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From the * Department of Medicine, Division of
Endocrinology, Harbor-UCLA Medical Center and Research and Education
Institute, Torrance, California; the Departments of
Physiology,
Surgery, and||
Pediatrics, University of Turku, Turku, Finland;
and the Department of Microbiology and Institute
of Cancer Research, Columbia University, New York, New York.
| Correspondence to: Dr Wael A. Salameh, Division of Endocrinology, Harbor–UCLA Medical Center, Box 446, 1000 W Carson St, Torrance, CA 90509 (e-mail: wsalameh{at}gcrc.rei.edu). |
| Received for publication September 5, 2002; accepted for publication January 10, 2003. |
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Abstract |
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was ubiquitous in the seminiferous tubules,
GSK-3ß was expressed in premeiotic type B spermatogonia, in both meiotic
preleptotene and leptotene spermatocytes, as well as in Sertoli cells in both
the mouse and rat. Thus, GSK-3ß is expressed in germ cells entering
meiosis. In addition, intense immunoreactivity was detected in rat step 6
though 11 spermatids. In situ hybridization (ISH) in rat testis confirmed the
immunostaining pattern in leptotene and spermatids and showed a GSK-3ß
messenger RNA (mRNA) signal in some pachytene spermatocytes. The restricted
pattern of expression suggests cell-specific regulation of Gsk-3ß mRNA.
To determine whether GSK-3 is required for meiosis entry, rat stage VIIa
seminiferous tubule segments were cultured with selective small-molecule GSK-3
inhibitors. These compounds markedly and dose-dependently suppressed meiotic
synthesis (S)-phase DNA. Since a yeast GSK-3 homolog, Rim11p (regulator of
inducer of meiosis), is pivotal to meiosis entry, we tested whether
GSK-3ß complements Rim11p function in meiosis. Rim11p phosphorylates
transcription factors Ume6p (unscheduled meiotic gene expression) and Ime1p
(inducer of meiosis) to induce meiosis entry. Overexpression of murine
GSK-3ß in a rim11 mutant yeast failed to rescue the sporulation defect.
Our finding that GSK-3ß interacted only with Ume6p but not with IME1 in a
yeast 2-hybrid assay suggests that noncomplementation reflects partial
divergence in substrate specificity. This work provides the basis for future
studies of GSK-3ß signaling in mammalian meiosis and spermatogenesis.
Key words: Testis, meiosis, tubule culture, Rim11p
In the past few years, glycogen synthase kinase-3 (GSK-3) has been
implicated in myriad fundamental processes in many species. These processes
range from embryonic cell fate determination
(Siegfried et al, 1994; Klein and Melton, 1996;
Moon et al, 1997) and glycogen
and glucose metabolism (Cross et al,
1997; Summers et al,
1999) to Alzheimer disease
(Imahori and Uchida, 1997),
oncogenesis (Hart et al, 1998;
Sparks et al, 1998;
Yost et al, 1998), and
apoptosis (Hoeflich et al,
2000; Cross et al,
2001; Lucas et al,
2001). GSK-3 is conserved in all eukaryotes examined and exists as
and ß isoforms in mammals, with GSK-3ß being more thoroughly
studied. In unstimulated cells, GSK-3 stays active and, through
phosphorylation, inhibits many of its targets, including transcription factors
such as the bipartite protein complex ß-catenin/T-cell factor protein
complex, the Period/Timeless protein complex, and the c-Jun protein family, as
well as the Even-skipped protein, the CCAAT enhancer binding proteins, and the
nuclear factor of the activated T-cell protein complex
(Woodgett, 2001). Because of
its negative regulation on growth, it is inactivated by distinct sets of
growth factor signals, such as phosphatidylinositol 3'-kinase signaling
and Wnt signaling (ibid). In addition to the antagonism of mitogenic
signaling, GSK-3ß may directly control cell cycle mechanisms by
phosphorylating cyclin D1, thus prompting the latter's nuclear exit and
subsequent proteolysis (Diehl et al,
1998). Recent evidence also suggests that GSK-3ß mediates
cell cycle arrest in Xenopus oocytes
(Fisher et al, 1999).
