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Isoform of CREB3L4 in Mouse Spermatids
From the * Molecular Endocrinology and Oncology
Research Center, Université Laval Medical Research Center,
Québec, Canada; and the Department of
Anatomy and Physiology, Faculty of Medicine, Université Laval,
Québec, Canada.
| Correspondence to: Dr Claude Labrie, CHUL Research Center, 2705 Laurier Boul, Ste-Foy, QC, G1V 4G2, Canada (e-mail: Claude.Labrie{at}crchul.ulaval.ca). |
| Received for publication January 10, 2006; accepted for publication May 13, 2006. |
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Abstract |
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, a truncated
(315-aa) form of Tisp40ß. In the mouse testis, Creb3l4
transcripts are known to be expressed exclusively in postmeiotic spermatids
but the presence of CREB3L4 protein in spermatids has not been formally
demonstrated. We produced an antibody directed against the carboxy terminus of
mouse CREB3L4 and used it in immunostaining experiments to document that
CREB3L4 protein accumulates in post-meiotic spermatids in a stage-specific
manner. Moreover, we show that Atce1/Tisp40 is the major form of
CREB3L4 in mouse testis. These findings suggest that testis-specific isoforms
of Creb3l4 could play an important role in spermatid
differentiation.
Key words: AIbZIP, endoplasmic reticulum, unfolded protein response
The molecular processes that result in the formation of mature spermatozoa are not fully understood, but they ultimately rely on the timely production of proteins that are involved in spermatid differentiation. One transcription factor that has been shown to play a crucial role in spermiogenesis is the bZIP (basic region-leucine zipper) transcription factor CREM (cAMP-response-element modulator). Inactivation of the Crem gene in mice causes an arrest of spermatogenesis at the round spermatid stage and, consequently, infertility (Blendy et al, 1996; Nantel et al, 1996). Infertility in these mice has been attributed to the failure of the germ cells to express CREM-responsive genes, such as the protamines that replace histones to allow DNA compaction (Kimmins et al, 2004).
The early phases of spermiogenesis are characterized by intense transcriptional activity (Eddy, 2002), which implies that the endoplasmic reticulum (ER) must function optimally to ensure that the required proteins are properly synthesized and processed. An important regulatory mechanism implicated in the maintenance of ER function is the "unfolded protein response" (UPR) (also known as "ER stress response"), an adaptive response that serves to re-establish normal ER function following disruptions such as the accumulation of unfolded or misfolded proteins (Zhang and Kaufman, 2004). This is achieved by promoting the degradation of misfolded proteins, by attenuating protein translation to reduce the amount of nascent proteins, and by increasing the production of the ER chaperones that are required to ensure the proper processing of nascent proteins.
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In humans, CREB3L4 is most abundant in prostate
(Qi et al, 2002), but standard
as well as quantitative RT-PCR assays have revealed the presence of
appreciable amounts of CREB3L4 mRNA in testis
(Cao et al, 2002; Cunha et al,
2005). In the mouse, on the other hand, CREB3L4 mRNA is most abundant in
testis (Nagamori et al, 2005)
and CREB3L4 transcription is up-regulated during mouse spermiogenesis
(Fujii et al, 2002). Together
these observations support the concept that CREB3L4 could potentially
play an important role in assuring ER function during spermiogenesis. However,
it is important to note that the Creb3l4 gene encodes at least 2
distinct transcripts that initiate from different transcription start sites
(Figure 1). The first
transcript to be identified in mouse testis initiates in intron 3 of the gene
and contains 8 of the 9 coding exons
(Stelzer and Don, 2002;
Nagamori et al, 2005). This
mRNA codes for a 315-residue protein designated Atce1/Tisp40 which
contains codons 56 to 370 of the larger Tisp40ß protein. Transcripts that
encode Tisp40ß initiate upstream of exon 1 or 2 and contain all 9 coding
exons of the gene.
