally,
syndecan-4 mRNA expression is not regulated by this
pathway.
The present study was extended during Sertoli cell devel-
opment. Indeed, Sertoli cells undergo extensive changes
during the postnatal period both in structure and function.

both bFGF and TGF-b1 in fibroblasts [11] and by bFGF in
mature oligodendrocytes [12], but the mechanisms that
account for the regulated expression of the glypican are
almost completely unknown. In contrast, syndecan expres-
sion is modulated by growth factors and cytokine [13,14]. In
most cells, levels of syndecan synthesis correlate well with
syndecan mRNA levels, suggesting that the regulation is
mainly transcriptional [8].
Syndecans and glypicans bind a variety of extracellular
matrix molecules and growth factors in a heparan sulfate
dependent manner [15]. Consequently, they take part in the
regulation of various biological events. In addition, synde-
cans associate with the actin cytoskeleton through a
mechanism dependent on their cytoplasmic domains [16].
Syndecan-4, which presents the widest expression pattern, is
localized to the focal adhesions of a range of cells in a
protein kinase C (PKC)-dependent manner and may
function as a coreceptor with integrins [17]. The variable
cytoplasmic region unique to syndecan-4 interacts with the
catalytic domain of PKCa and stimulates its activity [18].
These observations strongly suggest the participation of
syndecans in signal transduction mechanisms while that of
glypicans is still poorly understood.
In the mammalian testis, Sertoli cells are epithelia somatic
cells associated, by a basement membrane, to peritubular
cells surrounding seminiferous tubules. They play a crucial
Correspondence to S. Brucato, Laboratoire de Biochimie IRBA,
UPRES A 2608, Universite
´
de Caen, Esplanade de la Paix,

cells. FSH is the main regulator of Sertoli cells functions.
Although no FSH effect on the whole proteoglycans
synthesis has been described [26], FSH regulation on specific
HSPG mRNAs expression can not be excluded. The
binding of FSH onto specific receptors on Sertoli cells leads
to an intracellular cyclic adenosine monophosphate (cAMP)
increase [27,28]. In addition, the entry of extracellular
calcium is involved in the signal transduction triggered by
FSH in Sertoli cells [29–31]. It has been clearly demonstra-
ted that calcium influx occurs through voltage-independent
and -dependent calcium channels [30,31]. The latter have
beenidentifiedasbothLandNtype[30,32,33].
During testicular development, the physiology of Sertoli
cells is modified. The cell proliferation decreases and ceases
allowing the establishment of the hematotesticular barrier
(BHT) at around day 20 postpartum. Sertoli cells progres-
sively lose FSH responsiveness. In addition, some enzymatic
activities are modulated as the aromatase activity decrease
or the cAMP phosphodiesterase increase upon ontogenesis
[19].
The present work aims to investigate whether FSH,
intracellular cAMP level increase and alteration of trans-
membrane calcium influx induce changes on glypican-1,
syndecans-1 and -4 mRNA expression in developing rat
Sertoli cells.
MATERIALS AND METHODS
Reagents
All reagents were of analytical or molecular biology grade.
Dulbecco’s modified Eagle’s medium (DMEM), Ham’s F12
medium, Trypsin (USP Grade), trizol reagent and DNA

)2
in 75 cm
2
plastic flasks and cultured 48 h in
Ham’s F12/DMEM (1 : 1, v/v) supplemented with 2%
Ultroser SF in order to attach the Sertoli cells in a
humidified atmosphere of 5% CO
2
in air at 32 °C. Culture
medium was renewed after 48 h. Three days after plating,
residual germinal cells were removed by brief hypotonic
treatment using 20 m
M
Tris/HCl (pH 7.4) [35]. The culture
flasks were washed with fresh medium without Ultroser SF.
Monolayer Sertoli cells were used on day 5 after plating.
They were incubated for 24 h either in absence or in
presence of various treatments before RNA extraction.
Extraction of total RNA
Total RNA was extracted from rat Sertoli cells by single
step method of Chomczynski & Sacchi [36] using trizol
reagent.
The integrity and quality of purified RNA were con-
trolled by 1% agarose gel electrophoresis and measure of
the absorbance at 260 and 280 nm.
Semi-quantitative RT-PCR
Denatured total RNA (500 ng, 55–60 °C, 5 min) was added
to a reverse transcription reaction mixture containing the
reaction buffer (50 m
M