Interestingly, several pieces of evidence indicate that some of the signals governing meiosis entry may be conserved from yeasts to mammals. This is first borne out by a genome-wide profiling of temporal gene expression patterns during yeast meiosis (Chua and Roeder, 1998), which uncovered several yeast meiotic genes with vertebrate homologs that might play conserved roles in gametogenesis, and second, by biochemical evidence showing that a key member of the nutritional cascade governing yeast meiosis, Rim11p (regulator of inducer of meiosis), is a homolog of GSK-3ß (Bowdish et al, 1994). A more distant yeast homolog of GSK-3ß called Mck1p (meiosis centromere kinase) also plays a role at the level of the nutritional regulation of meiotic entry, along with other roles in vegetative growth and centromere segregation (Neigeborn and Mitchell, 1991). Under conditions of nitrogen limitation, which promote meiotic entry, both Rim11p and Mck1p contribute to the phosphorylation of a common substrate, a DNA-binding protein/transcription factor called Ume6p (unscheduled meiotic gene expression), thus promoting early meiotic gene expression and sporulation (Xiao and Mitchell, 2000). Given the importance of GSK-3ß in cell fate determination, its expression in the testis, and its homology to Rim11p and Mck1p, we decided to study its function during spermatogenesis.
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Materials and Methods |
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Animals and Sources of Tissues
Animal protocols and procedures were approved by both the Research and
Education Institute Animal Care and Review Committee and the Turku University
Committee on Ethics of Animal Experimentation. Adult male Sprague-Dawley rats
and C57Bl/6 mice were used. Animals were housed at 21°C with a constant
light-dark cycle and allowed food and water ad libitum. For RNA and protein
extraction, rats were sacrificed by CO2 asphyxiation, and mice were
killed by rapid cervical dislocation. The testis was quickly dissected,
decapsulated, snap frozen in liquid nitrogen, and stored at -70°C. For
tissue fixation, animals were anesthetized with pentobarbital (Sigma Chemical
Co, St Louis, Mo) prior to intraventricular perfusion with Bouin fixative.
Testes were removed and postfixed in Bouin solution overnight, transferred to
70% ethanol, and embedded in paraffin blocks. Tissues were sectioned at 5
µm and mounted on Probe-on slides (Fisher Scientific, Pittsburgh, Pa).
Northern Blots
Total RNA was isolated from tissues by TRIzol reagent (Life Technologies).
RNA samples (20 µg/lane) were electrophoresed on denaturing 1.2%
agarose/2.2 M formaldehyde gels, transferred to MagnaGraph nylon membranes
(Micron Separations, Westboro, Mass), and ultraviolet light crosslinked. A
BamHI cDNA restriction fragment from the 3'RACE product of
Gsk-3ß was 32P labeled by random priming (Prime-a-gene, Promega,
Madison, Wis) and used as a probe. Hybridization was performed in ExpressHyb
solution (Clontech) at 68°C for 1 hour. Blots were washed to a final
stringency of 0.1x SSC, 0.1% sodium dodecyl sulfate at 50°C, and
were exposed to Fuji x-ray film (Fisher Scientific) at -70°C
overnight.
