From the Department of Obstetrics and Gynecology and
¶ Department of Molecular and Human Genetics, Baylor College
of Medicine, Houston, Texas 77030
Received for publication, October 28, 2002, and in revised form, January 27, 2003
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ABSTRACT |
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The germ cell-deficient (gcd)
mutation is a recessive transgenic insertional mutation leading to a
deficiency of primordial germ cells (PGCs). We have recently shown that
the gene underlying this mutation is Pog, which is
necessary for normal proliferation of PGCs. Here we show that
Pog is also involved in spermatogenesis in that meiosis is
impaired in Pog-deficient mice. Yeast two-hybrid screening
revealed that POG interacted with GGN1 and GGN3, two proteins formed by
alternate splicing of the same gene, gametogenetin (Ggn).
Ggn had more than 10 different splice variants giving rise to three proteins, GGN1, GGN2, and GGN3. The three proteins had different subcellular localizations, with GGN1, GGN2, and GGN3 localized along the nuclear membrane, in the cytoplasm, and in the
nucleus/nucleoli respectively. The expression of Ggn was
confined to late pachytene spermatocytes and round spermatids, a time
window concomitant with the occurrence of meiosis. Mouse
Ggn and Pog were both expressed in primary
spermatocytes. Co-expression of POG with GGN1 or GGN3 in HeLa cells
changed the localization of POG to the perinuclear localization or the
nucleoli, respectively. Our data showed that in addition to functioning
in proliferation of primordial germ cells, POG also functioned in
spermatogenesis. Two spatial and temporal regulated proteins, GGN1 and
GGN3, interacted with POG, regulated the localization of POG, and
played a role in spermatogenesis.
Mouse primordial germ cells
(PGCs)1 are generated ~6
days after fertilization by the epiblast cells along the border of the epiblast and extraembryonic ectoderm (1). Bmp4 and
Bmp8 secreted by the neighboring extraembryonic ectoderm
cells are two important signals for this induction (2, 3). Thereafter,
the PGCs move out of the epiblast, and at 7.5-8 days postcoitum (dpc)
they are at the base of the allantois as a pool of about 100 tissue-nonspecific alkaline phosphatase-positive cells (4). A process
of proliferation and migration of the PGCs is followed until, at 13.5 dpc, they number about 20,000 in each gonad (5). Several genes are
known to function in this process. Mutations in Kit and its
ligand Kitl affect the proliferation and migration of PGCs
(6-8), deletion of Tial1 (mTIAR) affects their survival
(9), and Itgb1 (integrin After 13.5 dpc, germ cells in the male and female take different
developmental pathways (12). In the male, PGCs are arrested in mitosis
at 13.5 dpc. They resume mitosis after birth, the prospermatogonia, establish a stem cell pool, and start spermatogenesis (13). In the
female, the germ cells enter meiosis and are arrested at the diplotene
stage of meiosis I, 5 days after birth. Following a growth period, the
oocytes resume meiosis at puberty and arrest again at metaphase II.
Only after fertilization do the oocytes complete the meiosis. Unlike in
the male line, female germ cells do not form a self-renewing stem cell
population. They exist as a finite population, the number of which is
fixed at birth.
The prophase of meiosis I in both sexes is notably long, which includes
stages of leptotene, zygotene, pachytene, and diplotene (13). In the
prophase, a series of meiosis-unique events take place. For example,
chromosomal axes begin to condense in leptonema, chromosome pairing and
synapsis initiate in zygonema, synapsis completion and genetic
recombination occur in patchnema, and desynapse happens in diplonema
(14). A number of meiosis-specific proteins are known to express during
this period, these including the synaptonemal complex proteins (SYCP1
to -3) (15), germ cell-specific transcription factor SPRM1, and germ
cell-specific histone protein H1t (H1Ft) (16, 17). Identifying all of
the players in the process is necessary for the better understanding of meiosis.
The germ cell-deficient (gcd) mutant is a transgenic
insertional mutation showing a reduced number of PGCs in genital ridges of homozygotes from 9.5 dpc (18-20). Recently, we have shown that the
gene responsible for this phenotype is Pog
(proliferation of germ cells),
which encodes a novel protein containing a plant homeodomain (PHD)
motif at its C terminus (19). During the embryonic stage,
Pog is involved in proliferation but not migration of PGCs. It is also involved in other aspects of the embryonic development, since in certain genetic backgrounds deletion of Pog leads
to lower embryonic viability. Further investigation of the
gametogenesis of Pog-deficient mice revealed that
Pog-deficient females were sterile throughout their lives,
whereas Pog-deficient males eventually became fertile at the
age of 3-4 months due to the population of the tubules with
spermatogonial stem cells and the resumption of the spermatogenesis
(21). Here we show that in Pog In an attempt to identify interacting protein partners of POG in the
testis, we carried out yeast two-hybrid screening using an adult testis
cDNA library. A novel germ cell-specific gene, gametogenetin
(Ggn), was found to encode proteins interacting with POG.
Consistent with the functioning of Pog in spermatogenesis, Ggn was highly expressed in the adult gonad, specifically in
germ cells from the late pachytene spermatocyte to the round spermatid stage. Single spermatocyte RT-PCR showed that Ggn and
Pog were both expressed in the primary spermatocytes.
Multiple splicing of Ggn pre-mRNA gave rise to at least
three different proteins, GGN1, GGN2, and GGN3, showing a perinuclear,
cytoplasmic, and nucleolar localization, respectively. When POG was
co-expressed with GGN3 in HeLa cells, the localization of POG switched
from a ubiquitous intracellular localization to an essential nucleolar specific localization, whereas when POG was co-expressed with GGN1, it changed to a perinuclear localization. Our data suggested that
Pog also functioned in spermatogenesis. GGN1 and GGN3, two proteins produced from Ggn by alternative splicing,
interacted with POG and regulated its subcellular localization.
