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Acta Biochim |
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doi: 10.1093/abbs/ gmp055. |
Glycerol-3-phosphate acyltransferase 4 gene is
involved in mouse spermatogenesis
Qingming Qiu1,2, Gang Liu1, Weina Li1, Qiuwen Shi1,
Fuxi Zhu1, and Guangxiu
Lu1*
1Institute of Human Reproduction and Stem Cell Engineering,
Hospital,
Glycerol-3-phosphate acyltransferase
(GPAT) catalyzes the first committed step of de novo triacylglycerol synthesis by
converting glycerol-3-phosphate to lysophosphatidic
acid (LPA). LPA is a mitogen that mediates multiple
cellular processes including cell proliferation. Four GPAT isoforms have been cloned
to date. GPAT4 is strongly expressed in the mouse testis. Reverse transcription –polymerase chain reaction (PCR), real-time PCR, and in situ hybridization
(ISH) were used to analyze the GPAT4 expression and to localize the expressing cell types in
the mouse testis during postnatal development. GPAT4 cDNA was inserted
into pcDNA4/His to construct a recombinant vector, which was transfected into a mouse spermatogonial
cell line (GC-1spg). GPAT4 was first expressed in mice at 2 weeks postnatally. Expression was abundant from the third week, plateaued at week 5–6 and then maintained at a high level in the adult. ISH
revealed that GPAT4 gene was expressed abundantly in spermatocytes
and around spermatids during meiosis but not in
elongated spermatids during later spermiogenesis.
GC-1spg cells showed a marked increase in proliferation after transfection with GPAT4; cell cycle analysis showed a
decrease in the percentage of cells in the G0/G1 phase and an increase in the S
phase. Thus, GPAT4 might play an important role in spermatogenesis, especially in
mid-meiosis.
Keywords GPAT4; in situ hybridization; lysophosphatidic acid; meiosis, spermatogenesis
Received: March 15, 2009 Accepted: April 27, 2009
Introduction
Glycerol-3-phosphate acyltransferase (GPAT) is
involved in triacylglycerol (TAG) and phospholipid synthesis, catalyzing the first committed step
of de novo TAG synthesis by
converting glycerol-3-phosphate to lysophosphatidic
acid (LPA) [1–3]. To date, four GPAT isoforms have been cloned and
are designated GPAT1, GPAT2, GPAT3, and GPAT4. GPAT1 and GPAT2 isoforms exist in mitochondria, but GPAT3 and GPAT4 are
distributed in microsomes. GPAT1 was the first
mammalian isoform to be cloned, which was the major
mitochondrial isoform and was resistant to sulfhydrylmodifying reagents, such as N-ethylmaleimide
(NEM) [4–6]. GPAT2 was a minor, NEM-sensitive mitochondrial isoform
and GPAT3 was a microsomal NEMsensitive
enzyme [1,7,8]. GPAT4 was initially identified and cloned as a
novel human gene from human cDNA databases [9].
Previous researches revealed that GPAT4 was involved in lipid biosynthesis in mouse epithelia [10,11]. Northern blotting analysis showed that GPAT4 was highly expressed
not only in adipose tissue and liver, but also in testis. Mass spectroscopy
also demonstrated that the GPAT4 enzyme could promote the incorporation of
oleic acid into LPA [12].
In our previous study, we cloned 24 expressed sequence tags (ESTs) related to apoptosis in mouse spermatogenesis using a
cryptorchidism model and suppression subtractive
hybridization [13]. Using the EST BE644543, one of the ESTs
identified, we cloned a fulllength mouse cDNA sequence by using GeneScan
software and polymerase chain reaction (PCR) technology. Similarity searches on
nucleotide sequences were carried out using BLAST at the NCBI GenBank database (http:// www.ncbi.nlm.nih.gov) and found
that this putative gene had been designated as GPAT4. Although the roles of GPAT
Materials and Methods
Animals
Adult BALB/c male and female mice were obtained from Shanghai SLAC
Laboratory Animal Co. Ltd. (
Generation of pups and obtaining testis from different
postnatal stages
Four or five female mice were caged with a male for one night for mating.