In Situ Hybridization
A 414-bp HindIII-EcoRI fragment from the cloned
Gsk-3ß 3'RACE product was subcloned into pBluescript II
KS(-) (Stratagene, La Jolla, Calif). This fragment, corresponding to the amino
acid (aa) 333–420 region and an additional 3' untranslated region
of the Gsk-3ß mRNA, was chosen to avoid cross-hybridization with
Gsk-3. Sense and antisense riboprobes were generated by the
incubation of either a HindIII or EcoRI linearized template
with digoxigenin-labeled uridine triphosphate in the presence of T7 phage
(sense) or T3 phage (antisense) RNA polymerases according to the
manufacturer's recommendations (Boehringer Mannheim, Indianapolis, Ind). The
labeled cRNAs were precipitated and quantitated by comparison to a
digoxigenin-labeled RNA standard. Except for the following modifications, ISH
was performed as previously described
(Millar et al, 1993): 1)
dithiothreitol was omitted from the (pre)hybridization buffer, and RNase A
treatment was omitted during the posthybridization washes; 2) the
hybridization temperature was 50°C; and 3) color development lasted 5
hours. The sections were counterstained with nuclear fast red (Sigma-Aldrich,
Milwaukee, Wis) for 10 minutes. Sections were photomicrographed by a camera
mounted on an Olympus BH2 light microscope (Olympus Optical, Tokyo,
Japan).
Western Analysis
Protein extraction and blotting were performed as previously described
(Yamamoto et al, 2000), except
that prior to sample loading, the mouse lysate was precleared of endogenous
immunoglobulin G (IgG) by protein A/G agarose beads (Santa Cruz Biotechnology,
Santa Cruz, Calif). The primary antibody was anti–GSK-3ß monoclonal
Ab (Transduction Laboratories, Lexington, Ky) at 1:2500, and the secondary
antibody was a horseradish peroxidase–conjugated goat anti-mouse IgG
(Biorad, Hercules, Calif) at 1:2000.
Rat Immunohistochemistry
We applied a tyramide signal amplification (TSA) method to accentuate the
signal in the basal compartment of the seminiferous tubule, which was
overwhelmed by the intense signal in the haploid lineage in conventional
immunohistochemistry (IHC). Tissue sections were deparaffinized in xylene and
hydrated through a graded ethanol series. Endogenous peroxidase activity was
quenched with DAKO Peroxidase Blocking Reagent (DAKO, Carpinteria, Calif) for
20 minutes. Sections were blocked in TNB Blocking Buffer (TSA-Indirect Kit,
NEN Life Science Products, Boston, Mass). They were incubated with
anti–GSK-3ß at 1:50 in TNB Blocking Buffer for 2 hours at 37°C
and then incubated with a biotinylated horse anti-mouse IgG secondary antibody
(Oncogene Research Products, Cambridge, Mass) at 1:100 in TNB Blocking Buffer
for 1 hour at 37°C. TSA was employed for chromogenic signal enhancement
according to the manufacturer's instructions. All subsequent incubations were
carried out at room temperature. Briefly, sections were washed in a TNT buffer
and incubated in Streptavidin Horseradish Peroxidase solution at 1:100 for 30
minutes. Biotinyl tyramide (1:50) was added and incubated for 10 minutes, and
then Streptavidin-Alkaline Phosphatase (Biorad) was added at 1:200 and
incubated for 30 minutes. After several washes in TNT buffer, sections were
preequilibrated for 3 minutes in 0.1 M Tris-buffered saline, pH 9.5,
containing 50 mM MgCl2 (TBS-MgCl2). Color development,
which lasted 5 to 20 minutes, was performed in the dark with the addition of a
mixture of 4-nitroblue tetrazolium chloride (337 µg/mL),
5-bromo-4-chloro-3-indolyl-phosphate (175 µg/mL), and 1 mM levamisole in
TBS-MgCl2, pH 9.5. The slides were counterstained, mounted, and
viewed as described in "In Situ Hybridization." For the
GSK-3 protein, a conventional IHC method was used. The primary sheep
antibody (Upstate Biotechnology, Lake Placid, NY) was used at a 1:50 dilution,
and the secondary biotinylated donkey antibody (Sigma) was used at a 1:500
dilution.
Murine IHC
In the absence of haploid staining, a conventional IHC method was
sufficient to detect GSK-3ß in the mouse testis. Vectastain ABC-AP and
avidin-biotin blocking kits were used (Vector Labs, Burlingame, Calif).