Testis Histology--
Generation of Pog-deficient
mice has been previously described (19). Tissues were fixed overnight
at 4 °C in either 4% paraformaldehyde or Bouin's solution and were
dehydrated, embedded in Paraplast X-tra (Fisher). 8-µm sections were
stained with hematoxylin/eosin or periodic acid-Schiff reagent.
Russell's system was adopted for staging of the seminiferous tubules
(13).
Flow Cytometry Analysis of Testicular Cells--
A monocellular
suspension of testicular cells was prepared as described (22). Briefly,
the tunica albuginea was removed, and the seminiferous tubules were
minced in PBS (calcium- and magnesium-free) to release the testicular
cells. The minced tissue was gently aspirated for 2 min, and the cells
were washed in PBS and spun down at 800 × g for 10 min. The cells were resuspended in PBS, filtered through 80-µm nylon
mesh, fixed in cold 70% ethanol, and kept at 4 °C until further
analysis. For propidium iodide staining, 2 × 106
cells were washed twice with PBS and incubated in 300 µl of 0.5% pepsin in 0.9% saline, pH 2.0, at 37 °C for 10 min. After spinning down, the cells were incubated with propidium iodide staining solution
(25 µg/ml propidium iodide, 40 µg/ml RNase, 0.3% Nonidet P-40 in
PBS) at 37 °C for 30 min. Flow cytometry was performed with a
Coulter EPICS cytometer (Coulter, Krefeld, Germany). 20,000 cells were
counted for each sample.
Yeast Two-hybrid Screening--
Full-length Pog
cDNA was cloned in the EcoRI site of pGBKT7
(Clontech) to make a fusion protein between the
yeast GAL4 DNA binding domain and POG. The primers for amplifying
full-length Pog cDNA were PogF1
(5'-acgtgaattcatggacgaagcagaagcaag-3') and PogR1
(5'-ggttgaattcaaggttttctcccagaca-3'). To delete the C-terminal PHD
domain from the fusion protein, pGBKT7/Pog was cut with
BamHI, and the large fragment was religated with T4 DNA
ligase to obtain plasmid pGBKT7/Pog In Situ Hybridization--
The 3' 300 bp of the Ggn1
cDNA coding region was amplified and cloned into the
EcoRI site of pBluescript KS(II). Digoxigenin-labeled antisense and sense RNA probes were prepared using a digoxigenin RNA-labeling kit (Roche Molecular Biochemicals). Hybridization was
carried out at 65 °C for 18 h. After stringent washing, bound probe was detected by alkaline phosphatase-conjugated anti-digoxigenin antibody and BM purple (Roche Molecular Biochemicals). Serial sections
were stained with periodic acid-Schiff and staged using Russell's
system to define the stage and cell type that express Ggn1.
Single Primary Spermatocyte RT-PCR Analysis--
Testicular germ
cells were dissociated as described above. Single primary spermatocytes
were picked under a Leica microscope by virtue of their large cell
size. Single cell RT-PCR was performed as described (23) with
modifications. Briefly, each cell was lysed in 8 µl of first strand
cDNA synthesis buffer, which was made by mixing 96 µl of
cDNA/lysis buffer (1× alternate first strand buffer from the
Ambion Retroscript kit, with 0.52% Nonidet P-40), 2 µl of RNase
inhibitor (Invitrogen), 1.33 µl of 2.5 mM dNTPs, and 0.67 µl of 50 µM random decamers (Ambion, Austin, TX). The
lysate was incubated at 65 °C for 3 min and cooled at room temperature for 3 min. 4 µl of lysate from each cell was incubated with 0.5 µl of reverse transcriptase at 37 °C for 30 min, and the
other 4 µl of lysate from the same cell was similarly treated without
reverse transcriptase to serve as a control. 1.5 µl of the RT product
(with or without reverse transcriptase) was used as the template for
both Ggn and Pog PCR. Primers for Pog
PCR were GCD2F (5'-TCCAACAGAGAATGAAGCACTC-3') and SplicingR
(5'-CAGATTCCACAGTCCATGCT-3'). Primers for Ggn PCR were 215F
(5'-GGCAGTGATCTGATCTTTGGTCG-3') and 481R (5'-AGTTGATGGTGCTGGCGGTAG-3').
PCR was performed using the following cycle profile: one cycle of
94 °C for 4 min and 40 cycles of 94 °C for 30 s, 55 °C
for 30 s, and 72 °C for 30 s. For Ggn, the
first round PCR product was used for electrophoresis. For
Pog, a second round of PCR (30 cycles) was performed using the product of the first PCR as the template.
Co-immunoprecipitation--
Full-length Pog cDNA
was cloned in the EcoRI site of pM
(Clontech, CA) and pcDNA3.1/HisC (Invitrogen,
CA), to express the GAL4 DNA binding domain-tagged POG and
Xpress-tagged POG in mammalian cells. Ggn1 cDNA was
cloned in the EcoRI site of pcDNAmyc/HisA to express
Myc-tagged GGN1. 8 µg of plasmid DNA was transfected into 70%
confluent COS-1 or HeLa cells (grown in 10-cm tissue culture dishes)
using Fugene-6 (Roche Molecular Biochemicals). 60 h after
transfection, the cells were washed with ice-cold PBS and lysed with
0.8 ml of Nonidet P-40 lysis buffer (150 mmol/liter sodium chloride,
1.0% Nonidet P-40, 50 mmol/liter Tris, pH 8.0) including a protease
inhibitor mixture (Roche Molecular Biochemicals) and 50 µg/ml
phenylmethylsulfonyl fluoride. Anti-Myc antibody (Invitrogen) or
anti-GAL4 DNA binding domain antibody (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA) was used in the precipitation. 300 µl of each clear
lysate was incubated with the suitable antibody and 50 µl protein A/G
beads in a volume of 600 µl at 4 °C for 2 h. The beads were
spun down at 4 °C and washed three times with ice-cold PBS. The
beads were then resuspended in 40 µl of SDS loading buffer and used
for Western blotting analysis.
