Regulation by pH of the Alternative Splicing of the Stem Cell
Factor Pre-mRNA in the Testis*
Claire
Mauduit
§,
Gilles
Chatelain¶,
Solange
Magre
,
Gilbert
Brun¶,
Mohamed
Benahmed
, and
Denis
Michel¶**
From
INSERM U407, Communications Cellulaires en
Biologie de la Reproduction, Faculté de Médecine
Lyon-Sud, BP 12, 69 921 Oullins cedex, ¶ CNRS/ENS UMR 49,
Laboratoire de Biologie Moléculaire et Cellulaire de l'Ecole
Normale Supérieure de Lyon, 46 allée d'Italie, 69 364 Lyon cedex 07, and ** URA256, Biologie Cellulaire et
Reproduction, Université de Rennes, Rennes 1, Bât 13, 35042 Rennes cedex, and
Laboratoire de Physiologie de la Reproduction,
Université Pierre et Marie Curie, CNRS URA 1449, 7 quai St.
Bernard, Bât A, 75 005 Paris, France
 |
ABSTRACT |
Proliferation and differentiation of progenitor
stem cells are mainly controlled by diffusible and adhesion molecules.
Stem cell factor (SCF), an essential regulator of spermatogenesis
produced by Sertoli cells, utilize both modes of cell to cell
communication. Indeed, SCF exists in soluble (SCFs) and membrane-bound
(SCFm) forms, which are required for a complete spermatogenesis, and are generated by alternative splicing of optional exon 6, encoding sites of proteolysis. We show that in the mouse testis, the alternative splicing of SCF is developmentally regulated. SCFs predominates in
fetal and neonatal gonads and is then replaced by SCFm in the prepubertal and adult gonads. By sequencing SCF exon 6, we show that
the flanking intronic sequences perfectly follow the gt-at rule,
suggesting that the basal splicing machinery might not be responsible
by itself for exon 6 skipping. Moreover, freshly isolated Sertoli cells
mainly express SCFm, but a switch to SCFs occurs after 48 h of
culture. We found that this change can be prevented by acidification of
the culture medium at pH 6.3 or by addition of lactate. The sustained
synthesis of SCFm at low pH was no longer observed in the presence of
cycloheximide, suggesting that SCF exon 6 skipping requires de
novo protein synthesis. Accordingly, UV cross-linking experiments
show that nuclear Sertoli cell protein(s) bind in a sequence-specific
manner to exon 6. Together, our data allow the proposal of an
integrated mechanism in which the synthesis of lactate by Sertoli cells
is used in the same time as an energetic substrate for germ cells and
as a promoter of their survival/proliferation through the production of SCFm.
 |
INTRODUCTION |
Stem cells (from hematopoietic, nervous, and gonadal systems) are
the subject of increasing interest because of their biological and
medical importance (1). Specifically, in the testis, the conversion of
stem cell spermatogonia into differentiated haploid spermatozoa is a
complex process highly dependent upon the somatic Sertoli cells. One of
the major questions is the identification of the Sertoli cell-derived
factors driving quiescent stem cells into proliferation. Although these
factors are largely unknown, some growth factors and cytokines have
been suggested to be at play (2-5). Among them, the stem cell factor
(SCF)1 appears as a key
factor, as demonstrated by the pleiotropic effects of the SCF gene
mutation, leading to the depletion of three embryonic migratory
lineages: hematopoietic stem cells, neural crest-derived melanocytes,
and primordial germ cells (6).
SCF has been detected both in membrane-bound (SCFm)
or soluble (SCFs) forms (7, 8). The soluble form is generated from an
integral membrane protein precursor, by proteolytic cleavage at a site
located in the proximal extracellular domain (9). Because the main
proteolytic site involved in this process is encoded by a short 84-base
pair-long alternative exon (exon 6), the final localization of SCF is
ultimately dictated by differential splicing (10). Although SCFm and
SCFs transcripts are equally abundant in spleen and heart (9-11), SCFs
is predominant in all other organs tested, including brain, bone
marrow, kidney, lung, liver, and thymus of adult mouse (9-11).
Interestingly, in the testis, where SCF and its cognate receptor
c-kit are produced, respectively, by Sertoli cells (12-13)
and spermatogonia (14-17), the major form of SCF found in the adult
mouse is SCFm (10, 11). Accordingly, SCFm has been shown to be of major
functional importance for germ cells, as demonstrated by the
infertility of mice homozygous for a SCF gene mutation named
Steel-Dickie (Sld) (6, 18). This mutation consists in an
intragenic deletion of the transmembrane- and cytoplasmic domain-coding
regions, resulting in the constitutive production of SCFs (10, 19).
In the present study, we demonstrate that SCF pre-mRNA splicing is
developmentally regulated in the male gonad and that an acidic
microenvironment, probably because of the high amounts of lactate in
testicular Sertoli cells, might be responsible for the switch of SCF
splicing in favor of SCFm, which can promote germ cell survival in the
contact of Sertoli cells and proliferation of spermatogonia type A.