Sections were incubated for 1 hour at room temperature with 1.5% normal goat
serum in phosphate-buffered saline (NGS in PBS, pH 7.4) premixed with a 1:6
dilution of avidin solution. They were then incubated overnight at 4°C
with a 1:25 dilution of anti–GSK-3ß in 1% NGS in PBS premixed with
a 1:6 dilution of biotin solution. For negative controls, both nonimmune mouse
IgG (Sigma) and omission of the primary antibody gave adequate results. The
next day, sections were each washed 3 times in PBS for 5 minutes, and then a
biotinylated goat anti-mouse IgG1 antibody was incubated for 30 minutes at
room temperature (Santa Cruz Biotechnology) in 1% NGS in PBS at a dilution of
1:150. After 3 more PBS washes, sections were incubated with a premixed ABC-AP
reagent for 30 minutes at room temperature. The sections were washed once in
PBS and twice in TBS (pH 7.5) and then transferred to TBS-MgCl2.
Color development was performed as described above.
Transillumination-Assisted Microdissection of Rat Seminiferous Tubule
Segments
Stage VIIa of the seminiferous epithelial cycle was selected, since
premeiotic cells are in the gap 1 (G1) phase at this point, and the transition
to the meiotic synthesis (S) phase can be studied during a 72-hour culture
(Parvinen et al, 1991).
Two-millimeter seminiferous tubule segments were isolated under a
transilluminating stereomicroscope for in vitro analyses of DNA synthesis.
Stages were identified as described earlier
(Parvinen and Vanha-Perttula,
1972).
Tissue Cultures and Thymidine Incorporation Measurement
Tubule segments were transferred to 96-well plates in 10 µL of PBS and
incubated at 34°C for 72 hours in 100 µL of Ham F12/Dulbecco MEM (Life
Technologies) supplemented with 0.1% bovine serum albumin (Sigma),
G-penicillin 60 mg/L (Sigma), and streptomycin 500 mg/L (Sigma) in a
humidified atmosphere containing 5% CO2 in air. GSK-3 inhibitors
SB-216763 and SB-415286 (GlaxoSmithKline Pharmaceuticals, Harlow, Essex,
United Kingdom) dissolved in dimethylsulfoxide (DMSO) were added to the
culture media at 10 or 100 µM. Additional tubules were incubated without
the inhibitors or with the solvent DMSO alone. Additional controls were
lithium chloride (LiCl) or potassium chloride (KCl) at 10, 20, and 40 mM.
Tubules were pulse labeled during the last 4 hours of the culture by adding 20
kilobecquerels of [methyl-3H]thymidine (TRK 120, 185
gigabecquerels/mmol, Amersham). The cultures were harvested on filter discs
(Whatman 934-AH) with a continuous flow of distilled water for 1 minute. A
scintillation wax (MeltiLexA 1450-441, Wallac Oy, Turku, Finland) was melted
on the filters, and the radioactivity was measured by a flatbed liquid
scintillation counter equipped with 2 parallel detectors (1450 Microbeta,
Wallac Oy). Data (mean ± SEM) from 4 separate experiments, in which
each point was assayed in sextuplicate, were analyzed by three-way analysis of
variance with a Tukey test using SigmaStat (SPSS, Chicago, Ill). The level of
statistical significance was .05.
Yeast 2-Hybrid Interactions Between GSK-3ß and Inducer of
Meiosis or Ume6p
In order to determine whether GSK-3ß was able to interact with the
known and well-characterized substrates of its yeast homolog Rim11p, we used a
yeast two-hybrid protein functional assay
(Brent and Finley, 1997).
Briefly, in this assay, 2 plasmids are constructed to express 2 distinct
fusion proteins in 2 distinct yeast strains. The first plasmid encodes the
GAL4 DNA-binding domain (GBD) cloned in frame and upstream from GSK-3ß.