Subcellular Localization--
pEGFP-C2 and pEGFP-N2 vectors
(Clontech, Palo Alto, CA) were used to make green
fluorescent protein (GFP) fusion proteins with the target proteins. In
some cases both C-terminal and N-terminal fusion constructs were made
to ensure the GFP did not interfere with the subcellular localization.
The constructs were transfected into HeLa cells, COS-1 cells, or GC-1
cells (grown on glass cover slides). 36 h after transfection the
cells were washed twice with PBS and fixed in 4% phosphonoformic
acid at 4 °C for 30 min. The cells were mounted with
Vectashield (Vector Laboratories, Inc.), and localization of the GFP
signal was checked by confocal microscopy (Zeiss LSM 510). For checking
the nucleolar localization, the same field was examined under UV for
the GFP signal and under bright field for the nucleolus.
To check the co-localization of POG and GGN3, pEGFP-C2/Pog
and pCMV-HA/Ggn3 were co-transfected into HeLa cells at a
ratio of 1:1. For the co-localization of POG and GGN1,
pEGFP-N2/Ggn1 and pcDNA3.1HisC/Pog were
similarly cotransfected. HA-GGN3 was stained with goat anti-HA antibody
(Santa Cruz Biotechnology), and Xpress-POG was stained with anti-Xpress
monoclonal antibody. Co-transfected HeLa cells were fixed with 4%
phosphonoformic acid in PBS at 4 °C for 30 min, permeabilized
at room temperature with 0.1% Triton X-100/PBS for 30 min, and blocked
at room temperature with 3% bovine serum albumin/PBS for 30 min. The
cells were then incubated with primary antibody at room temperature for
1 h. After washing three times for 5 min each in PBS, the cells
were incubated with a rhodamine-conjugated secondary antibody (Santa
Cruz Biotechnology) and checked under a fluorescent microscope.
Pictures of the same field were taken using different filters to obtain
the EGFP and rhodamine signals, respectively. Merging of the EGFP and
rhodamine views was done with standard image manipulation software
(Adobe Photoshop).
Impaired Spermatogenesis in Pog-deficient
Mice--
Pog-deficient males eventually become fertile at 3-4 months
of age due to the population of the tubules with spermatogonial stem
cells and the resumption of spermatogenesis (21). Since Pog
is highly expressed in the testis, we were interested in determining whether the spermatogenesis in Pog-deficient males was
affected. A careful examination of periodic acid-Schiff's
stained testis sections revealed that although spermatogenesis appeared
qualitatively normal in Pog
Flow cytometry was used to quantitatively examine the germ cell
populations in normal and Pog-deficient mice. Five
populations could be distinguished according to the amount of propidium
iodide the cells bound. They were elongating and elongated spermatid (HC, H indicating hypostainability of compacted DNA during
spermiogenesis), round spermatid (1C), spermatogonia and somatic cells
(2C), spermatogonia and preleptotene spermatocytes synthesizing DNA
(S), and primary spermatocyte (4C). Since in the
Pog-deficient mouse, the lumens of the seminiferous tubules
were not completely normal, possibly affecting spermiation, the
elongating and elongated spermatids were not included in the
comparison. The data clearly showed that Pog-deficient mice
had a lower percentage of 1C (round spermatids) and 4C (majority
primary spermatocytes) cells (Fig. 1C), whereas they had the
same percentage of testicular cells in S phase and a higher percentage
of 2C cells. The higher percentage of 2C cells in
Pog-deficient mice could be the result of hyperproliferation of somatic cells in the testis. Since in the adult testis, germ cells
are the only cell type to synthesize DNA, the 4C/S and 1C/S ratios were
used to compare the spermatogenesis of Pog-deficient and
normal mice. Both ratios were significantly lower in
Pog-deficient mice (Fig. 1D), indicating that
some primary spermatocytes are lost and that the transition from
primary spermatocytes to round spermatids (meiosis) is impaired. These
data indicate that Pog is involved in spermatogenesis in
addition to its function in proliferation of primordial germ cells.
POG Interacts with GGN1 and GGN3 in Yeast Two-hybrid
Screening--
Since POG is a novel protein, little is known about its
function in the cell. The yeast two-hybrid system was used to search for proteins interacting with POG in the adult testis in order to shed
some light on its potential role. Since the presence of the POG
C-terminal PHD domain in the bait caused autoactivation of the reporter
gene upon transformation into AH109, the PHD domain-coding region was
deleted from the bait construct and used to screen a pretransformed
mouse adult testis cDNA library (Clontech).
Four million clones were screened using SD-Trp-Leu-His selection, and six independent clones were identified and sequenced. Three matched the
cDNA sequence of mouse RanbpM (24), and the other three matched the
mouse Unigene Mm.63529. Further testing the interaction by
co-transformation of the bait and pray plasmid into yeast AH109 revealed that interaction with RanbpM plasmids was nonspecific, whereas
activation of the reporter gene by the other three plasmids was
specific and dependent on the presence of POG. Since no homologous full-length cDNA sequences were found in the public data base, we
used the primer extension strategy to get the complete coding region of
the cDNA based on the genomic DNA sequence and the expressed sequence tag records in the data base. We named this gene gametogenetin (Ggn) because of its germ cell-specific expression and its
involvement in gametogenesis (see below).
In RT-PCR experiments using several primer sets aligned to the 5'- and
3'-end of the mouse Ggn cDNA, we consistently obtained more than 10 DNA bands of different sizes from the testis and the
ovary. Six major bands were sliced out of the gel and sequenced directly or after subcloning. Eight sequences were obtained (three sequences were from the smallest band after subcloning), showing all to
be specific products of variant splicing of the Ggn
pre-mRNA (Fig. 2A). Three
related proteins could be deduced from the cDNAs sequenced and were
named GGN1, GGN2, and GGN3. GGN1 has two potential trans-membrane
domains in the N terminus (amino acids 45-64) and the middle of the
protein (amino acids 268-287) and two arginine/lysine-rich domains at
the C terminus (Fig. 2B). GGN3 has the C-terminal 137 amino
acids of GGN1 together with the two arginine/lysine-rich domains. The
Ensemble mouse genome assembly indicated that mouse Ggn is
located on Chr 7 and is relatively small, spanning about 5 kb in the
mouse genome.