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EXPERIMENTAL PROCEDURES |
Materials--
OF1 mice were obtained from Iffa Credo
(L'arbresle, France). Dulbecco's modified Eagle's (DME)/Ham's F-12
medium, TRIzol®, and reverse transcriptase (Moloney murine
leukemia virus) were obtained from Life Technologies, Inc. (Eragny,
France). Collagenase/dispase was obtained from Boehringer Mannheim.
Sigma was the source for transferrin, gentamicin, nystatin, insulin,
-tocopherol, HEPES, random hexanucleotides, and deoxynucleotides
triphosphate (dNTP). Taq polymerase was purchased from
Appligene-Oncor (Illkirch, France). Amersham Pharmacia Biotech was the
source for [
-32P]UTP. In vitro
transcription kit was purchased from Promega (Madison, WI), and ABI
Prism® dye terminator kit was from Perkin-Elmer.
Sertoli Cell Isolation and Culture--
Sertoli cells were
prepared from 16- to 18-day-old mice, as described by Dorrington
et al. (20). Sertoli cells were plated in 3-cm diameter
plates in DME/F-12 medium supplemented with transferrin (5 µg/ml),
insulin (2 µg/ml), gentamicin (20 µg/ml), nystatin (20 IU/ml), and
-tocopherol (10 µg/ml) and cultured at 32 °C.
Sequence Analysis of SCF Splice Sites--
SCF intron 5 and 6 were amplified using a mouse DNA library as a template and
Expand® long template PCR system (Boehringer Mannheim).
Intron 5 was amplified using the following primers (see Fig.
1A): S5 (5'-TGGTGGCATCTGACACTAGTGA-3') and S6b
(5'-GCTACTGCTGTCATTCCTAAGG-3'); intron 6 was amplified using the
primers: S6a (5'-CCAGAGTCAGTGTCACAAAACC-3') and S7
(5'-CTTCCAGTATAAGGCTCCAAAAGC-3'). PCRs were done in the presence of 300 nM primers in the following conditions: 94 °C for 5 min;
10 cycles of 94 °C for 10 s, 60 °C for 30 s, 68 °C
for 8 min; 20 cycles of 94 °C for 10 s, 60 °C for 30 s,
68 °C for 8 min (with an increment of 20 s at each cycle); and
68 °C for 14 min; 90 ng of purified PCR products were used as
template for DNA sequencing using the ABI Prism dye terminator kit in a
DNA Thermal Cycler (Perkin-Elmer) for 30 cycles: 96 °C for 10 s, 50 °C for 5 s, 60 °C for 4 min. After cycling, PCR
products were purified and dried, and just before loading, the sample
was resuspended in 4.0 µl of deionized formamide, 50 mM
EDTA, pH 8.0 (5:1). Fluorescence-based DNA sequence analyses were
obtained on an ABI 373 DNA sequencer fitted with a 6% polyacrylamide
gel using the manufacturer's version 2.1.0 software.
RT-PCR Analysis--
Total RNAs were extracted from mouse testis
or cultured Sertoli cells with TRIzol® reagent, a
monophasic solution of phenol and guanidine isothiocyanate.
Single strand cDNA was synthesized by reverse transcription
starting from 3 µg of total RNA and using 5 µM random
hexanucleotides, 0.2 mM dNTP, and 1 units/µl Moloney
murine leukemia virus. PCR reactions were done using Taq
polymerase (0.01 units/µl), 0.2 mM dNTP, and 0.01 µg/µl S5 and S7 primers. The mixture was first heated at 94 °C
for 5 min and then 20 cycles of 94 °C for 40 s, 57 °C for 1 min (with 0.02 s and 0.3 °C decrease each cycle), 72 °C for
40 s followed with 12 cycles of 94 °C for 40 s, 51 °C for 15 s, 72 °C for 40 s, then 72 °C for 5 min. SCF
amplification products containing exon 6 (SCFs) or lacking exon 6 (SCFm) were, respectively, 251 bp and 167 bp long (Fig. 1A).
They were analyzed on 2% agarose gel and visualized by ethidium-UV
staining. For
-actin RT-PCR we used the primers:
5'-GACAGGATGCAGAAGGAGAT-3' and 5'-TTGCTGATCCACATCTGCTG-3', and
-actin cDNAs were amplified according the following conditions
94 °C for 2 min, 28 cycles of 94 °C for 30 s, 59 °C for
30 s, 72 °C for 30 s and 72 °C for 5 min.
Sertoli Cell Nuclear Extract Preparation--
Buffers A, C, and
D used for preparation of nuclear extracts were described by Dignam
et al. (21). All steps were done on ice, and proteolysis was
minimized by addition of 0.5 mM phenylmethysulfonyl fluoride and 0.3 µg/ml antipain and leupeptin to all buffers. Aprotinin (0.5 µg/ml) was also added to buffers A and C. Nuclear extracts were prepared as described by Dehbi et al. (22).