This is designated the "bait" vector. The second plasmid en codes
for another fusion protein containing the GAL4 activation domain (GAD) cloned
in frame and upstream of the "prey" sequence (in our case, either
a yeast inducer of meiosis [Ime1p] or the C-terminal of Ume6p). The 2 yeast
strains expressing either the "bait" or the "prey"
plasmids are then mated, and diploid strains coexpressing both plasmids are
selected through nutritional markers. The generated diploid yeast strains
harbor a reporter gene (such as ß galactosidase) under the control of the
GAL4 promoter. Mated diploid yeast strains coexpressing the bait and prey
plasmids are then cultured in media either unfavorable to the interaction of
Rim11p and its substrates and, thus, meiosis (such as glucose) or favorable to
such an interaction and to meiosis initiation (acetate). A positive
interaction between the 2 fusion proteins (GBD–murine GSK-3ß
[mGSK-3ß] with either GAD-IME1 or GAD-Ume6p) will bring in proximity the
DNA-binding domain and activation domains of the GAL4 promoter in the diploid
yeast strain, leading to expression of the reporter gene and giving a blue
color. The intensity of the color is proportional to the intensity of the
interaction. To generate the GBD–mGSK-3ß bait vector
(pAS2/mGSK-3ß, "GBD–mGSK-3ß" hereafter), we cloned
a reverse transcriptase (RT)-PCR fragment corresponding to the aa region
2–420 of mGSK-3ß into the NcoI site of pAS2. The frame of
translation and entire sequence were verified. The following GAD containing
prey vectors pACTII (empty), pYX132 (harboring the well-characterized minimum
sequence required for Rim11p–Ume6p interaction: N-terminal aa:
1–161 Ume6p), and pYX133 and pYX134 (harboring the mutations T99N and
Ala5 of Ume6p, which block Rim11p–Ume6p interactions, respectively
[Malathi et al, 1997,
1999]) were transformed into
yeast strain Y187 ( gal4 gal80 his3 trp1–901 ade2–101
leu2–3112 ura3–52::URA3-GAL1-lacZ), and the transformants
were selected on synthetic complete(SC)-Leu plates. GBD fusion plasmids
GBD–Rim11p (Malathi et al,
1999) or GBD–mGSK-3ß, described above, were transformed
into yeast strain Y190 ( gal4 gal80 his3 trp1 ade2 ura3 leu2
URA3::GAL1-lacZ LYS2::GAL1-HIS3 cyhr), and the transformants
were selected on SC-Trp plates. Strains were mated, and diploids were selected
on SC-Trp-Leu plates. Independent transformants were grown overnight in
SC-Trp-Leu medium, diluted 1:30 into acetate (yeast peptone acetate medium) or
glucose (yeast peptone dextrose medium), and harvested after 2 to 3 doublings
(8 to 12 hours). Yeast lysates were subjected Western blotting, and a
monoclonal antibody against GBD (Clontech) was used at a 1:5400 dilution to
detect the GBD–GSK-3ß fusion protein. ß-Galactosidase assays
were conducted on permeabilized cells, and quantitative determinations
expressed in Miller units were averages from 3 independent transformants.
Interactions between GSK-3ß and wild-type Ume6p or Ime1p were also
verified through transformation of yeast strain Y190 carrying
GBD–GSK-3ß with either pAC-Ime1p (GAD–Ime1p fusion plasmid)
or pYX132. Colonies growing on SC-Leu-Trp were tested for the LacZ
reporter gene expression in a filter lift assay after 3 days.
Functional Complementation Assay for a rim11 Mutant
In order to test for the ability of mGSK-3ß to complement a
rim11 mutation, we generated a yeast "knockout" for
rim11. The lack of expression of Rim11p in this diploid strain would
hamper its ability to sporulate (undergo meiosis), leading to the lack of
tetrad formation (4 daughter haploid yeast cells enclosed in asci). So, tetrad
analysis allows testing for the occurrence of successful meiotic completion
(ie, sporulation). If GSK-3ß is able to fully complement or substitute
for the function of Rim11p, then transformation of GSK-3ß into a
rim11 mutant strain would allow for the resumption of meiosis and
tetrad formation. To this end, we tested for sporulation in a
rim11-/rim11- diploid strain
designated YX425x445 after transformation with 1 of 3 plasmids at a time: GBD,
GBD-Rim11p, or GBD-GSK-3ß. Sporulation was measured for 3 independent
transformants after incubation on sporulation plates for 5 days at
30°C.