Interaction between POG and GGN Proteins in Yeast and Mammalian
Cells--
The three clones obtained from the yeast two-hybrid
screening all contained the cDNA coding the C-terminal part of
GGN1, with the shortest coding the C-terminal 122 amino acids. Thus,
both GGN1 and GGN3 contained the sequences mediating the interaction with POG in yeast. To further confirm the interaction between POG and
GGN3 in yeast, the Pog cDNA and cDNA coding for GGN3
were switched between the DNA binding domain vector pGBKT7 and the activation domain vector pACT2. The resulting two constructs were co-transformed into yeast strain AH109, and the interaction was tested
again. Only cells transformed with both pGBKT7/Ggn3 and pACT2/Pog survived the selection, whereas cells transformed
with pGBKT7 and pACT2/Pog did not (Fig.
3A). These data indicate that the interaction between the POG and GGN3 proteins is specific in
yeast.
Co-immunoprecipitation was used to further confirm the interaction in
mammalian cells. In one direction, C-terminal Myc-tagged GGN1
co-immunoprecipitated with GAL-4-tagged POG when the two proteins were
co-expressed in COS-1 cells (Fig. 3B). In the other direction, Xpress-tagged POG co-immunoprecipitated with Myc-tagged GGN1
when they were co-expressed in HeLa cells (Fig. 3C). These experiments provide further evidence that the two proteins interact specifically in mammalian cells.
To determine which part of POG mediated the interaction with GGN3,
cDNA coding for amino acids 182-274 of POG was tested in the yeast
two-hybrid system and was found to be sufficient to mediate the
interaction (Fig. 3A). Using a similar strategy, it could be
shown that the C-terminal 65 amino acids of GGN3 was not able to
mediate the interaction. Sequences further N-terminal to this region
were therefore needed for GGN3 to interact with POG.
In addition to binding to POG, GGN3 was able to form a homodimer with
itself and a heterodimer with GGN1 in yeast two-hybrid experiments
(Fig. 3D). The region mediating the homodimerization was
mapped to the C-terminal 65 amino acids of GGN3, a region that could
not mediate the interaction with POG. When pACT2/Ggn3 was
co-transformed into AH109 cells with either pGBKT7/Ggn1 or pGBKT7/Ggn3, the transformed clones could grow on selection
medium. In the case of pACT2/GgnC-65, only when it was
co-transformed with pGBKT7/Ggn3 but not
pGBKT7/Ggn1 could the cells survive the selection. It
appears that although the C-terminal 65 amino acids of GGN3 are enough
to mediate the interaction, the presence of the N-terminal sequences
added further strength to the interaction. With the construct
pGBKT7/Ggn1, the larger protein size and the potential
trans-membrane domain of GGN1 might limit the amount of protein getting
into the nucleus; thus, a stronger protein-protein interaction was
needed to activate the reporter gene.
Ggn and Pog Were Both Expressed in Primary Spermatocytes--
The
expression of Ggn in the adult tissues was first analyzed by
RT-PCR. A specific product could only be amplified from testis and
ovary RNA but not from adult liver, kidney, lung, heart, spleen, or
brain (data not shown). In situ hybridization was used to
check the cell type and stage that expressed Ggn in the
adult testis using a probe containing 300 bp of the 3' coding region,
present in all splice variants. Ggn was expressed only in
the germ cells and not in the somatic, Sertoli, or Leydig cells (Fig.
4A). In the germ cells,
Ggn expression is tightly related to the developmental stage. The expression started in stage VIII pachytene spermatocytes (Fig. 4, B and C), increased in stage IX and X
pachytene spermatocytes, and culminated in stage XI diplotene
spermatocytes and the meiotic cells in stage XII (Fig. 4, D
and E). Expression decreased slightly in step 1-3
spermatids, further decreased in step 4-11 spermatids, and was no
longer detectable in step 12 spermatids and beyond (Fig.
4E). The expression of Ggn in the testis is
summarized in Fig. 4G.
Consistent with the expression pattern in the adult testis, the
expression of Ggn in the developing postnatal gonad is also developmentally regulated. It is not expressed in 6-day-old testes in
which the germ cells are almost exclusively spermatogonia or 14-day
testis in which the most advanced germ cells are early pachytene
spermatocytes. However, it is expressed in 21-day testis tubules
containing late pachytene spermatocytes or spermatids (Fig.
4F). Thus, in the postnatal male testis, Ggn
expression is strictly confined to late pachytene spermatocytes through
spermatids, a time during which meiosis takes place.
Pog has been shown to expressed in the adult testis and the
ovary (19), although it is not known which individual cell types express Pog. For unknown reasons, we have been unable to
obtain a POG antibody suitable for immunohistochemistry; nor have we been able to obtain clear in situ hybridization data using
several different Pog probes and multiple different
techniques. Thus, to test whether Pog was also expressed in
the same cell type that expressed Ggn, we picked single
primary spermatocytes by virtue of their morphology (large cell size)
and performed single cell RT-PCR. As shown in Fig. 4H, three
cells (cell numbers 2, 3, and 5) were positive for both Ggn
and Pog, which indicated that Ggn and
Pog are expressed in primary spermatocytes. From the
sequenced splicing variations shown in Fig. 2, three DNA products of
922, 600, and 452 bp would be expected from the testis cDNA using
primer pair 215F and 481R for PCR, which was also confirmed in our
experiments (data not shown). In primary spermatocytes, only the 600- and 452-bp products could be amplified from the primary spermatocytes. This suggested that either the splicing of Ggn in 4N, 2N,
and 1N germ cells was regulated or the 922-bp product was inefficiently amplified due to the longer template and/or the lower copy number in
single cell. Nevertheless, these data clearly show that Ggn and Pog are both expressed in primary spermatocytes.
