Novel role for a sterol response element binding protein in directing spermatogenic cell-specific gene expression.
ABSTRACT Sperm are highly specialized cells, and their formation requires the synthesis of a large number of unique mRNAs. However, little is known about the transcriptional mechanisms that direct male germ cell differentiation. Sterol response element binding protein 2gc (SREBP2gc) is a spermatogenic cell-enriched isoform of the ubiquitous transcription factor SREBP2, which in somatic cells is required for homeostatic regulation of cholesterol. SREBP2gc is selectively enriched in spermatocytes and spermatids, and, due to its novel structure, its synthesis is not subject to cholesterol feedback control. This suggested that SREBP2gc has unique cell- and stage-specific functions during spermatogenesis. Here, we demonstrate that this factor activates the promoter for the spermatogenesis-related gene proacrosin in a cell-specific manner. Multiple SREBP2gc response elements were identified within the 5'-flanking and proximal promoter regions of the proacrosin promoter. Mutating these elements greatly diminished in vivo expression of this promoter in spermatogenic cells of transgenic mice. These studies define a totally new function for an SREBP as a transactivator of male germ cell-specific gene expression. We propose that SREBP2gc is part of a cadre of spermatogenic cell-enriched isoforms of ubiquitously expressed transcriptional coregulators that were specifically adapted in concert to direct differentiation of the male germ cell lineage.
- [Show abstract] [Hide abstract]
ABSTRACT: As germ cells divide and differentiate from spermatogonia to spermatozoa, they share a number of structural and functional features that are common to all generations of germ cells and these features are discussed herein. Germ cells are linked to one another by large intercellular bridges which serve to move molecules and even large organelles from the cytoplasm of one cell to another. Mitochondria take on different shapes and features and topographical arrangements to accommodate their specific needs during spermatogenesis. The nuclear envelope and pore complex also undergo extensive modifications concomitant with the development of germ cell generations. Apoptosis is an event that is normally triggered by germ cells and involves many proteins. It occurs to limit the germ cell pool and acts as a quality control mechanism. The ubiquitin pathway comprises enzymes that ubiquitinate as well as deubiquitinate target proteins and this pathway is present and functional in germ cells. Germ cells express many proteins involved in water balance and pH control as well as voltage-gated ion channel movement. In the nucleus, proteins undergo epigenetic modifications which include methylation, acetylation, and phosphorylation, with each of these modifications signaling changes in chromatin structure. Germ cells contain specialized transcription complexes that coordinate the differentiation program of spermatogenesis, and there are many male germ cell-specific differences in the components of this machinery. All of the above features of germ cells will be discussed along with the specific proteins/genes and abnormalities to fertility related to each topic.Microscopy Research and Technique 11/2009; 73(4):364-408. · 1.59 Impact Factor
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ABSTRACT: Extra-embryonic tissue-spermatogenesis-homeobox gene 1 (Esx1) encodes an X-linked homeobox protein. Despite the fact that the temporal and spatial mRNA expression pattern of the protein has been studied extensively in the testis, specific localisation of ESX1 in the testis remains to be determined. In the present study, we generated ESX1 antiserum to investigate the stage- and tissue-specific expression of ESX1 in the mouse. Western blotting and immunofluorescent analyses revealed that general localisations of ESX1 were consistent with its RNA expression patterns; that is, it was restricted mainly to the placenta and testis. Immunofluorescent studies demonstrated that ESX1 existed in the testes after 3 weeks of age, coincident with the appearance of round spermatids in the seminiferous tubules. Moreover, ESX1 expression became more abundant in the luminal regions of the seminiferous tubules as the development of round spermatids progressed into spermatozoa. In contrast, reduced expression of ESX1 was observed in experimentally induced cryptorchid testes. The later expression of ESX1 suggests a role in post-meiotic germ cell development. To further understand ESX1 expression in sperm with respect to X chromosome-bearing sperm, we used ESX1 antiserum to immunostain sperm by confocal laser microscopy. Approximately half the sperm population was recognised by the ESX1 antiserum. On the basis of results of the present study, we suggest that ESX1 could be used as a protein marker for X chromosome-bearing sperm.Reproduction Fertility and Development 02/2005; 17(4):447-55. · 2.58 Impact Factor
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ABSTRACT: The molecular mechanism of how cells maintain cholesterol homeostasis has become clearer for the understanding of complicated association between sterol regulatory element-binding proteins (SREBPs), SREBP cleavage-activating protein (SCAP), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) and Insuin induced-genes (Insigs). The pioneering researches suggested that SREBP activated the transcription of genes encoding HMG-CoA reductase and all of the other enzymes involved in the synthesis of cholesterol and lipids. However, SREBPs can not exert their activities alone, they must form a complex with another protein, SCAP in the endoplasmic reticulum (ER) and translocate to Golgi. Insigs are sensors and mediators that regulate cholesterol homeostasis through binding to SCAP and HMG-CoA reductase in diverse tissues such as adipose tissue and liver, as well as the cultured cells. In this article, we aim to review on the dual functions of Insig protein family in cholesterol homeostasis.Lipids in Health and Disease 12/2012; 11(1):173. · 2.31 Impact Factor
MOLECULAR AND CELLULAR BIOLOGY, Dec. 2004, p. 10681–10688
0270-7306/04/$08.00?0 DOI: 10.1128/MCB.24.24.2004.10681–10688.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 24, No. 24
Novel Role for a Sterol Response Element Binding Protein in
Directing Spermatogenic Cell-Specific Gene Expression
Hang Wang,1Jovenal T. San Agustin,2George B. Witman,2and Daniel L. Kilpatrick1*
Department of Physiology1and Department of Cell Biology,2University of Massachusetts Medical
School, Worcester, Massachusetts
Received 1 July 2004/Returned for modification 1 August 2004/Accepted 26 September 2004
Sperm are highly specialized cells, and their formation requires the synthesis of a large number of unique
mRNAs. However, little is known about the transcriptional mechanisms that direct male germ cell differen-
tiation. Sterol response element binding protein 2gc (SREBP2gc) is a spermatogenic cell-enriched isoform of
the ubiquitous transcription factor SREBP2, which in somatic cells is required for homeostatic regulation of
cholesterol. SREBP2gc is selectively enriched in spermatocytes and spermatids, and, due to its novel structure,
its synthesis is not subject to cholesterol feedback control. This suggested that SREBP2gc has unique cell- and
stage-specific functions during spermatogenesis. Here, we demonstrate that this factor activates the promoter
for the spermatogenesis-related gene proacrosin in a cell-specific manner. Multiple SREBP2gc response
elements were identified within the 5?-flanking and proximal promoter regions of the proacrosin promoter.
Mutating these elements greatly diminished in vivo expression of this promoter in spermatogenic cells of
transgenic mice. These studies define a totally new function for an SREBP as a transactivator of male germ
cell-specific gene expression. We propose that SREBP2gc is part of a cadre of spermatogenic cell-enriched
isoforms of ubiquitously expressed transcriptional coregulators that were specifically adapted in concert to
direct differentiation of the male germ cell lineage.