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Results |
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Gsk-3ß mRNA Testis Expression
Northern blotting indicates that Gsk-3ß mRNA appears as a
single 1.6-kb transcript in both the rat and mouse testis
(Figure 2A). We further
demonstrate by in situ hybridization (ISH) that Gsk-3ß mRNA has
a restricted pattern of expression in male germ cells. In the rat, the signal
for Gsk-3ß mRNA is close to the background in type B
spermatogonia and preleptotene spermatocytes (Figure
3D and
E). The expression is strong in
leptotene spermatocytes (Figure
3F and
G) but becomes minimal in
zygotene and early-to-midpachytene spermatocytes (Figure
3G and
H and
C through
E). There is a transient surge
of expression in late pachytene cells (stages IX to XI), but it is
substantially diminished at the execution of meiosis in stage XIV
(Figure 3H). Postmeiotically,
Gsk-3ß mRNA expression is highest in step 7 to 12 spermatids.
Indeed, this spermatid expression appears strongest in the whole testis
(Figure 3E and
F). A schematic representation
of this pattern of expression is shown in
Figure 3, panel Q. No staining
is present in the interstitium except for occasional blood vessel wall
staining. Thus, the cyclical pattern of expression suggests a coordinated
transcriptional upregulation at the middle-to-late stages of the seminiferous
epithelial cycle, when events such as meiosis initiation and spermatid
elongation occur.
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Western Analysis and IHC
Western blotting using a monoclonal antibody recognized specifically a
47-kd band corresponding to GSK-3ß in both rat and mouse testis lysates
(Figure 2B). This antibody is
also used for IHC.
For GSK-3ß IHC of rat testis, there is cytoplasmic immunostaining in type B spermatogonia and preleptotene spermatocytes (Figure 3L through N), even though the mRNA signal is barely detectable by nonradioactive ISH in these cells (Figure 3D and E). This difference in signal strength could be a reflection of the lower sensitivity of the ISH procedure or a reflection of the differences in RNA vs protein turnover. Strong staining is observed in leptotene but not in zygotene, pachytene, diplotene, or dividing spermatocytes. Step 5 through 11 spermatids are also highly immunoreactive (Figure 3L through P). In addition, GSK-3ß is expressed in Sertoli cells (Figure 3M). The germ cell expression pattern is schematized in Figure 3, panel R. In the mouse testis, robust signals are observed in type B spermatogonia and preleptotene and leptotene spermatocytes (Figure 4A through D). A conspicuous difference in the rat results is that mouse haploid cells lack staining. Results are summarized in Figure 4E.
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We have also examined the expression of GSK-3 for comparison. In
contrast to GSK-3ß, GSK-3 is ubiquitously and abundantly expressed
(Figure 4F) in rat seminiferous
tubules, with no discernible stage or cell type specificity. Similar results
(data not shown) are found in mouse testes.
Evidence That GSK-3 May Stimulate Meiotic DNA Synthesis in Tubule
Culture
Because GSK-3ß is expressed early during meiosis I, we sought to
determine whether it might be required for the meiotic S phase. We examined
DNA synthesis in cultured stage VIIa rat seminiferous tubules, which included
preleptotene spermatocytes at the G1 phase of meiosis I
(Parvinen et al, 1991). During
the 72-hour culture, preleptotene cells activated and entered into the meiotic
S phase. Active DNA synthesis was detected by measuring radioactive thymidine
incorporated after pulse labeling the samples during the last 4 hours of
culture (Figure 5). To
determine whether GSK-3 regulates this process, we used 2 small-molecule
inhibitors of GSK-3, SB-216763 and SB-415286. These compounds have been found
to be selective inhibitors of GSK-3 among 25 protein kinases that have been
tested (Coghlan et al, 2000).