Birth occurred about 21 days later and testis from the male mice were removed
surgically at 1 day and 1, 2, 3, 4, 5, 6, 7, 8, and 9 weeks postpartum
following euthanasia.
Multiple-tissue reverse
transcription–PCR analysis of GPAT4 gene expression
Reverse transcription (RT)–PCR was carried out to
determine whether GPAT4 mRNA was detectable in tissues from 6-week old adult males. Total RNAs from the whole testis, liver, adipose tissue, heart,
brain, lung, and spleen were isolated using Trizol (Invitrogen, Carlsbad, USA), and first strand cDNA synthesis was carried out using first strand cDNA synthesis kit (Roche Diagnostics GmbH, Mannheim,
Germany) according to the manufacturer’s protocols. PCRs
were conducted in an Eppendorf MasterCycler
(Eppendorf AG,
Semi-quantitative RT–PCR and real-time quantitative PCR analysis of GPAT4 expression levels
in mouse testis during postnatal development
GPAT4 gene expression in the mouse testis during postnatal development was
analyzed using semi-quantitative RT–PCR and real-time
quantitative PCR. As described above, RT–PCR products were subjected to electrophoresis on 1.5% agarose
gels stained with ethidium bromide and were quantified using the Bio-Profil/Bio-1D++ Windows Application V.99.03 program (VILBER LOURMAT,
In situ hybridization to confirm GPAT4 location in different cell types
In situ hybridization (ISH) was used to determine the cell types expressing GPAT4 mRNA in the mouse
testis. ISH was performed according to the manufacturer’s directions (Roche).
Briefly, mice (6-week old) were injected with 0.9% physiologic saline (50
mg/kg, i.p.) and 30 min later they were euthanized
with an overdose of pentobarbital (50 mg/kg, i.p.)
and perfused transcardially
with 10 ml of 0.9% saline followed by 4% paraformaldehyde
in 50 ml of phosphate-buffered saline (PBS; pH 7.2). The testis was frozen in isopentane at 240ºC and stored at –80ºC. Frozen tissue sections (20 mm) were thaw-mounted onto gelatin-coated glass
slides and stored at –80ºC. Following fixation,
deproteination and acetylation,
slides were hybridized with sense and antisense RNA probes for GPAT4. Digoxigeninlabeled
sense and antisense RNA probes were generated from partial cDNA
clones in the pGEM-T vector encoding GPAT4 (GenBank
accession No. NM_018743) using the T7/SP6 RNA transcription
system (Roche) as recommended by the manufacturer. Sections were
incubated overnight at 37ºC and then subsequently washed three times
in 60% formamide/0.2 ´ saline-sodium citrate buffer. Hybridization was visualized with the
substrate 3,3‘-diaminobenzidine
tetrahydrochloride dehydrate (Sigma-Aldrich,
ISH for different developmental stages in the mouse testis
To investigate GPAT4 expression status in three different development phases, 21- and
42-day-old male mice were used for ISH. The procedures were the same as above,
except that the neonatal mice were sacrificed directly after birth and their
testes were excised immediately.
Construction of pcDNA4/GPAT4/His expression vectors
To construct an eukaryotic expression vector
encoding the GPAT4 gene for investigating cell proliferation, one pair of primers was
designed according to the published nucleotide sequences of the GPAT4 cDNA. The 5‘ primer (5‘-GCGAATTCCCATGTTCCTGTTGCTACCT-3‘) contained a recognition site for EcoRI (underlined; New
England Biolabs;
Cell culture and transfection
Mouse spermatogonial cell lines (GC-1spg cells)
were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The cells
were maintained at 37ºC in a humidified atmosphere of 95% air and 5% CO2. Cells were transfected with plasmid DNA in six-well plates using Lipofectamine 2000 (Invitrogen)
according to the manufacturer’s instructions. Stable transfected cell lines
were established by selecting out transfected cells
with 250 mg/ml of zeocin (Invitrogen)
for 3 weeks. Cells were passaged twice a week using
0.25% trypsin with 0.1% EDTA.