Sperm are highly differentiated cells that are uniquely
adapted to their function as motile cells mediating fertilization.
As such, they serve as an important model for exploring reg-
ulatory programs responsible for cellular differentiation (17).
Spermatogenesis consists of a complex interplay between cell-
specific gene transcription, RNA processing, and translational
regulation (8, 17). It occurs in a series of proliferation and
differentiation stages, which can be subdivided into mitotic,
meiotic, and spermiogenic phases. Each phase is characterized
by distinct cell types, namely, spermatogonia, spermatocytes,
and spermatids, respectively. The highly specialized nature of
sperm is reflected in the large number of cell-specific tran-
scripts and proteins they express (8), many of which are asso-
ciated with unique sperm structures such as the acrosome,
sperm tail, and the highly compacted sperm chromosomal
DNA. Unique proteins also are required to meet specialized
requirements for energy metabolism, meiosis, and the matu-
ration of haploid cells, including cell-specific proteins that
compensate for X chromosome inactivation (e.g., phospho-
glycerate kinase 2 [pgk-2]) (9). These various gene products
also must be expressed at the appropriate time to ensure nor-
mal development. Thus, sperm formation requires both the
generation of a large number of cell-specific gene products and
the coordination of this differentiation program in a stepwise,
stage-appropriate manner. A key question is the nature of the
transcriptional network that controls the elaboration of this
Cell-specific transcription from alternative promoters or
unique genes plays a predominant role in directing male germ
cell differentiation (8). Numerous spermatogenic cell-enriched
transcription factors have been identified, many of which are
selectively expressed during meiotic and/or early haploid stages
(4, 6, 25, 33). For example, the spermatogenic cell-specific
factor CREM? is an activator of several genes expressed in
haploid spermatids and is required for completion of spermio-
genesis (5, 28). CREM? also interacts with a germ cell-specific
coactivator termed ACT (11), and unique germ cell isoforms of
basal transcription factors have been identified (15, 27). All
this indicates that spermatogenic cells have evolved a highly
specialized transcriptional program. However, functional iden-
tification of transcription factors responsible for controlling
spermatogenic cell differentiation has been elusive. In partic-
ular, CREM? is the only spermatogenic cell-enriched tran-
scription factor for which a physiological role and specific germ
cell-specific target genes have been determined (7). Moreover,
nothing is currently known about the cell-specific regulators of
gene promoters expressed in spermatocytes.
Sterol response element binding protein 2gc (SREBP2gc) is
a 55-kDa, germ cell-enriched form of the basic helix-loop-helix
leucine zipper (bHLHZip) transcription factor SREBP2 (50).
Its expression is highly up-regulated during late meiosis and in
early-round spermatids, suggesting stage-specific functions. In
somatic cells, SREBP2 regulates genes involved mainly in cho-
lesterol synthesis (19), and its transcriptional activity is highly
dependent on the function of coregulatory factors, such as
CREB/CREM, NF-Y, Sp1, and the SREBP antagonist YY1
(10). SREBPs are synthesized as membrane-bound precursor
proteins that are proteolytically processed in the Golgi appa-
ratus to generate a cytoplasmic, transcriptionally active mature
SREBP. Sterols regulate this processing step as part of a ho-
meostatic, inhibitory feedback mechanism by blocking the
* Corresponding author. Mailing address: Department of Physiol-
ogy, University of Massachusetts Medical School, 55 Lake Ave. N,
Worcester, MA 01655-0127. Phone: (508) 856-6274. Fax: (508) 856-
5997. E-mail: Daniel.Kilpatrick@Umassmed.edu.
transport of the SREBP precursor from the endoplasmic re-
ticulum to the Golgi apparatus (19). In contrast to this, trans-
lation of the alternatively spliced SREBP2gc mRNA generates
a soluble, constitutively active transcription factor that conse-
quently is insensitive to cholesterol feedback control (50).
These observations suggested that SREBP2gc performs novel
functions during spermatogenesis, not restricted to cholesterol
metabolism alone. The present studies demonstrate that
SREBP2gc regulates the transcription of a spermatogenic cell-
specific gene proacrosin, which is expressed in both spermato-
cytes and round spermatids. This factor likely regulates multi-
ple gene targets as part of a global transcriptional program
directing meiotic and postmeiotic stages of spermatogenic cell
MATERIALS AND METHODS
Plasmid DNA constructs. An ?1-kb genomic fragment containing 5?-flanking,
exon 1, intron 1, and partial exon 2 sequences for the rat proacrosin gene
(GenBank accession number X58550) was generated by PCR (primer sequences
are available upon request). This was inserted into pGEM-T Easy vector (Pro-
mega, Madison, Wis.) and then released with SacI and SacII and subcloned into
the pGL3-Basic vector by using SacI and SmaI sites. This step eliminates a
polylinker region within the pGL3-Basic plasmid that contains an E box respon-
sive to SREBPs (3). Additional proacrosin promoter constructs containing mu-
tations in SREBP2gc binding sites were generated by PCR. Detailed procedures
and conditions and various primer sequences are available upon request. The
wild-type and SRE-1 site mutant squalene synthase (SQS) gene promoter con-
structs were previously described (14).
RNA and protein analyses. Total RNAs were prepared and analyzed by North-
ern analysis and reverse transcription-PCR (RT-PCR) as previously described
(50). A 1.8-kb rat SREBP2gc cDNA was used as the probe for Northern analysis.
Nuclear extracts were prepared from cell lines and enriched mouse spermato-
genic cells by high salt extraction (26). Western blotting was performed as
described previously (50) using antiserum raised against mouse SREBP2. The
oligodeoxynucleotides used for generating various DNA probes and competitors
for electrophoretic mobility shift assays (EMSAs) as well as primers for RT-PCR
are available upon request. EMSAs were performed using nuclear extracts and
an SRE-1 probe, as in previous studies of SREBP2gc (50).
Cell cultures and transfections. Cell lines were cultured in Dulbecco’s mod-
ified Eagle’s medium (DMEM) containing 100 U of penicillin-streptomycin
(PS)/ml and 10% fetal bovine serum (FBS), except for GC-1spc cells, which were
cultured in 13% FBS. One percent nonessential amino acids (AA) also was
included for GC-4spc and GC-1spc cells. All cells were incubated with 5% CO2
at 37°C. For sterol depletion studies, cells were freshly plated in DMEM-PS-AA
medium containing 10% FBS. Twenty-fours later, they were rinsed with 1?
phosphate-buffered saline and then were cultured for an additional 10 h in
DMEM-PS-AA containing 5% lipoprotein-deficient FBS (Sigma, St. Louis,
Mo.), 50 ?M compactin, and 50 ?M sodium mevalonate with (sterol loaded) or
without (sterol depleted) cholesterol (10 ?g/ml) and 25-hydroxycholesterol (1
?g/ml). ALLN protease inhibitor (Calbiochem, La Jolla, Calif.) at 25 ?g/ml was
added to the culture medium 1 h prior to extraction of nuclear proteins.