The discovery of 2 distinct CREB3L4 isoforms in mouse is significant
because Tisp40ß is a potent transcriptional activator, whereas
Atce1/Tisp40 is not (Nagamori et al,
2005). In order to understand the role of CREB3L4 in mammalian
reproduction, it is therefore of paramount importance to determine precisely
which CREB3L4 isoform is present in testis. In this report, immunostaining
experiments performed using an antibody raised against the carboxy terminal
portion of mouse CREB3L4 documented that the CREB3L4 protein is expressed in a
stage-specific manner in differentiating spermatids. Moreover, we found that
the most abundant forms of CREB3L4 present in mouse testis lack the amino
terminal activation domain of mouse CREB3L4. These findings have important
implications for future studies of CREB3L4 in reproduction.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Antibody Production and Purification
A peptide corresponding to amino acids 354–367 (KARPPGQIRGMVHT) of
mouse CREB3L4 (GenBank accession number NM_030080) was synthesized in our
facility and conjugated to KLH via a cysteine residue appended to its amino
terminus. This particular peptide was selected because its sequence is not
present in other proteins of the mouse CREB3 family and it did not match any
other known mouse protein in the GenBank database. Antibodies were produced by
injecting the conjugated peptide to 3 rabbits using standard procedures.
Antiserum from rabbit number 1462 (AB1462) was selected on the basis of its
performance in immunoblotting experiments and subsequently purified by
affinity chromatography after immobilizing the immunogenic peptide to
Sulfo-Link Coupling Gel (Pierce Biotechnology, Rockford, Ill). All the
immunostaining and immunoblotting experiments presented in this report were
performed using affinity-purified AB1462 antibody.
Mouse Tissue Preparation
For immunostaining experiments, 4 3-month-old and 2 6-week-old male mice
were perfused through the left ventricle with 10% buffered formalin for 15
minutes. Following perfusion, the testes were collected and immersed in the
same fixative for 24 hours and then embedded in paraffin blocks. Testes used
for immunoblotting were collected from sexually mature male mice, snap-frozen
in liquid nitrogen, and homogenized using a tissue grinder in lysis buffer (6
mol urea, 20 mmol Tris pH 6.8, 1% SDS) supplemented with protease inhibitors
(Roche Applied Science, Indianapolis, Ind). Protein extracts were stored at
–80°C prior to use.
Immunostaining
Paraffin sections (4 µm) were deparaffinized in toluene and rehydrated
through ethanol. Endogenous peroxidase activity was eliminated by
preincubation in 3% H2O2 in methanol for 30 minutes. A
microwave retrieval technique using citrate buffer was applied
(Tacha and Chen, 1994) and
nonspecific binding was blocked using 10% (v/v) goat serum diluted in Dako
antibody diluent (DakoCytomation California, Carpinteria, Calif). The sections
were incubated with antibody 1462 (diluted 1:1000) for 75 minutes at room
temperature, washed in PBS buffer, and incubated with biotinylated anti-rabbit
secondary antibody for 10 minutes and then with streptavidin-peroxidase for
another 10 minutes. Under microscope monitoring, diaminobenzidine was used as
the chromogen to visualize the biotin/streptavidin-peroxidase complexes.
Counterstaining was performed using #2 Gill hematoxylin. As a negative
control, an excess (5-fold) of the synthetic peptide was coincubated with the
primary antibody for 3 hours at room temperature.
Expression Vectors
The full-length mouse CREB3L4 open reading frame (ORF) was isolated from
mouse prostate RNA by reverse transcription–polymerase chain reaction
(PCR) amplification. The PCR product was then cloned in frame with sequences
encoding a C-terminal haemagglutinin (HA) epitope in a modified pcDNA3
(Invitrogen, Carlsbad, Calif) expression plasmid. The recombinant protein
contains amino acids 1–370 of CREB3L4 (GenBank accession number
NM_030080) followed by amino acids SRGP and the HA epitope (YPYDVPDYASL). This
plasmid was then used as a template for PCR amplification to generate plasmids
producing an untagged form of Tisp40ß as well as HA-tagged and untagged
forms of Atce1/Tisp40 (mouse CREB3L4 codons 56–370).
Tisp40HA also contains the SRGPYPYDVPDYASL extension at its C terminus.