Establishment of stable transfected
GC-1spg cell lines
GC-1spg cells were transfected with
pcDNA4/GPAT4/ His and the empty vectors pcDNA4/His, respectively, using Lipofectamine 2000 under serum-free conditions as described
above. Selection was performed with 250 mg/ml of zeocin in DMEM media containing 10% FBS for ~3 weeks from the day after transfection. A
stable transfected cell line designated as GC-1spg/
pcDNA4/GPAT4/His was obtained. Likewise, a stable transfected
cell line with the empty vector pcDNA4/His (GC-1spg/pcDNA4/His) was also constructed.
Western blotting
analysis of GPAT4 expression Protein samples were extracted from stable transfected
cells using RIPA buffer (
Cell growth analysis
Stable transfected cells (GC-1spg cells containing
pcDNA4/GPAT4/His or pcDNA4/His), were plated into 24-well cell culture plates
at 5 ´ 104 cells/well in 500 ml DMEM supplemented with 10% FBS. Cells
were incubated for 1, 2, 3, 4, 5, 6, and 7 days, harvested by trypsinization and counted using a SYSMEX XT-1800I cytometer (Sysmex,
Cell cycle analysis
The stable transfected cell DNA content was
determined by flow cytometry. Cells were trypsinized, washed twice with PBS and suspended in 500 ml
PBS containing 0.1% FBS for 15 min on ice. The cell suspension was mixed with 5
ml of cold 70% ethanol fixation for 12 h at –20ºC. Fixed cells were stained with propidium
iodide containing 5 mg/ml of RNase A for 30 min at room temperature
in dark and subsequently analyzed using a flow cytometer
(BD Biosciences, Franklin Lake, USA). Histograms were generated and cell cycle
analysis was carried out using CellQuest software (BD
Biosciences).
Statistical analysis
All results are presented as the mean±SD. Student’s t test was used to
analyze the significance of any differences and P<0.05 was taken as statistically
significant.
Results
Tissue RT–PCR analysis of GPAT4 gene expression levels
As shown in Fig. 1, GPAT4 mRNA was
strongly expressed in the testis, adipose tissue, and liver, but was less
abundant in the heart, lung, and brain, and no expression was detected in the
spleen. Gene expression analysis of GPAT
GPAT4 mRNA expression in spermatocytes
and round spermatids
To examine which cell type in the testis was responsible for expression of
the GPAT4 gene, we used ISH, in
which frozen sections of testis were probed with a nonradioactive antisense
mRNA for GPAT4. As
shown in Fig. 3, the
results were unequivocal. No reaction product was seen in sections reacted with
the sense probe [Fig. 3(A)]. Using the antisense probe, brown reaction product indicating the
presence of GPAT4 mRNA was mainly observed in spermatocytes, round
spermatids but not in elongated spermatids
[Fig. 3(B)].
GPAT4 mRNA expression in different development stages
To further examine GPAT4 mRNA expression in testicular germ cells from different development
stages, testis sections were tested by ISH on day 0 (the day of birth) and in
21- and 42-day-old mice (Fig. 4). No positive signal was found in the neonatal mouse testis [Fig. 4(B)]. Positive staining
(brown staining) was seen in the cytoplasm of spermatocytes
and round spermatids from 21- and 42-day-old mice
testis sections [Fig. 4(D,F)]. No reaction product
was seen in sections reacted with the sense probe [Fig. 4(A,C,E)]. Western blotting
analysis of GPAT4 fusion protein To test whether GC-1spg expressed the GPAT4 protein after transfection, fusion proteins from stably transfected GC-1spg cell extracts were analyzed on 10% SDS– PAGE and then transferred to polyvinylidene difluoride membranes for western blotting analysis. Results
demonstrated that a recombinant His-tagged GPAT4 protein, with a molecular
weight of ~56 kDa,
was expressed successfully in the GC-1spg cells (Fig. 5). Effect of GPAT4 expression on GC-1spg
cell growth Flow cytometry was used to investigate the
effect of GPAT4 expression
on the proliferation of GC-1spg cells in vitro. Cells were counted daily per well over 7 days. The stably transfected GC-1spg cells demonstrated an accelerated cell
growth rate compared with control cells (untransfected
or transfected with an empty vector) by day 3 (Fig. 6). Thus, overexpression of GPAT4 accelerated cell growth in vitro.