For promoter studies, DNAs for promoter constructs (0.5 ?g), pCMV7 or
pCMV-BP2gc (10 ng), and pRL-null normalization plasmid (0.1 ?g) were co-
transfected with Trans-Fast reagent (Promega). Cell extracts were then analyzed
40 to 48 h later with the Dual Luciferase reporter assay system (Promega). All
promoter data (expressed as relative firefly luciferase light units [RLU]) were
normalized with Renilla luciferase activity and are reported as the means ?
standard errors of four to eight independent experiments. The expression vector
pKAc (0.1 ?g) for the protein kinase A c subunit also was included in proacrosin
promoter studies. Student’s t test was used to evaluate data significance.
Transgenic mice. Transgenes containing wild-type or mutant rat proacrosin
promoter-luciferase sequences as well as a simian virus 40 poly(A) signal were
released from their parent pGL3 vectors with SalI and ApaI and gel purified
prior to injection. The genotype of offspring was determined by PCR for lucif-
erase sequences (data available on request). Testes and somatic tissues from
adult (2 to 3 months) male transgenic founders or F1mice were extracted and
assayed for luciferase activity. Protein concentration was determined with Brad-
ford reagent (Bio-Rad Laboratory, Hercules, Calif.).
Immunohistochemistry. Immunostaining was performed on paraffin-embed-
ded sections of adult mouse testes as described in a previous study (2) with slight
modifications. Briefly, deparaffinized testis sections (5 ?m) were rehydrated and
subjected to antigen retrieval and blocking with the biotin blocking system
(DakoCytomation, Carpenteria, Calif.) and 20% normal swine serum–5% fatty
acid-free bovine serum albumin. Sections were incubated with a rabbit antilucif-
erase antibody (0.5 ?g/ml; Cortex Biochem Inc., San Leandro, Calif.), and bound
antibody was detected with biotinylated swine anti-rabbit immunoglobulin G and
alkaline phosphatase-conjugated streptavidin together with the Fuchsin sub-
strate system (DakoCytomation). Hematoxylin was used as a counterstain.
Promoter sequence analysis. To identify possible SREBP2gc response ele-
ments within the rat, mouse, and human proacrosin promoters, sequences ob-
tained from GenBank were searched for known sterol response element (SRE)
half-sites with OMIGA, version 2.0, software (Oxford Molecular Ltd.). These
were also compared to an NNCNNNCNAN motif often associated with SREs
SREBP2gc is expressed in a spermatogenic cell line. To test
the hypothesis that SREBP2gc mediates spermatogenic cell-
specific gene expression, we first examined whether it was
expressed in cell lines derived from male germ cells. GC-4spc
cells were originally selected with a neomycin resistance ex-
pression vector driven by the human pgk-2 promoter (47). They
express several spermatocyte-related genes, including proacro-
sin and pgk-2, but not various markers for testicular somatic
cells or a spermatogonium-associated gene promoter. North-
ern and RT-PCR analyses of GC-4spc cells detected an
SREBP2gc mRNA that was identical in size (?2.5 kb) and
similar in amount to that for the adult mouse germ cell tran-
script (Fig. 1A and B). They also detected an abundant
SREBP2 transcript (?5 kb) corresponding to the precursor
mRNA (Fig. 1B). Further, GC-4spc cells also contain substan-
tial amounts of sequence-specific SRE binding activity based
on EMSAs (Fig. 1C). Western blotting of nuclear extracts
confirmed the presence of a 55-kDa SREBP2 protein corre-
sponding in size to SREBP2gc (Fig. 1D). SREBP2 precursor
protein (?125 kDa) also was detected in GC-4spc cells (data
not shown), consistent with the presence of its mRNA in this
Due to SREBP2gc’s unique structure, SREBP2gc protein
levels in spermatogenic cells are unaffected by sterol concen-
trations that suppress formation of transcriptionally active
SREBPs in somatic cells by feedback inhibition of precursor
processing (50). Thus, we examined whether SREBP2 protein
levels were affected by sterols in GC-4spc cells. In Western
blots, amounts of the 55-kDa SREBP2gc protein under sterol-
loaded and sterol-depleted culture conditions were the same
(Fig. 2A). Similarly, SRE DNA binding activity in GC-4spc
cells was unaltered by sterol load (Fig. 2B). Thus, SREBP2
protein and DNA binding activity in GC-4spc cells are insen-
sitive to sterols, consistent with the properties of SREBP2gc
from spermatogenic cells.
Interestingly, we did not detect significant formation of the
mature, 66-kDa protein derived from the SREBP2 precursor
upon sterol depletion (Fig. 2A). This suggests that SREBP2
precursor processing is defective or suppressed in GC-4spc
cells. This is reminiscent of the sterol-resistant SRD-3 mutant
cell line that expresses a constitutively active SREBP2 protein
analogous to SREBP2gc (54). This novel isoform repressed
proteolytic processing of endogenous SREBP precursors.
SREBP2gc may have similar effects on the generation of ma-
10682WANG ET AL.MOL. CELL. BIOL.
ture SREBP2 protein in GC-4spc cells, as well as in pachytene
spermatocytes and round spermatids which express small
amounts of the SREBP2 precursor mRNA (50).
To examine whether GC-4spc cells express endogenous
SRE-dependent transcriptional activity, they were transfected
with SQS gene promoter-luciferase plasmids (13). The SQS
gene is responsive to SREBPs and is expressed in spermato-
cytes as well as spermatids (44). We observed much higher
basal SQS gene promoter activity in GC-4spc cells than in
somatic cell lines such as 3T3L1 (Fig. 3A), which lack detect-
able SREBPs under serum-containing conditions (50). Impor-
tantly, basal promoter activity in GC-4spc cells was highly
dependent on a functional SRE site (?10-fold difference be-
tween wild-type and SRE mutant constructs), which was not
the case in transfected 3T3L1 cells (Fig. 3A). This indicated
the presence of endogenous SREBP transcriptional activity
selectively in GC-4spc cells. Further, cotransfected SREBP2gc
dramatically increased SQS promoter activity in this cell line,
which also required the SRE site (Fig. 3B). Thus, GC-4spc
cells express active SREBP2gc protein and are suitable for
studying its transcriptional activity in a spermatogenic cell-like
environment, including its possible regulation of germ cell-
specific gene expression.
SREBP2gc activates a spermatogenic cell-specific promoter.