Since POG interacted with GGN1 and GGN3, and they were both expressed
in the primary spermatocytes, we performed in situ
hybridization with Ggn probe on
Pog GGN1, GGN2, and GGN3 Had Different Subcellular
Localization--
In the absence of suitable antibodies, we used EGFP
(green fluorescent protein) fusion proteins to determine the
subcellular localization of GGN1-3 and POG in in vitro
transfected HeLa cells. GGN1 was localized to the perinuclear region
when it was fused to either the C terminus or the N terminus of EGFP
(Fig. 5B). The same
localization was found when the protein was transiently expressed in
HeLa, COS-1, or GC-1 cells (a germ cell line resembling type B
spermatogonia). When the C terminus of GGN1 containing the two
nucleolar targeting signals (NTS) was deleted, the protein was confined
to the cytoplasm (Fig. 5C).
GGN2 shares the N-terminal 217 amino acids with GGN1. When GGN2 was
fused to the N terminus of GFP, the fusion protein was found to
localize exclusively to the cytoplasm (Fig. 5D), consistent with the cytoplasmic localization of GGN1 after the two
arginine/lysine-rich domains were deleted.
A GFP-GGN3 fusion protein was found exclusively in the nucleus, with
the majority of the protein accumulating in the nucleoli (Fig. 5,
E-G). GGN3 also had two arginine/lysine-rich domains. Similar domains have been found in other nucleoli-located proteins such
as TERT, FGF3, and TAT. All of these sequences contain NTSs (25), which
have the ability to target the protein to the nucleus and nucleolus.
GGN1 and GGN3 Determined the Localization of POG in Mammalian
Cells--
POG was an intracellular protein; it localized to both the
cytoplasm and the nucleus whether it was fused to a short Xpress tag or
the N or C terminus of EGFP (Fig.
6A and data not shown). Since
GGN1 and GGN3 were localized to the perinuclear region and the
nucleoli, respectively, we wanted to determine what would happen if POG
was co-expressed with GGN1 or GGN3 in HeLa cells. When EGFP-POG and
HA-GGN3 were co-expressed, EGFP-POG changed from a ubiquitous
intracellular localization to a nucleolar specific localization in some
cells (Fig. 6B). Depending on the ratio and the amount of
the two plasmids used, it was possible that only a part of the
transfected cells harbored both plasmids and that the cells showing
nucleolar specific GFP signal expressed both EGFP-POG and HA-GGN3,
whereas the cells showing a ubiquitous intracellular GFP signal
expressed only EGFP-POG. To confirm this hypothesis and to check
whether POG and GGN3 co-localized in the cell, we stained HA-GGN3 with
anti-HA antibody. After treating the cells with 0.1% Triton X-100, the
GFP signal from the cells showing ubiquitous distribution disappeared,
indicating that EGFP-POG was a soluble protein and could not withstand
the treatment. The GFP signal from cells showing nucleolar specific
distribution persisted (Fig. 6C). These cells expressed
HA-GGN3, and the HA-GGN3 signal colocalized with EGFP-POG (Fig. 6,
D and E). These data strongly supported the view
that the two proteins interacted with each other.
EGFP-GGN1 was similarly co-expressed with X-press-tagged POG. In this
experiment, POG showed a perinuclear localization similar to that of
GGN1 and co-localized with GGN1 (Fig. 6, F-H). Thus, GGN1
and GGN3 determined the subcellular localization of POG. The ability of
GGN1 and GGN3 to change the localization of POG in the cell suggested
that the interaction between POG and GGN1/GGN3 might serve as a means
to regulate the subcellular localization of POG and thus regulate the
activity of POG in the cell.
Here we have described a novel function in gametogenesis for
Pog, a gene previously shown to underlie the germ
cell-deficient mutation, gcd, and to be involved in the
proliferation of primordial germ cells (19). We have also described the
identification and characterization of a novel germ cell-specific gene
Ggn, which is involved in gametogenesis and encodes several
proteins that interact with POG.
We have recently shown that Pog is necessary for primordial
germ cell proliferation (19). Here we further show that Pog is also involved in gametogenesis in adulthood. We show that
spermatogenesis in Pog POG was shown here to interact with GGN1 and GGN3, two proteins from a
single germ cell-specific gene, Ggn. The germ cell-specific expression of Ggn, especially its spatio-temporal regulated
expression in the developing and adult testis, strongly suggested that
Ggn could be involved in gametogenesis. Furthermore, the
expression of the two genes in primary spermatocytes, the interaction
between POG and GGN1/GGN3, the change in the localization of POG upon interaction with GGN1 and GGN3, and the coincidence of the time window
of Ggn expression with the occurrence of the lesion in gametogenesis of Pog How the interaction between POG and GGN1/GGN3 affects gametogenesis is
not known at present. POG and GGN1/GGN3 are novel proteins, and the
biochemical pathways they are involved in remain to be identified. POG
is a PHD domain-containing protein and may have the ability to interact
with chromatin to exert its activity (27). Without the co-expression of
GGN1 or GGN3, POG is distributed in both the cytoplasm and the nucleus
in transfected HeLa cells. While in the presence of GGN1 or GGN3, POG
is localized near the nuclear membrane or in the nucleoli,
respectively. Whereas this is the result from co-transfected HeLa
cells, it is likely that the same process exists in germ cells in the
testis, since the two genes are both expressed in some of the germ
cells. Thus, the interaction between GGN1/GGN3 and POG may serve as a
means to regulate the localization and thus the activity of POG.
Ggn spans only about 5 kb of genomic DNA, but it has
more than 10 different splice variants and generates multiple proteins with different subcellular localizations. Variant splicing can be found
in about 40% of the human genes, particularly in the testis (28, 29).