The sequences of all PCR products and cloning junctions were verified using an
automated sequencer. Plasmid DNA for transfection experiments was purified by
gravity-flow anion exchange (Qiagen, Valencia, Calif).
Transient Transfections and Immunoblotting
Mouse CREB3L4 expression plasmids were transfected into human kidney 293
cells using ExGen 500 transfection reagent (Fermentas Life Sciences, Hanover,
Md). The cells were harvested 48 hours posttransfection and whole-cell
extracts were prepared in lysis buffer (see above). Protein extracts from
mouse testis (30 µg/lane) and transfected 293 cells (4 µg/lane) were
immobilized on nitrocellulose following electrophoresis through 10% denaturing
polyacrylamide gels. The blots were preincubated for 1 hour at room
temperature in TBS (0.9% NaCl, 10 mmol Tris-HCl, pH 8.0) containing 5% (w/v)
powder milk and then incubated overnight at 4°C in fresh TBS/milk
containing a 1:1000 dilution of AB1462. The blots were washed in TBS
containing 0.05% Tween-20 and 0.05% NP-40 (4 times 15 minutes at room
temperature) and then incubated for 1 hour at room temperature in TBS/milk
containing a 1:10 000 dilution of peroxidase-conjugated AffiniPure goat
antirabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, Pa). After
another series of washes, antigen-antibody complexes were revealed using the
Western Lightning Chemiluminescence Reagent Plus (Perkin Elmer, Wellesley,
Mass).
Deglycosylation Experiments
Extracts from mouse testis and from transfected 293 cells prepared in lysis
buffer were denatured in 1X Glycoprotein denaturing buffer (5% (w/v) SDS, 0.4
mol DTT) at 100°C for 10 minutes. Denatured testis (30 µg) and 293 cell
(4 µg) extracts were then incubated in 50 mmol sodium citrate buffer alone
or buffer containing 1000 units of Endoglycosidase H (New England BioLabs,
Ipswich, Mass) for 16 hours at 37°C prior to electrophoresis.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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in
immunostaining experiments but should be able to discriminate between the 2
polypeptides in immunoblotting experiments. Testes were collected from sexually mature 3-month-old C57BL/6 mice, and cross sections were processed for immunostaining using affinity-purified antiserum number 1462 (AB1462). As shown in Figure 2, AB1462 stained the epithelium of the seminiferous tubules, whereas interstitial cells and Leydig cells were unlabeled. The staining reaction was completely abolished when an excess of the immunogenic peptide was used to immunoabsorb the antiserum. Close examination of stained seminiferous tubules revealed that only the postmeiotic cells (round or elongated spermatids) were labeled, whereas spermatocytes, spermatogonia, and Sertoli cells were not labeled. Interestingly, some seminiferous tubules displayed little or no staining, whereas other tubules showed a strong staining reaction.
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To verify if both Tisp40ß and Atce1/Tisp40 are present in the
testes of sexually mature mice we performed immunoblotting experiments using
AB1462. Two abundant polypeptides with apparent molecular weights of 42 and 36
kd, hereafter referred to as p42 and p36, were detected in mouse testes
(Figure 4a). The apparent
molecular weights of these proteins are similar to the 44- and 38-kd
polypeptides that were previously detected in mouse testis extracts using a
polyclonal antibody raised against the N-terminal portion common to
Tisp40 and Tisp40ß (Nagamori et
al, 2005). Based on the electrophoretic mobility of in
vitro–synthesized Tisp40 proteins, Nagamori et al deduced that the 44-
and 38-kd polypeptides correspond to Tisp40ß and Tisp40,
respectively. However, the 42–44-kd polypeptide is much more abundant
than the 36–38-kd polypeptide in testis extracts, which is inconsistent
with the observation that the Tisp40ß mRNA is less abundant than the
Tisp40 mRNA (Nagamori et al,
2005). Moreover, Nagamori et al also showed that recombinant
Tisp40 proteins are glycosylated when they are transiently produced in human
HeLa cells, suggesting that p42–44 and p36–38 might correspond to
differentially glycosylated forms of the same polypeptide.