The extract was removed and stored at
80 °C. Protein concentration was determined by the method of Bradford at 545 nm, using bovine serum
albumin as a standard (23).
UV Cross-linking Analysis--
A SCF cRNA containing the last 57 nucleotides of exon 5, whole exon 6, and the first 46 nucleotides of
exon 7 (named SCF 567) was synthesized by in vitro
transcription from a pGEM-T plasmid containing the corresponding
cDNA fragment after linearization with NcoI and using
the SP6 RNA polymerase. A SCF cRNA containing the last 57 nucleotides
of exon 5 and 110 nucleotides of exon 7 but lacking exon 6 (named SCF
57) was transcribed using the T7 RNA polymerase after linearization of
the pGEM-T vector with NotI. A 241-nucleotide-long cRNA Trk
B, corresponding to a mRNA species produced by Sertoli cells but
unrelated to SCF, was used as a control and generated by transcription
from a pGEM-T vector containing the trkB cDNA using the SP6 RNA
polymerase after linearization with NcoI. Radiolabeled RNAs
were obtained by adding 50 µCi of [
-32P]UTP in the
in vitro transcription mixtures. The specific activity of
32P-SCF 567 is 7.9 × 108 cpm/µg.
Proteins interacting with radiolabeled precursor RNAs were detected by
UV cross-linking methods (24). Radiolabeled RNAs were incubated with 20 µg of nuclear protein extracts for 10 min at 30 °C in binding
buffer (1 mg/ml poly(G), 0.5 mg/ml tRNA, 0.1 mM
dithiothreitol, 50 mM KCl, 10 mM Tris, pH 8.0, 3 mM MgCl2). After binding, samples were
cross-linked at 2 J/cm2 using an Appligene UV cross-linker.
Then, RNA was digested for 15 min at 37 °C in the presence of RNase
A (1 mg/ml). After the addition of SDS sample buffer, the samples were
heated to 100 °C for 5 min and then run on SDS, 10% PAGE. Gels were
dried and exposed 1-2 days with Fuji RX film.
 |
RESULTS |
Alternative Splicing Pattern of SCF during Mouse Testicular
Development--
SCFs and SCFm mRNAs were discriminated by exon
connection RT-PCR, using primers defined in exons 5 and 7 and named S5
and S7, respectively, as shown in Fig.
1A. The SCFs cDNA fragment appears longer, with an additional 84-bp-long exon 6. Using this method, we have first determined the evolution of the SCFm/SCFs ratio
during mouse testicular development (Fig. 1B). Mouse testes were removed at different key stages of testicular development i.e. fetal (13, 15, 18 days post-coitum (dpc); day 0 post-coitum was the day where the vaginal plug was detected), neo-natal
(2 and 8 days post-partum (dpp), pre-pubertal (10, 15, and 21 dpp), pubertal (28, and 42 dpp), and adult (60 dpp). SCFm (lacking exon 6)
appears predominant in 13 dpc fetal testis, whereas SCFm and SCFs
(containing exon 6) mRNAs appear equally abundant in 15 dpc testis,
and SCFs becomes predominant at the end of gestation (18 dpc) and in
neonatal testis (2 dpp) (Fig. 1B). At the pre-pubertal and
adult periods, male gonad contains predominantly SCFm. At 42 and 60 dpp, the total level of expression of SCF was lower because of the
dilution of Sertoli cells (which are in a limited number) by the
increasing amounts of adult germ cells (Fig. 1B).

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Fig. 1.
Alternative splicing of SCF
pre-mRNA. A, exon 6, encoding proteolysis sites
(bold vertical bars) is either maintained or eliminated from
the cytoplasmic SCF mRNAs, generating, respectively, SCFs or SCFm.
The localization of the RT-PCR primers used in this study, S5, S6a,
S6b, and S7, is shown. S5-S7 RT-PCR products corresponding to SCFs and
SCFs were 251 and 167 bp long, respectively. The region of exon 7 encoding the transmembrane domain is represented with horizontal
lines. B, SCF alternative splicing pattern during
development. Expression of SCF mRNAs was analyzed by RT-PCR in
testis from fetus (13, 15, 18 dpc) and from 2- to 60-day-old mice.
-Actin mRNA was measured as an internal control from the same
input of reverse transcription mixtures.
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Sequence Analysis of SCF Gene Exon 6 and Flanking Intronic
Sequences--
Because most cases of alternative splicing are the
consequence of suboptimal splice sites, we determined the genomic
sequences surrounding exon 6. Surprisingly, this analysis showed that
5' and 3' splice sites of introns 5 and 6 perfectly follow the gt-ag rule, but in addition, exactly fit mammalian 5' and 3' splice site
consensus motifs, which are, respectively, GTRAGT and YAG preceded by a
polypyrimidine stretch (Fig. 2). The
presence of these optimal splice sites suggests that the basal splicing
machinery can not be responsible by itself for exon 6 skipping. Hence,
we have looked for the presence of other cis elements that may be the
targets for more specific RNA binding factors. The particularly high
conservation degree of the exon 6 nucleotide sequence, when compared
with upstream and downstream exons (Table
I), suggest that, in addition to its
coding capacity, the nucleotide sequence of exon 6 may contain a cis
element involved in its own splicing. Scanning of the intronic
sequences against data bases revealed that only the intronic region
flanking exon 6 in 3' (accession number AF083887) is related to a
previously identified cDNA sequence. This cDNA (accession
number A50814) corresponds to a human SCF mRNA species likely to
have retained intron 6 during splicing.