As an extreme example, cAMP-response element-binding protein (CREB) and
cAMP-response element mediator (CREM) are known to have more
than 20 isoforms resulting from multiple promoters, alternative
polyadenlyation, and multiple alternative splicing (29). Ggn
is another unusual example of a small testis-specific gene with many
(>10) different splice variants. At least three different proteins
with different subcellular localizations are produced from the same
Ggn gene. GGN1 is a perinuclear protein; GGN2 is localized
in the cytoplasm, and GGN3 is confined to the nucleus/nucleoli. Since
Ggn is expressed in 4N primary spermatocytes, 2N secondary
spermatocytes, and 1N spermatids, it is possible that the splicing is
regulated and that the production of GGN1 and GGN3 is related to the
stage of the germ cells. Thus, the localization of POG in the cell
would be determined by the availability and the amount of GGN1 and GGN3.
GGN1 has two trans-membrane domains and two nucleolar-targeting
signals. The additional findings that the N-terminal portion of GGN1
localized to the cytoplasm and the C-terminal part of GGN1 to the
nucleus suggested that GGN1 could be a nuclear membrane protein. The
topology of GGN1 is that the N terminus is cytoplasmic, the C terminus
containing the two arginine/lysine-rich domains is in the nucleus, and
the two trans-membrane domains span the two layers of the nuclear
membrane. Since in the testis Ggn has its highest expression
level in diplotene spermatocytes and meiotic germ cells, where the
nuclear membrane breaks down and the nucleolus is disorganized (30),
the fate and role of GGN1 and GGN3 during this process will be
intriguing. We have recently found another novel gene,
Ggnbp, showing a similar testis expression profile as
Ggn and encoding a protein interacting specifically with the N terminus of GGN1.2 Thus,
GGNBP, GGN1, and POG form a novel protein complex functioning in spermatogenesis.
In conclusion, we provide data to show that Pog is involved
in gametogenesis in addition to functioning in PGC development. We have
identified and characterized two germ cell-specific interacting proteins, GGN1 and GGN3, generated from Ggn by alternate
splicing. In addition, we present data localizing these proteins in the cell and show that Pog and Ggn are both expressed
in primary spermatocytes. Our work provides a basis for the eventual
dissection of the biological functions of these proteins in regulating
gametogenesis and human disease.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1) and
Cdh1 (E-cadherin) function in migration (10, 11).
/
males,
although spermatogenesis is qualitatively normal after age 9-12 weeks,
it is abnormal quantitatively. Pog
/
testes
have a lower percentage of primary spermatocytes and round spermatids
than normal controls, although they have the same percentage of
proliferating spermatogonia. The impaired transition from the primary
spermatocytes to spermatids in Pog
/
testis
indicates that Pog is also involved spermatogenesis in adult testis.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
.
pGBKT7/Pog
was transformed into reporter yeast strain
AH109 and mated with a pretransformed mouse testis cDNA library
cloned in pACT2 and carried in yeast strain Y87. The diploid colonies
were plated on SD-Leu-Trp-Lys agar plates to screen for interacting
partners according to the manufacturer's instructions. For testing
protein interactions in yeast, the cDNAs coding for the two test
proteins were cloned into pGBKT7 and pACT2. The two plasmids were then
co-transformed into yeast strain AH109, and the growth and color were
tested on
SD-Leu-Trp-Lys-Ade/5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-gal) agar plates.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice, in that
four waves of spermatogenesis could be seen in all populated tubules,
it was quantitatively abnormal. In
Pog
/
testis, there were consistently
fewer round spermatids in the seminiferous epithelium than
stage-matched tubules from normal littermates (Fig.
1, A and B).
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Fig. 1.
Spermatogenesis in 12-16-week
Pog-deficient mice. A and
B, seminiferous epithelia in 16-week
Pog-deficient mice (B) have fewer round
spermatids than those at the same stage in their normal
littermates (A). C, flow cytometric
analysis of testicular cell populations in Pog-deficient
testes. Four Pog-deficient mice (12 weeks) and three normal
littermates were analyzed. 20,000 events were counted for each sample.
Inset, a typical histogram with five populations of
testicular cells. D, comparison of the 1C/S and 4C/S ratios
between Pog-deficient mice and normal control.
Pog-deficient mice had significantly lower 1C/S and 4C/S
ratios than the normal control. Student's t test was used
in the statistic analysis. *, significant difference between
Pog-deficient mice and normal mice (p < 0.01).
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Fig. 2.
Multiple-splicing of mouse Ggn.
A, multiple splicing of mouse Ggn pre-mRNA.
Eight different splice variants were sequenced. The predicted
transcription start site was numbered nucleotide 1. Splice donor
(SD) and acceptor sites (SA) are listed
above and below the line, which
represents the pre-mRNA. Solid black
boxes, exons; dashed lines, introns.
The positions of primer 215F and 481R used in single cell RT-PCR are
indicated. B, sequences of mouse GGN1, GGN2, and GGN3. The
two trans-membrane domains are highlighted. Two nucleolar
targeting signals (NTS1 and NTS2) are underlined.
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Fig. 3.
Interaction between POG and GGN1/GGN3.
A, interaction between POG and GGN3 in yeast. Amino acids
182-274 of POG were enough to mediate the interaction with GGN3. The
interaction still existed after switching the Pog and
Ggn3 cDNA between the two vectors. B and
C, co-immunoprecipitation experiments to confirm the
interaction between POG and GGN1 in mammalian cells. B,
GAL4-tagged POG and MYC-tagged GGN1 expressing plasmids were
transfected into COS-1 cells. A specific band of 40 kDa (possibly the N
terminus-degraded protein generated in the process of
immunoprecipitation; since the Myc tag was in the C terminus of the
protein, the degradation of the N terminus did not interfere with the
co-immunoprecipitation) could be seen in co-transfection
immunoprecipitation but not in single transfection immunoprecipitation.
