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Departments of Physiology and Pediatrics, University of Turku, Kiinamyllynkatu 10, Turku 20520, Finland
Address all correspondence and requests for reprints to: Dr. Jorma Toppari, Department of Physiology, University of Turku, Kiinamyllynkatu 10, Turku 20520, Finland. E-mail: jorma.toppari{at}utu.fi
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, tumor necrosis
factor-, and activin] on SCF gene expression by using a
transillumination-assisted microdisection technique, a seminiferous
tubule culture system, and Northern hybridization. Our results showed
that FSH (10 ng/ml) increased steady state levels of SCF messenger RNA
(mRNA) in a stage-specific and time-dependent manner. 8-Bromo-cAMP
could increase the SCF mRNA level in a similar way as FSH, whereas
phorbol 12-myristate 13-acetate had no effect. Actinomycin D could
abolish the stimulatory effect of FSH, whereas cyclohexamide could not.
The half-life of SCF mRNA was apparently prolonged after FSH
stimulation (FSH-treated tubules, 15.6 ± 1.2 h; controls,
8.6 ± 2.7 h). Nuclear run-on assay revealed 5- and 10-fold
increases in the transcription rate after FSH stimulation for 8 and
30 h, respectively. Neither testosterone nor estradiol had
significant effects on SCF gene expression in our tissue culture
system. Activin, TGFß, TGF, and tumor necrosis factor- had no
effect on SCF gene expression in vitro. In conclusion,
SCF gene expression in the rat seminiferous tubule is regulated by FSH
through the cAMP/protein kinase A pathway. FSH regulates SCF gene
expression at both transcriptional and posttranscriptional levels
involving the increase in transcription rate and prolongation of
half-life of SCF mRNA, but is independent of de novo
protein synthesis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previously, it was shown that FSH was able to stimulate SCF expression
in a primary Sertoli cell culture system, and this effect was more
prominent in cells isolated from the 13-day-old than in cells from
18-day-old rat testis (10). However, other experimental models failed
to show links between FSH and SCF (12). To assess the potential
regulation of SCF gene expression during spermatogenesis, we tested the
effects of hormones (FSH, testosterone, and 17ß-estradiol) and some
growth factors [transforming growth factor-ß (TGFß), TGF, tumor
necrosis factor- (TNF), and activin] on SCF gene expression by
using a transillumination-assisted microdisection technique (13), a
seminiferous tubule culture system (14) and Northern hybridization.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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were purchased from Genzyme (Cambridge, MA). Recombinant
human TGF was from Immugenex (Immugenex, Los Angeles, CA).
Recombinant human activin was obtained from the National Hormone and
Pituitary Distribution Program, NIDDK, NIH. Testosterone,
17ß-estradiol, actinomycin D, and cyclohexamide were purchased from
Sigma Chemical Co. (St. Louis, MO).
Experimental animals
Sprague-Dawley rats at 2–3 months of age were housed in a
constant temperature (20 C) and light-dark cycle (lights on, 0600–2000
h) with free access to food and water.
Transillumination-assisted microdisection of seminiferous
tubules
Rats were killed by CO2 asphyxiation, and the testes
were decapsulated. Seminiferous tubule segments were isolated in the
medium of DMEM-Ham’s F-12 medium (1:1; DMEM/F12; Life Technologies, Paisley, Scotland, UK) supplemented with 15
mM HEPES, 1.25 µg/liter sodium bicarbonate, 10
mg/liter gentamicin sulfate, 60 mg/liter G-penicillin, 1 µg/liter
BSA, and 0.1 mM 3-isobutyl-1-methylxanthine (Aldrich
Chemie, Steinheim, Germany) under a stereomicroscope by
transillumination-assisted microdisection technique as described
previously (13).
Tissue culture and stimulation
Twenty pieces of 5-mm long seminiferous tubule segments were
incubated in 1 ml of the above-mentioned culture medium in the presence
and absence of FSH (10 ng/ml), testosterone (106
M), 17ß-estradiol (10-8 M),
activin (10 and 100 ng/ml), TGF (10 ng/ml), TGFß (1 ng/ml), and
TNF (12.5 ng/ml) for 8 and 30 h. Seminiferous tubule segments
from stages II–VI were cultured and stimulated with or without
8-bromo-cAMP (8Br-cAMP; 1 mM), the phorbol ester (100
nM) phorbol 12-myristate 13-acetate (TPA), actinomycin D
(0.5 µg/ml), and cyclohexamide (10 µg/ml) for 8 and 30 h.