Proacrosin is an acrosomal zymogen for a protease implicated
in sperm competition and sperm-oocyte interactions (1, 29)
and in the dispersal of acrosomal components upon onset of
the acrosome reaction (53). It is encoded by a spermatogenic
cell-specific gene first expressed in spermatocytes and then
highly up-regulated in spermatids (21), at which time mRNA
translation occurs (31). Since both the proacrosin and pgk-2
promoters are transcribed in pachytene spermatocytes and in
GC-4spc cells, we examined their potential regulation by
SREBP2gc in cotransfection studies. pgk-2 promoter activity
was not stimulated by SREBP2gc in any of the cell lines tested
(results not shown). However, strong activation of the proacro-
sin promoter was observed in GC-4spc cells (Fig. 3C). In con-
FIG. 1. GC-4spc cells express SREBP2gc. (A) RT-PCR analysis of
SREBP2gc mRNA. One microgram of total RNA from adult mouse
spermatogenic cells (G) and liver (L) and GC-4spc cells was analyzed.
—, no RNA template and no reverse transcriptase negative controls;
M, DNA size ladder. Primers that specifically detect the mouse
SREBP2gc transcript were used (50) (available on request). (B) North-
ern analysis using total RNA from GC-4spc cells (15 ?g), 21-day-old
mouse testis (MT; 20 ?g), and purified mouse pachytene spermato-
cytes (PS; 20 ?g). Arrow, SREBP2gc mRNA. Ethidium bromide stain-
ing is shown below the Northern analysis results. (C) EMSA of
SREBPs in GC-4spc cells. Lanes 1 to 3, 2 ?g of GC-4spc nuclear
extract; lanes 4 to 6, 2 ?g of adult mouse germ cell nuclear extract.
Lanes 1 and 4, no competitor; lanes 2 and 5, wild-type SRE-1 com-
petitor; lanes 3 and 6, mutated SRE-1 competitor. Arrow, specific
SREBP complex. (D) Western analysis of nuclear extracts (30 ?g)
from GC-4spc cells for SREBP2 proteins. A single, major band of ?55
kDa (arrow), identical in size to that for SREBP2gc, was detected.
FIG. 2. Levels of SREBP2gc protein and SRE DNA binding activ-
ity are insensitive to sterols. GC-4spc cells were cultured in either
sterol-loaded (?) or sterol-depleted (?) medium (see Materials and
Methods). Nuclear protein was then assayed by Western analysis (A;
30 ?g per lane) or EMSAs (B; 6 ?g per lane) using the SRE-1 probe.
Arrows, SREBP2gc protein or specific DNA binding complex in each
FIG. 3. Expression of SREBP transcriptional activity in GC-4spc
cells. (A) GC-4spc and 3T3L1 cells were transfected with human SQS
gene promoter constructs containing either wild-type or mutated
(MSQS) SRE sites, and luciferase activity was determined. (B) Co-
transfection of GC-4spc cells with wild-type or mutant SQS gene pro-
moter plasmids together with either an expression vector for
SREBP2gc (BP2GC) or the empty parent plasmid (CMV7). (C) Co-
transfection of the rat proacrosin promoter together with SREBP2gc or
pCMV7 expression plasmids in different cell lines. (D) Cell lines were
cotransfected with either SQS (NIH 3T3 and GC-1spg) or CYP51
(JEG3 and GC-4spc) gene promoter plasmids and expression vectors.
Data are shown as the increases in activity in the presence of
SREBP2gc relative to that for pCMV7.
VOL. 24, 2004 SREBP2gc REGULATES A GERM CELL-SPECIFIC PROMOTER10683
trast, this promoter was poorly expressed or undetectable in
somatic cell lines, and no significant stimulation by SREBP2gc
was observed in any of these (Fig. 3C and data not shown).
SREBP2gc also did not activate the proacrosin promoter in a
different spermatogenic cell-derived cell line, GC-1spg (data
not shown), which resembles late spermatogonial stages and
does not express the proacrosin promoter (18, 47). More gen-
erally expressed SREBP target promoters (CYP51  and
SQS gene promoters) were strongly activated by SREBP2gc in
all cell lines tested (Fig. 3D), demonstrating that the cotrans-
fected factor is transcriptionally active in each case. Thus,
SREBP2gc potently activates the proacrosin promoter in a
The proacrosin promoter contains SREBP2gc response ele-
ments. The rat and mouse proacrosin promoters are highly
homologous and contain a number of conserved trans-factor
consensus elements (24, 39) (Fig. 4A). These include sites for
known SREBP coregulators: Y boxes, cyclic AMP response
elements (CREs), YY1 sites, and GC boxes. A search for
SRE-like sequences identified five potential SREBP2gc re-
sponse elements within the rat and mouse proacrosin promot-
ers that were conserved in their locations and general sequence
FIG. 4. Identification of SREBP2gc binding sites within the proacrosin promoter. (A) Organization of SREs within the rat, mouse, and human
proacrosin promoters. GC, E, and Y boxes as well as CREs and YY1 sites also are shown. (B to D) Competitive EMSAs using adult mouse germ
cell nuclear extracts (2 ?g) and rat proacrosin SRE sites. Lanes: 0, no extract; 1, extract without competitor; 2, wild-type SRE-1 competitor; 3,
SRE-1mut; 4 to 6, wild-type SREpa2; 7 to 9, mutated SREpa2; 10, no competitor; 11, mutated SREpa3; 12, wild-type SREpa3; 13, mutated
SREpa4; 14, wild-type SREpa4; 15, wild-type SREpa5; 16, SRE-1mut; 17, wild-type SRE-1; 18, no competitor; 19, no extract. The mutated SREpa5
and SREpa4 competitors were identical (see Table 1). Arrows, specific SREBP complexes. (E) Southwestern analysis using SREpa2. Five
micrograms of nuclear extract from adult mouse germ cells (lanes 1 and 3) and adult mouse liver (lanes 2 and 4) was probed with either wild-type
SREpa2 (lanes 1 and 2) or SRE-1 (lanes 3 and 4) sequences. Arrow, germ cell-specific, 55-kDa SREBP2gc protein.
TABLE 1. Sequences of putative SREs in the mouse, rat, and humanaproacrosin promoters
GCACTTCAGCACAGATCAG (?123, ?141)
TGGCACCTCAGCG (?133, ?145)
CTCATGAGTACCTCACCACCCTGAGGCGG (?170, ?198)
GGCTGGCCAA (?240, ?249)
GGCTCGCCAA (?239, ?248)
ACCTTTCCATACTAT (?782, ?796)
GCCTTTCCATGCTATAAGAGG (?763, ?784)
CTGGATGGGTAGGA (?844, ?857)
CTCGATGGGTAGGA (?822, ?837)
aHuman SREs: TTGCAGGCCAGGC (?13, ?24), ACCTGGCCTGACT (?97, ?109), GGGTGATGTGGGG (?262, ?274), and GTCTGCAGTGGAC (?333,
?345). The significance of underlining and italics is indicated in footnote c.
bM, mouse, R, rat.
cSRE half-sites are underlined, and NNCNNNCNAN motifs are italicized. Numbers in parentheses indicate the positions of the first and last bases within respective
proacrosin promoter nucleotide sequences relative to the translational start site (?1).
dSubstitution or deletion mutations of rat sites.