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Fig. 2.
DNA sequence of 5' and 3' splice sites of SCF
intron 5 and 6. Introns 5 and 6, enclosing the optional exon 6, were cloned and sequenced as described under "Experimental
Procedures." The intron boundaries sequences close to exon 6 are
represented in bold letters, and polypyrimidine stretches
are underlined. GenBank accession numbers are AF083885 and
AF083886, for beginning and end of intron 5, respectively, and AF083887
and AF083888 for beginning and end of intron 6, respectively.
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Table I
Nucleotide sequence conservation of SCF gene exons among mammals
The % of identity between mouse and human SCF ("Human") and
between mouse and pig SCF ("Pig") are compared for each exon. Note
that only the exon 6 sequence is 100% conserved.
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Modulation of the SCF Alternative Splicing Pattern in Cultured
Sertoli Cells--
We wanted then to test the ability of Sertoli cells
to synthesize SCF when isolated from the whole testis context. As shown in Fig. 3, freshly isolated Sertoli cells
(from 16-18-day-old animals) predominantly expressed SCFm mRNA, as
observed in vivo. By contrast, during the culture, Sertoli
cell SCF alternative splicing pattern changed in favor of the
expression of SCFs mRNA (Fig. 3). For example, at 0.5 and 2.5 h of culture, SCFm mRNA is still predominantly expressed (ratio
SCFm/SCFs > 1), whereas at 48 and 72 h of culture, SCFs
becomes predominant (ratio SCFm/SCFs < 1). The point of
equivalence of SCFm and SCFs mRNAs is around 24 h (ratio
SCFm/SCFs
1).

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Fig. 3.
Progressive change of the SCF pre-mRNA
splicing in cultured Sertoli cells. Sertoli cells were freshly
isolated (0) or cultured for different times (0.5 to 72 h) in defined medium (DME/F12, pH 7.38, with HEPES 15 mM
and bicarbonate 1.2 g/liter) and supplemented as described under
"Experimental Procedures." Exon connection analysis was performed
using the S5 and S7 primers defined in exons 5 and 7 located apart from
the optional exon 6. The histogram in the lower
panel represents the SCFm/SCFs ratio.
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Role of Low pH and Lactate in the Regulation of SCF Alternative
Splicing--
The shift of SCF alternative splicing pattern observed
in cultured Sertoli cells has led us to look for the mechanisms
regulating the presence of exon 6 in mature SCF mRNAs. While
testing various culture conditions for their ability to maintain the
predominance of SCFm synthesis, we found that the SCFm mRNA
synthesis is strikingly dependent on pH. Lowering the culture medium pH
by HCl addition or sodium bicarbonate withdrawal prevents the shift to
the SCFs mRNA form. Sertoli cells cultured for 48 h at acidic
pH (6.3) predominantly expressed SCFm, whereas those cultured at pH 7.2 and pH 7.6 presented an alternative processing in favor of SCFs (Fig.
4). Furthermore, kinetic studies show
that at pH 6.3, SCFm predominates at each time tested (2 to 72 h),
whereas it is progressively replaced by SCFs after culture shift at pH
7.6 (Fig. 5). Indeed, at pH 6.3, at each
time tested, the SCFm/SCFs ratio is higher than 1, whereas at pH 7.6, the ratio decreased progressively until 0.6 at 72 h (Fig. 5) The
action of low pH on SCFm expression was detected at 12 h and
maintained during the other times tested.

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Fig. 4.
Effect of low pH on SCF alternative splicing
pattern. Sertoli cells were cultured in DME/F12 medium for 48 h, and the pH was adjusted from 6.3 to 7.6. The histogram in
the lower panel represents the SCFm/SCFs ratio.
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Fig. 5.
Time course of SCF alternative splicing
following pH changes. Sertoli cells were cultured for different
times (2 to 72 h) in DME/F12 medium, and the pH was adjusted to
6.3 or 7.6. The histogram in the lower panel
represents the SCFm/SCFs ratio.
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As Sertoli cells are known to produce lactate, an acidic metabolite in
the context of Sertoli cell-germ cell metabolic cooperation, this
metabolite was tested. As shown in Fig.
6, lactate in cultured Sertoli cells
maintained the SCFm expression. Indeed, after 48 h of culture in
the presence of lactate, the SCFm/SCFs ratio is higher than 1, whereas
in the absence of lactate, the ratio is lower than 1 (Fig. 6).