Measurement of the half-life of SCF mRNA
Seminiferous tubule segments from stages II–VI were
preincubated in the medium with or without FSH (10 ng/ml) for 8 h,
and then actinomycin D (0.5 µg/ml) was added. At 0, 6, 12, and
24 h after the addition of actinomycin D, the seminiferous tubule
segments were harvested, and SCF mRNA levels were detected by Northern
blot hybridization.
Riboprobe preparation and Northern blot hybridization
The pGEM 3Z plasmid containing a 560-bp long insert from the rat
SCF complementary DNA (cDNA; provided by Amgen, Inc.,
Thousand Oaks, CA) was linearized with HindIII as the
template for preparation of the antisense riboprobe. The riboprobe was
synthesized using a Riboprobe system II kit (Promega Corp., Madison, WI) and [32P]UTP (Amersham,
Aylesbury, UK). Total RNA was isolated from seminiferous tubules by a
single step method (15). Ten micrograms of total RNA were
size-fractionated in 1% denaturing agarose gel and transferred onto
Hybond-N+ nylon membrane (Amersham). The prehybridization
and hybridization were performed as described previously (16). After
hybridization with the SCF probe, the blots were stripped by pouring
the boiling 0.1% SDS onto the membrane and incubated until the
solution reached room temperature. The stripped membrane was
subsequently used for hybridization with glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) cDNA probe labeled with
[32P]deoxy-CTP (Amersham) by random priming method
(Prime-a-Gene Kit, Promega Corp.) at 45 C overnight.
Washing and autoradiography were performed similarly to SCF
probing.
Preparation of nuclei and nonradioactive nuclear run-on
assay
Seminiferous tubule segments of pooled stages II–VI that were
cultured in the medium with or without FSH for 8 and 30 h were
isolated by solubilizing the tubules in an isotonic solution containing
10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 3
mM CaCl2, 1 mM dithiothreitol
(DTT), 2 mM MgCl2, 0.32 M sucrose,
and 0.1% (vol/vol) Nonidet P-40 and homogenizing sufficiently with a
disposable syringe with a 22-gauge needle. The lysate containing the
crude nuclei was mixed with 1 vol sucrose cushion solution containing
10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 3
mM CaCl2, 1 mM DTT, 2
mM MgCl2, and 2 M sucrose, and then
layered on 2 vol of the sucrose cushion. The gradient was centrifuged
at 30,000 x g (20,000 rpm in TLS-55 rotor, TL-100
Ultracentrifuge, Beckman Coulter, Inc., Palo Alto, CA) for
45 min at 4 C, and the nuclear pellet was suspended in a glycerol
storage buffer containing 50 mM Tris-HCl (pH 8.0), 5
mM MgCl2, 0.1 mM EDTA, and 40%
(vol/vol) glycerol and frozen in liquid nitrogen. Nuclear run-on
experiments were performed according to the method described previously
(17) with modification. Briefly, frozen nuclei (100 µl containing
1–3 x 107 nuclei) were thawed and mixed with 1 vol
transcription 2 x buffer containing 10 mM Tris-HCl
(pH 8.0); 5 mM MgCl2; 0.3 M KCl; 1
mM DTT; 0.2 mM EDTA; 5 mM ATP, GTP,
and CTP; and 4 µl digoxigenin-UTP (10 nmol/µl; Boehringer Mannheim,
Mannheim, Germany), and the mixture was incubated at 32 C for 2 h.
Twenty microliters of RQ1 ribonuclease-free deoxyribonuclease I (1
U/µl; Promega Corp.) and 2 µl 1 M
CaCl2 were added to the reaction, and the incubation was
continued for 15 min at 37 C. Three volumes of denaturing solution [4
M guanidinium thiocyanate, 25 mM sodium citrate
(pH 7.0), 0.5% sodium lauroylsarcosine, and 0.1 M
ß-mercaptoethanol] was added to the reaction, and total RNA was
isolated by the single step method (15). The yield of the synthesized
RNA was measured by both spectrophotometer and the dot blot-based
detection assay according to the manufacturer’s instruction (DIG
System User’s Guide for Filter Hybridization, Boehringer Mannheim).