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In an attempt to resolve this issue we transiently produced HA
epitope-tagged versions of Tisp40ß and Atce1/Tisp40 in human 293
cells and compared their apparent molecular weights to those of p42 and p36.
As shown in Figure 4A, the
apparent molecular weight of Tisp40ßHA was considerably greater than that
of p42. In addition, transiently expressed Tisp40ßHA also generated
polypeptides with apparent molecular weights similar to those observed in
cells transfected with Tisp40 or Tisp40HA. It is possible that
these could result from internal initiation or from degradation. Most
importantly, the high-molecular-weight forms observed with Tisp40ßHA were
not present in cells transfected with Tisp40HA or in testis. The
apparent molecular weight of HA-tagged Atce1/Tisp40 was only slightly
greater than that of p42, suggesting that p42 might correspond to
Atce1/Tisp40. In agreement with this prediction, a recombinant untagged
form of Atce1/Tisp40 comigrated precisely with p42. Interestingly,
transiently expressed Tisp40 also produced a 36-kd polypeptide.
The results of these immunoblotting experiments suggested that
Atce1/Tisp40 could give rise to the 42- and 36-kd proteins present in
mouse testis, but they did not clarify the nature of these polypeptides. The
Tisp40 mRNA contains 2 internal methionines (see
Figure 1) which could
potentially give rise to a 36-kd protein. We therefore inactivated methionine
codons 123 and 156 to determine if internal initiation could explain the
production of p36 in cells transfected with Tisp40 mRNA, but the
mutated expression plasmid still produced p42 and p36 (data not shown).
To determine if p42 corresponds to a glycosylated form of p36, we incubated
testis extracts as well as extracts of 293 cells expressing untagged
recombinant Tisp40 or Tisp40ß with endoglycosidase H. As shown in
Figure 4b, transiently
expressed Tisp40ß produced a migration pattern distinct from that
observed in testis extracts, whereas the migration pattern obtained with
transiently expressed Tisp40 was similar to that seen in testis.
Addition of endoglycosidase H to testis extracts converted p42 to p36,
indicating that p42 is indeed a glycosylated form of p36. Endoglycosidase H
also converted the 42-kd protein produced by Tisp40 to the 36-kd form.
In cells producing Tisp40ß, endoglycosidase H reduced the abundance of
high-molecular-weight forms. The lower-molecular-weight polypeptides seen in
cells transfected with Tisp40ß mRNA could correspond to degradation
products or the products of internal initiation (this has not been
investigated further). In fact, lysates programmed with Tisp40ß mRNA give
rise to a polypeptide that co-migrates with in vitro–translated
Tisp40 (Nagamori et al,
2005). Whether this occurs in vivo remains to be determined. Taken
together, these results indicate that Tisp40 is the major CREB3L4
protein product in mouse testis and that Tisp40 exists as an abundant
glycosylated form and a less abundant unglycosylated form.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Previous studies employing in situ hybridization detected CREB3L4 mRNA exclusively in spermatids (Stelzer and Don, 2002; Nagamori et al, 2005). However, the distribution and relative abundance of CREB3L4 protein(s) in mouse testis had not yet been examined. The immunostaining data presented here confirm that CREB3L4 proteins are produced in differentiating spermatids and provide direct evidence that the abundance of CREB3L4 proteins varies during spermatid differentiation. The abundance of CREB3L4 increases gradually through steps 1–6, peaks at step 7, and decreases thereafter through step 12. During spermiogenesis, transcription occurs until the midpoint of the postmeiotic phase, ie, steps 7–8 (Eddy, 2002). Thus, the presence of CREB3L4 coincides with a phase during which the ER would be solicited to process a large number of proteins. The fact that CREB3L4 is not detected in spermatocytes and in elongated spermatids indicates that the protein is dispensable at these stages of male germ cell differentiation.