M
Tris/HCl, pH 9, 0.1%
Triton X-100), 2.5 UI Taq DNA polymerase, MgCl
2
1.5 m
M
,10pmolofeachprimerina20-lL reaction
volume. The sequences of 5¢ and 3¢ primers were 5¢-AGGT
GCTTTGCCAGATATGACT-3¢ and 5¢-CTCTTTGATG
ACAGAAGTGCCT-3¢ for syndecan-1; 5¢-GAGTCGAT
TCGAGAGACTGA-3¢ and 5¢-AAAAATGTTGCTGCC
CTG-3¢ for syndecan-4; 5¢-GAATGACTCGGAGCGTAC
ACTG-3¢ and 5¢-CCTTTGAGCACATTTCGGCAA-3¢
for glypican-1; 5¢-ACAGACTACCTCATGAAGAT-3¢
and 5¢-AGCCATGCCAAATGTCTCAT-3¢ for b-actin.
The PCR was started at 94 °Cfor1minandfollowedby
up to 27 cycles of amplification for the three proteoglycans
and 20 cycles for the internal control, b-actin as described
3462 S. Brucato et al. (Eur. J. Biochem. 269) Ó FEBS 2002
previously [24] which consisted of a denaturating step
(at 94 °C for 1 min), an annealing step (at 55 °Cfor1min)
andanextensionstep(at72°C for 2 min) then a final
elongation step (at 72 °C for 10 min) in RobocyclerÒ
Gradient 40 (Stratagene).
Optimum RT-PCR conditions were established in order to
further determine possible regulations of the HSPG mRNA
expression (constant input cDNA, determination of optimal
cycle number) [24]. An RT-PCR was performed without
AMV reverse transcriptase in order to check for contamin-
ating genomic DNA (data not shown). In all negative PCR

M
KH
2
PO
4
, DNA was
quantified in a Kontron spectrofluorimeter using Hoescht
33258 as fluorescent probe and calf thymus as standard.
RESULTS
FSH inhibits glypican-1 mRNA expression
in Sertoli cells from 20-day-old rats
Sertoli cells from 20-day-old rats were incubated for 24 h
with increasing concentrations of FSH (10–200 ngÆmL
)1
).
FSH did not modify significantly syndecan-1 and -4
mRNAs expression (Fig. 1). In contrast, glypican-1 mRNA
expression decreased in a dose-dependent manner. The
optimal effect was obtained from 100 ngÆmL
)1
of FSH
corresponding to a 45% decrease in the glypican-1 mRNA
expression (Fig. 1).
Glypican-1 and syndecans-1 and -4 mRNA expression
are regulated by the increase of intracellular cyclic AMP
and calcium level in Sertoli cells from 20-day-old rats
FSH stimulates at least two signaling pathways in the
Sertoli cells. This hormone induces the increase of intracel-
lular cyclic AMP and calcium levels.
Effect of intracellular cAMP level increase

one of syndecan-1 (+50%) but had no significant effect on
syndecan-4 mRNA expression (Fig. 3).
Finally, RO-20 1724, a specific cAMP phosphodiesterases
inhibitor used at 250 l
M
, led to similar results as the ones
described in Figs 2 and 3. The glypican-1 and syndecan-1
mRNAs expression were significantly decreased ()39%) and
increased (+36%), respectively, whereas syndecan-4
mRNA was not significantly affected by the treatment
(Fig. 4).
The optimal doses of chemical compounds used in this
study are doses that induce various effects in Sertoli cell
culture as proteoglycan synthesis [53] or estradiol
production [25].
As a first conclusion, in immature Sertoli cells, the
increase of intracellular cAMP level (a) regulates the
glypican-1 and syndecan-4 mRNA expression as FSH does;
and (b) modulates syndecan-1 mRNA expression in
contrast to FSH effect. Nevertheless, the intracellular
calcium increase induced by the FSH in Sertoli cells
[30,31,40] could explain this difference.
Fig. 1. Dose-dependent effect of FSH on glypican-1, syndecan-1 and
syndecan-4 mRNAs expression. Sertoli cells from 20-day-old rats were
incubated for 24 h in the presence of increasing concentrations
(0–200 ngÆmL
)1
) of FSH. Total RNA was extracted as described in
Material and methods. Then RNA (500 ng) was reverse transcribed
and amplified by relative quantitative RT-PCR as described previously