10684 WANG ET AL.MOL. CELL. BIOL.
features (SREpa1 to -5; Fig. 4A and Table 1). In most in-
stances, two or more previously identified SRE half-sites were
present, and several contained an NNCNNNCNAN motif
found in several SREs (45). The presence of multiple SREs
within a target promoter is not uncommon (20). Interestingly,
the SREpas for the rat and mouse were segregated into up-
stream (SREpa4 and -5) and downstream (SREpa1, -2, and -3)
groups that were closely adjacent to consensus sequences for
known SREBP coregulators (Fig. 4A). Such close proximity of
SREs and coregulator sites is typical for SREBP-responsive
promoters (40). Multiple SRE-like sequences along with
neighboring coregulator sites also were identified in the human
proacrosin promoter (Fig. 4A; Table 1), suggesting conserva-
tion of promoter organization in humans.
Competition EMSAs were performed on candidate SREs
for the rat proacrosin promoter using mouse germ cell extracts.
All but one (SREpa1; data not shown) exhibited good binding
to native SREBP2gc (Fig. 4B to D). Mutated versions of these
four rat SREpas showed greatly diminished binding. South-
western analysis previously demonstrated that the 55-kDa
SREBP2gc protein in spermatogenic cells bound to SRE se-
quences (50). This assay confirmed the binding of rat proacro-
sin SRE sites by endogenous SREBP2gc in mouse germ cell
extracts (Fig. 4E).
We next examined the functional importance of the SREpa
sites by promoter mutation analysis. Three different rat
proacrosin promoter constructs were generated, two in which
either the upstream sites (SREpa4,5mut) or the downstream
sites (SREpa2,3mut) were mutated and a third containing mu-
tations of all four sites (SREpa2-5mut). These promoters were
then tested in GC-4spc cells for basal and SREBP2gc-stimu-
lated activities (Fig. 5A). Mutation of the two upstream
SREpas reduced basal activity approximately threefold, while
activation by SREBP2gc was only modestly affected. In con-
trast, mutation of the downstream SREpa2 and -3 sites re-
sulted in complete loss of SREBP2gc-induced activation (Fig.
5A). The combined upstream and downstream mutant pro-
moter also showed no SREBP2gc-dependent stimulation.
Thus, SREpa2 and -3 are critical for SREBP2gc induction of
the proacrosin promoter in GC-4spc cells. SREpa4 and -5 have
only a modest role in this but appear to be required for optimal
basal promoter activation by endogenous SREBP2gc. Muta-
tion of either SREpa2 and -3 alone or of all four sites caused
a small increase in basal activity (Fig. 5A).
In vivo expression of the proacrosin promoter depends on
SREBP2gc response elements. To test the importance of
FIG. 5. SREBP2gc binding sites are required for proacrosin pro-
moter activation in vitro and in vivo. (A) Activities of different proacro-
sin promoter plasmids in GC-4spc cells cotransfected with either
empty pCMV7 (blue bars) or SREBP2gc (red bars) expression vectors.
?, significantly different from basal activity for the wild-type (WT)
promoter (P ? 0.01); ??, significantly different from basal activity for
the respective promoter construct (P ? 3.00 ?10?5). (B) Luciferase
activities for wild-type and SREpa2-5mut (MUT) rat proacrosin-lucif-
erase constructs in testicular extracts from male transgenic mice. Num-
bers along the x axis indicate independent transgenic lines. Mean
activity for the mutant construct (1.6 ? 105RLU) was significantly
different from that for the wild-type promoter (6.4 ? 105RLU) (P ?
0.014). (C) Staining for luciferase protein in testes of adult transgenic
mice expressing wild-type (line 46; WT46) or SREpa2-5mut (line 11;
MUT11) proacrosin promoter constructs. Luciferase staining is distin-
guishable in spermatids by its cytoplasmic localization. Scales are
shown for photomicrographs in the upper row as well as the lower two
panels, respectively. NO AB, no primary antibody control.
VOL. 24, 2004 SREBP2gc REGULATES A GERM CELL-SPECIFIC PROMOTER10685
SREBP2gc-induced activation in proacrosin promoter expres-
sion during spermatogenesis, we generated transgenic mice
harboring wild-type or SREpa mutant proacrosin-luciferase
fusion genes. The SREpa2-5mut promoter was examined to
test the cumulative role of all SREBP2gc response elements.
Previous studies showed that the ?1-kb rat proacrosin pro-
moter used here directed faithful cell-specific gene expression
in spermatocytes and spermatids of transgenic mice (30). As
observed in numerous earlier studies (22, 34, 57), there was no
correlation between transgene copy number and luciferase ac-
tivity for either promoter construct (data not shown). Out of
six transgenic males containing wild-type rat proacrosin pro-
moter sequences, four expressed moderate-to-high levels of
luciferase activity in the testis, while two exhibited low activity
(Fig. 5B). This expression frequency (67%) is typical for active
transgene promoters (26, 34). No activity was detected in so-
matic tissues from any transgenic mice (data not shown). In
contrast, the SREpa2-5 mutant promoter was expressed at
much lower levels in testes of founder males, with only 18% (2
of 11) having moderate testicular expression and none showing
Immunohistochemical staining confirmed the presence of
luciferase protein in the cytoplasm of round spermatids ex-
pressing the wild-type proacrosin promoter, with the strongest
staining occurring in spermatid stages VI and VII (Fig. 5C).
Weaker cytoplasmic staining was observed in tubules contain-
ing spermatids at other phases of development, including late,
condensing spermatids. No obvious staining was discernible in
spermatocytes, consistent with stage-dependent translational
regulation of endogenous proacrosin mRNA and proacrosin
transgene-derived transcripts (30, 31). Transgene expression
was undetectable in testicular somatic cell types (peritubular,
Sertoli, and interstitial cells). In contrast, expression of the
SREpa2-5mut proacrosin promoter was reduced in the cyto-
plasm of all spermatid stages of mouse line 11 (Fig. 5C), which
exhibits lower but detectable luciferase activity (Fig. 5B). Thus,
SREBP2gc response elements are critical for proacrosin pro-
moter expression during spermatogenesis.
The importance of SREBPs in the homeostatic control of
cholesterol and fatty acid synthesis in somatic cells is well
established (19). However, the finding of a constitutively ac-
tive, sterol-insensitive form of SREBP2 that is expressed in a
developmentally regulated manner during spermatogenesis in-
dicated a broader role for this factor not limited to lipid me-
tabolism alone (50). The present findings directly implicate
SREBP2gc in the stage-dependent expression of the spermat-
ogenic cell-specific gene proacrosin, which is expressed in both
spermatocytes and spermatids. SREBP2gc is only the second
spermatogenic cell-enriched transcription factor (CREM? is
the first) shown to regulate a germ cell-specific promoter, and
it is the first such factor shown to activate a gene expressed
during male meiosis. Further, it is likely that SREBP2gc reg-
ulates multiple spermatogenic-cell-specific genes, not proacro-
sin alone. Thus, this factor may be an integral part of a more
global differentiation program, and defining additional target
promoters for SREBP2gc in male germ cells is an important
future goal. In particular, disruption of SREBP2gc function
during spermatogenesis will establish the extent to which this
factor is involved in directing spermatogenic differentiation as
well as the nature of its gene targets. It also should provide the
first insight into the cell-specific transcriptional mechanisms
operating in meiotic spermatocytes.