C, Myc-tagged GGN1 and Xpress-tagged POG-expressing plasmids
were transfected into COS-1 cells. Xpress-tagged POG
co-immunoprecipitated with Myc-tagged GGN1. D, mapping the
region critical for GGN3 homodimerization. Amino acids 66-132 of GGN3
were able to mediate the homodimerization.
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Fig. 4.
Expression of Ggn and
Pog in mouse testis. A, in
situ hybridization using a 300-bp Ggn probe in
adult mouse testis. The Ggn expression was confined to the
germ cells and was stage-specific. Note that diplotene spermatocytes in
stage XI and meiotic cells in stage XII had the highest expression.
Purple granules represent the Ggn
mRNA signal. B, Ggn is expressed in round
spermatids but not spermatocytes in stage VII seminiferous epithelium.
C, Ggn starts expression in pachytene
spermatocytes of stage VIII seminiferous epithelium. D,
Ggn has highest expression in diplotene spermatocytes of
stage XI seminiferous epithelium. E, Ggn is
highly expressed in spermatocytes of stage XII seminiferous epithelium
but is no longer expressed in step 12 spermatids. F,
in situ hybridization of Ggn probe on 21-day
mouse testis. Ggn is only expressed in tubules containing
late pachytene spermatocytes and round spermatids. G,
summary of the expression of Ggn in the adult testis.
Russell's staging of the spermatogenesis cycle was adopted (13). The
thickness of the line covering the cell types
indicated the approximate expression level, the thicker the
line the higher the expression. In, intermediate
spermatogonia; B, type B spermatogonia; PI,
preleptotene spermatocytes; L, leptotene spermatocytes;
Z, zygotene spermatocytes; P, pachytene
spermatocytes; D, diplotene spermatocytes; 2°,
secondary spermatocytes; Sd, spermatid. Roman
numerals indicate the stages of the associations, and
Arabic numerals indicate the steps of the sperma tids. H,
Ggn and Pog were both expressed in primary
spermatocytes. In cells 2, 3, and 5, both of Gng and
Pog were positive in RT-PCR. Two major bands corresponding
450 and 600 bp were obtained from Ggn PCR because of
alternative splicing. A single 700-bp band was obtained in
Pog RT-PCR. The positions of Ggn PCR primer 215F
and 481R were shown in Fig. 2A. RT, reverse
transcriptase.
/
testis sections to see whether there
was any change in Ggn expression compared with the
expression in normal testis sections. No difference was noticed between
normal and Pog
/
testis in terms of
Ggn expression (data not shown), which indicated that
deletion of Pog did not affect the expression of
Ggn.
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Fig. 5.
Subcellular localization of GGN
proteins. A, control GFP was localized to the nucleus
and the cytoplasm in HeLa cells. B, GFP-GGN1 fusion protein
localized to the nuclear membrane. C, cytoplasmic
localization of GFP-GGN1 after the C-terminal NTSs of GGN1 were
deleted. D, cytoplasmic localization of GFP-GGN2 fusion
protein. E-G, nucleus/nucleolar localization of GFP-GGN3.
E, GFP-GGN3 was exclusively localized inside the nucleus
stained with 4',6-diamidino-2-phenyindole. The fusion protein was
enriched in the nucleoli (E) when the same cell was checked
under bright field to visualize the nucleoli (G).
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Fig. 6.
GGN1 and GGN3 determined the localization of
POG. A, EGFP-POG was localized to the cytoplasm and the
nucleus in HeLa cells. B, EGFP-POG showed nucleolar specific
localization upon co-expression with HA-GGN3 (pointed
arrows). C, EGFP-POG signal in the nucleoli
resisted the 0.1% Triton X-100 treatment. D, rhodamine
signal representing HA-GGN3 from the same field in as C. E, merging of C and D indicated the
co-localization of EGFP-POG and HA-GGN3. F, green
fluorescence signal from EGFP-GGN1 co-expressed with Xpress-tagged POG.
G, the same field as in F revealing the
localization of POG stained by anti-Xpress antibody. H,
merging of F and G, showing the co-localization
of POG and GGN1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
testis is
qualitatively normal but quantitatively abnormal. The lower 4C/S and
1C/S ratios in Pog
/
testis suggested a
deficiency in the differentiation of the primary spermatocytes and
round spermatids. Thus, in the Pog
/
male, in
addition to PGC deficiency, spermatogenesis is also impaired. Since
Ggn and Pog are expressed in the ovary, it is possible that they may also function in oogenesis. However,
Pog-deficient females have very few oogonia at birth, and
since females do not form a stem cell population from which late onset
population can occur, the role of Pog in the female could
not be directly addressed. Female sl17H/sl17H
(Kitl mutant) mice have about 6% of the PGC of normal mice,
and they are fertile (26). The fact that
Pog
/
females have a similar degree of PGC
deficiency (data not shown) but are infertile is consistent with
Pog
/
females having an additional defect in
oogenesis and that Pog may play a role in oogenesis in
addition to functioning in PGC development.
/
mice all suggest that
the two genes are involved in this process.
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ACKNOWLEDGEMENTS |
---|
We thank Cavatina Truong for excellent technical assistance and Michael Mancini and David Stenoien for help with the confocal microscopy.
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FOOTNOTES |
---|
* This work was supported by Grant HD36289 from the National Institutes of Health (to C. E. B.).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/EBI Data Bank with accession number(s) Ggn1-Ggn3 (mouse AF538032-AF538034) and AF538035, BK000550, AF538036 and AF538037 (human Ggn1a, Ggn1b, Ggn2, and Ggn3).
§ Present address: Beijing Institute of Biotechnology, Beijing 100071, China.
To whom correspondence should be addressed: Dept. of
Obstetrics and Gynecology, Baylor College of Medicine, 6550 Fannin St. (#880), Houston, TX 77030. Tel.: 713-798-8221; Fax: 713-798-5074; E-mail: bishop@bcm.tmc.edu.