Altogether, these results suggested that the trans-acting factors
regulating exon 6 skipping are somehow regulated by acidity. In this
respect, one must notice that pH remained neutral at the end of the
experiment shown in Fig. 3, suggesting that an intracellular factor
responsible for the production of SCFm is lost from the Sertoli cells
maintained in culture unless an experimental acidification is
imposed.

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Fig. 6.
Effect of lactate on SCF alternative splicing
pattern. Sertoli cells were cultured for 48 h in DME/F12
medium in the absence ( ) or presence (+) of lactate (10 mM). The histogram in the lower panel
represents the SCFm/SCFs ratio.
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Requirement of Protein(s) Neosynthesis for SCF Alternative
Splicing--
As shown in Fig. 7,
Sertoli cells cultured at pH 6.3 for 48 h expressed predominantly
SCFm (SCFm/SCFs ratio >1), whereas a progressive increase of the SCFs
is observed when cycloheximide is added into the culture medium
(SCFm/SCFs ratio = 1). This result indicates that protein
synthesis is required for sustained production of SCFm at low
pH.

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Fig. 7.
Effect of cycloheximide on SCF alternative
splicing pattern. Sertoli cells were cultured for 48 h in
DME/F12 medium (pH 6.3) in the absence (-) or presence (+) of
cycloheximide (10 µg/ml). The histogram in lower panel represents the
SCFm/SCFs ratio.
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Exon 6 RNA Recognition by Proteins from Sertoli Cell
Extracts--
To test whether exon 6 may be bound by a specific RNA
binding factor, [
-32P]UTP RNAs containing exon 6 (SCF567) were cross-linked by UV to nuclear protein extracts from
Sertoli cells freshly isolated. A single cross-linked protein-RNA
complex was detected, with an apparent molecular mass of 69 kDa (Fig.
8A, lane 1).
Competition experiments showed that the 69-kDa complex formation can be
prevented by previous addition of an excess of cold RNA containing exon 6 in a dose-dependent manner (Fig. 8A,
lanes 2-4) but not with SCF lacking exon 6 (SCF57) or the
unrelated trkB mRNA (Fig. 8B). The 69-kDa complex is no
longer obtained when using RNA probes unrelated to exon 6 as SCF57 or
TrkB RNA (data not shown). Altogether, these results suggested the exon
6 sequence specificity of the 69-kDa RNA-binding protein(s).

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Fig. 8.
Cross-link experiments. A, in the
competition studies, nuclear extracts from freshly isolated Sertoli
cells (20 µg) were allowed to interact with 32P RNA
SCF 567 (30,000 cpm) without (lane 1) or with increasing
concentrations of cold SCF 567 RNA: 0.04 µg, molar excess of 1.18 (lane 2), 0.4 µg, molar excess of 11.8 (lane
3), 4.5 µg, molar excess of 118 (lane 4).
B, to test the specificity of cross-linking experiments,
nuclear extract from freshly isolated Sertoli cells (20 µg) were
allowed to interact with 32P RNA SCF 567 (30,000 cpm)
alone (lane 1) or with cold TrkB RNA, molar excess of 68 (lane 2) or with cold SCF57 RNA, molar excess of 120 (lane 3).
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DISCUSSION |
The key role of SCF action in spermatogenesis is dramatically
dependent on its membrane-bound localization, as demonstrated by
several observations. (i) Adult mutant mice, which express only SCF
soluble form (Steel-Dickie mutation (19)) or an abnormal SCFm devoid of
cytoplasmic domain (Steel 17H mutation with a skipping of
exon 8, which encodes the cytoplasmic tail of SCF (25)) are infertile;
ii) Disruption of spermatogenesis using a Sertoli cell toxicant such as
2,5-hexanedione was accompanied with a switch in alternative splicing
from SCFm to SCFs mRNA (26); iii) In vitro experiments
have shown that SCFm promotes anchoring of germ cells to Sertoli cells
(11) as well as proliferation of germ cells (19). SCF thus provides a
striking example for the biological importance of certain
post-transcriptional regulations. The aim of the present study was to
examine the splicing responsible for the synthesis of SCFm and SCFs
during gonadal development and to identify the regulatory mechanisms
involved in alternative splicing of SCF pre-mRNA in Sertoli cells.