Coghlan et al (2000) reported
the inhibitory concentration at 50% (IC50) values of these
compounds at 0.01 mM adenosine triphosphate (ATP) to be less than 100 nM. They
further suggested that the concentrations of these compounds should be roughly
100-fold higher than the IC50 values in order to observe effects in
cells where the ATP concentration is 1 to 2 mM. On the basis of their studies,
we tested concentrations of 10 and 100 µM for our experiment. For
comparison, we also tested LiCl, a known inhibitor of GSK-3, or its monovalent
cation control, KCl, at concentrations widely used to inhibit GSK-3: 10, 20,
and 40 mM.
To make sure there was no toxic effect during culture, tubule viability was examined by phase-contrast microscopy. In cell squash preparations, healthy tubules readily extrude their cell contents, which spread into a monolayer (Parvinen and Hecht, 1981). Perished tubules are visualized by their blighted appearance and the inability to extrude their contents. Significantly, whereas tubules incubated with the SB (SmithKline Beechum group) compounds or the vehicle DMSO or K+ were as healthy as the untreated tubules, they were not viable when cultured with Li+, even at the lowest concentration. Since Li+ inhibits a number of related phosphatases such as inositol monophosphatase (IMPase) at a Ki less than that of GSK-3 (Phiel and Klein, 2001), it is possible that the toxic effect of Li+ is due to the attenuation of multiple pathways, including inositol-1,4,5-triphosphate signaling, owing to inositol depletion stemming from IMPase inhibition (Atack, 1996). Thus, lithium is not suitable in this model of tubule culture to assess GSK-3 function.
In contrast, the treatment of tubules with SB-415286 reduced DNA synthesis
to 35% and 22% of the vehicle-treated controls at 10 and 100 µM,
respectively (Figure 5).
SB-216763 had a milder effect, inhibiting DNA synthesis to 57% of control
levels at the 100-µM concentration. This is due to its insolubility, which
was observed in this experiment as had been reported previously
(Coghlan et al, 2000). The
finding that both GSK-3 inhibitors cause a reduction in DNA synthesis provides
support for the hypothesis that GSK-3 promotes meiotic DNA synthesis, though
our results cannot distinguish between GSK-3 and GSK-3ß
inhibition.
Functional Relationship Between GSK-3ß and Yeast Rim11p
The yeast GSK-3 homolog Rim11p is essential for the initiation of meiosis
(Bowdish et al, 1994). It is
expressed during meiotic prophase, and then expression levels decline
(Chua and Roeder, 1998). Through its regulation of IME2 transcription, it may also indirectly
regulate the meiotic S phase. These similarities between GSK-3ß and
Rim11p led us to examine whether GSK-3ß might substitute for Rim11p in
yeast. For this, we used a biological assay, which consisted of the production
of meiotic tetrads, and a protein functional assay, which consisted of the
interaction between GSK-3ß and the Rim11p substrates Ime1p and Ume6p.
GSK-3ß was introduced into the yeast as GBD–GSK-3ß, which was
readily detected by Western blotting
(Figure 6). In the biological
assay, we found that the expression of GBD–GSK-3ß did not permit
tetrad production by a rim11-/rim11-
mutant strain (<0.1%). A control GBD–Rim11p fusion promoted efficient
tetrad production (92%). These results suggest that GSK-3ß cannot fully
substitute for Rim11p. Rim11p interacts with the N-terminal portion of Ume6p
and the C-terminal portion of Ime1p
(Malathi et al, 1997;
Xiao and Mitchell, 2000), and
both of these interactions are vital for meiosis. Two-hybrid assays revealed
that murine GSK-3ß interacts with Ume6p but not with Ime1p. The
GSK-3ß–Ume6p interaction has the same physical requirements as the
Rim11p–Ume6p interaction, because mutations in Ume6p that
attenuate its interaction with Rim11p also attenuate interaction with
GSK-3ß (Table). The
interactions also respond to the same nutritional signals: interaction is
stronger in cells grown in acetate medium than in cells grown in glucose
medium (Table). Thus,
GSK-3ß and Rim11p have very similar interactions with Ume6p. Our findings
suggest that GSK-3ß cannot substitute for Rim11p in yeast meiosis because
they differ in their interaction with Ime1p.