Published, JBC Papers in Press, February 5, 2003, DOI 10.1074/jbc.M211023200
2 B. Lu and C. E. Bishop, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are: PGC, primordial germ cell; dpc, days postcoitum; PHD, plant homeodomain; RT, reverse transcription; PBS, phosphate-buffered saline; GFP, green fluorescent protein; EGFP, enhanced GFP; NTS, nucleolar targeting signal.
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REFERENCES |
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---|
1. | Lawson, K. A., and Hage, W. J. (1994) Ciba Found. Symp. 182, 68-84[Medline] [Order article via Infotrieve] |
2. |
Lawson, K. A.,
Dunn, N. R.,
Roelen, B. A.,
Zeinstra, L. M.,
Davis, A. M.,
Wright, C. V.,
Korving, J. P.,
and Hogan, B. L.
(1999)
Genes Dev.
13,
424-436 |
3. |
Ying, Y.,
Liu, X. M.,
Marble, A.,
Lawson, K. A.,
and Zhao, G. Q.
(2000)
Mol. Endocrinol.
14,
1053-1063 |
4. | Ginsburg, M., Snow, M. H., and McLaren, A. (1990) Development 110, 521-528[Abstract] |
5. | Tam, P. P., and Snow, M. H. (1981) J. Embryol. Exp. Morphol. 64, 133-147[Medline] [Order article via Infotrieve] |
6. | Chabot, B., Stephenson, D. A., Chapman, V. M., Besmer, P., and Bernstein, A. (1988) Nature 335, 88-89[CrossRef][Medline] [Order article via Infotrieve] |
7. | Geissler, E. N., Ryan, M. A., and Housman, D. E. (1988) Cell 55, 185-192[Medline] [Order article via Infotrieve] |
8. | Zsebo, K. M., Williams, D. A., Geissler, E. N., Broudy, V. C., Martin, F. H., Atkins, H. L., Hsu, R. Y., Birkett, N. C., Okino, K. H., and Murdock, D. C. (1990) Cell 63, 213-224[Medline] [Order article via Infotrieve] |
9. |
Beck, A. R.,
Miller, I. J.,
Anderson, P.,
and Streuli, M.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
2331-2336 |
10. |
Anderson, R.,
Fassler, R.,
Georges-Labouesse, E.,
Hynes, R. O.,
Bader, B. L.,
Kreidberg, J. A.,
Schaible, K.,
Heasman, J.,
and Wylie, C.
(1999)
Development
126,
1655-1664 |
11. | Bendel-Stenzel, M. R., Gomperts, M., Anderson, R., Heasman, J., and Wylie, C. (2000) Mech. Dev. 91, 143-152[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Byskov, A. G.
(1986)
Physiol. Rev.
66,
71-117 |
13. | Russell, L. D., Ettlin, R. A., Sinha Hikim, A. P., and Clegg, E. D. (1990) Histological and Histopathological Evaluation of the Testis , 1st Ed. , Cache River Press, Clearwater, FL |
14. | Cobb, J., and Handel, M. A. (1998) Semin. Cell Dev. Biol. 9, 445-450[CrossRef][Medline] [Order article via Infotrieve] |
15. | Heyting, C., Dettmers, R. J., Dietrich, A. J., Redeker, E. J., and Vink, A. C. (1988) Chromosoma 96, 325-332[CrossRef][Medline] [Order article via Infotrieve] |
16. | Andersen, B., Pearse, R. V., 2nd, Schlegel, P. N., Cichon, Z., Schonemann, M. D., Bardin, C. W., and Rosenfeld, M. G. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11084-11088[Abstract] |
17. | Kremer, E. J., and Kistler, W. S. (1991) Exp. Cell Res. 197, 330-332[Medline] [Order article via Infotrieve] |
18. | Duncan, M. K., Lieman, J., and Chada, K. K. (1995) Mamm. Genome 6, 697-699[Medline] [Order article via Infotrieve] |
19. |
Agoulnik, A. I.,
Lu, B.,
Zhu, Q.,
Truong, C.,
Ty, M. T.,
Arango, N.,
Chada, K. K.,
and Bishop, C. E.
(2002)
Hum. Mol. Genet.
11,
3047-3053 |
20. | Pellas, T. C., Ramachandran, B., Duncan, M., Pan, S. S., Marone, M., and Chada, K. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 8787-8791[Abstract] |
21. | Lu, B., and Bishop, C. E. (2003) Biol. Reprod., in press |
22. |
Krishnamurthy, H.,
Weinbauer, G. F.,
Aslam, H.,
Yeung, C. H.,
and Nieschlag, E.
(1998)
J. Androl.
19,
710-717 |
23. | Brady, G., and Iscove, N. N. (1993) Methods Enzymol. 225, 611-623[Medline] [Order article via Infotrieve] |
24. | Nishitani, H., Hirose, E., Uchimura, Y., Nakamura, M., Umeda, M., Nishii, K., Mori, N., and Nishimoto, T. (2001) Gene (Amst.) 272, 25-33[CrossRef][Medline] [Order article via Infotrieve] |
25. | Hatanaka, M. (1990) Bioessays 12, 143-148[Medline] [Order article via Infotrieve] |
26. | 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] |
27. | Aasland, R., Gibson, T. J., and Stewart, A. F. (1995) Trends Biochem. Sci. 20, 56-59[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Modrek, B.,
Resch, A.,
Grasso, C.,
and Lee, C.
(2001)
Nucleic Acids Res.
29,
2850-2859 |
29. | Venables, J. P. (2002) Curr. Opin. Genet. Dev. 12, 615-619[CrossRef][Medline] [Order article via Infotrieve] |
30. | Moss, S. B., Burnham, B. L., and Bellve, A. R. (1993) Mol. Reprod. Dev. 34, 164-174[Medline] [Order article via Infotrieve] |