Two other laboratories (11, 27) have reported, by using RT-PCR
approach, the testicular expression of SCFs and SCFm during the
post-natal period (but not to the fetal period). These data were
conflicting. Rossi et al. (27) showed that testis from 8 to
13 dpp expressed predominantly SCFm transcripts, whereas 18- to
60-day-old testis expressed the SCFs form. By contrast, Marziali
et al. (11) showed that at 6 dpp, SCFs and SCFm were equally
abundant, whereas SCFm form was predominantly expressed from 12 dpp to
the adult period. Because of these discrepancies, we first investigated
the developmental pattern in SCF expression during the testicular
fetal, postnatal, prepubertal pubertal, and adult periods. Our results,
at least in adult animal, appears to correlate with those of
Marziali et al. (11). In the fetal mouse testes,
we have shown that SCFm mRNAs are predominant at 13 dpc when
primordial germ cells proliferate. Indeed, primordial germ cells
proliferate from 7.5 to 13 dpc (28). Then SCFs mRNAs are
predominant at 18 dpc to 2 dpp, when germ cells are quiescent (28). In
this context, the two SCF forms are likely to play important and
distinctive roles during the fetal gonad development. SCFs may promote
migration and survival of primordial germ cells, whereas SCFm allows
survival and proliferation of primordial germ cells (29, 30). Finally,
from 8 dpp to adulthood, the SCFm form predominates. The switch from
SCFs to SCFm coincides with the first wave of multiplication of
spermatogonia, occurring at 6/8 dpp (31).
However, our data indicate that as soon as Sertoli cells were isolated
from their in vivo context, they exhibit a dramatic decline
in their ability to synthesize SCFm, suggesting that a factor(s) from
their local in vivo environment is required for active exon
6 skipping. We propose that low pH might initiate this process. Indeed,
acidity (pH 6.3) was able to maintain SCFm synthesis by cultured
Sertoli cells. Our findings were consistent with examples in the
literature showing that some cases of pre-mRNA splicing are
regulated by pH (32, 33). For instance, in the case of the ATP synthase
that is involved in energy production in heart and skeletal muscle
tissues, an exon skipping in ATP synthase
subunit is specifically
observed in heart and skeletal muscle but not in the liver (33).
Whether an acidic (micro)environment may occur in Sertoli cells in
physiological conditions is still to be established. However, it is of
interest to note that, in the testis, lactate is produced in very large
amounts by Sertoli cells, to fulfill the energetic requirements of germ
cells that are unable to metabolize glucose (34). Lactate is an
energetic substrate preferentially used by post-meiotic germ cells and
particularly spermatids (35). Recently, we have shown that these germ
cells control and direct glucose metabolism in Sertoli cells toward lactate formation through some signaling molecules that increase lactate dehydrogenase A (an enzyme that favors the convertion of
pyruvate into lactate (36)). Based on the present observation that in
acidic conditions, Sertoli cells preferentially express the SCFm, we
hypothesize that at certain specific stages during the seminiferous
epithelium cycle, which particularly associates Sertoli cells and
spermatids, an acidic microenvironment might be generated by an
elevated concentration of lactate. Therefore, lactate synthesis, in
addition to supplying spermatids with their energetic substrate, can
also, by lowering the pH, allow the synthesis of SCFm that stimulates
through c-kit the proliferation of spermatogonia and
therefore triggers a new spermatogenic cycle. The hypothesis that
terminally differentiated germ cells (spermatids) might send signals to
stem germ cells to initiate a new spermatogenic cycle, has been
proposed some 40 years ago by Roosen-Runge (37). Although some
candidates (e.g. residual bodies (37)) have been suggested as such signals between differentiated and stem germ cells, the precise
determining factor remains unknown. This report proposes that an
increased acidity through an increase in lactate production at the
level of Sertoli cells in contact to spermatids may represent the
triggering signal for SCFm expression.
To investigate the mechanisms involved in SCF exon 6 skipping in the
male gonad, we used as an experimental model, purified mouse Sertoli
cells cultured in defined medium and have taken advantage from the
observation related to a switch occurring from SCFm to SCFs after
12 h of culture of Sertoli cells. With regard to the mechanisms
involved in alternative splicing of pre-mRNA, examples available in
the literature indicate that exon skipping is most often the
consequence of suboptimal cis elements at the exon-intron boundaries.
These weak sites fail to bind components of the basal splicing
machinery, including U1 small nuclear RNP (38, 39) and
U2AF65 (39), thus leading to constitutive exon exclusion.
Exon inclusion then requires additional trans-acting factors, which
specifically recognize the weak exons at the level of so-called
splicing enhancers (40). Interestingly, an opposite situation is likely
to occur in the case of SCF. Because our determination of intron
sequences surrounding the alternative exon 6 has revealed the presence
of 5' and 3' intron sequences, exactly fitting the splice site
consensus for the major splicing pathway of nuclear pre-mRNA
introns (41), exon 6 of SCF would be constitutively retained in mature
SCF mRNAs by the basal spliceosome. In such a situation, one may
hypothesize that an exclusion factor is involved to specifically
eliminate exon 6 during the synthesis of SCFs mRNA. This
possibility is strongly supported by the fact that de novo
protein synthesis is required for predominant synthesis of SCFm,
i.e. for exon 6 elimination. The particularly high degree of
sequence conservation of exon 6 during evolution raises the hypothesis
that exon 6 RNA sequence may be the target for a sequence-specific RNA
binding factor(s). Consistently, cross-linking experiments show that a nuclear protein(s) specifically interacts with SCF alternative exon.