Based on the present results, it appears that a ubiquitous
somatic factor (SREBP2) was adapted by spermatogenic cells
to function in an entirely new manner as a trans regulator of
germ cell-specific genes. In fact, precedent for this notion al-
ready exists in the form of CREM?: analogous to SREBP2gc,
it is a spermatogenic cell-specific variant of a generally ex-
pressed transcription factor family generated by alternative
splicing. Both factors also possess unique properties that cir-
cumvent regulatory mechanisms operating in somatic cells and
that are critical for their function as spermatogenic cell trans
regulators. For CREM?, alternative splicing converts the
CREM repressor into a germ cell-specific activator of CREs
(12). Further, phosphorylation mechanisms normally required
for interactions with the CREB coactivator CBP do not appar-
ently operate in spermatids. Instead, CREM? interacts with
the phosphorylation-independent coactivator ACT, which is
expressed only in haploid spermatogenic cells along with
CREM? (11). This alternative pathway apparently evolved to
provide for both stage- and cell-specific activation of CRE-
dependent promoters in germ cells. Similarly, alternative RNA
processing in spermatogenic cells generates an SREBP2 iso-
form that bypasses sterol-dependent inhibitory mechanisms,
permitting stage-dependent up-regulation of a constitutively
active factor and its target promoters in late spermatocytes and
It is of interest that SRE- and CRE-binding proteins act
together to regulate numerous promoters in somatic cells (40).
It therefore seems likely that SREBP2gc and CREM? coordi-
nately regulate common spermatogenic cell-specific promoters
in spermatids. This may reflect coevolution of functionally
related transcription factors, in which interacting partners take
on cell-specific functions in parallel. In fact, these two proteins
may be members of a larger group of factors, including
Y/CAAT- and GC box binding factors, as well as YY1-like
proteins, specifically arising from more generally expressed
trans-regulator families to control gene expression in the male
germ line. Such adaptation may be an efficient means for
generating germ cell-specific transcription factors since it uti-
lizes generally expressed, and perhaps ancient (52), trans fac-
tors as well as response elements commonly found in RNA
polymerase II promoters. Notably, many germ cell-specific
promoters expressed in late spermatocytes and/or round sper-
matids contain CRE, YY1, and Y- and GC-box elements (23,
38, 55, 56), and unique, spermatogenic-cell- or testis-enriched
nuclear factors that bind these sites have been previously iden-
tified (16, 32, 35, 39, 42, 43, 49). Additional, novel coregulator
isoforms also may function in late spermatogenesis.
Analysis of the proacrosin gene, which contains binding sites
for all major SREBP coregulators and which is expressed in
both of these stages, provides an excellent opportunity to ex-
plore the role of coregulators in both cell- and stage-dependent
activation by SREBP2gc. Such analyses ultimately will expand
our understanding of the transcriptional network regulating
spermatogenesis and the unique placement of SREBP2gc
within it. GC-4spc cells should prove useful in this regard due
10686 WANG ET AL.MOL. CELL. BIOL.
to their expression of SREBP2gc as well as the cell-specific
regulation of proacrosin promoter activity that they exhibit.
Finally, what is the significance of SREBP2gc expression for
cholesterol synthesis during spermatogenesis? Recent studies
have shown that loss or inhibition of the function of dhcr24, a
terminal reductase in the cholesterol biosynthetic pathway,
disrupts spermatogenesis (41, 51). Several cholesterol biosyn-
thesis genes also are specifically up-regulated during late sper-
matogenesis (46, 48), which likely involves trans activation by
SREBP2gc. However, a number of observations indicate that
enhancement of cholesterol synthesis per se is not the role of
this transcription factor in meiotic and haploid germ cells. For
one thing, not all cholesterol biosynthetic genes are coordi-
nately up-regulated during late spermatogenesis (46). Accord-
ingly, cholesterol synthesis actually declines in pachytene sper-
matocytes and round spermatids (36), as does testicular
cholesterol content during sexual maturation (46). These facts
further argue that SREBP2gc has major functions distinct from
cholesterol synthesis and are consistent with the switch to a
sterol-independent mechanism of SREBP2 production in
these spermatogenic stages. While this may involve an in-
creased synthesis of certain cholesterol intermediates, such as
T-MAS (46), it is likely that a major role of SREBP2gc is to
regulate a totally new set of promoters uniquely expressed in
spermatocytes and spermatids.
This work was supported by Public Service Grant RO1 DK36468
and Center Grant DK32520.
We thank George Gagnon and Rachel Stock for their excellent
assistance with several aspects of this work. The mouse pgk-2 promot-
er-LacZ plasmid was provided by Y. Nakanishi (Kanazawa University,
Ishikawa, Japan), SQS gene promoter plasmids were obtained from I.
Shechter (Uniformed Services University of the Health Sciences, Be-
thesda, Md.), and the human CYP51 gene promoter construct was
provided by D. Rozman (University of Ljubljana, Ljubljana, Slovenia).
GC-4spc cells were kindly provided by Wolfgang Engel (University of
Go ¨ttingen, Go ¨ttingen, Germany).
1. Adham, I. M., K. Nayernia, and W. Engel. 1997. Spermatozoa lacking acrosin
protein show delayed fertilization. Mol. Reprod. Dev. 46:370–376.
2. Agustin, J. T., C. G. Wilkerson, and G. B. Witman. 2000. The unique
catalytic subunit of sperm cAMP-dependent protein kinase is the product of
an alternative C? mRNA expressed specifically in spermatogenic cells. Mol.
Biol. Cell 11:3031–3044.
3. Annicotte, J. S., K. Schoonjans, C. Haby, and J. Auwerx. 2001. An E-box in
pGL3 reporter vectors precludes their use for the study of sterol regulatory
element-binding proteins. BioTechniques 31:993–994
4. Bellefroid, E. J., M. Sahin, D. A. Poncelet, M. Riviere, C. Bourguignon, J. A.
Martial, P. L. Morris, T. Pieler, C. Szpirer, and D. C. Ward. 1998. Kzf1—a
novel KRAB zinc finger protein encoding gene expressed during rat sper-
matogenesis. Biochim. Biophys. Acta 1398:321–329.
5. Blendy, J. A., K. H. Kaestner, G. F. Weinbauer, E. Nieschlag, and G. Schutz.
1996. Severe impairment of spermatogenesis in mice lacking the CREM
gene. Nature 380:162–165.
6. Cunliffe, V., S. Williams, and J. Trowsdale. 1990. Genomic analysis of a
mouse zinc finger gene, Zfp-35, that is up- regulated during spermatogenesis.