Ten micrograms of the labeled RNA were used for hybridization. The pGEM
3Z plasmid containing the SCF insert and the pGEM 3Z without any insert
were heat denatured and blotted onto Hybond-N+ nylon
membrane (Amersham). The blots were hybridized in 15 ml hybridization
solution containing 50% formamide, 3 x SSC (1 x SSC =
150 mM NaCl and 15 mM sodium citrate, pH 7.0),
5 x Denhart’s solution, 1% SDS, 0.1 µg/liter heat-denatured
calf thymus DNA, and 100 mg/liter yeast transfer RNA at 65 C overnight.
After hybridization, the blots were washed twice in 2 x SSC-0.1%
SDS at 65 C for 15 min each and twice in 0.1 x SSC-0.1% SDS at
65 C for 20 min each. The digoxigenin-chemiluminescent detection
procedure was performed according to the manufacturer’s protocol. The
exposure time of x-ray films (Fuji Photo Film Co., Ltd.,
Tokyo, Japan) was between 1–4 h.
Densitometric analysis
The x-ray films of Northern blotting and run-on assay results
were first scanned by a UMAX scanner (UMAX Technologies, Inc., Fremont, CA) and a Binuscan Photoperfect software package
(Binuscan Inc., New York, NY). The images were saved as TIFF-type files
(1.tif, Microsoft Corp. and Aldus Co., New York, NY) and
then quantified by TINA 2.0 densitometric analytical system (Raytest
Isotopenmesgerate GmbH, Straubenhardt, Germany) according to the
manufacturer’s instruction.
Replication of experiments and statistical analysis
All the experiments were repeated independently three times. In
all Northern hybridization analyses, the densitometric values of the
signals of SCF mRNA were first normalized to GAPDH signals and then the
highest densitometric value was designated as 100%. Other values were
expressed as the percentages of the highest one. The values from all
the experiments were pooled for the calculation of the means, and their
SEs and for one-way ANOVA and Duncan’s new multiple range
test to determine the significant differences between different
experimental groups by using StatView 4.51 statistic program (Abacus
Concepts, Inc., Berkeley, CA). P < 0.05 was considered
statistically significant. For half-life determination, a simple
regression test was performed by using the same statistic program as
mentioned above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). SCF mRNA level in stages II–VI
increased about 1.5-fold (P < 0.05) and 5-fold
(P < 0.01) after 8- and 30-h stimulation,
respectively. The extents to which the SCF mRNA level increased were
different among the four pooled stages of the rat seminiferous tubules
(P < 0.05). In stages II–VI, SCF mRNA level had an
approximately 5-fold (P < 0.01) increase after 30-h
FSH stimulation, whereas in stages VII–VIII, only about a 1.5-fold
(P < 0.05) increase was observed after the same period
of stimulation. At stages IX–XII and stages XIII–I, SCF mRNA level
increased 3.0- (P < 0.01) and 3.5-fold
(P < 0.01), respectively.
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Lack of effect of TGFß, TGF, TNF, and activin on SCF gene
expression
TGFß, TGF, TNF, and activin are growth factors that have
been found to be expressed in the seminiferous epithelium and supposed
to play important roles during spermatogenesis (18, 19, 20). However, the
detailed mechanisms by which their function is coordinated in space and
time are largely unknown. To test whether they are involved in the
regulation of SCF gene expression, we tested the effects of these
factors as well. However, no effects were observed at the tested dose
levels (data not shown).
Effects of 8Br-cAMP and TPA on SCF gene expression
Because of the highest stimulation of SCF mRNA level at stages
II–VI, we selected the pooled seminiferous tubule segments at these
stages for further studies on the mechanisms by which FSH up-regulates
SCF gene expression. FSH binds to specific FSH receptors on Sertoli
cell membranes and causes an increase in intracellular cAMP level or
activation of protein kinase C to induce gene expression in the Sertoli
cells (21). The induction of the gene expression by FSH can be
reproduced by cAMP analogs, such as 8Br-cAMP, when it is mediated
through cAMP/protein kinase A pathway or by the phorbol ester, TPA, a
protein kinase C activator, when it is mediated via the protein kinase
C (PKC) pathway. To test whether FSH stimulation of SCF gene expression
is mediated by cAMP/protein kinase A (PKA) pathway or PKC pathway,
seminiferous tubule segments from stages II–VI were incubated in the
presence or the absence of 8Br-cAMP or TPA for 8 and 30 h,
respectively. SCF mRNA level was increased by 8Br-cAMP in a similar
manner to FSH, whereas TPA had no effects (Fig. 2).