Cell cycle analysis by flow cytometry
To examine whether the effect of GPAT4 on cell proliferation was related
to alterations in the cell cycle, GC-1spg cells stably transfected
with GPAT4 were analyzed using flow cytometry. The
percentages of cells in the G0/G1, S, and G2/M phases of the cell cycle were
calculated. As shown in Fig. 7, the proportions of cells in S and G2/M phases increased and the
proportions in G0/G1 phase decreased, compared with GC-1spg and GC-1spg/pcDNA4
controls. This was a statistically significant difference (P<0.05).
Discussion
To our knowledge, this is the first study on GPAT4 expression during mouse testicular
maturation and spermatogenesis. These data provide a useful basis for new
hypotheses about the physiological functions of the GPAT4 enzyme and will help
to elucidate the molecular mechanisms of spermatogenesis. Spermatogenesis is a
continuous of process of cellular differentiation involving spermatogonial
renewal and proliferation, meiosis, and spermiogenesis.
About 8 days after birth, diploid spermatogonial stem
cells pass through self-renewal to proliferation and produce spermatogonia types A and B. The meiotic prophase is
initiated by 10 days of age in the mouse. Type B spermatogonia
differentiate into primary spermatocytes and produce
round spermatids after the second meiotic division at
about 20 days. Finally, after a series of biochemical and morphological
changes, the spermatids elongate and then the fully
differentiated spermatozoa are released into the lumen of the seminiferous tubule [15,16].
In the present study, we investigated GPAT4 expression patterns by RT–PCR and real-time quantitative PCR. Our data showed that GPAT4 expression level was
related to testicular maturation: it was slight in immature 2-week-old mice,
was abundant from week 3, plateaued at week 5–6 and maintained at a high level in adults. This trend in GPAT4 expression was
consistent with the onset and progress of spermatogenesis and this suggested
that the GPAT4 might
contribute to this complex process.
ISH is a powerful technique for localizing the expression of specific
genes in tissues and results can provide important clues to gene function.
Using sections from three different age testes for ISH, we found that seminiferous tubules taken at the day of birth did not show
positive staining. However, there were clearly positive cells from 21- and
42-day-old mouse testis. There are only primitive type
A spermatogonia in the neonatal mouse testis and
round spermatids first appear in the seminiferous epithelium at 20 days postpartum [15]. Because
the GPAT4 gene was mainly
expressed in spermatocytes and round spermatids, our results were consistent with the idea that GPAT4 played a role in the
meiosis. GPAT4 expression
levels were low on the day of birth and at week 1 (premeiotic
division phase), but there was abundant after 3 weeks (beginning of meiotic
division). On the other hand, positive signals detected by ISH were expressed
in spermatocytes and round spermatids,
both of them appearing in the seminiferous epithelium
after the meiotic division phase, but not in elongating spermatids
(spermiogenesis phase). Meiotic division is an
important period for spermatogenesis, and involves a number of genes expressed
specifically in the mouse testis. For example, SRG4 and PigM are newly cloned and
specifically expressed gene in the mouse testis that play essential roles in
spermatogenesis [17,18]. Interestingly, their gene
expression patterns were similar to GPAT4 to some extent. To gain further insights into the potential functions of GPAT4 during mouse
testicular development, additional experiments were performed by overexpressing GPAT
ISH performed on mouse testis taken at different developmental stages provide
evidence that GPAT4 might play an important role in spermatogenesis. The localization of its
transcripts in the spermatocytes as well as around spermatids suggested that this gene acted mainly during
meiosis in spermatogenesis. The overexpression experiments
suggested that the GPAT4 enzyme stimulated mitogenic
activity through its product LPA. However, additional experiments will be
necessary to explore the pathway leading to cell proliferation.
Acknowledgements
We thank Prof. Changqi Li (
Funding
This work was supported by a grant from the National
Basic Research Program of
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