7. Delmas, V., F. van der Hoorn, B. Mellstrom, B. Jegou, and P. Sassone-Corsi.
1993. Induction of CREM activator proteins in spermatids: down-stream
targets and implications for haploid germ cell differentiation. Mol. Endocri-
8. Eddy, E. M. 1998. Regulation of gene expression during spermatogenesis.
Semin. Cell Dev. Biol. 9:451–457.
9. Eddy, E. M., and D. A. O’Brien. 1998. Gene expression during mammalian
meiosis. Curr. Top. Dev. Biol. 37:141–200.
10. Edwards, P. A., D. Tabor, H. R. Kast, and A. Venkateswaran. 2000. Regu-
lation of gene expression by SREBP and SCAP. Biochim. Biophys. Acta
11. Fimia, G. M., D. De Cesare, and P. Sassone-Corsi. 1999. CBP-independent
activation of CREM and CREB by the LIM-only protein ACT. Nature
12. Foulkes, N. S., B. Mellstrom, E. Benusiglio, and P. Sassone-Corsi. 1992.
Developmental switch of CREM function during spermatogenesis: from
antagonist to activator. Nature 355:80–84.
13. Guan, G., P. Dai, and I. Shechter. 1998. Differential transcriptional regula-
tion of the human squalene synthase gene by sterol regulatory element-
binding proteins (SREBP) 1a and 2 and involvement of 5? DNA sequence
elements in the regulation. J. Biol. Chem. 273:12526–12535.
14. Guan, G., P. H. Dai, T. F. Osborne, J. B. Kim, and I. Shechter. 1997.
Multiple sequence elements are involved in the transcriptional regulation of
the human squalene synthase gene. J. Biol. Chem. 272:10295–10302.
15. Han, S. Y., L. Zhou, A. Upadhyaya, S. H. Lee, K. L. Parker, and J. DeJong.
2001. TFIIA?/?-like factor is encoded by a germ cell-specific gene whose
expression is up-regulated with other general transcription factors during
spermatogenesis in the mouse. Biol. Reprod. 64:507–517.
16. He, F., S. Narayan, and S. H. Wilson. 1996. Purification and characterization
of a DNA polymerase beta promoter initiator element-binding transcription
factor from bovine testis. Biochemistry 35:1775–1782.
17. Hecht, N. B. 1998. Molecular mechanisms of male germ cell differentiation.
18. Hofmann, M.-C., S. Narisawa, R. A. Hess, and J. L. Millan. 1992. Immor-
talization of germ cells and somatic testicular cells using the SV40 large T
antigen. Exp. Cell Res. 201:417–435.
19. Horton, J. D., J. L. Goldstein, and M. S. Brown. 2002. SREBPs: activators of
the complete program of cholesterol and fatty acid synthesis in the liver.
J. Clin. Investig. 109:1125–1131.
20. Ikeda, Y., J. Yamamoto, M. Okamura, T. Fujino, S. Takahashi, K. Takeuchi,
T. F. Osborne, T. T. Yamamoto, S. Ito, and J. Sakai. 2001. Transcriptional
regulation of the murine acetyl-CoA synthetase 1 gene through multiple
clustered binding sites for sterol regulatory element-binding proteins and a
single neighboring site for Sp1. J. Biol. Chem. 276:34259–34269.
21. Kashiwabara, S., Y. Arai, K. Kodaira, and T. Baba. 1990. Acrosin biosyn-
thesis in meiotic and postmeiotic spermatogenic cells. Biochem. Biophys.
Res. Commun. 173:240–245.
22. Khillan, J. S., A. Schmidt, P. A. Overbeek, B. de Crombrugghe, and H.
Westphal. 1986. Developmental and tissue-specific expression directed by
the alpha 2 type I collagen promoter in transgenic mice. Proc. Natl. Acad.
Sci. USA 83:725–729.
23. Kistler, M. K., P. Sassone-Corsi, and W. S. Kistler. 1994. Identification of a
functional cyclic adenosine 3?-5?-monophosphate response element in the
5?-flanking region of the gene for transition protein 1 (TP1), a basic chro-
mosomal protein of mammalian spermatids. Biol. Reprod. 51:1322–1329.
24. Kremling, H., S. Keime, K. Wilhelm, I. M. Adham, H. Hameister, and W.
Engel. 1991. Mouse proacrosin gene: nucleotide sequence, diploid expres-
sion, and chromosomal localization. Genomics 11:828–834.
25. Lee, C. H., L. Chang, and L. N. Wei. 1996. Molecular cloning and charac-
terization of a mouse nuclear orphan receptor expressed in embryos and
testes. Mol. Reprod. Dev. 44:305–314.
26. Liu, F., J. Tokeson, S. P. Persengiev, K. Ebert, and D. L. Kilpatrick. 1997.
Novel repeat elements direct rat proenkephalin transcription during sper-
matogenesis. J. Biol. Chem. 272:5056–5062.
27. Martianov, I., G. M. Fimia, A. Dierich, M. Parvinen, P. Sassone-Corsi, and
I. Davidson. 2001. Late arrest of spermiogenesis and germ cell apoptosis in
mice lacking the TBP-like TLF/TRF2 gene. Mol. Cell 7:509–515.
28. Nantel, F., L. Monaco, N. S. Foulkes, D. Masquiller, M. LeMeur, K. Hen-
riksen, A. Dierich, M. Parvinen, and P. Sasson-Corsi. 1996. Spermiogenesis
deficiency and germ-cell apoptosis in CREM-mutant mice. Nature 380:159–
29. Nayernia, K., A. Meinhardt, B. Drabent, I. M. Adham, C. Muller, M. Steckel,
U. Sancken, and W. Engel. 2003. Synergistic effects of germ cell expressed
genes on male fertility in mice. Cytogenet. Genome Res. 103:314–320.
30. Nayernia, K., S. Nieter, H. Kremling, H. Oberwinkler, and W. Engel. 1994.
Functional and molecular characterization of the transcriptional regulatory
region of the proacrosin gene. J. Biol. Chem. 269:32181–32186.
31. Nayernia, K., K. Reim, H. Oberwinkler, and W. Engel. 1994. Diploid expres-
sion and translational regulation of rat acrosin gene. Biochem. Biophys. Res.
32. Nikolajczyk, B. S., M. T. Murray, and N. B. Hecht. 1995. A mouse homo-
logue of the Xenopus germ cell-specific ribonucleic acid/deoxyribonucleic
acid-binding proteins p54/p56 interacts with the protamine 2 promoter. Biol.
33. Noce, T., Y. Fujiwara, M. Ito, T. Takeuchi, N. Hashimoto, M. Yamanouchi,
T. Higashinakagawa, and H. Fujimoto. 1993. A novel murine zinc finger
gene mapped within the tw18 deletion region expresses in germ cells and
embryonic nervous system. Dev. Biol. 155:409–422.