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, FSH stimulation was abolished after
8- and 30-h treatments with actinomycin D. The steady state levels of
SCF mRNA decreased dramatically after RNA synthesis was blocked by
actinomycin D for 30 h.
Independence of FSH up-regulation of de novo protein
synthesis
To assess whether the up-regulation of SCF by FSH involves
de novo protein synthesis, cyclohexamide, a protein
synthesis inhibitor (22), was used to treat the samples together with
FSH. As shown in Fig. 2, cyclohexamide did not affect the FSH
stimulation.
Prolongation of the half-life of SCF mRNA after FSH
stimulation
The elevated SCF mRNA level can be contributed either by an
increase in transcription or a decrease in mRNA degradation. To study
whether the stability of SCF mRNA was increased after FSH stimulation,
the half-life of SCF mRNA with and without FSH stimulation was
determined by the actinomycin D-blocking method (23). It has been
reported that actinomycin D may prolong the half-life of some unstable
mRNAs and long lasting blocking of transcription can also kill the
cells (24). In our study, we tried different concentration of
actinomycin D (0.1, 0.5, 1, 5, and 10 µg/ml) and different incubation
time points (8, 24, and 30 h) by using four pools of seminiferous
tubule segments because the stage-specific expression pattern of SCF
mRNA served as a good marker of the viability of the cells. We found
that the concentration used in the main experiments (0.5 µg/ml) was
the optimal one at which SCF gene transcription was completely blocked,
and the characteristic of the stage-specific expression pattern of SCF
mRNA was still observed after 30 h of incubation (data not shown).
As both control and FSH-stimulated samples were treated with
actinomycin D, we could compare transcript half-lives of both groups.
As shown in Fig. 3, the decrease in SCF
mRNA was slower when the seminiferous tubules were pretreated with FSH
for 8 h compared with the control. The SCF mRNA half-lives, as
calculated by regression analysis, were 15.6 ± 1.2 h for the
FSH-treated samples and 8.6 ± 2.7 h for the control (n
= 3; P < 0.05).
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, the transcription rate of SCF gene was
increased significantly in a time-dependent manner (n = 3;
P < 0.01). Five- and 10-fold increases in
transcription rates were observed after 8- and 30-h FSH stimulation,
respectively. PGEM 3Z plasmid as a negative control gave very weak
signals and was used for correction of the nonspecific binding.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, TGFß, TNF,
and activin on the SCF gene expression. In this tissue culture system,
germ cells and Sertoli cell are cocultured, and the communication
between germ cells and Sertoli cells was maintained intact (14). Our
previous study showed that SCF was expressed at the highest level in
stages II–VI, at the lowest level in stages VII–VIII, and at an
intermediate level in stages IX–I (Hakovirta H., et al.,parallel manuscript). In the present study, we used the
stage-specific expression pattern of SCF as an internal control to
monitor the culture condition to assure the validity of our data.
In the hormonal stimulation experiments, we found a significant
induction of SCF mRNA level after 8 and 30 h of FSH stimulation.
It is of interest to note that FSH differentially stimulated SCF gene
expression at different stages of the seminiferous epithelial cycle.
The maximal increase in SCF mRNA level was found at stages II–VI,
implicating that more SCF might be needed in these stages than in other
stages. The minimum increase was observed at stages VII–VIII. It has
previously been documented that many genes expressed in Sertoli cells,
show stage-specific expression pattern, e.g. activin (25, 26), inhibin- (27), and proenkephalin (28). Our results, for the
first time, show that SCF is not only expressed differentially in
Sertoli cells, but also is responsive to FSH differentially at
different stages of the seminiferous epithelial cycle. We suggest that
the stage-specific steady state level of SCF mRNA might result from the
stage-specific responsiveness of SCF gene in Sertoli cells to FSH at
different stages of the seminiferous epithelial cycle.