Although we have not yet obtained direct evidence that the exon
6-binding 69-kDa complex is the one actually involved in the
acidity-mediated splicing, this possibility is highly suggested by the
strict sequence conservation of exon 6 between mammals and even birds
(42), in parallel with the evolutionary conservation of the roles of
SCFs and SCFm. Identifying the nuclear protein(s) interacting with SCF
exon 6 would be of great interest for elucidating a central turning
point of the testis development.
 |
ACKNOWLEDGEMENT |
We thank Dr. O. Chassande for his generous
gift of DNA mouse library.
 |
FOOTNOTES |
*
This work was supported by INSERM U 407, Ministère de
l'Education Nationale, de l'Enseignement Supérieur et de la
Recherche Scientifique (MENESRS), CNRS, Association pour la Recherche
contre le Cancer (ARC) (to D. M.), and in part by European Society for Pediatric Endocrinology (ESPE) Research Fellowship, sponsored by Novo
Nordisk A/S (to C. M.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF083885, AF083886, AF083887, and AF083885.
§
To whom correspondence should be addressed. Tel.:
33-4 78 86 16 07; Fax: 33-4 78 86 59 22; E-mail:
mauduit{at}lsgrisn1.univ-lyon1.fr.
The abbreviations used are:
SCF, stem cell
factor; SCFs, soluble SCR; SCFm, membrane-bound SCF; DME, Dulbecco's
modified Eagle's medium; PCR, polymerase chain reaction; RT, reverse
transcription; dpc, days post-coitum; dpp, days post-partum.
 |
REFERENCES |
-
Morrison, S. J.,
Shah, N. M.,
and Anderson, D. J.
(1997)
Cell
88,
287-298[Medline]
[Order article via Infotrieve]
-
Benahmed, M.
(1996)
Male Infertility: Clinical Investigation, Cause, Evaluation, and Treatment, pp. 55-96, Chapman & Hall, London
-
Robertson, D. M.,
Risbridger, G. P.,
Hedger, M.,
and McLachlan, R. I.
(1993)
Molecular Biology of the Male Reproductive System, pp. 411-438, Academic Press, San Diego
-
Gnessi, L.,
Fabbri, A.,
and Spera, G.
(1997)
Endocr. Rev.
18,
541-609[Abstract/Free Full Text]
-
Griswold, M. D.
(1995)
Biol. Reprod.
52,
211-216[Abstract]
-
Bernstein, S. E.
(1960)
Mouse News Lett.
23,
33-34
-
Anderson, D. M.,
Lyman, S. D.,
Baird, A.,
Wignall, J. M.,
Eisenman, J.,
Rauch, C.,
March, C. J.,
Boswell, H. S.,
Gimpel, S. D.,
Cosman, D.,
and Williams, D. E.
(1990)
Cell
63,
235-243[Medline]
[Order article via Infotrieve]
-
Toksoz, D.,
Zsebo, K. M.,
Smith, K. A.,
Hu, S.,
Brankow, D.,
Suggs, S. V.,
Martin, F. H.,
and Williams, D. A.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
7350-7354[Abstract]
-
Huang, E. J.,
Nocka, K. H.,
Buck, J.,
and Besmer, P.
(1992)
Mol. Biol. Cell
3,
349-362[Abstract]
-
Flanagan, J. G.,
Chan, D.,
and Leder, P.
(1991)
Cell
64,
1025-1035[Medline]
[Order article via Infotrieve]
-
Marziali, G.,
Lazzaro, D.,
and Sorrentino, V.
(1993)
Dev. Biol.
157,
182-190[CrossRef][Medline]
[Order article via Infotrieve]
-
Motro, B.,
Van der Kooy, D.,
Rossant, J.,
Reith, A.,
and Bernstein, A.
(1991)
Development
102,
1207-1221
-
Nakayama, H.,
Kuroda, H.,
Onoue, H.,
Fujita, J.,
Nishimune, Y.,
Matsumoto, K.,
Nagano, T.,
Suzuki, F.,
and Kitamura, Y.
(1988)
Development
102,
117-126[Abstract]
-
Orr-Urtreger, A.,
Avivi, A.,
Zimmer, Y.,
Givol, D.,
Yarden, Y.,
and Lonai, P.
(1990)
Development
109,
911-923[Abstract]
-
Manova, K.,
Nocka, K.,
Besmer, P.,
and Bachavrova, R. F.
(1990)
Development
110,
1057-1069[Abstract]
-
Dym, M.,
Jia, M. C.,
Dirami, G.,
Price, J. M.,
Rabin, S. J.,
Mocchetti, I.,
and Ravindranath, R.
(1995)
Biol. Reprod.
52,
8-19[Abstract]
-
Orth, J. M.,
Jester, W. F.,
and Qiu, J.
(1996)
Mol. Reprod. Dev.
45,
123-131[CrossRef][Medline]
[Order article via Infotrieve]
-
Kuroda, H.,
Nakayama, H.,
Namiki, M.,
Matsumoto, K.,
Nishimune, Y.,
and Kitamura, Y.