34. Palmiter, R. D., and R. L. Brinster. 1986. Germ-line transformation of mice.
Annu. Rev. Genet. 20:465–499.
35. Persengiev, S. P., P. J. Raval, S. Rabinovitch, C. F. Millette, and D. L.
Kilpatrick. 1996. Transcription factor Sp1 is expressed by three different
VOL. 24, 2004SREBP2gc REGULATES A GERM CELL-SPECIFIC PROMOTER10687
developmentally regulated messenger ribonucleic acids in mouse spermato-
genic cells. Endocrinology 137:638–646.
36. Potter, J. E., C. F. Millette, M. J. James, and A. A. Kandutsch. 1981.
Elevated cholesterol and dolichol synthesis in mouse pachytene spermato-
cytes. J. Biol. Chem. 256:7150–7154.
37. Rozman, D., M. Fink, G. M. Fimia, P. Sassone-Corsi, and M. R. Waterman.
1999. Cyclic adenosine 3?,5?-monophosphate(cAMP)/cAMP-responsive ele-
ment modulator (CREM)-dependent regulation of cholesterogenic lanos-
terol 14?-demethylase (CYP51) in spermatids. Mol. Endocrinol. 13:1951–
38. Schulten, H. J., W. Engel, K. Nayernia, and P. Burfeind. 1999. Yeast one-
hybrid assay identifies YY1 as a binding factor for a proacrosin promoter
element. Biochem. Biophys. Res. Commun. 257:871–873.
39. Schulten, H. J., K. Nayernia, K. Reim, W. Engel, and P. Burfeind. 2001.
Assessment of promoter elements of the germ cell-specific proacrosin gene.
J. Cell. Biochem. 83:155–162.
40. Shimano, H. 2001. Sterol regulatory element-binding proteins (SREBPs):
transcriptional regulators of lipid synthetic genes. Prog. Lipid Res. 40:439–
41. Singh, S. K., and S. Chakravarty. 2003. Antispermatogenic and antifertility
effects of 20,25-diazacholesterol dihydrochloride in mice. Reprod. Toxicol.
42. Sogawa, K., H. Imataka, Y. Yamasaki, H. Kusume, H. Abe, and Y. Fujii-
Kuriyama. 1993. cDNA cloning and transcriptional properties of a novel GC
box-binding protein, BTEB2. Nucleic Acids Res. 21:1527–1532.
43. Stelzer, G., and J. Don. 2002. Atce1: a novel mouse cyclic adenosine 3?,5?-
monophosphate-responsive element-binding protein-like gene exclusively
expressed in postmeiotic spermatids. Endocrinology 143:1578–1588.
44. Stromstedt, M., M. R. Waterman, T. B. Haugen, K. Tasken, M. Parvinen,
and D. Rozman. 1998. Elevated expression of lanosterol 14?-demethylase
(CYP51) and the synthesis of oocyte meiosis-activating sterols in postmeiotic
germ cells of male rats Endocrinology. 139:2314–2321. Erratum 139:3771.
45. Swinnen, J. V., P. Alen, W. Heyns, and G. Verhoeven. 1998. Identification of
diazepam-binding inhibitor/acyl-CoA-binding protein as a sterol regulatory
element-binding protein-responsive gene. J. Biol. Chem. 273:19938–19944.
46. Tacer, K. F., T. B. Haugen, M. Baltsen, N. Debeljak, and D. Rozman. 2002.
Tissue-specific transcriptional regulation of the cholesterol biosynthetic
pathway leads to accumulation of testis meiosis-activating sterol (T-MAS). J.
Lipid Res. 43:82–89.
47. Tascou, S., K. Nayernia, A. Samani, J. Schmidtke, T. Vogel, W. Engel, and
P. Burfeind. 2000. Immortalization of murine male germ cells at a discrete
stage of differentiation by a novel directed promoter-based selection strat-
egy. Biol. Reprod. 63:1555–1561.
48. Teruya, J. H., E. C. Salido, P. A. Edwards, and C. F. Clarke. 1991. Testis-
specific transcripts of rat farnesyl pyrophosphate synthetase are developmen-
tally regulated and localized to haploid germ cells. Biol. Reprod. 44:663–671.
49. Vanden Heuvel, G. B., S. E. Quaggin, and P. Igarashi. 1996. A unique variant
of a homeobox gene related to Drosophila cut is expressed in mouse testis.
Biol. Reprod. 55:731–739.
50. Wang, H., F. Liu, C. F. Millette, and D. L. Kilpatrick. 2002. Expression of a
novel, sterol-insensitive form of sterol regulatory element binding protein 2
(SREBP2) in male germ cells suggests important cell- and stage-specific
functions for SREBP targets during spermatogenesis. Mol. Cell. Biol. 22:
51. Wechsler, A., A. Brafman, M. Shafir, M. Heverin, H. Gottlieb, G. Damari, S.
Gozlan-Kelner, I. Spivak, O. Moshkin, E. Fridman, Y. Becker, R. Skaliter, P.
Einat, A. Faerman, I. Bjorkhem, and E. Feinstein. 2003. Generation of
viable cholesterol-free mice. Science 302:2087.
52. Worgall, T. S., S. R. Davis-Hayman, M. M. Magana, P. M. Oelkers, F.
Zapata, R. A. Juliano, T. F. Osborne, T. E. Nash, and R. J. Deckelbaum.
2004. Sterol and fatty acid regulatory pathways in a Giardia lamblia derived
promoter: evidence for SREBP as an ancient transcription factor. J Lipid
53. Yamagata, K., K. Murayama, M. Okabe, K. Toshimori, T. Nakanishi, S.
Kashiwabara, and T. Baba. 1998. Acrosin accelerates the dispersal of sperm
acrosomal proteins during acrosome reaction. J. Biol. Chem. 273:10470–
54. Yang, J., M. S. Brown, Y. K. Ho, and J. L. Goldstein. 1995. Three different
rearrangements in a single intron truncate sterol regulatory element binding
protein-2 and produce sterol-resistant phenotype in three cell lines. Role of
introns in protein evolution. J. Biol. Chem. 270:12152–12161.
55. Yiu, G. K., and N. B. Hecht. 1997. Novel testis-specific protein-DNA inter-
actions activate transcription of the mouse protamine 2 gene during sper-
matogenesis. J. Biol. Chem. 272:26926–26933.
56. Zhang, L. P., J. Stroud, C. A. Eddy, C. A. Walter, and J. R. McCarrey. 1999.
Multiple elements influence transcriptional regulation from the human tes-
tis-specific PGK2 promoter in transgenic mice. Biol. Reprod. 60:1329–1337.
57. Zinn, S. A., K. M. Ebert, N. D. Mehta, J. Joshi, and D. L. Kilpatrick. 1991.
Selective transcription of rat proenkephalin fusion genes from the spermat-
ogenic cell-specific promoter in testis of transgenic mice. J. Biol. Chem.
10688 WANG ET AL.MOL. CELL. BIOL.