Thus, the increase of intracellular calcium was appreci-
ated indirectly by incubating Sertoli cell cultures with both
FSH (100 ngÆmL
)1
)andH8(5 l
M
), a specific protein kinase
A inhibitor. Figure 6 indicates that glypican-1 and synde-
can-1 mRNAs expression was downregulated by the
resulting intracellular calcium increase ()26 and )30%,
respectively), whereas syndecan-4 mRNA was not. Thus,
the effect of the increased intracellular calcium level on
HSPG mRNA expression confirmed the results obtained
with verapamil and EGTA.
Our results suggest that: (a) the increase of intracellular
cAMP and intracellular calcium levels contributes similarly
to the FSH-induced inhibition of glypican-1 mRNA
expression, whereas (b) the absence of FSH on syndecan-1
mRNA expression results from an antagonistic effect of
increased intracellular cAMP and intracellular calcium
levels.
FSH, cAMP and intracellular calcium effects
on glypican-1 and syndecans-1 and -4 mRNA expression
during Sertoli cell development
In Sertoli cells from 10-day-old rats, FSH (100 ngÆmL
)1
)
downregulated the glypican-1 mRNA expression ()30%)
whereas it did not modify syndecans mRNA expression
(Table 1). The dbcAMP (1 m

)1
cholera toxin (CT) during 24 h. Total RNA was
extracted as described in Materials and methods. Then, RNA (500 ng)
was reverse transcribed and amplified by relative quantitative RT-PCR
as described previously [24]. Glyp-1, glypican-1; synd-1, syndecan-1;
synd-4, syndecan-4. (A) Agarose gel of one representative experiment.
(B) Densitometry data are representative of seven different experi-
ments (mean ± SE). Each relative HSPG mRNA level under treat-
ment is expressed vs. control which is arbitrarily set to 100%.
3464 S. Brucato et al. (Eur. J. Biochem. 269) Ó FEBS 2002
expression by )33 and )30%, respectively (Table 1),
whereas intracellular calcium increase did not influence
glypican-1, syndecans-1 and -4 mRNAs expression. In
contrast, FSH and dbcAMP upregulated the syndecan-1
and syndecan-4 mRNA expression in 30-days-old rat Sertoli
cells (Tables 2 and 3). Indeed, FSH increased syndecan-1
and syndecan-4 mRNAs expression by +40 and +53%,
respectively, and dbcAMP stimulated them by +35 and
+55%, respectively (Tables 2 and 3).
DISCUSSION
This report shows, for the first time, the FSH regulation of
HSPG mRNA expression in rat Sertoli cells. The effects of
FSH, main effector of Sertoli cell functions, and its second
messengers (increase in intracellular cyclic AMP and
intracellular calcium levels) were evaluated on glypican-1,
syndecan-1 and -4 mRNAs expression.
Our data indicate the existence of a HSPG-specific
regulation depending on the Sertoli cell developmental
stage. FSH induces the inhibition of the glypican-1 mRNA
expression in all studied Sertoli cell developmental stages

sible for regulating the synthesis of these HSPG are complex
and that cAMP effect could be cell type-dependent.
Fig. 5. Effect of verapamil on glypican-1, syndecans-1 and )4 mRNAs
expression. Sertoli cells from 20-day-old rats were incubated in the
presence (+) or in the absence (–) of 100 l
M
verapamil during 24 h.
Total RNA was extracted as described in Materials and methods.
Then, RNA (500 ng) was reverse transcribed and amplified by relative
quantitative RT-PCR as described previously in [24]. Glyp-1,
glypican-1; synd-1, syndecan-1; synd-4, syndecan-4. (A) Agarose gel of
one representative experiment. (B) Densitometry data are representa-
tive of seven different experiments (mean ± SE). Each relative HSPG
mRNA level under treatment is expressed vs. control which is arbi-
trarily set to 100%.
Fig. 4. Action of a cAMP phosphodiesterase inhibitor, RO-20-1724 on
mRNAs expression. Sertoli cells from 20-day-old rats were incubated in
the presence (+) or in the absence (–) of 250 l
M
RO-20-1724 during
24 h. Total RNA was extracted as described in Material and Methods.
Then, RNA (500 ng) was reverse transcribed and amplified by relative
quantitative RT-PCR as described previously in [24]. Glyp-1,
glypican-1; synd-1, syndecan-1; synd-4, syndecan-4. (A) Agarose gel of
one representative experiment. (B) Densitometry data are representa-
tive of three different experiments (mean ± SE). Each relative HSPG
mRNA level under treatment is expressed vs. control which is arbi-
trarily set to 100%.
Ó FEBS 2002 FSH regulation of HSPG expression (Eur. J. Biochem. 269) 3465
The FSH-binding to Sertoli cells activates the cAMP-