In view of the fact that genes can give rise to multiple isoforms, some of
which are endowed with unique functional properties, it is important that we
know the tissue-specific distribution of such isoforms. The presently
available antibodies do not allow us to distinguish between the 2 Tisp40
isoforms. We therefore used transiently expressed recombinant Tisp40 proteins
combined with deglycosylation experiments to determine, with a reasonable
degree of certainty, that the 315-aa Atce1/Tisp40 protein is the major
CREB3L4 protein produced in mouse testis and that it exists as glycosylated
and unglycosylated forms. The observation that Atce1/Tisp40, rather
than Tisp40ß, is detected in mouse testis is consistent with the fact
that several genes encode testis-specific transcripts which result from the
use of alternative promoters (Kleene,
2001; Sassone-Corsi,
2002).
Our findings appear to contradict a recent study which concluded that both
Tisp40ß and Tisp40 are present in testis
(Nagamori et al, 2005).
Although Nagamori et al demonstrated that Tisp40 proteins are glycosylated
when they are expressed in HeLa cells, they did not specifically examine the
glycosylation state of endogenous Tisp40 proteins in testis. Because the
testis proteins detected by Nagamori et al are indistinguishable in size from
p42 and p36 reported in this study, we would anticipate that their p44 is a
glycosylated form of p38. Interestingly, Nagamori et al recently reported that
Creb3l4 is a target of CREM and that CREM activates transcription of the
Tisp40 mRNA via response elements located between the transcription
start sites of the Tisp40ß and Tisp40 mRNAs
(Nagamori et al, 2006). In
their assays, CREM did not activate the promoter region located upstream of
the Tisp40ß mRNA. Taken together, these data from Hiroshi Nojima's
laboratory, combined with those reported here, support the conclusion that
Tisp40 is the major CREB3L4 protein in testis.
The realization that Tisp40ß is not produced in differentiating
spermatids has important implications regarding the role of CREB3L4 in male
reproduction. Indeed, Atce1/Tisp40 lacks a large portion of the
transcription activation domain present in Tisp40ß, and a recent report
demonstrated that Tisp40ß could activate transcription via an unfolded
protein response element, whereas Tisp40 failed to activate
transcription via this same element
(Nagamori et al, 2005). Thus,
one might be tempted to conclude that Atce1/Tisp40 (and p36) actually
serves as a transcriptional repressor, possibly by interfering with the
activity of other bZIP transcription factors. On the other hand, it is also
possible that Atce1/Tisp40 could regulate gene expression via other
unidentified response elements, or that it could only act as a transcriptional
activator when dimerized with another bZIP protein.
Hiroshi Nojima's laboratory recently reported that the nuclear form of
Tisp40 is capable of dimerizing with CREM and that this interaction
augments the interaction between CREM and a CRE
(Nagamori et al, 2006). Such
an association seems somewhat counterintuitive, as a bZIP protein that is
implicated in ER stress would not be expected to contribute to the activity of
a transcription factor whose actions could ultimately stress the ER. Possibly,
the CREM-Tisp40 dimer regulates a subset of genes that serve to protect
the ER and/or the CREM-Tisp40 dimer fulfills a specialized function in
spermatids. In fact, Nojima's laboratory discovered that the
CREM-Tisp40 dimer recruits a histone chaperone. The functional
significance of this interaction remains to be determined.
The importance of CREB3L4 in mammalian reproduction is supported by a recent report which described alterations in the reproductive system of male mice in which the Creb3l4 gene was inactivated by replacement with a green fluorescent protein (Adham et al, 2005). Although the resulting mice were fertile, inactivation of Creb3l4 resulted in a significant reduction in the number of spermatozoa in the epididymis. This reduction was attributed to increased apoptosis of haploid spermatids, which is consistent with a protective role of CREB3L4 in ER function.
In summary, the data presented herein indicate that the Atce1/Tisp40
isoform of CREB3L4 is expressed in a stage-specific manner during spermatid
differentiation in mice. Future studies focussing on the regulation and action
of this testis-specific CREB3L4 variant will allow us to better understand the
function of this potentially important gene in mammalian reproduction.
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Acknowledgments |
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Footnotes |
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DOI: 10.2164/jandrol.106.000596
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