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Discussion |
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Prior studies have shown that the expression of GSK-3 RNAs and proteins is
fairly ubiquitous among tissues in the rat, with the brain having the highest
levels (Woodgett, 1990;
Takahashi et al, 1994). Generally, the isoform is more abundant than the ß isoform
(ibid). Both isoforms have been detected in the bovine testis
(Vijayaraghavan et al, 1996).
We have extended those observations with the finding that GSK-3ß is
tightly regulated in a cell-specific manner at the mRNA level in both the
mouse and rat testis. The most important finding from this work is that
GSK-3ß is expressed in rodent type B spermatogonia and preleptotene
spermatocytes, an observation that agrees well with the hypothesis that
GSK-3ß has a role in meiosis initiation. Also, the fortuitous difference
between the mouse and rat—that mouse haploid cells express
GSK-3ß—permits us to suggest that the most conserved function of
GSK-3ß may be confined to the early events of meiosis, while this
expression during haploid differentiation suggests other species-specific
functions such as in spermatid elongation.
Expression data alone cannot provide detailed functional information, so we
have used the pharmacological inhibitors SB-216783 and SB-415286 to test
whether GSK-3ß may be required for meiotic DNA synthesis in tubule
culture. The inhibitors inhibit both GSK-3 and GSK-3ß in vitro
(Coghlan et al, 2000), so we
cannot ascribe their effects to functions of GSK-3ß alone. However, the
inhibitors we have used are nontoxic, are more selective than Li+,
and are also useful at a 10- to 20-fold lower dose
(Cross et al, 2001). They
clearly can inhibit GSK-3ß activity in cultured cells, as indicated by
the stabilization of ß-catenin (Cross
et al, 2001). We observed that both SB-216783 and SB-415286
inhibited meiotic DNA synthesis. This observation permits us to refine our
hypothesis: we suggest that GSK-3ß functions in a manner that promotes
meiotic DNA synthesis.
Finally, we have examined the GSK-3ß function in a heterologous system, the budding yeast Saccharomyces cerevisiae, to see whether its function in meiotic regulation might be conserved. While a budding yeast is only distantly related to humans and animals, it is noteworthy that many functions in DNA metabolism, cell cycle regulation, and intracellular protein trafficking are conserved between humans and this simple eukaryote. We have observed that murine GSK-3ß is capable of interaction with Ume6p, a key regulator of early meiotic transcription in yeast (Vershon and Pierce, 2000). The yeast GSK-3ß homolog interacts with both Ume6p and Ime1p to facilitate the activation of early meiotic genes and meiotic DNA synthesis. Our findings are consistent with the idea that murine GSK-3ß has diverged sufficiently from Rim11p that the interaction with Ime1p is lost. However, the finding that interaction with Ume6p is conserved is consistent with some functional conservation of GSK-3ß and Rim11p.
We suggest that these findings together should motivate more concise tests of the hypothesis that GSK-3ß is required for mammalian spermatogenesis. Among many possible approaches, an accessible strategy is to determine whether spermatogenesis-specific promoter/enhancer regions might be stimulated by GSK-3ß activity, either in cell culture or in transgenic animals. Ultimately, we believe that transgenic alteration of GSK-3ß expression will yield the clearest evidence to confirm or refute this hypothesis. Because a GSK-3ß knockout mutation is an embryonically lethal approach (Hoeflich et al, 2000), we will need to create conditional expression constructs for the analysis of a specific GSK-3ß function in spermatogenesis.
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70-kd band on a Western blot that is detected by a monoclonal antibody
against the GBD. It is absent in the untransformed parent strain.