same conclusion concerning the calcium effect.
Fig. 6. Action of H8, a specific inhibitor of protein kinase A. Sertoli cells
from 20-day-old rats were incubated without (–) or with (+) FSH
(100 ngÆmL
)1
) or in combination of FSH (100 ngÆmL
)1
)andH8
(5 l
M
) for 24 h. Total RNA was extracted as described in Materials
and methods. Then, RNA (500 ng) was reverse transcribed and
amplified by relative quantitative RT-PCR as described in [24]. Glyp-1,
glypican-1; synd-1, syndecan-1; synd-4, syndecan-4. (A) Agarose gel of
one representative experiment. (B) Densitometry data are representa-
tive of three different experiments (mean ± SE). Each relative HSPG
mRNA level under treatment is expressed vs. control which is arbi-
trarily set to 100%.
Table 1. FSH, cAMP and calcium increase effects on glypican-1 mRNA
expression during Sertoli cells development. Sertoli cells from 10-, 20-
and 30-day old rats were incubated for 24 h with 100 ngÆmL
)1
FSH,
1 mM dbcAMP (intracellular cAMP increase), or 100 ngÆmL
)1
FSH
plus 5 l
M
H8 (increase in intracellular calcium). Each relative HSPG
mRNA level under treatment is expressed versus control which is

mRNA expression relative to control (%)
Sertoli cells
10-days-old
Sertoli cells
20-day-old
Sertoli cells
30-day-old
FSH +10 ± 4 +3 ± 1 +40 ± 3*
dbcAMP )4 ± 3 +50 ± 5* )35 ±5*
Calcium )5±3 )30 ± 2* )6±3
Table 3. FSH, cAMP and calcium increase effects on syndecan-4
mRNA expression during Sertoli cells development. Sertoli cells from 10,
20 and 30-day-old rats were incubated for 24 h with 100 ngÆmL
)1
FSH, 1 m
M
dbcAMP (intracellular cAMP increase), or 100 ngÆmL
)1
FSH plus 5 l
M
H8 (increase in intracellular calcium). Each relative
HSPG mRNA level under treatment is expressed versus control which
is arbitrarily set to 100%. Each percentage is obtained from densi-
tometry data representative of at least three different experiments
(mean ± SE). *, Significant values.
Glypican-1
mRNA expression relative to control (%)
Sertoli cells
10-days-old
Sertoli cells

in intracellular calcium concentrations alone are not suffi-
cient to induce syndecan expression. Further experiments
will be necessary to understand calcium regulation on
glypican-1 and syndecans-1 and -4 in rat Sertoli cell
development.
The physiological significance of the FSH regulation of
glypican-1 and syndecans-1 and -4 mRNA remains to be
elucidated. FSH stimulates the postnatal and pubertal
development of Sertoli cells [61]. This age dependency is
described for all FSH-stimulated intracellular events in
isolated Sertoli cells [62–64]. Thus, increased cAMP but also
inhibition of phosphodiesterase, activation of protein kin-
ase, RNA and protein synthesis or mitotic activity present a
peak of activity around 20 days of age which corresponds to
the tight junctions formation between in vivo Sertoli cells
[63,65].
During Sertoli cell ontogenesis, the lack of FSH
responsiveness could be the consequence of cAMP
inactivity by phosphodiesterase activity increase [19] rather
than a reduced FSH receptors number as these receptors
are increased in the same time. Phamanthu et al. [23]
suggests a possible involvement of cell HSPG in the age-
related increases in Sertoli cell phosphodiesterase activity.
The increase of syndecans-1 and -4 expression induced by
FSH in 30-day-old rat Sertoli cells suggested that these
proteoglycans may be positive regulators of phosphodi-
esterase activity. Indeed, the syndecan-4 cytoplasmic
domain binds and regulates the PKC-a activity [66,67].
Considering these data, syndecans would regulate enzy-
matic activities confined in the plasma membrane. The

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