To understand the regulatory mechanism in detail, we further tested the effects of 8Br-cAMP, a cAMP analog and PKA stimulator, and TPA, a PKC activator, on the SCF gene expression. The similar effect of 8Br-cAMP as FSH and the lack of an effect of TPA on SCF mRNA level indicate that the up-regulation of SCF gene expression is mediated through the cAMP/PKA pathway and not through the PKC pathway. The elevation of the SCF mRNA level resulted from the accumulation of SCF mRNA, which can be due to many transcriptional and posttranscriptional regulatory mechanisms. The fact that actinomycin D abolished the stimulatory effect of FSH on SCF gene expression suggests that the up-regulation at least partially happened at transcriptional level. We then further measured the stability of SCF mRNA before and after FSH stimulation by using actinomycin D blocking method. The prolongation of the half-life of SCF mRNA after FSH stimulation indicates that the degradation rate of SCF mRNA was decreased after FSH stimulation. Cyclohexamide, a potent protein synthesis inhibitor, did not affect FSH stimulation. This result indicates that the FSH up-regulation of SCF mRNA level is independent of de novo protein synthesis. Transcription run-on assays make it possible to further analyze the changes in transcription rate before and after FSH stimulation. Although the nuclei isolated from the cultured seminiferous tubule segments are mixtures of the nuclei from Sertoli cells and germ cells, it is still possible to perform in vitro transcription run-on assays because SCF had been shown to be expressed only in Sertoli cells (7). Nuclei from at least 15 cm of seminiferous tubules were needed for a successful reaction in our study. Our run-on results revealed a 5-fold increase and a 10-fold increase after 8- and 30-h FSH stimulation, respectively. However, in the Northern analyses, after 8- and 30-h FSH stimulation, only 1.5- and 5-fold increase in steady state SCF mRNA level were observed. The discrepancy suggests that there are some factors that can repress the SCF steady state mRNA level at the posttranscriptional level during the FSH stimulation.
No consistent effects on SCF mRNA expression in the seminiferous epithelium were found after testosterone and estradiol stimulation. It is known that testosterone is needed in Sertoli cells for the production of cAMP response element (CRE)-binding protein (CREB), which acts in cAMP stimulation of gene expression (29, 30). A recent study (31) on the promoter of the human SCF gene showed that testosterone had no effect on human SCF promoter activity in the Sertoli cell line SF7, either by interaction with AR protein directly with the GRE sequence or by modulating expression of factors such as CREB, which might regulate the SCF promoter through CRE sequences. Our findings that testosterone had no effect on SCF gene expression further support the suggestion that the induction of SCF promoter activity by cAMP is not mediated by CRE sequences. No effect of estradiol was found in our study, although there is an activating protein-1 site on the SCF promoter according to the previous studies (31, 32). As mentioned earlier, TPA, which has been found to increase the synthesis of c-fos and AP-1 by a PKC-dependent pathway and inhibit gene transcription in the rat GnRH promoter (33, 34, 35), had no effect on SCF gene expression. This might suggest that estrogens are not the inhibitory regulators of SCF gene expression. No significant effects were found in growth factor stimulation experiments. It may be either the real situation in vitro or artificial due to the inappropriate time points or dosages we used. Thus, these negative findings may only represent the in vitro situation, and the possibility that the growth factors might have some effects on SCF gene expression in vivo cannot be ruled out.
In summary, SCF gene expression in the rat seminiferous epithelium was
up-regulated by FSH in a stage-specific and time-dependent manner. The
up-regulation of FSH was mediated through the cAMP/PKA pathway, not
through the PKC pathway. The up-regulation of the SCF mRNA level was
due to the increase in the transcription rate and the prolongation of
the mRNA half-life, but it was independent of de novo
protein synthesis. Testosterone, estradiol, TGF, TGFß, TNF, and
activin had no effect on SCF gene expression in rat seminiferous
tubules in vitro.
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Footnotes |
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Received May 19, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß-B subunits during the cycle
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 W Yan, J Suominen, and J Toppari Stem cell factor protects germ cells from apoptosis in vitro J. Cell Sci., January 1, 2000; 113(1): 161 - 168. [Abstract] [PDF]
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H. Hakovirta, W. Yan, M. Kaleva, F. Zhang, K. Vänttinen, P. L. Morris, M. Söder, M. Parvinen, and J. Toppari Function of Stem Cell Factor as a Survival Factor of Spermatogonia and Localization of Messenger Ribonucleic Acid in the Rat Seminiferous Epithelium Endocrinology, March 1, 1999; 140(3): 1492 - 1498. [Abstract] [Full Text]
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