(1989)
J. Cell. Physiol.
139,
329-334[Medline]
[Order article via Infotrieve]
-
Brannan, C. I.,
Lyman, S. D.,
Williams, D. E.,
Eisenman, J.,
Anderson, D. M.,
Cosman, D.,
Bedell, M. A.,
Jenkins, N. A.,
and Copeland, N. G.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
4671-4674[Abstract]
-
Dorrington, J. H.,
Roller, N. F.,
and Fritz, I. B.
(1975)
Mol. Cell. Endocrinol.
3,
57-70[CrossRef][Medline]
[Order article via Infotrieve]
-
Dignam, J. E.,
Lebovitz, R. M.,
and R
der, R. G.
(1983)
Nucleic Acids Res.
11,
1475-1489[Abstract] -
Dehbi, M.,
Mbiguino, A.,
Beauchemin, M.,
Chatelain, G.,
and Bedard, P.-A.
(1992)
Mol. Cell. Biol.
12,
1490-1499[Abstract]
-
Bradford, M. M.
(1976)
Anal. Biochem.
72,
248-254[CrossRef][Medline]
[Order article via Infotrieve]
-
Stolow, D. T.,
and Berget, S. M.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
320-324[Abstract]
-
Brannan, C. I.,
Bedell, M. A.,
Resnick, J. L.,
Eppig, J. J.,
Handel, M. A.,
Williams, D. E.,
Lyman, S. D.,
Donovan, P. J.,
Jenkins, N. A.,
and Copeland, N. G.
(1992)
Genes Dev.
6,
1832-1842[Abstract]
-
Allard, E. K.,
Blanchard, K. T.,
and Boekelheide, K.
(1996)
Biol. Reprod.
55,
185-193[Abstract]
-
Rossi, P.,
Dolci, S.,
Albanesi, C.,
Grimaldi, P.,
Ricca, R.,
and Geremia, R.
(1993)
Dev. Biol.
155,
68-74[CrossRef][Medline]
[Order article via Infotrieve]
-
Monk, M.,
and McLaren, A.
(1981)
J. Embryol. Exp. Morphol.
63,
75-84[Medline]
[Order article via Infotrieve]
-
Dolci, S.,
Williams, D. E.,
Ernst, M. K.,
Resnick, J. L.,
Brannan, C. I.,
Lock, L. F.,
Lyman, S. D.,
Boswell, H. S.,
and Donovan, P. J.
(1991)
Nature
352,
809-811[CrossRef][Medline]
[Order article via Infotrieve]
-
Matsui, Y.,
Toksoz, D.,
Nishikawa, S.,
Nishikawa, S.-I.,
Williams, D.,
Zsebo, K. M.,
and Hogan, B. L. M.
(1991)
Nature
353,
750-752[CrossRef][Medline]
[Order article via Infotrieve]
-
Bellvé, A. R.,
Cavicchia, J. C.,
Millette, C. F.,
O'Brien, D. A.,
Bhatnagar, Y. M.,
and Dym, M.
(1977)
J. Cell Biol.
74,
68-85[Abstract/Free Full Text]
-
Borsi, L.,
Balza, E.,
Gaggero, B.,
Allemanni, G.,
and Zardi, L.
(1995)
J. Biol. Chem.
270,
6243-6245[Abstract/Free Full Text]
-
Endo, H.,
Matsuda, C.,
and Kagawa, Y.
(1994)
J. Biol. Chem.
269,
12488-12493[Abstract/Free Full Text]
-
Robinson, R.,
and Fritz, I. B.
(1981)
Biol. Reprod.
24,
1032-1041[Medline]
[Order article via Infotrieve]
-
Jutte, N. H. P. M.,
Grootegoed, J. A.,
Rommerts, F. F. G.,
Clausen, O. O. F.,
and Van der Molen, H. J.
(1982)
J. Reprod. Fertil.
65,
431-438[Abstract]
-
Nehar, D.,
Mauduit, C.,
Boussouar, F.,
and Benahmed, M.
(1997)
Endocrinology
138,
1964-1971[Abstract/Free Full Text]
-
Roosen-Runge, E. C.
(1952)
Ann. N. Y. Acad. Sci.
55,
574-584
-
Kuo, H. C.,
Nassim, F.-U.,
and Grabowski, P. J.
(1991)
Science
251,
1045-1050[Medline]
[Order article via Infotrieve]
-
Chabot, B.
(1996)
Trends Genet.
12,
472-478[CrossRef][Medline]
[Order article via Infotrieve]
-
Berget, S. M.
(1995)
J. Biol. Chem.
270,
2411-2414[Free Full Text]
-
Tarn, W.-Y.,
and Steitz, J. A.
(1997)
Trends Biochem. Sci.
22,
132-137[CrossRef][Medline]
[Order article via Infotrieve]
-
Petitte, J. N.,
and Kulik, M. J.
(1996)
Biochim. Biophys. Acta
1307,
149-151[Medline]
[Order article via Infotrieve]
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